Chalkbrood-Infected Larvae

Or, Mummies at your Doorstep

Mummified larvae were the first sign of something wrong. My hive was only months old when it started placing little white-black bricks at the hive entrance. I didn’t know much at the time, so I panicked. What was going on?

Chalkbrood is caused by the fungus Ascosphaera apis. The fungal spores consume the bodies of young larvae, leaving the cadaver behind. The healthy, recovering colony removes these mummies before the spores can spread, and so far the best treatment for this condition is to rely on the bees’ hygienic behaviour.

How do colonies get infected? Why is temperature the driving factor, and what takes place inside the hive to drive recovery?

Lifecycle of A. apis

Adult bees are carriers of the chalkbrood spores. As nurse bees feed brood, they spread the fungi to the developing larvae. Larvae in the first 3-4 days of are most susceptible to infection as their hindgut is underdeveloped (elaborated below). Once ingested, the A. apis spores grow inside intestinal opening and develop their mycelia- threads that form a fungal network.

These threads (hyphae) branch out of the intestinal tube and into the peritrophic membrane- a protective barrier that lines the gut. Once breached, the fungus invades the body cavity, damaging and killing the larva. As hyphae continue spreading, they cover the corpse in a white layer, later darkening as they mate and form the fruiting bodies containing the next generation of spores¹.

An Underdeveloped Intestine Drives Infection Risk

A larva is most susceptible to chalkbrood in the first four days of its life. This can be due to a lack of stable gut bacteria, or increased sensitivity to temperature and nutritional changes. Once they get to five days of age, time becomes a limiting factor for establishing infection (Jensen et al. 2009; Kim et al., 2023).

Under normal conditions, the insect exoskeleton contains waxes and unsaturated fatty acids with strong antifungal activity; and their gut environment provides some defence against food-borne pathogens. For example, one byproduct of insect melanin production are reactive oxygen species, which display antimicrobial properties (Gullan & Cranston, the Insects, 2nd edition).

Developmentally, the end of the larval intestine is closed off until the cell caps. This leads to a buildup of food and wastes in the mid-intestine that is released as faeces when the mid- & hind-gut are connected. These faeces would contain the chalkbrood spores and thus the larva would be cured of infection.

However, delays in larval development occur as a result of stressful conditions, such as A. apis infections. Despite the delayed development, the workers continue capping cells on the seventh day with the stressed larva. The accumulating carbon dioxide concentrations and the release of protein-degrading enzymes from the fungi causing intestinal damage, further stressing the larva.

Starks and colleagues suggested that the worker bees can sense the stress from A. apis infection and begin increasing the brood nest temperature (Starks et al., 2000). This social fever has been confirmed in followup studies, however in the experimental colonies, it did not reduce the signs of chalkbrood disease (Goblirsch et al., 2020). Antimicrobial peptide expression increased in the worker bees, and one hypothesis from the authors suggests that temperature may impact mature spores, reducing their infectivity as the heat drives them A. apis outside its optimal range.

Emergence of the Fruiting Body

As the germ tube (reproductive organ) and hyphae emerge, the fungi successfully breaches the underdeveloped peritrophic matrix and into the gut wall, growing inside the body cavity (reviewed in Aronstein and Murray, 2010). Larval death occurred from a combination of physical and enzymatic damage, disrupted haemolymph (blood) circulation and general toxicity.

The growth of Ascosphaera apis “mushrooms” starts with the hyphae spreading from the posterior to anterior region of the larva. This covers the corpse in a thick layer of white mycelium. As the infection progresses and the spores mate, brown or black spots emerge due from the fruiting “ascomata”. This is when beekeepers find mummified larvae at their hive entrance, since the average colony tends to remove diseased brood.

Chalkbrood Infections are Most Common in Spring

Spring is a cool and usually-humid season. These conditions are ideal for the growth of A. apis, as the queen lays eggs enmass, and the low adult bee population cannot maintain the optimal brood nest temperature of ~36°C. In managed hives, it can be made worse by beekeepers who may open hives during colder or wetter days to assess overwintering progress (I plead guilty).

Infections outside of this period are possible, as with many diseases. If a colony is unable to regulate temperature and humidity, or is impacted by pests, diseases and pesticides, then the stressors make it easier for the fungus to infect brood cells.

