Centers for Disease Control and Prevention (CDC), 1600 Clifton Road, NE, Atlanta, GA, 30329-4027, MS G49, USA.
Centro de Estudios en Biotecnologia, Universidad del Valle de Guatemala, 18 Avenida 11-95, 01015, Guatemala City, Guatemala.
Parasit Vectors. 2019 Apr 15;12(1):170. doi: 10.1186/s13071-019-3437-4.
Mosquito-borne diseases affect millions worldwide, with malaria alone killing over 400 thousand people per year and affecting hundreds of millions. To date, the best strategy to prevent the disease remains insecticide-based mosquito control. However, insecticide resistance as well as economic and social factors reduce the effectiveness of the current methodologies. Alternative control technologies are in development, including genetic control such as the sterile insect technique (SIT). The SIT is a pivotal tool in integrated agricultural pest management and could be used to improve malaria vector control. To apply the SIT and most other newer technologies against disease transmitting mosquitoes, it is essential that releases are composed of males with minimal female contamination. The removal of females is an essential requirement because released females can themselves contribute towards nuisance biting and disease transmission. Thus, females need to be eliminated from the cohorts prior to release. Manual separation of Anopheles gambiae pupae or adult mosquitoes based on morphology is time consuming, is not feasible on a large scale and has limited the implementation of the SIT technique. The doublesex (dsx) gene is one of the effector switches of sex determination in the process of sex differentiation in insects. Both males and females have specific splicing variants that are expressed across the different life stages. Using RNA interference (RNAi) to reduce expression of the female specific (dsxF) variant of this gene has proven to have detrimental effects to the females in other mosquito species, such as Aedes aegypti. We tested oral RNAi on dsx (AgdsxF) in An. gambiae.
We studied the expression pattern of the dsx gene in the An. gambiae G3 strain. We knocked down AgdsxF expression in larvae through oral delivery of double stranded RNA (dsRNA) produced by bacteria and observed its effects in adults.
Our results show that feeding of AgdsxF dsRNA can effectively reduce (> 66%) the mRNA of female dsx transcript and that there is a concomitant reduction in the number of female larvae that achieve adulthood. Control groups produced 52% (± 3.9% SE) of adult males and 48% (± 4.0% SE) females, while AgdsxF dsRNA treated groups had 72.1% (± 4.0% SE) males vs 27.8% females (± 3.3% SE). In addition, the female adults produce fewer progeny, 37.1% (± 8.2% SE) less than the controls. The knockdown was sex-specific and had no impact on total numbers of viable male adults, in the male dsx transcripts or male fitness parameters such as longevity or body size.
These findings indicate that RNAi could be used to improve novel mosquito control strategies that require efficient sex separation and male-only release of An. gambiae by targeting sex determination genes such as AgdsxF. The advantages of using RNAi in a controlled setting for mosquito rearing are numerous, as the dose and time of exposure are controlled, and the possibility of off-target effects and the waste of female production would be significantly reduced.
蚊媒疾病影响着全球数百万人，仅疟疾每年就导致超过 40 万人死亡，数百万人患病。迄今为止，预防疾病的最佳策略仍是基于杀虫剂的蚊虫控制。然而，杀虫剂耐药性以及经济和社会因素降低了现有方法的效果。替代控制技术正在开发中，包括遗传控制，如不育昆虫技术（SIT）。SIT 是综合农业害虫管理的重要工具，可用于改善疟疾媒介控制。为了应用 SIT 和大多数其他较新技术来防治传播疾病的蚊子，释放的蚊子必须由雄性组成，且雌性污染最小。去除雌性是必不可少的要求，因为释放的雌性本身可能会导致骚扰性叮咬和疾病传播。因此，在释放之前，必须从群体中消除雌性。基于形态学的按蚊蛹或成蚊的手动分离既耗时又费力，在大规模情况下不可行，并且限制了 SIT 技术的实施。双性（dsx）基因是昆虫性别分化过程中性别决定的效应器开关之一。雌雄两性都有特定的剪接变体，在不同的生命阶段表达。在其他蚊子物种（如埃及伊蚊）中，使用 RNA 干扰（RNAi）来降低该基因的雌性特异性（dsxF）变体的表达已被证明对雌性具有有害影响。我们在冈比亚按蚊（An. gambiae）中测试了 dsx（AgdsxF）的口服 RNAi。
我们研究了 dsx 基因在冈比亚按蚊 G3 品系中的表达模式。我们通过细菌产生的双链 RNA（dsRNA）经口传递来降低幼虫中的 AgdsxF 表达，并观察其在成虫中的影响。
我们的结果表明，AgdsxF dsRNA 的摄食可有效降低（>66%）雌性 dsx 转录物的 mRNA，并且达到成年的雌性幼虫数量相应减少。对照组产生 52%（±3.9% SE）雄性和 48%（±4.0% SE）雌性，而 AgdsxF dsRNA 处理组产生 72.1%（±4.0% SE）雄性对 27.8%（±3.3% SE）雌性。此外，雌性成虫产生的后代数量减少了 37.1%（±8.2% SE），低于对照组。这种敲低是性别的特异性的，对总存活雄性成虫数量、雄性 dsx 转录物或雄性寿命或体型等适应性参数没有影响。
这些发现表明，RNAi 可用于通过靶向 AgdsxF 等性别决定基因来改善需要有效性别分离和仅释放雄性的新型冈比亚按蚊控制策略。在控制条件下使用 RNAi 进行蚊子饲养具有许多优势，因为可以控制剂量和暴露时间，并且可以显著减少脱靶效应和雌性产生的浪费。
