Effect of hygienic behavior on resistance to chalkbrood disease (Ascosphaera apis) in Africanized bee colonies (Apis mellifera)

Medina-Flores, Carlos Aurelio; Medina Medina, Luis Abdelmir; Guzmán-Novoa, Ernesto; Medina-Flores, Carlos Aurelio; Medina Medina, Luis Abdelmir; Guzmán-Novoa, Ernesto

doi:10.22319/rmcp.v13i1.5907

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Revista mexicana de ciencias pecuarias

versión On-line ISSN 2448-6698versión impresa ISSN 2007-1124

Rev. mex. de cienc. pecuarias vol.13 no.1 Mérida ene./mar. 2022 Epub 06-Jun-2022

https://doi.org/10.22319/rmcp.v13i1.5907

Articles

Effect of hygienic behavior on resistance to chalkbrood disease (Ascosphaera apis) in Africanized bee colonies (Apis mellifera)

Carlos Aurelio Medina-Flores^a

Luis Abdelmir Medina Medina^b

Ernesto Guzmán-Novoa^c

^{^a} Universidad Autónoma de Zacatecas. Unidad Académica de Medicina Veterinaria y Zootecnia, Zacatecas, México.

^{^b} Universidad Autónoma de Yucatán. Departamento de Apicultura, Campus de Ciencias Biológicas y Agropecuarias. Carretera Mérida-Xmatkuil Km. 15.5, Mérida, Yucatán, Mexico.

^{^c}School of Environmental Sciences, University of Guelph, Guelph, Canada.

Abstract

The objective was to evaluate the hygienic behavior (HB) of Africanized honeybees (Apis mellifera) and its impact on resistance to ascospherosis caused by Ascosphaera apis. The HB and the population of adult bees and brood of 50 colonies were evaluated. In addition, colonies with high (>95 %) and low (<50 %) HB were inoculated with A. apis and in them, the number of broods with signs of ascospherosis (mummies) was determined for 17 days, data that correlated with their degree of HB. The susceptibility to A. apis of larvae from colonies with high and low HB in a common environment was also evaluated to separate environmental from genotypic effects. The degree of HB between colonies varied significantly (CV>36 %), with 20 % of the colonies showing high HB (≥95 %) and this did not correlate with the population of adult bees and brood. Colonies with high HB had significantly fewer mummies than colonies with low HB and there was a negative correlation between HB and number of mummies (r= -0.63, P= 0.02). In addition, larvae from colonies with high or low HB were equally susceptible to the fungus. These results suggest that HB and larval susceptibility are not associated and that the main protection mechanism against A. apis in Africanized bee populations is HB. Therefore, the selection of colonies with high hygienic behavior could contribute to improving the health and productivity of honeybees.

Key words Apis mellifera; Ascosphaera apis; Hygienic behavior; Larval susceptibility; Africanized bees; Yucatán

Resumen

El objetivo fue evaluar el comportamiento higiénico (CH) de abejas melíferas (Apis mellifera) africanizadas y su impacto en la resistencia a la ascosferosis causada por Ascosphaera apis. Se evaluó el CH y la población de abejas adultas y de cría de 50 colonias. Además, colonias con alto (>95 %) y bajo (<50 %) CH fueron inoculadas con A. apis y en ellas se determinó el número de crías con signos de ascosferosis (momias) durante 17 días, datos que se correlacionaron con su grado de CH. También se evaluó la susceptibilidad a A. apis de larvas de colonias con alto y bajo CH en un medio ambiente común, para separar efectos ambientales de genotípicos. El grado de CH entre colonias varió significativamente (CV>36 %), con 20 % de las colonias mostrando alto CH ((95 %) y éste no se correlacionó con la población de abejas adultas y cría. Las colonias con alto CH tuvieron un número significativamente menor de momias que las colonias con bajo CH y hubo una correlación negativa entre el CH y el número de momias (r= -0.63, P= 0.02). Además, las larvas de colonias con alto o bajo CH fueron igualmente susceptibles al hongo. Estos resultados sugieren que el CH y la susceptibilidad de las larvas no están asociados y que el principal mecanismo de protección contra A. apis en poblaciones de abejas africanizadas es el CH. Por ello, la selección de colonias con alto comportamiento higiénico podría contribuir a mejorar la salud y productividad de las abejas melíferas.

