无乳链球菌感染对吉富罗非鱼行为的影响及其机制
|
摘要
在罗非鱼养殖过程中,无乳链球菌(Streptococcus agalactiae)感染会导致罗非鱼行为异常,并严重危害罗非鱼产业的健康发展。为了研究无乳链球菌感染后吉富罗非鱼(Oreochromis niloticus)行为变化的生理机制,本研究采用q RT-PCR和免疫荧光的方法,以人工腹腔注射感染无乳链球菌的吉富罗非鱼为研究对象,检测感染前后吉富罗非鱼脑组织和肠道组织中无乳链球菌含量、行为变化以及5-羟色胺(5-hydroxytryptamine, 5-HT)在其小脑、肠道和胃中的表达情况。结果表明,吉富罗非鱼脑组织和肠道组织中无乳链球菌的含量分别从人工感染后的第6和第0小时开始急剧增加,均在第18小时达到高峰,其中脑组织峰值61.2 copies/mg,肠道组织峰值1 800.5 copies/mg;实验鱼在人工感染后的第12小时出现无定向乱窜、持续旋转游动的异常行为,并且这种异常行为一直持续到实验结束或实验鱼死亡;在人工感染前和感染后的实验鱼小脑、肠道和胃中均发现5-HT阳性反应,但是小脑和肠道中5-HT的阳性反应随着病原感染时间延长呈现变弱的趋势,在实验第18小时时荧光强度分别为9.1和6.9,而胃组织中5-HT阳性反应随着病原感染时间延长呈现先升高后降低的趋势,在实验末期到达最低值3.1。因此可以推测,无乳链球菌感染罗非鱼后,通过调控脑中枢神经系统和外周消化道系统中的5-HT使罗非鱼产生无定向乱窜和持续旋转的异常行为。本研究从5-HT的角度解释无乳链球菌感染后罗非鱼的异常行为,可为研究感染病原后鱼类冲动行为的机制提供新的研究思路。
Streptococcus agalactiae infection may cause abnormal behavior of tilapia(Oreochromis niloticus)and seriously endangers the development of the tilapia industry. To investigate the impacts of S. agalactiae on behavior of GIFT tilapia and its mechanism, the q RT-PCR and immunofluorescence were used to examine the concentration of S. agalactiae in the brain and intestine, the behavior changes, as well as the expression of 5-hydroxytryptamine(5-HT) in the cerebellum, intestine and stomach of tilapia infected with by intraperitoneal injection. The results showed that the copies of S. agalactiae in the brain and intestinal tissues of tilapia increased sharply from the 6 th and 0 h after pathogen infection respectively, and reached the maximum at 18 th h(61.2 copies/mg in the brain and 1 800.5 copies/mg in the intestine). Furthermore, fish showed abnormal behavior from the 12 th h after pathogen challenge, and the abnormal behavior continued until the end of the experiment or the death of the fish. In addition, 5-HT positive reaction was found in the cerebellum, intestine and stomach of tilapia before and after S. agalactiae infection. While the positive reaction of 5-HT in cerebellum and intestine showed a weakening trend with the prolongation of experiment, and the fluorescence intensity of cerebellum and intestine were 9.1 and 6.9 at 18 th h, respectively. But the 5-HT positive reaction in stomach increased first and then decreased with experiment went on, reaching a minimum of 3.1 at the end of the experiment. Therefore, it could be speculated that after infection of S. agalactia, tilapia induces abnormal behavior of tilapia by regulating 5-HT in the central nervous system and the peripheral digestive system. The current study researched the abnormal behavior of tilapia after S. agalactiae infection from the perspective of5-HT, which could provide a new insight into the mechanism of impulsive behavior of fish infected with pathogen.
Streptococcus agalactiae infection may cause abnormal behavior of tilapia(Oreochromis niloticus)and seriously endangers the development of the tilapia industry. To investigate the impacts of S. agalactiae on behavior of GIFT tilapia and its mechanism, the q RT-PCR and immunofluorescence were used to examine the concentration of S. agalactiae in the brain and intestine, the behavior changes, as well as the expression of 5-hydroxytryptamine(5-HT) in the cerebellum, intestine and stomach of tilapia infected with by intraperitoneal injection. The results showed that the copies of S. agalactiae in the brain and intestinal tissues of tilapia increased sharply from the 6 th and 0 h after pathogen infection respectively, and reached the maximum at 18 th h(61.2 copies/mg in the brain and 1 800.5 copies/mg in the intestine). Furthermore, fish showed abnormal behavior from the 12 th h after pathogen challenge, and the abnormal behavior continued until the end of the experiment or the death of the fish. In addition, 5-HT positive reaction was found in the cerebellum, intestine and stomach of tilapia before and after S. agalactiae infection. While the positive reaction of 5-HT in cerebellum and intestine showed a weakening trend with the prolongation of experiment, and the fluorescence intensity of cerebellum and intestine were 9.1 and 6.9 at 18 th h, respectively. But the 5-HT positive reaction in stomach increased first and then decreased with experiment went on, reaching a minimum of 3.1 at the end of the experiment. Therefore, it could be speculated that after infection of S. agalactia, tilapia induces abnormal behavior of tilapia by regulating 5-HT in the central nervous system and the peripheral digestive system. The current study researched the abnormal behavior of tilapia after S. agalactiae infection from the perspective of5-HT, which could provide a new insight into the mechanism of impulsive behavior of fish infected with pathogen.
