Male and female Fundulus exposed to 575ppb arsenic, and their parentally exposed offspring. Fundulus heteroclitus

The present study investigated arsenic’s effects on mummichogs (Fundulus heteroclitus), while also examining what role that gender or age of exposure might play. Adult male and female mummichogs were exposed to 172ppb, 575ppb, or 1,720ppb arsenic as sodium arsenite for 10 days immediately prior to spawning. No differences were noted in the number or viability of eggs between the groups, but there was a significant increase in deformities in the 1,720ppb arsenic exposure group. Total RNA from adult livers or 6-week-old juveniles was used to probe custom macroarrays for changes in gene expression. In females, 3% of the genes were commonly differentially expressed in the 172 and 575ppb exposure groups. In the males, between 1.1-3% of the differentially expressed genes were in common between the exposure groups. Several genes, including apolipoprotein, serum amyloid precursor, lysozyme, and tributyltin-binding protein, were commonly expressed in either a dose-responsive or dose-specific, but across genders, manner. These patterns of regulation were confirmed by QPCR. These findings will provide us with a better understanding of the effects of dose, gender, and exposure age on the response to arsenic. Overall design: Adult mummichogs (Fundulus heteroclitus) were exposed to either 0 or 172ppb sodium arsenite for 10 days before the full moon. Each treatment group had 5 20-gallon plastic tanks with 2 male and 5 female fish per tank. The fish were exposed via a static renewal, with renewals every 48 hours. Mummichogs were housed at 25C in a 14/10 light/dark cycle at 18% salinity. On the 3 days surrounding the full moon, spawning substrates were provided and eggs collected. Adult males and females: After the last day of egg collection, the adults were euthanized. The livers were removed from the males and females, placed in TRI-Reagent® for RNA isolation (Sigma Chemical Co, St. Louis, MO), and then stored at -80C. Juveniles: Eggs were transferred to clean water and allowed to hatch. The juveniles were grown out in clean water for 6 weeks and then were euthanized. Whole juveniles (7-10 per exposure per tank) were placed in TRI-Reagent® for RNA isolation (Sigma Chemical Co, St. Louis, MO), and then stored at -80C.

本研究探究了砷对底鳉（*Fundulus heteroclitus*）的影响，同时分析暴露性别与暴露年龄在砷暴露响应中可能发挥的调控作用。实验选取成年雌雄底鳉，以亚砷酸钠形式暴露于浓度为172ppb、575ppb或1720ppb的砷环境中，于产卵前连续暴露10天。各组间的卵数量与卵存活率均无显著差异，但1720ppb砷暴露组的胚胎畸形率显著升高。分别提取成年个体肝脏及6周龄幼体的总RNA，利用定制化宏阵列检测基因表达变化。在雌性个体中，172ppb与575ppb暴露组共有3%的基因呈现差异表达；在雄性个体中，各暴露组间共有1.1%~3%的差异表达基因重合。载脂蛋白、血清淀粉样前体蛋白、溶菌酶以及三丁基锡结合蛋白等多个基因，均呈现出跨性别的剂量依赖性或剂量特异性表达模式，该调控模式经定量聚合酶链式反应（QPCR）验证。本研究结果将有助于更深入理解暴露剂量、性别以及暴露年龄对砷暴露响应的影响。

实验设计概况：
成年底鳉于满月前分别暴露于0ppb（对照组）与172ppb的亚砷酸钠环境中，连续暴露10天。每个处理组设置5个20加仑塑料养殖缸，每缸放养2尾雄鱼与5尾雌鱼。采用静态更新法进行暴露处理，每48小时更换一次暴露液。养殖环境设置为25℃、14小时光照/10小时黑暗循环，盐度18‰。在满月前后3天内，投放产卵基质并收集鱼卵。

成年雌雄个体处理：
末次收卵结束后，对成年个体实施安乐死。分别提取雌雄个体的肝脏组织，置于TRI-Reagent®中用于RNA提取（Sigma Chemical Co, 美国密苏里州圣路易斯市），随后于-80℃保存。

幼体处理：
将鱼卵转移至洁净水中孵化，幼体于洁净水中饲养6周后实施安乐死。取完整幼体（每缸每个暴露组取7~10尾），置于TRI-Reagent®中用于RNA提取（Sigma Chemical Co, 美国密苏里州圣路易斯市），随后于-80℃保存。