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微塑料通常指直径小于5 mm的塑料颗粒[1]。据统计,塑料垃圾占海洋垃圾总量的80%~85%,并以每年480~1270万吨的速度增长[2]。研究表明,在中国沿海城市的贻贝和养殖牡蛎中均发现了微塑料的存在[3]。微塑料尺寸较小,被贝类误食后进入消化组织,导致贝类机体出现能量和脂质等代谢异常,影响贝类的生殖、免疫和神经系统[4-5]。以欧洲市售的紫贻贝(Mytilus edulis)和太平洋牡蛎(Crassostrea gigas)中发现的微塑料数量进行估算,欧洲贝类消费者每年通过饮食可摄入11000个微塑料颗粒[6]。由此可见,贝类体内携带的微塑料和人类的生活及健康密切相关。
微塑料的比表面积较大,可作为微生物(细菌、病毒)的载体[7],使微生物具有更强的耐受力。研究表明,在微塑料中检出的弧菌约77%是具有潜在致病性的病原菌,如河口弧菌(Vibiro aestuarianus)和灿烂弧菌(V. splendidus)[8]。与其他海洋生物相比,贝类拥有的开放式循环系统和特殊摄食方式(过滤水体)使贝类更容易接触和富集环境中的污染物。因此,双壳贝类被广泛用于监测持久性环境污染物(如微塑料)[9]。近江牡蛎(C. ariakensis)属于典型的双壳贝类,是我国重要的经济品种,主要生长在我国河口水域,在贝类产业中占据重要的地位。因此,本研究选用近江牡蛎作为实验生物,将其进行微塑料与哈维弧菌(V. harveyi)联合暴露,通过累计死亡率以及鳃组织中的碱性磷酸酶(alkaline phosphatase,AKP)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、过氧化氢酶(catalase,CAT)、一氧化氮合成酶(nitric oxide synthase,NOS)和乙酰胆碱酯酶(acetylcholinesterase,A-chE)的活力变化,探究聚苯乙烯微塑料对近江牡蛎免疫力和抗菌力的影响,以期为微塑料对滤食性贝类的潜在风险评估提供参考数据。
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近江牡蛎(壳长约为6.6 cm) 采自广东省惠州市牡蛎养殖区。实验前暂养7 d,盐度为20,水温为(24±1)℃。暂养期间,在牡蛎侧边开一小孔以便后期推注聚苯乙烯微塑料溶液。
聚苯乙烯微塑料购于上海麦克林生化科技有限公司,粒径为4 μm。实验所用哈维弧菌为中国水产科学研究院南海水产研究所实验室保存菌种。
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使用BHI固体培养基平板(含1.5%的NaCl,下同)对受试病原哈维弧菌菌株进行活化,培养18 h后挑取单菌落于BHI斜面培养基上,培养18 h后用灭菌PBS洗涤沉淀,重复3次,经比浊法计数,调整菌浓度为5×108 CFU/mL。
实验组设置3个聚苯乙烯微塑料浓度:0.05 mg/L、0.5 mg/L、5 mg/L,以哈维弧菌菌悬液作为稀释液;另设一个空白对照组,以生理盐水作为稀释液,每组3个重复。
选取144只健康的近江牡蛎,在每个玻璃缸(容积为120 L)中随机放入12只牡蛎。通过侧边的小孔推注聚苯乙烯微塑料—菌悬液到牡蛎体腔,推注时注意不要伤及肌肉组织,推注剂量为1 mL/只。记录牡蛎死亡率,在干露胁迫24 h和72 h后,每组随机取3只牡蛎测定其酶活力。
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对牡蛎进行解剖,取其鳃组织,用预冷的PBS缓冲液清洗,经液氮速冻后转移至−80 ℃冰箱保存。
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按重量(g)∶体积(mL)=1∶9的比例加入9倍体积的匀浆介质(生理盐水)到待测样品中,制成10%的匀浆,3500 r/min离心10 min,取上清液。用试剂盒(购于南京建成生物工程研究所)分别检测近江牡蛎的总蛋白含量(用于计算相关酶活力)及AKP、MDA、SOD、CAT、NOS、A-chE的活力,实验操作均按照试剂盒说明书进行。
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用SPSS 22.0对数据进行统计分析,数据以平均值±标准偏差(x±SD)表示。微塑料对牡蛎各指标的影响采用单因素方差(One-way ANOVA)和LSD多重比较进行统计学分析,以P<0.05作为差异显著的标志。
