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2025, 46(1):161-182.
DOI: 10.19663/j.issn2095-9869.20241024002
Abstract:
This article reviews the importance of biosecurity in aquaculture and its role in promoting sustainable and high-quality development. As a strategic and comprehensive method based on risk analysis, the core objective of biosecurity is to prevent and control disease risks, which ensures the health and sustainable development of the industry. This article elaborates on the concept and historical development of biosecurity, distinguishing it from biosafety, disease prevention, aquaculture, and animal health while exploring the connections and differences between these concepts. The authors proposed that animal health needs three approaches, including biosecurity, animal welfare, and ecological intervention. Six elements for constructing a biosecurity system and implementing the biosecurity plan are identified. Subsequently, the article outlines the key scientific and technological issues involved in the biosecurity framework and introduces the current state of research and development in the fields of science, technology, and policy, both internationally and domestically. Furthermore, this article discusses the role of global and regional organizations in promoting aquaculture biosecurity strategies and the practices of different countries and regions in constructing aquaculture biosecurity regulatory systems. In particular, it introduces how international organizations such as the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH) emphasize the application of the biosecurity concept to promote the sustainable development of aquaculture. This article also discusses the evaluation of biosecurity in aquaculture enterprises and its technical content. Using the cultivation of specific pathogen-free (SPF) shrimp seedlings as an example, this article introduces the development of biosecurity in international enterprises and the practices employed by the authors in shrimp hatcheries. Finally, this article highlights the challenges and opportunities for China in the field of aquaculture biosecurity and discusses the future direction of biosecurity strategies. This includes constructing a roadmap for the long-term development strategy of the national biosecurity plan; strengthening research, education, and awareness of biosecurity; prioritizing the development of biosecurity for the aquatic seed industry; and building an aquatic seed industry system with a high standard of biosecurity to ensure the sustainable and high-quality development of aquaculture industry in China.
This article reviews the importance of biosecurity in aquaculture and its role in promoting sustainable and high-quality development. As a strategic and comprehensive method based on risk analysis, the core objective of biosecurity is to prevent and control disease risks, which ensures the health and sustainable development of the industry. This article elaborates on the concept and historical development of biosecurity, distinguishing it from biosafety, disease prevention, aquaculture, and animal health while exploring the connections and differences between these concepts. The authors proposed that animal health needs three approaches, including biosecurity, animal welfare, and ecological intervention. Six elements for constructing a biosecurity system and implementing the biosecurity plan are identified. Subsequently, the article outlines the key scientific and technological issues involved in the biosecurity framework and introduces the current state of research and development in the fields of science, technology, and policy, both internationally and domestically. Furthermore, this article discusses the role of global and regional organizations in promoting aquaculture biosecurity strategies and the practices of different countries and regions in constructing aquaculture biosecurity regulatory systems. In particular, it introduces how international organizations such as the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH) emphasize the application of the biosecurity concept to promote the sustainable development of aquaculture. This article also discusses the evaluation of biosecurity in aquaculture enterprises and its technical content. Using the cultivation of specific pathogen-free (SPF) shrimp seedlings as an example, this article introduces the development of biosecurity in international enterprises and the practices employed by the authors in shrimp hatcheries. Finally, this article highlights the challenges and opportunities for China in the field of aquaculture biosecurity and discusses the future direction of biosecurity strategies. This includes constructing a roadmap for the long-term development strategy of the national biosecurity plan; strengthening research, education, and awareness of biosecurity; prioritizing the development of biosecurity for the aquatic seed industry; and building an aquatic seed industry system with a high standard of biosecurity to ensure the sustainable and high-quality development of aquaculture industry in China.
2024, 45(2):257-266.
DOI: 10.19663/j.issn2095-9869.20221211002
Abstract:
Aeromonas hydrophila (Family: Aeromonadaceae) is a traditional aquatic animal pathogen. It has been widely prevalent in our country since the 1980s and 1990s, causing diseases in a series of freshwater fish, leading to serious economic losses. It has been confirmed to be one of the main pathogens in freshwater aquaculture worldwide. The American shad (Alosa sapidissima) is one of the biggest shad in the world and grows much faster than other shad. Because of its delicious taste as reeves shad (Tenualosa reevesii), American shad were introduced into China from the USA by the Shanghai Fisheries Research Institute for local farming in 1998 and is widely welcomed in Shanghai, Jiangsu, and Zhejiang Provinces. In recent years, with the rapid development of the American shad culturing, fish diseases have become a major threat to fish farming. However, because most of the American shad are cultured in extensive ponds, few diseases have been reported in China. In this study, we reported a case of A. hydrophila infection in American shad. In 2021, the disease outbreak was observed in American shad cultured in Linyi City, Shandong Province, with severe mortality. The daily mortality could be up to 2.5%. The fish were cultured in indoor ponds for breeding and outdoor ponds when fish reached 300 g. The American shad were cultured with underground water and water was changed 1–3 times daily. The water temperature was 18–20 ℃. The fish were fed largemouth bass (Micropterus salmoides) commercial feed, and the daily feeding rate was approximately 2%. The disease broke out in the indoor ponds first, and then in outdoor ponds. The cumulative mortality was approximately 90% in 2 months. Enrofloxacin was administered orally, but no effects were observed and the disease continued to progress. The typical disease symptoms in the American shad were furunculosis or ulceration, with shedding scales and surface bleeding, especially on the tail, sometimes swollen and pus-filled. In autopsies of the diseased fish, ascites were found in the fish abdomen, and dark red necrosis on the liver, with sepsis and enteritis. The liver, spleen, and kidney of American shad with typical symptoms were collected and cut into about 1 cm3 tissue blocks, immersed in Davidson's Fixative (Davidson's AFA) for 24 h, and preserved in 70% ethanol. The tissues were mounted onto glass slides with hematoxylin and eosin staining for histological analysis. Histopathological results showed swollen liver cells, vacuolar degeneration, basophilia, and diffuse necrosis; the spleen showed hemorrhagic anemic necrosis, nuclear rupture, and atrophy. Glomerular atrophy of the renal corpuscle, cells in the proximal and distal tubules cytoarchitectural loss, and necrosis and shedding of kidney lymphocytes was also observed. No parasite was found on the fish surface, fins, in the gills, or internal organs with the naked eye and a light microscope. Freshwater viruses, such as Cyprinus herpesvirus type 2, largemouth bass ranavirus, megalocytivirus, and rhabdovirus, were checked by polymerase chain reaction (PCR), and no viruses were detected. The liver, spleen, and kidney of the diseased fish were sampled and cultured in tryptic soy agar medium (TSA) and Luria-Bertani agar medium (LB) plate medium at 28 ℃ for 24 h. Several pure and dominant colonies with the same morphology were observed on all the plates. These colonies were purified and cultured. The 16S rRNA gene sequencing results of all the purified colonies showed that the dominant strains were of the same species. The typical isolate was purified and named AS-AH2101. The results of biochemical identification with Biolog GenⅢ showed that the isolate AS-AH2101 was negative to gentiobiose, stachyose, D-raffinose, α-D-lactose, D-melibiose, 3-methyl glucose, D-fucose, D-sorbitol, D-arabitol, and Myo-inositol, while positive to dextrin, D-maltose, D-trehalose, D-cellobiose, sucrose, D-turanose, and β-methyl-D-glucoside. According to the Biolog GenⅢ identification system database, the biochemical characterization of AS-AH2101 was similar to that of A. hydrophila, with a confidence of 0.999. The 16S rRNA gene sequence of AS-AH2101 was submitted to GenBank databases under the accession number OP787967 and blasted in GenBank and EzTaxon. Comparison of the 16S rRNA gene sequences showed 99%100% identity with those of A. hydrophila. The phylogenetic tree was constructed using Mega 7 with the Aeromonas typical strains 16S rRNA gene sequences obtained from GenBank, and the phylogenetic analysis also clustered AS-AH2101 with A. hydrophila. Thus, the molecular analysis results identified the SC18032201 strains as A. hydrophila, and the phenotype also supported this result. Because of the strong stress response of American shad, it is difficult to perform the experimental culturing and infection in the laboratory. As a classic pathogenic infection model organism in aquatic animals, blue gourami (Trichogaster trichopterus) is a traditional model for fish pathogen study and has been widely used in the research of A. hydrophilia and E. piscicida. Therefore, in this study, blue gouramis were used as the model organism for virulence evaluation of AS-AH2101 in the experimental infection. The results of the challenge experiment showed that the death of the blue gourami infected via intramuscular injection was observed on the third day post infection. The infected fish showed redness, bleeding, and scale shedding at the injection site, congestion or bleeding at the base of the fin, abdominal ascites, and liver necrosis, which were similar to the naturally infected American shad. The isolate strain AS-AH2101 showed high virulence to blue gouramis, with the median lethal dose (LD50) of 3.23×104 CFU/fish. The virulence genes of A. hydrophilia were also detected by PCR, and results indicated that AS-AH2101 possessed six virulence genes, including aerolysin (aerA), hemolysin (hlyA), extracellular protease (ahpA), anti-metalloproteinases (ast), enterotoxin (altA), and quorum sensing gene (luxS). Antibiotic sensitivity studies showed that AS-AH2101 was resistant to cefradine, amoxicillin, ampicillin, and erythromycin. These results provided important information for disease control and A. hydrophila prevention and control of American shad culturing in China.
Aeromonas hydrophila (Family: Aeromonadaceae) is a traditional aquatic animal pathogen. It has been widely prevalent in our country since the 1980s and 1990s, causing diseases in a series of freshwater fish, leading to serious economic losses. It has been confirmed to be one of the main pathogens in freshwater aquaculture worldwide. The American shad (Alosa sapidissima) is one of the biggest shad in the world and grows much faster than other shad. Because of its delicious taste as reeves shad (Tenualosa reevesii), American shad were introduced into China from the USA by the Shanghai Fisheries Research Institute for local farming in 1998 and is widely welcomed in Shanghai, Jiangsu, and Zhejiang Provinces. In recent years, with the rapid development of the American shad culturing, fish diseases have become a major threat to fish farming. However, because most of the American shad are cultured in extensive ponds, few diseases have been reported in China. In this study, we reported a case of A. hydrophila infection in American shad. In 2021, the disease outbreak was observed in American shad cultured in Linyi City, Shandong Province, with severe mortality. The daily mortality could be up to 2.5%. The fish were cultured in indoor ponds for breeding and outdoor ponds when fish reached 300 g. The American shad were cultured with underground water and water was changed 1–3 times daily. The water temperature was 18–20 ℃. The fish were fed largemouth bass (Micropterus salmoides) commercial feed, and the daily feeding rate was approximately 2%. The disease broke out in the indoor ponds first, and then in outdoor ponds. The cumulative mortality was approximately 90% in 2 months. Enrofloxacin was administered orally, but no effects were observed and the disease continued to progress. The typical disease symptoms in the American shad were furunculosis or ulceration, with shedding scales and surface bleeding, especially on the tail, sometimes swollen and pus-filled. In autopsies of the diseased fish, ascites were found in the fish abdomen, and dark red necrosis on the liver, with sepsis and enteritis. The liver, spleen, and kidney of American shad with typical symptoms were collected and cut into about 1 cm3 tissue blocks, immersed in Davidson's Fixative (Davidson's AFA) for 24 h, and preserved in 70% ethanol. The tissues were mounted onto glass slides with hematoxylin and eosin staining for histological analysis. Histopathological results showed swollen liver cells, vacuolar degeneration, basophilia, and diffuse necrosis; the spleen showed hemorrhagic anemic necrosis, nuclear rupture, and atrophy. Glomerular atrophy of the renal corpuscle, cells in the proximal and distal tubules cytoarchitectural loss, and necrosis and shedding of kidney lymphocytes was also observed. No parasite was found on the fish surface, fins, in the gills, or internal organs with the naked eye and a light microscope. Freshwater viruses, such as Cyprinus herpesvirus type 2, largemouth bass ranavirus, megalocytivirus, and rhabdovirus, were checked by polymerase chain reaction (PCR), and no viruses were detected. The liver, spleen, and kidney of the diseased fish were sampled and cultured in tryptic soy agar medium (TSA) and Luria-Bertani agar medium (LB) plate medium at 28 ℃ for 24 h. Several pure and dominant colonies with the same morphology were observed on all the plates. These colonies were purified and cultured. The 16S rRNA gene sequencing results of all the purified colonies showed that the dominant strains were of the same species. The typical isolate was purified and named AS-AH2101. The results of biochemical identification with Biolog GenⅢ showed that the isolate AS-AH2101 was negative to gentiobiose, stachyose, D-raffinose, α-D-lactose, D-melibiose, 3-methyl glucose, D-fucose, D-sorbitol, D-arabitol, and Myo-inositol, while positive to dextrin, D-maltose, D-trehalose, D-cellobiose, sucrose, D-turanose, and β-methyl-D-glucoside. According to the Biolog GenⅢ identification system database, the biochemical characterization of AS-AH2101 was similar to that of A. hydrophila, with a confidence of 0.999. The 16S rRNA gene sequence of AS-AH2101 was submitted to GenBank databases under the accession number OP787967 and blasted in GenBank and EzTaxon. Comparison of the 16S rRNA gene sequences showed 99%100% identity with those of A. hydrophila. The phylogenetic tree was constructed using Mega 7 with the Aeromonas typical strains 16S rRNA gene sequences obtained from GenBank, and the phylogenetic analysis also clustered AS-AH2101 with A. hydrophila. Thus, the molecular analysis results identified the SC18032201 strains as A. hydrophila, and the phenotype also supported this result. Because of the strong stress response of American shad, it is difficult to perform the experimental culturing and infection in the laboratory. As a classic pathogenic infection model organism in aquatic animals, blue gourami (Trichogaster trichopterus) is a traditional model for fish pathogen study and has been widely used in the research of A. hydrophilia and E. piscicida. Therefore, in this study, blue gouramis were used as the model organism for virulence evaluation of AS-AH2101 in the experimental infection. The results of the challenge experiment showed that the death of the blue gourami infected via intramuscular injection was observed on the third day post infection. The infected fish showed redness, bleeding, and scale shedding at the injection site, congestion or bleeding at the base of the fin, abdominal ascites, and liver necrosis, which were similar to the naturally infected American shad. The isolate strain AS-AH2101 showed high virulence to blue gouramis, with the median lethal dose (LD50) of 3.23×104 CFU/fish. The virulence genes of A. hydrophilia were also detected by PCR, and results indicated that AS-AH2101 possessed six virulence genes, including aerolysin (aerA), hemolysin (hlyA), extracellular protease (ahpA), anti-metalloproteinases (ast), enterotoxin (altA), and quorum sensing gene (luxS). Antibiotic sensitivity studies showed that AS-AH2101 was resistant to cefradine, amoxicillin, ampicillin, and erythromycin. These results provided important information for disease control and A. hydrophila prevention and control of American shad culturing in China.
2023, 44(3):235-244.
