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Volume 9 Issue 11
Nov.  2021
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Accumulation of nitrite and responses of energy metabolism exposed to nitrite stress in Litopenaeus vannamei

  • Corresponding author: SHAN Hongwei, shanhongwei@ouc.edu.cn
  • Received Date: 2020-11-03
    Accepted Date: 2020-12-22
    Available Online: 2021-04-01
  • Litopenaeus vannamei is one of the most important breeding economic varieties in the world because of its fast growth, strong environmental adaptability, and short growth cycle. Along with the rapid promotion of the intensive culture model of L. vannamei, large quantities of harmful substances accumulate in the culture environment which seriously affect the growth and health of L. vannamei. Nitrite enters through gills and accumulates in tissues, which has severely toxic effects on cultured L. vannamei. In the present study, to explore the accumulation of nitrite and changes in enzymes activities related to energy metabolism exposed to nitrite in L. vannamei [body length (6.8 ± 0.3) cm, weight (4.0 ± 0.6 g)], shrimps were exposed to four nitrite concentrations of 0 (control), 0.8 (11.2 mg/L NO2-N), 4.0 (56 mg/L NO2-N) and 8.0 mmol/L (112 mg/L NO2-N) for 96 h and then recovered for 12 h. Each experimental treatment included six replicates. Three replicates were used for sample collection, and the other three replicates were used to calculate the cumulative mortality of L. vannamei. At 0, 6, 12, 24, 48 and 96 h of the nitrite stress, the hemolymph, hepatopancreas, muscle, gill and intestine of 9 L. vannamei in each group were randomly selected for the determination of the index. Additionally, death number were recorded every 12 h. The results indicated that the shrimp mortality rate increased with nitrite concentrations. At the end of nitrite stress, shrimp mortality in control group, 0.8 mmol/L group, 4.0 mmol/L group and 8.0 mmol/L group were 10.7%, 32.0%, 42.7% and 52.0%, respectively. Within 6 hours of exposure, nitrite accumulated significantly in the gill, hemolymph, intestine, hepatopancreas, and muscle tissues of L. vannamei, and was positively correlated with stress concentrations. The maximum accumulation of nitrite in gills, hemolymph, hepatopancreas, intestine and muscle were 50.1 mg/kg, 43.2 mg/L, 20.7 mg/kg, 33.5 mg/kg and 14.9 mg/kg. In the same stress concentration group, nitrite accumulated the most in gill and the least in muscle, and the accumulation in gill was about 3 times greater than that in muscle. The activity of Na+-K+-ATPase in hepatopancreas and muscle of L. vannamei were significantly increased at 0.8 and 4.0 mmol/L, but significantly decreased in muscle of 8.0 mmol/L during the exposure period. The activity of AMPK in the hepatopancreas in the stress groups increased compared to the control group, and showed a positive correlation with the stress concentration. During the recovery period, except for hemolymph (8.0 mmol/L group), 1-hour recovery rate of nitrite in L. vannamei tissues in the stress groups were above 50%, and the hepatopancreas and gill showed the highest recovery efficiency exceeding 74%. The recovery time of hemolymph, gill and intestine were the shortest within 6 hours. In addition, the content of nitrite in water increased significantly. This study indicates that nitrite can be accumulated to shrimp tissues in a short time and can accelerate the process of energy metabolism. Nitrite would excrete from the body rapidly during recovery in order to reduce the toxic effects. The results of this study will provide reference for alleviating the toxic effects of nitrite on cultured shrimp.
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Accumulation of nitrite and responses of energy metabolism exposed to nitrite stress in Litopenaeus vannamei

    Corresponding author: SHAN Hongwei, shanhongwei@ouc.edu.cn
  • Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao    266003, China