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微塑料胁迫24 h后,近江牡蛎CAT、SOD、A-chE和NOS的活力变化如图1所示。随着微塑料浓度的增加,CAT活力虽呈现先上升再下降后上升的趋势,但所有实验组的CAT活力仍高于对照组,SOD活力显著上升(P<0.05)。SOD-CAT 系统是抗氧化防御系统的第一道防线,能增强吞噬细胞的防御能力和机体的免疫功能[10]。SOD催化超氧阴离子歧化为分子氧和过氧化氢,CAT则进一步催化过氧化氢分解成无毒害作用的氧和水,阻止其转化为活性更高的羟自由基。Webb[11] 等发现,绿唇贻贝(Perna canaliculus)暴露在添加有三氯生(triclosan)的微塑料环境中,机体SOD活力增强,同时伴随脂质过氧化现象。SOD 在清除超氧阴离子的过程中产生的过氧化氢会调动CAT 的活力,但大量的超氧阴离子会抑制CAT 活力[12],使CAT活力增长减缓甚至下降,从而维持机体内免疫酶的动态平衡。
图 1 微塑料胁迫24 h后近江牡蛎的免疫指标活力
Figure 1. Immune index in oysters (C. ariakensis) under microplastics stress for 24 h
本研究中,随着微塑料浓度的增加,A-chE活力呈现显著降低的趋势(P<0.05)。有报道指出,截形斧蛤(Donax trunculus)在聚乙烯和聚丙烯的联合暴露下,其鳃、消化腺以及肌肉中的A-chE活力显著下降[13];在河蚬(Corbicula fluminea)[14]、地中海贻贝(M. galloprovincialis)[15]中也发现微塑料抑制A-chE活力的现象,与本实验结果类似。这表明,微塑料确实可引起A-chE活力明显下降,并通过免疫细胞胞膜上的乙酰胆碱受体调节机体的免疫应答能力。
本实验进一步分析了诱导型(inducible nitric oxide synthase,iNOS)和结构型(structural nitric oxide synthase,cNOS)一氧化氮合成酶对微塑料浓度的响应。研究发现,当微塑料浓度高于0.5 mg/L时,iNOS和cNOS受到显著影响(P<0.05),NOS的活力随微塑料浓度的上升呈现降低的趋势。NOS能合成一氧化氮,发挥巨噬细胞的功能,参与贝类免疫防御。例如,厚壳贻贝(M. coruscus)在灿烂弧菌(V. splendidus)的刺激下,NOS含量显著升高[16]。NOS需要高浓度的Ca2+/CaM催化才可合成一氧化氮,但微塑料能降低细胞内Ca2+浓度[17],导致一氧化氮合成受阻。因此,本研究中,NOS活力随微塑料浓度升高而下降,牡蛎机体的免疫能力逐渐减弱。
微塑料胁迫24 h后,牡蛎体内AKP活力和MDA含量变化如图2所示。当微塑料浓度为0.5 mg/L时,牡蛎体内AKP活力与对照组存在显著差异(P<0.05)。AKP主要参与解毒、大分子的代谢和生物合成、调节膜运输、参与转磷酸作用等多种代谢过程,是海洋无脊椎动物重要的溶酶体酶[18]。溶藻弧菌(V. alginolyticus)感染三疣梭子蟹(Portunus trituberculatus)后,AKP活力随感染时间延长而逐渐减弱[19],黑海参(Holothuria atra)暴露在聚苯乙烯环境下,AKP活力呈现先升后降的趋势[20]。而本研究中,AKP活力呈先下降后上升再下降的趋势,与上述研究结果不同。推测在微塑料浓度较低时,AKP受弧菌因素影响较大,而随着微塑料浓度的增加,微塑料对牡蛎机体的影响逐渐扩大,导致AKP活力上调。
图 2 微塑料胁迫24 h后近江牡蛎体内AKP活力和MDA含量
Figure 2. Activities of AKP and content of MDA in oysters (C. ariakensis) under microplastics stress for 24 h
随着微塑料浓度的增加,牡蛎体内MDA含量持续上升。MDA是当生物体受到污染物胁迫时,产生的脂质过氧化代谢产物,可以反映生物体氧化损伤程度。已有研究证实,太平洋牡蛎(C. gigas)暴露在聚对苯二甲酸乙二醇酯和聚乙烯中,其体内的MDA含量升高[21];在翡翠贻贝(Perna canaliculus)[11]和贻贝属(Mytilus spp.)[22]中也发现微塑料使机体出现脂质过氧化的现象。本研究中,近江牡蛎体内MDA含量随微塑料浓度升高而持续增加,表明微塑料浓度的升高可进一步损伤牡蛎的脂质。
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微塑料胁迫72 h后,CAT、SOD、A-chE和NOS的活力变化如图3所示。随着微塑料浓度的升高,CAT活力变化不明显,但所有实验组的CAT活力均高于对照组,而SOD的活力则呈现缓慢上升趋势。有研究指出,贝类CAT和SOD等抗氧化酶的表达量会随聚苯乙烯微塑料浓度的增加而增加[23]。A-chE呈下降趋势,机体通过抑制A-chE的活力来调节自身免疫力。NOS活力呈先增加后下降的趋势,表明微塑料降低了机体抵抗病原弧菌的能力。
图 3 微塑料胁迫72 h后近江牡蛎的免疫指标活力
Figure 3. Immune index in oysters (C. ariakensis) under microplastics stress for 72 h
