Neonicotinoids are neuroactive insecticides commonly detected in freshwater ecosystems. Recent studies have indicated that these compounds are markedly toxic to Chironomidae, a widespread family of ecologically important aquatic insects. However, despite their sensitivity, the pharmacological mechanisms driving neonicotinoid toxicity have yet to be characterized in these insect species. Here, we used a combination of saturation and competition binding studies to characterize neonicotinoid binding properties to nicotinic acetylcholine receptors (nAChR) in two different Chironomidae (Chironomus riparius and Chironomus dilutus) at two different life stages (larval and adult). Using radiolabeled imidacloprid ([³H]-IMI), we characterized and compared receptor density (B_max), imidacloprid binding affinity (K_D), and receptor binding affinity (K_i) to three different neonicotinoid competitors (imidacloprid, clothianidin, and thiamethoxam). We then compared receptor density and binding affinity parameters derived for Chironomidae to data previously generated for other dipterans and agricultural pests. We found that there were limited differences in neonicotinoid binding between C. riparius and C. dilutus, with both organisms demonstrating high affinities for imidacloprid (K_D = 0.22−0.87 nM) and high receptor densities (B_max = 0.92–6.53 pmol/mg). However, there were significant differences between life-stages, with larvae expressing higher densities of nicotinic acetylcholine receptors and higher imidacloprid affinities than adults. Moreover, there were compound-specific differences in receptor affinity, with larval stages displaying relative affinities (K_i) that generally correlated with acute neonicotinoid toxicity (e.g. clothianidin ≥ imidacloprid >>> thiamethoxam). Finally, compared to other dipterans and agricultural pests, Chironomidae display very high densities of high affinity nicotinic acetylcholine receptors, which likely contribute to their sensitivity. Results indicated that receptor-level differences in neonicotinoid binding may be responsible for ecotoxicological differences amongst distinct insect species, and they likely play a role in life stage-, and compound-level toxicity differences previously observed for Chironomidae. Overall, this study highlights the value of understanding the toxicological mechanisms of action of neonicotinoids in sensitive, non-target aquatic insects, to better predict adverse effects associated with unintentional neonicotinoid exposure.

新烟碱是通常在淡水生态系统中发现的神经活性杀虫剂。最近的研究表明，这些化合物对Chironomidae（一种在生态上重要的水生昆虫广泛的科）有明显的毒性。然而，尽管它们具有敏感性，但在这些昆虫物种中尚未确定驱动新烟碱类毒性的药理机制。在这里，我们使用的饱和度和竞争结合研究的组合在两个不同的摇蚊（表征新烟碱类结合特性烟碱型乙酰胆碱受体（胆碱受体）摇riparius和摇dilutus在两个不同的生命阶段（幼虫和成虫））。使用放射性标记的吡虫啉（[ ³H] -IMI），我们表征并比较了受体密度（B _max），吡虫啉结合亲和力（K _D）和受体结合亲和力（K _i）对三种不同新烟碱类竞争剂（吡虫啉，可比安定和噻虫嗪）的影响。然后，我们将衍生自手足科的受体密度和结合亲和力参数与先前为其他二足动物和农业害虫生成的数据进行了比较。我们发现，有被限制在新烟碱类的结合差异C. riparius和C. dilutus，具有两种生物体展示吡虫啉高亲和力（K _d = 0.22-0.87 1nM）和高受体密度（B_最大= 0.92-6.53 pmol / mg）。然而，生命阶段之间存在显着差异，幼虫比成人表达更高的烟碱乙酰胆碱受体密度和吡虫啉亲和力。此外，受体亲和力存在化合物特异性差异，幼虫阶段显示出相对亲和力（K _i），通常与急性新烟碱类毒性有关（例如，可比尼丁≥吡虫啉>>>噻虫嗪）。最后，与其他dip虫和农业害虫相比，,科显示出很高的高亲和力烟碱型乙酰胆碱受体密度，这可能有助于它们的敏感性。结果表明，新烟碱类化合物结合的受体水平差异可能是不同昆虫物种之间生态毒理学差异的原因，并且它们可能在以前观察到的猫科动物的生命阶段和化合物水平的毒性差异中起作用。总的来说，这项研究强调了了解新烟碱在敏感的非目标水生昆虫中的毒理作用机制的价值，以更好地预测与无意新烟碱暴露相关的不良反应。