Seasonal plasticity in male courtship in Bicyclus anynana butterflies is due to variation in levels of the steroid hormone 20E (20-hydroxyecdysone) during pupation. Wet season (WS) males have high levels of 20E and become active courters. Dry season (DS) males, have lower levels of 20E and reduced courtship rates, although WS courtship rates can be achieved if DS male pupae are injected with 20E at 30% of pupation. Here we investigated the genes involved in male courtship plasticity and examine whether 20E plays an organizational role in the pupal brain that later influences the sexual behaviour of adults. We show that DS pupal brains have a 7-fold upregulation of the yellow gene relative to the WS and that knocking out yellow leads to increased male courtship. We find that injecting 20E into DS pupa reduced yellow expression although not significantly. Our results show that yellow is a repressor of the neural circuity for male courtship behaviour in B. anynana. 20E levels experienced during pupation could play an organizational role during pupal brain development by regulating yellow expression, however, other factors might also be involved. Our findings are in striking contrast to Drosophila where yellow is required for male courtship.

Behavioural assays (two males and two females) was conducted by recording the duration and frequency of different courtship elements (localisation, flickering, thrusting, attempting) in male Bicyclus anynana (Day 4 - 8). Courtship latency (time taken for first mating between male and female) and mating duration was recorded as well. All behavioural assays lasted one hour, or once the first mating occurred (i.e. even if one male has mated, the other male's behavior is still quantified until its own mating or one hour has lapsed). Assays are done in a temperature-controlled behavioural room set at 23 degrees Celcius.

There are two types of assays done: using live females and decapitated females. Data inputted into Microsoft Excel, analysed using R packages 'pscl' and 'tweedie' for count and continuous data respectively. Figures are also made in R using the 'ggplot2' package.

Data from live assays are all collapsed into the same excel sheet ("Compare ws and ds live.4grps"), while data from decapitation assays are collapsed into another excel sheet ("Compare ws and ds decap.4grps"). Included a "README.txt" file to better understand the variable names in dataset. Data was analysed using R software. 

For count/frequency and continuous/duration data, read R script "zipandtweediemodels".

For how figures were made, read R script "yellow_graphs_rscript".