Abstract: Litopenaeus vannamei is one of the most important breeding economic varieties in the world because of its fast growth, strong environmental adaptability, and short growth cycle. Along with the rapid promotion of the intensive culture model of L. vannamei, large quantities of harmful substances accumulate in the culture environment which seriously affect the growth and health of L. vannamei. Nitrite enters through gills and accumulates in tissues, which has severely toxic effects on cultured L. vannamei. In the present study, to explore the accumulation of nitrite and changes in enzymes activities related to energy metabolism exposed to nitrite in L. vannamei [body length (6.8 ± 0.3) cm, weight (4.0 ± 0.6 g)], shrimps were exposed to four nitrite concentrations of 0 (control), 0.8 (11.2 mg/L NO2-N), 4.0 (56 mg/L NO2-N) and 8.0 mmol/L (112 mg/L NO2-N) for 96 h and then recovered for 12 h. Each experimental treatment included six replicates. Three replicates were used for sample collection, and the other three replicates were used to calculate the cumulative mortality of L. vannamei. At 0, 6, 12, 24, 48 and 96 h of the nitrite stress, the hemolymph, hepatopancreas, muscle, gill and intestine of 9 L. vannamei in each group were randomly selected for the determination of the index. Additionally, death number were recorded every 12 h. The results indicated that the shrimp mortality rate increased with nitrite concentrations. At the end of nitrite stress, shrimp mortality in control group, 0.8 mmol/L group, 4.0 mmol/L group and 8.0 mmol/L group were 10.7%, 32.0%, 42.7% and 52.0%, respectively. Within 6 hours of exposure, nitrite accumulated significantly in the gill, hemolymph, intestine, hepatopancreas, and muscle tissues of L. vannamei, and was positively correlated with stress concentrations. The maximum accumulation of nitrite in gills, hemolymph, hepatopancreas, intestine and muscle were 50.1 mg/kg, 43.2 mg/L, 20.7 mg/kg, 33.5 mg/kg and 14.9 mg/kg. In the same stress concentration group, nitrite accumulated the most in gill and the least in muscle, and the accumulation in gill was about 3 times greater than that in muscle. The activity of Na+-K+-ATPase in hepatopancreas and muscle of L. vannamei were significantly increased at 0.8 and 4.0 mmol/L, but significantly decreased in muscle of 8.0 mmol/L during the exposure period. The activity of AMPK in the hepatopancreas in the stress groups increased compared to the control group, and showed a positive correlation with the stress concentration. During the recovery period, except for hemolymph (8.0 mmol/L group), 1-hour recovery rate of nitrite in L. vannamei tissues in the stress groups were above 50%, and the hepatopancreas and gill showed the highest recovery efficiency exceeding 74%. The recovery time of hemolymph, gill and intestine were the shortest within 6 hours. In addition, the content of nitrite in water increased significantly. This study indicates that nitrite can be accumulated to shrimp tissues in a short time and can accelerate the process of energy metabolism. Nitrite would excrete from the body rapidly during recovery in order to reduce the toxic effects. The results of this study will provide reference for alleviating the toxic effects of nitrite on cultured shrimp.

  • 凡纳滨对虾(Litopenaeus vannamei)因其生长快、环境适应性强、生长周期短、适合多种模式养殖等特点,已成为全球最主要的养殖经济品种之一。凡纳滨对虾也是我国主要的甲壳动物养殖种类[1-2],2018年其海水养殖产量已达111.8万t。近年来,随着市场对高养殖效益的追求,凡纳滨对虾集约化养殖模式迅速推广,而在集约化养殖中后期,环境中有害物质大量积累,严重影响对虾的生长和健康。

    亚硝态氮对水生动物具有较强毒性,其在养殖中后期极易积累,浓度可达到1.4 mmol/L (20 mg/L NO2-N),是影响水产动物健康的主要环境胁迫因子之一[3-5]。已有研究表明,高浓度亚硝态氮胁迫能降低凡纳滨对虾免疫力, 增加其对副溶血弧菌(Vibrio parahaemolyticus)的易感性[6]。Han等[7]和Duan等[8]以凡纳滨对虾为研究对象发现亚硝态氮会影响其正常的生长与成活,并造成氧化应激。

