The sterile insect technique (SIT) has been successfully used in many pest management programs worldwide. Some SIT programs release both sexes due to the lack of genetic sexing strains or efficient sex separation methods but sterile females are ineffective control agents. Transgenic sexing strains (TSS) using the tetracycline-off control system have been developed in a variety of insect pests, from which females die by either of two commonly used lethal effectors: overexpression of the transcription factor tetracycline transactivator (tTA) or ectopic expression of a proapoptotic gene, such as head involution defective (hid). The lethality from tTA overexpression is thought to be due to “transcriptional squelching”, while hid causes lethality by induction of apoptosis. This study aims to create and characterize a TSS of Lucilia cuprina, which is a major pest of sheep, by combining both lethal effectors in a single transgenic strain. Here a stable TSS of L. cuprina (DH6) that carries two lethal effectors was successfully generated, by crossing FL3#2 which carries a female-specific tTA overexpression cassette, with EF1#12 which carries a tTA-regulated LshidAla2 cassette. Females with one copy of the FL3#2 transgene are viable but up to 99.8% of homozygous females die at the pupal stage when raised on diet that lacks tetracycline. Additionally, the female lethality of FL3#2 was partially repressed by supplying tetracycline to the parental generation. With an additional LshidAla2 effector, the female lethality of DH6 is 100% dominant and cannot be repressed by maternal tetracycline. DH6 females die at the late-larval stage. Several fitness parameters important for mass rearing such as hatching rate, adult emergence and sex ratio were comparable to those of the wild type strain. Compared to the parental FL3#2 strain, the DH6 strain shows stronger female lethality and lethality occurs at an earlier stage of development. The combination of two tTA-dependent lethal effectors could improve strain stability under mass rearing and could reduce the risk of resistance in the field if fertile males are released. Our approach could be easily adapted for other pest species for an efficient, safe and sustainable genetic control program.

中文翻译：

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使用两个致死效应器构建用于遗传控制澳大利亚绵羊吹蝇 Lucilia cuprina 的转基因性别鉴定品系

昆虫不育技术 (SIT) 已成功用于全球许多害虫管理计划。由于缺乏遗传性别鉴定菌株或有效的性别分离方法，一些 SIT 计划释放两性，但不育雌性是无效的控制剂。已经在多种害虫中开发了使用四环素关闭控制系统的转基因性鉴定菌株 (TSS)，雌性会被两种常用的致死效应物之一杀死：转录因子四环素反式激活因子 (tTA) 的过度表达或异位表达一个促凋亡基因，如头部退化缺陷（hid）。tTA 过度表达的致死性被认为是由于“转录抑制”，而 hid 通过诱导细胞凋亡而导致致死性。本研究旨在创建和表征 Lucilia cuprina 的 TSS，这是绵羊的主要害虫，通过将两种致死效应物结合在一个转基因品系中。在这里，通过将携带女性特异性 tTA 过表达盒的 FL3#2 与携带 tTA 调节的 LshidAla2 盒的 EF1#12 杂交，成功生成了携带两个致死效应物的 L.cuprina (DH6) 的稳定 TSS。具有 FL3#2 转基因拷贝的雌性是有活力的，但在缺乏四环素的饮食中饲养时，高达 99.8% 的纯合雌性在蛹期死亡。此外，通过向亲代提供四环素，FL3#2 的雌性致死率被部分抑制。使用额外的 LshidAla2 效应器，DH6 的雌性致死率是 100% 显性的，并且不能被母体四环素抑制。DH6 雌性在幼虫后期死亡。孵化率、成虫羽化和性别比等对大规模饲养很重要的几个健康参数与野生型菌株的参数相当。与亲本 FL3#2 菌株相比，DH6 菌株显示出更强的雌性致死率，并且致死率发生在发育的早期阶段。两个依赖于 tTA 的致死效应物的组合可以提高大规模饲养下的应变稳定性，并且如果释放可育雄性，可以降低田间抗性的风险。我们的方法可以很容易地适用于其他害虫物种，以实现高效、安全和可持续的基因控制计划。两个依赖于 tTA 的致死效应物的组合可以提高大规模饲养下的应变稳定性，并且如果释放可育雄性，可以降低田间抗性的风险。我们的方法可以很容易地适用于其他害虫物种，以实现高效、安全和可持续的基因控制计划。两个依赖于 tTA 的致死效应物的组合可以提高大规模饲养下的应变稳定性，并且如果释放可育雄性，可以降低田间抗性的风险。我们的方法可以很容易地适用于其他害虫物种，以实现高效、安全和可持续的基因控制计划。