Norovirus(NoV)is a prevalent etiological agent of foodborne viral gastroenteritis worldwide,accounting for over 50％of all foodborne disease outbreaks and constituting a substantial public health burden across diverse populations.This non-enveloped,single-stranded RNA virus belongs to the Caliciviridae family and is categorized into at least seven genogroups(G Ⅰ-GⅦ),with GⅡ being the predominant genogroup responsible for most human infections.Reverse transcription quantitative real-time PCR(RT-qPCR)has been established as the gold standard for NoV molecular detection owing to its superior sensitivity,specificity,and capacity for quantitative analyses.However,numerous methodological challenges persistently compromise assay reliability,including complex RT-qPCR inhibitors in clinical and environmental matrices,suboptimal RNA recovery rates during nucleic acid extraction procedures,and spontaneous degradation of RNA templates prior to amplification.These technical variables frequently result in false-negative results,thereby undermining diagnostic accuracy and epidemiological surveillance efforts.Consequently,the implementation of robust quality control materials throughout the entire analytical workflow,from nucleic acid extraction through reverse transcription to final amplification,represents a critical safeguard to ensure result validity.To address the notable deficiency in biosafety standard reference materials specifically designed for monitoring NoV contamination within laboratory environments,we engineered a novel Qβbacteriophage-based armored RNA(AR)standard reference material.This innovative construct was designed to simultaneously encapsulate detection targets for the GⅡ genogroup of NoV and the giant panda(Ailuropoda melanoleuca)RPS15a gene,serving as an internal control for comprehensive biosafety monitoring.The development and rigorous characterization of this dual-target armored RNA standard,designated AR-NS15,enable a more thorough and dependable surveillance of potential biological safety hazards within laboratory settings,thereby substantially elevating the overall laboratory biosafety management standards. The full-length cDNA fragment was artificially synthesized through de novo gene assembly,comprising the Qβ bacteriophage maturase-encoding gene,capsid protein-encoding gene,packaging signal,and the concatenated detection targets(NoV GⅡ-specific region and RPS15a sequence).This synthetic cassette was subcloned into the pET-28a(+)expression vector using Ndel and Xhol restriction sites to construct the recombinant plasmid pET-QN-S15.The sequence-verified construct was transformed into Escherichia coli BL21(DE3)cells for high-level protein expression.Bacterial cultures were grown at 37 ℃ in LB medium containing 50 μg/mL kanamycin up to an optical density of 0.6 at 600 nm.Protein expression was induced using 1 mmol/L isopropyl β-D-l-thiogalactopyranoside(1PTG)for 3 h at 37 ℃ with vigorous shaking.The clarified lysate was subjected to ultracentrifugation at 85,000 r/min for 4 h at 4 ℃ to pellet the VLPs.The concentrated VLPs were further purified by size-exclusion chromatography using a Sephacryl S-500 HR gel filtration column equilibrated with phosphate-buffered saline.Purified AR-NS15 preparations were characterized using transmission electron microscopy(TEM)after negative staining with 2％uranyl acetate.For value assignment and stability assessment,absolute quantification was performed using a validated RT-PCR assay,with a standard curve generated from serial dilutions of linearized plasmid DNA at known concentrations.Homogeneity testing was conducted,analyzing 10 randomly selected units from the batch,while stability studies were performed under accelerated conditions(4 ℃ and-20 ℃)over defined time intervals. SDS-PAGE analysis of IPTG-induced E.coli lysates revealed a prominent protein band at approximately 14.2 kDa,corresponding to the expected molecular weight of the Qβ capsid protein monomer,confirming robust recombinant construct expression.Subsequent purification steps effectively eliminated host-derived contaminating proteins and residual nucleic acids,as evidenced by the absence of extraneous bands on Coomassie blue-stained gels and undetectable plasmid DNA by agarose gel electrophoresis.TEM demonstrated that the purified AR-NS15 formed uniform,non-aggregated spherical virus-like particles with structural integrity,measuring approximately 25 nm in diameter and resembling native Qβ phage morphology.The quantitative characterization indicated that AR-NS15 contained 9.59× 1010 copies/μL of encapsidated RNA targets,providing an abundant and consistent source of reference material.The homogeneity evaluation confirmed excellent batch uniformity.Stability studies demonstrated that AR-NS15 remained stable when stored at 4 ℃ for up to 15 days without significant degradation,whereas storage at-20 ℃ preserved