Young but not defenseless: Antifungal activity during embryonic development of a social insect

      Termites live in environments heavily colonized by diverse microbial communities, including pathogens. Eggs laid within the nest likely experience similar pathogenic pressures as their older nestmates. Consequently, they may have also been under selective pressures to be immune-competent. Through in vitro experiments, we tested the ontogeny, location and strength of embryos' antifungal activity against the fungus Metarhizium brunneum. Extraembryonic washes and intraembryonic components were incubated with fungal conidia, which was then scored for viability. Fungistatic activity was location- and stage-dependent. Extraembryonic washes had relatively weak antifungal activity. Conversely, intraembryonic contents were highly antifungal, exhibiting increased potency through development. Boiling both embryonic washes and intraembryonic contents rescued conidia viability, indicating the antifungal agent(s) is (are) heat-sensitive, and likely proteinaceous. Embryonic protein profiles shifted from putative vitellogenins in young embryos to multiple proteins in subsequent stages, suggesting that the increase in fungistatic activity may be due to the expression of endogenous proteins during embryogenesis. This study is the first to address embryonic antifungal activity in a hemimetabolous, eusocial taxon. Our results support the hypothesis that microbes have been significant agents of selection, fostering the evolution of antifungal properties in termite eggs, the most immature and supposed, most vulnerable stage of development.