Etiology, symptoms and prevention of chalkbrood disease: a literature review

Etiologia, sintomas e prevenção da doença cria giz: uma revisão bibliográfica

Guido Laércio Bragança CASTAGNINO Ana MATEOS Aránzazu MEANA Lucia MONTEJO Luis Vicente ZAMORANO ITURRALDE Maria Teresa CUTULI DE SIMÓN About the authors

ABSTRACT

The fungus Ascosphaera apis, responsible for causing the chalkbrood disease of honey bees, is widely present in temperate regions of the northern hemisphere, but has also spread to other regions of the world such as Brazil. Although it is not usually lethal for the colony, it can reduce its population, hampering its development. This study is a review on the disease that presents a broad overview of its development, identification methods as well as ways to control it. Research shows that chalkbrood is associated with several factors and is most frequently found in colonies of Apis bees during the spring, when there is excess humidity and sudden temperature changes in the hive. Other factors such as viral or bacterial infection, the presence of the ectoparasite Varroa destructor, pesticide poisoning and poor nutrition of nurse bees can also affect its incidence and severity. Field diagnosis is made based on the presence of hardened mummified brood in the pupal stage, of white or black color, in the cells and entrance. Affected cells show dead pupae covered with white mycelia, resembling cotton, or hardened, dry and brittle, resembling chalk pieces, which originated the name. To date, there are no efficient methods to reduce the damage caused by chalkbrood. Genetic selection of bees with higher hygienic behavior and disease resistance is recommended.

Key words:
Apis mellifera; Ascosphaera apis; bee pathology; chalkbrood

RESUMO

O fungo Ascosphaera apis, responsável por causar a doença apícola cria giz, ocorre amplamente nas regiões temperadas do hemisfério norte, estendendo-se a outras regiões do mundo como, no caso, do Brasil. Normalmente não chega a exterminar a colônia, pode reduzir a sua população, prejudicando o seu desenvolvimento. O objetivo deste trabalho foi realizar uma revisão sobre essa enfermidade, apresentando um amplo panorama sobre o seu desenvolvimento, métodos de identificação, bem como as formas de combatê-la. Estudos mostram que essa doença está associada a diversos fatores, sendo mais frequente em colônias de abelhas Apis na primavera quando ocorre excesso de umidade e trocas bruscas de temperatura na colmeia. Outros fatores como infecções por vírus, bactérias e a presença do ectoparasita Varroa destructor, envenenamento por pesticida e má alimentação das abelhas nutrizes também podem induzir a sua incidência e severidade. O diagnóstico de campo é identificado pela a presença de crias mumificadas na fase de pupa endurecidas de cor branca ou negras nos favos e no alvado. As células de crias operculadas nos favos apresentam pupas mortas cobertas por micélio branco semelhantes a algodão ou endurecidas, secas e quebradiças, semelhantes a pedaços de giz, o que deu origem ao seu nome. Até o momento, não existe uma forma eficaz para reduzir os prejuízos da cria giz e recomenda-se a seleção genética de colônias que apresentam maior comportamento higiênico e maior resistência a doenças.

Palavras-chave:
Apis mellifera; Ascosphaera apis; ascosferiose; patologia apícola

INTRODUCTION

Chalkbrood is a disease affecting the brood of Apis mellifera L. bees. The mycosis occurs virtually all across the world, though mainly present in the temperate regions of the northern hemisphere (BAILEY & BALL, 1991BAILEY, L.; BALL, B.V. Honey bee pathology. London: Academic Press, 1991.). It is caused by the fungus Ascosphaera apis, first identified in Apis bees in 1913 by Maassen, who named it Pericystis apis (Maassen, 1913) and then reclassified it as Ascosphaera apis in 1955 (SPILTOIR & OLIVE, 1955SPILTOIR, C.F.; OLIVE, L.S. A reclassification of the genus Pericystis Betts. Mycologia, v.47, n.2, p.238-244, 1955.). This fungus is characterized for being an opportunistic pathogen and causing mortality in broods during the pupal stage (KLINGER et al., 2013KLINGER, E.G.; JAMES, R.R.; YOUSSEF, N.N.; WELKER, D.L. A multi-gene phylogeny provides additional insight into the relationships between several, Ascosphaera species. Journal of Invertebrate Pathology, v.112, n.1, p.41-48, 2013.), thereby weakening the colony and reducing its capacity (EVISON, 2015EVISON, S. E. Chalkbrood: epidemiological perspectives from the host-parasite relationship. Current Opinion in Insect Science, v. 10, p. 65-70, 2015.).

The disease affects all castes of honeybee broods (DE JONG, 1976DE JONG, D. Experimental enhancement of chalk brood infections. Bee World, v.57, n.3, p.114-115, 1976.), which are infected after ingesting food contaminated with spores of the fungus Ascosphaera apis. The development of this fungus in the colony depends on a physiologically imbalanced medium or a stressful situation involving the brood during its development (ARONSTEIN & MURRAY, 2010ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010.). Chalkbrood is established in colonies infected by other diseases or by stress situations, aggravating bee mortality (POTTS et al., 2016POTTS, S. G.; IMPERATRIZ-FONSECA, V.; NGO, H. T.; AIZEN, M. A.; BIESMEIJER, J. C.; BREEZE, T. D.; DICKS, L. V.; GARIBALDI, L. A.; HILL, R. , SETTELE, J.; VANBERGEN A. J. Safeguarding pollinators and their values to human well-being. Nature, v. 540, n.7632, p. 220-229, 2016.), and hence the importance of a higher knowledgement on its symptoms, forms of diagnosis and prevention and control techniques. The present study is a literature update from a selection of the publications considered most representative of the topic in order to provide a broad overview of the etiological and morphological traits of Ascosphaera apis, factors causing its spread, symptoms, diagnosis and ways to prevent it.

ETIOLOGICAL AND MORPHOLOGICAL TRAITS

Genera of the fungus Ascosphaera are among the Apis mellifera pathogens. Twenty-eight species have been recorded around the world, most of which are saprophytes found in the pollen, honey and feces of larvae and in the wax within the colony (WYNNS et al., 2012WYNNS, A.A.; JENSEN, A. B.; EILENBERG, J.; JAMES, R. Ascosphaera sub globosa, a new spore cyst fungus from North America associated with the solitary bee Megachilerotundata. Mycologia, v.104, n.1, p.108-114, 2012.). However, only one Ascosphera apis species is associated with chalkbrood disease.