DOI: 10.19663/j.issn2095-9869.20221011001
Abstract:
Shrimp has become a highly traded global seafood product, with 8 million tons of shrimp produced annually. Acute hepatopancreatic necrosis disease (AHPND) is the most prevalent and severe disease affecting shrimp aquaculture, resulting in considerable economic losses. The AHPND incidence in shrimp farming was as high as 60%–80% in China, resulting in reduced farming capacity and unstable production. Vibrio parahaemolyticus has been identified as the main causative agent of AHPND. In addition, V. harveyi, V. cambelii, V. algolyticus, and V. owenii are capable of causing similar diseases, demonstrating a distinct pathogenic diversity. Previous studies have indicated that not all of the above-mentioned Vibrios species are capable of causing AHPND, and whole gene sequencing and knockout genes have revealed that pirA and pirB are the primary pathogenic factors responsible for AHPND in shrimp. Specifically, the causative agent for AHPND should be a specific strain of Vibrio carrying the binary toxins pirAVp and pirBVp on the extrachromosomal virulence plasmid pVA1. Among them, the pirB toxin mainly determines the pathogenicity of the bacterium, whereas the pirA virulence is relatively weak. Furthermore, it has been demonstrated that the virulence plasmids encoding the binary genes pirAVp and pirBVp are the main causative agents of AHPND. The virulence gene toxR is prevalent in Vibrio and plays an important role through the genetic diversity of 16S rRNA genes during shrimp infection. Real-time fluorescence quantitative PCR technology has less contamination, more accurate quantification, real-time monitoring, and greater automation than conventional PCR technology, which has been utilized in the fields of transgenic detection, environmental science, and medicine. However, this technique is time-consuming, involves multiple instruments and reagents, and requires personnel with extensive professional skills and experience. As a result of its small size, low sample and reagent consumption, rapid detection speed, miniaturization, and integration, microfluidic chip assay technology has emerged as a new focal point in assay technology. Therefore, in this study, we designed specific primers and established a microfluorescence quantitative PCR assay based on two genes, pirA and pirB, to address the genetic similarity of AHPND pathogens carrying a large plasmid encoding a binary toxin, pirA and pirB. The method was specific for the pathogenic pirA and pirB genes, and only when DNA from AHPND-infected samples was tested could the two genes be successfully amplified, while all other pathogenic bacteria were detected with negative results. The sensitivity was high, and the minimum detection limits for the pirA and pirB genes were 5.43×100 and 4.31×101 copies/μL, respectively. Standard curves for pirA and pirB were constructed and demonstrated good linearity in the concentration range of 5.43×109–5.43×104 copies/μL for pirA (y= –3.145x+6.63, R2=0.999) and 4.31×109–4.31×104 copies/μL for pirB (y= –3.015x+5.45, R2=0.999), with an average sample detection time of approximately 26 min. In order to evaluate the efficacy of the method in practice, artificial infection experiments with V. parahaemolyticus were performed. In this study, artificial infection experiments were induced by both injection and immersion, and samples were collected at different time periods to clinically validate the established method and compare its effectiveness in detecting different shrimp tissues, thereby facilitating a more thorough analysis of the pathogenic pathways of infection. The experimental group with injection as the mode of infection was found to be positive for all tissues in all time periods except the water test, which was negative. The experimental group that used immersion as the infection method showed different results for various time periods and with different genetic tests. In terms of the infection method, the tissues could be infiltrated within 2 h using the injection method, whereas the target genes were not detected in the hepatopancreas at 6 h using the immersion method. This indicated that the injection method infiltrated the tissues more rapidly than the immersion method. According to the comparison results of the three genes, pirB was only negative in the intestine at 2 h and positive in all tissues the rest of the time; pirA was negative in the hepatopancreas and intestine at 2 h, only the intestine was negative at 6 h, and all tissues were positive at 12 h; and toxR was negative in all tissues at 2 h. The rate of infestation from rapid to slow showed that pirB > pirA > toxR. Based on the rate of tissue infestation, pirA and pirB were detected in both cheek filaments and muscles at 2 h, making them the most rapid infiltration agents. Therefore, the strategy of using pirB as the primer and gill filament or muscle as the target tissue is more suitable for the rapid detection of AHPND in the field. In this study, we established a method for microfluidic fluorescent quantitative PCR that has the advantages of being rapid, sensitive, high throughput, less contaminated, on-site detectable, and integrated. The method is not only applicable to the laboratory but also meets the requirements of rapid field detection at hatcheries and farms, and can be used as a new technical method for shrimp fry quality detection and disease control.
Shrimp has become a highly traded global seafood product, with 8 million tons of shrimp produced annually. Acute hepatopancreatic necrosis disease (AHPND) is the most prevalent and severe disease affecting shrimp aquaculture, resulting in considerable economic losses. The AHPND incidence in shrimp farming was as high as 60%–80% in China, resulting in reduced farming capacity and unstable production. Vibrio parahaemolyticus has been identified as the main causative agent of AHPND. In addition, V. harveyi, V. cambelii, V. algolyticus, and V. owenii are capable of causing similar diseases, demonstrating a distinct pathogenic diversity. Previous studies have indicated that not all of the above-mentioned Vibrios species are capable of causing AHPND, and whole gene sequencing and knockout genes have revealed that pirA and pirB are the primary pathogenic factors responsible for AHPND in shrimp. Specifically, the causative agent for AHPND should be a specific strain of Vibrio carrying the binary toxins pirAVp and pirBVp on the extrachromosomal virulence plasmid pVA1. Among them, the pirB toxin mainly determines the pathogenicity of the bacterium, whereas the pirA virulence is relatively weak. Furthermore, it has been demonstrated that the virulence plasmids encoding the binary genes pirAVp and pirBVp are the main causative agents of AHPND. The virulence gene toxR is prevalent in Vibrio and plays an important role through the genetic diversity of 16S rRNA genes during shrimp infection. Real-time fluorescence quantitative PCR technology has less contamination, more accurate quantification, real-time monitoring, and greater automation than conventional PCR technology, which has been utilized in the fields of transgenic detection, environmental science, and medicine. However, this technique is time-consuming, involves multiple instruments and reagents, and requires personnel with extensive professional skills and experience. As a result of its small size, low sample and reagent consumption, rapid detection speed, miniaturization, and integration, microfluidic chip assay technology has emerged as a new focal point in assay technology. Therefore, in this study, we designed specific primers and established a microfluorescence quantitative PCR assay based on two genes, pirA and pirB, to address the genetic similarity of AHPND pathogens carrying a large plasmid encoding a binary toxin, pirA and pirB. The method was specific for the pathogenic pirA and pirB genes, and only when DNA from AHPND-infected samples was tested could the two genes be successfully amplified, while all other pathogenic bacteria were detected with negative results. The sensitivity was high, and the minimum detection limits for the pirA and pirB genes were 5.43×100 and 4.31×101 copies/μL, respectively. Standard curves for pirA and pirB were constructed and demonstrated good linearity in the concentration range of 5.43×109–5.43×104 copies/μL for pirA (y= –3.145x+6.63, R2=0.999) and 4.31×109–4.31×104 copies/μL for pirB (y= –3.015x+5.45, R2=0.999), with an average sample detection time of approximately 26 min. In order to evaluate the efficacy of the method in practice, artificial infection experiments with V. parahaemolyticus were performed. In this study, artificial infection experiments were induced by both injection and immersion, and samples were collected at different time periods to clinically validate the established method and compare its effectiveness in detecting different shrimp tissues, thereby facilitating a more thorough analysis of the pathogenic pathways of infection. The experimental group with injection as the mode of infection was found to be positive for all tissues in all time periods except the water test, which was negative. The experimental group that used immersion as the infection method showed different results for various time periods and with different genetic tests. In terms of the infection method, the tissues could be infiltrated within 2 h using the injection method, whereas the target genes were not detected in the hepatopancreas at 6 h using the immersion method. This indicated that the injection method infiltrated the tissues more rapidly than the immersion method. According to the comparison results of the three genes, pirB was only negative in the intestine at 2 h and positive in all tissues the rest of the time; pirA was negative in the hepatopancreas and intestine at 2 h, only the intestine was negative at 6 h, and all tissues were positive at 12 h; and toxR was negative in all tissues at 2 h. The rate of infestation from rapid to slow showed that pirB > pirA > toxR. Based on the rate of tissue infestation, pirA and pirB were detected in both cheek filaments and muscles at 2 h, making them the most rapid infiltration agents. Therefore, the strategy of using pirB as the primer and gill filament or muscle as the target tissue is more suitable for the rapid detection of AHPND in the field. In this study, we established a method for microfluidic fluorescent quantitative PCR that has the advantages of being rapid, sensitive, high throughput, less contaminated, on-site detectable, and integrated. The method is not only applicable to the laboratory but also meets the requirements of rapid field detection at hatcheries and farms, and can be used as a new technical method for shrimp fry quality detection and disease control.
2023, 44(5):172-181.
DOI: 10.19663/j.issn2095-9869.20220404001
Abstract:
Mud crab reovirus (MCRV) is one of the most fatal pathogens of the mud crab Scylla paramamonsain. The outbreak and epidemic of MCRV has seriously affected the healthy development of the mud crab aquaculture industry. To limit MCRV transmission from breeding crabs to larva, we attempt to establish a more sensitive and practical diagnostic method for screening MCRV-free crabs. The primers of the present diagnostic methods for MCRV are based on the VP1 gene (MCRV RNA polymerase gene), and the low expression level of this gene limits the sensitivity of the diagnostic method. Therefore, it is necessary to select the target gene with the highest expression level for the detection primer to improve the sensitivity of the diagnostic method. In addition, the current diagnostic methods require gill samples for virus detection, which requires killing the mud crabs before sampling. This sampling method is obviously not suitable for screening MCRV-free breeding crabs. Therefore, it is necessary to develop a less invasive sampling method for breeding crabs. In this study, a SYBR Green fluorescent quantitative diagnostic method was developed to screen MCRV-free breeding crabs. To improve the sensitivity of the detection, we initially analyzed the relative load of MCRV in the main tissues of infected mud crabs. The viral load in the hemolymph was the highest of all the tissues. The expression levels of 13 putative genes of MCRV were detected in the hemolymph. The relative expression level of the VP11 gene was the highest. Finally, specific primers were designed based on the conserved region of the VP11 gene sequence to establish a SYBR Green qRT-PCR (quantitative reverse-transcription PCR) detection method to accurately detect 50 copies/µL of viral nucleic acid in a sample. Considering the advantages of tissue and target gene selection, the sensitivity of this method should be significantly higher than that of preexisting detection methods. This diagnostic method is very specific for MCRV and no specific amplification was observed using nucleic acid samples containing 5 different kinds of common crustacean pathogens (MCDV, WSSV, DIV1, EHP, and Vibrio parahemolyticus). Compared to other methods of extracting RNA by killing and grinding the gill tissues of crabs, we can select MCRV-free crabs by sampling very small amounts of hemolymph (as low as 20 µL). All of the healthy crabs screened by this method were able to hold eggs that hatched normally. To test the effectiveness of this method, 22 breeding crabs and 20 commercial crabs were screened for MCRV. The positive rates were 54.55% and 85.00%, respectively. In addition, we analyzed the proliferation of MCRV in the mud crabs, and found that MCRV proliferates exponentially in the early stage, then enters a plateau phase, and no crabs died during the infection period of seven days. In conclusion, this study established a highly-sensitive and practical detection method for MCRV in breeding crabs, which can meet the requirements for MCRV-free breeding crab screening with low damage to the breeders. We also investigated the pathogenic infection mechanisms.
Mud crab reovirus (MCRV) is one of the most fatal pathogens of the mud crab Scylla paramamonsain. The outbreak and epidemic of MCRV has seriously affected the healthy development of the mud crab aquaculture industry. To limit MCRV transmission from breeding crabs to larva, we attempt to establish a more sensitive and practical diagnostic method for screening MCRV-free crabs. The primers of the present diagnostic methods for MCRV are based on the VP1 gene (MCRV RNA polymerase gene), and the low expression level of this gene limits the sensitivity of the diagnostic method. Therefore, it is necessary to select the target gene with the highest expression level for the detection primer to improve the sensitivity of the diagnostic method. In addition, the current diagnostic methods require gill samples for virus detection, which requires killing the mud crabs before sampling. This sampling method is obviously not suitable for screening MCRV-free breeding crabs. Therefore, it is necessary to develop a less invasive sampling method for breeding crabs. In this study, a SYBR Green fluorescent quantitative diagnostic method was developed to screen MCRV-free breeding crabs. To improve the sensitivity of the detection, we initially analyzed the relative load of MCRV in the main tissues of infected mud crabs. The viral load in the hemolymph was the highest of all the tissues. The expression levels of 13 putative genes of MCRV were detected in the hemolymph. The relative expression level of the VP11 gene was the highest. Finally, specific primers were designed based on the conserved region of the VP11 gene sequence to establish a SYBR Green qRT-PCR (quantitative reverse-transcription PCR) detection method to accurately detect 50 copies/µL of viral nucleic acid in a sample. Considering the advantages of tissue and target gene selection, the sensitivity of this method should be significantly higher than that of preexisting detection methods. This diagnostic method is very specific for MCRV and no specific amplification was observed using nucleic acid samples containing 5 different kinds of common crustacean pathogens (MCDV, WSSV, DIV1, EHP, and Vibrio parahemolyticus). Compared to other methods of extracting RNA by killing and grinding the gill tissues of crabs, we can select MCRV-free crabs by sampling very small amounts of hemolymph (as low as 20 µL). All of the healthy crabs screened by this method were able to hold eggs that hatched normally. To test the effectiveness of this method, 22 breeding crabs and 20 commercial crabs were screened for MCRV. The positive rates were 54.55% and 85.00%, respectively. In addition, we analyzed the proliferation of MCRV in the mud crabs, and found that MCRV proliferates exponentially in the early stage, then enters a plateau phase, and no crabs died during the infection period of seven days. In conclusion, this study established a highly-sensitive and practical detection method for MCRV in breeding crabs, which can meet the requirements for MCRV-free breeding crab screening with low damage to the breeders. We also investigated the pathogenic infection mechanisms.
ZHANG Xiang
,
GU Li
,
ZHENG Yudong
,
LI Chen
,
BAI Changming
,
XIN Lusheng
,
WANG Chongming
,
LIU Jinlan
2023, 44(2):174-185.