Palabras clave Apis mellifera; Ascosphaera apis; Cría calcárea; Comportamiento higiénico; Susceptibilidad de larvas; Abejas africanizadas; Yucatán

Introduction

Ascospherosis or chalkbrood, is a disease caused by the fungus Ascosphaera apis, which when it reproduces and sporulates in the larvae of honeybees (Apis mellifera) causes their mummification (black and white mummies), reduces the population size of their colonies, and in some regions can cause high losses in honey production¹^,².

In the presence of A. apis and other health problems of the brood, honeybees can respond with behavioral and physiological resistance mechanisms³^,⁴. An important behavioral mechanism is hygienic behavior (HB), which consists of the ability of workers to detect, uncap and remove from inside the cells the brood that is sick or dead⁵^,⁶. The evaluation of the HB level of a bee colony is based on sacrificing broods inside capped cells by puncture⁷ or freezing⁸ and determining the percentage of removal in a short period of time by the workers. It has been reported that colonies with very high HB (≥95 % removal of the dead brood in 24 to 48 h) show some degree of resistance to chalkbrood⁹^,¹⁰ and American foulbrood¹¹^-¹³, and there is some evidence of resistance of highly hygienic colonies against the parasitic mite Varroa destructor¹⁴ and the deformed wing virus¹⁵. It has also been argued that physiological mechanisms associated with the cellular and humoral immunity of bees could give them resistance against ascospherosis⁴. Therefore, it is not clear what is the contribution and relative importance of HB to the resistance of bees against chalkbrood, particularly in Africanized bee populations.

The HB has been reported to provide protection against the fungus A. apis in colonies of Africanized bees crossed with Europeans from South America¹⁰. However, it is unknown whether this also happens in Africanized bee populations in other countries. In addition, the relationship between HB expression and brood susceptibility in Africanized bees infected with the fungus A. apis has not been studied. Generating such information would be very useful to design control strategies against the disease, such as establishing selective breeding programs.

In Mexico in particular, it is known that in certain regions the prevalence of chalkbrood can exceed 50 %, especially in humid areas¹⁶, and that this disease can interact with others and cause bee colonies to collapse¹⁷. In addition, in Mexico, the relationships of HB with ascospherosis and the susceptibility of Africanized bee larvae to the fungus have not been studied.

Therefore, the objective of this study was to evaluate the degree of HB of Africanized bee colonies and its impact on the resistance and susceptibility of these insects to chalkbrood.

Material and methods

Place of study

This study was carried out in experimental apiaries of the Department of Beekeeping of the Campus of Biological and Agricultural Sciences of the Autonomous University of Yucatán, in Xmatkuil, Mérida, Yucatán, Mexico (20° 52’ 3.00” N, 89° 37’ 29.15” W). This region has a hot-subhumid climate with rainfall in summer (Awo), with annual rainfall of 985 mm, annual temperature of 26.8 ºC and annual relative humidity of 78 %¹⁸.

Hygienic behavior and bee population

The studies were carried out in 50 colonies of commercial bees to which a morphometric analysis was carried out in order to confirm that they were Africanized¹⁹. The colonies were housed in Langstroth-type hives, distributed in five apiaries, and had different population conditions and food reserves, but without clinical signs of diseases. Colony bee populations were determined for both the capped brood area and the number of adult bees. To determine the brood area of each colony, two operators estimated the percentage of the area on both sides of each comb occupied by capped brood. The percentage area of the brood areas was converted to cm², considering the area that has a Langstroth-type comb on both sides (1,760 cm²). In addition, the number of combs covered with bees was recorded and multiplied by the number of bees that occupy a Langstroth comb in the brood chamber on both sides (2,430 bees)²⁰. The measurements were made in the afternoons (> 1700 h) when most of the bees were inside the hives and the same operators participated in all the measurements.

The HB of the colonies was evaluated twice. The two assessments were conducted during July when there are no major blooms in the area and were made with an interval of 14 days. To estimate the HB level of each colony, a comb with capped brood with 3 to 4-day old pupae, identified by the white body and purple eyes²¹, the most appropriate stage to determine HB²², was selected. A galvanized sheet cylinder (8 cm diameter x 10 cm height) was placed over a compact capped brood area and 300 ml of liquid N₂ was poured to sacrifice the pupae inside the cells by freezing. When the N₂ evaporated, the cylinder was removed, and the frozen areas were photographed, and the comb was reintroduced into the colony being evaluated. The combs with the sacrificed brood of the experimental colonies were inspected 48 h after the previous procedure and the frozen areas were photographed again to record the number of dead pupae that were removed, and thus be able to determine the percentage of removal of the dead brood. The evaluations were carried out 48 hours after freezing because it is enough time to limit the reproduction and spread of an infectious agent, which is less strict than at 24 h and allows identifying the expression of HB in colonies not selected for this characteristic and naturally mated. Colonies that uncapped and removed 95 % or more of the frozen brood in the two tests were classified as highly hygienic (high HB) while colonies that removed 50 % or less of the frozen brood were considered to have low HB⁸^,¹². Subsequently, 10 colonies that presented a high HB and 10 colonies that presented a low HB were selected in order to evaluate their relative resistance to ascospherosis.