引文
段云峰,吴晓丽,王涛,等.2013.五羟色胺和多巴胺与攻击行为的关联研究进展[J].生命科学,25(10):1027-1035.(Duan Y F,Wu X L,Wang T,et al.2013.Research progress on the correlations of aggressive behavior with serotonin and dopamine[J].Chinese Bulletin of Life Sciences,25(10):1027-1035.)
方静,樊均德,陈玥.2012.齐口裂腹鱼脑内5-羟色胺免疫组织化学的定位观察[J].水生生物学报,36(1):143-147.(Fang J,Fan J D,Chen Y.2012.The Immunocytochemical study on the distribution of 5-hydroxytryptamine in the brain of Schizothorax prenantia[J].Acta Hydrobiological Sinica.36(1):143-147.).
黄威权,方永强,苏慧慈,等.1990.文昌鱼体内5-羟色胺免疫组织化学定位的研究[J].科学通报,35(15):1187-1189.(Huang W Q,Fang Y Q,Su H C,et al.1990.Immunocytochemical study on the distribution of 5-Hydroxytryptamine in the brain of Branchiostoma belcheri[J].Chinese Science Bulletin,35(15):1187-1189.)
阚延胜.2007.罗非鱼消化道5-HT内分泌细胞的免疫组织化学研究[J].安徽农学通报,13(17):27-28.(Kan Y S.2007.Studies on immunohistochemistry of 5-HT endocrine cells in the digestive tract of tilapia Mossamblca[J].Anhui Agricultural Science Bulletin,13(17):27-28.)
卢迈新.2010.罗非鱼链球菌病研究进展[J].南方水产科学,6(1):75-79.(Lu M X.2010.Review of research on streptococcosis in tilapia[J].South China Fisheries Science,6(1):75-79.)
卢迈新,黎炯,叶星,等.2010.广东与海南养殖罗非鱼无乳链球菌的分离、鉴定与特性分析[J].微生物学通报,37(5):766-774.(Lu M X,Li J,Ye X,et al.2010.Identification and characterizations of Streptococcus agalactiae isolated from tilapia cultured in Guangdong and Hainan provinces[J].Microbiology China,37(5):766-774.)
罗佳,金锋.2014.肠道菌群影响宿主行为的研究进展[J].科学通报,59(22):2169-2190.(Luo J,Jin F.2014.Recent advances in understanding the impact of intestinal microbiota on host behavior(in Chinese)[J].Chinese Science Bulletin(Chinese Version),59(22):2169-2190.)
燕晋媛.2015.细菌侵染线虫所诱发的逃避和免疫之间消长平衡的分子机制研究[D].博士学位论文,云南大学,导师:黄晓玮.pp.Ⅰ-Ⅱ.(Yan J Y.2015.Molecular mechanism underlying the counter balance between avoidance and immunne response in Caenorhabditis elegans[D].Thesis for Ph.D.,Yunnan University,Supervisor:Huang X W,pp.Ⅰ-Ⅱ.)
Airhart M J,Lee D H,Wilson T D,et al.2012.Adverse effects of serotonin depletion in developing zebrafish[J].Neurotoxicology&Teratology,34(1):152-160.
Alzghoul L,Bortolato M,Delis F,et al.2012.Altered cerebellar organization and function in monoamine oxidase Ahypomorphic mice[J].Neuropharmacology,63(7):1208-1217.
Bercik P,Verdu E F,Foster J A,et al.2010.Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry in mice[J].Gastroenterology,139(6):2102-2112.
Bortolato M,Pivac N,Seler D M,et al.2013.The role of the serotonergic system at the interface of aggression and suicide[J].Neuroscience,236:160-185.
