Molecular mechanisms of tolerance of Daphnia to microcystins

人为的湖泊富营养化导致蓝藻大量繁殖的频率增加，与此同时，浮游植物的主要食草动物水蚤的数量也减少了。水蚤丰度的减少是食物网操作经常只导致水质短期改善的主要原因。最常报道的蓝藻华毒素是微囊藻毒素，它对哺乳动物有肝毒性，并增加水蚤的死亡率。虽然经常有研究表明水蚤与蓝藻共存，对有毒蓝藻的耐受性增加，但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的遗传基础。将一个产生微囊藻毒素的菌株和该菌株的突变体（缺乏微囊藻毒素）喂给一个耐微囊藻毒素和一个敏感微囊藻毒素的大水蚤克隆。基于RNASeq的转录组分析将用于鉴定差异表达的基因，这些基因要么在两个水蚤克隆中组成性地表达不同，要么在摄入微囊藻毒素时表达不同。数据的生物信息学分析和代谢图谱将用于鉴定介导对微囊藻毒素耐受性的关键酶和基因。选择的差异表达基因的表达将在一个独立的实验中使用qPCR进行验证。通过研究饮食中微囊藻毒素浓度对基因表达的影响，我们将获得微囊藻毒素耐受基因介导的进一步证据。这将通过给水蚤喂食含有微囊藻毒素的脂质体来实现。了解其分子机制对于理解水蚤对微囊藻毒素耐受的进化是必不可少的，并且对有毒蓝藻水华湖泊的管理具有潜在的关键意义。

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.