Ayu （Plecoglossus altivelis） fish, which are an amphidromous species distributed in East Asia, live in brackish water （BW） during their larval stage and in fresh water （FW） during their adult stage. In this stud...Ayu （Plecoglossus altivelis） fish, which are an amphidromous species distributed in East Asia, live in brackish water （BW） during their larval stage and in fresh water （FW） during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendo- crinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase （ID）, pro-opiom- elanocortin （POMC）, betaine-homocysteine S-meth- yltransferase 1 （BHMT）, fructose-bisphosphate aldolase B （aldolase B）, tyrosine aminotransferase （TAT）, and Na＋-K＋ ATPase （NKA） were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriurefic peptide （BNP） and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.展开更多
Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value.To determine the expression stabilities of six commonly used internal reference genes in C.altivelis challenged by Vibrio ...Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value.To determine the expression stabilities of six commonly used internal reference genes in C.altivelis challenged by Vibrio harveyi and viral nervous necrosis virus(VNNV)through quantitative real-time PCR(qRT-PCR),the expression levels of selected genes in five immune organs stimulated with pathogenic infection were carefully evaluated using algorithms of geNorm,NormFinder,and BestKeeper.The results show that the expre ssion stabilities of the six candidate inte rnal reference genes were diffe re nt.Under no rmal physiological conditions,RPL13 were identified as the most stably expressed genes among five different immune organs(liver,spleen,kidney,intestine,and gill).After V.harveyi stimulation,RPL13,RPL13,EF1 A,RPL13,and EF1 A were identified by geNorm,NormFinder,and BestKeeper as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.Combining these three algorithms suggested that under stimulation of VNNV,RPL13,EF1 A,Actin,RPL13,and Actin were as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.These results suggest that specific experiment conditions and tissue types shall be considered when selecting the reference genes in qRT-PCR analysis.This study provided a solid foundation for future studies on gene expression of C.altivelis under different conditions.展开更多
Cathepsin D （CTSD） is a lysosomal acidic endoproteinase that plays an important role in immune response, In this study, we obtained sweetfish （Plecoglossus altivelis） CTSD （PaCTSD） via de-novo transcriptome sequ...Cathepsin D （CTSD） is a lysosomal acidic endoproteinase that plays an important role in immune response, In this study, we obtained sweetfish （Plecoglossus altivelis） CTSD （PaCTSD） via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17x 103. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.展开更多
The countable characters of Qingdao, Zhejiang, Liaoning and Japanese Ayu arecompared. There are no substantial different characters among the former three Ayu, so thecomprehensive values of them are taken as represent...The countable characters of Qingdao, Zhejiang, Liaoning and Japanese Ayu arecompared. There are no substantial different characters among the former three Ayu, so thecomprehensive values of them are taken as representative of Chinese Ayu, which is in turn comparedwith Japanese Ayu ( Plecoglossus altivelis altivelis and Plecoglossus altivelis ryukyuensis). By thecoefficient of difference test, 3 and 4 characters are found to be beyond the subspecies levelbetween Chinese Ayu and nominate subspecies (Plecoglossus altivelis altivelis) and between ChineseAyu (Plecoglossus altivelis chinensis) and Ryukyus subspecies (Plecoglossus altivelis ryukyuensis),respectively, which shows that they belong to different subspecies. The China mainland Ayu is a newsubspecies, Plecoglossus altivelis chinensis Wu and Shan, the establishment of which and its formingcauses are discussed.展开更多
Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease inclu...Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease include microscopy examination,quantitative real-time PCR,and loop-mediated isothermal amplification-lateral flow dipstick(LAMP-LFD).In this study,a recombinase polymerase amplification-lateral flow dipstick(RPA-LFD)method,targeting the beta-tubulin gene,was developed to detect G.plecoglossi,three sets of primers and probes were designed and screened,after which the initial reaction system was established.The RPA-LFD method for G.plecoglossi could complete nucleic acid amplification at 39℃ for 10 min,after which the amplification product was dropped on the LFD strip,and the results could then be observed within 5 min.A specificity assay revealed that there was no cross reactivity with other protozoa except G.plecoglossi.A sensitivity assay revealed that the detection limit was 9.38×10^(-6) ng/μL,which was more sensitive than that of conventional PCR.Compared with conventional detection methods,the novel RPA-LFD method has the advantages of simple operation,short operation time,high sensitivity,and high specificity for G.plecoglossi detection,indicating its potential use in rapid field detection of G.plecoglossi.展开更多