full viability for at least 60 days,with minimal loss of detectable RNA targets.These findings collectively validated the suitability of AR-NS15 as a long-term reference material under standard laboratory storage conditions. This study successfully developed a homogeneous and stable armored RNA standard reference material,AR-NS15,that simultaneously encapsulates detection targets for GⅡ norovirus and the giant panda RPS15a gene.This Qβ VLP-based delivery system ensured RNase resistance and biological safety,making it an ideal surrogate for native viral particles in molecular diagnostics.With its precisely assigned copy number,exceptional batch-to-batch consistency,and demonstrated thermal stability,AR-NS15 provides comprehensive quality control for nucleic acid extraction,reverse transcription,and amplification.This dual-target reference material enables laboratories to monitor both analytical variables and extrinsic contamination risks,thereby establishing a robust quality assurance framework that significantly improves the accuracy and reliability of NoV molecular detection,while enhancing overall laboratory biosafety protocols.
As an economically important aquaculture species in China,spotted sea bass(Lateolabrax maculatus)has flourished and made notable contributions to the national fishery economy over the past few decades.However,the"south-central-north relay"pattern of cross-regional farming in L.maculatus has led to germplasm mixing among farmed populations across China,posing a major constraint on the sustainable development of the industry.Therefore,understanding the phenotypic variations among populations in major production areas and assessing the shaping effects of aquaculture environments are crucial for improving the overall quality of L.maculatus.In this study,310 market-size L.maculatus strains from three representative production areas in Guangdong,Fujian,and Shandong were examined.Nineteen phenotypic indicators encompassing morphological and muscle texture traits were compared using principal component and hierarchical cluster analyses,and further integrated with nutritional quality evaluation to provide a comprehensive assessment of regional differences in morphology and quality.The results showed that(1)although principal component and cluster analyses confirmed the occurrence of germplasm admixture among populations,significant morphological and muscle texture trait differences were still observed among the regions.L.maculatus from Guangdong exhibited a phenotype characterized by higher fullness(1.90％±0.15％)and body height(9.64±0.38 cm),but shorter body length(35.21±1.45 cm)and caudal peduncle(7.03±0.41 cm),as well as a smaller head(10.13±0.57 cm)and stronger muscle adhesiveness(-25.05±6.73 g·s).L.maculatus from Fujian showed a more slender body shape with a well-developed caudal peduncle,and exhibited the highest values in key texture parameters,including hardness(7 069.34±536.63 g)and springiness(0.69±0.06).The Shandong population displayed a general body shape similar to that from Fujian,but with a more elongated caudal peduncle(9.06±0.74 cm);(2)the muscle nutritional profiles of L.maculatus from the three regions exhibited distinct characteristics,each with distinct advantages.L.maculatus from Guangdong had higher lipid content(3.61％±1.51％)and a more balanced amino acid composition.The Fujian population showed higher protein(20.71％±0.59％)and lower lipid content(1.80％±0.76％),along with a higher proportion of umami amino acids(5.89±0.21 g/100 g),resulting in superior flavor quality.The Shandong population was characterized by markedly higher eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)contents(7.46％±0.65％and 23.01％±1.16％,respectively),demonstrating the most prominent advantage in functional fatty acid nutrition;(3)environmental variation,especially in annual mean temperature and salinity,has emerged as the major driving force for the morphology,muscle texture traits,and nutritional divergence in L.maculatus.Pearson correlation analysis demonstrated that caudal peduncle length,EPA and DHA proportions,and crude protein were strongly negatively correlated with temperature but positively correlated with salinity.In contrast,fullness,∑ω-6 PUFA,and crude lipid content showed opposite correlations.Therefore,under the current aquaculture model,the stable production of L.maculatus products with specific quality advantages can still be achieved through scientific regulation of the aquaculture environment,even when germplasm sources are complex.These findings provide a scientific basis for differentiated brand development of L.maculatus across regions and offer theoretical support for environment-based quality improvement and targeted breeding of germplasm admixtures,which are of great significance for enhancing the product quality,efficiency,and sustainability of the L.maculatus industry.