Goehler L E,Su M P,Opitz N,et al.2008.Campylobacter jejuni infection increases anxiety-like behavior in the holeboard:Possible anatomical substrates for viscerosensory modulation of exploratory behavior[J].Brain Behavior&Immunity,22(3):354-366.
Hannon J,Hoyer D.2008.Molecular biology of 5-HT receptors[J].Behavioural Brain Research,195(1):198-213.
Hoover K,Grove M,Gardner M,et al.2011.A gene for an extended phenotype[J].Science,333(6048):1401.
Lyte M,Li W,Opitz N,et al.2006.Induction of anxiety-like behavior in mice during the initial stages of infection with the agent of murine colonic hyperplasia Citrobacter rodentium[J].Physiology&Behavior,89(3):350-357.
Nelson R J,Chiavegatto S.2001.Molecular basis of aggression[J].Trends in Neurosciences,24(12):713-719.
Norton W H J,Folchert A,Bally-Cuif L.2008.Comparative analysis of serotonin receptor(HTR1A/HTR1B families)and transporter(slc6a4a/b)gene expression in the zebrafish brain[J].Journal of Comparative Neurology,511(4):521-542.
Nowicki M,Tran S,Muraleetharan A,et al.2014.Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner[J].Pharmacology Biochemistry&Behavior,126:170-180.
Rapport M M,Green A A,Page I H.1948.Crystalline serotonin[J].Science,108(2804):329-330.
Rodriguez-Gomez F J,Rendon-Unceta M C,Sarasquete C,et al.2000.Localization of tyrosine hydroxylase-immunoreactivity in the brain of the Senegalese sole,Solea senegalensis[J].Journal of Chemical Neuroanatomy,19(1):17-32.
Honma S.1970.Presence of monoaminergic neurons in the spinal cord and intestine of the lamprey,Lampetra japonica[J].Archivum Histologicum Japonicum,32(4):383-393.
Schneider H,Fritzky L,Williams J,et al.2012.Cloning and expression of a zebrafish 5-HT2Creceptor gene[J].Gene,502(2):108-117.
Wang B,Gan Z,Wang Z,et al.2017.Integrated analysis neurimmirs of tilapia(Oreochromis niloticus)involved in immune response to Streptococcus agalactiae,a pathogen causing meningoencephalitis in teleosts[J].Fish&Shellfish Immunology,61:44-60.
Wang G,Zhang J,Shen Y,et al.2015.Transcriptome analysis of the brain of the silkworm Bombyx mori infected with Bombyx mori nucleopolyhedrovirus:A new insight into the molecular mechanism of enhanced locomotor activity induced by viral infection[J].Journal of Invertebrate Pathology,128:37-43.
Winberg S,Th?rnqvist P O.2016.Role of brain serotonin in modulating fish behavior[J].Current Zoology,62(3):317-323.
Zhang H,Wen W,Yan J.2017.Application of immunohistochemistry technique in hydrobiological studies[J].Aquaculture and Fisheries,2:140-144.
方静,樊均德,陈玥.2012.齐口裂腹鱼脑内5-羟色胺免疫组织化学的定位观察[J].水生生物学报,36(1):143-147.(Fang J,Fan J D,Chen Y.2012.The Immunocytochemical study on the distribution of 5-hydroxytryptamine in the brain of Schizothorax prenantia[J].Acta Hydrobiological Sinica.36(1):143-147.).
黄威权,方永强,苏慧慈,等.1990.文昌鱼体内5-羟色胺免疫组织化学定位的研究[J].科学通报,35(15):1187-1189.(Huang W Q,Fang Y Q,Su H C,et al.1990.Immunocytochemical study on the distribution of 5-Hydroxytryptamine in the brain of Branchiostoma belcheri[J].Chinese Science Bulletin,35(15):1187-1189.)
阚延胜.2007.罗非鱼消化道5-HT内分泌细胞的免疫组织化学研究[J].安徽农学通报,13(17):27-28.(Kan Y S.2007.Studies on immunohistochemistry of 5-HT endocrine cells in the digestive tract of tilapia Mossamblca[J].Anhui Agricultural Science Bulletin,13(17):27-28.)
卢迈新.2010.罗非鱼链球菌病研究进展[J].南方水产科学,6(1):75-79.(Lu M X.2010.Review of research on streptococcosis in tilapia[J].South China Fisheries Science,6(1):75-79.)
卢迈新,黎炯,叶星,等.2010.广东与海南养殖罗非鱼无乳链球菌的分离、鉴定与特性分析[J].微生物学通报,37(5):766-774.(Lu M X,Li J,Ye X,et al.2010.Identification and characterizations of Streptococcus agalactiae isolated from tilapia cultured in Guangdong and Hainan provinces[J].Microbiology China,37(5):766-774.)