微塑料胁迫72 h后, AKP活力和MDA含量变化如图4所示。AKP活力呈先降后升再降的趋势,但仍低于对照组,而MDA含量则呈上升的趋势。已有研究表明,微塑料影响溶酶体膜的稳定性[24],从而使AKP 活力受到限制。当微塑料持续作用于近江牡蛎时,其MDA含量不断上升,表明机体脂质损伤不断加重。
微塑料胁迫72 h后,CAT、AKP、A-chE和SOD的变化趋势与胁迫24 h的趋势大致相同,但活力显著降低(P<0.05)。近江牡蛎对短期的微塑料胁迫可以通过启动应激防御系统来降低微塑料对自身的伤害,但随着微塑料胁迫时间的延长,超出牡蛎的承受范围,机体则可能受到损伤;另外,牡蛎自身能量存储不足可能导致机体对酶的合成能力不断下降,从而影响机体正常的生理活动。
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微塑料胁迫对近江牡蛎抗菌力的影响如图5所示。随着微塑料浓度的增加,近江牡蛎的累计死亡率逐渐升高,并且结果显示,牡蛎的抗菌力与微塑料浓度呈正相关关系(R2为0.95)。其中,当微塑料浓度达到0.5 mg/L时,牡蛎的死亡率显著高于对照组(P<0.05)。
图 5 微塑料胁迫对近江牡蛎累计死亡率的影响
Figure 5. Effects of microplastic stress on cumulative mortality of oysters (C. ariakensis)
已有研究表明,在生物体内不断累积的微塑料不仅造成宿主组织损伤,还可成为条件致病菌的载体[25]。本研究分析了在微塑料胁迫下牡蛎机体对哈维弧菌的抵抗力变化情况。结果显示,随着微塑料浓度的增加,牡蛎体内的免疫酶活力下降,其死亡率升高,可见,当微塑料存在于牡蛎体内时,牡蛎机体对哈维弧菌的抵抗力下降。
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(1) 不同浓度(0.05 mg/L、0.5 mg/L、5 mg/L)的聚苯乙烯微塑料—菌悬液注入近江牡蛎壳腔内后,其体内MDA含量和SOD活力随微塑料浓度的升高而上升,但A-chE和NOS活力则随微塑料浓度的上升而显著下降,AKP活力仅在微塑料胁迫浓度为0.5 mg/L时显著上升。而CAT的活力呈先上升再下降然后再上升的趋势。结果表明,微塑料浓度的上升加剧了牡蛎的氧化应激反应。
(2)当微塑料胁迫时间由24 h延长至72 h时,CAT、AKP、A-chE和SOD的活力减弱,脂质损伤程度加剧,死亡率不断上升。以上现象表明,微塑料胁迫可导致牡蛎机体免疫力下降。
微塑料胁迫对近江牡蛎免疫及抗菌力的影响
Effects of microplastics stress on the immunity and antibacterial activity of Crassostrea ariakensis
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摘要: 为探究微塑料对近江牡蛎 (Crassostrea ariakensis)免疫及抗菌力的影响,本研究将不同浓度(0.05 mg/L、0.5 mg/L、5 mg/L)的聚苯乙烯微塑料—菌悬液注入牡蛎壳腔内,研究牡蛎在胁迫24 h和72 h后鳃组织中6种免疫指标的活力,同时记录死亡率。结果显示,微塑料胁迫24 h后,丙二醛(malondialdehyde,MDA)含量和超氧化物歧化酶(superoxide dismutase,SOD)活力随暴露浓度的升高而升高,但一氧化氮合成酶(nitric oxide synthase,NOS)和乙酰胆碱酯酶(acetylcholinesterase,A-chE)随暴露浓度升高被显著抑制,碱性磷酸酶(alkaline phosphatase,AKP)活力仅在0.5 mg/L处理下显著上升,过氧化氢酶(catalase,CAT)的活力呈现先上升再下降后上升的趋势。微塑料胁迫72 h后,CAT、AKP、A-chE和SOD的活力变化趋势与24 h的变化趋势大致相同,但活性显著减弱,牡蛎的累计死亡率与对照组相比显著上升。以上结果表明,随着微塑料浓度的增加和暴露时间的延长,牡蛎的抗菌能力和免疫能力均呈下降趋势。