    环境中的亚硝态氮主要以${\rm{HNO}}_2^ - $${\rm{NO}}_2^ - $两种形式存在,前者含量较少,可通过鳃细胞自由扩散进入体内;后者为亚硝态氮主要存在形式,主要通过与Cl竞争鳃上的吸收位点进入甲壳动物血淋巴,也有小部分通过肠道主动吸收[9-10]。进入体内的亚硝态氮会在不同组织中积累,在对斑节对虾 (Penaeus monodon) 的胁迫实验中发现,48 h后亚硝态氮主要在肠道中积累,为胁迫浓度的11.4倍;而在肌肉中积累最少,为1.1倍[11]。明确亚硝态氮被吸收、积累和排出的过程对了解其毒性影响具有重要意义。此外,水生动物在受到环境胁迫时会通过调节能量代谢来适应外部环境[12-14],而关于亚硝态氮胁迫下对虾能量代谢响应的相关研究有待丰富。

    本实验以凡纳滨对虾为材料,以累积死亡率为表征,通过研究亚硝态氮胁迫以及恢复期间对虾体内亚硝态氮的时空分布和胁迫下组织内能量代谢相关酶活性的变化规律,揭示亚硝态氮对凡纳滨对虾的毒性作用,为今后缓解亚硝态氮对养殖对虾毒性效应的研究提供参考。

1.   材料与方法
  • 用于实验的凡纳滨对虾购自山东省威海市乳山某对虾养殖场,初始体长为(6.8±0.3) cm,体质量为(4.0±0.6) g。对虾运回后在实验室内暂养2周,期间每天投喂4餐配合饲料(粗蛋白含量≥40%),每天换水50%,于胁迫实验开始前24 h停止投喂。

  • 将NaNO2溶解在1 L的蒸馏水中配置成714.3 mmol/L(10 g/L NO2-N)的母液,然后根据各组设定浓度用海水进行稀释。通过预实验得到对虾96 h半致死浓度(LC50)为8.1 mmol/L(113.7 mg/L NO2-N),据此设计4个实验组:0(对照组)、0.8 mmol/L(11.2 mg/L NO2-N)、4.0 mmol/L(56 mg/L NO2-N)及8.0 mmol/L(112 mg/L NO2-N)。每组设置6个平行,每个平行25尾虾,其中3个平行用于样品采集,另外3个平行用于记录对虾死亡情况。胁迫实验持续96 h,期间不投喂,并且每天换水50%后加入适量亚硝态氮母液以维持每组相应的胁迫浓度。胁迫期间水体指标:温度(25±1) °C,盐度30,DO≥5 mg/L,pH 7.8~8.1。

  • 在亚硝态氮胁迫实验结束后,将胁迫组的对虾放入不添加NaNO2的海水中进行恢复实验,实验持续12 h。每个胁迫组设置3个平行,每个平行12尾虾。

  • 在胁迫实验的0、6、12、24、48和96 h分别从各组中随机取9尾凡纳滨对虾进行取样,并每12 h记录对虾死亡情况。在恢复实验的0、1、6和12 h分别从各胁迫组中取9尾对虾进行取样,另取环境水样于−20 °C冰箱中冷冻保存,待测。

    抗凝剂(0.45 mmol/L NaCl、0.01 mmol/L KCl、0.01 mmol/L EDTA-Na2、0.01 mmol/L HEPES,pH值7.45)预冷(4 °C)后,使用1 mL注射器按照1∶3(血淋巴∶抗凝剂,体积比)的比例抽取对虾血淋巴,将混合物离心(1 531× g,10 min),取上清液放入−80 °C保存,待测。取鳃、肝胰腺、肌肉和肠道(排空粪便)组织,用预冷超纯水冲洗组织表面后,用吸水纸快速拭去表面水分后迅速放入液氮中,放入−80 °C保存,待测。

  • 称取对虾肝胰腺、肌肉、鳃及肠道组织0.1~0.2 g于预冷的生理盐水(样品质量与生理盐水体积比为1∶9)中冰上充分匀浆,然后离心(1 250 × g,10 min),取上清液用于各指标的测定。

    对虾血淋巴、鳃、肝胰腺、肌肉及肠道中亚硝态氮含量,肝胰腺、肌肉中Na+-K+-ATP酶活性均使用南京建成生物工程研究所试剂盒测定,指标按照相应试剂盒操作说明完成。肝胰腺及肌肉中AMP依赖的蛋白激酶(AMPK)活性使用Elisa试剂盒(泉州科诺迪生物科技有限公司)测定。水体中亚硝态氮采用萘乙二胺分光光度法测定[15]

