Schematic of experimental flow is shown (A). Flies are exposed to wasps for a period of four days prior to egg collection. The descendants from either wasp-exposed or unexposed treatment groups, termed ‘legacy’ flies, are separated from the previous generation and reared until maturity. Legacy flies are either used to propagate the next generation, or are assayed for ethanol preference. Flies from a particular generation are referred to as Fn, where n denotes the number of generations removed from the treatment. For example, the treatment group itself is F0, whereas their direct offspring are F1. Ethanol preference is quantified as proportion of eggs laid on ethanol food (B), illustrating that this behavior is heritable through the F5 generation. Flies with deficient long-term memory (Orb2[ΔQ]) were tested for transgenerational inheritance of ethanol along side the wild type control strain (CS) (C). Embryos (F1 legacy flies) were collected during wasp exposure or in the 24 hr period following the wasp exposure; both CS and Orb2[ΔQ] exposed legacy flies are able to inherit the ethanol preference. Asterisk indicates p-value of <0.05 from a Mann-Whitney U test. Error bars are bootstrap 95% confidence intervals. Color-coding of bar charts indicates treatment and generation; dark blue (unexposed), light blue (unexposed legacy), dark magenta (exposed), light magenta (exposed legacy).