Local adaptation of Daphnia to protease inhibitors in cyanobacteria

湖泊的人为富营养化导致蓝藻水华的频率增加，这伴随着浮游植物的主要食草动物--水蚤的数量减少。这种水蚤丰度的减少是通过食物网操作只能在短期内改善水质的主要原因。尽管与蓝藻共存的水蚤表现出对蓝藻更强的耐受性，但这种耐受性的遗传基础是完全未知的。最常见的蓝藻水华抑制物是蛋白酶抑制物(PI)，它已被证明能特异性地抑制水蚤的消化酶。在以前的工作中，我们已经证明了与蓝藻共存的大型水蚤种群对这些蓝藻PI是局部适应的：动物本身及其肠道蛋白水解酶比没有蓝藻的种群的动物更能耐受PI。对适应群体中消化酶基因的单倍型分析表明，这些基因座一直处于定点选择之下。这表明，具有这些选定位点的蛋白水解酶等位基因编码具有更高的抑制物耐受性的蛋白水解酶。然而，这些普遍存在的消化酶非同义突变导致了对PI更具耐受性的蛋白水解酶，这一点仍有待证明。此外，蛋白水解酶基因表达的增加可能有助于适应种群中耐受性的增强。在这里，我们将测试蛋白酶表达的可塑性和消化酶中氨基酸序列的差异是否有助于在适应群体中提高对饮食中蛋白酶抑制剂的耐受性。D.从适应群体和非适应群体建立的巨型克隆将比较消化酶基因表达对蓝藻蛋白酶抑制剂的响应变化。在适应种群中被选择的蛋白酶位置的适应价值将通过所选等位基因的异源表达和所产生的蛋白质对蛋白酶抑制剂的耐受性的酶动力学来评估。这些数据将用于优化模型，以预测水蚤消化酶的三维结构及其对PI的耐受性。这些三维结构将被用来预测种群水平上对PI的耐受性。总体而言，该项目的目的是首次了解水蚤种群对蓝藻抑制剂的进化适应。这一发现可能有助于理解昆虫对寄主植物毒素的抗性进化。

Man-made eutrophication of lakes has caused increasing frequencies of cyanobacterial blooms, which goes along with a reduction in abundance of Daphnia, the major herbivore of phytoplankton. This reduced abundance of Daphnia is a major cause for only short term improvement of water quality by food web manipulations. Although it has repeatedly been shown that Daphnia that coexist with cyanobacteria show increased tolerance to cyanobacteria, the genetic basis of this tolerance is entirely unknown. Among the most frequently reported inhibitors of cyanobacterial blooms are protease inhibitors (PIs), which have been demonstrated to specifically inhibit digestive proteases in Daphnia. In previous work, we have shown that a population of Daphnia magna that coexists with cyanobacteria is locally adapted to these cyanobacterial PIs: The animals themselves and their gut proteases are more tolerant to the PIs than animals from a population that exists without cyanobacteria. Haplotype analyses of the genes of the digestive proteases in the adapted population revealed that these loci have been under site-specific selection. This suggests that protease alleles with these selected sites are coding for proteases with increased inhibitor tolerance. However, it remains to be demonstrated that these prevalent non-synonymous mutations in digestive proteases have led to proteases that are more tolerant to PIs. Furthermore, an increased expression of protease genes might contribute to the enhanced tolerance in the adapted population. Here we are going to test whether or not plasticity in protease expression and differences in amino acid sequence in the digestive proteases contribute to the enhanced tolerance to dietary protease inhibitors in the adapted population.D. magna clones established from adapted and non-adapted populations will be compared for changes in gene expression of the digestive proteases in response to cyanobacterial protease inhibitors. The adaptive value of protease sites that have been under selection in the adapted population will be assessed by heterologous expression of chosen alleles and by enzyme-kinetics of the resulting proteins with respect to their tolerance to protease inhibitors. These data will be used for optimizing models for the prediction of 3D-structures of the digestive proteases and their tolerance to PIs in Daphnia. These 3D-structures will be used to predict tolerance to PIs at the population level of D. magna. Overall, the project aims at for the first time understanding the evolutionary adaptation of populations of Daphnia to cyanobacterial inhibitors. The findings might be relevant for understanding the evolution of resistance against host plant toxins in insects.