DOI: 10.19663/j.issn2095-9869.20210930001
Abstract:
Ostreid herpesvirus 1 (OsHV-1) has caused a serious economic loss to the global bivalve aquaculture industry. Given its wide host range and the frequent emergence of mutated strains, OsHV-1 infection threatens mollusk production. Unlike the common vertebrate herpesviruses that generally exhibit high host specificity, more than ten bivalve species have been identified as potentially susceptible to OsHV-1 infection, including members of Ostreoida, Pterioida, Arcoida, and Veneroida. A variety of OsHV-1 detection methods have been developed, such as PCR, in situ hybridization, in situ PCR, histopathology, transmission electron microscopy, ring-mediated isothermal nucleic acid amplification (LAMP), and recombinase polymerase amplification (RPA). The sensitivity and specificity of transmission electron microscopy detection are low, and specific complementary detection methods such as PCR are needed to confirm the occurrence of OsHV-1. In situ hybridization, although highly specific, has the disadvantages of low sensitivity, complicated procedures, and high investment of effort. PCR methods are still the most widely used for epidemiological investigation of OsHV-1. However, PCR cannot alone confirm infection, which has led to the emergence of unconfirmed cases of OsHV-1 infection in many species and regions. According to the World Organization for Animal Health´s (OIE) Aquatic Animal Manual, positive nucleic acid-specific detection combined with histopathology and transmission electron microscopy is required for the confirmed diagnosis of OsHV-1 infection. Other detection methods, such as LAMP and RPA, which use nucleic acid amplification alone to detect OsHV-1, can not confirm infection as the presence of nucleic acids is not equivalent to viral infection. As a result, the susceptibility of several bivalve species to OsHV-1 infection has not been evaluated. Instead, several obstacles remain with regards to the development of epidemiological surveillance programs and the implementation of quarantine, prevention, and control measures for OsHV-1 infection. Compared with traditional in situ hybridization and in situ PCR assays, in situ LAMP has the advantages of low nucleic acid amplification reaction temperature, less damage to tissue, constant temperature amplification that does not require special experimental equipment, and more convenient and efficient experimental design. In this study, we selected a set of LAMP primers designed for specific detection of OsHV-1, and a pair of loop primers were designed to improve the specificity and stability of the LAMP reaction on slides. An optimized in situ LAMP method for OsHV-1 detection was developed, which provides a rapid diagnostic method with high specificity and sensitivity to identify hosts that are potentially susceptible to OsHV-1 infection, and to characterize the distribution pattern and tissue affinity of the virus in new hosts. The optimized protocol and quantitative PCR (qPCR) were then used to detect OsHV-1 infection in bivalve samples (Crassostrea gigas, C. angulata, Chlamys farreri, Mizuhopecten yessoensis, Ruditapes philippinarum, and Scapharca subcrenata) collected from 2019 to 2021. OsHV-1 hybridization signals were observed in S. subcrenata samples alone, although positive qPCR results were obtained in more species. Further investigation of pathological characteristics and associated viral hybridization signals indicated that OsHV-1 infection always occurred in fibroblasts and hemocytes in the connective tissues of the mantle and hepatopancreas, nucleus of muscle cells in the foot, and adductor muscle. Signals were also occasionally observed in infiltrated hemocytes between and within the gill filaments. OsHV-1 hybridization signals were observed within the hemocytes infiltrating several organs of the furcula visceral cluster. The hemocytes of S. subcrenata seem to be particularly susceptible to OsHV-1 infection. Histopathological lesions and viral hybridization signals in the hepatopancreas organ were consistently observed in individuals with clinical signs, and we recommend the hepatopancreas as the preferred target organ for histopathological and in situ LAMP assays for OsHV-1 infection in S. subcrenata. In situ LAMP-based detection indicated that S. subcrenata was a susceptible host for OsHV-1, and the connective tissue, muscle tissue, and hemolymph cells had a strong affinity for the virus. At present, the susceptibility of many cultured and wild shellfish to OsHV-1 infection is still unknown. Since the emergence of mollusk mortalities associated with OsHV-1 infection occurred in China in the 1990s, its host range has expanded and changed along with the environment, which has complicated its prevention and control. In recent years, the scale of artificial breeding and cultivation of triploid C. gigas has expanded in China, and larval mortalities are frequently associated with OsHV-1 infection. The in situ LAMP detection method for OsHV-1 infection developed in this study proved to be convenient and fast and could be a valuable tool for the rapid detection and confirmation of OsHV-1 infection.
Ostreid herpesvirus 1 (OsHV-1) has caused a serious economic loss to the global bivalve aquaculture industry. Given its wide host range and the frequent emergence of mutated strains, OsHV-1 infection threatens mollusk production. Unlike the common vertebrate herpesviruses that generally exhibit high host specificity, more than ten bivalve species have been identified as potentially susceptible to OsHV-1 infection, including members of Ostreoida, Pterioida, Arcoida, and Veneroida. A variety of OsHV-1 detection methods have been developed, such as PCR, in situ hybridization, in situ PCR, histopathology, transmission electron microscopy, ring-mediated isothermal nucleic acid amplification (LAMP), and recombinase polymerase amplification (RPA). The sensitivity and specificity of transmission electron microscopy detection are low, and specific complementary detection methods such as PCR are needed to confirm the occurrence of OsHV-1. In situ hybridization, although highly specific, has the disadvantages of low sensitivity, complicated procedures, and high investment of effort. PCR methods are still the most widely used for epidemiological investigation of OsHV-1. However, PCR cannot alone confirm infection, which has led to the emergence of unconfirmed cases of OsHV-1 infection in many species and regions. According to the World Organization for Animal Health´s (OIE) Aquatic Animal Manual, positive nucleic acid-specific detection combined with histopathology and transmission electron microscopy is required for the confirmed diagnosis of OsHV-1 infection. Other detection methods, such as LAMP and RPA, which use nucleic acid amplification alone to detect OsHV-1, can not confirm infection as the presence of nucleic acids is not equivalent to viral infection. As a result, the susceptibility of several bivalve species to OsHV-1 infection has not been evaluated. Instead, several obstacles remain with regards to the development of epidemiological surveillance programs and the implementation of quarantine, prevention, and control measures for OsHV-1 infection. Compared with traditional in situ hybridization and in situ PCR assays, in situ LAMP has the advantages of low nucleic acid amplification reaction temperature, less damage to tissue, constant temperature amplification that does not require special experimental equipment, and more convenient and efficient experimental design. In this study, we selected a set of LAMP primers designed for specific detection of OsHV-1, and a pair of loop primers were designed to improve the specificity and stability of the LAMP reaction on slides. An optimized in situ LAMP method for OsHV-1 detection was developed, which provides a rapid diagnostic method with high specificity and sensitivity to identify hosts that are potentially susceptible to OsHV-1 infection, and to characterize the distribution pattern and tissue affinity of the virus in new hosts. The optimized protocol and quantitative PCR (qPCR) were then used to detect OsHV-1 infection in bivalve samples (Crassostrea gigas, C. angulata, Chlamys farreri, Mizuhopecten yessoensis, Ruditapes philippinarum, and Scapharca subcrenata) collected from 2019 to 2021. OsHV-1 hybridization signals were observed in S. subcrenata samples alone, although positive qPCR results were obtained in more species. Further investigation of pathological characteristics and associated viral hybridization signals indicated that OsHV-1 infection always occurred in fibroblasts and hemocytes in the connective tissues of the mantle and hepatopancreas, nucleus of muscle cells in the foot, and adductor muscle. Signals were also occasionally observed in infiltrated hemocytes between and within the gill filaments. OsHV-1 hybridization signals were observed within the hemocytes infiltrating several organs of the furcula visceral cluster. The hemocytes of S. subcrenata seem to be particularly susceptible to OsHV-1 infection. Histopathological lesions and viral hybridization signals in the hepatopancreas organ were consistently observed in individuals with clinical signs, and we recommend the hepatopancreas as the preferred target organ for histopathological and in situ LAMP assays for OsHV-1 infection in S. subcrenata. In situ LAMP-based detection indicated that S. subcrenata was a susceptible host for OsHV-1, and the connective tissue, muscle tissue, and hemolymph cells had a strong affinity for the virus. At present, the susceptibility of many cultured and wild shellfish to OsHV-1 infection is still unknown. Since the emergence of mollusk mortalities associated with OsHV-1 infection occurred in China in the 1990s, its host range has expanded and changed along with the environment, which has complicated its prevention and control. In recent years, the scale of artificial breeding and cultivation of triploid C. gigas has expanded in China, and larval mortalities are frequently associated with OsHV-1 infection. The in situ LAMP detection method for OsHV-1 infection developed in this study proved to be convenient and fast and could be a valuable tool for the rapid detection and confirmation of OsHV-1 infection.
YU Xingtong
,
WAN Xiaoyuan
,
JIA Tianchang
,
XIA Jitao
,
XU Tingting
,
YAO Liang
,
ZHAO Wenxiu
,
ZHANG Qingli
2024, 45(4):146-154.
DOI: 10.19663/j.issn2095-9869.20230215002
Abstract:
Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasitic microsporidian species that infects the hepatopancreas of shrimp and causes hepatopancreatic microsporidiosis (HPM). It was discovered in stunted Penaeus monodon in Thailand in 2004. It is a fungal pathogen that can infect various crustacean hosts. It affects farmed shrimp in Southeast Asia and South America, including Thailand, Vietnam, China, Indonesia, India, Malaysia, South Korea and Venezuela. An E. hepatopenaei infection does not cause death in shrimp, but can lead to necrosis and rupture of the hepatopancreatic epithelial cells. This results in reduced digestive and absorptive functions of the hepatopancreatic gland and impaired nutrient storage functions. Therefore, an EHP infection usually leads to shrimp growth retardation or stagnation that greatly reduces shrimp production and seriously affects the high-quality development of shrimp farming. E. hepatopenaei infections were detected in farmed shrimp in coastal provinces of China since 2013 and their high infection rate causes serious economic losses to the cultured shrimp industry. It is difficult to confirm EHP infections solely based on the symptoms of diseased shrimp in the field owing to the extremely small size of EHP and the lack of obvious symptoms in the early stages of EHP infection. Furthermore, the early stages of infection are difficult to accurately detect by light microscopy and histopathological sections. Pathological changes of EHP infection are only observed in the middle and later stages by histopathological sections. In addition, tissue section preparation is time- consuming and complex. This makes it unsuitable as a practical technical method to detect EHP. In contrast, molecular biology methods have become a common laboratory method to detect EHP. This study collected 936 shrimp samples from coastal areas in China from 2021 to 2022, detected EHP positive samples by TaqMan quantitative polymerase chain reaction (qPCR), and analyzed the morphology by histopathology. This study aims to clarify the prevalence of EHP in major shrimp farming species in coastal provinces and cities in China from 2021 to 2022, and to clarify the hazard risk of EHP in cultured shrimp. TaqMan qPCR assays showed that the positive detection rate of EHP in shrimp samples from coastal areas was 10.67% (54/506) in 2021. There was a decreasing trend compared with the national aquatic animal disease surveillance plan from 2017 to 2020. However, the positive rate of EHP in shrimp samples from coastal areas reached 13.72% (59/430) in 2022. There was an increase in the EHP positive detection rate compared with the national surveillance data from 2019 to 2020. The detection results of EHP in samples collected from different regions showed that the positive rate of EHP was high in samples collected from Liaoning (10%) and Shandong (18.80%) in 2021, and in samples collected from Liaoning (14.63%), Hebei (29.17%), Tianjin (28.57%), and Shandong (16.88%) in 2022. The results of samples collected from different shrimp varieties showed that EHP was mainly detected in P. vannamei in 2021 and 2022, with positive rates of 14.10% (54/383) and 16.71% (58/347), respectively. Among them, one sample of Exopalaemon carinicauda was positive for EHP, while no EHP positive sample was detected in Macrobrachium rosenbergii, Protocrayfish cruzi, Penaeus monodon, Marsupenaeus japonicus, and Penaeus chinensis. This study collected and analyzed 506 and 430 shrimp samples from coastal provinces and cities in China in 2021 and 2022, respectively by TaqMan qPCR. These detection results are more reliable than those of previous studies owing to the large sample size and the high specificity and sensitivity of the detection method. The results provided an important reference for a comprehensive understanding of the epidemic situation of EHP in major cultured shrimp in coastal areas of China from 2021 to 2022. Histopathological examination of EHP-positive P. vannamei revealed that scattered or clustered EHP spores and EHP protoplasts were observed in hepatopancreatic epithelial cells. The epidemiological survey and molecular epidemiological analysis of shrimp farmed in coastal provinces, municipalities, and some inland provinces of China in 2021 to 2022 showed that the prevalence rate of EHP is generally decreasing compared to previous years. This indicated that remarkable achievements were made to prevent and control EHP in China's shrimp farming industry in recent years. However, EHP is widely prevalent in farmed shrimp in coastal provinces in Northern China. Therefore, measures such as strict EHP quarantine of brood stock and seedlings and the promotion of non-EHP seedlings should be taken to further reduce the epidemic range and risk of EHP and promote the green and high-quality development of the shrimp culture industry in China.
Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasitic microsporidian species that infects the hepatopancreas of shrimp and causes hepatopancreatic microsporidiosis (HPM). It was discovered in stunted Penaeus monodon in Thailand in 2004. It is a fungal pathogen that can infect various crustacean hosts. It affects farmed shrimp in Southeast Asia and South America, including Thailand, Vietnam, China, Indonesia, India, Malaysia, South Korea and Venezuela. An E. hepatopenaei infection does not cause death in shrimp, but can lead to necrosis and rupture of the hepatopancreatic epithelial cells. This results in reduced digestive and absorptive functions of the hepatopancreatic gland and impaired nutrient storage functions. Therefore, an EHP infection usually leads to shrimp growth retardation or stagnation that greatly reduces shrimp production and seriously affects the high-quality development of shrimp farming. E. hepatopenaei infections were detected in farmed shrimp in coastal provinces of China since 2013 and their high infection rate causes serious economic losses to the cultured shrimp industry. It is difficult to confirm EHP infections solely based on the symptoms of diseased shrimp in the field owing to the extremely small size of EHP and the lack of obvious symptoms in the early stages of EHP infection. Furthermore, the early stages of infection are difficult to accurately detect by light microscopy and histopathological sections. Pathological changes of EHP infection are only observed in the middle and later stages by histopathological sections. In addition, tissue section preparation is time- consuming and complex. This makes it unsuitable as a practical technical method to detect EHP. In contrast, molecular biology methods have become a common laboratory method to detect EHP. This study collected 936 shrimp samples from coastal areas in China from 2021 to 2022, detected EHP positive samples by TaqMan quantitative polymerase chain reaction (qPCR), and analyzed the morphology by histopathology. This study aims to clarify the prevalence of EHP in major shrimp farming species in coastal provinces and cities in China from 2021 to 2022, and to clarify the hazard risk of EHP in cultured shrimp. TaqMan qPCR assays showed that the positive detection rate of EHP in shrimp samples from coastal areas was 10.67% (54/506) in 2021. There was a decreasing trend compared with the national aquatic animal disease surveillance plan from 2017 to 2020. However, the positive rate of EHP in shrimp samples from coastal areas reached 13.72% (59/430) in 2022. There was an increase in the EHP positive detection rate compared with the national surveillance data from 2019 to 2020. The detection results of EHP in samples collected from different regions showed that the positive rate of EHP was high in samples collected from Liaoning (10%) and Shandong (18.80%) in 2021, and in samples collected from Liaoning (14.63%), Hebei (29.17%), Tianjin (28.57%), and Shandong (16.88%) in 2022. The results of samples collected from different shrimp varieties showed that EHP was mainly detected in P. vannamei in 2021 and 2022, with positive rates of 14.10% (54/383) and 16.71% (58/347), respectively. Among them, one sample of Exopalaemon carinicauda was positive for EHP, while no EHP positive sample was detected in Macrobrachium rosenbergii, Protocrayfish cruzi, Penaeus monodon, Marsupenaeus japonicus, and Penaeus chinensis. This study collected and analyzed 506 and 430 shrimp samples from coastal provinces and cities in China in 2021 and 2022, respectively by TaqMan qPCR. These detection results are more reliable than those of previous studies owing to the large sample size and the high specificity and sensitivity of the detection method. The results provided an important reference for a comprehensive understanding of the epidemic situation of EHP in major cultured shrimp in coastal areas of China from 2021 to 2022. Histopathological examination of EHP-positive P. vannamei revealed that scattered or clustered EHP spores and EHP protoplasts were observed in hepatopancreatic epithelial cells. The epidemiological survey and molecular epidemiological analysis of shrimp farmed in coastal provinces, municipalities, and some inland provinces of China in 2021 to 2022 showed that the prevalence rate of EHP is generally decreasing compared to previous years. This indicated that remarkable achievements were made to prevent and control EHP in China's shrimp farming industry in recent years. However, EHP is widely prevalent in farmed shrimp in coastal provinces in Northern China. Therefore, measures such as strict EHP quarantine of brood stock and seedlings and the promotion of non-EHP seedlings should be taken to further reduce the epidemic range and risk of EHP and promote the green and high-quality development of the shrimp culture industry in China.
2024, 45(3):193-202.
