Butterflies feeling ‘the butterflies’

Abstract

Biologists have long been captivated by courtship rituals in the animal kingdom, in which (usually) males work tirelessly to advertise their mating desirability to the opposite sex. Some species woo their mates by building elaborate nests for seduction, while others choreograph and execute impressive dance routines. Squinting bush brown butterflies (Bicyclus anynana) attract a member of the opposite sex by rapidly opening and closing their wings, flashing gorgeous eyespots on their wings to their potential mate. Interestingly, the frequency at which these tropical male butterflies embark on courtship is strongly dependent on the time of year at which they enter the pupal stage, when they transition from caterpillar to butterfly. Males that transition during the wet season become active courters, whereas males that transition during the dry season have lower courtship rates. Biologists believe that these behavioural differences are mediated by levels of the hormone 20-hydroxyecdysone (20E), produced during the pupal stage, but how 20E acts on the brain to influence courting behaviour was unclear.In a new study led by Heidi Connahs [National University of Singapore, Singapore (NUS)] and Eunice Jingmei Tan (NUS and Yale-NUS College, Singapore), a team of researchers from NUS and Yale University, USA, questioned whether 20E alters the expression of the yellow gene in the brain of male butterflies – a gene that has been linked to courting behaviour in other insects. Wet season pupae tend to have very high levels of 20E in their blood, which the team suspected may ‘turn down’ the expression of the yellow gene, leading to elevated courtship rates in the adult male butterflies. To test this idea, the team reared squinting bush brown butterfly caterpillars under wet season (27°C) or dry season (17°C) conditions until the caterpillars created cocoons and became pupae. After a few days, the team collected the brains of the young pupae so that the expression of the yellow gene could be analysed and compared with previous measurements of 20E hormone levels in wet and dry season pupae, as well as with the courtship rates of adult butterflies raised under the same conditions.The researchers first discovered that the yellow gene was expressed at different levels in the brains of wet season and dry season pupae. As expected, wet season males with high 20E levels expressed the yellow gene at low levels and courted like love-struck teenagers as butterflies. In contrast, dry season males with low 20E levels expressed the yellow gene at high levels and courted half as much. Given this strong correlation, the researchers decided to take their study one step further and experimentally elevated hormone levels in dry season pupae by injecting them with a concentrated dose of 20E. When the team analysed the brains of the injected pupae, the expression of the yellow gene appeared to be reduced, leading to high courtship rates and providing further support for the team's idea.In a final experiment to solidify their findings, the researchers decided to use a cutting-edge gene editing method known as CRISPR-Cas9 to knock-out the yellow gene in these butterflies altogether. The team carefully observed the knock-out mutants and found that they courted at very high rates, providing the final compelling piece of evidence that the yellow gene regulates courting behaviour in the squinting bush brown butterfly.Most species in the animal kingdom – including the squinting bush brown butterfly – make their intentions for mating very clear, through goofy dances, thoughtful gift giving or some other wonderfully weird act. Human dating rituals, in contrast, can be confusing and not at all productive. So, the next time you're wondering whether you should go ask that special someone out on a date, simply turn down your metaphorical ‘yellow gene’ and maybe you'll get those butterfly feelings too.