Molecular impact of juvenile hormone agonists on neonatal Daphnia magna

Daphnia magna has been used extensively to evaluate organism- and population-level responses to pollutants in acute toxicity and reproductive toxicity tests. We have previously reported that exposure to juvenile hormone (JH) agonists results in a reduction of reproductive function and production of male offspring in a cyclic parthenogenesis, D. magna. Recent advances in molecular techniques have provided tools to better understand the responses to pollutants in aquatic organisms including D. magna. DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to JH agonists; methoprene (125, 250 and 500 ppb), fenoxycarb (0.5, 1 and 2 ppb), and epofenonane (50, 100 and 200 ppb). Exposure to these JH analogs resulted in chemical specific patterns of gene expression. The heat map analyses based on hierarchical clustering revealed a similar pattern between treatments with a high dose of methoprene and with epofenonane. In contrast, treatment with low to middle doses of methoprene resulted in similar profiles to fenoxycarb treatments. Hemoglobin and JH epoxide hydrolase genes clustered to be JH-responsive genes. These data suggest that fenoxycarb has high activity as a JH agonist, methoprene shows high toxicity and epofenonane works through a different mechanism compared with other JH analogs, agreeing with data of previously reported toxicity tests. In conclusion, D. magna DNA microarray is useful for classification of JH analogs and identification of JH-responsive genes. Juvenile hormone agonists induced gene expression in daphnids neonates was measured at 48 hours after exposure to methoprene (125, 250, 500 ppb), fenoxycarb (0.5, 1, 2 ppb), epofenonane (50, 100, 200) and DMF as a control. Three independent experiments were performed at each chemicals.

大型溞（Daphnia magna）已被广泛应用于急性毒性与生殖毒性试验，以评估污染物对生物体及种群层面的效应。我们此前曾报道，暴露于保幼激素（juvenile hormone, JH）激动剂会导致循环孤雌生殖的大型溞出现生殖功能下降，并产生雄性后代。近年来分子技术的进步为深入理解包括大型溞在内的水生生物对污染物的响应提供了研究工具。本研究采用DNA微阵列（DNA microarray）技术，分析暴露于保幼激素激动剂后新生大型溞的基因表达谱，所用试剂包括甲氧普烯（methoprene，浓度设置为125、250与500 ppb）、苯氧威（fenoxycarb，浓度设置为0.5、1与2 ppb）以及环氧虫啶（epofenonane，浓度设置为50、100与200 ppb）。暴露于上述保幼激素类似物后，可诱导产生具有化学物质特异性的基因表达模式。基于层级聚类的热图分析显示，高剂量甲氧普烯处理组与环氧虫啶处理组的基因表达模式相似；与之相反，低至中剂量的甲氧普烯处理组则与苯氧威处理组的基因表达谱高度相似。血红蛋白基因与保幼激素环氧水解酶基因被聚类为保幼激素响应基因。上述数据表明，苯氧威作为保幼激素激动剂具有较高活性，甲氧普烯则表现出较强毒性，而环氧虫啶的作用机制与其他保幼激素类似物存在差异，这与此前已发表的毒性试验结果相符。综上，大型溞DNA微阵列技术可有效应用于保幼激素类似物的分类与保幼激素响应基因的鉴定。本研究在暴露于甲氧普烯（125、250、500 ppb）、苯氧威（0.5、1、2 ppb）、环氧虫啶（50、100、200 ppb）以及作为溶剂对照的二甲基甲酰胺（DMF）后，于48小时检测了新生大型溞中保幼激素激动剂诱导的基因表达情况。每组化学处理均开展三次独立重复实验。