The application of new DNA-based fungal taxonomy methods has recently provided a new update to the mycological systematics, with the current taxonomic line of the chalkbrood fungus being reclassified as Ascomycota, Pezizomycotina, Eurotiomycetes, Eurotiomycetidae, Onygenales, Ascosphaeraceae and Ascosphaera apis (LUMBSCH & HUHNDORF, 2007LUMBSCH, H.T.; HUHNDORF, S.M. (Eds.). Outline of Ascomycota. Myconet, v.13, p.1-58, 2007.).

Ascosphaera apis is a heterothallic fungus, that is, a dioecious species (separate sexes) with two distinct mycelial forms that can reproduce sexually after interacting with another individual of a different sex (ANDERSON & GIBSON, 1998ANDERSON, D.L.; GIBSON, N.L. New species and isolates of spore-cyst fungi (Plectomycetes:Ascosphaerales) from Australia. Australian Systematic Botany, v.11, n.1, p.53-72. 1998.). Sexual reproduction results in the formation of fruits called fruiting bodies, which are usually 47-40 µm in diameter, depending on factors such as temperature, moisture and culture medium used (ARONSTEIN & MURRAY, 2010ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010.; JENSEN et al., 2013JENSEN, A. B.; ARONSTEIN, K.; FLORES, J.M.; VOJVODIC, S.; PALACIO, M.A.; SPIVAK, M. Standard methods for fungal broad disease research. Journal of Apicultural Research, v.52, n.1, p.1-20, 2013.).

According to Bissett (1988)BISSETT, J. Contribution towards a monograph of the genus Ascosphaera. Canadian Journal of Botany, v.66, n.12, p.2541-2560, 1988., those fruiting bodies are grey or brown, globose and contain sac-like spore balls 11-17 µm in diameter. Spore balls are produced in large amounts and, when mature, their color changes from white to brown and, finally, black.

The spore balls are coated by a thick transparent layer, and within them are the hyaline ascospores (1.2 × 2.5 µm), which have an ellipsoidal shape, containing several ribosomes and mitochondria. These provide the ascospores with great resistance to extreme temperatures, UV radiation and various types of disinfection products, allowing their survival for many years in the environment (ARONSTEIN & MURRAY, 2010ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010.). This disease affects worker bee larvae, drones and, at a lower frequency, the queen. Even though ascospores can affect all brood castes, susceptibility to the infection depends on the age of the larva.

Li et al. (2018)LI, Z.; YOU, X.L.; WANG, L.L.; YAN, Z.T.; ZHOU, Z.Y. Spore morphology and ultrastructure of an Ascosphaera apis strain from the honeybees (Apis mellifera) in southwest China. Mycologia, v.110, n. 2, p. 325-338, 2018. analyzed the morphology and the ultrastructure of spores of the A. apis CQ1 strain from southwestern China and found that mature spores are oval and long, with an average size of 2 × 1.2 µm, and firmly packed within spherical spheres. Ultrastructural analysis revealed that mature spores have two nuclei of distinct sizes. A large nucleus, with double nuclear membranes, was found within the spore, whereas the small nuclei, with only one-fifth of the volume of the large nuclei, was located near the end of the spore. The spore wall consists of an outer electron-dense surface layer, an electron-lucent layer and an inner plasma membrane. Chitin was also found to be the main component of the spore wall.

As stated by De Jong (1976)DE JONG, D. Experimental enhancement of chalk brood infections. Bee World, v.57, n.3, p.114-115, 1976., the best time for the spores to germinate are the first four days of age of the larvae. Jensen et al. (2009)JENSEN, A.; PEDERSEN, B.V.; EILENBERG, J. Differential susceptibility across honey bee colonies in larval chalk brood resistance. Apidologie, v.40, n.5, p.524-534, 2009. showed that, in five-day-old larvae, time is a limiting factor for the germination and penetration of the spores into the larva’s intestine. However, Gilliam, Taber, & Bray Rose (1978)GILLIAM M.; TABER S.; BRAY ROSE J. Chalkbrood disease of honey bees Apis mellifera L.: a progress report. Apidologie, v. 9, p. 75-89, 1978. also suggested previously that three- and four-day-old larvae are also susceptible to the fungus.

The larvae are infected after ingesting food contaminated with spores of A. apis that germinate in the lumen of their intestine, initiating the development and growth of mycelium in the terminal portion of the brood’s intestine (HEATH & GAZE, 1987HEATH, L.A.F.; GAZE, B.M. Carbon dioxide activation of spores of the chalk brood fungus Ascosphaera apis. Journal of Apicultural Research, v.26, n.4, p.243-246, 1987.; LOPES et al., 2015LOPES, L.Q.S.; QUATRIN, P.M.; DE SOUZA, M.E.; DE ALMEIDA VAUCHER, R.; VIANNA SANTOS, R.C. Fungal infections in honey bees. Fungal Genomics & Biology, v. 4, p. 118, 2015.). In this stage, the intestine of the larvae is closed in the caudal portion until the cell is capped (JAY, 1963JAY, C.S. The development of honey bees in their cells. Journal of Apicultural Research, v.2, n.2, p.117-134, 1963.). This causes the food ingested by the brood to accumulate in the mid-intestine, where the fungus spores may also be found occasionally. Under normal larval development conditions, the food (so the fungus) does not accumulate in the intestine, since, soon after capping the cell, it will be fully developed (midgut and hindgut are connected) and finally have its end opening, allowing the elimination of feces along with the spores.

However, any abnormal or stressful situation for the brood may delay larval development (the intestine will still be closed in the caudal portion) and the evacuation of feces with the spores also delayed (JAY, 1963JAY, C.S. The development of honey bees in their cells. Journal of Apicultural Research, v.2, n.2, p.117-134, 1963.). This fact, coupled with the presence of CO2 accumulated in the intestine of the larva and the enzymes produced by the fungus, causes lesions on the intestinal membrane, thus providing adequate conditions for it to germinate and initiate the formation of the germ tube and the hyphae (THEANTANA & CHANTAWANNAKUL, 2008THEANTANA, T.; CHANTAWANNAKUL, P. Protease and β-N acetylglucosaminidase of honey bee chalkbrood pathogen Ascosphaera apis. Journal of Apicultural Research, v.47, n.1, p.68-76, 2008.).

