Journal of Apicultural Research 53(5): 537-544 (2014) © IBRA 2014 DOI 10.3896/IBRA.1.53.5.02

ORIGINAL RESEARCH ARTICLE

Differentiation of Nosema apis and Nosema ceranae

spores under Scanning Electron Microscopy (SEM)

Aneta A Ptaszyńska1*, Grzegorz Borsuk2, Wiesław Mułenko1, Jerzy Demetraki-Paleolog2

1Department of Botany and Mycology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland. 2Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland. Received 10 November 2013, accepted subject to revision 27 June 2014, accepted for publication 11 September 2014. *Corresponding author: Email: aneta.ptaszynska@poczta.umcs.lublin.pl

Summary

Spores of the microsporidian gut pathogens of honey bees Nosema apis and Nosema ceranae, the causative agents of nosemosis, are difficult

to distinguish using the light microscope. This study aimed to find methods which could help to identify N. apis and N. ceranae spores, and to

define morphometric parameters in order to distinguish the differences between the spores of the two species. Our observation of spores

under Scanning Electron Microscopy (SEM) revealed that N. apis and N. ceranae spores differ in their surface structure. Generally, N. ceranae

spores seem to be more sculptured with deeper ornamentation than those of N. apis. Therefore, ornamentation of the spore cell wall with

special reference to the width of their exospore furrows and sculpture index allows unequivocal identification of N. apis and N. ceranae

species. Whole glands, organs, or various surfaces can be examined under SEM for the presence of spores, and consequently possible

reservoirs or niches for storage of Nosema spores can be detected. We observed the results of Nosema spp. infestation on bees’ intestines

under SEM. Our study revealed that even medium Nosema spp. infection causes the midgut to be completely covered with spores. This spore-

made layer can be the cause of bee malnutrition and higher mortality of foragers.

Diferenciación de esporas de Nosema apis y Nosema ceranae

bajo microscopio electrónico de barrido (MEB)

Resumen

Las esporas de Nosema apis y Nosema ceranae, los agentes causantes de nosemosis, son difíciles de distinguir usando el microscopio óptico.

Por lo tanto, este estudio tuvo como objetivo encontrar métodos que podrían ayudar a identificar esporas de N. apis y de N. ceranae y definir

parámetros morfométricos con el fin de distinguir las diferencias entre las esporas de estas dos especies de microsporidios. Nuestra

observación de esporas bajo microscopía electrónica de barrido (MEB) reveló que las esporas de N. apis y de N. ceranae difieren en su

estructura superficial. En general, las esporas de N. ceranae parecían estar más esculpidas con ornamentación más profunda que las de N.

apis. Por lo tanto, la ornamentación de la pared celular de las esporas, con especial referencia a la anchura de sus surcos exosporio y el índice

escultura permitió la identificación inequívoca de las especies N. apis y N. ceranae. Se pueden examinar las glándulas enteras, órganos o

varias superficies bajo MEB para detectar la presencia de esporas, y en consecuencia se podrán detectar los posibles reservorios o nichos para

el almacenamiento de esporas de Nosema. En primer lugar, observamos los resultados de la infestación por Nosema spp. en los intestinos de

las abejas bajo SEM. Nuestro estudio reveló que incluso una infección media de Nosema spp. puede cubrir completamente el intestino medio

con esporas. Esta capa hecha de esporas puede ser la causa de la desnutrición de abeja y de una mayor mortalidad de las pecoreadoras.

Keywords: honey bee; Apis mellifera; colony losses; nosemosis; intestines; SEM

Introduction

Nosemosis is a widespread, contagious disease of bees caused by

two species of Microsporidia (Fungi), Nosema apis and Nosema

ceranae. The first description of this disease in honey bees (Apis

mellifera) was published 100 years ago (Zander, 1909). Initially,

nosemosis was thought to be caused solely by one species, N. apis. At

the end of the twentieth century, however, a new species N. ceranae

538 Ptaszyńska et al.

(Fries et al., 1996) attacking the eastern honey bee (Apis cerana) was

described in Asia. The first infection of A. mellifera by N. ceranae was

recorded in Taiwan in 2005 (Huang et al., 2005), and soon after in

Europe (Higes et al., 2006;. Paxton et al., 2007), the USA (Chen et

al., 2008), China, and Vietnam (Liu et al., 2008).

