Chalk Brood Research Articles

Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen Ascosphaera apis.

The insect pathogenic fungus, Ascosphaera apis, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by A. apis in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box-Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (R2 = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl2 at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7-10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol/L and a Vmax of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of A. apis.

Relative Abundance and Status of Arthropod Pests and Diseases of Honey Bees at South Ari District of South Western Ethiopia

Aim: To determine the species composition, relative abundance and status of honey bee pests and diseases in South Ari district of South Omo Zone of Southern Western Ethiopia.
 Study Design: Four major honey producing peasant associations (PAs) representing diverse ecological zones were selected purposively, while the sub-localities, villages and beekeepers were selected randomly using a nested design.
 Place and Duration of Study: Survey was conducted between 1, August – 30, December 2019 with the interval of one month in major honey producing peasant associations representing diverse ecological zones of Ari district of South Western Ethiopia.
 Methodology: The assessment on abundance and status was made from of half kilogram of fresh and dry honey comb samples that were taken from 720 farmer’s hives of four peasant associations using taxonomic key of books related to arthropod pests and diseases of honey bee.
 Results: Five species of predators and parasitoids belonging to four orders were recorded. Of these 76.63% accounts for small hive beetles, followed ants (38.31%) and wasps (19.92%). Besides, six type’s diseases of honey bee caused by different pathogens were recorded. Of which, Chalk brood, other virosis, European foulbrood and American foulbrood, respectively were the most frequently occurring. In terms of the status, the diseases of honey bee were in the following orders, i.e., Chalk brood > other virosis > European foulbrood > American foulbrood > Sac Brood Virus, while their pests were in the following orders, i.e., Small hive beetles > Ants > Wasps > Wax moths > Spiders.
 Conclusion: Arthropod pests and diseases were the most important constraints of honey colony and honey production in the study areas. Thus, it is important to design and implement effective management strategies to reduce loss of honey colony and honey production in the study area.

AN EXPERIMENT FOR GENETIC TRANSMISSION OF HYGIENIC BEHAVIOR IN HONEYBEES

Next to productive performances and somehow direct economic value, the attention of queen breeders is recently oriented back to those c haracteristics which contribute to the increasing o f the bee colonies to the most infectious bee disease: Am erican foulbrood (AFB). It is already known for a f ew decades that the resistance to AFB is hereditary an d responds to artificial selection. The mechanisms that determines it are quite numerous and complex, but o ne of the most important and above all the easiest to be measured, is the hygienic behavior, defined as the capacity of bees to recognize, to uncover and evacu ate ill or dead larvae or pupae (nymphs). It represents a r are type of behavior mechanism of resistance to dis eases and it is considered the primary mechanism to self- defense in facing at least 2 of the diseases of the bee brood: AFB and chalk brood. In the last years, the researches carried out to verify this behavior are truly effective and aren't far to establish exactly the r ole in resistance to parasite. The hygienic behavio r represents the generalized behavior answer to elimi nate the pathogen agents and parasites from the nes t, minimizing the adaptation of specific pathogen agen ts to the adult bees' genotypes.

Heritability of hygienic behavior in grey honey bees (Apis mellifera carnica)

Breeding grey A. m. carnica queens for hygienic behavior is beneficial for commercial beekeeping in Serbia, since hygienic behavior is associated with the resistance to the mite Varroa destructor, as well as to American foulbrood and chalk brood. In this work, heritability of hygienic behavior was analyzed in autochthonous grey A. m. carnica honey bees from Sumadija region in order to ascertain if the expression of their hygienic behavior could be enhanced through selective breeding. Heritability of hygienic behavior was monitored through three queen generations and estimated by one-parent-offspring regression method (motherdaughter regression method). The heritability values for hygienic behavior were h2=0.63±0.02 in relationship between daughter queens (F1s) and breeder mother queens (Ps), h2=0.45±0.01 in relationship between grand-daughter queens (F2s) and Ps and h2=0.44±0.02 in relationship between F2s and F1s. These results show that the expression of hygienic behavior through selective breeding could be enhanced and the best results could be achieved in the F1 generation. It is important to emphasize that the F1 colonies bred for hygienic behavior in our study suffer no apparent costs in reproductive and productive performances.

Doença Cria Giz Ascosphaera apis (Maassen ex Claussen) Olive & Spiltoir em abelhas Apis mellifera L. na Depressão Central do Rio Grande do Sul

A ocorrência da doença apícola "cria giz", em colônias de abelhas Apis mellifera no apiário do Departamento de Zootecnia da Universidade Federal de Santa Maria (UFSM), RS, Brasil, foi constatada em 2004. Foram coletadas amostras de crias de abelhas duras e mumificadas em favos falhados nas colônias com pequena população. Essas amostras foram analisadas no Laboratório de Fitopatologia do Departamento de Defesa Fitossanitária da UFSM, onde se observou a presença de estruturas fúngicas com formação típica do fungo Ascosphaera apis, conhecido como causador da doença "cria giz". Até então não existiam notificações da presença dessa doença na região central do Estado do Rio Grande do Sul, o que pode caracterizar um avanço dessa enfermidade para o interior, a partir de regiões próximas à fronteira com o Uruguai e a Argentina.