The fungus produces mycelia formed by irregularly septate white hyphae with an average diameter of 4.5 µm. These hyphae penetrate the peritrophic membrane of the mid-intestine of the larva, killing it, spreading throughout the body and soon initiating the production of fruiting bodies (spore cysts), giving rise to a new generation of spores (JENSEN et al., 2013JENSEN, A. B.; ARONSTEIN, K.; FLORES, J.M.; VOJVODIC, S.; PALACIO, M.A.; SPIVAK, M. Standard methods for fungal broad disease research. Journal of Apicultural Research, v.52, n.1, p.1-20, 2013.; SARWAR, 2016SARWAR, M. Challenges due to bacterial infections of the honey bees and contributions to manage pest problems. International Journal of Entomological Research, v. 1, p.4-10, 2016.).

FACTORS THAT FAVOR THE SPREAD OF THE FUNGUS

Experiments show the great sensitivity of bee broods when exposed to a drop in temperature to values below normal within the hive (32-35°C) for a prolonged period (> 2 h), after the capping has been sealed (BAILEY & BALL, 1991BAILEY, L.; BALL, B.V. Honey bee pathology. London: Academic Press, 1991.). It only takes the colony brooding area to cool for adverse conditions or stresses to trigger the disease in the larvae, since the ideal temperature for the development of fruit bodies is approximately 30 °C (ARONSTEIN & MURRAY, 2010ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010.).

In cold regions, this brood cooling may occur naturally, when the fall or winter seasons begin and there is a sudden temperature decrease. Another possibility is in early spring, when the queen lays many eggs and there are not yet enough worker bees to produce heat and keep the brood warm (MORAWETZ et al., 2019MORAWETZ, L.; KÖGLBERGER, H.; GRIESBACHER, A.; DERAKHSHIFAR, I.; CRAILSHEIM, K.; BRODSCHNEIDER, R.; MOOSBECKHOFER, R. Health status of honey bee colonies (Apis mellifera) and disease-related risk factors for colony losses in Austria. PLoS One, v. 14, n. 7, p. e0219293, 2019.).

According to Fries (2010)FRIES, I. Nosema ceranae in European honey bees (Apis mellifera). Journal of Invertebrate Pathology, v. 103 (Suppl. 1), p. S73-S79, 2010., Goulson et al. (2015)GOULSON, D.; NICHOLLS, E.; BOTÍAS, C.; ROTHERAY, E.L. Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science, v. 347, n. 6229, p. 1435 -1444, 2015. doi: 10.1126/science.1255957.
https://doi.org/10.1126/science.1255957...
and Dosselli et al. (2016)DOSSELLI, R.; GRASSL, J.; CARSON, A.; SIMMONS, L.W.; BAER, B. Flight behaviour of honey bee (Apis mellifera) workers is altered by initial infections of the fungal parasite Nosema apis. Scientific Reports, v. 6, p. 1-11, 2016. doi: 10.1038/srep36649.
https://doi.org/10.1038/srep36649...
, additional factors such as exposure of the foraging bees to pesticides and to the microsporidium Nosema ceranae may predispose the colony to diseases. These causes may also be associated with the same effects of cooling and increase disease severity as well as infection by viruses, bacteria and by the ectoparasite V. destructor. Puerta et al. (1994)PUERTA, F. ; FLORES, J.M.; BUSTOS, M.; PADILLA, F.; CAMPANO, F. Chalk brood development in honey bee brood under controlled conditions. Apidologie, v.25, p.540-546, 1994. mentioned that, in tropical regions, chalkbrood can be triggered by the low temperatures at night, with the drone larvae being subjected to greater risks of cooling, as they are located in the periphery of brood combs.

Inadequate handling practices by beekeepers, such as the transfer of combs between colonies, especially if a colony is weakened, may also be related to transmission of the fungus Ascophaera apis between colonies (VOJVODIC et al., 2011VOJVODIC, S.; JENSEN, A. B.; JAMES, R.R.; BOOMSMA, J.J.; EILENBERG, J. Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood. Veterinary Microbiology, v. 149, p. 200-205, 2011.; SIMONE-FINSTROM et al., 2018SIMONE-FINSTROM, M.; ARONSTEIN, K.; GOBLIRSCH, M.; RINKEVICH, F.; DE GUZMAN, L. Gamma irradiation inactivates honey bee fungal, microsporidian, and viral pathogens and parasites. Journal of Invertebrate Pathology, v. 153, p. 57-64, 2018.).

Padilla et al. (2014)PADILLA, F.; FLORES, J.M.; CAMPANO, F. Control de la ascosferosis (Ascosphaera apis) mediante el uso de fondos higiénicos de rejilla. Actas Iberoamericanas de Conservación Animal, v. 4, p. 289-291, 2014. mentioned that other factors can also contribute for the spread of the disease, such as opening the hives at low-temperature times, inducing the queen to lay eggs or dividing the colonies with artificial feed when the bee population is still low and its ability to maintain the thermal homeostasis of the colony-which is essential for its survival-is low. Factors such as sudden changes in temperature, humidity and poor ventilation in the colony predispose to the growth of the fungus, especially if there are few adult bees (NATSOPOULOU et al., 2016NATSOPOULOU, M.E.; MCMAHON, D.P., PAXTON, R.J. Parasites modulate withincolony activity and accelerate the temporal polyethism schedule of a social insect, the honey bee. Behavioral Ecology and Sociobiology, v.70, n.7, p. 1019-1031, 2016. doi: 10.1007/s00265-015-2019-5.
https://doi.org/10.1007/s00265-015-2019-...
; NAZZI & LE CONTE, 2016NAZZI, F.; LE CONTE, Y. Ecology of Varroa destructor, the major ectoparasite of the western honey bee, Apis mellifera. Annual Review of Entomology, v. 61, p.417-32, 2016. doi: 10.1146/annurev-ento-010715-023731.
https://doi.org/10.1146/annurev-ento-010...
).

Arranging the hives too close to each other may induce drifting of contaminated foraging bees upon return, spreading the disease in the apiary. Although adult bees are not susceptible to the disease, they may transport the fungus A. apis upon performing trophallaxis during the foraging stage, contaminating the nursing bees, which, in turn, contaminate the larvae by feeding them (ARONSTEIN & MURRAY, 2010ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010.). Contaminated drones are another possible direct contamination source, since they do not possess glands of identification and may enter healthy hives, contaminating them.