    根据恢复实验0和1 h各组织中亚硝态氮含量,计算各组织1 h恢复效率(1-hour recovery rate,1HRR),计算公式:

    式中,C0表示恢复实验0 h时亚硝态氮含量(mg/L,mg/kg); Ct表示恢复实验1 h时亚硝态氮含量(mg/L,mg/kg)。

  • 实验数据采用mean±SD表示,凡纳滨对虾96 h LC50通过线性内插法计算得到[16]。利用单因素方差分析(One-Way ANOVA)比较同一时间点不同处理组间各指标的差异性。在数据分析之前首先检验数据的正态性(Shapiro-Wilk 检验)和方差齐性(Levene's 检验),对不符合的数据在进行对数转换后再进行检验,如仍不符合将对数据进行非参数检验(Kruskal-Wallis 检验)。使用Tukey氏多重比较法分析各组之间的差异性,P<0.05即表示差异显著,数据的统计与分析采用软件SPSS 25.0进行。

2.   结果
  • 随着亚硝态氮胁迫浓度的升高,对虾死亡率呈上升趋势。8.0 mmol/L组在胁迫12 h时出现大量死亡,并且在胁迫96 h时,8.0 mmol/L组对虾累积死亡率显著高于0 mmol/L组(P<0.05)。胁迫结束时,各组对虾死亡率分别为10.7% (0 mmol/L组)、32.0% (0.8 mmol/L组)、42.7% (4.0 mmol/L组)、52.0%(8.0 mmol/L组)(图1)。

    Figure 1.  Cumulative mortality rate of L. vannamei in different groups during nitrite exposure

  • 各组织亚硝态氮含量明显积累,且与胁迫浓度呈正相关。鳃中亚硝态氮最大积累量为50.1 mg/kg、血淋巴中为43.2 mg/L、肝胰腺中为20.7 mg/kg、肠道为33.5 mg/kg、肌肉中为14.9 mg/kg(图2)。

    Figure 2.  Nitrite concentration of different tissues during nitrite exposure

  • 胁迫组对虾肝胰腺中Na+-K+-ATP酶活性呈现先上升后下降的趋势,其中在胁迫6和12 h时的活性显著高于对照组(P<0.05),而在96 h时下降至0 mmol/L组水平(图3-a)。肌肉中除8.0 mmol/L组外,其余各组Na+-K+-ATP酶活性呈现升高趋势,且4 mmol/L组酶活性在6~48 h显著高于0 mmol/L组(P<0.05)。8.0 mmol/L组酶活性呈先上升后下降的趋势,但始终低于0 mmol/L组,在6、24和48 h时达到显著差异水平(P<0.05,图3-c)。

    Figure 3.  Different enzyme activity in different groups during nitrite exposure

    肝胰腺中AMPK活性随胁迫浓度增加呈现升高趋势(图3-b),且各胁迫组在6、12和96 h均显著高于0 mmol/L组(P<0.05),4.0 mmol/L组在24和48 h显著高于0 mmol/L组(P<0.05)。各组肌肉中AMPK活性在胁迫6 h时升高,然后下降,在胁迫12至96 h过程中呈现升高趋势,然而各组之间没有显著差异(图3-d)。

  • 各组织中亚硝态氮含量均出现明显下降(图4)。恢复1 h后,除4.0 mmol/L组外,胁迫组对虾鳃中亚硝态氮含量与0 mmol/L组无显著性差异(P>0.05),在恢复6 h后,各胁迫组对虾鳃中亚硝态氮含量与0 mmol/L组无显著差异(P>0.05,图4-a)。恢复1 h后,4和8 mmol/L组对虾血淋巴和肠道中亚硝态氮含量仍显著高于0 mmol/L组(P<0.05),但恢复6 h后,各胁迫组与0 mmol/L组无显著差异(P>0.05,图4-b, c)。恢复1 h后,肝胰腺中亚硝态氮含量与0 mmol/L组相比无显著差异(P>0.05),恢复6~12 h过程中,0.8 mmol/L组有升高趋势,且显著高于其他各组(P<0.05, 图4-d)。胁迫组对虾肌肉中的亚硝态氮含量高于对照组(图4-e),且8 mmol/L组达到显著水平,0.8 mmol/L和4.0 mmol/L组分别在6~12 h、1~6 h达到显著水平(P<0.05)。

