Laboratório de Bioquímica de Resposta Ao Estresse, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal Do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia Em Entomologia Molecular (INCT-EM), Rio de Janeiro, RJ, Brazil.
Laboratório de Bioquímica de Resposta Ao Estresse, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal Do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia Em Entomologia Molecular (INCT-EM), Rio de Janeiro, RJ, Brazil.
Anal Biochem. 2019 Jul 1;576:33-41. doi: 10.1016/j.ab.2019.04.005. Epub 2019 Apr 8.
Aedes aegypti is the most important and widespread vector of arboviruses, including dengue and zika. Insect dispersal through the flight activity is a key parameter that determines vector competence, and is energetically driven by oxidative phosphorylation in flight muscle mitochondria. Analysis of mitochondrial function is central for a better understanding of cellular metabolism, and is mostly studied using isolated organelles. However, this approach has several challenges and methods for assessment of mitochondrial function in chemically-permeabilized tissues were designed. Here, we described a reliable protocol to assess mitochondrial physiology using mechanically permeabilized flight muscle of single A. aegypti mosquitoes in combination with high-resolution respirometry. By avoiding the use of detergents, high respiratory rates were obtained indicating that substrate access to mitochondria was not limited. This was confirmed by using selective inhibitors for specific mitochondrial substrates. Additionally, mitochondria revealed highly coupled, as ATP synthase or adenine nucleotide translocator inhibition strongly impacted respiration. Finally, we determined that pyruvate and proline induced the highest respiratory rates compared to other substrates tested. This method allows the assessment of mitochondrial physiology in mosquito flight muscle at individual level, and can be used for the identification of novel targets aiming rational insect vector control.
埃及伊蚊是最重要和分布最广泛的虫媒病毒载体，包括登革热和寨卡病毒。昆虫通过飞行活动扩散是决定媒介能力的一个关键参数，由飞行肌肉线粒体中的氧化磷酸化提供能量。分析线粒体功能对于更好地了解细胞代谢至关重要，并且主要使用分离的细胞器进行研究。然而，这种方法存在一些挑战，因此设计了用于评估化学渗透组织中线粒体功能的方法。在这里，我们描述了一种使用机械渗透的单个埃及伊蚊飞行肌来评估线粒体生理学的可靠方案，并结合高分辨率呼吸测定法。通过避免使用去污剂，可以获得高呼吸速率，表明线粒体对底物的进入不受限制。这通过使用特定线粒体底物的选择性抑制剂得到了证实。此外，线粒体显示出高度偶联，因为 ATP 合酶或腺嘌呤核苷酸转运蛋白的抑制强烈影响呼吸。最后，与测试的其他底物相比，我们确定丙酮酸和脯氨酸诱导的呼吸速率最高。该方法允许在个体水平上评估蚊子飞行肌中的线粒体生理学，并且可以用于鉴定新的目标，旨在实现合理的昆虫媒介控制。