Food sources highly frequented by bees are also a risk factor. As stated by Castagnino et al. (2006a)CASTAGNINO, G.L.B.; FUNARI, S.R.; BLUME, E.; ARBOITTE, M.Z.; WEBER, M.N. Doença Cria Giz Ascosphaera apis (Maassenex Claussen) Olive & Spiltoir em abelhas Apis mellifera L. na Depressão Central do Rio Grande do Sul. Ciência Rural, v.36, n.6, p.1909-1911, 2006a., the import of honey and pollen with the presence of A. apis spores from locations where chalkbrood is frequent might have introduced and disseminated it to regions where the disease does not yet exist. The visit of foraging bees to contaminated flowers as well as collective drinkers and feeders and other sites highly accessed by bees has been mentioned as a possible cause of contamination (KOENIG et al., 1987KOENIG, J.P.; BOUSH, G.M.; ERICKSON JR., E.H. Effect of spore introduction and ratio of adult bees to brood on chalk brood disease in honeybee colonies. Journal of Apicultural Research, v.26, p.191-195, 1987.), though possibly of less importance.

Another noteworthy fact is that the ascospores of the fungus A. apis are resistant to environmental factors, possibly surviving long periods in unfavorable conditions such as high salinity, extreme temperature and sunlight. For those reasons, it is often difficult to eliminate the spores once it is established in an environment (GABRIEL et al., 2018GABRIEL, K.T.; KARTFOROSH, L.; CROW, S.A.J.R.; CORNELISON, C.T. Antimicrobial Activity of Essential Oils Against the Fungal Pathogens Ascosphaera apis and Pseudogymnoascus destructans. Mycopathologia, v. 183, n. 6, p. 921-934, 2018.).

The fungus spores can also contaminate honey, the melted wax and stored pollen. This highlights the risks of beekeepers when using these products from non-certified sources.

SYMPTOMS IN THE BROOD

The disease symptoms in the colonies can be seen by the presence of mummies in front of the entrance on the floor or on the ground, beneath the hive (HEATH, 1982HEATH, L.A.F. Development of chalk brood in a honey bee colony: a review. Bee World, v.63, n.3, p.119-130, 1982.). Perforated cappings with mummified broods and empty cells can be observed in the combs (Figure 1), indicating removal of dead brood by the hygienic behavior of worker bees (SARWAR, 2016SARWAR, M. Challenges due to bacterial infections of the honey bees and contributions to manage pest problems. International Journal of Entomological Research, v. 1, p.4-10, 2016.).

Figure 1
Pupae of mummified bees (white and black) within the brood combs.

According to Bailey & Ball (1991)BAILEY, L.; BALL, B.V. Honey bee pathology. London: Academic Press, 1991., infected larvae generally die within the first two days after being capped, in the pupal stage, and, soon after dying, they dilate and start to be covered by the white mycelia of the fungus. In the subsequent stage of the disease, some pupae remain white, resembling a piece of chalk (hence the name chalkbrood), while others start to turn brown or gray and, finally, black, due to the production of fruiting bodies that may vary in size or color (Figure 2). In this development stage, the mummified pupae are found enlarged, hardened and brittle, in the vertical position.

Figure 2
Magnified image showing dead pupae; a white mummy (third) still without spores fruiting and a black mummy with the presence of spores.

DIAGNOSIS OF THE FUNGUS ASCOSPHAERA APIS

Laboratory diagnosis is obtained by visualizing the dead pupa (mummy) under a stereoscopic microscope with 200A magnification (ANDERSON & GIBSON, 1998ANDERSON, D.L.; GIBSON, N.L. New species and isolates of spore-cyst fungi (Plectomycetes:Ascosphaerales) from Australia. Australian Systematic Botany, v.11, n.1, p.53-72. 1998.). Spore balls can be seen in black mummies (with spores) and in white mummies when they only have mycelia. Through this observation, the diagnosis can be made to visually identify other fungi of the genus Aspergillus that occur sporadically in the colonies but produce symptoms similar to those of A. apis, though of lesser damage to the colony. It can also be observed that the reproduction of A. apis is sexual and that the result of the reproduction of the fungus mycelia with the opposite sex will form spherical bodies called ascospores (spores).

Unlike other pathogenic fungi of insects that form asexual conidia, which are the infectious units of the fungus Aspergillus, those conidiophores are situated at the extremities of the hyphae rather than in the mycelial mass, as occurs in chalkbrood (FLORES et al., 2000FLORES, J.M.; FUNARI, S.R.C.; RUIZ, J.A.; RUZ, J.M.; PUERTA, F.; CAMPANO, F. Ascosferiose (Ascosphaera apis): causas predisponentes, medidas de controle e prevenção. Boletim de Indústria Animal, v.57, n.2, p.201-209, 2000.). Since A. apis performs sexed reproduction, some authors have observed different formations of different types of strains (LEE et al., 2013LEE, G.M.; MCGEE, P.A.; OLDROYD, B.P. Variable virulence among isolates of Ascosphaera apis: testing the parasite-pathogen hypothesis for the evolution of polyandry in social insects. Naturwissenschaften, v.100, n.3, p.229-234, 2013.). Diverse culture media can be used to cultivate the fungus, but it develops very well in media with high sugar contents such as Sabouraud agar (RAPER & FENNELL, 1965RAPER, K.B.; FENNEL, D.I. The genus Aspergillus. Baltimore: Williams and Wilkins Company, 1965.).

Initially, to prevent contamination by other types of microorganisms, the contaminated pupa is treated with a 1% hypochlorite solution and, subsequently, washed three times with distilled water. The culture medium should be in an environment with an average temperature of 30 ºC and 40% moisture (ARONSTEIN & MURRAY, 2010ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010.), forming dense and white mycelium that contains aerial, septate, hyaline hyphae 2.5-8 µm in diameter. These mycelia present superficial hyphae with very pronounced dichotomous ramifications (JENSEN et al., 2013JENSEN, A. B.; ARONSTEIN, K.; FLORES, J.M.; VOJVODIC, S.; PALACIO, M.A.; SPIVAK, M. Standard methods for fungal broad disease research. Journal of Apicultural Research, v.52, n.1, p.1-20, 2013.).

For more than 15 years, new and more precise methods of identifying the presence of A. apis have been applied based on molecular diagnostics using the segment of the internal transcript region (ITS) of the nuclear ribosomal repeat unit (NILSSON et al., 2008NILSSON, R.H.; KRISTIANSSON, E.; RYBERG, M.; HALLENBERG, N.; LARSSON, K.H. Intraspecific ITS variability in the Kingdom Fungi as expressed in the International Sequence Databases and its implications for molecular species identification. Evolutionary Bioinformatics, v.4, p.193-201, 2008.) and two pairs of primers [3-F1 and 3-R1, mentioned by James & Skinner (2005)JAMES, R.R.; SKINNER, J.S. PCR diagnostic methods for Ascosphaera infections in bees. Journal of Invertebrate Pathology, v.90, n.2, p.98-103, 2005.; and AscosF3 and AapisR3, mentioned by Murray et al. (2005)MURRAY, K.D.; ARONSTEIN, K.A.; JONES, W.A. A molecular diagnostic method for selected Ascosphaera species using PCR amplification of internal transcribed spacer regions of rDNA. Journal of Apicultural Research, v.44, n.2, p.61-64, 2005.] as sample.

