Environmental RNA as a Noninvasive Tool for Assessing Toxic Effects in Fish: A Proof-of-concept Study Using Japanese Medaka Exposed to Pyrene

Although environmental RNA (eRNA) is emerging as a noninvasive tool to assess the health status of aquatic macroorganisms, the potential of eRNA in assessing chemical hazards remain largely untested. In this study, we investigated the ability of eRNA to detect changes in gene expression in Japanese medaka fish (Oryzias latipes) in response to sublethal pyrene exposure, as a model toxic chemical. We performed standardized acute toxicity tests and collected eRNA from tank water and RNA from fish tissue after 96 h of exposure. Our results showed that over 1000 genes were detected in eRNA and the sequenced read counts of these genes correlated with those in fish tissue (r = 0.50). Moreover, eRNA detected 86 differentially expressed genes in response to pyrene, some of which were shared by fish RNA, including the suppression of collagen fiber genes. These results suggest that eRNA has the potential to detect changes in gene expression in fish in response to environmental stressors without the need for sacrificing or causing pain to fish. However, we also found that the majority of sequenced reads of eRNA (>99%) were not mapped to the reference medaka genome and they originated from bacteria and fungi, resulting in low sequencing depth. In addition, eRNA, in particular nuclear genes, was highly degraded with a median transcript integrity number (TIN) of <20. These limitations highlight the need for future studies to improve the analytical methods of eRNA application.尽管环境RNA（environmental RNA, eRNA）作为一种无创评估水生大型生物健康状态的新兴工具逐渐得到应用，但其在化学危害评估方面的潜力仍未得到充分验证。本研究以模式有毒化学物质芘（pyrene）为受试物，以日本青鳉（Oryzias latipes）为研究对象，探究了eRNA检测其在亚致死剂量芘暴露下基因表达变化的能力。本研究开展了标准化急性毒性试验，并在暴露96小时后从养殖水体中采集eRNA，同时从鱼体组织中提取总RNA。研究结果显示，eRNA中可检测到超过1000个基因，且这些基因的测序读段计数与鱼体组织中的对应基因呈现显著相关性（r=0.50）。此外，eRNA可检测到86个响应芘暴露的差异表达基因，其中部分基因与鱼体组织RNA检测到的差异表达基因重合，包括胶原纤维相关基因的表达抑制。上述结果表明，eRNA有望在无需处死或给鱼类造成痛苦的前提下，检测鱼类响应环境胁迫的基因表达变化。但本研究同时发现，eRNA的大部分测序读段（>99%）无法比对至青鳉参考基因组，其来源多为细菌和真菌，导致测序深度偏低。此外，eRNA（尤其是核基因来源的转录本）降解程度极高，其转录本完整性指数（transcript integrity number, TIN）的中位数低于20。这些局限性凸显了未来研究中亟需优化eRNA应用相关分析方法的必要性。