How Chalkbrood Spreads

Spores persist for more than 15 years in the soil near infected apiaries, and remain viable in stored honey, pollen and used beekeeping equipment. Larvae must swallow spores in order to contract the infection. Additional stressors such as exposure of the foraging bees to pesticides and to Nosema disease can make chalkbrood infections more likely.

Mycology of Ascosphaera apis

The non-technical reader is welcome to skip this section

Ascosphaera apis is a heterothallic fungus, requiring two compatible partners for mating, unlike the self-fertile homothallic fungi (reviewed in Ni et al., 2011). Two distinct mycelilal forms exist, and sexual reproduction occurs when they interact a mycelium of a different sex². Reproduction forms the ascomata, whose size is influenced by temperature, pH, moisture and growth medium – available at optimal levels in the gut of the developing larva.

The individual spore balls are covered by a thick proteinaceous layer. In this layer are the translucent ascospores, containing ribosomes and mitochondria. The structures are what provide A. apis spores with resistance to extreme temperatures, UV radiation and chemical exposures, enabling them to survive for extended periods (years) in the environment (reviewed in Aronstein and Murray, 2010).

Ascospores affect all stages of the honey bee brood and castes. However the infection only progresses in young larva, as mentioned above.

Detecting Chalkbrood Disease

In the field, the presence of mummified cadavers at the hive entrance is a strong indicator of chalkbrood infection. Laboratory-based methods involve microscopic detection of the spore cysts on the mummified larvae, and in rarer cases, culturing the samples in a petri dish to confirm the pathogen’s presence (Mraz et al., 2021).

Microscopic observations only identify a fungal infection. Other fungi from the genus Aspergillus (causative agents of stonebrood) produce symptoms similar to those of A. apis, but are much rarer. For those set on a certain analysis, consider confirmations with PCR testing.

Chalkbrood Symptoms – Swollen & Dried Larvae

Dead larvae are stretched out in the cells and swollen to the size of cell walls, eventually drying out into the hard mummies. The mummies stay white only if one strain of the fungus is present, and upon mating with a compatible strain, form the fruiting bodies that causes gray or black colouration (Puerta et al., 1994). Although fatal to individual larvae, the disease does not usually destroy a honey bee colony³.

Subspecies-Dependent Resistance

Research by Jensen & colleagues showed variations in chalkbrood susceptibility between A. mellifera subspecies (Jensen et al., 2008). The brood of the Italian bees A.m. ligustica were found to be less susceptible to Ascosphaera apis infection compared to the European dark bees A.m. mellifera and the Carolinian bees A.m. carnica.

The authors suggest that the results be taken with a grain of salt (grain of pollen?). The low sample size (3 colonies from each subspecies) and large variations in spore dosage (the number of spores required to kill half the larval population ranged from 55 to 905) are too variable to draw conclusions. Further, colonies of A. m. carnica showed large dose-related fluctuations between colonies – some larvae required ten times as much chalkbrood spores to die out (Jensen et al., 2008).

Host genotype certianly plays a role in the resistance, as honey bee colonies in Australia were infected with multiple strains of A. apis (Gerdts et al., 2021)

Prevention and Control of Chalkbrood

Worker bees appear to recognise chalkbrood infection and react by increasing temperature in the brood area (Goblirsch et al., 2020). This temporary heating aims to reduce moisture and mycelial growth to prevent healthy larvae from infection, as the fungi cannot develop at temperatures above 35ºC. Given that ~35°C is already the ideal temperature for brood rearing, this highlights the importance of proper insulation and timing hive inspections to maintain heating.

Outside of feeding sugar syrup supplemented with essential oils or proprietary blends, chalkbrood controls have been mostly tested in laboratory settings (Ansari et al., 2017). Effective treatments have been found in the essential oils of plants like may chang (Litsea cubeba), and spearmint (Mentha spicata), as well as compounds found in the oils compounds formed by the oils of lemongrass (Cymbopogon flexuosus) and cinnamon (Cinnamomum zeylanicum). Note that these essential oils have been longed recommended by beekeepers in sugar feeders due to their colony-boosting health benefits.

Supplementing Hives with Propolis for Chalkbrood Prevention

In the natural beehive, propolis is used by bees to seal cracks and holes in the tree cavity and preserve wood. Thus propolis is one method to maintain an optimal temperature and its antimicrobial activity reduces the risk of harbouring pathogens. Since bees gather propolis from many types of trees, there is variability in the effectiveness based on the sample source.