Garrido-Bailon et al. (2013)GARRIDO-BAILÓN, E.; HIGES, M.; MARTÍNEZ-SALVADOR, A.; ANTÚNEZ, K.; BOTÍAS, C.; MEANA, A.; PRIETO, L.; MARTÍN-HERNÁNDEZ, R. The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcusplutonius in Spanish apiaries determined with a new multiplex PCR assay. Microbial Biotechnology, v.6, n.6, p.731-739, 2013. developed the PCR method, which is able to detect multiple pathogenic bacteria in bee, such as P. larvae and M. plutonius and, simultaneously, A. apis. Although the complete genome of the fungus was sequenced by Qin et al. (2006)QIN, X.; EVANS, J.D.; ARONSTEIN, K. A.; MURRAY, K.D.; WEINSTOCK, G.M. Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis. Insect Journal of Molecular Biology, v.15, n.5, p.715-718, 2006., little is known about the diversity of this pathogen.

SPREAD OF THE FUNGUS ASCOSPHAERA APIS

Chalkbrood occurs in virtually all regions of the planet, and clinical symptoms usually appear in a short time, most commonly in cold seasons with high humidity, including hot and dry climates (ARONSTEIN y MURRAY, 2010ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010.). According to Jensen et al. (2013)JENSEN, A. B.; ARONSTEIN, K.; FLORES, J.M.; VOJVODIC, S.; PALACIO, M.A.; SPIVAK, M. Standard methods for fungal broad disease research. Journal of Apicultural Research, v.52, n.1, p.1-20, 2013. and Seyedmousavi et al. (2015)SEYEDMOUSAVI, S.; GUILLOT, J.; ARNÉ, P.; DE HOOG, G.S.; MOUTON, J.W.; MELCHERS, W.J.; VERWEIJ, P.E. Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease. Medical Mycology, v. 53, p. 765-797, 2015., in several countries, chalkbrood is considered a notifiable disease that should be reported to the authorities when observed in colonies.

Epidemiological surveys carried out in Spain in 2006 and 2007 revealed a low incidence of diseases in bee broods, with chalkbrood causing the highest mortality rates (5.0%) and surpassing the American foulbrood, caused by the bacillus Paenibacillus larvae (3.0%). In comparing the origin of positive samples, chalkbrood was found to be more frequent in warmer areas, whose climatic characteristics thus far have not been considered favorable for the development of the disease (GARRIDO-BAILÓN et al., 2013GARRIDO-BAILÓN, E.; HIGES, M.; MARTÍNEZ-SALVADOR, A.; ANTÚNEZ, K.; BOTÍAS, C.; MEANA, A.; PRIETO, L.; MARTÍN-HERNÁNDEZ, R. The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcusplutonius in Spanish apiaries determined with a new multiplex PCR assay. Microbial Biotechnology, v.6, n.6, p.731-739, 2013.). This observation is in line with the results of Aronstein & Murray (2010)ARONSTEIN, K.A.; MURRAY, K.D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, v.103, n.1, p.20-29, 2010., who mentioned that Ascosphaera apis appears for a short period and is generally associated with cold and high humidity climates. According to Garrido-Bailón et al. (2013)GARRIDO-BAILÓN, E.; HIGES, M.; MARTÍNEZ-SALVADOR, A.; ANTÚNEZ, K.; BOTÍAS, C.; MEANA, A.; PRIETO, L.; MARTÍN-HERNÁNDEZ, R. The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcusplutonius in Spanish apiaries determined with a new multiplex PCR assay. Microbial Biotechnology, v.6, n.6, p.731-739, 2013., a possible explanation for this high prevalence in those regions is stress in the colonies caused by Nosema ceranae, which occurs more frequently in the warmer region of Spain.

