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Journal of Tropical Oceanography >

2021 , Vol. 40 >Issue 3: 114 - 123

DOI: https://doi.org/10.11978/2020026

Marine Biology

Effects of salinity changes on serum and kidney immune status associated with injection of Aeromonas hydrophila in Scatophagus argus

  • LU Mengying , 1 ,
  • SU Maoliang 2, 3 ,
  • ZHANG Junbin , 1, 2
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  • 1. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
  • 2. Shenzhen Key Laboratory of Marine Bioresource & Eco-Environmental Science, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China
  • 3. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
ZHANG Junbin. email:

Copy editor: LIN Qiang

Received date: 2020-03-09

  Request revised date: 2020-04-07

  Online published: 2020-04-09

Supported by

National Natural Science Foundation of China(41806177)

National Natural Science Foundation of China(41976108)

China Postdoctoral Science Foundation(2019M653010)

Copyright

Copyright reserved © 2021. Office of Acta Agronomica Sinica All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.
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Abstract

The salinity in coastal waters fluctuates frequently due to seasonal rainfall and ocean currents. Salinity change makes fish more susceptible to pathogens, leading to disease and death. As the main lymphoid tissue of teleosts, kidney is closely related to fish’s immune function. This study aims to explore effects of environmental salinity on the kidney immune function of Scatophagus argus. In order to analyze the relationship between the change of environmental salinity and immune situation in fish’s kidney after Aeromonas hydrophila injection, we compared the changes of superoxide dismutase (SOD) concentration in serum and kidney, and immune-related parameters Complement 4 (C4), Interleukin-6 (IL-6), and Immunoglobulin M (IgM) concentrations in kidney before and after infection within 96 h of each salinity group. Our results show that except for 96 hpi, SOD concentrations in the serum and kidney tissues of the freshwater group and low-salinity (?‰) group were higher than those of the 25‰ salinity group (P<0.01), and that the maximum difference between low-salinity group and 25‰ salinity group was about 150 ng·mL-1. The concentration of C4 in the serum and kidney of the freshwater group and low-salinity group was about 100-600 μg·mL-1 higher than that of the 25‰ salinity group within 96 hpi (P<0.01). The concentration of IL-6 in the kidney tissues of S. argus in the 25‰ salinity group was higher than that in the freshwater group and low-salinity group (P<0.01), although IL-6 concentrations in kidney of the three groups were significantly lower than that of the control group (14.2±0.1 pg·mL-1, in freshwater; 17.9±0.0 pg·mL-1, in low-salinity; 17.9±0.0 pg·mL-1, in 25‰ salinity) at 96 hpi (P<0.01), while the serum IL-6 concentration in the freshwater group was significantly increased to 56.9±1.0 pg·mL-1 (P<0.01). The serum and kidney IgM levels of fish in the 25‰ salinity grop (71.8±2.9 μg·mL-1 and 6.3±0.4 μg·mL-1) were about 1-20 g·mL-1 higher than those of the freshwater group and low-salinity group (P<0.05). The time of IgM production in serum and kidney tissues of the 25‰ salinity group (12 hpi) was earlier than that of the freshwater group and low-salinity group (24 hpi). In summary, the 25‰ salinity group suffered less damage after bacterial infection, and the immune status of kidney and serum was better than that of the freshwater group and low-salinity group; and the immune response was faster in the 25‰ salinity group. Therefore, we speculate that a decrease of salinity would lead to a decrease of kidney immune state of S. argus. This research provides a reference value for S. argus mariculture.

Key words： Scatophagus argus; salinity; Aeromonas hydrophila; immunity; kidney

Cite this article

LU Mengying , SU Maoliang , ZHANG Junbin . Effects of salinity changes on serum and kidney immune status associated with injection of Aeromonas hydrophila in Scatophagus argus[J]. Journal of Tropical Oceanography, 2021 , 40(3) : 114 -123 . DOI: 10.11978/2020026

