SNP Array for Small-Shrimp (Genus Acetes) Origin Determination Using Machine Learning.

Accurate origin determination of seafood is crucial for consumer trust and safety. This study was performed to develop a machine learning-based single-nucleotide polymorphism (SNP) analysis technique to determine the origin of Acetes species in salted small-shrimp products. Mitochondrial DNA (COI and 16S rRNA) analysis revealed genetic variations among species and origins. Eight candidate SNPs were identified, six of which were developed into markers for genotyping analysis. Using the developed markers, an SNP array was created and SNP data from salted small-shrimp samples were obtained. Machine learning analysis using a supervised learning algorithm achieved 100% accuracy in classifying the origin of Acetes based on SNP data. This method offers a reliable method for regulatory bodies to combat food fraud and ensure product integrity. The approach can be further improved by expanding the data set to encompass a wider range of species and origins. This study highlights the potential of SNP analysis and machine learning for ensuring seafood authenticity and promoting sustainable practices.

Clinical Assessment of Thermotherapy Applications during Hepatectomy and Laparotomy in Sturgeon (Acipenser ruthenus): Impact on Bioparameter Variations Based on Liver Condition.

Surgical techniques are gaining attention for treating physical diseases in aquaculture and aquarium fish. Sturgeon is a suitable species for surgical experiments due to its industrial significance. Maintaining homeostasis is crucial during surgical procedures, and the liver plays a major role in immune regulation. High temperature is suggested to improve physiological activity and wound healing. This study investigated differences in hepatectomy sturgeons' tolerance and histopathological responses of internal organs. Moreover, this study investigated the effects of high temperatures on wound healing and hematopoietic recovery in fish undergoing surgical procedures. The liver condition was found to play a pivotal role in the analysis, and cortisol levels were affected by anesthesia. The results showed that high temperature facilitated hematopoietic recovery and wound healing, but excessive induction of physiological activity caused damage. Managing high temperatures and liver conditions induced a remarkable improvement in wound healing. However, anesthesia itself can be a significant stressor for fish, and wound healing requires a greater amount of energy. Further research is needed to understand the stress factors caused by surgical procedures and anesthesia and to promote animal welfare in fishery products.

Identification of Potential Hazards Associated with South Korean Prawns and Monitoring Results Targeting Fishing Bait.

This study detected two potential pathogens, Vibro parahaemolyticus, which causes acute hepatopancreatic necrosis disease (AHPND), and white spot syndrome virus (WSSV), in fishing bait in South Korea. However, their infectious nature was not confirmed, possibly due to the degradation caused by freezing/thawing or prolonged storage under frozen conditions. While infectivity was not confirmed in this study, there is still a significant risk of exposure to these aquatic products. Furthermore, fishing bait and feed should be handled with caution as they are directly exposed to water, increasing the risk of disease transmission. In Australia, cases of WSSV infection caused by imported shrimp intended for human consumption have occurred, highlighting the need for preventive measures. While freezing/thawing is a method for inactivating pathogens, there are still regulatory and realistic issues to be addressed.

Draft genome of the Korean smelt Hypomesus nipponensis and its transcriptomic responses to heat stress in the liver and muscle.

Pond smelt (Hypomesus nipponensis) is a cold-freshwater fish species and a winter economic aquaculture resource in South Korea. Because of its high susceptibility to abnormal water temperature from global warming, a large number of smelt die in hot summers. Here, we present the first draft genome of H. nipponensis and transcriptomic changes in molecular mechanisms or intracellular responses under heat stress. We combined Illumina and PacBio sequencing technologies to generate the draft genome of H. nipponensis. Based on the reference genome, we conducted transcriptome analysis of liver and muscle tissues under normal (NT, 5°C) vs. warm (HT, 23°C) conditions to identify heat stress-induced genes and gene categories. We observed a total of 1987 contigs with N50 of 0.46 Mbp, with the largest contig (3.03 Mbp) in the assembled genome. A total of 20,644 protein-coding genes were predicted, and 19,224 genes were functionally annotated: 15,955 genes for Gene Ontology terms and 11,560 genes for KEGG Orthology. We conducted the lost and gained genes analysis compared with three species that: human, zebrafish, and salmon. In the lost genes analysis, we detected that smelt lost 4461 (22.16%), 2825 (10.62%), and 1499 (3.09%) genes compare with above three species, respectively. In the gained genes analysis, we observed that smelt gained 1133 (5.49%), 1670 (8.09%), and 229 (1.11%) genes compared with the above species, respectively. From transcriptome analysis, a total of 297 and 331 differentially expressed genes (DEGs) with a false discovery rate <0.05 were identified in the liver and muscle tissues, respectively. Gene enrichment analysis of DEGs indicates that upregulated genes were significantly enriched for lipid biosynthetic process (GO:0008610, P < 0.001) and regulation of apoptotic process (GO:0042981, P < 0.01), and genes were downregulated by immune responses such as myeloid cell differentiation (GO:0030099, P < 0.001) in the liver under heat stress. In muscle tissue, upregulated genes were enriched for hypoxia (GO:0001666, P < 0.05), transcription regulator activity (GO:0140110, P < 0.001), and calcium-release channel activity (GO:0015278, P < 0.01), and genes were downregulated for a nicotinamide nucleotide biosynthetic process (GO:0019359, P < 0.01). The results of KEGG pathway analysis were similar to that of gene enrichment analysis. The draft genome and transcriptomic of H. nipponensis will be a useful genetic resource for functional and evolutionary studies. Our findings will improve understanding of molecular mechanisms and heat responses and be useful for predicting survival of the smelt and its closely related species under global warming.

