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Centro Andaluz de Apicultura Ecológica, Universidad de Córdoba, Campus Universitario de Rabanales, 14071 Córdoba, Spain
R. Espejo
Departamento de Estadística e I. O. Universidad de Córdoba, Spain
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ABSTRACT |
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Chalkbrood in honeybees (Apis mellifera L. Himenoptera: Apidae) is a fungal disease caused by Ascosphaera apis (Maassen ex Claussen) Olive and Spiltoir. This disease requires the presence of fungal spores and a predisposing condition in the susceptible brood for the disease to develop. In this study we examined the role of pollen in the development of chalkbrood disease under two experimental conditions: (i) pollen combs were transferred from infected to uninfected beehives and (ii) colonies were deprived of adequate pollen supplies to feed the brood. The results of both treatments confirmed that pollen is an element that should be taken into account when controlling this honeybee disease.
Key words: Ascosphaera apis, fungal disease, epidemiology, honeybee
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Larvae in the fifth stage, before and some hours after sealing, are most susceptible to the possible stress that triggers the disease (Bayley 1967, Puerta et al 1994, Flores et al 1996).
Due to the lack of effective treatments and the need to preserve the wholesome and natural characteristics of beekeeping products (Puerta et al 1995, Flores et al 2001), alternative methods must be developed to control chalkbrood. To determine the best control measures, it is important to know more about how the disease is transmitted and the possible predisposing conditions that trigger the disease. However, until techniques were developed that allowed the experimental expression of clinical symptoms in a controlled way while maintaining the natural conditions of the beehives as far as possible, the various hypotheses that had been put forward regarding this disease could not be confirmed (Puerta et al 1994, Flores et al 1996).
In previous research (Puerta et al 1994) we experimentally induced clinical symptoms of chalkbrood by chilling the susceptible brood, the starting point for further research on the disease. Later, using this same technique, we studied other possible predisposing conditions, such as excess dampness (Flores et al 1996). Furthermore we confirmed the lack of effective products to control the disease (Flores et al 2001). Nevertheless, further research was required on other topics, namely the role of pollen in the transmission of fungal spores and whether insufficient pollen supply could be a predisposing condition.
Pollen is harvested by the honeybee as a source of protein and is consumed by adults so they can feed the brood, making it a key factor in the development of the colonies. Pollen combs may be agents of transmission of A. apis spores and other possible bee diseases because beekeepers frequently use pollen combs from other beehives or weakened, ill or dead colonies to feed other colonies (reviewed by Heath 1982, Puerta et al 2001).
In addition pollen is harvested by beekeepers by placing a pollen trap at the entrance to the beehives. The overuse of pollen traps may lead to an inadequate pollen supply in the beehives and has been proposed as another possible predisposing condition in the development of brood diseases such as chalkbrood (Cardenal et al 1990).
Thus pollen may play an important role in this disease of the bee brood. In this study we used chilled brood to examine the role of pollen combs from infected colonies as a source and agent of transmission of chalkbrood, as well as insufficient pollen supply as a predisposing condition in the development of the infection.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The risk of becoming infected with chalkbrood disease was evaluated in newly capped brood (worker brood sealed 14 h). Unsealed fifth instar larvae (Rembold et al 1980) were marked on plastic sheets. The brood combs subsequently were returned to the beehives and again removed after 14 h. Portions of combs with newly sealed brood were removed and maintained in incubators at 25 ± 1 C and 60% relative humidity. Five days after sealing, the cells were opened and the percentage of mummified larvae was checked (Flores et al 1996). The number of investigated cells are provided (TABLE I).
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).
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Six days after applying the treatments, clinical symptoms of the disease were evaluated. Sections of susceptible brood combs were removed weekly from each colony and the disease was induced as described in Experiment 1, for a total of three times (18 and 25 Nov and 2 Dec). The availability of brood decreased after the third evaluation, making it necessary to conclude the experiment. The number of investigated cells are provided (TABLE II). The data obtained were evaluated statistically using descriptive parameters, analysis of variance (one-way ANOVA and univariate analysis of variance) and post-hoc tests (Duncan’s multiple range test, P < 0.05) (SPSS 8.0).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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After introducing infected pollen combs in the initially healthy beehives (Group B), the disease was observed to increase. In contrast, the percentage of mummification in the initially sick colonies decreased. Finally the disease remained at a low level in the healthy colonies that had not received infected pollen combs (FIG. 1
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Statistical analysis (univariate analysis of variance) showed that the applied treatments had a significant effect (F = 12.146; df = 2; P = 0.000), resulting in three statistically different groups (Duncan’s multiple range test, P < 0.05): the initially diseased colonies with a larger percentage of mummified brood; a second group composed of the initially healthy colonies in which infected pollen combs had been introduced; and a third group comprising the healthy colonies that did not receive infected pollen combs and which had the smallest percentage of mummification. A significant effect was not observed among evaluations within each treatment (F = 0.6487; df = 3; P = 0.591).