The global aquaculture product industry is undergoing a strategic shift from"quality-driven"to"efficiency-driven"development,with consumer demand increasingly focused on premium seafood characterized by superior flavor and nutritional value.As the most productive marine shellfish in China,oyster yield reached 7.252 million tons in 2024.Glycogen content is a crucial determinant of oyster sweetness,flavor,and texture,and plays vital roles in energy metabolism and stress tolerance.Consequently,it has emerged as a key target trait for oyster germplasm innovation.Substantial genetic breeding research has been conducted on the Pacific oyster(Crassostrea gigas)with established heritability estimates for both growth traits and glycogen content,demonstrating significant potential for genetic improvement.In contrast,the Jinjiang oyster(Crassostrea ariakensis),an economically and ecologically important estuarine species in China,lags considerably behind.Although valued for their large size,attractive shell morphology,and sweet flavor,particularly in high-end markets,systematic data on the heritability of their growth traits are scarce,and crucially,no genetic parameter estimates for glycogen content have been reported to date.This knowledge gap severely impedes the development of high-quality fast-growing C.ariakensis strains through scientific breeding.To enable the effective selective breeding of high-quality,fast-growing strains,this study aimed to systematically estimate the genetic parameters of shell height,shell length,and glycogen content in C.ariakensis. A nested mating design(one male × four females)was used to create 72 full-sib families.All families were reared in a common pond environment to minimize non-genetic effects.Twenty families(600 individuals)were sampled at 330 days of age.Shell height and length were measured using a Vernier caliper.The glycogen content of dry soft tissues was determined using near-infrared spectroscopy(NIRS).Data were analyzed using ANOVA and Tukey's multiple range test to examine family differences.Genetic parameters were estimated in a reduced individual animal model using the ASReml package in R. Descriptive statistics for the 600 individuals revealed a mean shell height of(77.58±13.38)mm,shell length of(63.02±12.41)mm,and glycogen content of(208.10±67.61)mg/g.Glycogen content exhibited the highest variability among the traits,with a coefficient of variation(CV)of 0.32,compared to 0.17 and 0.20 for shell height and shell length,respectively.Substantial phenotypic variation within a population is a prerequisite for successful selective breeding. Analysis of variance indicated significant differences(P＜0.05)in all three traits among the half-sib families,but not among full-sib families within the same half-sib group.Regarding growth traits,families F53 and F55(sired by male 14)achieved the highest shell height(86.79 mm),and families F66 and F68(sired by male 17)achieved the longest shell length(70.02 mm).Pronounced variation in glycogen content was observed among families.Families F36 and F34(sired by male 9)displayed the highest glycogen levels,at 270.64 mg/g and 260.08 mg/g,respectively,representing an increase of 30.05％and 24.97％over the population mean.In contrast,families F09 and F10(sired by male three)had the lowest glycogen content,averaging 145.94 mg/g.Clear stratification of family performance underscores the potential for identifying and selecting superior genetic lines. Heritability estimates derived from individual animal models were moderate to high for all traits.Shell height heritability was 0.37±0.12,shell length was 0.48±0.14,and glycogen content was 0.45±0.13.The bivariate animal model revealed significant positive genetic(rg=0.74±0.12)and phenotypic(rp=0.62±0.04)correlations between shell height and shell length.The results demonstrate that key growth and quality traits in C.ariakensis are under moderate to strong additive genetic control.The heritability estimates for shell height and shell length were consistent with those of previous reports on C.gigas,confirming their suitability for selective breeding.The strong genetic correlation between these growth traits suggests that they are influenced by a shared set of genes or closely linked genetic loci,allowing for concurrent genetic improvements and simplifying breeding objectives. Most significantly,this study provided the first estimate of glycogen content heritability in C.ariakensis.The value of 0.45 falls within the range reported for C.gigas(0.29-0.61),firmly establishing glycogen content as a heritable trait in Jinjiang oysters.The identification of high-glycogen families,such as F34 and F36,which significantly outperformed the population average,provides invaluable core breeding material for establishing elite lines.This study represents the first systematic assessment of genetic parameters for growth and glycogen content in C.ariakensis,filling a critical knowledge gap. In conclusion,our findings provide crucial genetic parameters necessary to launch scientific breeding programs for C.ariakensis.Moderate to high heritability indicates that significant genetic gain can be achieved through family-or individual-based selection.The identified high-glycogen families provide valuable core breeding materials.Collectively,these findings fill a critical gap in the genetic knowledge of C.ariakensis and provide the essential parameters required to initiate a formal breeding program aimed at developing new high-quality varieties that meet the evolving demands of the premium seafood market.