A comparison of methods to experimentally induce chalk brood disease in honey bees

Chalk brood in honey bee is a fungal brood disease caused by Ascosphaera apis. It is very difficult to reproduce chalk brood in a homogeneous way in a large number of colonies, therefore appropriate methods to experimentally induce and evaluate the disease would be useful for research. We inoculated experimentally the colonies, using three treatments: spores sprayed over combs, spores mixed with pollen and sugar syrup with spores. The collection of mummies in traps in the hive entrance, bottom board and combs showed a lack of efficacy in the quantification of the disease. A controlled chilling of the brood yielded very satisfactory results. Using this last technique, spores mixed with water sprayed over combs reached 90.63% of mummification, while colonies fed with spores mixed up with pollen reached 86.32%. The use of sugar syrup with spores (60.13% of mummification) proved to be less effective.

Influence of Wood, Paper, and Plastic Nesting Units on Efficacy of Three Candidate Fungicides for Control of Chalk brood in the Alfalfa Leaf cutting Bee (Hymenoptera: Megachilidae)1

Field trials were conducted for three fungicides (Captan, Botran, and Carbendazim) to determine their influence on chalk brood of the alfalfa leaf cutting bee, Megachile rotundata (F.) in paper, wood, and plastic nesting units. Captan reduced disease incidence significantly in bee nests in both wood and plastic; Carbendazim had no effect; and Botran repelled adults. Bees preferred to nest in plastic units, and percentage of cells infected with chalk brood was higher in plastic. Numbers of cells per nest and disease incidence were related; as nest size increased, percentage of chalk brood increased in both wood and plastic nest units. Percentage of live larvae was related to nest size in wood units, but not in plastic units. When bees were given a choice, paper nesting units were unattractive.

Effect of Type of Brood Comb on Chalk Brood Disease in Honeybee Colonies

SummaryThe incidence of chalk brood infection was compared among groups of 10 honeybee (Apis mellifera) colonies maintained on each of 5 different comb types: comb drawn from foundation, honey-super comb that had been used for storing honey but not for brood rearing, comb that had been used as brood comb for 5–30 and 30–45 years, and old brood comb that had been fumigated with ethylene oxide prior to use. The level of chalk brood disease ranged from negligible to over 6% and was lowest in the colonies with foundation or super comb, intermediate in the colonies with fumigated comb, and highest in the colonies with the two types of older brood comb. The results indicate that old brood comb contributes to the development of chalk brood disease in honeybee colonies, possibly by serving as a reservoir for Ascosphaera apis spores or by promoting the growth of the pathogen.

Effects of Selected Chemical Treatments on Spore Germination of Ascosphaera aggregata (Ascosphaerales)

Alfalfa leafcutting bee larvae become infected with chalk brood via the gut. Chemotherapy requires that any chemical used systemically as a fungicide must be non-lethal to the developing bee. The objective of this study was to determine if components of a chemical formulation patented in Romania for honeybee chalkbrood are fungicidal or inhibitory toward the alfalfa leafcutting bee chalkbrood in vitro. The fungicide contains 0.075 parts methyl-p-hydroxybenzoate (methyl paraben), 0.300 parts sodium benzoate, 0.300 parts sodium salicylate, 1.00 part sodium bisulfite, and 8,325.00 parts sucrose. The combined chemicals without sucrose were fungicidal to Ascosphaera aggregata in vitro at a concentration of 0.30%. Methyl paraben was totally effective at 0.06%, sodium benzoate at 0.20%, sodium salicylate at 0.06%, and sodium bisulfite at 0.08%. Sucrose was not tested.

TOXICITY EFFECTS AND CHALKBROOD INCIDENCE IN HONEY BEE1 COLONIES FED CONTROLLED DOSES OF FUNGICIDES2,3

Folpet and a combination with folpet, benomyl, citral, sodium propionate, and sorbic acid were fed or exposed to honey bee, Apis mellifera L., field colonies to: 1) determine their long-term toxic effects on the bees; and 2) to determine if chalkbrood (CB) disease, Ascosphaera apis (Maassen ex Claussen) Olive et Spiltor, is inhibited or controlled by the compounds. When folpet was fed to honey bee colonies in sucrose syrup, the group fed the highest rate (1000 ppm) never differed significantly (P > 0.05) from the control colonies. There was an equal number of adult bees, an equal amount of sealed brood, and mortality was identical. Analysis indicated significantly (P < 0.05) fewer CB mummies in treatment groups fed 10 or 1000 ppm folpet, but this apparent benefit may have been due to seasonal changes of reduced CB infection that occur in late summer. Folpet or a combination of folpet, benomyl, citral, sodium propionate, and sorbic acid (1000 ppm each, total 5000 ppm) incorporated into lipid/sucrose extender patties produced no significant (P > 0.05) effect of any kind on colonies to which they were applied, including CB infection. However, when the combination of five fungicides (5000 ppm) was impregnated into beeswax foundation and exposed to honey bee colonies, only a small amount of comb was drawn on the test foundation, indicating a repellent effect. Otherwise, the test foundation had no significant (P > 0.05) effect on the honey bees or the CB infection.

