Departamento de Agronomia-Entomologia, Universidade Federal Rural de Pernambuco - UFRPE, Recife, Brazil.
Universidade Católica de Brasília - UCB, Brasília, Brazil.
Pestic Biochem Physiol. 2022 Aug;186:105166. doi: 10.1016/j.pestbp.2022.105166. Epub 2022 Jul 3.
The efficiency of RNAi technology in insects varies considerably, particularly in lepidopterans. An important limiting factor of RNAi-mediated gene silencing is the degradation of dsRNA by insect nucleases before cellular uptake. To date, few studies have reported effective gene knockdown in the sugarcane borer Diatraea saccharalis. However, yielding contradictory results when using oral delivery. Further, the RNAi efficiency in D. saccharalis and presumed activity of gut nucleases remain poorly understood. Therefore, we investigated whether gene silencing was feasible via dsRNA feeding in D. saccharalis. Two different genes were tested, juvenile hormone esterase (DsJHE) and chitin synthase 1 (DsCHS1). Discrete knockdown was verified only for DsCHS1 with high dsRNA dosages and long exposure times. Neither mortality nor abnormal phenotypes were observed after treatment with any tested dsRNA. It was also verified that dsRNAs were quickly degraded when incubated with gut juice. Furthermore, we identified four possible nucleases that could reduce the knockdown efficiency in D. saccharalis. Three of them had the endonuclease_NS domain (DsNucleases), and one had the PIN domain (DsREase), with REase-like genes being scarcely represented in databanks. We further remark that DsNuclease1 and DsREase are highly expressed in the larval gut, and DsREase was upregulated as insects were fed with artificial diet (without dsRNA), and also when injected with dsRNA. Conversely, no nuclease was triggered when insects were fed with a sucrose droplet containing dsRNA. Thus, our findings suggest that nuclease activity within the gut is one of the possible reasons for the inefficiency of RNAi in D. saccharalis. Our data may shed light on the challenges to overcome when introducing RNAi as a strategy for controlling lepidopteran pests.
RNAi 技术在昆虫中的效率差异很大，尤其是在鳞翅目昆虫中。RNAi 介导的基因沉默的一个重要限制因素是 dsRNA 在细胞摄取之前被昆虫核酶降解。迄今为止，很少有研究报道在甘蔗螟 Diatraea saccharalis 中有效基因敲低。然而，口服递送时会产生矛盾的结果。此外，D. saccharalis 中的 RNAi 效率和推测的肠道核酶活性仍知之甚少。因此，我们研究了是否可以通过 D. saccharalis 的 dsRNA 喂养实现基因沉默。测试了两种不同的基因，即保幼激素酯酶（DsJHE）和几丁质合酶 1（DsCHS1）。只有在用高剂量的 dsRNA 和长时间暴露时，才观察到 DsCHS1 的明显敲低。用任何测试的 dsRNA 处理后，既没有观察到死亡率也没有观察到异常表型。还验证了当与肠液孵育时，dsRNA 很快被降解。此外，我们鉴定了四种可能的核酶，它们可以降低 D. saccharalis 的敲低效率。其中三种具有内切酶_NS 结构域（DsNucleases），一种具有 PIN 结构域（DsREase），而数据库中很少有 REase 样基因。我们进一步指出，DsNuclease1 和 DsREase 在幼虫肠道中高度表达，当昆虫以人工饲料（不含 dsRNA）喂养时，以及当注射 dsRNA 时，DsREase 被上调。相反，当昆虫以含有 dsRNA 的蔗糖液滴喂养时，没有触发任何核酶。因此，我们的研究结果表明，肠道内的核酶活性是 RNAi 在 D. saccharalis 中效率低下的可能原因之一。我们的数据可能为克服将 RNAi 作为控制鳞翅目害虫的策略所面临的挑战提供了一些线索。
