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.
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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
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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.
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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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