盐度作为水体重要环境因素之一, 对水生生物的生命生理活动具有重要影响(Jeffries et al, 2019)。海水的蒸发降雨、洋流运动、及全球变暖引起的冰川融化使得近几年近岸海水盐度的变化越来越剧烈(Chesney et al, 2000; Nielsen et al, 2012; Duggan et al, 2014; Van Wijk et al, 2014)。环境盐度发生变化时, 鱼体内环境稳态将会发生改变, 进一步影响其正常的生理机制和机体免疫能力(Geven et al, 2017; El-Leithy et al, 2019)。长期以来, 人们一直认为环境条件的改变可能会抑制鱼体免疫力, 从而对鱼类机体健康有害, 然而也有一些学者持有不同的观点, 认为环境条件改变在鱼类受到病原的感染其他环境胁迫时似乎能够增强免疫力减轻机体受到的损伤(Dhabhar, 2002)。例如: 已有文献证实盐度能够提高免疫相关基因(IgM、HSP70等)的表达来影响鱼类机体免疫能力(Deane et al, 2004; Huang et al, 2015)。此外, 有研究表明盐度的升高能够增强动物上皮细胞对某些病原体的黏附作用, 使病原体更易进入并感染机体, 增强机体吞噬效率, 同时增加涉及先天免疫和炎症几种途径的蛋白丰度, 进而增强机体清除病原体的能力, 增强鱼体免疫力(Piertney et al, 2006; Schmitz et al, 2017)。因此, 本文将要探究盐度变化对于金钱鱼免疫系统应对细菌感染的影响。
细菌广泛分布在自然水体中, 常常是导致水产养殖物种爆发疾病的重要病原体之一(Laith et al, 2014)。近年来, 水产养殖业爆发了大量的细菌性疾病, 对养殖业造成了不可估量的损失(Van Hai, 2015)。嗜水气单胞菌(Aeromonas hydrophila)是近年鱼类爆发的细菌性疾病中常见致病菌之一, 多被用作科学研究中进行动物感染实验的病原(Zhang et al, 2016; Abdelhamed et al, 2017; Baumgartner et al, 2017; Peatman et al, 2018)。Altinok等(2001)发现斑点叉尾鮰、金鱼、条纹鲈鱼和墨西哥湾鲟在面对柱状黄杆菌感染时, 死亡率随着盐度上升所导致的细菌体外粘附力下降而降低。但嗜水气单胞菌对鱼体免疫状态受盐度影响的研究尚少, 与盐度相互作用的规律尚不明确。因此, 本文将采用嗜水气单胞菌进行动物感染实验以便分析比较鱼类免疫学状态。
金钱鱼(Scatophagus argus)隶属于鲈形目金钱鱼属金钱鱼科, 是一种广盐性硬骨鱼类, 主要分布在印度—太平洋沿岸(Bardach et al, 1972; Ghazilou et al, 2011)。在我国主要生活在江河入海口的咸淡水交融水域近海岩礁处。金钱鱼常常在初春时节游至近岸产卵, 产卵后的成鱼游向外海, 而幼鱼则在近岸沿海岩礁区觅食生长, 随着幼鱼的生长发育成熟逐渐迁徙至远岸海水区, 直至性成熟再回到近岸进行下一轮产卵活动(梁雪梅, 2018)。就其生活史来看, 金钱鱼具有典型的生殖洄游习性, 在其完整的生长周期中必定会经历海水不同的盐度变化, 生活盐度范围从5‰~30‰不等(Mookkan et al, 2014)。由于金钱鱼对盐度拥有较强的耐受性, 抗逆能力强, 在我国东南沿海地区又极受人们的喜爱, 近年来人们开始将金钱鱼经淡水驯化后进行大规模的淡水化养殖(杨尉 等, 2018)。因此盐度在金钱鱼生活史中是必不可少的重要环境因子。
鱼类机体中存在多种免疫相关成分, 它们能够在鱼类受到环境因素和病原体的影响时保护机体, 起到一定的免疫作用。例如超氧化物歧化酶(superoxide dismutase, SOD)能够防止由于盐度变化引起机体活性氧(reactive oxygen species, ROS)产生对机体造成的损伤(Martínez-Álvarez et al, 2002; Regoli et al, 2014)。补体Complement 4(C4)(Boshra et al, 2006)、细胞因子Interleukin-6(IL-6)(Dannevig et al, 1994; Brattgjerd et al, 1996)和抗体Immunoglobulin M(IgM)(De Lima et al, 2011; Gallani et al, 2019)能够通过单独或联合作用一起杀死并清除机体中入侵的病原体, 在机体应对病原微生物的感染时起到重要的防御作用。肾脏组织作为主要的免疫器官, 存在大量能够吞噬细菌、释放免疫相关分子并促进抗体产生的巨噬细胞(MacArthur et al, 1983; Dannevig et al, 1994; Brattgjerd et al, 1996)。在非特异性免疫、清除碎片和损伤细胞等方面, 肾脏组织也起着重要的作用(Meseguer et al, 1995)。因此, 本文将利用嗜水气单胞菌进行动物感染实验, 通过比较不同盐度组金钱鱼血清和肾脏组织中SOD浓度以及免疫相关成分C4、IL-6和IgM浓度变化来分析盐度变化对于感染嗜水气单胞菌时金钱鱼血清和肾脏免疫学功能的影响。这为金钱鱼最佳养殖盐度提供了科学的参考, 也对水产养殖业有着重要的参考价值。

