To date, the use of CRISPR/Cas9 technology in ecological-model species for validating genotype to phenotype connections has focused primarily on visual phenotypes using G0 mutations coupled with analyses of resulting mosaic phenotypes. However, studies of physiological phenotypes necessitate germline mutations in order to assess non-visible phenotypic effects, thus dedicated efforts to developing efficient germline mutations in ecological model species are needed. Here we applied the CRISPR/Cas9 technology to an ecological model species, the speckled wood butterfly (Pararge aegeria). We targeted yellow-y, which is required for the production of black melanin, as yellow-y loss of function (LOF) mutations are not lethal and easy to phenotype, affording efficient assessment of F0 and germline mutations. To explore what factors may affect efficiency of transformation, we employed four alternative treatments, including variation in sgRNAs and their concentrations. Color changes in the head capsule of first larval instar as well as adult wing color were used as indicators of successful knockouts. Individuals with wings that were at least 50% transformed were mated, with their F1 offspring assessed for the presence of germline mutations. Our CRISPR/Cas9 technique was highly efficient at generating LOF mutations in yellow-y. Across all treatments, nearly 80% of adults exhibited mosaic LOF phenotypes, of which nearly 30% appeared to have 100% LOF phenotypes. Crosses between adults exhibiting at least 50% LOF phenotypes resulted in fully transformed offspring, revealing high incidence of germline LOF mutations in yellow-y. We provide a detailed protocol on how to obtain high germline LOF mutation efficiency in order to advance the study of genotype-phenotype connections for non-visible physiological traits across natural populations of this and other model ecological species.

Many insects possess the plastic ability to either develop directly to adulthood, or enter diapause and postpone reproduction until the next year, depending on environmental cues (primarily photoperiod) that signal the amount of time remaining until the end of the growth season. These two developmental pathways often differ in co-adapted life history traits, e.g. with slower development and larger size in individuals headed for diapause. The developmental timing of these differences may be of adaptive importance: if pathways diverge late, the scope for phenotypic differences is smaller, whereas if pathways diverge early, the risk is higher of expressing a maladaptive phenotype if the selective environment changes. Here we explore the effects of changes in photoperiodic information during life on pupal diapause and associated life history traits in the butterfly Pararge aegeria. We find that both pupal diapause and larval development rate are asymmetrically regulated: while exposure to long days late in life (regardless of earlier experiences) was sufficient to produce nondiapause development and accelerate larval development accordingly, more prolonged exposure to short days was required to induce diapause and slow down pre-diapause larval development. While the two developmental pathways diverged early in development, development rates could be partially reversed by altered environmental cues. Meanwhile, pathway differences in body size were more inflexible, despite emerging late in development. Hence, in P. aegeria several traits are regulated by photoperiod, along subtly different ontogenies, into an integrated phenotype that strikes a balance between flexibility and phenotype-environment matching.