DOI: 10.19663/j.issn2095-9869.20221216001
Abstract:
Flinders technology associates (FTA) card (Whatman®) is a paper-based matrix designed to fix, purify, and store genetic material from various biological sources. It can conveniently and quickly preserve nucleic acids and may fulfil the requirements of long-distance and cross-border sample transportation. The FTA card can store and transport tissue, nucleic acid, and other sample types at room temperature (20–25 ℃). Nucleic acid can be extracted directly for detection and be sent by express as an ordinary parcel without being treated as dangerous or as special goods, eliminating tedious processes, saving time, and ensuring sample quality. It is widely used in the human and animal medicine field. It has been successfully used for the storage and transportation of livestock pathogens and viral nucleic acids. In terms of aquatic animals, the FTA card has been used by researchers to store white spot syndrome virus (WSSV) and the shrimp Enterozoon hepatopoaei (EHP): However, there is no relevant research report on the elution effect of the nucleic acid stored in the FTA card, which affects the application of the FTA card. The infectious hypodermal and haematopoietic necrosis virus (IHHNV) is an important shrimp pathogen and severely impacts the shrimp culture industry. It was first found in Hawaii, United States, in 1981 and then spread to several countries, including to Australia, Singapore, Malaysia, South Korea, Brazil, and China. The IHHNV infecting Penaeus vannamei does not cause high mortality, but growth would become slow and deformed, resulting in great economic losses. Early detection and prevention management are particularly important in current situations, which lack effective control measures for the disease. Several IHHNV detection methods have been established that use molecular biological methods, including conventional polymerase chain reaction (PCR) and real-time PCR (these are recommended in the aquatic animal disease diagnosis Manual of the World Organization for Animal Health). Nucleic acid extraction by the above methods meets the requirements for samples, usually frozen, ethanol, or other nucleic acid preservation reagents. Low temperature preservation conditions and composition restrictions of preservation solutions present certain difficulties in disease investigation, surveillance, and monitoring of shrimp farming. It is particularly important to address this dilemma. To find a fast method for the preservation and separation of IHHNV DNA and provide complete nucleic acid materials for subsequent research, we selected FTA cards as the preservation medium, and designed seven kinds of FTA cards with attached DNA elution methods based on the FTA purification reagent, TE buffer, and deionized water. We evaluated the elution and separation effects of different nucleic acids and the minimum amount of dot FTA card nucleic acids through real-time PCR detection. The appropriate solution was spotted onto FTA cards according to the manufacturer´s protocol, labeled, and air-dried for 1 day at room temperature. The result shows that on the 4 mm2 FTA card, the sample volume was 2.5 μL. When the eluent is used as the template, the minimum FTA card nucleic acid concentration needs to be 1.47×104 copies/μL above the best detection sensitivity, and 100% detection rate can be obtained by washing the FTA card with 50 μL TE buffer solution at 95 ℃ for 5 min. Using the FTA card as the template, the nucleic acid concentration of the dot FTA card needs to be above 1.82×103 copies/μL, eluted with FTA purified reagent thrice at room temperature, and then eluted with TE buffer twice. Each elution time is 5 min as this can obtain the best detection sensitivity and demonstrates 100% accuracy. The elution effect of the above two schemes was better than that of the other five schemes. The nucleic acids of WSSV, EHP, decapod iridescent virus 1, covert mobility Noda virus, and Vibrio parahaemolyticus causing acute hepatopancreatic necrosis were preserved using FTA card to test the efficiency of the established elution method. It is assumed that this method is universal for the elution of other shrimp pathogenic nucleic acids. At present, research on the application of FTA card is mostly seen in the nucleic acid effect of its preservation and transportation of tissue samples. There are few reports on the relationship between the amount of preserved nucleic acid, separation methods, and detection effect. This study shows that FTA cards used to preserve pathogenic nucleic acid requires a specific amount of nucleic acid in the sample and directly affects the detection results of the sample with different FTA card elution methods. This study provides a feasible scheme for the preservation and elution of IHHNV DNA with FTA cards. The application of this technology has potential use as storage and transport strategy for surveillance programs and can enhance biosecurity in shrimp culture, which provides a scientific basis for the preservation and transportation conditions for the collection of wild shrimp samples and the regional transmission of viral nucleic acid samples.
Flinders technology associates (FTA) card (Whatman®) is a paper-based matrix designed to fix, purify, and store genetic material from various biological sources. It can conveniently and quickly preserve nucleic acids and may fulfil the requirements of long-distance and cross-border sample transportation. The FTA card can store and transport tissue, nucleic acid, and other sample types at room temperature (20–25 ℃). Nucleic acid can be extracted directly for detection and be sent by express as an ordinary parcel without being treated as dangerous or as special goods, eliminating tedious processes, saving time, and ensuring sample quality. It is widely used in the human and animal medicine field. It has been successfully used for the storage and transportation of livestock pathogens and viral nucleic acids. In terms of aquatic animals, the FTA card has been used by researchers to store white spot syndrome virus (WSSV) and the shrimp Enterozoon hepatopoaei (EHP): However, there is no relevant research report on the elution effect of the nucleic acid stored in the FTA card, which affects the application of the FTA card. The infectious hypodermal and haematopoietic necrosis virus (IHHNV) is an important shrimp pathogen and severely impacts the shrimp culture industry. It was first found in Hawaii, United States, in 1981 and then spread to several countries, including to Australia, Singapore, Malaysia, South Korea, Brazil, and China. The IHHNV infecting Penaeus vannamei does not cause high mortality, but growth would become slow and deformed, resulting in great economic losses. Early detection and prevention management are particularly important in current situations, which lack effective control measures for the disease. Several IHHNV detection methods have been established that use molecular biological methods, including conventional polymerase chain reaction (PCR) and real-time PCR (these are recommended in the aquatic animal disease diagnosis Manual of the World Organization for Animal Health). Nucleic acid extraction by the above methods meets the requirements for samples, usually frozen, ethanol, or other nucleic acid preservation reagents. Low temperature preservation conditions and composition restrictions of preservation solutions present certain difficulties in disease investigation, surveillance, and monitoring of shrimp farming. It is particularly important to address this dilemma. To find a fast method for the preservation and separation of IHHNV DNA and provide complete nucleic acid materials for subsequent research, we selected FTA cards as the preservation medium, and designed seven kinds of FTA cards with attached DNA elution methods based on the FTA purification reagent, TE buffer, and deionized water. We evaluated the elution and separation effects of different nucleic acids and the minimum amount of dot FTA card nucleic acids through real-time PCR detection. The appropriate solution was spotted onto FTA cards according to the manufacturer´s protocol, labeled, and air-dried for 1 day at room temperature. The result shows that on the 4 mm2 FTA card, the sample volume was 2.5 μL. When the eluent is used as the template, the minimum FTA card nucleic acid concentration needs to be 1.47×104 copies/μL above the best detection sensitivity, and 100% detection rate can be obtained by washing the FTA card with 50 μL TE buffer solution at 95 ℃ for 5 min. Using the FTA card as the template, the nucleic acid concentration of the dot FTA card needs to be above 1.82×103 copies/μL, eluted with FTA purified reagent thrice at room temperature, and then eluted with TE buffer twice. Each elution time is 5 min as this can obtain the best detection sensitivity and demonstrates 100% accuracy. The elution effect of the above two schemes was better than that of the other five schemes. The nucleic acids of WSSV, EHP, decapod iridescent virus 1, covert mobility Noda virus, and Vibrio parahaemolyticus causing acute hepatopancreatic necrosis were preserved using FTA card to test the efficiency of the established elution method. It is assumed that this method is universal for the elution of other shrimp pathogenic nucleic acids. At present, research on the application of FTA card is mostly seen in the nucleic acid effect of its preservation and transportation of tissue samples. There are few reports on the relationship between the amount of preserved nucleic acid, separation methods, and detection effect. This study shows that FTA cards used to preserve pathogenic nucleic acid requires a specific amount of nucleic acid in the sample and directly affects the detection results of the sample with different FTA card elution methods. This study provides a feasible scheme for the preservation and elution of IHHNV DNA with FTA cards. The application of this technology has potential use as storage and transport strategy for surveillance programs and can enhance biosecurity in shrimp culture, which provides a scientific basis for the preservation and transportation conditions for the collection of wild shrimp samples and the regional transmission of viral nucleic acid samples.
ZHAO Weizhi
,
WANG Yingeng
,
YU Yongxiang
,
ZHANG Zheng
,
ZHU Hongyang
,
PANG Minghai
,
WANG Chunyuan
2024, 45(4):175-186.
DOI: 10.19663/j.issn2095-9869.20230226002
Abstract:
Penaeus vannamei is an important breeding shrimp species in China. Moreover, the quality of the larvae is the cornerstone for the sustainable development of the shrimp industry. In recent years, shrimp postlarva bacterial vitrified syndrome (BVS) has caused huge economic losses in the shrimp postlarva breeding industry, with epidemiological characteristics of rapid onset and high mortality, occurring at 2–3 days post-hatch. The diseased postlarva symptoms include emaciation, decreased activity, and an empty intestinal tract and stomach. The hepatopancreas shows atrophy, blurring of contour, paleness, and even vitrified syndrome. The causative agents of the disease are Vibrio alginolyticus and Vibrio parahaemolyticus. Furthermore, antibiotics are often used to treat BVS; however, frequent and inappropriate use of antibiotics can lead to bacterial resistance and drug residues. With the advantages of antibacterial activity, immunity regulation, low toxicity, few side effects, and lack of drug resistance and drug residues, Chinese herbal medicines play an important role in aquaculture. There are currently no effective drugs available to prevent and treat BVS. It is urgent and important that effective Chinese herbal compounds are developed for BVS prevention and treatment. In this study, first, an antibacterial test was carried out in vitro by the Oxford cup and double dilution methods. Overall, 50 types of Chinese herbal medicines were screened, and those with a good bacteriostasis effect on V. alginolyticus and V. parahaemolyticus were identified. Then, a pathological model of BVS was constructed with different concentrations of V. alginolyticus. The efficacies of the Chinese herb compounds were evaluated based on the mortality, histopathology and ultrastructural pathology characteristics of the postlarvae. The bacterial inhibition test showed that the inhibition zone of V. alginolyticus could be > 12 mm with the application of one of six types of Chinese herbs, including Schisandrae chinensis fructus, Verbenae herba, and Granati pericarpium. The inhibition zone of V. parahaemolyticus could be > 13 mm with the application of one of five types of Chinese herbs, including Chebulae fructus, S. chinensis fructus, Moutan cortex and Sanguisorbae radix. Then, 11 types of antibacterial and two types of immunological drugs were selected for further analysis. Moreover, the MIC and MBC of G. chinensis, C. fructus, Caryophylli flos, and S. chinensis fructus against V. alginolyticus and V. parahaemolyticus were ≤ 12.5 mg/mL and ≤ 50 mg/mL, respectively. Then, three types of compounds were used for in vivo pharmacodynamics tests by combining Chinese antibacterial medicine with Chinese immunological medicine (Cyperi rhizoma and Gardeniae fructus), which were named prescription 1 (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g), prescription 2 (C. fructus 40 g, G. pericarpium 30 g, C. flos 30 g), and prescription 3 (C. fructus 40 g, G. chinensis 30 g, G. fructus 30 g), respectively. Florfenicol was used as the positive drug control, and V. alginolyticus infection without treatment was used as the positive control. The BVS model indicated that V. alginolyticus caused the same vitrification symptoms in shrimp postlarvae as a natural infection. The LD50 of shrimp postlarvae was 2.82 × 105 CFU/mL after 72 h of infection with V. alginolyticus. The pathological model of BVS was constructed by immersing shrimp postlarvae with 5 × 104 CFU/mL V. alginolyticus. Based on this, the infected shrimp postlarvae were treated with drugs for 7 days. After this period, the mortality rates of each group from low to high were: blank control < prescription 1 < florfenicol < prescription 3 < prescription 2 < positive control. The mortality rate of the shrimp postlarvae in the prescription 1 group was significantly lower than that in the positive control group (P < 0.05). The mortality rate of the shrimp postlarvae in the prescription 2 and 3 groups and the florfenicol group was lower than that in the positive control group, but the difference was not significant (P > 0.05). Thus, prescription 1 can significantly reduce the mortality of BVS-positive shrimp postlarvae. Histopathology observation showed that after 7 days of administration, the hepatopancreas and intestinal epithelium of larvae in the prescription 1 group were more intact, with more hepatic tubules, less exfoliation and necrosis of the epithelial cells, and more abundant and well-arranged intestinal epithelial cells. Ultrastructural pathology observation showed that after 7 days of administration, the cell membrane and nucleus of the liver tubule epithelial cells of the diseased shrimp postlarvae in the prescription 1 group were normal, and the mitochondria and endoplasmic reticulum were abundant. Histopathology observation showed that the hepatopancreas and intestinal tract lesions of the shrimp postlarvae in the prescription 1 treatment were significantly less than those in the other groups. In summary, Chinese herbal compounds (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g) had the best effect for preventing and treating shrimp postlarva BVS caused by V. alginolyticus. The results of this study lay a scientific foundation for the development of specialized herbal compounds for the prevention and treatment of bacterial infections and contribute to the green and high-quality development of the shrimp industry.
Penaeus vannamei is an important breeding shrimp species in China. Moreover, the quality of the larvae is the cornerstone for the sustainable development of the shrimp industry. In recent years, shrimp postlarva bacterial vitrified syndrome (BVS) has caused huge economic losses in the shrimp postlarva breeding industry, with epidemiological characteristics of rapid onset and high mortality, occurring at 2–3 days post-hatch. The diseased postlarva symptoms include emaciation, decreased activity, and an empty intestinal tract and stomach. The hepatopancreas shows atrophy, blurring of contour, paleness, and even vitrified syndrome. The causative agents of the disease are Vibrio alginolyticus and Vibrio parahaemolyticus. Furthermore, antibiotics are often used to treat BVS; however, frequent and inappropriate use of antibiotics can lead to bacterial resistance and drug residues. With the advantages of antibacterial activity, immunity regulation, low toxicity, few side effects, and lack of drug resistance and drug residues, Chinese herbal medicines play an important role in aquaculture. There are currently no effective drugs available to prevent and treat BVS. It is urgent and important that effective Chinese herbal compounds are developed for BVS prevention and treatment. In this study, first, an antibacterial test was carried out in vitro by the Oxford cup and double dilution methods. Overall, 50 types of Chinese herbal medicines were screened, and those with a good bacteriostasis effect on V. alginolyticus and V. parahaemolyticus were identified. Then, a pathological model of BVS was constructed with different concentrations of V. alginolyticus. The efficacies of the Chinese herb compounds were evaluated based on the mortality, histopathology and ultrastructural pathology characteristics of the postlarvae. The bacterial inhibition test showed that the inhibition zone of V. alginolyticus could be > 12 mm with the application of one of six types of Chinese herbs, including Schisandrae chinensis fructus, Verbenae herba, and Granati pericarpium. The inhibition zone of V. parahaemolyticus could be > 13 mm with the application of one of five types of Chinese herbs, including Chebulae fructus, S. chinensis fructus, Moutan cortex and Sanguisorbae radix. Then, 11 types of antibacterial and two types of immunological drugs were selected for further analysis. Moreover, the MIC and MBC of G. chinensis, C. fructus, Caryophylli flos, and S. chinensis fructus against V. alginolyticus and V. parahaemolyticus were ≤ 12.5 mg/mL and ≤ 50 mg/mL, respectively. Then, three types of compounds were used for in vivo pharmacodynamics tests by combining Chinese antibacterial medicine with Chinese immunological medicine (Cyperi rhizoma and Gardeniae fructus), which were named prescription 1 (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g), prescription 2 (C. fructus 40 g, G. pericarpium 30 g, C. flos 30 g), and prescription 3 (C. fructus 40 g, G. chinensis 30 g, G. fructus 30 g), respectively. Florfenicol was used as the positive drug control, and V. alginolyticus infection without treatment was used as the positive control. The BVS model indicated that V. alginolyticus caused the same vitrification symptoms in shrimp postlarvae as a natural infection. The LD50 of shrimp postlarvae was 2.82 × 105 CFU/mL after 72 h of infection with V. alginolyticus. The pathological model of BVS was constructed by immersing shrimp postlarvae with 5 × 104 CFU/mL V. alginolyticus. Based on this, the infected shrimp postlarvae were treated with drugs for 7 days. After this period, the mortality rates of each group from low to high were: blank control < prescription 1 < florfenicol < prescription 3 < prescription 2 < positive control. The mortality rate of the shrimp postlarvae in the prescription 1 group was significantly lower than that in the positive control group (P < 0.05). The mortality rate of the shrimp postlarvae in the prescription 2 and 3 groups and the florfenicol group was lower than that in the positive control group, but the difference was not significant (P > 0.05). Thus, prescription 1 can significantly reduce the mortality of BVS-positive shrimp postlarvae. Histopathology observation showed that after 7 days of administration, the hepatopancreas and intestinal epithelium of larvae in the prescription 1 group were more intact, with more hepatic tubules, less exfoliation and necrosis of the epithelial cells, and more abundant and well-arranged intestinal epithelial cells. Ultrastructural pathology observation showed that after 7 days of administration, the cell membrane and nucleus of the liver tubule epithelial cells of the diseased shrimp postlarvae in the prescription 1 group were normal, and the mitochondria and endoplasmic reticulum were abundant. Histopathology observation showed that the hepatopancreas and intestinal tract lesions of the shrimp postlarvae in the prescription 1 treatment were significantly less than those in the other groups. In summary, Chinese herbal compounds (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g) had the best effect for preventing and treating shrimp postlarva BVS caused by V. alginolyticus. The results of this study lay a scientific foundation for the development of specialized herbal compounds for the prevention and treatment of bacterial infections and contribute to the green and high-quality development of the shrimp industry.