Classical Fc receptors (FcRs) mediate the binding to and recognition of the Fc portion of antibodies and play an important role during immune responses in mammals. Although proteins similar to soluble FcRs have been i...Classical Fc receptors (FcRs) mediate the binding to and recognition of the Fc portion of antibodies and play an important role during immune responses in mammals. Although proteins similar to soluble FcRs have been identified in fish, little is known about the role of such proteins in fish immunity. Here, we cloned a cDNA sequence encoding a soluble Fc receptor for an immunoglobulin G (FcγR) homolog from ayu (Plecoglossus altivelis)(PaFcγRl). The predicted protein was composed of two immunoglobulin C2-like domains but lacked a transmembrane segment and a cytoplasmic tail. The PaFcγRl transcripts were distributed at low levels in all tested tissues, but significantly increased after Vibrio anguillarum infection. The PaFcγRl protein was expressed in the head kidney, trunk kidney, and neutrophils. Recombinant PaFcγRl (rPaFcγRl) was secreted when transfected into mammalian cells and the native protein was also detected in serum upon infection. rPaFcγRl was also demonstrated to bind to ayu IgM, as assessed by cell transfection. Suppressive activity of the recombinant mature protein of PaFcγRl (rPaFcγRlm) on in vitro anti-sheep red blood cell (SRBC) responses was detected by a modified hemolytic plaque forming cell assay. In conclusion, our study revealed that PaFcγRl is closely involved in the negative regulation of IgM production in the ayu spleen.展开更多
The aim of this study was to explore the potency of N. oculata extracts as antibacterial, antioxidant and antiviral on grouper Cromileptes altivelis （C. altivelis） infected by Vibrio alginolyticus （V. alginolyticus...The aim of this study was to explore the potency of N. oculata extracts as antibacterial, antioxidant and antiviral on grouper Cromileptes altivelis （C. altivelis） infected by Vibrio alginolyticus （V. alginolyticus） and Viral Nervous Necrotic （VNN）. Dilution test was used to measure antibacterial activity of N. oculata extracts. Antioxidant activity of extracts was expressed by levels of MDA （Malondialdehyde） and SOD （superoxide dismutase） in the brain and kidneys of fish. Antiviral capability of N. oculata was determined by the expression of cellular immune cells Major Histocompatibility Complex （MHC） Class I for proliferation and inhibition of VNN in blood cells with immunocytochemistry. The results showed that N. oculata extract was able to suppress the growth of V. alginolyticus at a concentration of 40%. Increasing levels of SOD and reducing level of MDA indicated that N. oculata extracts may serve as an antibacterial and antioxidant. Providing cellular response of MHC class I cell expressed on C. altive/is blood cells, N. oculata demonstrated its antiviral activity.展开更多
基金Foundation items： The project was supported by the Program for the National Natural Science Foundation of China （31201970） and the KC Wong Magna Fund in Ningbo University
文摘Ayu （Plecoglossus altivelis） fish, which are an amphidromous species distributed in East Asia, live in brackish water （BW） during their larval stage and in fresh water （FW） during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendo- crinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase （ID）, pro-opiom- elanocortin （POMC）, betaine-homocysteine S-meth- yltransferase 1 （BHMT）, fructose-bisphosphate aldolase B （aldolase B）, tyrosine aminotransferase （TAT）, and Na＋-K＋ ATPase （NKA） were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriurefic peptide （BNP） and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.
基金the Key Research Project of Hainan Province(No.ZDKJ2019011)the Natural Science Foundation of Hainan Province(No.2019RC078)the Nanhai Famous Youth Project。
文摘Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value.To determine the expression stabilities of six commonly used internal reference genes in C.altivelis challenged by Vibrio harveyi and viral nervous necrosis virus(VNNV)through quantitative real-time PCR(qRT-PCR),the expression levels of selected genes in five immune organs stimulated with pathogenic infection were carefully evaluated using algorithms of geNorm,NormFinder,and BestKeeper.The results show that the expre ssion stabilities of the six candidate inte rnal reference genes were diffe re nt.Under no rmal physiological conditions,RPL13 were identified as the most stably expressed genes among five different immune organs(liver,spleen,kidney,intestine,and gill).After V.harveyi stimulation,RPL13,RPL13,EF1 A,RPL13,and EF1 A were identified by geNorm,NormFinder,and BestKeeper as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.Combining these three algorithms suggested that under stimulation of VNNV,RPL13,EF1 A,Actin,RPL13,and Actin were as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.These results suggest that specific experiment conditions and tissue types shall be considered when selecting the reference genes in qRT-PCR analysis.This study provided a solid foundation for future studies on gene expression of C.altivelis under different conditions.
基金Foundation items： The project was supported by the Program for the National Natural Science Foundation of China （31201970, 31372555）, Zhejiang Provincial Natural Science Foundation of China （LZ13C- 190001, LQ13C190002）
文摘Cathepsin D （CTSD） is a lysosomal acidic endoproteinase that plays an important role in immune response, In this study, we obtained sweetfish （Plecoglossus altivelis） CTSD （PaCTSD） via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17x 103. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.