Some studies have shown that both N. apis and N. ceranae are

not tissue-specific and their spores can be found in the midgut

epithelium, Malpighian tubule system, hypopharyngeal glands, salivary

glands, and venom sacs (Copley and Jabaji, 2012). Other research

indicated that the spores of N. apis can only be found in the gut of

bees, while N. ceranae spores in other tissues and glands. (Fries,

1988, 2010; Graaf and Jacobs, 1991; Chen et al., 2009). Recently,

Huang and Solter (2013) claimed that Nosema spp. spores found in

places other than honey bee ventricular cells was circumstantial and

probably a consequence of contamination of samples during

dissection. However, the small dimensions of the spores can enable

them to move through e.g. ducts of the hypopharyngeal glands and

to stay there without development. During liquid food ingestion, the

action of the muscles attached to the cibarium and clypeus causes

that cibarium to act like a sucking pump (Dade, 2009). The negative

pressure, which is formed during sucking, can cause small particles

(e.g. microsporidian spores) to be transmitted not only to the

digestive tract but also through the two openings of the

hypopharyngeal glands’ ducts, which lie on either side of the

hypopharyngeal plate, and are about 30 µm in diameter, straight to

the hypopharyngeal gland acini, with a diameter over 100 µm

(Crailsheim and Stolberg, 1989; Malone et al., 2004; Deseyn and

Billen, 2005). So far, there has been no evidence that Nosema spp.

can complete the full life cycle beyond the intestine. Consequently,

the life stages of this parasite can be found only in intestinal epithelial

cells, while spores can be found in various tissues and glands,

including the hypopharyngeal glands. In addition, spores remaining in

the glands are potential reservoirs of infection (Ptaszyńska et al.,

2012). This may interfere with the function of the glands, which are

related to the production of royal jelly, honey, wax, and bee bread.

Nosemosis is traditionally regarded as an infection of ventricular

cells caused by N. apis and classified as nosemosis type A.

Conversely, dry nosemosis or nosemosis type C is exclusively assigned

to the N. ceranae bee infection (Higes et al., 2010, Huang and Solter

2013). Environmental spores of two Nosema species differ very

slightly when observed under the light microscope. The fresh mature

spores of N. apis are 4-6 µm in length and 2-4 µm in width, while N.

ceranae spores are 3.3-5.5 µm and 2.3-3.0 µm, respectively (Fries et

al., 1996; Higes et al., 2010). Therefore, the largest N. ceranae

spores have the same dimensions as the smallest spores of N. apis

(Higes et al., 2007).

It is crucial to be able to determine the type of nosemosis quickly

and reliably. Until now, spores have been distinguished under TEM

(Transmission Electron Microscopy), by techniques based on PCR

(Fries, 2010), and related: as duplex PCR (Martín-Hernández et al.,

2007), real-time PCR (Bourgeois et al., 2010; Burgher-MacLellan et

al., 2010) or LAMP (Ptaszyńska et al., 2014).

Techniques based on DNA analysis are clear but sometimes smear

samples contain inhibitors of PCR, which prevents all investigations by

means of these methods. Due to the Nosema spp. life cycle, the only

easily accessible stage that could be examined under the light

microscope is the spore, but the differentiation between spores of N.

apis and N. ceranae is very difficult. Spores can be distinguished

under TEM, but using this method, only a small area of a sample can

be studied (Fries et al., 2006). Therefore, it is crucial to find new

methods of Nosema spore observation and differentiation.

