Abstract
Vitellogenin is a yolk precursor protein in most oviparous females. In the advanced eusocial honeybee, Apis mellifera (Hymenoptera: Apidae), vitellogenin has recently attracted much interest as this protein, in addition to a classical function in oocyte development in the reproductive queen caste, has evolved functions in the facultatively sterile female worker caste not documented in other species. However, research on the spatial dynamics of vitellogenin in various tissues is not easily performed with available tools. Here we present an immunogold staining procedure that visualizes honeybee vitellogenin in resin embedded tissue. To establish the protocol, we used ovaries of worker bees from colonies with and without a queen. Under the first condition, vitellogenin is assumed not to be present in the workers' ovaries. Under the second condition, the ovaries of worker bees become vitellogenic, with abundant opportunities for detection of complex patterns of vitellogenin uptake and storage. By use of this experimental setup, the staining method is shown to be both sensitive and specific. To demonstrate the functional significance of the protocol, it was subsequently used to identify vitellogenin protein in the hypopharyngeal glands (brood food producing head glands) of nursing worker bees and in adjacent head fat body cells for the first time. Localization of vitellogenin in these tissues supports previously hypothesized roles of vitellogenin in social behavior. This protocol thus provides deeper insights into the functions of vitellogenin in the honeybee.