An analysis was conducted on the antimicrobial properties of propolis from 12 regions in the United States against the pathogens of Paenibacillus larvae (the causative agent of American foulbrood) and Ascophaera apis (Wilson et al., 2015). The results showed that porpolis did inhibit the growth of these pathogens, however the rate of inhibiton was dependant on the region and tree type.

More generally, the colonies living in Langstroth hives containing a propolis envelope that mimicked the natural tree hives, had less active immune systems compared to those bees living without one (reviewed in Simone-Finstrom et al., 2017;Dalenberg et al., 2020). Hives with propolis envelopes also have decreased levels of pathogens in the hive.

Could coating hive walls with propolis cause bees to spend less energy in maintaining an immune system and thereby allocate those resources elsewhere?

Hygienic Bees for Chalkbrood

In any discussion of managing bee diseases, there is always “keep hygienic bees!” and the same applies for chalkboard. Bees in more hygienic colonies bees detect infected larvae more quickly, removing the dead pupae before they become infectious mummies. Chalkbrood-specific hygienic tests showed a barbell-like response to infection:

• At high hygienic behaviour, the sick brood was removed quickly and sometimes before it became infectious, and
• At low hygienic behaviour, the sick brood remained inside the sealed cells, (Invernizzi et al., 2011, Al Toufailia et al., 2018).

The trigger for bees to remove diseased brood is in phenethyl acetate (Swanson et al., 2009). However in some locations like Australia, this chemical signal is missing, and the same hygienic behaviour listed above does not apply here (Gerdts et al., 2018, Gerdts, 2020).

Heating Honey at High Temperatures Removes Chalkboard Spores

Heating honey can be done to remove chalkbrood spores, but this impacts the honey composition. Honey can be incubated for 8 hours at 65 °C or at 70 °C for 2 hours in water baths to remove the spores (Anderson et al., 1997).

Actionable Advice for Beekeepers

In addition to supplemental feeding and keeping hygienic bees:

• Do not inspect hives in wet or colder conditions to avoid chilling the brood cells.
  • Avoid humidity and low temperatures during the night by placing hives on elevated positions to avoid direct ground contact.
• Replace old honeycombs or combs known to have symptoms of A. apis to remove spores from the hive.
  • The replacement with new wax in the combs also has the advantage of inducing the queen to lay eggs, increasing the bee population.
• Clean and disinfect equipment such as hive tools and gloves after handling sick hives. Hives with symptoms of any disease should be the last to be managed, to reduce the possibility of transmission into healthy colonies.
• Avoiding feeding the colonies with pollen from unknown apiaries, as it can be a source of contamination and spread of pathogens beyond chalkbrood (Pereira et al., 2019).
• Remove abandoned hives from the apiary to avoid robbing, which can spread infectious material.
• Requeen a hive that shows recurring symptoms of chalkbrood with queens from stronger, more hygienic stock.
• If the colony population is small in late autumn, consider feeding them with thick syrup or powdered sugar to boost their stores, making making them stronger in spring.
• Where possible, arranging hives further apart to reduce the change of drifting foragers entering and spreading the disease in the hive.

A (Very) Brief Mention of Stonebrood

Stonebrood is a rare brood diseases caused by several fungi from the genus Aspergillus, with A. flavus being the most prevalent (Jensen et al., 2013). Unlike chalkbrood, the disease infects larvae from the gut AND contact with the exoskeleton’s cuticle, infecting both brood and adult bees. The primary cause of death in infected bees is from poisoning via fungal-produced aflatoxins. The fungus is found in soil, and can infect other hosts like the lungs and eyes of immune-compromised humans (Gefter, 1992).

Difference between Chalkbrood and AFB Symptoms

We conclude by comparing the two diseases. both are brood diseases, and the differences are presented in a table below.

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Footnotes

1. Results from Cheng & colleagues showed that the presence of Aspergillus tubingensis could enhance chalkbrood onset by A. apis, and that both fungi act as vectors for the acute & chronic bee paralysis viruses (Cheng et al., 2022). ↩︎
2. It is not the two mycelial forms that are sexually reproducing with each other, rather than the interaction of male and female forms from the same “mycelilal form” interacting that leads to reproduction. An oversimplification, I know. ↩︎
3. Note that a strain that infects Asian honey bees also exists – Ascosphera cerana. The interested reader is referred to the literature for further reading. ↩︎