1 材料与方法

1.1 材料与主要试剂

健康金钱鱼200±50g购自广东省湛江市某养殖场, 体长约13±0.5cm, 于自然水体盐度25‰盐度水系统中暂养一周后将其分别转至0‰、10‰和25‰盐度水体中分别驯化, 增氧机增氧, 水温维持在28±1℃。早晚各投喂一次红虫, 实验开始前三天停止喂食, 驯化两周后开始实验。普通肉汤培养基购自上海生工, 补体C4含量检测试剂盒购自南京建成生物有限公司, SOD、IL-6和IgM含量检测试剂盒购自Cusabio公司。

1.2 方法

1.2.1 菌悬液的制备
将嗜水气单胞菌接种至普通肉汤培养基, 置于28℃恒温摇床培养18~24h, 将培养所得菌液置于离心机中以4000rpm离心10min, 收集沉淀。用0.85%无菌生理盐水将其制成菌悬液, 参考张庆华 等(2016)的实验方法稀释成3×105、3×106、3×107、3×108、3×109、3×1010CFU·mL-L共6个浓度, 腹腔注射6组, 每组12尾鱼, 根据预实验结果确定注射剂量为每尾0.2mL, 对照组注射等剂量0.85%无菌生理盐水。实验鱼分组养在不同水箱中, 实验中连续充氧, 水温控制在28±1℃。连续观察96h内金钱鱼死亡情况, 记录数据并根据结果计算LD50。
1.2.2 动物感染实验
根据上述LD50实验结果, 确定动物感染实验注射浓度。重新制备新鲜3×108CFU·mL-1菌悬液, 腹腔注射0‰、10‰和25‰实验组金钱鱼, 每尾注射3×108 CFU·mL-1细菌悬液0.2mL, 60尾每组。对照组5尾每组, 注射等剂量0.85%无菌生理盐水。实验鱼分组养在不同水箱中, 在感染后的6、12、24、48、96h对实验组进行取样, 对照组于0h取样, 每次每组取样5尾。用MS-222 麻醉后解剖, 尾柄取血收集血液, 置于4℃静置8h, 7000rpm离心 10min, 收集血清, 将5份样本均等随机混样成三份, 得到3个生物学平行样本, 置于-20℃保存待检测。每尾鱼剪取肾脏组织后于2mL离心管称重并按照1∶19的比例加入无菌磷酸缓冲盐溶液(phosphate buffer saline, PBS)后, 置于-20℃冷冻保存。
1.2.3 组织悬液制备
将存放于-20℃冰箱的组织样品取出, 经过反复两次冻融后, 在离心管中加入匀浆钢珠。将每个样品管简单配平放置于匀浆容器中, 设置匀浆条件为60Hz, 每次20s, 匀浆3次左右。取出样品, 置于离心机中1000×g离心5min, 收集上清液。按照血清混样规律, 将每个盐度组, 每个时间点的5份样本混样成3份生物学平行样本
1.2.4 超氧化物歧化酶浓度检测
超氧化物歧化酶含量检测试剂盒购自武汉华美生物有限公司, 按照试剂盒说明书检测血清及肾脏组织中SOD浓度, 单位为ng·mL-1。取适量血清样品经200倍稀释后得到110μL待测样品稀释液备用, 吸取110μL组织样品原液置于冰台待检测。按照说明书指示逐级稀释标准品得到标准品工作液。每个样品及标准品分别设置两个重复孔作为技术平行, 按照试剂盒说明书操作步骤进行操作, 加入反应终止液后利用酶标仪在450nm处检测吸光度。
1.2.5 C4、IL-6和Ig M浓度检测
根据武汉华美生物公司C4、IL-6和Ig M试剂盒说明书对血清和肾脏组织中各免疫相关指标浓度进行检测, C4和Ig M浓度单位为μg·mL-1, IL-6浓度单位为pg·mL-1。血清4倍稀释, 组织液使用原液进行酶联免疫吸附测定(enzyme-linked immuno sorbent assay, ELISA)检测。
1.2.6 数据处理
实验所得数据用EXCEL软件进行计算, 结果以平均值±标准误(mean±SE)表示。用SPSS软件对数据进行单因素方差分析(ANOVA), P<0.05表示差异显著。利用Curve Expert拟合标准曲线, 将样品吸光度代入标准曲线得到样品浓度, 运用Origin 8.5.0软件对数据进行图标的绘制。