The yellowtail kingfish(Seriola aureovittata)is a large,fast-swimming fish species characterized by high and aggressive feeding activity,rapid growth rate,and tender flesh.Feed intake is a key factor that influences fish growth and production performance,and appetite is a major determinant of feed intake.To explore the role and potential mechanisms of orexin and its receptor ox2r in the regulation of feeding behavior in SS.aureovittata,we performed homologous cloning to obtain the open reading frame(ORF)sequences of the orexin and ox2r genes in S.aureovittata.Quantitative real-time PCR was used to analyze the expression of these genes across various tissues and developmental stages,as well as their regulatory roles in response to starvation and subsequent refeeding. The results showed that the ORF of orexin in S.aureovittata is 450 bp long,encoding 149 amino acids,including a 46-amino acid signal peptide,a 43-amino acid Orexin A mature peptide,and a 28-amino acid Orexin B mature peptide.The ox2r gene ORF is 1,269 bp,encoding 422 amino acids.Amino acid sequence alignment revealed that orexin and ox2r are highly conserved among teleosts.Phylogenetic analysis indicated that S.aureovittata orexin and ox2r are most closely related to those of Seriola dumerili.The mRNA expression of orexin and ox2r was detected in all 12 examined tissues,with the highest levels observed in the brain,followed by the pituitary gland and stomach.Expression of orexin and ox2r mRNA was also detected in the sperm and eggs,indicating parental inheritance.Both genes are involved in the embryonic and larval development of S.aureovittata,with significant expression changes observed during the transition from endogenous to exogenous nutrition.Starvation stimulated the upregulation of orexin and ox2r mRNA in the brain and pituitary,with expression levels returning to control group levels after 7 d of refeeding. These results suggest that the orexin system may be involved in organ development and feeding regulation during the early growth and feeding of S.aureovittata,thereby providing valuable insights into the mechanisms of feeding regulation in this species.
