Dense, small and male-biased cages exacerbate male–male competition and reduce female choosiness in Bicyclus anynana

• Extreme sex ratio and density in the lab bias sexual selection in Bicyclus anynana.
• Male competition increased with density, specifically under a male-biased sex ratio.
• Female choosiness decreased with density under a male-biased sex ratio.
• Small cages and very high density promoted sexual harassment of females by males.

Sexual selection is increasingly recognized to depend upon, and to fluctuate with, major ecological factors in natural environments. The operational sex ratio (OSR) and population density can affect the opportunity for, and strength of, sexual selection but their effects are rarely taken into account in laboratory behavioural studies. In Bicyclus anynana, a model butterfly for mate choice studies, the experimental set-up widely used in laboratories often involves very high densities compared to the field, male-biased sex ratios and small cage volumes. We hypothesized that these conditions impede the proper expression of female mating preference by promoting male competition and sexual harassment of females. Using various cage volumes, we separately manipulated OSR and density to cover the range of values used in B. anynana laboratory mating experiments and to approach field values. Male competition, quantified by the number and duration of courtships aborted by males, became stronger with increasing densities, specifically under more male-biased sex ratios, and decreasing cage volumes. Thus, male eagerness to mate was essentially due to the use of small cage volumes and very high experimental densities. Concomitantly, female choosiness, quantified by the proportion of rejections of male mating attempts, decreased with increasing densities under a male-biased sex ratio. Females also accepted more matings with decreasing cage volume, and mated more rapidly with increasing density. We conclude that the laboratory social environments frequently used to test mate choice in B. anynana, with unnaturally high densities and male-biased sex ratios, exacerbate male–male competition and strongly hinder female choice, biasing the estimates of the strength and direction of sexual selection that were shaped under very different natural environments in the wild. We propose a set-up for B. anynana mate choice studies that allows the assessment of female choosiness in statistically robust mating experiments.