2 结果

2.1 血清和肾脏组织SOD浓度

血清SOD浓度结果显示(图1a): 除25‰盐度组经嗜水气单胞菌注射后第96h外, 三个盐度实验组其他时间点血清SOD浓度分别为989.4±57.8、897.0±32.2和849.7±23.5ng·mL-1, 均低于对照组。在淡水组中金钱鱼经嗜水气单胞菌注射后96h内血清SOD浓度均低于对照组但差异不显著; 在低盐组中金钱鱼经嗜水气单胞菌注射后96h内血清SOD浓度均低于对照组(897.0±32.2ng·mL-1), 在注射后6h(739.2±6.8ng·mL-1)和12h(743.1±3.5ng·mL-1)差异极显著, 至24~96h内浓度稍有升高; 在25‰盐度组中除注射后96h(1182.9±13.1ng·mL-1)高于对照组(849.7±23.5ng·mL-1)外, 其余时间点均显著低于对照组。与此同时, 除注射后6h和96h外, 淡水组和低盐组血清SOD浓度均高于25‰盐度组。
图1 不同盐度金钱鱼注射嗜水气单胞菌后96h内血清(a)和肾脏(b)组织中SOD浓度(n=5)

**P < 0.01; * P < 0.05(与对照组相比较)

Fig.1 Concentrations of SOD in serum (a) and kidney (b) among three salinity experimental groups after A. hydrophila injection at 6, 12, 24, 48, and 96 h (n=5).

**P < 0.01; *P < 0.05 (compared to the control group)

肾脏组织中SOD浓度结果显示(图1b), 除25‰盐度组第6h(81.6±0.6ng·mL-1)外, 三个实验组金钱鱼经嗜水气单胞菌注射后肾脏组织SOD浓度均高于对照组, 分别为61.9±0.9、84.9±4.0和90.8± 0.7ng·mL-1, 且于注射后24h开始与对照组差异极显著。淡水组和低盐组肾脏组织SOD浓度均高于25‰盐度组, 与血清结果基本一致。

2.2 血清和肾脏组织C4浓度

不同盐度下金钱鱼注射嗜水气单胞菌后血清C4浓度显示(图2a), 淡水组血清C4含量显著高于对照组(537.5±25.0μg·mL-1), 且呈现上升趋势。低盐组和25‰盐度组血清C4浓度均在注射后12h达到最低值, 分别为370.3±16.4和197.3±13.6μg·mL-1; 而后升高, 直至96h时浓度达到523.8±27.9和507.6±27.2μg·mL-1, 均回升至近似对照组(分别为440.7±28.1和554.3±18.2·mL-1)水平。淡水组和低盐组血清C4浓度趋势虽不同, 但浓度均高于25‰盐度组。
图2 不同盐度金钱鱼注射嗜水气单胞菌后96h内血清(a)和肾脏(b)中C4浓度变化(n=5)

**P < 0.01; * P < 0.05(与对照组相比较)

Fig. 2 Concentrations of C4 in serum (a) and kidney (b) among three salinity experimental groups after A. hydrophila injection at 6, 12, 24, 48, and 96 h (n=5).

