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Descriptions of Microsporidia spp. in Bombus

Bumble bees host lots of parasites including one microsporidium: Nosema bombi Fantham and Porter, 1914. The Pollinator Parasites project has documented this species in a large number of different bumble bee hosts. Numerous publications have treated various aspects on the ecology of this microsporidium and the impact on the hosts, but few have dealt with the cytology and none has done so in depth.

Nosema bombi infected bumble bees were collected in southern Sweden and Denmark from B. hortorum, B. hypnorum, B. lapidarius, B. lucorum, B. pascuorum, B. pratorum, B. ruderarius, B. subterraneus and B. terrestris. Specimens were either dissected directly, killed by decapitation or freezing, or stored frozen for some time. Light microscopy were conducted on unstaied and fixed preparations using standard techniques. Ultrastructure was studied using standard techniques for electron microscopy but including the improved fixation of spores developed in the Pollinator Parasites project.

Nosema bombi was most regularly observed in tissues samples from the Malpighian vessels. Small foci of developing microsporidia were often found in all regions of the gut wall, from the ventriculus and backwards. It was not observed that gut cells completely filled with microsporidia were released into the gut lumen, like is the case of Nosema apis, but in the regions of the gut posteriorly to the Malpighian vessels microsporidian spores were regularly observed in the lumen. In heavily infected hosts practically all Malpigian vessels, in their entire length, were infected. When squashed entire spore-filled cells were released. Presporal stages were most commonly seen in epithelium of the Malpighian vessels. In cases of advanced infection also the walls of the tracheal system were invaded. In two specimens, one male of B. pascuorum and one worker of B. lapidarius, microsporidian spores were also found in the musculature. In several specimens of B. hypnorum, B. lucorum, B. pascuorum and B. terrestris infection was restricted to peripheral fat cells, which means that samples from the Malpighian vessels and other tissues were negative.

The presporal development was of Nosema type. The cells measured up to 6.1 µm in sections, the greatest diameter of sectioned nuclei was 1.9 µm. The cells had a typical, about 7 nm thick, plasma membrane. Cytoplasm and nuclei were both granular, but the nuclei were slightly more dense. When the merozoites matured to sporonts vesicular areas appeared in the cytoplasm, interpreted as a primordial Golgi apparatus. The thick external layer of the sporont wall, the primordium of the exospore, was added over large areas simultaneously, not as the regularly spaced strands typical for the genus Nosema. The sporogony was disporoblastic with the two elongated sporoblasts arranged like a short chain. In the sporoblasts the polar filament was initiated and extended forwards from a system of Golgi vesicles at the posterior pole. The earliest signs of a polaroplast became visible late in the maturation process, but prior to the first signs of the endospore layer. The diplokaryon, with distinct nucleoli in the nuclei, was located to the anterior half of the immature spore until it was pushed backwards by the developing polaroplast.

The recorded length of living spores ranged from 2.73 to 6.63µm long. In one specimen of B. terrestris where two distinct classes of morphs were seen, the oval spores measured 1.95-2.60 x 2.77-5.85µm, the elongated spores 1.69-2.47 x 4.42-6.24µm. Spores were either completely uncoupled, or, when dealing with the more elongated morph, appeared pair-wise or arranged to strands of spores. The spores further differed in the willingness to eject the polar filament.

The mature spores exhibited cytology of the most common microsporidian type. With exception of the spores found in one specimen of B. pratorum, the lowest number of polarfilament coils observed was 12 (spore length 3.1 µm), the greatest 24 coils (spore length 3.98 µm). Two intervals of coils were dominant, 13-15 and 18-20, the greatest number in the elongated spore morph. The coils were normally arranged as a single layer of coils, but with more than 15 coils, the last 2-4 coils were normally displaced to form an internal layer (Fig. 6 A, inset). Occasionally two almost complete layers of coils were observed (20-21 coils). The filament was isofilar, measuring about 100 nm wide (96-105 nm).

The results presented complete the previous incomplete descriptions of N. bombi and document an unusual morphological variability. This documentation is useful to avoid future confusions on Microsporidia infections in bumblebees.

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University of Lund
Department of Cell and organism BiologyHelgonavaegen 3
S-223 62 Lund
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