1.1. introduction

Chalkbrood, the most common fungal bee brood disease, is caused primarily by the fungus Ascosphaera apis (Maassen ex Claussen) Olive and Spiltoir (Spiltoir, 1955). It was recognized in the honey bee in the early 20th Century (Maassen, 1913). The field diagnosis of chalkbrood is based on visual detection of diseased, mummified brood, commonly known as “chalkbrood mummies”. Chalkbrood can reduce colony productivity by lowering the number of newly emerged bees, and in some cases may lead to colony losses. The disease is found infecting honey bee brood in most regions of the world, including warm and dry climates. Clinical symptoms of chalkbrood often appear for only a short time, typically under cold and damp weather conditions (Aronstein and Murray, 2010).

The genus Ascosphaera spp. comprises species that are adapted to eusocial and solitary bees and their habitats. Some of the species are saprophytic, growing on nest materials, such as stored food, faecal matter, and nest debris; others have evolved as opportunistic and / or obligate bee pathogens (Bissett, 1988). In honey bees Ascosphaera major and Arrhenosphaera cranae (both belonging to Ascospheracea) have been reported only a few times from chalkbrood infected colonies, but Koch’s Postulates have never been demonstrated for them, so they might be secondary invaders.

Ascosphaera apis primarily infects honey bee brood by entering the host through the gut lining. In an infected larva, hyphae penetrate the gut wall, and mycelium develops inside the body cavity. After a few days, mycelium breaks out of the posterior end of the larva leaving the head unaffected (Maurizio, 1934). Fruiting bodies with the new ascospores are formed on aerial hyphae outside the dead larvae. The ascospores are a result of sexual reproduction of fungal mycelia with the opposite sex, unlike most other insect pathogenic fungi where asexually conidia are the infective units, as e.g. seen in Aspergillus. Ascospores of A. apis (hereafter referred to as spores) are believed to be adapted to the harsh gut environment of the host. Ascosphaera apis can only infect the host larvae if ingested, unlike other insect pathogenic fungi which infect through the external cuticle. Spores of A. apis can stay dormant and viable for years, but upon exposure to CO2 the spores becomes activated, resulting in spore swelling and subsequently germ tube formation that extends to form hyphae (Bamford and Heath, 1989). Presumably, CO2 produced by the larval tissues accumulates in the closed hindgut of the larvae aiding in spore germination (Heath and Gaze, 1987). However additional factors that may be involved in spore germination within the host still remain to be discovered. In addition, it has been shown that chilling of the brood below the optimal rearing temperature increases the number of diseased larvae (Vojvodic et al., 2011a; Flores et al., 1996a). All these specific adaptations have to be taken into account when working with A. apis, and specific protocols have been developed.