Experiment 2.— –
On the first evaluation date (4 Nov) significant differences in percentage of mummification were not found among the three groups of beehives (one-way ANOVA, F = 0.046; df = 2; P = 0.955) (FIG. 2). The first evaluation after removing the stored pollen of groups A and B (18 Nov) showed that chalkbrood decreased in the control group (C), levels that then were maintained during the rest of the experiment. In the group of beehives from which the stored pollen was removed (Group A), the disease remained at high levels until the last evaluation (2 Dec), at which time it decreased. Finally, in the group of colonies from which the stored pollen was removed and in which a pollen trap was placed to prevent the entrance of fresh pollen (Group B), chalkbrood initially decreased, then rose to the highest level and then dropped of the experiment (FIG. 2).
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). However significant differences were not observed across dates within each treatment (F = 1.584; df = 2; P = 0.213). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Gilliam 1990). Moeller and Williams (1976) and Nelson and Gochnauer (1982) suggested that bees could harvest pollen contaminated with A. apis spores from flowers after they found mycelial growth on this pollen in specific culture medium. Nevertheless it is possible for pollen to be contaminated by spores from forager bees (Koenig et al 1987). However we suggest that the small number of spores entering the beehives in this manner does not significantly increase the risk of infection because the amount of spores has a direct influence on the development of chalkbrood (Puerta et al 1990). It is likely therefore that the most important agent in the transmission of this disease is the transference of either live or inert contaminated material from infected hives (reviewed by Heath 1982, Gilliam 1990).
Pollen stored as beebread in infected beehives may be a source of chalkbrood spores (reviewed by Heath 1982). Nevertheless, due to the lack of appropriate techniques, it has not been possible to confirm this hypothesis. On the other hand research has been carried out along these lines with artificially infected pollen (Mehr et al 1976, Moffett et al 1978, Befus-Nogel et al 1992). In our research we used pollen combs from contaminated hives, approaching what could be considered common beekeeping practice.
Our results initially would seem to suggest that contaminated pollen has an immediate effect, leading to an increase in chalkbrood. Nevertheless this was possible in our experiment due to the fact that the susceptible brood was artificially chilled, immediately triggering the disease. However this does not normally occur in the apiaries of beekeepers, in which a delay in the performance of a possible predisposing condition may lead to the development of the disease at a later time. This gives rise to confusion of the beekeepers who do not associate the subsequent development of chalkbrood with poor management practices carried out at an earlier time.
In a second reading the results of our experiment have shown how the viability of spores increases the risk of chalkbrood. These findings agree with those by Hale and Menapace (1980) who found that the A. apis spores can remain viable at least 12 mo in beebread. When this is the case, the existence of a predisposing condition will trigger chalkbrood, producing new spores during the normal cycle of the disease. In contrast the absence of a predisposing condition will permit infected material to be eliminated gradually, thus decreasing the risk of chalkbrood. This is similar to what has been observed in the group of initially sick colonies, where the disease decreased, aided by the advantageous environmental conditions of the experiment (mild and stable temperatures and heavy nectar flow). It is possible that, if this experiment was carried out in fall, under worse climatic conditions and inferior nectar flow, the reduction of chalkbrood could be slower or the disease could even be increased. The same thing could happen in treatment B.
In addition 2.82% of the brood showed chalkbrood symptoms in treatment C (FIG. 1). We hypothesize that the expression of chalkbrood in this treatment was due to a small number of circulating spores in each colony.
Finally we cannot overlook the fact that pollen combs contaminated with A. apis spores constitute a reserve of infectious material in the beehive. Thus a measure of control in infected beehives should include either the substitution of infected pollen combs for pollen combs from healthy beehives or a pollen substitute.
On the other hand the deficit of proteins repeatedly has been considered a possible predisposing condition in chalkbrood (reviewed by Heath 1982, Gilliam 1990). It also has been suggested that the disease is aggravated as much by the supply of old pollen (reviewed by Heath 1982) as by the use of pollen traps (Cardenal et al 1990). These latter authors, however, obtained contradictory results because pollen traps were found to be associated with the disease in a given beekeeping season, but not in the following one.
In our study we have examined two circumstances in which the deficit of pollen could act as a predisposing condition. In the first we removed the stored pollen, but let the bees harvest fresh pollen (Experiment 2; Group A), leading us to determine that this deficit has only an occasional impact on the development of the disease. In contrast, in the second, we removed the stored pollen and placed a pollen trap at the entrance to the beehive, thus achieving a long-term protein deficit (Experiment 2; Group B). In both cases, chalkbrood was aggravated compared to the control colonies, although the tendency seems to indicate that the risk of developing the disease remained higher in the colonies in which the pollen trap impeded the harvesting of fresh pollen. For this reason we recommend that beekeepers use pollen traps with caution, do not leave them on the beehives for long periods and closely supervise any pollen deficit. It is possible that, if this experiment was carried out in spring (mild and stable temperatures and heavy pollen entrance and nectar flow), the reduction of chalkbrood in treatments A and C could be quicker.