文摘The countable characters of Qingdao, Zhejiang, Liaoning and Japanese Ayu arecompared. There are no substantial different characters among the former three Ayu, so thecomprehensive values of them are taken as representative of Chinese Ayu, which is in turn comparedwith Japanese Ayu ( Plecoglossus altivelis altivelis and Plecoglossus altivelis ryukyuensis). By thecoefficient of difference test, 3 and 4 characters are found to be beyond the subspecies levelbetween Chinese Ayu and nominate subspecies (Plecoglossus altivelis altivelis) and between ChineseAyu (Plecoglossus altivelis chinensis) and Ryukyus subspecies (Plecoglossus altivelis ryukyuensis),respectively, which shows that they belong to different subspecies. The China mainland Ayu is a newsubspecies, Plecoglossus altivelis chinensis Wu and Shan, the establishment of which and its formingcauses are discussed.
基金Supported by the Visiting and Training Foundation of Teachers in Ordinary Undergraduate Universities of Shandong Province,the Qingdao Agricultural University Doctoral Start-Up Fund(No.6631122030)the Advanced Talents Foundation of QAU(No.6651118016)+2 种基金the Fish Innovation Team of Shandong Agriculture Research System(No.SDAIT12-06)the Shandong Engineering Research Center for Prevention and Control of Aquatic Animal Disease,the“First Class Fishery Discipline”Program[(2020)3]of Shandong Provincethe Key R&D Program(Soft Science Project)of Shandong Province,China(No.2023 RKY 06004)。
文摘Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease include microscopy examination,quantitative real-time PCR,and loop-mediated isothermal amplification-lateral flow dipstick(LAMP-LFD).In this study,a recombinase polymerase amplification-lateral flow dipstick(RPA-LFD)method,targeting the beta-tubulin gene,was developed to detect G.plecoglossi,three sets of primers and probes were designed and screened,after which the initial reaction system was established.The RPA-LFD method for G.plecoglossi could complete nucleic acid amplification at 39℃ for 10 min,after which the amplification product was dropped on the LFD strip,and the results could then be observed within 5 min.A specificity assay revealed that there was no cross reactivity with other protozoa except G.plecoglossi.A sensitivity assay revealed that the detection limit was 9.38×10^(-6) ng/μL,which was more sensitive than that of conventional PCR.Compared with conventional detection methods,the novel RPA-LFD method has the advantages of simple operation,short operation time,high sensitivity,and high specificity for G.plecoglossi detection,indicating its potential use in rapid field detection of G.plecoglossi.
基金supported by the Program for the National Natural Science Foundation of China(3177287631402323+6 种基金31372555)Natural Science Foundation of Zhejiang Province(LZ18C190001LY14C190007)Scientific Innovation Team Project of Ningbo(2015C110018)Natural Science Foundation of Ningbo City of China(2018A610225)Ningbo Science and Technology “Fumin Engineering”Project(2017C10037)K.C.Wong Magna Fund in Ningbo University
文摘Classical Fc receptors (FcRs) mediate the binding to and recognition of the Fc portion of antibodies and play an important role during immune responses in mammals. Although proteins similar to soluble FcRs have been identified in fish, little is known about the role of such proteins in fish immunity. Here, we cloned a cDNA sequence encoding a soluble Fc receptor for an immunoglobulin G (FcγR) homolog from ayu (Plecoglossus altivelis)(PaFcγRl). The predicted protein was composed of two immunoglobulin C2-like domains but lacked a transmembrane segment and a cytoplasmic tail. The PaFcγRl transcripts were distributed at low levels in all tested tissues, but significantly increased after Vibrio anguillarum infection. The PaFcγRl protein was expressed in the head kidney, trunk kidney, and neutrophils. Recombinant PaFcγRl (rPaFcγRl) was secreted when transfected into mammalian cells and the native protein was also detected in serum upon infection. rPaFcγRl was also demonstrated to bind to ayu IgM, as assessed by cell transfection. Suppressive activity of the recombinant mature protein of PaFcγRl (rPaFcγRlm) on in vitro anti-sheep red blood cell (SRBC) responses was detected by a modified hemolytic plaque forming cell assay. In conclusion, our study revealed that PaFcγRl is closely involved in the negative regulation of IgM production in the ayu spleen.
文摘The aim of this study was to explore the potency of N. oculata extracts as antibacterial, antioxidant and antiviral on grouper Cromileptes altivelis （C. altivelis） infected by Vibrio alginolyticus （V. alginolyticus） and Viral Nervous Necrotic （VNN）. Dilution test was used to measure antibacterial activity of N. oculata extracts. Antioxidant activity of extracts was expressed by levels of MDA （Malondialdehyde） and SOD （superoxide dismutase） in the brain and kidneys of fish. Antiviral capability of N. oculata was determined by the expression of cellular immune cells Major Histocompatibility Complex （MHC） Class I for proliferation and inhibition of VNN in blood cells with immunocytochemistry. The results showed that N. oculata extract was able to suppress the growth of V. alginolyticus at a concentration of 40%. Increasing levels of SOD and reducing level of MDA indicated that N. oculata extracts may serve as an antibacterial and antioxidant. Providing cellular response of MHC class I cell expressed on C. altive/is blood cells, N. oculata demonstrated its antiviral activity.