Effect of hygienic behavior on ascospherosis

The 20 colonies with high and low HB selected from the previous experiment were relocated to an isolated apiary and their queens were marked with indelible ink on the thorax for their identification. The bee populations of both groups of colonies were homogenized based on the colony that had the least number of bees, brood area, honey and pollen reserves. Therefore, at the beginning of the experiment, the 20 colonies had approximately the same number of combs covered with adult bees (8), capped brood (3), open brood (2), honey (2) and pollen (1). Additionally, it was corroborated that no selected colony presented clinical signs of chalkbrood (mummies present in the combs, floor, or entrance).

To obtain the fungus A. apis, black mummies (sporulated fungus) were initially identified from colonies outside the experiment. This clinical sign is pathognomonic of ascospherosis so there is certainty of obtaining the fungus from these mummies. Additionally, the sporocysts of the fungus were observed under a microscope in mummy samples²³. To induce A. apis infection in brood of the experimental colonies, the protocol of Flores et al²⁴ was followed. Three mummies per milliliter of distilled water were macerated. Each of the 20 experimental colonies was inoculated with 6 ml of the macerate diluted in 120 ml of sucrose syrup (1:1), which was supplied to each colony by means of Boardman-type feeders. In addition, two combs containing young larvae (open brood) on at least 80 % of their area, as well as the bees present in those combs, were sprayed with 6 ml of the same macerate diluted in 14 ml of sucrose syrup (1:1), supplying 5 ml of the inoculum on each side of each comb. The colonies were reviewed to record the number of black and white mummies present in the comb cells on days 3, 5, 7, 9, 12 and 17, post-exposure²⁵.

Susceptibility of larvae from high and low HB colonies to ascospherosis

In order to assess whether the differences in the number of mummies in the combs between colonies with high and low HB from the previous experiment were due to any extent to differences in the susceptibility of their larvae to the fungus A. apis, larvae from five colonies of each type selected at random were used to inoculate them and allow their development in a common environment. From each colony, a comb section (7 x 7 cm) containing an average of 264 ± 3.4 larvae 3 to 4 days old was cut with a knife. Immediately afterwards, racks were assembled, each containing a section from a colony with high HB and another from a colony with low HB, these sections and five receiving colonies that had a low HB were inoculated with the fungus A. apis as described above. Each comb assembled in this way was placed in the center of the brood chamber of a receiving colony in order to give the larvae the same nest environment, and that the probability of being removed by the HB of the bees from the receiving colonies was similar for both types of larvae. Making larvae and bees cohabitate in the same brood nest has been used successfully in the past to separate environmental from genotypic effects in studies of various bee behaviors, including HB²⁶^-²⁹. The number of chalkbrood mummies in the comb sections was recorded on d 5, 9 and 13, post-exposure to the fungus.

Statistical analysis

Measures of central tendency and dispersion were obtained for the data from HB evaluations and population conditions of the 50 colonies. Pearson’s correlation tests were also performed between the data from the first and second test of HB, as well as between those from the HB and those from the bee population, brood areas, and the number of mummies recorded in the combs. The percentage of HB, brood areas and bee population of colonies with high and low HB that were selected for testing the effect of HB on chalkbrood, as well as the proportion of larvae clinically affected by ascospherosis of the two types of colonies that were used in the susceptibility experiment, were analyzed with Student’s t-tests. The number of mummies from colonies with high and low HB and the effect of time on this variable were analyzed by means of repeated measures variance and Newman-Keuls comparison of means tests. Prior to the analyses, the percentage values of HB and mummified broods (susceptibility test) were transformed to square root of the arcsine and the number of mummies to logarithm, to ensure a normal distribution of the data. Statistical analyses were performed in the SAS program³⁰.

