Exploring the mysterious behavior of the critically endangered South African Wolkberg Zulu butterfly ( <i>Alaena margaritacea</i> ) using micro-CT scanning and morphological analysis (Lycaenidae: Liptenini)

Background The Critically Endangered endemic Wolkberg Zulu butterfly (Lycaenidae: Alaena margaritacea Eltringham, 1929) is known from only two colonies in the Wolkberg mountain range in north-east South Africa. An unusual behavior of this species was reported in 2020: the female climbs and criss-crosses grass blades while rubbing her abdomen against them and curling her abdomen. The function of the unusual behavior is unknown, but it might attract males by depositing a pheromone. Although long-range female sex pheromones are rare in butterflies (Papilionoidea), this conjecture is supported by a male flying to a tussock containing a female, where mating occurred directly. Methods We used non-destructive micro-CT scanning and morphological analysis to uncover the potential abdominal and reproductive structures, including glands, that could explain the female’s behavior. We illustrate the structures on A. margaritacea examined using micro-CT scanning, including rendered 2-D surface photos, rendered surface videos, a video showing the entire slide stack, video showing the density differences, and focus-stacked 2-D photos. Results We found a sclerotized area mid-ventrally on the 7th sternite, a ventral pouch-shaped opening on the 9th segment, and tiny, spherical, superficially gland-like structures inside the 7th abdominal segment. The sclerotized area on the 7th sternite may be associated with the observed behavior, and the gland-like structures may have a pheromone-producing function, but our data do not allow unambiguous confirmation of either of these explanations. However, we confirm the presence of a waxy mating plug sealing the ostium bursae, the first confirmation of this structure in a lycaenid. It corresponds to a protosphragis, extending into the posterior part of the ductus bursae and onto the ventral part of the 9th sternite. The mating plug could offer an alternative explanation for why the female rubs her abdomen against grass blades: she may be trying to remove the mating plug mechanically to allow remating. The closely related Alaena amazoula was also found to have a sclerotized area on the 7th sternite. We illustrate the female genitalia of A. margaritacea and A. amazoula using morphological analysis to explain their structures.