The female Bicyclus anynana butterfly (left) features larger eyespots on their wings than its male counterpart (right). New research shows that the hormone ecdysone regulates the eyespot size in B. anynana, both between males and females and between wet- and dry-season morphs. (Photo credit: William H. Piel and Antónia Monteiro)

By Viviane Callier

The butterfly Bicyclus anynana has beautiful eyespots on its wings, which function in sexual signaling and predator deflection. For years researchers believed that the differences between male and female wing patterns were regulated cell-autonomously by a gene called doublesex. Indeed, gynandromorph butterflies, which are exactly half male and half female, had suggested to researchers long ago that sex in butterflies was determined cell by cell.

Now, new research from Antónia Monteiro’s lab at the University of Singapore shows that male and female Bicyclus butterflies have different levels of the hormone ecdysone and that ecdysone levels regulate the different-sized eyespots on male and female Bicyclus butterflies.

“This work is the first to conclusively show that sexual differences in hormone titers can regulate dimorphism in sexual traits in insects, without the need of cell-autonomous factors also being expressed in the trait,” Monteiro said. The research, conducted by Monteiro and colleagues at the University of Singapore and Yale University, was published this week in Molecular Biology and Evolution.

The B. anynana butterfly, native to Africa and also known as the “squinting bush brown,” has seasonal forms: In the dry season, food is scarce. Dry-season males have small eyespots, to blend into the background like a dry leaf. Dry-season males invest a lot in reproduction, making metabolically expensive spermatophores which are highly sought after by the females. The dry-season females have big eyespots—ornaments to court the males to obtain a spermatophore. (For more, see “How Flashy Wings, UV Light, and Seasons All Play a Role in One Butterfly Species’ Mating Rituals.”)

In the wet season, resources are abundant, and the males don’t need to invest as much in reproduction. In the wet season, females are the choosy sex, and both males and females sport big eyespots, which function for predator deflection: Predators like mantids are abundant and frequently attack the big eyespots, but the butterflies can escape the attack. However, in butterflies with small eyespots, the mantids attack the head and thorax, killing the butterfly.

The question, for many years, has been to understand how the eyespot size differences between males and females and seasonal forms is regulated.

To investigate this question, Monteiro and her team first looked at the window of developmental time that is critical for eyespot size regulation and found that the wandering stage, right before metamorphosis, was the temperature-sensitive stage during which the male butterfly determines whether it will become a wet- (27 degrees C) or dry-season (17 degrees C) morph.

Although previous work suggested that the gene doublesex regulates sexual traits, the researchers found that doublesex was not expressed in the eyespot centers. That led them to investigate whether differences in hormone levels could be controlling the wing ornaments. Indeed, they found that ecdysone peaks during the wandering stage, and females have higher titers than males. In addition, the wet season titers are higher than dry season titers.

The researchers found that the ecdysone receptor was expressed in the developing wing at the location of the future eyespot, and it was expressed to a similar extent in each sex and seasonal form.

When ecydsone binds the ecdysone receptor, it triggers cell division locally. In dry-season males, the low ecdysone levels meant that the cells in the eyespot did not divide, and the eyespot stayed small. In contrast, in the females, high ecdysone titers triggered more local cell division, producing a bigger eyespot.

The researchers then manipulated the hormone levels to see if that alone could control eyespot size. They injected ecdysone into the dry-season males and lowered ecydsone signaling by injecting an ecdysone receptor antagonist, cucurbitacin, in the females and wet-season males. That increased the size of the eyespots in dry-season males and significantly reduced the eyespot size in the other groups.

“We have shown that sex differences in levels of a steroid hormone, during a brief period of development, control a very localized pattern of division in cells that express the hormone receptor, which later develop into the bright UV-reflective scale cells that make up a sexual ornament in adult butterflies,” the researchers wrote. Monteiro’s team now is working to understand how temperature and sex determine the ecdysone titer.

Viviane Callier