**P < 0.01; *0.01 < P < 0.05 (compared to the control group)

肾脏组织中C4浓度结果如图2b所示, 肾脏组织中C4浓度均呈现先增加后降低的趋势, 且淡水组与低盐组浓度均高于25‰盐度组。其中, 淡水组与低盐组差异极显著, 均于注射后24h达到最高值, 分别为218.3±1.2和186.3±6.0μg·mL-1, 于96h降至99.6±3.2和70.3±5.9μg·mL-1, 接近对照组水平(58.3±0.8和74.3±1.1μg·mL-1)。25‰盐度组在注射后6h的C4浓度为63.1±0.7μg·mL-1, 在12h为81.2± 3.6μg·mL-1, 显著低于对照组(95.6±2.1μg·mL-1); 后期呈增加趋势, 于48h达到最大值(119.9± 1.5μg·mL-1)后下降。

2.3 血清和肾脏组织IL-6浓度

不同盐度下金钱鱼注射嗜水气单胞菌后血清IL-6浓度结果如图3a所示, 淡水组血清IL-6浓度显著高于低盐组和25‰盐度组。淡水组金钱鱼注射嗜水气单胞菌后血清IL-6浓度显著升高, 在注射后12h达到峰值(56.9±1.0pg·mL-1), 之后呈现下降趋势, 于96h达到43.5±0.7pg·mL-1, 与对照组水平(42.1± 0.5pg·mL-1)相似。淡水组血清IL-6浓度相比对照组波动不大, 但25‰盐度组血清IL-6浓度(38.8± 1.0pg·mL-1)显著低于对照组。
图3 不同盐度金钱鱼注射嗜水气单胞菌后96h内血清和肾脏中IL-6浓度变化(n=5)

**P < 0.01; *P < 0.05(与对照组相比较)

Fig. 3 Concentrations of IL-6 in serum (a) and kidney (b) among three salinity experimental groups after A. hydrophila injection at 6, 12, 24, 48, and 96 h (n=5).

**P < 0.01; * P < 0.05 (compared to the control group)

肾脏组织中IL-6浓度如图3b所示, 25‰盐度组与对照组浓度均高于淡水组和低盐组与两盐度对照组浓度。在淡水组中, 金钱鱼经嗜水气单胞菌注射后96h内肾脏组织中IL-6浓度低于对照组浓度(15.9±0.2pg·mL-1), 且差异极显著; 在10‰和25‰盐度组中, 金钱鱼经嗜水气单胞菌注射后第6h的肾脏组织IL-6浓度为17.9和17.9pg·mL-1, 显著高于对照组(17.3±0.1和17.1±0.1pg·mL-1); 但从12h(15.3± 0.2和15.5pg·mL-1)开始至48h三个盐度组均显著低于对照组, 且呈上升趋势。

2.4 血清和肾脏组织IgM浓度

不同盐度组金钱鱼经嗜水气单胞菌感染后血清免疫球蛋白IgM浓度结果如图4a所示, 三个盐度组血清IgM浓度均呈现上升趋势, 但25‰盐度组血清IgM浓度显著高于淡水组和低盐组, 同时低盐组血清IgM浓度高于淡水组。
图4 不同盐度金钱鱼注射嗜水气单胞菌后96h内血清(a)和肾脏(b)中IgM浓度变化(n=5)

**P < 0.01; *P < 0.05(与对照组相比较)

Fig. 4 Concentrations of IgM in serum (a) and kidney (b) among three salinity experimental groups after A. hydrophila injection at 6, 12, 24, 48, and 96 h (n=5).

**P < 0.01; * P < 0.05 (compared to the control group)

不同盐度实验组金钱鱼肾脏组织中免疫球蛋白IgM浓度在嗜水气单胞菌注射后96h内均呈现先升高后下降的趋势(图4b)。其中25‰盐度组肾脏IgM浓度高于淡水组和低盐组, 且25‰盐度组金钱鱼肾脏组织中IgM浓度在注射后12h达到最大值(6.3± 0.4μg·mL-1), 而淡水组和低盐组晚于且低于25‰盐度组, 在24h和48h才达到最大值, 分别为5.2±0.4和4.9±0.0μg·mL-1(图4b)。