Since chalkbrood is an opportunistic pathology, it finds appropriate conditions to develop in weakened colonies, regardless of ideal environmental conditions. According to Cavigli et al. (2016)CAVIGLI, I.; DAUGHENBAUGH, K. F.; MARTIN, M.; LERCH, M.; BANNER, K.; GARCIA, E.; BRUTSCHER, L. M.; FLENNIKEN, M. L. Pathogen prevalence and abundance in honey bee colonies involved in almond pollination. Apidologie, v. 47, n. 2, p. 251-266, 2016., this observation shows how vulnerable the colonies may be to the attack of the fungus A. apis, since they are constantly under the action of stress inducers such as pesticides, migration, lack of food, parasites and other diseases (SIMONE-FINSTROM et al., 2016SIMONE-FINSTROM, M.; LI-BYARLAY, H.; HUANG, M. H.; STRAND, M. K.; RUEPPELL, O.; TARPY D. R. Migratory management and environmental conditions affect lifespan and oxidative stress in honey bees. Scientific Reports, v. 6, p. 1-9, 2016.). According to Heath (1985)HEATH, L.A.F. Occurrence and distribution of chalk brood disease of honey bees. Bee World, v.66, n.1, p.9-15, 1985., from the 1970s, chalkbrood was detected in countries like Japan, the Philippines and Mexico, where it was considered the disease whose frequency most increased among Apis mellifera L. species (WILSON et al., 1984WILSON, W.T.; NUNAMAKER, R.A.; MAKI, D. The occurrence of brood diseases and the absence of the Varroa mite in honeybees from Mexico. American Bee Journal, v.124, p.51-53, 1984.). In South America, it was initially reported in Argentina, in 1978, by Rossi & Carranza (1980)ROSSI, C.; CARRANZA, M. Momificación de larvas de abejas (Apis mellifera L.) provocada por Ascosphaera apis. Revista de la Facultad de Agronomía, v. 56, p.11-15, 1980. in the province of Buenos Aires. From that period, it spread rapidly and is currently present in all areas where beekeeping is practiced (ALBO & REYNALDI, 2010ALBO, G.N.; REYNALDI, F.J. Ascosphaera Apis, agente etiológico de la cría yesificada de las abejas. Revista Argentina de Microbiologia, v.42, n.1, p.80, 2010.), with an incidence of around 11% in the colonies (REYNALDI et al., 2003REYNALDI, F.J.; LÓPEZ, A. C.; ALBO, G.N.; ALIPPI, A.M. Differentiation of Ascosphaera apis isolates by rep-PCR fingerprinting and determination of chalk brood incidence in Argentinean honey samples. Journal of Apicultural Research, v.42, p.68-76, 2003.). In Brazil, four isolated cases were initially reported: one in the state of São Paulo (ROCHA et al., 1998ROCHA, H.C.; FUNARI, S.R.C.; BAGLAGI, E. Identificação do fungo Ascosphaera apis em colmeias de abelhas Apis mellifera L. no Estado de São Paulo. In: CONGRESSO BRASILEIRO DE APICULTURA, 1998. Salvador. Proceedings... Salvador: [s.n.], 1998.), two in Rio Grande do Sul (SATTLER et al., 1998SATTLER, A.; DISCONZI, M.S.; DUARTE, V.J.; SILVEIRA, R.P. Ocorrência de cria giz (Ascosphaera apis) em apiários no Rio Grande do Sul. In: CONGRESSO BRASILEIRO DE APICULTURA, 1998. Salvador. Proceedings... Salvador: [s.n.], 1998.; CASTAGNINO et al., 2006aCASTAGNINO, G.L.B.; FUNARI, S.R.; BLUME, E.; ARBOITTE, M.Z.; WEBER, M.N. Doença Cria Giz Ascosphaera apis (Maassenex Claussen) Olive & Spiltoir em abelhas Apis mellifera L. na Depressão Central do Rio Grande do Sul. Ciência Rural, v.36, n.6, p.1909-1911, 2006a.) and one in Minas Gerais (CASTAGNINO et al., 2006bCASTAGNINO, G.L.B.; MESSAGE, D.; BARRETO, R.W. Primeiro relato da doença “cria giz” em abelhas Apis mellifera no estado de Minas Gerais. Revista Ceres, v.53, n.306, p. 234-236, 2006b.). Castagnino et al. (2006a)CASTAGNINO, G.L.B.; FUNARI, S.R.; BLUME, E.; ARBOITTE, M.Z.; WEBER, M.N. Doença Cria Giz Ascosphaera apis (Maassenex Claussen) Olive & Spiltoir em abelhas Apis mellifera L. na Depressão Central do Rio Grande do Sul. Ciência Rural, v.36, n.6, p.1909-1911, 2006a. described that the fungus A. apis was widespread in southern Brazil from 1998, the year of its first report. The authors suggested that the introduction and expansion of the disease in the state of Rio Grande do Sul was due to the migration of colonies to cultivation fields located on the border with Uruguay and Argentina, where the disease had been frequent for many years. Teixeira et al. (2018)TEIXEIRA, E.W.; GUIMARÃES-CESTARO, L.; ALVES, M.L.T.M.F.; MESSAGE, D.; MARTINS, M.F.; LUZ, C.F.P.; SERRÃO, J.E. Spores of Paenibacillus larvae, Ascosphaera apis, Nosema ceranae and Nosema apis in bee products supervised by the Brazilian Federal Inspection Service. Revista Brasileira de Entomologia, v. 62, p. 188-194, 2018. analyzed 41 samples of bee products marketed in the state of São Paulo and identified the presence of spores of the pathogen A. apis in 73.17% of them, in addition to other investigated pathogens.

FORMS OF PREVENTION AND CONTROL OF THE FUNGUS ASCOSPHAERA APIS

According to Wilson et al. (2015)WILSON, M.B.; BRINKMAN, D.; SPIVAK, M.; GARDNER, G.; COHEN, J.D. Regional variation in composition and antimicrobial activity of US propolis against Paenibacillus larvae and Ascosphaera apis. Journal of Invertebrate Pathology, v.124, p.44-50, 2015., the A. apis fungi are resistant to biocides like sulfuric acid, iodinated substances and other products that are commonly used to disinfect hives infected by diseases. Thus, eliminating the disease focus becomes a hard task once it is already established in the apiary.

Studies led by Starks et al. (2000)STARKS, P.T.; BLACKIE, C.A.; SEELEY, T.D. Fever in honeybee colonies. Naturwissenschaften, v.87, p.229-231, 2000. showed an additional behavior of the colony in an attempt to reduce the possibility of infection and control of A. apis. Bees seem to recognize the attack of this disease and react to the infestation by producing a temperature increase in the brooding area. According to those authors, this temporary heating aims to reduce mycelial growth and prevent infection of healthy larvae, since A. apis are sensitive and do not develop at temperatures above 35 ºC. This shows the importance of keeping colonies in suitable places and providing good-quality hives for the colony to maintain its homeostasis. However, to date, there is no effective technique to eliminate the spores of A. apis or a pharmacological treatment to minimize its effects on the hive (PUERTA et al., 1995PUERTA, F.; FLORES, J.M.; BUSTOS, M. Efecto del ácido tricloroisocianúrico sobre la germinación de las esporas de Ascosphaera apis. Revista Iberoamericana de Micología, v. 12, p. 49-51, 1995.).

Researchers have tried new ways to control chalkbrood by using natural products. Many of these studies, carried out in vitro, focused on the use of essential oils of aromatic plants in the control of A. apis and showed effective results, with emphasis on the oils from the plants Litsea cubeba, Pelargonium graveolens, Croton bonplandianus and Mentha spicata and compounds formed by oils of L. cubeba, C. zeylanicum, Cymbopogon flexuosus and L. cubeba, C. zeylanicum, P. graveolens and C. flexuosus (Saleem et al., 2015SALEEM, M.; BHATTI, H.N.; JILANI, M.I.; HANIF, M.A. Bioanalytical evaluation of Cinnamomum zeylanicum essential oil. Natural Product Research, v. 29, 1857-1859, 2015.; Ansari et al., 2015ANSARI, M.J.; AL-GHAMDI, A.; USMANI, S.; KHAN, K.A.; ALQARNI, A.S.; KAUR, M.; AL-WAILI N. In vitro evaluation of the effects of some plant essential oils on Ascosphaera apis, the causative agent of Chalkbrood disease. Saudi Journal of Biological Sciences, v. 24, n. 5, p. 1001-1006. 2015.; Nardoni et al., 2018NARDONI, S.; D'ASCENZI, C.; ROCCHIGIANI, G.; PAPINI, R.A.; PISTELLI, L.; FORMATO, G.; NAJAR, B.; MANCIANTI, F. Stonebrood and chalkbrood in Apis mellifera causing fungi: in vitro sensitivity to some essential oils. Natural Product Research, v. 32, n. 4, p. 385-390, 2018.). Chaimanee et al. (2017)CHAIMANEE, V.; THONGTUE, U.; SORNMAI, N.; SONGSRI, S.; PETTIS, J.S. Antimicrobial activity of plant extracts against the honeybee pathogens, Paenibacillus larvae and Ascosphaera apis and their topical toxicity to Apis mellifera adults. Journal of Applied Microbiology, v. 123, n. 5, 1160-1167, 2017. also tested the antimicrobial activity of 37 plant extracts against A. apis in vitro. Seven species-Amomum krervanh, Allium sativum, Cinnamomum spp., Piper betle, Piper ribesioides, Piper sarmentosum and Syzygium aromaticum-exhibited inhibitory effect against the fungus.

