African Honey Bees (Apis Mellifera Scutellata) and Nosema (Nosema Apis) Infections

AFRICAN HONEY BEES (APIS MELLIFERA SCUTELLATA) AND NOSEMA (NOSEMA APIS) INFECTIONS

Ingemar FRIES

Department of Entomology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden

ABSTRACT Nosema apis has been found on all continents where there is beekeeping using Apis mellifera. However, there is very little data on the prevalence and impact of Nosema apis in honey bee colonies in tropical climates and it may be uncertain if all records of Microsporidia in honey bees actually are records of the same parasite. Also, the development of N. apis has not been documented in tropical races of honey bees. We have sampled honey bees from five different colonies in two apiaries on a weekly basis for a full year in Zimbabwe and investigated the samples for N. apis. In infection experiments the development of the parasite has been monitored. The gene sequence of the 16S small subunit ribosomal RNA gene of Zimbabwean isolates of Microsporidia from honey bees have been sequenced and compared to sequences for N. apis registered in GenBank. The molecular results demonstrate that the Zimbabwean isolates of Microsporidia are N. apis. Sampling results show that N. apis may occur at high levels of prevalence at colony level under tropical conditions and develop similar to European records in individual bees. Thus, the investigated parasite probably carry no further risks to beekeeping if transported with live bees between different regions.

INTRODUCTION

N. apis has a world-wide distribution (Matheson, 1993) but is not considered an important problem in tropical and sub-tropical climates (Wilson and Nunamaker, 1983). However, there is not enough information available to evaluate the impact of the parasite in warm climates. In contrast, infections by N. apis are considered to be detrimental, in temperate climates. N. apis reduces the honey yield in heavily infected colonies of honey bees in temperate climates (Farrar, 1947; Fries, et al., 1984) and the survival of the colony during winter is affected by the disease (Farrar, 1942; Fries, 1988a). The meager amount on information available on the prevalence and impact of nosema disease in tropical climates makes it impossible to evaluate the seriousness of infections by this pathogen in the tropics.

The quantitative spore production of N. apis isolated in Europe in honey bees from temperate climates is well described (Fries, 1988b; Lotmar, 1943) as well as the intracellular development of the parasite (Fries, et al., 1992). However, there are no similar records from tropical or sub-tropical climates, nor any information on genetic variations between isolates of N. apis from temperate and tropical climates.

This paper aims to investigate the seasonal prevalence of N. apis in one sampling site in tropical Africa where beekeeping is practiced using Langstroth hives. Further, the objective is to describe the development of an African isolate of the parasite in individual bees of A. mellifera scutellata and to investigate if there are sequence variations in the 16S ribosomal RNA gene (SSUrRNA) in an African isolate compared to isolates from temperate climates.

MATERIAL AND METHODS Sampling for prevalence Live honey bee samples were collected weekly from honey bee colonies of A. mellifera scutellata in two different apiaries located in Zimbabwe a few kilometres northwest of Harare where 3 and 2 colonies respectively were sampled. Bee samples were collected from the top of the bee cluster and kept frozen for further investigations. From the live bee samples, 60 bees were investigated as a composite sample for nosema spores. The bees were squashed thoroughly using 1 ml water per bee and the fluid examined in a haemacytometer to determine number of spores per bee (Cantwell, 1970).

Parasite development For infection experiment to investigate the development of the parasite in individual bees, spores were collected from infected foragers found on a feeding tray. The ventriculi of collected foragers (N=100) were examined under a compound microscope and when infected specimens were found, these were used to prepare spore solutions. Two different spore solutions were prepared using a sugar:water solution (1:1 w:v), one containing 106 spores per ml, the other 105 spores per ml. Each spore solution was individually fed to 60 bees using a 10 µl constriction pipette, yielding spore doses of 104 and 103 per bee respectively. Similarily, 60 bees were fed sugar solution only. After feeding, each group of bee was incubated at + 30 °C, 50% Rh and supplied with sugar solution and water ad lib. Any bee mortality in the cages was recorded and samples of 4 live bees from each group were extracted daily for 10 days and examined for N. apis. In each bee to be investigated, the ventriculus was removed and squashed in 0.5 ml of water and the resulting fluid examined in a haemocytometer to make spore counts. As young spores refract light slightly differently compared to mature spores (Lotmar, 1943), separate counts were made for both types of spores.