3 讨论

由于近几年全球变暖等气候问题日渐严重, 造成了大量的冰川融化以及气候降水等问题, 致使近岸海水盐度剧烈波动(Nielsen et al, 2012; Duggan et al, 2014; Van Wijk et al, 2014)。盐度是影响水生生物生命活动的重要环境因子, 不仅影响鱼类机体渗透调节, 还能间接影响鱼类机体免疫功能(Geven et al, 2017; El-Leithy et al, 2019)。肾脏是硬骨鱼类重要的免疫器官之一, 其状态与鱼类机体免疫系统密切相关(Guo et al, 2017)。肾脏中免疫相关因子主要有特异性免疫和非特异性两大类, 这些免疫相关因子的含量是检测其应对病原入侵时免疫状态的重要指标(Magnadottir et al, 2005; Ni et al, 2016)。在目前的研究中常以SOD、C4及IL-6含量等非特异性免疫指标(Whyte, 2007; Zhu et al, 2013; Rombout et al, 2014)和IgM含量等特异性免疫指标(Larrieta et al, 2012; Rombout et al, 2014)来反映机体免疫状态。因此本文以生活在近岸海水交汇区的金钱鱼为研究对象, 以嗜水气单胞菌为感染病原, 结合上述免疫指标的检测结果, 来研究海水盐度变化对鱼类血清和肾脏免疫状态的影响。
众所周知, 盐度变化引起的氧化应激反应与ROS的产生有关(Liu et al, 2007; Lushchak, 2011)。SOD是抗氧化防御系统中第一个遇到超氧阴离子自由基的抗氧化酶, 能通过直接或间接的机制与非酶自由基清除剂共同作用来避免ROS造成的损伤(Regoli et al, 2014; Ghanavatinasab et al, 2019)。因此, SOD在盐度与鱼类机体的氧化应激关系中起着十分重要的作用, 能够直接反映鱼类受到盐度胁迫后机体应激反映强弱, 可作为评判机体免疫能力的重要指标。在目前已发表的文章中, 学者们普遍证实盐度降低将会导致SOD活性升高, 以中和盐度胁迫造成的损伤(An et al, 2010a, b; Zeng et al, 2017)。本实验观察到, 除96hpi外, 血液和肾脏组织中, 淡水组和低盐组金钱鱼体内SOD浓度总体均比25‰盐度组高约2~150ng·mL-1, 其中血清结果差异极显著(P<0.01)。由于淡水组和低盐组金钱鱼机体血清及肾脏组织SOD浓度大量升高, 推测大量 SOD的激活是由于外界刺激造成的损伤较严重, 需要大量SOD来修复损伤(Zeng et al, 2017)。因此, 相比25‰盐度组, 淡水组和低盐组中的金钱鱼在受到感染后鱼体损伤较重, 肾脏组织免疫状态也将随之降低。
补体C4在补体系统的三种激活途径中起关键作用(Sirotkina et al, 2013)。病原体入侵机体时, 补体通过识别蛋白与病原体结合将会促进巨噬细胞吞噬作用, 并进一步促进细胞因子的释放和抗体的产生(Boshra et al, 2006; Gallani et al, 2019)。此外, 病原体被三种补体激活途径的特异性识别分子识别结合后将会激活C4, 而C4进一步裂解能够促进抗体的形成来杀死病原体(Mortensen et al, 2015)。本文通过比较三个盐度组血清和肾脏组织C4浓度, 发现淡水组与低盐组金钱鱼血清和肾脏组织C4浓度总体均比25‰盐度组高100~600μg·mL-1(P<0.01)左右, 且淡水组和低盐组C4水平均呈现先显著上升后缓慢下降的趋势, 25‰盐度组在血清和肾脏中的C4水平分别为38.6±1.0和95.6±2.1μg·mL-1(P<0.01), 显著低于对照组, 后期才缓慢上升。以往的研究表明, 草鱼幼鱼C4含量的增加, 能够促进其他免疫相关指标的增加, 共同作用提高机体免疫功能(Liu et al, 2018)。但在本实验中, 淡水组和低盐组显著提高了血清和肾脏中C4浓度, 却并未显著激活下游抗体IgM的产生。由于C4作为补体激活途径中关键成分, 关系到吞噬细胞对免疫因子和抗体的释放和促进作用, 推测是由于淡水组和低盐组金钱鱼补体激活途径的上游未受到抑制, 促进了C4的激活, 但C4未能成功裂解导致下游细胞因子和抗体激活效率或活性下降(Legendre et al, 2017)。因此, 淡水组和低盐组的补体裂解效率可能受到了抑制, 阻碍了免疫应答的进程。