Results

Hygienic behavior and bee population

Table 1 shows the degree of HB and population conditions of the 50 colonies, as well as the variation for these parameters. Clearly, there was a wide range and variability for the degree of HB between the colonies evaluated (CV >36 %). However, it is noteworthy that 20 % of them had a high HB (≥95 %), 30 % had a low HB (≤50 %) and 50 % had an intermediate level of HB (51-94 % of frozen brood removal). The adult bee population was also highly variable (CV >37 %), but not the amount of brood in the colonies (CV <18 %).

Table 1 Mean and dispersion values of hygienic behaviour, estimated bee population and brood areas in two tests to 50 honeybee colonies

Descriptive statistics	Hygienic behavior (%)	Brood areas (cm²)	Adult bee population
Mean	66.12	9,574.4	32,683
Standard error	0.48	33.8	245.43
Coefficient of variability, %	36.2	17.5	37.5
Minimum value	12.5	5,820	9,720
Maximum value	100	12,320	53,460
Range	87.5	6,500	43,740

There was a positive and significant correlation between the HB level of the first and second evaluation (r= 0.60, P= 0.0001), so the pupae freezing test showed repeatability. On the other hand, no relationship was found between the HB level and the number of adult bees (r= -0.03, P= 0.84) or with the capped brood area of the colonies (r= 0.02, P= 0.87), so it is presumed that these factors did not significantly influence the degree of HB of the colonies.

Effect of hygienic behavior on ascospherosis

The colonies with high and low degree of HB that were selected for the test of relative resistance to chalkbrood differed significantly in their level of HB (t= 8.71, P<0.0001), but did not differ in terms of population of adult bees (t= 0.10, P= 0.75) or brood (t= 2.02, P= 0.17). Mean HB levels were 31 ± 0.81 and 97 ± 0.20 %, for colonies with low and high HB, respectively.

After being exposed to A. apis, the clinical manifestation of chalkbrood (presence of mummies in combs) was observed at 3 d post-exposure, and although the amount of affected brood was similar in both colony groups until day five post-exposure, colonies of high HB had significantly fewer white (F_1,76= 32.1, P<0.0001) and black mummies (F_1,76= 10.8, P<0.001) in the combs than the colonies of low HB. In addition, the number of black and white mummies in the combs of both colony groups decreased significantly and progressively between days 9 and 17 post-exposure (F_4,76= 3.2, P= 0.01 and F_4,76= 2.6, P= 0.03, respectively), but there was no interaction effect between the degree of HB and the days post-exposure to the fungus in white (F_4,76= 0.7, P= 0.61) and black mummies (F_4,76= 0.95, P= 0.45; Figures 1 and 2).

^ab Different literals indicate significant differences (P<0.05) based on a repeated-measures analysis of variance and the Newman-Keuls comparison of means test, after transformation of the data to logarithm. Untransformed values are displayed.

Figure 1 Number (mean ± SE) of white mummies recorded in the combs of colonies with high (■) and low (▲) hygienic behavior (HB) after inoculation with the fungus A. Apis

Figure 2 Number (mean ± SE) of black mummies recorded in the combs of colonies with high (■) and low (▲) hygienic behavior (HB) after inoculation with the fungus A. Apis

In addition to the above, a negative and significant correlation was found between the level of HB and the number of total mummies of the colonies inoculated with A. apis for the tests of resistance to chalkbrood (r= -0.63, P= 0.02). This result indicates that, the higher HB, the fewer mummies in the combs of the colonies, so this behavior seems to give resistance to honeybees against ascospherosis.

Susceptibility of larvae from colonies with high and low HB to ascospherosis

Regarding the susceptibility of larvae from colonies with high and low HB to the fungus A. apis, it was found that the proportion of mummies in the combs was not statistically different between both groups of larvae throughout the evaluation period (Table 2). In addition, there was no significant correlation between the HB level of the larval donor colonies and the percentage of mummies found in the combs post-exposure of the larvae with A. apis (r= 0.21, P= 0.55).

Table 2 Percentage (mean ± SE) of larvae from colonies with high and low hygienic behavior (HB) that clinically manifested ascospherosis post-exposure with A. apis

Post-exposure days with A. apis	High HB (n= 5)	Low HB (n= 5)	t	P
5	6.54 ± 0.77	5.32 ± 0.33	3.93	0.21
9	5.32 ± 3.24	4.14 ± 1.60	1.30	0.80
13	1.34 ± 1.81	1.46 ± 1.01	1.91	0.54

The t and P values were obtained from the analysis of data on the percentage of mummified broods transformed to the square root of the arcsine.