罗佳,金锋.2014.肠道菌群影响宿主行为的研究进展[J].科学通报,59(22):2169-2190.(Luo J,Jin F.2014.Recent advances in understanding the impact of intestinal microbiota on host behavior(in Chinese)[J].Chinese Science Bulletin(Chinese Version),59(22):2169-2190.)
燕晋媛.2015.细菌侵染线虫所诱发的逃避和免疫之间消长平衡的分子机制研究[D].博士学位论文,云南大学,导师:黄晓玮.pp.Ⅰ-Ⅱ.(Yan J Y.2015.Molecular mechanism underlying the counter balance between avoidance and immunne response in Caenorhabditis elegans[D].Thesis for Ph.D.,Yunnan University,Supervisor:Huang X W,pp.Ⅰ-Ⅱ.)
Airhart M J,Lee D H,Wilson T D,et al.2012.Adverse effects of serotonin depletion in developing zebrafish[J].Neurotoxicology&Teratology,34(1):152-160.
Alzghoul L,Bortolato M,Delis F,et al.2012.Altered cerebellar organization and function in monoamine oxidase Ahypomorphic mice[J].Neuropharmacology,63(7):1208-1217.
Bercik P,Verdu E F,Foster J A,et al.2010.Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry in mice[J].Gastroenterology,139(6):2102-2112.
Bortolato M,Pivac N,Seler D M,et al.2013.The role of the serotonergic system at the interface of aggression and suicide[J].Neuroscience,236:160-185.
Goehler L E,Su M P,Opitz N,et al.2008.Campylobacter jejuni infection increases anxiety-like behavior in the holeboard:Possible anatomical substrates for viscerosensory modulation of exploratory behavior[J].Brain Behavior&Immunity,22(3):354-366.
Hannon J,Hoyer D.2008.Molecular biology of 5-HT receptors[J].Behavioural Brain Research,195(1):198-213.
Hoover K,Grove M,Gardner M,et al.2011.A gene for an extended phenotype[J].Science,333(6048):1401.
Lyte M,Li W,Opitz N,et al.2006.Induction of anxiety-like behavior in mice during the initial stages of infection with the agent of murine colonic hyperplasia Citrobacter rodentium[J].Physiology&Behavior,89(3):350-357.
Nelson R J,Chiavegatto S.2001.Molecular basis of aggression[J].Trends in Neurosciences,24(12):713-719.
Norton W H J,Folchert A,Bally-Cuif L.2008.Comparative analysis of serotonin receptor(HTR1A/HTR1B families)and transporter(slc6a4a/b)gene expression in the zebrafish brain[J].Journal of Comparative Neurology,511(4):521-542.
Nowicki M,Tran S,Muraleetharan A,et al.2014.Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner[J].Pharmacology Biochemistry&Behavior,126:170-180.
Rapport M M,Green A A,Page I H.1948.Crystalline serotonin[J].Science,108(2804):329-330.
Rodriguez-Gomez F J,Rendon-Unceta M C,Sarasquete C,et al.2000.Localization of tyrosine hydroxylase-immunoreactivity in the brain of the Senegalese sole,Solea senegalensis[J].Journal of Chemical Neuroanatomy,19(1):17-32.
Honma S.1970.Presence of monoaminergic neurons in the spinal cord and intestine of the lamprey,Lampetra japonica[J].Archivum Histologicum Japonicum,32(4):383-393.
Schneider H,Fritzky L,Williams J,et al.2012.Cloning and expression of a zebrafish 5-HT2Creceptor gene[J].Gene,502(2):108-117.
Wang B,Gan Z,Wang Z,et al.2017.Integrated analysis neurimmirs of tilapia(Oreochromis niloticus)involved in immune response to Streptococcus agalactiae,a pathogen causing meningoencephalitis in teleosts[J].Fish&Shellfish Immunology,61:44-60.
Wang G,Zhang J,Shen Y,et al.2015.Transcriptome analysis of the brain of the silkworm Bombyx mori infected with Bombyx mori nucleopolyhedrovirus:A new insight into the molecular mechanism of enhanced locomotor activity induced by viral infection[J].Journal of Invertebrate Pathology,128:37-43.
Winberg S,Th?rnqvist P O.2016.Role of brain serotonin in modulating fish behavior[J].Current Zoology,62(3):317-323.
Zhang H,Wen W,Yan J.2017.Application of immunohistochemistry technique in hydrobiological studies[J].Aquaculture and Fisheries,2:140-144.