Local adaptation of Daphnia to protease inhibitors in cyanobacteria

人为造成的湖泊富营养化导致蓝藻水华的频率增加，这沿着浮游植物的主要食草动物水蚤的丰度减少。这种减少丰富的水蚤是一个主要原因，只有短期改善水质的食物网操纵。虽然已经多次表明，水蚤与蓝细菌共存，表现出增加的耐蓝细菌，这种耐受性的遗传基础是完全未知的。其中最常报道的蓝藻水华抑制剂是蛋白酶抑制剂（PI），已被证明能特异性抑制水蚤的消化蛋白酶。在以前的工作中，我们已经表明，与蓝藻共存的大型蚤种群局部适应这些蓝藻PI：动物本身和他们的肠道蛋白酶比没有蓝藻存在的种群中的动物更耐受PI。单倍型分析的消化蛋白酶的基因在适应人口显示，这些位点已根据位点特异性选择。这表明具有这些选择位点的蛋白酶等位基因编码具有增加的抑制剂耐受性的蛋白酶。然而，仍有待证明的是，消化蛋白酶中这些普遍的非同义突变导致了对PI更耐受的蛋白酶。此外，蛋白酶基因表达的增加可能有助于适应群体中耐受性的增强。在这里，我们将测试蛋白酶表达的可塑性和消化蛋白酶中氨基酸序列的差异是否有助于适应人群对饮食蛋白酶抑制剂的耐受性增强。将比较从适应和非适应群体建立的Magna克隆对蓝藻蛋白酶抑制剂应答的消化蛋白酶基因表达的变化。在适应群体中已经选择的蛋白酶位点的适应值将通过所选等位基因的异源表达和通过所得蛋白质的酶动力学（关于它们对蛋白酶抑制剂的耐受性）来评估。这些数据将用于优化模型的预测的三维结构的消化蛋白酶和他们的耐受性，在水蚤。这些3D结构将用于预测D群体水平下PI的耐受性。magna.总体而言，该项目旨在首次了解水蚤种群对蓝藻抑制剂的进化适应。这些发现可能有助于了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.