Potential aquatic environmental risks of trifloxystrobin: Enhancement of virus susceptibility in zebrafish through initiation of autophagy
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摘要: 水生生态系统中存在的长期污染会对水生环境以及水生生物产生多种不利影响，其中包括提高水生生物对病原体的敏感性。肟菌酯（TFS）是一种在亚洲广泛用于防治大豆锈病的甲氧基丙烯酸酯类农药。然而，在长期使用的过程中它有可能进入并污染水生生态系统导致鱼类对病毒抵抗力降低。为了研究TFS潜在的水环境风险，我们检测了长期暴露于TFS的宿主在鲤春病毒血症病毒（spring viraemia of carp virus，SVCV）感染下抗病能力的变化。实验结果表明，尽管环境浓度25 μg/L暴露下的TFS对细胞和斑马鱼没有明显毒性，但SVCV增殖却随着暴露时间的增长显著增加，最高病毒载量甚至相对于对照组呈现100倍以上的增加。先前的研究表明，鱼类出现自噬将有利于SVCV在机体内增殖。而我们则发现，长时间TFS暴露下的EPC细胞和斑马鱼出现自噬体增加、LC3蛋白转化、Beclin-1累积、P62蛋白降解及mTOR表达和磷酸化水平下降，这些说明TFS通过诱导宿主自噬提高宿主的病毒易感性。综上所述，该研究提出了TFS对非靶标水生生物致毒机制的新见解，提醒我们应关注甲氧基类农药在水环境中蓄积可能会诱发鱼类病毒病暴发，从而危及生态环境的安全。Abstract: Chronic pollution in aquatic ecosystems can lead to many adverse effects, including a greater susceptibility to pathogens among resident biota. Trifloxystrobin (TFS) is a strobilurin fungicide widely used in Asia to control soybean rust. However, it has the potential to enter aquatic ecosystems, where it may impair fish resistance to viral infections. To explore the potential environmental risks of TFS, we characterized the antiviral capacities of fish chronically exposed to TFS and subsequently infected with spring viraemia of carp virus (SVCV). Although TFS exhibited no significant cytotoxicity at the tested environmental concentrations during viral challenge, SVCV replication increased significantly in a time-dependent manner within epithelioma papulosum cyprini (EPC) cells and zebrafish exposed to 25 μg/L TFS. Results showed that the highest viral load was more than 100-fold that of the controls. Intracellular biochemical assays indicated that autophagy was induced by TFS, and associated changes included an increase in autophagosomes, conversion of LC3-II, accumulation of Beclin-1, and degradation of P62 in EPC cells and zebrafish. In addition, TFS markedly decreased the expression and phosphorylation of mTOR, indicating that activation of TFS may be associated with the mTOR-mediated autophagy pathway. This study provides new insights into the mechanism of the immunosuppressive effects of TFS on non-target aquatic hosts and suggests that the existence of TFS in aquatic environments may contribute to outbreaks of viral diseases.
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Key words:
- Trifloxystrobin /
- Autophagy /
- SVCV /
- Chronic toxicity /
- Susceptibility
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Figure 1. Cytotoxicity of TFS in EPC cells
Cytotoxicity data for both TFS and DMSO are shown. No TFS cytotoxicity was detected up to 125 μg/L over 4 d of exposure. Each value represents mean±SD, normalized to values for no treatment. Statistical significance for each study was determined between ControlDMSO and TFS treatments by ANOVA with Tukey’s post hoc tests. **: P<0.01; *: P<0.05. n=4.
Figure 2. Antiviral effects of TFS in vitro
A: Expression of SVCV nucleoprotein (N) increased significantly in TFS-treated groups. B: TFS had no effect on SVCV infection in vitro by altering virus binding to host cells. TFS at 25 μg/L was preincubated with 1×103 TCID50/mL SVCV for 15-60 min. C–F: SVCV replication was detected in TFS pre-exposed cells at 1, 3, 5, and 7 d. EPC cells were pre-exposed to 2.5 μg/L and 25 μg/L TFS for up to 7 d, followed by SVCV infection at each time point. SVCV loads were determined via qRT-PCR analysis of N gene expression. Each value represents mean±SD normalized to values for no treatment. P-value for each study was determined by ANOVA with Tukey’s post hoc tests. **: P<0.01; *: P<0.05; ns: No significance. n=4 for A–B, and n=8 for C–F.
Figure 3. Ultrastructural features of TFS-exposed cells observed via transmission electron microscopy (TEM)
Red arrows point to autophagosomes.
Figure 4. Expression of LC3B regulated by TFS in EPC cells
A: Western blot analyses of LC3B and LC3I/LC3II in EPC cells. B–E: Immunocytochemical results showing intracellular location and number of LC3B at 1, 3, 5, and 7 d, respectively.
Figure 5. Total amounts of Beclin-1, P62, and mTOR, and phosphorylation level of pmTOR in EPC cells, as analyzed by western blotting
Grayscale values were analyzed by Image J. Data are mean±SD of one representative experiment performed in triplicate. P-value for each study was determined by ANOVA with Tukey’s post hoc tests. **: P<0.01; *: P<0.05.
Figure 6. SVCV replication in TFS pre-exposed zebrafish at 1–5 d (A), 7 d (B), and 14 d (C)
Each value represents mean±SD, normalized to values for no treatment. P-value for each study was determined by ANOVA with Tukey’s post hoc tests. **: P<0.01; *: P<0.05. n=12 fish in each group.
Figure 7. Alteration in autophagy-related gene and protein expression levels in TFS-exposed zebrafish
A: Expression of autophagy-related genes was changed by TFS exposure in zebrafish. Expression levels of four autophagy-related mRNA genes, i.e., gabarap, atg5, wipi5, and ambra1, were analyzed by qRT-PCR. B: Total amount of autophagy-related proteins in zebrafish were analyzed by western blotting. Grayscale values were analyzed by Image J. Experiments were performed in triplicate, and each value represents mean±SD. P-value for each study was determined by ANOVA with Tukey’s post hoc tests. **: P<0.01; *: P<0.05. n=12 fish in each group.
Table 1. Sequences of primer pairs used for analysis of gene expression by qRT-PCR
Gene Primer sequence (from 5' to 3') β-actin (EPC cells) Forward GCTATGTGGCTCTTGACTTCGA Reverse CCGTCAGGCAGCTCATAGCT SVCV nucleoprotein (N) Forward AACAGCGCGTCTTACATGC Reverse CTAAGGCGTAAGCCATCAGC ambra1 (zebrafish) Forward TCTTTCGAGAAATGGCACCT Reverse CTCTCTGCGTTAGGGACAGG wipi1 (zebrafish) Forward GTGAGAGGGTAGAGAACAG Reverse GTAACAACGACCCAACATC atg5 (zebrafish) Forward AGAGAGGCAGAACCCTACTATC Reverse CCTCGTGTTCAAACCACATTTC gabarap (zebrafish) Forward GTCTGACCTCACAGTTGGGC Reverse TCCTGGTAGAGCAGTCCCAT 18S (zebrafish) Forward ACCACCCACAGAATCGAGAAA Reverse GCCTGCGGCTTAATTTGACT 
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