Since Fries et al. (2006) compared the internal structure of N.

ceranae and N. apis spores, it has been noticed that the sculpture of

the exospore (or exosporium) of these two species is different. In

TEM images of N. apis and N. ceranae spores (e.g. Fries et al., 1996;

Higes et al., 2007), the surface of the exospore was seen as a line

defining the spore shape. Due to the small size of the microsporidian

spores the details of their surface can be visible only under large

magnifications such as Scanning Electron Microscopy (SEM).

Therefore, in this study, SEM was used to verify whether there are

differences in the sculpture of the N. apis and N. ceranae spore

exosporium.

Material and methods

During a three-year study (2011-2013), Nosema spp. infected Apis

mellifera carnica and “Buckfast” hybrid bees (Brother Adam, 1983)

were examined. Bees were collected from local apiaries (about 60

colonies) both in the spring and during overwintering from the

autumn, to prepare water homogenates as pooled samples. The

homogenates were examined for the presence of Nosema spp. spores

under an Olympus BX61 light microscope (Cantwell, 1970; Hornitzky,

2008). When Nosema spp. spores were found in the bee homogenate,

the colony such bees were sampled from was regarded as infected

with Nosema spp. and designated for further sampling of bees during

summer. The worker bees sampled in this way were used to obtain

faecal, pooled, and intestine samples for PCR and SEM analysis.

Additionally, bees sampled from 10 infected colonies were frozen at -

20○C and kept for 1.5 years in order to determine whether freezing

influences the exospore sculpture. After that time, they were

subjected to PCR and SEM analysis as pooled samples.

Furthermore, the brood combs taken from colonies identified as

Nosema spp. uninfected, were incubated to obtain one day old

healthy honey bees. On the third day, these bees were inoculated

either with N. apis or with N. ceranae spore solutions, according to

aluminium foil to a 1.5 ml microcentrifuge tube. The total genomic

DNA from each faeces sample was extracted using the QIAamp DNA

Stool Mini Kit following the manufacturer’s procedure. Every isolate

was used as a template for detection of N. apis and N. ceranae

specific 16S rDNA by PCR.

Isolation of DNA from the pooled samples

100 μl of each H2O homogenate was added to 180 μl of lysis buffer

and 20 μl of proteinase K, and the total DNA was isolated by the

DNeasy Blood and Tissue Kit (QIAGEN) according to the

manufacturer’s instruction. Every isolate was used as a template for

detection of N. apis and N. ceranae specific 16S rDNA by PCR.

Isolation of DNA from the intestines samples

Each piece of midgut (about 0.4 cm) was individually immersed into

180 μl of lysis buffer and 20 μl of proteinase K, and the total DNA was

isolated by the DNeasy Blood and Tissue Kit (QIAGEN) according to

the manufacturer’s instruction. Every isolate was used as a template

for detection of N. apis and N. ceranae specific 16S rDNA by PCR.

Molecular detection of Nosema apis and N.

ceranae

Duplex PCR was carried out to confirm the presence of N. apis and N.

ceranae spores in the faecal, pooled and intestine samples. PCR

cocktails were prepared using the QIAGEN Taq PCR Core Kit (QIAGEN

Inc.) and PCR was conducted in a thermal cycler in 25 μl of cocktails

containing 2.5 μl PCR buffer, 5 μl Q solution, 0.1 mM dNTP mix, 0.7 U

Taq DNA polymerase, 0.2 μM of each primer, approximately 0.15 μg

of DNA template, and ddH2O added to a final reaction volume of 25

μl. For DNA amplification, the following PCR cycling conditions were

used: 1 min at 94°C, 1 min at 61.8°C, and 1 min at 72°C, repeated

for 30 cycles, and 10 min at 72°C. The primers selected to

differentiate between both Nosema species were 321-APIS for N. apis

and 218-MITOC for N. ceranae (Martín-Hernández et al., 2007). The

PCR products were visualised on agarose gel under UV light using a

CCD camera coupled with a computer with the ScionImage software.