Electropositive Redox State of the Fifth-Instar Larval Gut ofApis Mellifera

SummaryA platinum and calomel electrode combination was used to determine the in vivo redox potential (Eh) of the blind-ending midgut of pathogen-free 5th-instar honeybee larvae. In a sample of 20 larvae the mean Eh at 0, 5 and 10 min after electrode insertion was 147 ± 79, 134 ± 44 and 156 ± 56 mV, respectively. The pH of the gut contents was 7·5. The results suggest that anaerobic conditions do not exist in the honeybee midgut, and thus that anaerobiosis is not the stimulus which causes ascospores of the chalk brood fungus Ascosphaera apis to germinate at this site. It is suggested that an alternative stimulus or an alternative route of entry for the fungus should be considered.

Occurrence and Distribution of Chalk Brood Disease of Honeybees

(1985). Occurrence and Distribution of Chalk Brood Disease of Honeybees. Bee World: Vol. 66, No. 1, pp. 9-15.

Life Cycle of the Chalk Brood Fungus, Ascosphaera aggregata, in the Alfalfa Leafcutting Bee, Megachile rotundata, and Its Associated Symptomatology

The life cycle of Ascosphaera aggregata in Megachile rotundata and the symptomatology of the resultant disease are described. The formation of the ascogonium, and the process by which the asci are formed within it, are unlike other Ascomycetes. Variable numbers of asci develop within a membrane-bound “chamber,” which is derived from the ascogenous hyphae within the nutriocyte. Following sporogenesis, the newly developed ascospores are retained within the membrane-bound chambers where they form tightly packed, spore-containing spheres, the spore balls.

Effect of Fungicide Treatments on Incidence of Chalkbrood Disease in Nests of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae)

A fungicide (Vitavax, carboxin with captan) was field tested to determine its effect on chalk brood disease of Megachile rotundata (F.). The fungicide was blown into the holes of nesting materials. Disease incidence was significantly reduced when nesting units were treated once a week. Fungicide application had no effect on nesting behavior (number of cells per nest) or immature mortality (from causes other than chalkbrood) when treated and untreated nests were compared. Thus, fungicide applications reduced fungal growth but did not disturb nesting of the bees.

蜜蜂のChalk broodの人工感染試験と防除試験

蜜蜂のChalk broodの人工感染試験と防除試験

Pathogenicity ofAscosphaeraSpecies for Larvae of Megachile Rotundata

SummarySeveral Ascosphaera species were tested for their pathogenicity for larvae of the alfalfa leafcutting bee, Megachile rotundata. Larvae were reared aseptically on a sterile pollen-based diet and inoculated at 6 days of age with spores of the fungi. A. aggregata caused the distinctive syndrome called ‘chalk brood’. Both A. apis and A. proliperda were pathogenic for larvae and produced syndromes similar to chalk brood in Apis mellifera. Chalk brood in M. rotundata was characterized by colour changes and sporulation of A. aggregala beneath the host cuticle. A. apis and A. proliperda caused no colour changes in infected larvae; white mycelia erupted through the cuticle before sporulation occurred. A. atra and A. major grew saprophytically on provisions of leafcutting-bee cells and did not cause larval disease. Spores taken from larvae killed by A. apis or A. proliperda grew less readily on the pollen diet, and appeared to be more virulent, than spores taken from subcultures.

Development of Chalk Brood in a Honeybee Colony: A Review

Development of Chalk Brood in a Honeybee Colony: A Review

Emission of Volatile Sulphide from Residues of Diseased Honeybee Larvae

SummaryAmerican foul brood scales and chalk brood mummies produced a volatile sulphide which was detected by lead acetate strips. A positive reaction required a minimum of 0·03–0·1 g larval residue per ml volume of container and an exposure time of 2–4 weeks. However, the method proved to be non-specific and is not sufficiently sensitive for detection of disease in honeybee colonies.

岐阜県下で確認された密蜂のChalk Brood

An outbreak of chalk brood disease in honeybees was seen in Gifu in March, 1979. Diseased larvae found in sealed or unsealed cells. Most of them were in the upright stage. They were grayish white or had black spots.The fungus Ascosphaera apis was isolated from white mummified larvae about 4 days old.

Transmission of Chalk Brood Disease of Honeybees by Infected Queens, and Worker Brood and Adults

SummaryChalk brood infections were produced in disease-free nuclei by transferring honeybee queens, adult workers, and unsealed or sealed brood from diseased colonies. Bees fed Wheast patties remained disease free for 7 weeks, but all nuclei except the controls were infected 2 weeks after they were fed 2-year-old pollen. Bees that removed the diseased larvae most readily recovered from the disease and reared as much brood to the sealed stage as the controls. This suggests that selection for the best hygienic behaviour may be one way to control chalk brood. The disease disappeared in all nuclei during the study, but we were able to recover the fungus in either macerated queens or workers taken from the infected nuclei of bees by streaking suspensions onto plates of potato dextrose agar fortified with 4 g/litre yeast extract.