2023, 44(5):137-152.
DOI: 10.19663/j.issn2095-9869.20220424002
Abstract:
Procambarus clarkii is commonly known as crayfish and has become one of the main species of freshwater aquaculture in China because of its delicious meat and strong adaptability to the environment. The incredible demand promotes the rapid development of the crayfish breeding industry. Viral diseases caused by white spot syndrome virus (WSSV) are widely spread in crustaceans, including P. clarkii. WSSV has become a serious threat to the crayfish breeding industry because of its extremely fast transmission and associated high mortality. Virus infection can directly induce autophagy mechanisms. Autophagosomes can wrap virus particles and transport them to lysosomes for degradation. As a highly conserved cellular defense mechanism, autophagy plays an important role in the regulation of virus infections. However, many viruses have evolved special mechanisms to resist autophagy regulation or use the membrane structure produced by autophagy body formation to complete their own replication. In this study, WSSV in susceptible P. clarkii were explored to determine how autophagy related genes of P. clarkii participate in the regulation of virus infection. To study the role of the autophagy related gene (Atg2) in the innate immunity of P. clarkii, the full-length sequence of the Atg2 gene in P. clarkii (named PcAtg2) was cloned using the total RNA of P. clarkii hepatopancreas as a template with the rapid-amplification of cDNA ends technique (RACE). The bioinformatic analysis showed that the total length of the PcAtg2 gene sequence in P. clarkii was 9 966 bp, including a 582 bp 5' non coding region, 2 817 bp 3' non coding region, and 6 567 bp open reading frame. We speculate it encodes 2 189 amino acids. Multiple sequence alignments showed the PcAtg2 gene had the characteristic sequence of the Atg family, with 65 serine phosphorylation sites, and 48 glycosylation sites. The amino acid sequence of PcAtg2 in P. clarkii had the highest homology with the Homarus americanus Atg2 gene. The distribution of the PcAtg2 gene in the gill, heart, midgut, hepatopancreas, stomach, muscle, hemocyte, epidermis, testis, ovary, abdominal ganglion, and eyestalk of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that there was no significant difference in the expression of the PcAtg2 gene between male and female individuals. However, there were variations in expression in the different tissues. PcAtg2 was expressed in all tissues of P. clarkii, with the highest expression in the hepatopancreas and the lowest expression in the eyestalk. Under WSSV infection, PcAtg2 was initially up-regulated and then down-regulated in the different tissues, after induced expression. These findings suggest that PcAtg2 is involved in the regulation of autophagy in P. clarkii infected with the WSSV virus, and also plays an important regulatory role in the immune response. RNA interference (RNAi) technology was used to further explore the autophagy related genes PcAtg2 of P. clarkii and their role in WSSV infection. In the WSSV infection experiment with P. clarkii, the copy number of the WSSV virus in the dsPcAtg2 injection group was significantly lower than that in the control group and the dsGFP injection group, indicating that the replication of the WSSV virus was inhibited to some extent during the gene silencing of PcAtg2. The mortality results also showed that silencing PcAtg2 could reduce the mortality of P. clarkii infected with WSSV. In this experiment, after PcAtg2 was silenced, the transmission electron microscope images showed that after 24 and 48 hours of WSSV stress, autophagy vacuoles began to appear in the lysosomes in the hepatopancreas of P. clarkii in the control group, the injected dsPcAtg2 group, and the dsGFP injected group. More autophagosomes appeared and accumulated near the nucleus, indicating that P. clarkii can activate the regulation of cell autophagy under WSSV stress. Among them, more autophagosomes appeared in the hepatopancreas of P. clarkii in the dsPcAtg2 injection group, indicating the PcAtg2 gene promoted the formation of autophagosomes. WSSV virus proliferation can take advantage of autophagy. To avoid using the virus, cells will relatively down regulate the expression of autophagy related genes, and reduce the level of autophagy. In this experiment, by silencing the expression of the PcAtg2 gene, P. clarkii can promote autophagy regulation by up-regulating the expression of other autophagy related genes. In conclusion, the full-length sequences of the autophagy related gene PcAtg2 in P. clarkii were obtained for the first time, allowing us to reveal the effect and mechanism of WSSV infection on autophagy in P. clarkii. The effect of regulating autophagy on WSSV replication was analyzed, and the mechanism of the PcAtg2 gene acting on virus replication by regulating the formation of autophagosome was clarified. The PcAtg2 gene plays an important role in anti-virus immune defense in P. clarkii. We provide a theoretical basis for investigating anti-virus strategies from the perspective of autophagy. Further research on the host defense mechanism regulated by autophagy will provide new antiviral strategies.
Procambarus clarkii is commonly known as crayfish and has become one of the main species of freshwater aquaculture in China because of its delicious meat and strong adaptability to the environment. The incredible demand promotes the rapid development of the crayfish breeding industry. Viral diseases caused by white spot syndrome virus (WSSV) are widely spread in crustaceans, including P. clarkii. WSSV has become a serious threat to the crayfish breeding industry because of its extremely fast transmission and associated high mortality. Virus infection can directly induce autophagy mechanisms. Autophagosomes can wrap virus particles and transport them to lysosomes for degradation. As a highly conserved cellular defense mechanism, autophagy plays an important role in the regulation of virus infections. However, many viruses have evolved special mechanisms to resist autophagy regulation or use the membrane structure produced by autophagy body formation to complete their own replication. In this study, WSSV in susceptible P. clarkii were explored to determine how autophagy related genes of P. clarkii participate in the regulation of virus infection. To study the role of the autophagy related gene (Atg2) in the innate immunity of P. clarkii, the full-length sequence of the Atg2 gene in P. clarkii (named PcAtg2) was cloned using the total RNA of P. clarkii hepatopancreas as a template with the rapid-amplification of cDNA ends technique (RACE). The bioinformatic analysis showed that the total length of the PcAtg2 gene sequence in P. clarkii was 9 966 bp, including a 582 bp 5' non coding region, 2 817 bp 3' non coding region, and 6 567 bp open reading frame. We speculate it encodes 2 189 amino acids. Multiple sequence alignments showed the PcAtg2 gene had the characteristic sequence of the Atg family, with 65 serine phosphorylation sites, and 48 glycosylation sites. The amino acid sequence of PcAtg2 in P. clarkii had the highest homology with the Homarus americanus Atg2 gene. The distribution of the PcAtg2 gene in the gill, heart, midgut, hepatopancreas, stomach, muscle, hemocyte, epidermis, testis, ovary, abdominal ganglion, and eyestalk of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). The results showed that there was no significant difference in the expression of the PcAtg2 gene between male and female individuals. However, there were variations in expression in the different tissues. PcAtg2 was expressed in all tissues of P. clarkii, with the highest expression in the hepatopancreas and the lowest expression in the eyestalk. Under WSSV infection, PcAtg2 was initially up-regulated and then down-regulated in the different tissues, after induced expression. These findings suggest that PcAtg2 is involved in the regulation of autophagy in P. clarkii infected with the WSSV virus, and also plays an important regulatory role in the immune response. RNA interference (RNAi) technology was used to further explore the autophagy related genes PcAtg2 of P. clarkii and their role in WSSV infection. In the WSSV infection experiment with P. clarkii, the copy number of the WSSV virus in the dsPcAtg2 injection group was significantly lower than that in the control group and the dsGFP injection group, indicating that the replication of the WSSV virus was inhibited to some extent during the gene silencing of PcAtg2. The mortality results also showed that silencing PcAtg2 could reduce the mortality of P. clarkii infected with WSSV. In this experiment, after PcAtg2 was silenced, the transmission electron microscope images showed that after 24 and 48 hours of WSSV stress, autophagy vacuoles began to appear in the lysosomes in the hepatopancreas of P. clarkii in the control group, the injected dsPcAtg2 group, and the dsGFP injected group. More autophagosomes appeared and accumulated near the nucleus, indicating that P. clarkii can activate the regulation of cell autophagy under WSSV stress. Among them, more autophagosomes appeared in the hepatopancreas of P. clarkii in the dsPcAtg2 injection group, indicating the PcAtg2 gene promoted the formation of autophagosomes. WSSV virus proliferation can take advantage of autophagy. To avoid using the virus, cells will relatively down regulate the expression of autophagy related genes, and reduce the level of autophagy. In this experiment, by silencing the expression of the PcAtg2 gene, P. clarkii can promote autophagy regulation by up-regulating the expression of other autophagy related genes. In conclusion, the full-length sequences of the autophagy related gene PcAtg2 in P. clarkii were obtained for the first time, allowing us to reveal the effect and mechanism of WSSV infection on autophagy in P. clarkii. The effect of regulating autophagy on WSSV replication was analyzed, and the mechanism of the PcAtg2 gene acting on virus replication by regulating the formation of autophagosome was clarified. The PcAtg2 gene plays an important role in anti-virus immune defense in P. clarkii. We provide a theoretical basis for investigating anti-virus strategies from the perspective of autophagy. Further research on the host defense mechanism regulated by autophagy will provide new antiviral strategies.
WANG Yingeng
,
YU Yongxiang
,
CAI Xinxin
,
ZHANG Zheng
,
WANG Chunyuan
,
LIAO Meijie
,
ZHU Hongyang
,
LI Hao
2024, 45(3):171-181.
DOI: 10.19663/j.issn2095-9869.20221124002
Abstract:
Acute hepatopancreatic necrosis disease (AHPND) is widely prevalent, has a rapid onset, and has high mortality in shrimp culture, making it a key limiting factor affecting shrimp aquaculture development in recent years, resulting in massive economic losses to the industry worldwide. Systematic studies that investigate which factors significantly correlate with the occurrence of AHPND, and further establishment of a prediction model for the occurrence of shrimp AHPND, are important for preventing and controlling the disease. In this study, Penaeus vannamei in pond culture were preliminarily analyzed for the coupling relationship between the occurrence and prevalence of AHPND in shrimps and pathogens, and for environmental and host autoimmune factors by assessing the environmental factors, pathogen abundance, and host health indicators under AHPND incidence. Then, a mathematical early warning model of AHPND occurrence in pond-cultured P. vannamei was constructed using Deep Forest algorithm. The occurrence of AHPND and its environment, pathogen, and shrimp immunity factors in pond-cultured P. vannamei were continuously monitored to explore the relationship between the occurrences of AHPND in relation to these factors. A total of 18 parameters were assessed, including the air and water temperature, salinity, pH, dissolved oxygen (DO), ammonia nitrogen (NH4+-N) and nitrite (NO2-N) concentrations, culturable bacteria and Vibrio in water, culturable bacteria and Vibrio in the shrimp hepatopancreas, the proportion of Vibrio in water and the shrimp hepatopancreas, and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LZM), and phenol oxidase (PO) in shrimp muscles. The parameter simulation prediction data based on the P. vannamei AHPND occurrence-related factor sequence (environmental factor, microbial factor, and shrimp health indicator) were constructed for the first time. The one-dimensional sequence was mapped into the three-dimensional space, different kernel functions were selected in combination with the actual classification problem to compare the model fitting accuracy, and the test algorithm optimized the parameters in the model. A total of 140 relevant data groups were collected under the same mode, and the groups of additional exogenous inputs during the breeding process were eliminated. After deleting invalid data, there were 100 groups of classified monitoring data, including 25 groups of morbidity data and 75 groups of health data. Moreover, the model was affected due to the dimensional and quantitative differences among different factors. In order to improve the speed of subsequent experimental training and prediction accuracy, the 100 groups of training test data processed by the mapminmax function were normalized for data processing. The relationship between 18 parameters and the occurrence of AHPND in P. vannamei was analyzed using Pearson´s correlation, and the main influencing factors were further screened using pairwise analysis between the factors. Pearson´s correlation analysis indicated that the incidence of AHPND positively correlated (P<0.05) with salinity, the number of culturable bacteria and Vibrio in the shrimp, the proportion of Vibrio in the shrimp, the number of culturable bacteria and Vibrio in water, and the activities of LZM, ACP, and PO in shrimp muscles. The correlation coefficients were 0.350 1, 0.574 1, 0.521 1, 0.391 1, 0.374 7, 0.238 3, 0.438 2, 0.257 1, and 0.228 9, respectively, indicating that AHPND was more likely to occur with an increase of these parameter values within a certain range. The incidence of AHPND negatively correlated with water temperature (P<0.05), and the correlation coefficient was –0.227 9. Moreover, the water temperature, pH, DO, NH4+-N and NO2-N concentrations, Vibrio proportion in water, AKP, and SOD had a weak correlation with the incidence of AHPND (P>0.05). Furthermore, parameters were removed in the model construction process according to the correlation between parameters and factors. The occurrence of AHPND in P. vannamei directly and significantly correlated with seven parameters, including the total number of shrimp bacteria, the total number of shrimp Vibrio, LZM, the proportion of shrimp Vibrio, the total number of water bacteria, salinity, and the total number of water Vibrio. The prediction performance of three popular integrated learning method algorithms based on decision tree, Deep Forest, LightGBM, and XGBoost was evaluated using Python language programming, and, finally, a four-dimensional vector early warning prediction model based on the Deep Forest algorithm for the total number of shrimp bacteria, the proportion of Vibrio shrimp, the total number of water bacteria, and salinity was established (accuracy: 89.00%). Although the prediction performance of the Deep Forest model decreased somewhat compared with that of the support vector machine model established in this study, the algorithm was gradually screened out based on the correlation between factors, including the effects of all factors. It was proven that the Deep Forest model established in this study was the ideal prediction model for predicting the occurrence of AHPND in P. vannamei among the 10 dimension parameters tried, and the superiority of the Deep Forest algorithm was also further verified. The results provide basic data and technical support for shrimp AHPND disease prediction, prevention and control, and lay a theoretical foundation for further establishment of aquaculture animal disease early warning theory.