Another natural product non-toxic to bees that constitutes an alternative to the use of synthetic drugs is propolis from Apis bees, considered an important component of immunity for the hive due to its antimicrobial activity. Wilson et al. (2015)WILSON, M.B.; BRINKMAN, D.; SPIVAK, M.; GARDNER, G.; COHEN, J.D. Regional variation in composition and antimicrobial activity of US propolis against Paenibacillus larvae and Ascosphaera apis. Journal of Invertebrate Pathology, v.124, p.44-50, 2015. characterized the metabolic profiles of propolis from 12 different climatic regions in the USA by comparing the antimicrobial activity of samples in culture medium against the pathogens of P. larvae and Ascophaera apis bees. The authors verified their viability to control the growth of these pathogens, but with important differences in inhibition capacity depending on the region of the United States. More recently, Wilson et al. (2017)WILSON, M.B.; PAWLUS, A.D.; BRINKMAN, D.; GARDNER, G.; HEGEMAN, A.D.; SPIVAK, M.; COHEN, J.D. 3-Acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis. Phytochemistry, v. 138, p. 83-92, 2017. researched the botanical drug profile of propolis compounds in North America by in vitro testing and proved their inhibitory activity against the same pathogens.

Simone-Finstrom et al. (2018)SIMONE-FINSTROM, M.; ARONSTEIN, K.; GOBLIRSCH, M.; RINKEVICH, F.; DE GUZMAN, L. Gamma irradiation inactivates honey bee fungal, microsporidian, and viral pathogens and parasites. Journal of Invertebrate Pathology, v. 153, p. 57-64, 2018. studied the potential of gamma irradiation for the inactivation of Ascosphaera apis, among other pathogens, and confirmed the effectiveness of this technique against the fungus. The authors also stressed that this treatment may help reduce colony losses and the spread of pathogens through exchanges of combs between colonies.

Another way to control the spread of the fungus without using fungicides and avoid contamination of colony products would be the genetic selection of bees with greater hygienic behavior. Gilliam et al. (1988)GILLIAM, M.; TABER, S.; LORENZ, B.J.; PREST, D.B. Factors affecting development of chalk brood disease in colonies of honey bee Apis mellifera, fed pollen contaminated with Ascosphaera apis. Journal of Invertebrate Pathology, v.52, n.2, p.314-325, 1988. found that hygienic behavior is correlated with the resistance of the colonies and with the chalkbrood disease and the population of the fungus A. apis in the colonies. The same authors found that, in colonies that exhibited reduced hygienic behavior, there was a wide range of spores distributed in the brooding area and in the honeycombs. This finding shows that the use of selected queens, with greater hygienic behavior, should be a more usual tool for beekeepers.

According to Invernizzi et al. (2011)INVERNIZZI, C.; RIVAS, F.; BETTUCCI, L. Resistance to Chalk brood Disease in Apis mellifera L. (Hymenoptera: Apidae) Colonies with Diferent Hygienic Behaviour. Neotropical Entomology, v.40, n.1, p.28-34, 2011., the advantage of more-hygienic over less-hygienic colonies is that bees detect infected larvae more quickly. They remove dead pupae from the hive before they become infectious mummies, reducing the possibility of the disease establishing in the colony. Al Toufailia et al. (2018)AL TOUFAILIA, H.; EVISON, S.E.F.; HUGHES, W.O.H.; RATNIEKS FLW. Both hygienic and non-hygienic honeybee, Apis mellifera, colonies remove dead and diseased larvae from open brood cells. Philosophical Transactions of the Royal Society, v. 19, n. 373, p. 1751, 2018. quantified the removal of larvae killed by freezing and infected with chalkbrood in uncapped cells in 20 colonies and concluded that there are two adaptive peaks that confer disease resistance: high hygienic behavior (the sick brood is removed quickly and, in some cases, before it becomes infectious) and poor hygienic behavior, when the sick brood remains isolated within the sealed cells.

However, Gerdts et al. (2018)GERDTS, J.; DEWAR, R.L.; SIMONE-FINSTROM, M.; EDWARDS, T.; ANGOVE, M. Hygienic behaviour selection via freeze-killed honey bee brood not associated with chalkbrood resistance in eastern Australia. PLoS One, v. 13, n. 11, p. e0203969, 2018. found that hygienic behavior was not a significant predictor of the presence of A. apis infection in colonies. Lui et al. (2016)LIU, Y.; YAN, L.; LI, Z.; HUANG, W.F.; POKHREL, S.; LIU, X.; SU, S. Larva-mediated chalkbrood resistance-associated single nucleotide polymorphism markers in the honey bee Apis mellifera. Insect Molecular Biology, v. 25, n. 3, p. 239-50, 2016. drew attention to the fact that there is not yet an easy method to select and keep resistant bees. For this reason, their study aimed to find the genes involved in the development of resistance and to identify single nucleotide polymorphisms (SNPs), which can be used as molecular markers of resistance. The results showed that the SNP C2587245T can be useful as a genetic marker for the selection of bees resistant to chalkbrood.

