= Solution
The target is genetic variation in a <reaction norm>: how pigmentation produced by a <genotype> changes with developmental temperature. A <gene> affecting pigmentation at every temperature is not necessarily a <gene> affecting plasticity. The design must estimate inherited differences in responsiveness separately from differences in baseline colour.
Raise replicated <genotypes> or families across several controlled developmental temperatures, distributing members of the same <genetic background> among environments. Standardize sex, age, nutrition, density and the body region scored; female posterior abdominal pigmentation is a useful defined trait. Randomize position and scoring order and quantify colour from calibrated images. Record viability and development time, since selective mortality can change the composition of survivors and imitate a plastic response. A parental common-garden generation and reciprocal crosses can help separate inherited nuclear effects from maternal or carry-over effects.
A simple <mixed-effects model> is
$$
z_{ijk}=\mu+G_i+E_j+(GE)_{ij}+b_k+\epsilon_{ijk},
$$
where <genotype> and environment effects describe baseline differences and the shared temperature response, their interaction describes genotype-specific responses, and $b_k$ represents an appropriate block effect. Treating temperature as a continuous predictor gives genotype-specific slopes, but a curved response should not be forced into one line. Fit <reaction norms> with uncertainty and test whether their shapes differ.
\Image[/past-exam-of-the-genetics-course-of-the-university-of-cambridge/2024/paper-4-pigmentation-reaction-norms.png]
{title=Illustrative pigmentation reaction norms separating baseline genetic differences from genotype-by-temperature interaction}
<Quantitative trait locus mapping> in segregating crosses can locate loci affecting the temperature interaction. A <genome-wide association study> in a diverse panel can complement it, with controls for relatedness and population structure. Analyse the joint <phenotype> across temperatures, rather than comparing a significant association at one temperature with a nonsignificant association at another and calling the difference an interaction. Mapping resolution, power and multiple-testing correction determine how many candidate regions can be distinguished.
Follow candidates with staged expression measurements, <chromatin> assays and functional perturbations. A coding change might alter enzyme temperature sensitivity; a cis-regulatory change might alter where or when the enzyme is produced; a trans-acting <chromatin> or hormonal regulator could affect several pathways. Allele-specific expression in a shared trans environment can help distinguish cis from trans effects. Replacing an <allele> or rescuing a perturbation and then measuring the entire <reaction norm> tests causality more strongly than expression correlation alone.
The expectation is a context-dependent genetic network, potentially including pigmentation enzymes, developmental regulators and <chromatin> regulation. <Jean-Michel Gibert> and colleagues found temperature-sensitive regulation involving <Abdominal-B>, <bric-à-brac> and <chromatin> regulators in female abdominal pigmentation. That supports a distributed mechanism rather than a universal dedicated plasticity <gene>. It does not imply that the same causal variants explain every natural population or all body regions.<Phenotypic Plasticity in Drosophila Pigmentation Caused by Temperature Sensitivity of a Chromatin Regulator Network by Jean-Michel Gibert and others>[Temperature, regional developmental regulation and pigmentation-enzyme expression]{ref}
Thus one may find variants affecting baseline colour, slope or curvature, as well as environment-dependent dominance and <epistasis>. Some apparent interactions may disappear after controlling development stage or survival. Demonstrating heritable plasticity does not establish that it is adaptive: that further claim requires fitness comparisons in the relevant environments.
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