Molecular mechanisms of tolerance of Daphnia to microcystins

人为的湖泊富营养化导致蓝藻水华频率增加，这沿着浮游植物的主要食草动物水蚤的丰度减少。水蚤丰度的减少是食物网操作经常只会导致水质短期改善的主要原因。蓝藻水华最常见的毒素是微囊藻毒素，它对哺乳动物有肝毒性，并增加水蚤的死亡率。虽然它经常被证明，水蚤与蓝细菌共存显示出对有毒蓝细菌的耐受性增加，这种耐受性的遗传基础是完全未知的。本文将探讨水蚤对微囊藻毒素耐受性的遗传基础。将产生微囊藻毒素的菌株和该菌株的微囊藻毒素缺乏的突变体喂给大型蚤的微囊藻毒素耐受性和微囊藻毒素敏感性克隆。基于RNASeq的转录组分析将用于鉴定差异表达的基因，这些基因在两个水蚤克隆中的表达组成性不同，或者在摄入微囊藻毒素后差异表达。对数据的生物信息学分析和代谢图谱将用于确定介导微囊藻毒素耐受性的关键酶和基因。 将使用qPCR在独立实验中验证所选差异表达基因的表达。通过研究微囊藻毒素浓度对基因表达的影响，将进一步获得微囊藻毒素耐受性介导的差异表达基因的证据。这将通过向水蚤喂食载有微囊藻毒素的脂质体来实现。了解的分子机制是必不可少的理解微囊藻毒素的耐受性在水蚤的演变，是潜在的关键重要性与有毒蓝藻水华的湖泊管理。

Man-made eutrophication of lakes has caused increased frequencies of cyanobacterial blooms, which go along with a reduction in abundance of Daphnia, the major herbivore of the phytoplankton. This reduced abundance of Daphnia is a major cause for the fact that food web manipulations frequently cause only short term improvements of water quality. The most frequently reported toxins of cyanobacterial blooms are microcystins, which are hepatotoxic to mammals and increase mortality in Daphnia. Although it has frequently been shown that Daphnia coexisting with cyanobacteria show increased tolerance to toxic cyanobacteria, the genetic basis of this tolerance is entirely unknown. Here the genetic basis of tolerance to microcystins in Daphnia will be investigated. A microcystin-producing strain and a mutant of that strain, which is deficient in microcystins, will be fed to a microcystin-tolerant and a microcystin-sensitive clone of Daphnia magna. Transcriptome analysis based on RNASeq will be used to identify differentially expressed genes that either differ constitutively in expression among the two Daphnia clones or are differentially expressed upon ingestion of microcystins. Bioinformatic analysis of the data and metabolic mapping will be used to identify key enzymes and genes that mediate tolerance to microcystins. Expression of selected differentially expressed genes will be validated in an independent experiment using qPCR. Further evidences for a mediation of microcystin tolerance by selected differentially expressed genes will be gained by investigating how the concentration of dietary microcystins affect gene expression. This will be achieved by feeding liposomes that are loaded with microcystins to Daphnia. Understanding the molecular mechanisms is indispensable for the understanding of the evolution of microcystin tolerance in Daphnia and is of potentially pivotal importance for the management of lakes with toxic cyanobacterial blooms.