Discussion

Evaluations of the HB and strength of the population of the Africanized bee colonies studied showed wide variation as expected, but showed no correlation, which suggests that it is possible to identify bee colonies that vary in their HB regardless of their strength. These results coincide with what was reported in previous studies¹⁰^,¹²^,³¹. In addition, the N₂ test used to measure HB showed high correlation between repetitions, which indicates that it is reliable as previously demonstrated³² and allows the categorization of colonies with different degrees of expression for this behavior. The frequency (20 %) of colonies that expressed a high degree of HB is within the ranges shown by European and Africanized bee colonies (10-31.5 %) in other regions¹²^,³³^-³⁵. And this low frequency could be increased by breeding queens from colonies with high HB, allowing their free fertilization, as Spivak and Reuter¹² and Bigio et al³¹ have shown in practice. This is based on the fact that part of the variability of HB is of genetic origin³⁶^,³⁷, that HB is a highly heritable behavior³⁸^-⁴¹ and that this is inherited through the mother²⁸.

Colonies selected for their high HB had significantly fewer broods with clinical signs of ascospherosis in the combs than colonies selected for their low HB. This was presumably because they removed more diseased brood from the combs than the colonies with low HB, and they did so progressively as the evaluation time passed. These results coincide with those of studies carried out in colonies with bees of European origin⁴²^,⁴³ and with those of a study carried out in colonies of Africanized bees crossed with Europeans¹⁰. The detection of larvae infected by A. apis depends on the perception of volatile compounds by bees⁴⁴^-⁴⁶, with variation in the detection thresholds of these compounds in workers. Bees of strains selected for high HB are more sensitive and react to the smell of these compounds more frequently than bees not selected for this behavior⁴⁷. It has also been suggested that the efficiency of HB against chalkbrood depends on the early detection of larvae infected by the fungus prior to their mummification and sporulation, which limits the spread of the infection in the colony¹⁰. Timely removal of diseased broods reduces the risk of sporulation and spread of the fungus⁹. This may explain why in colonies with high HB there were significantly fewer black and white mummies in combs compared to colonies with low HB. This conclusion is reinforced by the results of the correlation analysis that showed that, at a higher the degree of HB, the number of broods infected with A. apis in the combs of the bee colonies studied decreased significantly.

It has been speculated that the resistance of bees to brood diseases depends on the interaction of physiological resistance factors such as the immune response to different pathogens, the antimicrobial activity of the microbiota and larval foods, as well as HB and other defense mechanisms⁴⁸^,⁴⁹. This study analyzed for the first time the susceptibility of larvae to A. apis in a common nest environment and found that larvae from colonies with high and low HB were equally susceptible to ascospherosis. Therefore, the results of the present study allow inferring that the level of HB expression and the susceptibility of larvae to A. apis are not associated, and support the hypothesis that although various factors and mechanisms could contribute to the resistance of bees against ascospherosis⁵⁰, HB is the most important of them. Therefore, it is suggested that efforts for the development of colonies resistant to brood diseases, such as ascospherosis, be focused on the selection of colonies with high HB.

The frequency of colonies with high HB from genetic improvement programs is higher than that found in unselected populations¹² and it has been reported that this behavior is associated with greater honey production¹²^,³⁴^,⁵¹ and greater resistance to diseases⁹^,¹²^-¹⁵^,³⁶; the latter is consistent with the results of the present study. Favorably, the selection and reproduction of queens from colonies with high HB seems to be sufficient to increase the frequency of colonies with high HB in bee populations³¹^,³⁹^,⁴¹, because HB is inherited mainly through the mother²⁸, so the implementation of selection and reproduction programs of queens whose colonies express a high HB would contribute to improving the health and productivity of the colonies.

Conclusions and implications

It is concluded that the degree of HB between colonies is variable, and that the population of bees and broods do not significantly influence the HB degree of the colonies. The frequency of colonies with high HB in the Africanized bee population analyzed would allow genotypes to be selected to increase the HB degree of bee populations in genetic improvement programs. Colonies with high HB had greater control of ascospherosis than colonies with low HB. The susceptibility of larvae to ascospherosis and HB do not appear to be associated factors. These results suggest that the main protective mechanism against A. apis in Africanized bee populations is HB. Future research is needed to study the humoral and cellular immunity of bees against A. apis, as well as the mechanisms for its identification and associated factors, so that they are integrated into genetic selection and improvement programs in order to improve the health and production of honeybee populations.

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Received: December 18, 2020; Accepted: April 29, 2021

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