Abstract

Insects typically acquire their beneficial microbes early in development. Endosymbionts housed intracellularly are commonly integrated during oogenesis or embryogenesis, whereas extracellular microbes are only known to be acquired after hatching by immature instars such as larvae or nymphs. Here, however, we report on an extracellular symbiont that colonises its host during embryo development. Tortoise leaf beetles (Chrysomelidae: Cassidinae) host their digestive bacterial symbiont Stammera extracellularly within foregut symbiotic organs, and in ovary-associated glands to ensure its vertical transmission. We outline the initial stages of symbiont colonization and observe that while the foregut symbiotic organs develop three days prior to larval emergence, they remain empty until the final 24 hours of embryo development. Infection by Stammera occurs during that timeframe, and prior to hatching. By experimentally manipulating symbiont availability to embryos in the egg, we describe a 12-hour developmental window governing colonization by Stammera. Symbiotic organs form normally in aposymbiotic larvae, demonstrating that these Stammera-bearing structures develop autonomously. In adults, the foregut symbiotic organs are already colonized following metamorphosis and host a stable Stammera population to facilitate folivory. The ovary-associated glands, however, initially lack Stammera. Symbiont abundance subsequently increases within these transmission organs, thereby ensuring sufficient titers at the onset of oviposition [~]29 days following metamorphosis. Collectively, our findings reveal that Stammera colonization precedes larval emergence, where its proliferation is eventually decoupled in adult beetles to match the nutritional and reproductive requirements of its host.