    Figure 4.  Nitrite content of different tissues in different groups during nitrite recovery period

    恢复期间对虾组织中亚硝态氮1 h恢复效率(1HRR)结果显示,1 h内对虾组织中亚硝态氮含量降低明显,除血淋巴(8 mmol/L组) 1HRR为39%,其余各组织均在50%以上,其中,鳃和肝胰腺的恢复效率最高,并且4.0和8.0 mmol/L组恢复效率显著高于0.8 mmol/L组(P<0.05)(表1)。此外,血淋巴、鳃、肠道6 h恢复到对照组水平,肌肉组织12 h内没有恢复到对照组水平。

    亚硝态氮浓度/(mmol/L)
    nitrite concentration
    血淋巴
    hemolymph
    肝胰腺
    hepatopancreas
    肌肉
    muscle

    gills
    肠道
    intestine
    0.8 98.4±2.8a 54.6±12.0a 50.8±11.9a 87.9±7.2a 54.3±27.6a
    4.0 78.3±0.9ab 89.4±2.1a 67.4±7.5bc 73.5±4.6b 59.1±5.8c
    8.0 39.1±3.0c 94.9±1.3a 83.0±1.9b 98.2±0.4a 83.8±2.0b
    注:同一浓度,不同字母表示组织间存在显著差异(P<0.05)
    Notes: at each nitrite concentration, different letters mean significant differece among different tissues (P<0.05)

    Table 1.  1-hour recovery rate (1HRR) of tissues in different groups during itrite recovery period %

  • 胁迫各组水体中亚硝态氮含量高于0 mmol/L组,且8.0 mmol/L组在6和12 h时达到显著水平(P<0.05)(图5)。

    Figure 5.  Nitrite content of water sample in different groups during nitrite recovery period

3.   讨论
  • 本实验发现,亚硝态氮胁迫能够引起对虾的死亡,并随着胁迫浓度的升高而升高,表明亚硝态氮的毒性影响具有剂量依赖效应,这与黄翔鹄等[17]和Seneriches-Abiera等[18]的研究结果一致。只是在不同的实验中,由于环境因子和实验动物的差异,半数致死的剂量会有较大差异,如研究发现,盐度[19]、pH[9]、溶解氧[20]等环境因素,以及实验动物规格、发育阶段等自身因素都会使实验动物在亚硝态氮胁迫下表现出不同的耐受能力[10, 18, 21]

  • 胁迫6 h内亚硝态氮在对虾各组织中大量积累,表明胁迫环境下亚硝态氮在对虾体内的蓄积速率较快。Hong等[22]研究发现,暴露在高浓度亚硝态氮环境下3 h,中华绒螯蟹(Eriocheir sinensis)血淋巴中亚硝态氮的积累几乎达到蓄积峰值。此外,各部位的积累量与胁迫浓度成正相关关系,说明亚硝态氮在对虾体内的积累符合剂量依赖效应,这与重金属的蓄积表现一致[23]

    亚硝态氮在不同组织内的积累程度呈现显著差异,鳃和血淋巴内积累的峰值最高,在肌肉中最低。由于鳃是对虾直接与外界进行水体交换的组织,而血淋巴是体内物质运输的关键通道,因此,当外界的亚硝态氮进入后,鳃和血淋巴成为其蓄积的主要部位。当然,由于血淋巴也直接连接着其他组织,亚硝态氮也会随之转移[24],本实验中肝胰腺和肠道内也均发现了亚硝态氮的大量积累。亚硝态氮在肌肉中积累量较低,可能与其内部结构复杂导致渗透速率较慢有关,具体原因还需进一步研究。综上表明,胁迫12 h后,亚硝态氮含量在鳃和血淋巴中有所下降,而在其他组织中持续升高,从侧面印证亚硝态氮在对虾体内转移路径可能为鳃吸收—血淋巴转运—组织存储。