Genotyping-by-Sequencing of the regional Pacific abalone (Haliotis discus) genomes reveals population structures and patterns of gene flow.

Continuous monitoring of the present genetic status is essential to preserve the genetic resource of wild populations. In this study, we sequenced regional Pacific abalone Haliotis discus samples from three different locations around the Korean peninsula to assess population structure, utilizing Genotyping-by-Sequencing (GBS) method. Using PstI enzyme for genome reduction, we demonstrated the resultant library represented the whole genome region with even spacing, and as a result 16,603 single nucleotide variants (SNVs) were produced. Genetic diversity and population structure were investigated using several methods, and a strong genetic heterogeneity was observed in the Korean abalone populations. Additionally, by comparison of the variant sets among population groups, we were able to discover 26 Korean abalone population-specific SNVs, potentially associated with phenotype differences. This is the first study demonstrating the feasibility of GBS for population genetic study on H. discus. Our results will provide valuable data for the genetic conservation and management of wild abalone populations in Korea and help future GBS studies on the marine mollusks.

Classification of Takifugu rubripes, T. chinensis and T. pseudommus by genotyping-by-sequencing.

Takifugu rubripes is more expensive than other species of the genus because of its high protein content and special flavor. However, it is easily confused with imported T. chinensis and T. pseudommus because they have similar morphological characteristics. We identified single nucleotide polymorphism (SNP) markers of T. rubripes by genotyping-by-sequencing (GBS) and evaluated their ability to distinguish among T. rubripes, T. chinensis, and T. pseudommus. In all, 18 polymorphic SNPs were subjected to phylogenetic analyses of the three Takifugu species. Additionally, we subjected a second set of samples to Sanger sequencing to verify that the polymorphic SNPs could be used to evaluate the genetic variation among the three Takifugu species. A phylogenetic tree that included the analyzed sequence of set A, which is referred to as the reference sequence, and a validation sequence of set B with 18 SNPs were produced. Based on this phylogenetic tree and STRUCTURE analyses, T. rubripes, T. chinensis and T. pseudommus have low genetic variation and should be considered the same gene pool. Our findings suggest that further studies are needed to estimate the genetic association of the three Takifugu species.

Expression of Heat Shock Proteins in Thermally Challenged Pacific Abalone Haliotis discus hannai.

Summer mortality, caused by thermal conditions, is the biggest threat to abalone aquaculture production industries. Various measures have been taken to mitigate this issue by adjusting the environment; however, the cellular processes of Pacific abalone (Haliotis discus hannai) have been overlooked due to the paucity of genetic information. The draft genome of H. discus hannai has recently been reported, prompting exploration of the genes responsible for thermal regulation in Pacific abalone. In this study, 413 proteins were systematically annotated as members of the heat shock protein (HSP) super families, and among them 26 HSP genes from four Pacific abalone tissues (hemocytes, gill, mantle, and muscle) were differentially expressed under cold and heat stress conditions. The co-expression network revealed that HSP expression patterns were tissue-specific and similar to those of other shellfish inhabiting intertidal zones. Finally, representative HSPs were selected at random and their expression patterns were identified by RNA sequencing and validated by qRT-PCR to assess expression significance. The HSPs expressed in hemocytes were highly similar in both analyses, suggesting that hemocytes could be more reliable samples for validating thermal condition markers compared to other tissues.

Quantitative analysis of Alaska pollock in seafood products by droplet digital PCR.

A highly accurate quantitative method, based on the new technique, droplet digital PCR (ddPCR), was applied to determine the content of Alaska pollock (Gadus chalcogrammus) in seafood products. Using this method, we found a linear relationship among raw sample weight, DNA concentration and DNA copy number. We also established a formula to calculate the raw sample weight, based on the number of DNA copies. To confirm the accuracy and applicability of this method, mixed samples of known composition were analyzed. Results from this study indicated that the ddPCR method described is suitable for quantifying Alaska pollock in seafood products and has the potential applied to a variety of tasks in food quality certification.

Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus Takifugu.

Pufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene.

Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China.

Isolation and characterization of novel microsatellite markers for the northern mauxia shrimp, Acetes chinensis, using pyrosequencing.

Among the 14 species in the Acetes genus, Acetes japonicus and Acetes chinensis, are the only and also abundant two species around the Korean Peninsula, and are used in traditional recipes. These species are of great economic importance, but little is known about their population genetics, despite the fact that information of this kind is important for stock assessment, fisheries management and identification of origin. A total of 9 microsatellite (MS) markers for A. chinensis were developed using pyrosequencing techniques. Polymorphisms of these markers were evaluated in 96 wild individuals collected from the Yellow Sea off the coast of Korea. A total of 133 alleles were detected at nine loci, with a cross-species transferability of 56% with A. japonicus. These markers will facilitate assessment of population genetic diversity in the genus Acetes.