Finally the joint effect of both experiments must be considered. Although in Experiment B we placed a pollen trap on the beehive before removing the stored pollen, thereby causing a greater deficit of protein in the colonies, this would not occur in normal beekeeping practice. Beekeepers normally do not remove the pollen combs before using pollen traps, therefore letting the bees resort to this reserve supply to continue feeding the brood. This can cause the spores remaining in the beebread to recirculate and lead to two possible effects in the infected colonies. First, if the increased number of circulating spores does not coincide with a predisposing condition for chalkbrood to develop, it probably will have a beneficial effect by reducing the spores of the fungus in the colonies. On the contrary, if it coincides with a predisposing condition, the disease could increase in the colonies, producing more spores. This situation could be aggravated further by a persistent deficit of pollen, which then would act as a predisposing condition. Nonetheless further research must be carried out along these lines if we are to confirm these last suppositions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: ba1flsej{at}Uco.es
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Befus-Nogel J, Nelson DL, Lefkovitch LP. 1992. Observations on the effect of management procedures on chalkbrood levels in honey bee (Apis mellifera L.: Hymenoptera: Apidae) colonies. Bee Sci 2:20–24.
Cardenal JA, Alonso JM, Antón JM, Hermoso J, Hermoso M, Naranjo G, Rey JM. 1990. Ascosferiosis, análisis de los factores que favorecen y desencadenan la enfermedad. Vida Apí 40:54–58.
Flores JM, Puerta F, Gutiérrez I, Arrebola F. 2001. Estudio de la eficacia del Apimicos-B® en el control y la prevención de la ascosferiosis en la abeja de la miel. Rev Iberoam Micol 18:187–190.[Medline]
———, Ruiz JA, Ruz JM, Puerta F, Bustos M, Padilla F, Campano F. 1996. Effect of temperature and humidity of sealed brood on chalkbrood development under controlled conditions. Apidologie 27:185–192.[CrossRef]
Gilliam M. 1990. Chalkbrood disease of honey bees, Apis mellifera, caused by the fungus, Ascosphaera apis: a review of past and current research. In: Proceedings, 5th International Colloquium on Invertebrate Pathology and Microbial Control. The XXIII Annual Meeting of the Society for Invertebrate and Pathology, 20–24 Aug. Adelaide, Australia, p 398–402.
Hale PJ, Menapace DM. 1980. Effect of time and temperature on the viability of Ascosphaera apis. J Invertebr Pathol 36:429–430.[CrossRef]
Heath LAF. 1982. Development of chalkbrood in a honeybee colony: a review. Bee World 63:119–130.
Koenig JP, Mallory G, Erickson EH. 1987. Isolation of the chalkbrood pathogen, Ascosphaera apis, from honeybee (Apis mellifera) surfaces, pollen loads, and a water source. Am Bee J 127:581–583.
Mehr Z, Menapace DM, Wilson WT, Sackett RR. 1976. Studies on the initiation and spread of chalkbrood within an apiary. Am Bee J 116:266–268.
Moeller FE, Williams PH. 1976. Chalkbrood research at Madison, Wisconsin. Am Bee J 116:484–495.
Moffett JO, Wilson WT, Stoner A, Wardecker A. 1978. Feeding commercially purchased pollen containing mummies caused chalkbrood. Am Bee J 118:412–414.
Nelson DL, Gochnauer TA. 1982. Field and laboratory studies on chalkbrood disease of honeybees. Am Bee J 122:29–34.
Puerta F, Flores JM, Bustos M, Padilla F. 1995. Efecto del ácido tricloroisocianúrico sobre la germinación de las esporas de Ascosphaera apis. Rev Iberoam Micol 12:49–51.
———, ———, ———, ———, Campano F. 1994. Chalkbrood development in honeybee brood under controlled conditions. Apidologie 25:540–546.[CrossRef]
———, ———, Ruíz JA, Ruz JM, Campano F. 2001. Enfermedades fúngicas. In: Enfermedades de las abejas: prevención, diagnóstico y tratamiento. Ed. COAG Andalucía. Consejería de Agricultura y Pesca. Junta de Andalucía, Sevilla. Spain. p 89–106.
———, Pellin P, Flores JM, Bustos M, Padilla F. 1990. Influencia de la dosis infectante en la aparición de la ascosferiosis en Apis mellifera y notas sobre su desar-rollo. Rev Iberoam Micol 7:11–14.
Rembold H, Kremer JP, Ulrich GM. 1980. Characterization of postembryonic developmental stages of the female castes of the honeybee, Apis mellifera L. Apidologie 11:29–38.[CrossRef]
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