Acute hepatopancreatic necrosis disease (AHPND) is widely prevalent, has a rapid onset, and has high mortality in shrimp culture, making it a key limiting factor affecting shrimp aquaculture development in recent years, resulting in massive economic losses to the industry worldwide. Systematic studies that investigate which factors significantly correlate with the occurrence of AHPND, and further establishment of a prediction model for the occurrence of shrimp AHPND, are important for preventing and controlling the disease. In this study, Penaeus vannamei in pond culture were preliminarily analyzed for the coupling relationship between the occurrence and prevalence of AHPND in shrimps and pathogens, and for environmental and host autoimmune factors by assessing the environmental factors, pathogen abundance, and host health indicators under AHPND incidence. Then, a mathematical early warning model of AHPND occurrence in pond-cultured P. vannamei was constructed using Deep Forest algorithm. The occurrence of AHPND and its environment, pathogen, and shrimp immunity factors in pond-cultured P. vannamei were continuously monitored to explore the relationship between the occurrences of AHPND in relation to these factors. A total of 18 parameters were assessed, including the air and water temperature, salinity, pH, dissolved oxygen (DO), ammonia nitrogen (NH4+-N) and nitrite (NO2-N) concentrations, culturable bacteria and Vibrio in water, culturable bacteria and Vibrio in the shrimp hepatopancreas, the proportion of Vibrio in water and the shrimp hepatopancreas, and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), lysozyme (LZM), and phenol oxidase (PO) in shrimp muscles. The parameter simulation prediction data based on the P. vannamei AHPND occurrence-related factor sequence (environmental factor, microbial factor, and shrimp health indicator) were constructed for the first time. The one-dimensional sequence was mapped into the three-dimensional space, different kernel functions were selected in combination with the actual classification problem to compare the model fitting accuracy, and the test algorithm optimized the parameters in the model. A total of 140 relevant data groups were collected under the same mode, and the groups of additional exogenous inputs during the breeding process were eliminated. After deleting invalid data, there were 100 groups of classified monitoring data, including 25 groups of morbidity data and 75 groups of health data. Moreover, the model was affected due to the dimensional and quantitative differences among different factors. In order to improve the speed of subsequent experimental training and prediction accuracy, the 100 groups of training test data processed by the mapminmax function were normalized for data processing. The relationship between 18 parameters and the occurrence of AHPND in P. vannamei was analyzed using Pearson´s correlation, and the main influencing factors were further screened using pairwise analysis between the factors. Pearson´s correlation analysis indicated that the incidence of AHPND positively correlated (P<0.05) with salinity, the number of culturable bacteria and Vibrio in the shrimp, the proportion of Vibrio in the shrimp, the number of culturable bacteria and Vibrio in water, and the activities of LZM, ACP, and PO in shrimp muscles. The correlation coefficients were 0.350 1, 0.574 1, 0.521 1, 0.391 1, 0.374 7, 0.238 3, 0.438 2, 0.257 1, and 0.228 9, respectively, indicating that AHPND was more likely to occur with an increase of these parameter values within a certain range. The incidence of AHPND negatively correlated with water temperature (P<0.05), and the correlation coefficient was –0.227 9. Moreover, the water temperature, pH, DO, NH4+-N and NO2-N concentrations, Vibrio proportion in water, AKP, and SOD had a weak correlation with the incidence of AHPND (P>0.05). Furthermore, parameters were removed in the model construction process according to the correlation between parameters and factors. The occurrence of AHPND in P. vannamei directly and significantly correlated with seven parameters, including the total number of shrimp bacteria, the total number of shrimp Vibrio, LZM, the proportion of shrimp Vibrio, the total number of water bacteria, salinity, and the total number of water Vibrio. The prediction performance of three popular integrated learning method algorithms based on decision tree, Deep Forest, LightGBM, and XGBoost was evaluated using Python language programming, and, finally, a four-dimensional vector early warning prediction model based on the Deep Forest algorithm for the total number of shrimp bacteria, the proportion of Vibrio shrimp, the total number of water bacteria, and salinity was established (accuracy: 89.00%). Although the prediction performance of the Deep Forest model decreased somewhat compared with that of the support vector machine model established in this study, the algorithm was gradually screened out based on the correlation between factors, including the effects of all factors. It was proven that the Deep Forest model established in this study was the ideal prediction model for predicting the occurrence of AHPND in P. vannamei among the 10 dimension parameters tried, and the superiority of the Deep Forest algorithm was also further verified. The results provide basic data and technical support for shrimp AHPND disease prediction, prevention and control, and lay a theoretical foundation for further establishment of aquaculture animal disease early warning theory.
LIU Dingyuan
,
WANG Chunyuan
,
YU Yongxiang
,
WANG Yingeng
,
LI Jingze
,
ZHANG Xiaosong
,
QIN Lei
,
ZHANG Zheng
2024, 45(3):182-192.
DOI: 10.19663/j.issn2095-9869.20230211001
Abstract:
As a pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed widely in the marine environment, and the host species of PDD are diverse. Although in the past decade the number of reports on the pathogenicity of PDD to marine animals has gradually increased, it is necessary to study this further in order to establish targeted prevention and technical control measures to reduce harm to marine organisms. In this study, a high-virulence PDD strain (PDD1608) was selected to explore the effect of the virulence gene dly on the biological characteristics and pathogenicity. The dly-deleted mutant strain Δdly PDD1608::Cm was successfully constructed using the λRed recombination technique. The biological characteristics of the wild-type and mutant strains were compared, including growth, swarming motility, drug susceptibility, physiological and biochemical characteristics, biofilm formation ability, and hemolytic and phospholipase activity of extracellular products (ECP). Meanwhile, Oryzias melastigma was used as the target host, and the pathogenicity of wild-type and mutant strains and their ECPs to O. melastigma was detected by the artificial infection test. There was a positive correlation between the results of the bacterial solution and ECP challenge, indicating that deletion of the dly gene affects the virulence of the PDD strain. Deletion of the virulence gene dly resulted in slower growth of the PDD mutant strain; reduced swarming and hemolytic and phospholipase activity; no change in the drug susceptibility or physiological and biochemical characteristics of the wild type and mutant strains; compared with the wild type strain, significant different (P<0.05) biofilm formation ability of the mutant strain; the artificial infection test showed that the pathogenicity of the mutant strain and its ECP decreased. The results of this study revealed that the virulence gene dly affects many biological characteristics of PDD strains, such as growth, swarming motility, and hemolytic and phospholipase activity, and it was found to be closely related to the pathogenicity of the PDD strain and ECP. The results of this study contribute to further study of the influence of the virulence gene dly on the biological characteristics of the PDD strain in order to determine the appropriate prevention and control measures to curtail its spread and infection. The artificial infection test of the model organism O. melastigma helps provide an animal reference model for further exploration of the pathogenic mechanism and infection process of the PDD strain on the host. The results of the ECP experiments provide a theoretical reference for the research and development of PDD subunit vaccines.
As a pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed widely in the marine environment, and the host species of PDD are diverse. Although in the past decade the number of reports on the pathogenicity of PDD to marine animals has gradually increased, it is necessary to study this further in order to establish targeted prevention and technical control measures to reduce harm to marine organisms. In this study, a high-virulence PDD strain (PDD1608) was selected to explore the effect of the virulence gene dly on the biological characteristics and pathogenicity. The dly-deleted mutant strain Δdly PDD1608::Cm was successfully constructed using the λRed recombination technique. The biological characteristics of the wild-type and mutant strains were compared, including growth, swarming motility, drug susceptibility, physiological and biochemical characteristics, biofilm formation ability, and hemolytic and phospholipase activity of extracellular products (ECP). Meanwhile, Oryzias melastigma was used as the target host, and the pathogenicity of wild-type and mutant strains and their ECPs to O. melastigma was detected by the artificial infection test. There was a positive correlation between the results of the bacterial solution and ECP challenge, indicating that deletion of the dly gene affects the virulence of the PDD strain. Deletion of the virulence gene dly resulted in slower growth of the PDD mutant strain; reduced swarming and hemolytic and phospholipase activity; no change in the drug susceptibility or physiological and biochemical characteristics of the wild type and mutant strains; compared with the wild type strain, significant different (P<0.05) biofilm formation ability of the mutant strain; the artificial infection test showed that the pathogenicity of the mutant strain and its ECP decreased. The results of this study revealed that the virulence gene dly affects many biological characteristics of PDD strains, such as growth, swarming motility, and hemolytic and phospholipase activity, and it was found to be closely related to the pathogenicity of the PDD strain and ECP. The results of this study contribute to further study of the influence of the virulence gene dly on the biological characteristics of the PDD strain in order to determine the appropriate prevention and control measures to curtail its spread and infection. The artificial infection test of the model organism O. melastigma helps provide an animal reference model for further exploration of the pathogenic mechanism and infection process of the PDD strain on the host. The results of the ECP experiments provide a theoretical reference for the research and development of PDD subunit vaccines.
ZHAO Wenxiu
,
WAN Xiaoyuan
,
XIA Jitao
,
YAO Liang
,
XU Ruidong
,
WANG Wei
,
YU Xingtong
,
ZHANG Qingli
2024, 45(5):195-203.
DOI: 10.19663/j.issn2095-9869.20230601002
Abstract:
Viral covert mortality disease (VCMD) in cultured shrimp is caused by covert mortality nodavirus (CMNV). The infected shrimp mainly die successively in the deep-water areas at the bottom of farming ponds, which is difficult to observe; as such, the term "covert mortality disease" was coined by local farmers. The disease is known as running mortality syndrome by Southeast Asian farmers because a small number of diseased shrimp die daily in the infected ponds, and more continue to die until harvest time. The infected shrimp mostly show symptoms such as carapace softening, hepatopancreas color fading, atrophy, empty stomach, slow growth, etc. In the acute infection stage, the infected shrimp show opaque abdominal muscles. The prevalence of VCMD has caused severe economic losses in the shrimp farming industry over the past decade. CMNV can infect many major cultured shrimp species, including Penaeus vannamei, P. japonicus, P. chinensis, Macrobrachium rosenbergii, and Exopalaemon carinicauda. Moreover, the virus infects the co-inhabiting organisms in shrimp ponds, which makes it difficult to remove from shrimp farming areas due to its wide host range among invertebrates and its transport by many vectors. To elucidate the prevalence of CMNV in the main cultured shrimp species in China in recent years, an epidemiological investigation of CMNV, including clinical sample collection from shrimp and other organisms inhabiting the shrimp ponds, was carried out in the main shrimp culture areas of China from 2021 to 2022, and the collected samples were systematically analyzed using molecular biology, histopathology, and transmission electron microscopy approaches. During this period, 1,299 samples were collected from Tianjin, Shandong, Jiangsu, Hainan, Hubei, and Xinjiang and included P. vannamei, M. rosenbergii, P. japonicus, P. chinensis, Procambarus clarkia, and other organisms living in shrimp culture ponds. The collected samples were analyzed using CMNV TaqMan probe real-time quantitative reverse transcription PCR (TaqMan RT-qPCR). The results showed that CMNV could be detected in the main cultured shrimp species, including P. vannamei, M. rosenbergii, and P. japonicus. A percentage of the samples collected from Shandong, Jiangsu, Hainan, Xinjiang, Guangxi, and Tianjin were observed to be CMNV-positive in the TaqMan RT-qPCR assays. In addition to cultured shrimp, CMNV was detected in organisms living in the shrimp ponds, such as Artemia collected in 2021 and Perinereis aibuhitensis collected in 2022. The CMNV-positive rate of samples collected in 2021 was 10.04% (69/687), and that of samples collected in 2022 was 11.44% (70/612). Epidemiological investigation showed that CMNV infection in outdoor pond culture usually caused a certain degree of cumulative death, whereas CMNV infection in indoor culture mainly caused the softening and reduced growth of infected shrimp and did not cause a large number of infected shrimp to die. High CMNV loads were detected in samples of Artemia used as fresh bait for shrimp and in samples of P. aibuhitensis living in shrimp pond soils. Accordingly, a high CMNV infection rate was observed in cultured P. vannamei feeding on CMNV-positive Artemia and in that farmed in ponds containing CMNV-positive P. aibuhitensis. The results indicated that CMNV vectors such as Artemia and P. aibuhitensis may pose a high risk of CMNV transition, threatening the health of the shrimp in the culture system. Recent studies on the pathogenesis of CMNV infection indicated that the virus mainly infected epithelial tissues, nerve tissues, and connective tissues in the hepatopancreas, appendages, mid-gut, gills, eye stalks, and nerves of shrimp. Therefore, histopathological and in situ hybridization analyses were performed on samples identified as CMNV-positive using TaqMan RT-qPCR in this study. Eosinophilic virus inclusion bodies were observed in the hepatopancreas and foremidgut tissues, and pathological lesions were observed in the appendicular nerves of diseased Penaeus shrimp. Moreover, obvious purple hybridization signals from the CMNV RNA probe were seen in tissues with lesions, which further confirmed the positive detection results for CMNV in shrimp samples obtained using TaqMan RT-qPCR in this study. The results of this study showed that, compared with previous years, there was still a high CMNV-positive detection rate in many areas of crustacean culture in 2022, and the epidemic risk of this virus was still high. Multiple CMNV-positive results were obtained in the co-inhabiting and feed organisms in crustacean farming ponds. These results remind fishery authorities and stakeholders in the prawn breeding industry to further strengthen the monitoring and early detection of CMNV to prevent and control the spread of the virus in seedlings, reduce the risk of epidemic hazards, and promote the sustainable and healthy development of the crustacean aquaculture industry.
