Physiological and molecular mechanisms of dessication resistance in embryos of the killifish, Fundulus heteroclitus

Northern killifish, Fundulus heteroclitus macrolepidotus, spawn in estuaries at high tides. Embryos may be stranded in air at stream margins as the water recedes. These aerially incubated embryos are exposed to desiccation stress and may survive and develop normally. At ∼14 days post-fertilization (dpf), they hatch when flooded by spring tides. We investigated the physiological mechanisms by which killifish embryos tolerate desiccation stress. Using a microbalance system, we measured the kinetics of water loss from single embryos at various relative humidities (RHs). Mid-stage (7 dpf) embryos had the longest periods of resistance to water loss compared to early-stage (2 dpf) and late-stage (14 dpf) embryos. Mid-stage embryos survived acute desiccation (2 hrs at 23% RH) that killed early- and late-stage embryos. We also measured the osmolyte composition and osmotic pressure of embryos. Key osmolyte concentrations (K+, Na+, NH4+ and Cl--) and free amino acids (FAAs) increased in desiccated embryos approximately as predicted by reduced water content and did not vary dramatically with embryo age. Unusually high Cl-- activity was found in 14 dpf embryos and we speculate that Cl-- may bind to macromolecules elaborated near hatching. FAAs (∼60 mM) are likely to serve as compatible solutes that stabilize cellular proteins during desiccation stress. Quantitative real-time PCR (Q-RT-PCR) measurements of mRNA expression of the water channel protein, aquaporin3 (AQP3), showed significant down-regulation in mid-stage embryos compared with late-stage but not with early-stage embryos. Q-RT-PCR measurements of mRNA expression of heat shock proteins (HSC70, HSP70, HSP90-alpha, and HSP90-beta) indicated that HSP expression was largely constitutive with no large changes after desiccation stress compared with controls, and no significant differences among embryo ages. The key physiological responses of aerially incubated killifish embryos: sustained resistance to water loss, presence of compatible solutes, down-regulation of AQP3 and the expression of HSPs, help explain the unusual ability of these embryos to survive.