MANAGEMENT STRATEGIES TO REDUCE RISK OF COLONY LOSSES

In addition to negatively affecting the growth and production of colonies, pathogens and diseases cause significant economic losses for beekeepers (Panasiuk et al., 2014PANASIUK, B.; BIEŃKOWSKA, M.; GERULA, D.; WĘGRZYNOWICZ, P. Susceptibility of bee larvae to chalkbrood in relation to hygienic behaviour of worker bees in colonies of chosen races of honeybee (Apis mellifera). Journal of Apicultural Science, v. 58, n. 1, p. 119-126, 2014.), and some management practices can be used to mitigate colony losses. Berry & Delaplane (2001)BERRY, J.A.; DELAPLANE, K.S. Effects of comb age on honeybee colony growth and brood survivorship. Journal of Apicultural Research, v.40, n.1, p.3-8, 2001. suggested replacing old honeycombs or those with symptoms of A. apis as an initial management technique to eliminate spores from the hive. The replacement with new wax in the combs has the advantage of reducing the presence of spores and inducing the queen to lay eggs, promoting an increase in the bee population. It is recommended to replace one third of the hive combs annually and to clean and disinfect equipment such as chisels, gloves and fumigator after handling sick hives. Hives with symptoms of any disease should be the last to be managed, thus reducing the possibility of healthy colonies being contaminated by the beekeeper's equipment (FLORES et al., 2000FLORES, J.M.; FUNARI, S.R.C.; RUIZ, J.A.; RUZ, J.M.; PUERTA, F.; CAMPANO, F. Ascosferiose (Ascosphaera apis): causas predisponentes, medidas de controle e prevenção. Boletim de Indústria Animal, v.57, n.2, p.201-209, 2000.). Castagnino et al. (2006a)CASTAGNINO, G.L.B.; FUNARI, S.R.; BLUME, E.; ARBOITTE, M.Z.; WEBER, M.N. Doença Cria Giz Ascosphaera apis (Maassenex Claussen) Olive & Spiltoir em abelhas Apis mellifera L. na Depressão Central do Rio Grande do Sul. Ciência Rural, v.36, n.6, p.1909-1911, 2006a. recommended avoiding feeding the colonies with pollen from unknown apiaries, as it can be a source of contamination and spread of pathogens.

Another recommended measure would be the immediate removal of abandoned hives from the apiary to avoid robbing and because it is a source of infectious material. Flores et al. (1996)FLORES, J.M.; RUIZ, J.A.; RUZ, J. M. Effect of temperature and humidity of sealed brood on chalk brood development under controlled conditions. Apidologie, v.27, n.4, p.185-192, 1996. suggested avoiding manipulating the colonies at times of lower temperature to avoid cooling the offspring, while Castagnino et al. (2006a)CASTAGNINO, G.L.B.; FUNARI, S.R.; BLUME, E.; ARBOITTE, M.Z.; WEBER, M.N. Doença Cria Giz Ascosphaera apis (Maassenex Claussen) Olive & Spiltoir em abelhas Apis mellifera L. na Depressão Central do Rio Grande do Sul. Ciência Rural, v.36, n.6, p.1909-1911, 2006a., Flores et al. (2000)FLORES, J.M.; FUNARI, S.R.C.; RUIZ, J.A.; RUZ, J.M.; PUERTA, F.; CAMPANO, F. Ascosferiose (Ascosphaera apis): causas predisponentes, medidas de controle e prevenção. Boletim de Indústria Animal, v.57, n.2, p.201-209, 2000. and De Jong (1976)DE JONG, D. Experimental enhancement of chalk brood infections. Bee World, v.57, n.3, p.114-115, 1976. recommended not placing the hives directly on the ground to avoid humidity and low temperatures during the night. Another strategy, according to Castagnino et al. (2006a)CASTAGNINO, G.L.B.; FUNARI, S.R.; BLUME, E.; ARBOITTE, M.Z.; WEBER, M.N. Doença Cria Giz Ascosphaera apis (Maassenex Claussen) Olive & Spiltoir em abelhas Apis mellifera L. na Depressão Central do Rio Grande do Sul. Ciência Rural, v.36, n.6, p.1909-1911, 2006a., would be to replace the queens of hives that had clinical symptoms of chalkbrood with queens from colonies that show a higher hygienic behavior and to avoid the migration of hives to regions where a high prevalence of disease is present (Traynor et al., 2016TRAYNOR, K. S.; PETTIS, J. S.; TARPY, D. R.; MULLIN, C. A.; FRAZIER, J. L.; FRAZIER, M.; VAN ENGELSDORP, D. In-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States. Scientific Reports, v. 6, p. 1-16, 2016.; Guimarães-Cestaro et al., 2017GUIMARÃES-CESTARO, L. G.; ALVES, M. L. T. M. F.; SILVA, M. V. G. B.; TEIXEIRA, E. W. Honey bee (Apis mellifera) health in stationary and migratory apiaries. Sociobiology, v. 64, p. 42-49, 2017. doi: 10.13102/sociobiology.v64i1.1183.
https://doi.org/10.13102/sociobiology.v6...
). In the summer, Evison & Jensen (2018)EVISON, S.E.; JENSEN, A. B. The biology and prevalence of fungal diseases in managed and wild bees. Current Opinion in Insect Science, v. 26, p. 105-113, 2018. suggest that colonies with a small population should be fed before the winter begins to induce their increase and maintain thermal homeostasis, making them stronger and better able to recover.

FINAL REMARKS

The fungus Ascosphaera apis is a pathogen that attacks Apis mellifera bees during the pupal stage. Though not highly lethal, its impact on hives can be significant, as it reduces the colony brood. Contamination occurs when the brood eats food infected with spores of the fungus. For the fungus to affect the colony, the physiological environment must be out of balance or the brood must have faced some stressful situation during its development.

Because chalkbrood is an opportunistic disease, its presence may be an indication that the hives are infected by other diseases such as the ectoparasite Varroa destructor, which could greatly weaken the colonies. In this regard, the importance of knowing the symptoms of this fungus, forms of diagnosis, prevention and control techniques is reinforced.

Additional factors associated with management practices of beekeepers may be related to the transmission of Ascophaera apis between colonies, such as the introduction of contaminated combs from other hives. The ascospores of this fungus can also contaminate the stored honey, melted wax and pollen, which shows the importance of the beekeeper buying these products only from certified sources.

There is no consensus among the developed studies as to how to combat this fungus. For this reason, further research is warranted on more effective ways that can effectively reduce the incidence of this disease.

ACKNOWLEDGMENTS

The first author thanks the Complutense University of Madrid and the Marchamalo Apiculture Center for the opportunity to deepen his knowledge of the fungus Ascosphaera apis; and the Coordination for the Improvement of Higher Education Personnel (Capes) for the doctoral fellowship grant.

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Publication Dates

  • Publication in this collection
    01 June 2020
  • Date of issue
    2020

History

  • Received
    26 Nov 2018
  • Accepted
    04 Feb 2020
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