In addition to spore counts, the ventriculus from two bees on two sampling occasions, three and six days post infection, were fixed for transmission electron microscopy and light microscopy using 4 % glutaraldehyde (v/v) in 0.067 M cacodylate buffer, pH 7.4, for three weeks. The material was kept refrigerated (+7 °C) during prefixation. After washing in cacodylate buffer, the specimens were post fixed for 2 h. in 2 % OsO4 (w/v) in 0.1 M S-colloidine buffer. After dehydration in an ascending concentration series of ethanol solutions, followed by a propylene oxide solution, the tissue pieces were embedded in Epoxy resin (Agar 100) by routine procedures for electron microscopy. Sections of the embedded material were mounted for light microscopy after contrast coloring with toluidine blue. Thin sections of the embedded specimens were mounted on copper grids and stained with uranyl acetate followed by lead citrate. The preparations were examined in a Philips 420 electron microscope at an accelerating voltage of 60-80 kV.

DNA analysis For DNA analysis, a portion of spores collected for infection experiments were stored in 70% ethanol. DNA extraction, PCR amplification, cloning, and sequencing of the SSUrRNA sequence was done as described previously (Visvesvara, et al., 1995). Briefly, spores were disrupted with glass beads (Cat. No. G-9139 Sigma, St. Louis, MO) in a buffer containing proteinase K and 1% lauryl alcohol polyether (Laureth 12, PPG Industries Inc., Gurnee, IL). After an overnight incubation at 55° C, proteinase K was inactivated by heating the sample at 95° C for 10 min. This DNA preparation was stored at 4° C until used. For amplification of the entire N. apis SSUrRNA coding region, PCR primers

MICRO-F and MICRO-R were used (Visvesvara, et al., 1995). The obtained SSUrRNA sequence was compared to sequences retrieved from the GenBank databasefor N. apis (U26534)??????

RESULTS Prevalence The level of nosema infections presented as average number of spores per bee each week for the two apiaries can be seen in Figure 1.

Parasite development In Figure 2 the quantitative spore production of the parasite is seen at different times post infection for two different spore doses. Figure 3 demonstrates the presence of emptied spores within the host cell cytoplasm, often seen in European infections of N. apis.. This is interpreted as means of intercellular spread of the parasite (Fries et al., 1992)

DNA analysis Comparisons with GeneBank entries demonstrated that there was a single base pair substitution of A for G at position 45 of the SSUrRNA coding region when compared to an American and a Swedish isolate of N. apis (GenBank Accession number U26534). The SSUrRNA coding region from an isolate from New Zealand (GenBank Accession number U97150) was identical to the Zimbabwean isolate.

DISCUSSION It is obvious from the presented results that the prevalence of N. apis may be quite high also under tropical conditions (Figure 1). The general perception of N. apis epidemiology is that of a disease primarily spread through wax combs soiled with bee feaces (Bailey, 1953). Although there are periods of confinement for the bees during rainy periods, it is unusual for the experimental sites that bees are confined completely more than a few days due to weather conditions. Even during the rain periods, the mornings are usually sunny with rain falling in the afternoon. Under the circumstances of the present experiment, it seems unlikely that soiled comb should be the primary source of infection since bees may fly and defecate outside the hives most days of the year.

We have no explanation for the comparatively high incidence of the nosema parasite demonstrated in this experiment. One hypothesis that should be tested is if the use of open air feeding trays where bees are fed small amounts of sugar solution during periods with little or no honey flow or before handling the bees, may aid as agents for spreading disease (Figure 3). This system with open air feeding trays has been practiced in the investigated apiaries for many years. It should also be investigated if N. apis infections have any measurable negative effect on colony vitality and production under tropical conditions. In temperate climates, the impact from infection is mostly notable when winter bees need (and fail if infected) to regenerate their hypopharyngeal glands when brood rearing is initiate in the spring (Bailey and Ball, 1991). In areas where brood rearing occurs throughout the year, this effect should be less pronounced and negative effects, even at the levels recorded here, may not be measurable.

Parasite development and DNA analysis The limited data presented here suggests that the intracellular development of the parasite is similar both quantitatively (Fries, 1988b) and qualitatively (Fries, 1989; Fries, et al., 1992) compared to European races of A. mellifera.

Measurable spore numbers are observed three days post infection and the typical features seen in European infections with emptied spores within the host cytoplasm, interpreted as means of intercellular parasite spread, was also recorded in this investigation (Figure 2, 3).

The sequence of the 16S small subunit ribosomal RNA coding region was identical to a New Zealand isolate and differed by one base pair only from Swedish and American isolates. Together with other information this is enough to conclude that it is likely that the species N. apis probably occur throughout the world where A. mellifera beekeeping is practiced. Thus, species designation using light microscopy observations of Microsporidia in this host species may be correct when there is a match in gross morphology. This is in contrast to observations of Microsporidia in the Asian honey bee, A. cerana, where such observations often may be due another species of Microsporidia, Nosema ceranae (Fries et al., 1996)

ACKNOWLEDGEMENTS The dedicated help in sampling and documenting by Cecil J. Coleman is highly appreciated. The study had financial support from the Swedisch International Development Agency (SISA).

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