Viral covert mortality disease (VCMD) in cultured shrimp is caused by covert mortality nodavirus (CMNV). The infected shrimp mainly die successively in the deep-water areas at the bottom of farming ponds, which is difficult to observe; as such, the term "covert mortality disease" was coined by local farmers. The disease is known as running mortality syndrome by Southeast Asian farmers because a small number of diseased shrimp die daily in the infected ponds, and more continue to die until harvest time. The infected shrimp mostly show symptoms such as carapace softening, hepatopancreas color fading, atrophy, empty stomach, slow growth, etc. In the acute infection stage, the infected shrimp show opaque abdominal muscles. The prevalence of VCMD has caused severe economic losses in the shrimp farming industry over the past decade. CMNV can infect many major cultured shrimp species, including Penaeus vannamei, P. japonicus, P. chinensis, Macrobrachium rosenbergii, and Exopalaemon carinicauda. Moreover, the virus infects the co-inhabiting organisms in shrimp ponds, which makes it difficult to remove from shrimp farming areas due to its wide host range among invertebrates and its transport by many vectors. To elucidate the prevalence of CMNV in the main cultured shrimp species in China in recent years, an epidemiological investigation of CMNV, including clinical sample collection from shrimp and other organisms inhabiting the shrimp ponds, was carried out in the main shrimp culture areas of China from 2021 to 2022, and the collected samples were systematically analyzed using molecular biology, histopathology, and transmission electron microscopy approaches. During this period, 1,299 samples were collected from Tianjin, Shandong, Jiangsu, Hainan, Hubei, and Xinjiang and included P. vannamei, M. rosenbergii, P. japonicus, P. chinensis, Procambarus clarkia, and other organisms living in shrimp culture ponds. The collected samples were analyzed using CMNV TaqMan probe real-time quantitative reverse transcription PCR (TaqMan RT-qPCR). The results showed that CMNV could be detected in the main cultured shrimp species, including P. vannamei, M. rosenbergii, and P. japonicus. A percentage of the samples collected from Shandong, Jiangsu, Hainan, Xinjiang, Guangxi, and Tianjin were observed to be CMNV-positive in the TaqMan RT-qPCR assays. In addition to cultured shrimp, CMNV was detected in organisms living in the shrimp ponds, such as Artemia collected in 2021 and Perinereis aibuhitensis collected in 2022. The CMNV-positive rate of samples collected in 2021 was 10.04% (69/687), and that of samples collected in 2022 was 11.44% (70/612). Epidemiological investigation showed that CMNV infection in outdoor pond culture usually caused a certain degree of cumulative death, whereas CMNV infection in indoor culture mainly caused the softening and reduced growth of infected shrimp and did not cause a large number of infected shrimp to die. High CMNV loads were detected in samples of Artemia used as fresh bait for shrimp and in samples of P. aibuhitensis living in shrimp pond soils. Accordingly, a high CMNV infection rate was observed in cultured P. vannamei feeding on CMNV-positive Artemia and in that farmed in ponds containing CMNV-positive P. aibuhitensis. The results indicated that CMNV vectors such as Artemia and P. aibuhitensis may pose a high risk of CMNV transition, threatening the health of the shrimp in the culture system. Recent studies on the pathogenesis of CMNV infection indicated that the virus mainly infected epithelial tissues, nerve tissues, and connective tissues in the hepatopancreas, appendages, mid-gut, gills, eye stalks, and nerves of shrimp. Therefore, histopathological and in situ hybridization analyses were performed on samples identified as CMNV-positive using TaqMan RT-qPCR in this study. Eosinophilic virus inclusion bodies were observed in the hepatopancreas and foremidgut tissues, and pathological lesions were observed in the appendicular nerves of diseased Penaeus shrimp. Moreover, obvious purple hybridization signals from the CMNV RNA probe were seen in tissues with lesions, which further confirmed the positive detection results for CMNV in shrimp samples obtained using TaqMan RT-qPCR in this study. The results of this study showed that, compared with previous years, there was still a high CMNV-positive detection rate in many areas of crustacean culture in 2022, and the epidemic risk of this virus was still high. Multiple CMNV-positive results were obtained in the co-inhabiting and feed organisms in crustacean farming ponds. These results remind fishery authorities and stakeholders in the prawn breeding industry to further strengthen the monitoring and early detection of CMNV to prevent and control the spread of the virus in seedlings, reduce the risk of epidemic hazards, and promote the sustainable and healthy development of the crustacean aquaculture industry.
HAO Wenyue
,
WANG Jinjin
,
GE Jianlong
,
LI Bin
,
WANG Yingeng
,
LIAO Meijie
,
RONG Xiaojun
,
ZHAO Hongjing
,
JIANG Minqi
,
ZHAO Wenguang
,
NIU Licheng
,
PAN Jiao
2025, 46(3):183-193.
DOI: 10.19663/j.issn2095-9869.20240322002
Abstract:
Boveria disease affects the aquaculture efficiency of sea cucumbers. Boveria labialis causes Boveria disease in Apostichopus japonicus. Owing to the lack of rapid and accurate detection methods, analyzing the transmission route of the pathogen remains complicated. Based on the partial mitochondrial genome sequence of B. labialis obtained using sequencing, primers were designed to amplify nad10 and a SYBR GreenⅠreal-time fluorescent quantitative PCR detection method was established. The concentration of B. labialis in different culture areas and different culture modes of A. japonicus was detected and analyzed. The standard curve established by the designed primers had a good linear relationship in the range of the plasmid standard of 4.05×101–4.05×109 copies/µL, with a reliability (R2) of 0.997. The melting curve showed a single peak, without primer dimers or nonspecific amplification. The minimum detection limit of sensitivity test was 40.5 copies/µL. The designed primers showed specific amplification for B. labialis only and exhibited no cross reaction to Uronema sp., chaenea sp., Colpoda sp. and Paramecium sp. In the repeatability test, the homogeneity of Ct values in the intra-assay and inter-assay of each concentration was high, the CV values of intra- and inter-assay tests were 0.32%–0.82% and 0.40%–0.88%, respectively, indicating good stability. This method was used to detect environmental samples and feeds in different culture areas of four kinds of A. japonicus culture modes. Combined with the comparative analysis of microscopic examination results, there was a moderate positive correlation (R=0.563) between the DNA load of B. labialis in sea water and the infection degree of B. labialis in A. japonicus. A high positive correlation (R=0.931) was found between the DNA load of B. labialis in sediment and attachment samples and the infection degree of B. labialis in A. japonicus. Fresh sea mud was an important carrier for B. labialis transmission. The relevant research results provide reference for the rapid detection, transmission route analysis, and B. labialis prevention and control.
Boveria disease affects the aquaculture efficiency of sea cucumbers. Boveria labialis causes Boveria disease in Apostichopus japonicus. Owing to the lack of rapid and accurate detection methods, analyzing the transmission route of the pathogen remains complicated. Based on the partial mitochondrial genome sequence of B. labialis obtained using sequencing, primers were designed to amplify nad10 and a SYBR GreenⅠreal-time fluorescent quantitative PCR detection method was established. The concentration of B. labialis in different culture areas and different culture modes of A. japonicus was detected and analyzed. The standard curve established by the designed primers had a good linear relationship in the range of the plasmid standard of 4.05×101–4.05×109 copies/µL, with a reliability (R2) of 0.997. The melting curve showed a single peak, without primer dimers or nonspecific amplification. The minimum detection limit of sensitivity test was 40.5 copies/µL. The designed primers showed specific amplification for B. labialis only and exhibited no cross reaction to Uronema sp., chaenea sp., Colpoda sp. and Paramecium sp. In the repeatability test, the homogeneity of Ct values in the intra-assay and inter-assay of each concentration was high, the CV values of intra- and inter-assay tests were 0.32%–0.82% and 0.40%–0.88%, respectively, indicating good stability. This method was used to detect environmental samples and feeds in different culture areas of four kinds of A. japonicus culture modes. Combined with the comparative analysis of microscopic examination results, there was a moderate positive correlation (R=0.563) between the DNA load of B. labialis in sea water and the infection degree of B. labialis in A. japonicus. A high positive correlation (R=0.931) was found between the DNA load of B. labialis in sediment and attachment samples and the infection degree of B. labialis in A. japonicus. Fresh sea mud was an important carrier for B. labialis transmission. The relevant research results provide reference for the rapid detection, transmission route analysis, and B. labialis prevention and control.
2023, 44(2):186-195.
DOI: 10.19663/j.issn2095-9869.20220426001
Abstract:
China is the largest producer of Neopyropia yezoensis, ranking first in the world for cultivation area and yield production. In N. yezoensis production, diseases occur frequently every year due to increased farming density, environmental deterioration, and germplasm degeneration, resulting in serious economic losses to farmers. Red rot disease is caused by Pythium sp. and is one of the most common diseases during N. yezoensis farming, leading to empty nets and harvest loss. Air-dry, cold storage, and acid wash are common methods to counteract red rot disease in N. yezoensis farming. These physical or chemical disinfection methods, however, are not completely effective, and some have serious consequences. For example, refrigeration equipment and space will greatly increase costs, and acid wash treatments can cause environmental pollution. Although research has attempted to select or cultivate disease-resistant strains of laver, there remains no laver strain completely immune to red rot disease. Biocontrol is an effective method that is widely used in disease control of land crops. Biocontrol is potentially an environment-friendly and effective control method for macroalgal diseases. However, limited information exists on biocontrol in macroalgal diseases. During the growth and development of macroalgae, a variety of metabolites are produced on their surfaces, which provide suitable substrates for microbial colonization. The microbial community attached to the surface of algae is highly diverse and can produce many kinds of biologically active compounds. These compounds not only play a major role in normal morphology, growth, and development of algae, but also have antibacterial, antiviral, antiparasitic, and other activities to protect the host from harmful organisms. Therefore, the epiphytic microorganisms of algae provide good sources of microorganisms for biological screening. This study aimed to screen and identify bacteria with antagonistic ability towards Pythium sp.. A total of 385 bacterial strains, isolated from farming algae and their culturing environments, were screened. In the first round of screening, the plate confrontation method was used and repeated twice and confirmed that nine strains had antagonistic effects on the growth of Pythium sp.. The diameter of the bacteriostatic zone was approximately 1.65–16.54 mm. In the second round of screening, three strains (assigned as P3, P6, and P19) were further investigated using the toxic medium method for inhibitory activities in their extracellular products. Repeated experiments showed that the bacteriostatic rate was approximately 20.04%–30.09%. The antibacterial spectrum was determined by the plate confrontation method. Strains P3, P6, and P19 all had antagonistic effects on the eight tested strains of Pythium preserved in our laboratory. The inhibition rates reached 52.09%–97.95% for P3, 26.81%–78.04% for P6, 10.47%–41.91% for P19, respectively. The Pythium hyphae on the confrontation edge were further investigated by lactic acid phenol cotton blue staining. When compared with Pythium hyphae in a control group, the density and color of Pythium hyphae against strains P3 and P19 became sparse and lighter. There were no significant changes in Pythium hyphae against strain P6. Strains P3 and P6 were identified as Pseudoalteromonas piscicida, and P19 as P. peptidolytica, based on 16S rRNA gene identification and multilocus sequences analysis of 16S rRNA-dnaA-dnaN-recA. The bacterial strains of P3, P6, and P19 had significant antagonistic capabilities against the pathogenic Pythium strains. This indicates they are potential biocontrol probiotics for the control of red rot disease in N. yezoensis. The present study provides the foundation for research on the evaluation and application of antagonistic bacterial strains in the biocontrol of red rot disease of N. yezoensis.
China is the largest producer of Neopyropia yezoensis, ranking first in the world for cultivation area and yield production. In N. yezoensis production, diseases occur frequently every year due to increased farming density, environmental deterioration, and germplasm degeneration, resulting in serious economic losses to farmers. Red rot disease is caused by Pythium sp. and is one of the most common diseases during N. yezoensis farming, leading to empty nets and harvest loss. Air-dry, cold storage, and acid wash are common methods to counteract red rot disease in N. yezoensis farming. These physical or chemical disinfection methods, however, are not completely effective, and some have serious consequences. For example, refrigeration equipment and space will greatly increase costs, and acid wash treatments can cause environmental pollution. Although research has attempted to select or cultivate disease-resistant strains of laver, there remains no laver strain completely immune to red rot disease. Biocontrol is an effective method that is widely used in disease control of land crops. Biocontrol is potentially an environment-friendly and effective control method for macroalgal diseases. However, limited information exists on biocontrol in macroalgal diseases. During the growth and development of macroalgae, a variety of metabolites are produced on their surfaces, which provide suitable substrates for microbial colonization. The microbial community attached to the surface of algae is highly diverse and can produce many kinds of biologically active compounds. These compounds not only play a major role in normal morphology, growth, and development of algae, but also have antibacterial, antiviral, antiparasitic, and other activities to protect the host from harmful organisms. Therefore, the epiphytic microorganisms of algae provide good sources of microorganisms for biological screening. This study aimed to screen and identify bacteria with antagonistic ability towards Pythium sp.. A total of 385 bacterial strains, isolated from farming algae and their culturing environments, were screened. In the first round of screening, the plate confrontation method was used and repeated twice and confirmed that nine strains had antagonistic effects on the growth of Pythium sp.. The diameter of the bacteriostatic zone was approximately 1.65–16.54 mm. In the second round of screening, three strains (assigned as P3, P6, and P19) were further investigated using the toxic medium method for inhibitory activities in their extracellular products. Repeated experiments showed that the bacteriostatic rate was approximately 20.04%–30.09%. The antibacterial spectrum was determined by the plate confrontation method. Strains P3, P6, and P19 all had antagonistic effects on the eight tested strains of Pythium preserved in our laboratory. The inhibition rates reached 52.09%–97.95% for P3, 26.81%–78.04% for P6, 10.47%–41.91% for P19, respectively. The Pythium hyphae on the confrontation edge were further investigated by lactic acid phenol cotton blue staining. When compared with Pythium hyphae in a control group, the density and color of Pythium hyphae against strains P3 and P19 became sparse and lighter. There were no significant changes in Pythium hyphae against strain P6. Strains P3 and P6 were identified as Pseudoalteromonas piscicida, and P19 as P. peptidolytica, based on 16S rRNA gene identification and multilocus sequences analysis of 16S rRNA-dnaA-dnaN-recA. The bacterial strains of P3, P6, and P19 had significant antagonistic capabilities against the pathogenic Pythium strains. This indicates they are potential biocontrol probiotics for the control of red rot disease in N. yezoensis. The present study provides the foundation for research on the evaluation and application of antagonistic bacterial strains in the biocontrol of red rot disease of N. yezoensis.
WANG Chunyuan
,
WANG Yingeng
,
ZHAO Weizhi
,
YU Yongxiang
,
ZHANG Zheng
,
RONG Xiaojun
,
LIU Dingyuan
2025, 46(3):212-221.
