THE DEVELOPMENT OF THE INFECTION OF APIS MELLIFERA LARVAE BY ASCOSPHAERA APIS

Paweł Chorbiński

ABSTRACT

The fungus Ascosphaera apis (Plectomycetes, Ascosphaeraceae) (Maassen ex Claussen), is the causative agent of chalkbrood disease in honey bees (Apis mellifera L.). By using the histological studies, the process of infection of honey bee brood by the Ascosphaera apis was observed, by feeding 3rd instar larvae with a dose of 5 x 10⁵ ascospores in 5 µl of 35% (w/v) sucrose solution. The spores were seen in the midgut lumen. The young hyphae penetrated the peritophic membranes, epithelial cells and basal membrane. After 3 days, the hyphae grew in haemocoel, the fat body and other larval tissues. During the following 2 days, the hyphae penetrated the integument and grew aerially. The first spore cysts were observed on 6th day after the penetrating of the integument of infected larvae. The hyphae of Ascosphaera apis was not observed inside the trachea.

Key words: Ascosphaera apis, honeybee, brood cells, infection, histology.

INTRODUCTION

Ascosphaera apis (Maassen ex Claussen) Olive & Spiltoir is an etiologic factor causing encystic mycosis (ascosphaeriosis) in honey bee larvae Apis mellifera L. The infection of honey bee larvae proceeds per os, during feeding with a mixture of pollen honey. A.apis spores, entering the alimentary tract of the larvae, germinate, colonize the gut and then grow into hemocel and internal organs. The developing fungus damages tissues leading to the death of the larvae which, with time, become covered with white mycelium, producing characteristic, dark colored, fructifications.

There are relatively few research papers using histological techniques concerning fungus development in the bee larvae organisms in the available literature [2,3]. These authors describe the generation of A.apis fructifications only on the surface of Apis mellifera larvae bodies as opposed to Ascosphaera aggregata in miesiarka lucernówka larvae (Megachile rotundata), in which the production of the fungus fructifications takes place both inside the bodies of the larvae and on their surface [9].

This research aimed at observing the development of invasion of Ascosphaera apis fungus in the organism of bee larva in case of artificial infection.

MATERIAL AND METHODS

Laboratory inoculation of larvae

From the A. mellifera colonies free of the chalkbrood disease, the cuttings of beeswax (10x10cm) were collected, including the 3rd-instar larvae. The wax cuttings were put into the cages with the honey workers and bee bread. Each larva was fed individually for 3 days with 5 µl of 35% (w/v) sucrose solution containing spores A.apis (a dose of 5 x 10⁵ spores) obtained from cultures of this fungus [2,8]. In the experiment two strains of A.apis were used: A1 and A6. The strains was grown on Sabouarda’s medium (SDY-YE) with 0.2 % yeast extract and 0.1% chloramphenicol (BioMerieux, France) incubated in compliance with Glinski’s procedure [7]. The honey workers were fed the 35% sugar syrup during the experiment.

The infected larvae were kept during the first 24 hours at 22°C, and for the next days at 28°C [1,6]. At 24 h, and daily thereafter, after the first feeding with infected food, living larvae were collected and fixed for histological processing, using Carnoy’a fluid. Fixed samples were embedded in paraffin wax and cut of 6 µm in thickness. The sections were examined microscopically for evolution of infection, compared with that section from uninfected larvae. The sections were fixed to glass slides coated with egg albumen, and stained with haematoxylin/eosin, or azane.

RESULTS AND DISCUSSION

Out of two strains of Ascosphaera apis used for infection, growth was obtained only in the group of bee larvae which received A1 strain. Germination of the fungus spores takes place in midgut along its entire length. The spores are visible in the gut’s lumen and surrounded with peritrophic membranes during this stage of infection. A delicate growth of A. apis hyphae, which slowly begin to grow into the peritrophic membranes is visible after 48th hour of feeding the bee larvae with the infected food. Midgut epithelium of the infected larvae and healthy ones shows no differences in the histological picture (Fig. 1, 2).