Abstract: Oyster Crassostrea ariakensis is one of the typical bivalve molluscs and is an important economic species in China. It mainly grows in the estuary in China and occupies an important position in the bivalve industry in Guangdong. In this study, C. ariakensis was selected as the experimental organism to explore the impact of the polystyrene microplastics on the immunity of C. ariakensis. In order to study the effects of microplastics immunity and antibacterial activity of oyster, the oysters (average shell length: 6.6 cm) were injected with different concentrations of polystyrene microplastics, respectively (0.05 mg/L, 0.5 mg/L, 5 mg/L, with sterilized saline water as diluent) by intra-shell injection (without damage to the soft tissue of oyster). The gills were sampled after being treated with microplastics for 24 h and 72 h, respectively, activities of six immune related indexes, including alkaline phosphatase (AKP), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), nitric oxide synthase (NOS), acetylcholinesterase (A-chE) and the mortality rates were recorded. The results showed that the activities of SOD and A-chE in the gill of oysters were significantly affected by microplastic concentration after 24 h (P<0.05). The activities of AKP increased first and then decreased, while SOD and MDA showed an upward trend, A-chE and NOS showed a downward trend. After 72 h of microplastic treatment, the enzyme activity was significantly lower than those of 24 h (P<0.05). With the increase of microplastic concentration, the trend of changes in enzyme activity is roughly consistent with 24 h trend. When the concentration of microplastics was above 0.5 mg/L, the activity of NOS was lower than that of the control group, and the cumulative mortality of oysters was significantly higher than that of the control group (P<0.05). The above results showed that with the increase of microplastic concentration, the oxidative stress of oysters was more intense. With the extension of exposure time, the antibacterial ability and immune ability of oysters decreased. It is expected to provide reference data for the analysis and evaluation of the potential risks of microplastics to the filter-feeding bivalves.
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Key words:
- oyster /
- microplastics /
- Vibrio harveyi /
- antioxidant enzyme /
- immune index
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