DOI: 10.19663/j.issn2095-9869.20240312002
Abstract:
Vibrio alginolyticus is a primary etiological agent for aquatic animal death. V. alginolyticus can infect fish, causing bleeding, ulcer, and blood poisoning; shrimps, causing shrimp post-larvae bacterial vitrified syndrome, acute hepatopancreatic necrosis disease, rotted gill disease, and white fecal syndrome; crabs, causing milk and toothpaste diseases; sea cucumbers, causing skin ulcer syndrome; and shellfish, thereby leading to substantial economic loss in the marine aquaculture industry. Antibiotics, disinfectants, and microecologics are usually used in the aquaculture to prevent or cure such infectious diseases however, their outcomes remain unsatisfactory. In addition, irrational drug use increases risks, such as environmental pollution, bacterial drug resistance, and drug residue. Compared with antibiotics, traditional Chinese medicines (TCMs) present certain advantages, such as antibacterial properties, immunoregulation, slight toxic and side effect, and low drug resistance or residue. Therefore, TCMs have garnered increasing attention in recent years. Using V. alginolyticus and V. parahemolyticus isolated from penaeid shrimp larvae with bacterial vitrified syndrome as research objects, we screened the bacteriostatic activity of 50 TCMs. Four kinds of TCM, namely Terminalia chebula, Galla chinensis, pomegranate peel, and Sanguisorba officinalis, demonstrated good bacteriostatic effects. Gallic acid (GA) is a main active component of these four TCM compounds. GA has good antibacterial, antiviral, anti-inflammatory, and antioxidative properties and can protect the liver and improve the immunological function of the body, thus it may be used to treat and prevent multiple animal diseases. Although GA has antibacterial effects on various bacteria, the bacteriostatic activity and possible mechanism against V. alginolyticus remains unelucidated. By measuring the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and growth curve of GA against V. alginolyticus, we evaluated the bacteriostatic activity of GA against V. alginolyticus. Moreover, the changes in the AKP activity and electrical conductivity of the supernatant fluid of bacteria solution and biofilms, moveability, and aggregation capacity of V. alginolyticus were determined before and after GA treatment to investigate the bacteriostatic mechanism of GA against V. alginolyticus. The MIC and MBC of GA against V. alginolyticus were 4 mg/mL and 8 mg/mL, respectively. These two GA concentrations could completely inhibit V. alginolyticus growth, while 2 mg/mL GA significantly suppressed V. alginolyticus growth, suggesting that the inhibitory effect of GA on V. alginolyticus was dose-dependent. Thus, GA concentrations greater than 4 mg/mL should be selected and used to achieve an effective bacteriostatic effect on V. alginolyticus. The bacterial cell wall is an important structure that maintains cell morphology and facilitates cell protection. Biofilms comprise various extracellular materials, such as proteins, exopolysaccharides, lipids, and extracellular DNA. Biofilms not only enhance the resistance of bacteria to adverse external environment, but also increase their resistance to antibacterial agents. However, TCM can restrain bacterial biofilm formation and development, destroy their cell wall and membrane, affect protein and nucleic acid synthesis, promote oxidative stress, and inhibit virulent factor expression to ultimately suppress or kill bacteria. The changes in AKP and electrical conductivity of bacterial culture serve as an index to verify whether the bacterial cell wall was destroyed or whether its permeability increased. GA (1, 2, 4, and 8 mg/mL) destroyed the cell wall within 2 h and caused AKP leakage. Furthermore, the degree of V. alginolyticus cell wall destruction was positively correlated with GA concentrations; 2, 4, and 8 mg/mL GA could remarkably increase electrical conductivity of the supernatant fluid of V. alginolyticus; compared with the positive control group, 4 and 8 mg/mL GA had a significant inhibitory effect on the formation of the biofilms of V. alginolyticus, with an inhibition ratio of 83.26% (P<0.05) and 77.80% (P<0.05), respectively. Meanwhile, 4 and 8 mg/mL GA notably eliminated mature biofilms, with an elimination ratio of 68.01% (P<0.05) and 67.54% (P<0.05), respectively; 4 and 8 mg/mL GA completely suppressed V. alginolyticus growth on LB swimming motility agar plates; and 1, 2, 4, and 8 mg/mL GA significantly inhibited V. alginolyticus aggregation capacity, with aggregation rates reduced to 18.68% (P<0.05), 19.19% (P<0.05), 25.70% (P<0.05), and 37.41% (P<0.05), respectively. In conclusion, GA has a strong inhibitory effect on V. alginolyticus by growth restraint, cell wall destruction, cell membrane permeability increase, biofilm formation suppression, mature biofilm elimination, and moveability and aggregation inhibition. This study presents a foundation for exploring the action mechanism of GA in suppressing V. alginolyticus growth and provides a theoretical basis for GA in preventing and curing infectious diseases caused by V. alginolyticus in aquatic animals.
Vibrio alginolyticus is a primary etiological agent for aquatic animal death. V. alginolyticus can infect fish, causing bleeding, ulcer, and blood poisoning; shrimps, causing shrimp post-larvae bacterial vitrified syndrome, acute hepatopancreatic necrosis disease, rotted gill disease, and white fecal syndrome; crabs, causing milk and toothpaste diseases; sea cucumbers, causing skin ulcer syndrome; and shellfish, thereby leading to substantial economic loss in the marine aquaculture industry. Antibiotics, disinfectants, and microecologics are usually used in the aquaculture to prevent or cure such infectious diseases however, their outcomes remain unsatisfactory. In addition, irrational drug use increases risks, such as environmental pollution, bacterial drug resistance, and drug residue. Compared with antibiotics, traditional Chinese medicines (TCMs) present certain advantages, such as antibacterial properties, immunoregulation, slight toxic and side effect, and low drug resistance or residue. Therefore, TCMs have garnered increasing attention in recent years. Using V. alginolyticus and V. parahemolyticus isolated from penaeid shrimp larvae with bacterial vitrified syndrome as research objects, we screened the bacteriostatic activity of 50 TCMs. Four kinds of TCM, namely Terminalia chebula, Galla chinensis, pomegranate peel, and Sanguisorba officinalis, demonstrated good bacteriostatic effects. Gallic acid (GA) is a main active component of these four TCM compounds. GA has good antibacterial, antiviral, anti-inflammatory, and antioxidative properties and can protect the liver and improve the immunological function of the body, thus it may be used to treat and prevent multiple animal diseases. Although GA has antibacterial effects on various bacteria, the bacteriostatic activity and possible mechanism against V. alginolyticus remains unelucidated. By measuring the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and growth curve of GA against V. alginolyticus, we evaluated the bacteriostatic activity of GA against V. alginolyticus. Moreover, the changes in the AKP activity and electrical conductivity of the supernatant fluid of bacteria solution and biofilms, moveability, and aggregation capacity of V. alginolyticus were determined before and after GA treatment to investigate the bacteriostatic mechanism of GA against V. alginolyticus. The MIC and MBC of GA against V. alginolyticus were 4 mg/mL and 8 mg/mL, respectively. These two GA concentrations could completely inhibit V. alginolyticus growth, while 2 mg/mL GA significantly suppressed V. alginolyticus growth, suggesting that the inhibitory effect of GA on V. alginolyticus was dose-dependent. Thus, GA concentrations greater than 4 mg/mL should be selected and used to achieve an effective bacteriostatic effect on V. alginolyticus. The bacterial cell wall is an important structure that maintains cell morphology and facilitates cell protection. Biofilms comprise various extracellular materials, such as proteins, exopolysaccharides, lipids, and extracellular DNA. Biofilms not only enhance the resistance of bacteria to adverse external environment, but also increase their resistance to antibacterial agents. However, TCM can restrain bacterial biofilm formation and development, destroy their cell wall and membrane, affect protein and nucleic acid synthesis, promote oxidative stress, and inhibit virulent factor expression to ultimately suppress or kill bacteria. The changes in AKP and electrical conductivity of bacterial culture serve as an index to verify whether the bacterial cell wall was destroyed or whether its permeability increased. GA (1, 2, 4, and 8 mg/mL) destroyed the cell wall within 2 h and caused AKP leakage. Furthermore, the degree of V. alginolyticus cell wall destruction was positively correlated with GA concentrations; 2, 4, and 8 mg/mL GA could remarkably increase electrical conductivity of the supernatant fluid of V. alginolyticus; compared with the positive control group, 4 and 8 mg/mL GA had a significant inhibitory effect on the formation of the biofilms of V. alginolyticus, with an inhibition ratio of 83.26% (P<0.05) and 77.80% (P<0.05), respectively. Meanwhile, 4 and 8 mg/mL GA notably eliminated mature biofilms, with an elimination ratio of 68.01% (P<0.05) and 67.54% (P<0.05), respectively; 4 and 8 mg/mL GA completely suppressed V. alginolyticus growth on LB swimming motility agar plates; and 1, 2, 4, and 8 mg/mL GA significantly inhibited V. alginolyticus aggregation capacity, with aggregation rates reduced to 18.68% (P<0.05), 19.19% (P<0.05), 25.70% (P<0.05), and 37.41% (P<0.05), respectively. In conclusion, GA has a strong inhibitory effect on V. alginolyticus by growth restraint, cell wall destruction, cell membrane permeability increase, biofilm formation suppression, mature biofilm elimination, and moveability and aggregation inhibition. This study presents a foundation for exploring the action mechanism of GA in suppressing V. alginolyticus growth and provides a theoretical basis for GA in preventing and curing infectious diseases caused by V. alginolyticus in aquatic animals.
2014, 35(4):1-6.
DOI: 10.11758/yykxjz.20140401
Abstract:
In this study we evaluated the biomass and distribution of Sthenoteuthis oualaniensis in South China Sea based on the data collected by Bdstar Navigation fishery information collection network that was mounted on the light falling net vessels. Considering the operation process of the light falling vessel, we built a light fishing stock assessment model and introduced a probability function to calculate the sweeping area and the biomass of S. oualaniensis. We then used kriging method to predict the density of S. oualaniensis and the CPUE. We subsequently generated a map of the distribution of S. oualaniensis and estimated the total biomass and the allowable catch. Our analysis showed that S. oualaniensis were widely distributed in South China Sea with high density (4 t/km2) in the area of 110.5°–111.5°E, 11°–12°N and 115.5°–116.5°E, 9.5°–11.5°N; in the area of 112°–112.5°E, 14.5°–15°N and 113°–115°E, 15°–16.5°N, the value of CPUE was as high as 1 kg/(kW•d•km2). The results of Kriging interpolation suggested that in the area of 108°–118°E, 9°–20°N there was a biomass of 2.05 million tons and an allowable catch of 994,000 tons in 359 fishing areas. The annual allowable catch could be 392,000 tons in 105 fishing areas inferred from CPUE. We assessed that there were 630,700 tons of S. oualaniensis in the area of Nansha Islands and it could be one of the future target species in the deep-sea fisheries. Here we only provided a crude estimate because all the parameters in our model were obtained by the sample vessels. To make an accurate estimate, further investigation will be needed on fishing vessels and fishing ground. It was found that the enhanced machine and light power did not necessarily increase the fishing efficiency. Although a higher light power could enlarge the illuminated area, davits could not support a larger falling net. Moreover, our model could also be used to assess the light arrangement, practice distance, and cost effectiveness in light fisheries.
In this study we evaluated the biomass and distribution of Sthenoteuthis oualaniensis in South China Sea based on the data collected by Bdstar Navigation fishery information collection network that was mounted on the light falling net vessels. Considering the operation process of the light falling vessel, we built a light fishing stock assessment model and introduced a probability function to calculate the sweeping area and the biomass of S. oualaniensis. We then used kriging method to predict the density of S. oualaniensis and the CPUE. We subsequently generated a map of the distribution of S. oualaniensis and estimated the total biomass and the allowable catch. Our analysis showed that S. oualaniensis were widely distributed in South China Sea with high density (4 t/km2) in the area of 110.5°–111.5°E, 11°–12°N and 115.5°–116.5°E, 9.5°–11.5°N; in the area of 112°–112.5°E, 14.5°–15°N and 113°–115°E, 15°–16.5°N, the value of CPUE was as high as 1 kg/(kW•d•km2). The results of Kriging interpolation suggested that in the area of 108°–118°E, 9°–20°N there was a biomass of 2.05 million tons and an allowable catch of 994,000 tons in 359 fishing areas. The annual allowable catch could be 392,000 tons in 105 fishing areas inferred from CPUE. We assessed that there were 630,700 tons of S. oualaniensis in the area of Nansha Islands and it could be one of the future target species in the deep-sea fisheries. Here we only provided a crude estimate because all the parameters in our model were obtained by the sample vessels. To make an accurate estimate, further investigation will be needed on fishing vessels and fishing ground. It was found that the enhanced machine and light power did not necessarily increase the fishing efficiency. Although a higher light power could enlarge the illuminated area, davits could not support a larger falling net. Moreover, our model could also be used to assess the light arrangement, practice distance, and cost effectiveness in light fisheries.
2014, 35(4):7-12.
DOI: 10.11758/yykxjz.20140402
Abstract:
In this study, we examined the phytoplankton community in the artificial reef areas of Laoshan Bay, and analyzed the relationships between phytoplankton and environment factors. Hence we provided references for the construction and evaluation of artificial reefs. The data and water samples were collected from four surveys in three artificial reef areas (Fengshan, Yangkou and Gangdong) of Laoshan Bay from March to December in 2011. By using Primer 5.0, we calculated the Shannon-Wiener, Margalef and Pielou of the phytoplankton community. We monitored the quarterly changes of phytop¬lankton community with cluster analysis, and applied Canonical Correspondence Analysis (CCA) to explore the relationship between phytoplankton species and the environmental factors using Canoco 4.5. In four surveys we identified a total of 69 species with microscope. We found that the dominant species was Skeletonema costatum in March, September and December, and it was Navicula spp in June. The density of phytoplankton in December was 49.78×105 cell/m3, which was the lowest in this survey. The density of phytoplankton in September was up to 408.65×105 cell/m3, which was the highest among all months. Shannon-Wiener and Margalef varied from 2.034 to 2.83, and from 5.27 to 7.25 respectively, both of which reached the maximum in September and the minimum in December. The range of Pielou was 0.580.78, and it reached the maximum in June and the minimum in December. According to the cluster analysis, the degree of similarity between March and June was 43.02%, and it was up to 42.38% between September and December. The species-environment bi-plots were drawn based on the results of CCA. The results showed that phosphate, temperature and silicate were the most important factors that influenced the distribution of phytoplankton species.
In this study, we examined the phytoplankton community in the artificial reef areas of Laoshan Bay, and analyzed the relationships between phytoplankton and environment factors. Hence we provided references for the construction and evaluation of artificial reefs. The data and water samples were collected from four surveys in three artificial reef areas (Fengshan, Yangkou and Gangdong) of Laoshan Bay from March to December in 2011. By using Primer 5.0, we calculated the Shannon-Wiener, Margalef and Pielou of the phytoplankton community. We monitored the quarterly changes of phytop¬lankton community with cluster analysis, and applied Canonical Correspondence Analysis (CCA) to explore the relationship between phytoplankton species and the environmental factors using Canoco 4.5. In four surveys we identified a total of 69 species with microscope. We found that the dominant species was Skeletonema costatum in March, September and December, and it was Navicula spp in June. The density of phytoplankton in December was 49.78×105 cell/m3, which was the lowest in this survey. The density of phytoplankton in September was up to 408.65×105 cell/m3, which was the highest among all months. Shannon-Wiener and Margalef varied from 2.034 to 2.83, and from 5.27 to 7.25 respectively, both of which reached the maximum in September and the minimum in December. The range of Pielou was 0.580.78, and it reached the maximum in June and the minimum in December. According to the cluster analysis, the degree of similarity between March and June was 43.02%, and it was up to 42.38% between September and December. The species-environment bi-plots were drawn based on the results of CCA. The results showed that phosphate, temperature and silicate were the most important factors that influenced the distribution of phytoplankton species.
2014, 35(2):22-29.
DOI: 10.11758/yykxjz.20140204
Abstract:
Collecting acoustic data from fishing vessels is an important trend in fishery resources study, yet the quality of the data may be seriously compromised by inter-instrument acoustic interferences due to the lack of signal synchronization apparatus for onboard acoustic instruments. In order to fully use the contaminated acoustic data, a method of extracting swarm signals from echogram with strong interference was established based on the relevant modules in the Echoview,an acoustic data post-processing software. The methods were then applied to the acoustic data on Antarctic krill collected onboard a fishing vessel operated around South Orkney Islands during February 2011, and the interference was effectively removed from the echogram. Distinct diel vertical migration was observed in the krill swarms. The geometrical center of the krill swarms was distributed in layers with a mean depth of 77.4 m and a mean thickness of 41.2 m in daytime, while the swarms tended to migrate upward nocturnally at a mean depth of 34.9 m and a mean thickness of 8.8 m.
Collecting acoustic data from fishing vessels is an important trend in fishery resources study, yet the quality of the data may be seriously compromised by inter-instrument acoustic interferences due to the lack of signal synchronization apparatus for onboard acoustic instruments. In order to fully use the contaminated acoustic data, a method of extracting swarm signals from echogram with strong interference was established based on the relevant modules in the Echoview,an acoustic data post-processing software. The methods were then applied to the acoustic data on Antarctic krill collected onboard a fishing vessel operated around South Orkney Islands during February 2011, and the interference was effectively removed from the echogram. Distinct diel vertical migration was observed in the krill swarms. The geometrical center of the krill swarms was distributed in layers with a mean depth of 77.4 m and a mean thickness of 41.2 m in daytime, while the swarms tended to migrate upward nocturnally at a mean depth of 34.9 m and a mean thickness of 8.8 m.
