Live imaging of symbiosis: spatiotemporal infection dynamics of GFP-labelled Burkholderia symbiont in the bean bug Riptortus pedestris

Many insects possess endosymbiotic bacteria inside their body, wherein intimate interactions occur between the partners. While recent technological advancements have deepened our understanding of metabolic and evolutionary features of the symbiont genomes, molecular mechanisms underpinning the intimate interactions remain difficult to approach because the insect symbionts are generally uncultivable. The bean bug Riptortus pedestris is associated with the betaproteobacterial Burkholderia symbiont in a posterior region of the midgut, which develops numerous crypts harbouring the symbiont extracellularly. Distinct from other insect symbiotic systems, R. pedestris acquires the Burkholderia symbiont not by vertical transmission but from the environment every generation. By making use of the cultivability and the genetic tractability of the symbiont, we constructed a transgenic Burkholderia strain labelled with green fluorescent protein (GFP), which enabled detailed observation of spatiotemporal dynamics and the colonization process of the symbiont in freshly prepared specimens. The symbiont live imaging revealed that, at the second instar, colonization of the symbiotic midgut M4 region started around 6 h after inoculation (hai). By 24 hai, the symbiont cells appeared in the main tract and also in several crypts of the M4. By 48 hai, most of the crypts were colonized by the symbiont cells. By 72 hai, all the crypts were filled up with the symbiont cells and the symbiont localization pattern continued during the subsequent nymphal development. Quantitative PCR of the symbiont confirmed the infection dynamics quantitatively. These results highlight the stinkbug-Burkholderia gut symbiosis as an unprecedented model for comprehensive understanding of molecular mechanisms underpinning insect symbiosis.

诸多昆虫体内均携带有内共生细菌（endosymbiotic bacteria），宿主与共生菌之间存在紧密的相互作用。尽管近年来技术进步加深了我们对共生菌基因组代谢与进化特征的认知，但由于昆虫共生菌大多难以体外培养，解析这类紧密互作背后的分子机制仍极具挑战。豆缘蝽（Riptortus pedestris）的中肠后部与β-变形菌门伯克霍尔德菌（Burkholderia）共生菌形成共生关系，其肠道此处发育出大量可胞外定植共生菌的隐窝。与其他昆虫共生系统不同，豆缘蝽并非通过垂直传播获得伯克霍尔德菌共生菌，而是每一代均从环境中获取该共生菌。借助该共生菌可体外培养且具备遗传可操作性的优势，我们构建了带有绿色荧光蛋白（GFP）标记的转基因伯克霍尔德菌菌株，得以在新鲜制备的样本中详细观察共生菌的时空动态与定殖过程。共生菌活体成像结果表明，在二龄若虫阶段，共生中肠M4区域的定殖始于接种后约6小时（hai）。接种后24小时（hai），共生菌细胞已出现在M4区域的主肠道与部分隐窝中。接种后48小时（hai），大部分隐窝已被共生菌细胞定殖。接种后72小时（hai），所有隐窝均被共生菌细胞填满，且共生菌的定位模式在后续若虫发育阶段保持稳定。针对共生菌的定量PCR实验从定量层面验证了该感染动态过程。上述研究结果表明，蝽类-伯克霍尔德菌肠道共生系统可作为前所未有的模型，用于全面解析昆虫共生关系背后的分子机制。