  • 水生动物对环境因子的变化较为敏感,通过调节能量代谢以适应外部环境的变化,在正常的生理条件下机体能量调控有序,而一旦稳定被打破,会直接体现在与调控相关的酶活性发生改变。Na+-K+-ATP酶是一种广泛存在于真核动物中的膜蛋白酶,在对虾渗透压调节和ATP分解供能方面起重要作用[25-26]。Huong等[27]发现凡纳滨对虾在低盐度胁迫时会显著提高Na+-K+-ATP酶活性,Pinto等[28]将马氏沼虾(Macrobrachium amazonicum)暴露在氨氮环境72 h发现Na+-K+-ATP酶活性升高。本研究中,亚硝态氮胁迫前24 h,随着胁迫浓度的提高,对虾肝胰腺和肌肉中Na+-K+-ATP酶活性与对照组相比显著升高,表明对虾在应激后会加快ATP的分解,促进体内外渗透压维持平衡。然而,可能由于8 mmol/L的胁迫浓度过高,肌肉中Na+-K+-ATP酶活性低于其他各组,并且肝胰腺中该酶活性在胁迫24 h之后也维持在较低水平。由此可见,亚硝态氮诱导的Na+-K+-ATP酶活性的升高不仅在组织上具有差异性,而且对胁迫浓度的响应也不一致。

    AMPK能够促进体内能源物质的分解,维持应激下的能量平衡,作为真核生物进化过程中新陈代谢和应激传感器,已成为近年来的研究热点[29-30]。大量研究表明,环境因子如温度[31]、盐度[32]、氨氮[33]的胁迫都会使虾蟹类动物AMPK的调节失衡。本研究也发现,亚硝态氮会使凡纳滨对虾肝胰腺中AMPK活性显著升高,并且会随着胁迫浓度的升高而提高,而在肌肉中,这种差异性消失。以上结果提示,胁迫期间,ATP被大量使用,从而诱导肝胰腺中的AMPK活性提高,持续向机体发送需能信号。此外,Xu等[32]报道凡纳滨对虾在急性低盐度胁迫6 h后,肝胰腺中AMPK 2个亚基mRNA表达也出现显著上调。关于亚硝态氮胁迫对对虾肌肉中AMPK活性的影响还需要进一步的探究。

  • 环境中亚硝态氮水平能够影响水生动物体内亚硝态氮含量,将淡水螯虾(Pacifastacus leniusculus)和(Astacus astacus)放入无亚硝态氮的水中几天后,亚硝态氮能够从体内完全清除 [34-35]。对鲤(Cyprinus carpio)的恢复实验中也发现,存活的鱼中大部分亚硝态氮的诱导作用是可逆的[36]。本实验得到了相似的结果,即在恢复期1 h内,胁迫组对虾各组织中亚硝态氮含量明显降低,12 h基本可恢复至对照组水平。推测可能是体内外浓度差较大,亚硝态氮由高浓度向低浓度迅速转移,排出体外。此外,以上结果提示,换水的方式能够在短时间内有效解除对虾体内亚硝态氮超标的问题。鳃中亚硝态氮的恢复时间和恢复效率都要优于肌肉,推测可能与鳃直接和外部环境相连,利用亚硝态氮迅速排出有关。而肌肉作为亚硝态氮的蓄积部位,其复杂的构造不易于亚硝态氮的彻底排出。除鳃外,肠道也是亚硝态氮进入体内的主要累积部位,也有研究发现进入鱼体2/3的亚硝态氮是通过肠上皮吸收的[37],鳃与肠道的恢复时间表明,体内亚硝态氮的转移过程在6 h内即可完成。此外,环境水体中亚硝态氮含量在恢复期间明显升高也证实了体内积累的亚硝态氮排至体外的猜测。

    综上,亚硝态氮胁迫条件下,亚硝态氮在凡纳滨对虾各组织内快速积累,鳃和血淋巴是积累的主要部位。亚硝态氮的积累引起组织分解供能进程加快,诱导肝胰腺中AMPK活性升高从而向机体发送需能信号。胁迫环境解除后,对虾组织内亚硝态氮被迅速排除体外以减轻毒性影响,其中鳃和肝胰腺的恢复效果最好。

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