Often referred to as renowned"ecosystem engineers",oyster reefs perform crucial ecological functions,such as water purification,providing habitat and shelter for other organisms,and stabilizing coastlines.As important components of blue carbon sinks,oyster reefs sequester carbon by absorbing carbon dioxide from seawater and undergoing calcification,biological assimilation,and sedimentation,resulting in carbon fixation.However,global wild oyster resources have declined sharply,leading to the destruction or loss of the ecological structure and functions of oyster reef habitats.Currently,most studies on oyster reefs in China have focused on temperate regions,with relatively few studies on tropical waters,such as the coastal waters of Hainan Island.Investigating the community structure and carbon stocks of oyster reefs along the coast of Hainan Island will be conducive to the protection and restoration of coastal ecosystems in China. To fill this knowledge gap,we conducted sampling and environmental monitoring across 12 oyster reefs along the coast of Hainan Island.Based on preliminary surveys,reef areas were categorized into four habitat types:Estuarine oyster reefs,intertidal natural oyster reefs,mangrove oyster reefs,and coastal engineering area oyster reefs.The research methodology comprised the following steps to ensure comprehensive and accurate data collection.First,aerial imagery captured by drones was used to photograph the sampling sites,and specialized software was used to delineate the reef boundaries and areas.These measurements were integrated with satellite imagery to estimate total reef area and oyster coverage.For specific stations requiring validation,on-site surveys were conducted to verify spatial data.Second,a multi-parameter water quality analyzer was used to determine seawater temperature,salinity,pH,and chlorophyll-a concentration to characterize the aquatic environment of each reef area.Third,depending on habitat types,quadrats of appropriate sizes were established for quantitative sampling,supplemented by qualitative collection in the surrounding areas.After rinsing and screening,the samples were stored at low temperature and transported to the laboratory for further analysis.Fourth,all reef-dwelling organisms were identified and quantified using standard taxonomic guidelines.Oyster samples were identified to the species level using molecular methods,whereas other reef-dwelling organisms were identified at the family level.Species composition,oyster diversity,and proportional abundance,as well as the abundance and wet weight of live oysters and other reef-dwelling taxa were recorded for subsequent community analysis.Lastly,the dried shells,soft tissues,and shell debris from reef-dwelling shellfish were ground,sieved,and stored separately.Carbon content was measured using an elemental analyzer,and oyster reef carbon storage was quantified by applying the standard shellfish carbon sequestration equation. The results of this study were as follows:(1)A total of 53 species of reef-dwelling organisms were identified,belonging to seven phyla,nine classes,18 orders,and 28 families,with Bivalvia and Gastropoda being the dominant classes.(2)Eight oyster species were identified.Saccostrea malabonensis was the dominant species at most survey stations(except Longlou and Guoheyuan)with relative abundances ranging from 6.25％to 100％.Saccostrea mordax was primarily found in intertidal natural oyster reef areas,accounting for 93.75％and 50.00％of its relative abundance in Longlou and Huiwen Town,respectively.Crassostrea iredalei was distributed across all surveyed habitats,except for the intertidal natural oyster reefs.(3)Among the four habitats,the species diversity index(H')and richness index(d)of intertidal natural oyster reefs were higher than those of other habitats,with the highest values recorded at Haitou Town(1.83 for H'and 3.28 for d),followed by estuarine oyster reefs and mangrove oyster reefs,while coastal engineering area oyster reefs had the lowest indices,indicating that intertidal natural oyster reefs provide a more favorable habitat for reef-dwelling organisms.(4)The composition of reef-dwelling organisms and the content of shell remains varied significantly among stations:intertidal oyster reefs were mainly composed of live oysters(12.8％-77.9％)and shell remains(8.7％-86.7％);estuarine oyster reefs and coastal engineering area oyster reefs contained higher proportions of shell remains(67.2％-79.6％);and mangrove oyster reefs had higher biomass of other reef-dwelling shellfish(e.g.,mussels and beach snails,12.4％-28.9％).(5)In terms of biomass and carbon stock of reef-dwelling organisms in different oyster reef habitats,intertidal natural oyster reefs had significantly higher total biomass and total carbon stock than other habitats,with Haitou Town showing the highest value(total live biomass:49,760.86 t;shell remains:46,503.71 t;total carbon stock:14,844.48 tC). Through field surveys and measurements of carbon content in the shells and soft tissues of reef-dwelling shellfish,this study systematically investigated species composition,community structure,and their relationships with environmental factors across different oyster reef habitats.We also quantified the biomass and carbon storage capacities of reef-dwelling shellfish in these habitats.These results provide valuable data to support the protection and restoration of oyster reef ecosystems,particularly in understudied tropical coastal regions such as Hainan Island.The results of this study enhance our understanding of coastal ecosystem dynamics and strengthen the scientific basis for ecological conservation and restoration efforts in China's coastal zones.