The increase of length and diameter of the hyphae as well as the beginning of their penetration into the gut wall are visible in 72nd hour of observation. The hyphae grow through the basal membrane and grow into the hemocel of the larvae. Midgut epithelial cells are subject to slow changes; the number of granules increases, and a slight swelling of nuclei is visible (Fig. 3, 4).

A significant proliferation of the fungus hyphae in hemocel and their growing into fat body structures take place in 96th hour after infection, which causes a slow degeneration of trophocytes, blurring of their shape and decomposition. Besides, the changes in enocytes, which are also grown through with the hyphae (fig 5 and 6), are also visible. The increase of dystrophy level takes place the midgut epithelial cells as well as the beginning of cytoplasm and nuclei vacuolization. Some larvae were invaded completely by the fungus’ hyphae, others only in part.

Bamford and Heath in their study of 1989 did not find fat body growing through by A. apis. hyphae. However, high proteolytic and lipolytic activity (production of C4 esterase, C8 lipase-esterase, cysteine and valine arylamidase) of A. apis strains proves high adaptation of this fungus to develop and colonize larva’s body [4]. Carrera et al. [3] in their research proved that hyphae damages all larvae tissues, except trachea. A1 strain did not grow into the respiratory tract of the experimentally infected larva, either. The hyphae developing in the fat body and hemolymph were considerably thicker and shorter that those obtained from cultures on Sabouraud base (SDA-YE) with 0.2% addition of yeast extract and 0.1% of chloramphenicol).

After colonizing of the larva’s visceral cavity, the hyphae grew through the cuticle (on 5th-6th day after infection) and covered the entire body surface with white mycelium. Though, the A. apis does not produce chitinase, but the A1 strain used for the experiment is characterized with high N-acetyl-βglycosaminidase, the enzyme which is related to breaking of protective insect body barriers, and which replaces the action of chitinase [1,2,4]. A. apis produces characteristic fructifications (fig. 7) 6 days after the moment of aerial hyphae appearance on the surface of the infected larvae. It is manifested in the form of characteristic darkening of the hyphae. The production of fructifications in A1 strain took place as soon as on the 7th day after inoculation at 25°C in the cultures on artificial base.

The biochemical characteristics of Ascosphaera apis indicate adaptation to the development inside the bee larvae organism. The process takes place in aerobic as well as in anaerobic environment. A. apis easily breaks through the immunological intestinal barrier and grows intensively at the cost of the larvae organisms, leading to their death. It also actively grows through the cuticle, decomposing - thanks to the above mentioned enzymes - chitin, while its ripe mycelium produces numerous fructifications (spore cysts) containing a huge amount of ascospores [4,5]. The larva killed by ascosphaeriosis is removed from the cells by working bees. While cleaning the cells of the dead larva, the cleaning bees spread the spores in the hive environment. The spores then get into food supplies – honey and bee bread (pollen), which is fed to subsequent bee larvae generations infecting them simultaneously, which, consolidates the disease process of the bee family.

REFERENCES

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2. Bamford S., Heath L.A.F. 1989. The infection of Apis mellifera by Ascosphaera apis. J. Apic. Res. 28, 30-35.

3. Carrera P., Sommaragua A., Vailiti G. 1987. The development of Ascosphaera apis within larvae of Apis mellifera ligustica. J. Apic Res. 26, 59-63.

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6. Flores J.M., Ruiz J.A., Ruz J.M., Puerta F., Bustos M., Padilla F., Campano F. 1996. Effect of temperature and humidity of sealed brood on chalkbrood development under controled conditions. Apidologie, 27, 185-192.

7. Gliński Z, Wolski T., Chmielewski M. 1988. The “in vitro” studies on antifungal action or Archangelica officinalis Hoffm. seed extract against Ascosphaera apis. Medycyna Wet. 44, 552-556. [in Polish].

8. Puerta F., Flores J.M., Bustos M., Padilla F., Campano F. 1994. Chalkbrood development in honeybee brood under controlled conditions. Apidologie, 25, 540-546.

9. Vandenberg J.D., Stephen W.P. 1983. Pathogenesis of chalkbrood in the alfaalfa leafcutting bee, Megachile rotundata. Apidologie, 14, 333-341.

¹Investigation paper elaborated within research project financed by KBN No 5 PO6K 027 19

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