IL-6是一种多效性细胞因子, 在免疫系统炎症反应的调节中起着至关重要的作用(Hunter et al, 2015)。IL-6主要是由单核细胞/巨噬细胞受微生物刺激后产生的一种细胞因子(Zou et al, 2016)。在病毒或细菌入侵的过程中, 上述补体C4促进了吞噬细胞的吞噬和细胞因子的释放, IL-6迅速而短暂地上调, 成为感染的重要标志(Narazaki et al, 2018)。在哺乳动物中, IL-6在感染和组织发生损伤时迅速产生, 参与宿主防御机制(Rose-John, 2018)。硬骨鱼类中, IL-6被证实参与感染后抗体的产生(Wei et al, 2018)。此外, IL-6能够促进鱼类吞噬细胞的吞噬作用, 提高杀菌活性(Zhu et al, 2019)。在本实验中虽然三个盐度感染嗜水气单胞菌后组肾脏组织IL-6浓度显著低于对照组(14.2±0.0、17.9±0.0和17.9±0.0pg·mL-1)(P<0.01), 25‰盐度组肾脏组织IL-6水平仍显著高于淡水组和低盐组(P<0.01)。推测低盐驯化组金钱鱼体内吞噬细胞对细菌的吞噬作用或吞噬活性低于25‰盐度组, 低盐驯化可能会降低金钱鱼肾脏组织吞噬细胞对病原的清除率, 降低免疫状态。然而, 血清结果与肾脏存在些许差异, 淡水组的血清IL-6浓度在96hpi内显著增加至56.9±1.0pg·mL-1(P<0.01), 但肾脏组织中IL-6浓度却低于对照组(15.9±0.2pg·mL-1), 且差异极显著(P<0.01)。这可能是由于金钱鱼受到感染后, 外周血单核细胞开始产生IL-6用于清除病菌, 但不能成功转运至肾脏组织, 导致肾脏组织中IL-6浓度显著降低(Inturri et al, 2017)。
血清免疫球蛋白是体液免疫系统的主要组成部分, IgM是鱼体内存在的主要免疫球蛋白(Wilson et al, 1995)。在机体遭遇抗原的刺激下, 浆细胞产生具有免疫功能的抗体IgM等免疫球蛋白, IgM能够识别和中和体外入侵的病毒和细菌(Cerezuela et al, 2012; 张媛媛 等, 2018)。IgM是初次体液免疫应答中最早出现的抗体, 抗体产生量的多少不仅与C4含量与裂解量相关, 同时还与细胞因子的促进作用相关(侯月娥 等, 2010; Legendre et al, 2017)。IgM浓度的增加, 能够促进罗非鱼自身免疫系统的联级反应, 增强机体免疫功能(Tellez-Bañuelos et al, 2010)。本文研究结果显示, 25‰盐度组金钱鱼血清和肾脏组织IgM浓度(71.8±2.9和6.3±0.4μg·mL-1)比淡水组和低盐组高约1~20μg·mL-1, 且差异显著(P<0.05)。同时, 25‰盐度组血清和肾脏组织大量产生抗体IgM时间(12 hpi)早于淡水组和低盐组(24hpi)。根据Domingueza等(2004)和何杰 等(2014)等的研究结果显示, 短期的应急刺激可能有助于增加循环蛋白, 导致IgM水平呈现出上升趋势, 但是随着应激时间的延长, 蛋白开始用于能量需求导致合成减少, 抑制机体免疫活性。因此, 推测本实验中淡水组和低盐组血清和肾脏IgM浓度低于25‰盐度组的原因可能是由于为期两周的低盐驯化导致金钱鱼IgM合成减少, 使机体免疫状态受到抑制。综合上述免疫相关指标(C4、IL-6和IgM)的结果与SOD结果, 证实了低盐驯化导致金钱鱼血清和肾脏组织免疫状态和功能受到抑制。
本研究通过ELISA检测等方法, 比较了不同盐度条件下血清和肾脏组织SOD浓度以及免疫相关指标(C4、IL-6和IgM)浓度。结果表明, 淡水组与低盐组免疫状态与25‰盐度组相比较弱。因此, 我们推测金钱鱼在低盐状态下血清和肾脏组织的免疫功能将会减弱或受到抑制, 使得细菌更易感染鱼体并造成更严重的损伤。综上所述, 我们建议在金钱鱼淡水化养殖中能够维持一定盐度, 保护金钱鱼肾脏组织免疫机能以避免疾病的发生。同时, 尽量减少露天养殖, 以避免降雨对池塘盐度造成的影响, 或者在暴雨过后适当添加海盐来增加水体盐度, 以减轻降水导致的水体盐度波动对金钱鱼的影响。

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