Differences in the amplicon lengths were the criterion to differentiate

between both Nosema taxa in the sample. To confirm the validity of

Nosema species identification, some amplicons were sequenced using

ABI 3100 Avant and amplified N. apis and N. ceranae 16S rDNA

sequences were submitted to GenBank (KC707997─KC708007).

Scanning Electron Microscopy (SEM)

All samples were divided into two parts. The first part was immersed

in lysis buffer for DNA isolation and PCR analysis, and second one was

fixed during 24h in 5% gluteraldehyde (v/v) in 0.1 M phosphate

buffer pH 7.3 for SEM.

the methodology described in Forsgren and Fries (2010), caged (6

cages, 40 workers in each), and kept under the laboratory conditions

in complete darkness (30oC; RH=65%). On the 12th day of the

infection, bees were taken from the cages to PCR and SEM analysis as

pooled samples.

The faecal sampling protocol

Faeces were collected in accordance with the methodology described

by Czekońska (2001) for queen bees. To obtain environmental spores

of N. apis and N. ceranae, each worker bee was individually placed on

a piece of aluminium foil and covered with a watch glass, after which

the bee was not able to turn away or move. Within 20 minutes, most

of the bees discharged faeces. Each sample of the faeces was divided

into two portions: the first portion was subjected to PCR analysis and

was washed from the piece of aluminium using a stool lysis buffer

(Qiagen), in order to isolate DNA and the other portion was

immediately fixed in 5% gluteraldehyde (v/v) in 0.1 M phosphate

buffer pH 7.3 for SEM preparation.

The pooled sampling protocol

The homogenates were obtained by grinding 10 whole bees in 10 ml

of distilled water and the number of Nosema spp. spores was counted

according to Cantwell (1970) and Hornitzky (2008). The same

samples were observed under the bright field and differential

interference contrast (DIC) using an Olympus BX 61 microscope.

When spores of Nosema spp. were observed in the sample, two

portions per 100 μl of the homogenate from such pooled sample were

taken; one for isolation of the total DNA and PCR analysis, and the

other for SEM preparation.

The intestine sampling protocol

Bees from the naturally Nosema spp.-infected colony (number of

spores were counted according to Cantwell, 1970 and Hornitzky, 2008

from pooled samples) were taken to prepare intestines for SEM

analysis. The alimentary tracts of 6 bees were carefully and

individually removed under the stereoscopic Olympus SZX 16

microscope and gently washed in distilled water to prevent

contamination by haemolymph (Borsuk et al., 2013). Each midgut was

divided into two parts (each about 0.4 cm) for PCR and SEM analysis.

First sample was immediately immersed in lysis buffer (DNeasy Blood

and Tissue Kit, QIAGEN) and the other in 5% gluteraldehyde (v/v) in

0.1 M phosphate buffer pH 7.3.

Isolation of DNA from the individual faecal

samples

Each faeces sample, taken from the individual bee specimen, was

washed using a stool lysis buffer (QIAGEN) from the piece of

Differentiation of N. apis and N. ceranae spores under SEM 539

540 Ptaszyńska et al.

Fig. 1. A.-F. Scanning electron micrographs of Nosema apis and Nosema ceranae spores. A. N. apis spore, the beginning of extrusion of the

polar tubule. B. N. ceranae spore. C. N. ceranae spore with shown distances among exospore furrows (a) and widths of furrows (b). D. the

intestine of Apis mellifera carnica infected by nosemosis, with the infestation range of 59 x 106 spores per bee. E. spores of two species (ap –

N. apis, cer – N. ceranae) from intestine samples. F. a germinated N. ceranae spore. G. Light micrograph of Nosema spp. spores observed in

differential interference contrast (DIC), the asterisk (*) indicates the germinated spore.

Only faeces and pooled samples infected by one Nosema species

(solely by N. apis or N. ceranae), and additionally all 6 intestines

samples were selected for further SEM preparation. After selection,

the samples were washed in phosphate buffer prior to post fixation in

1% osmium tetraoxide in 0.1 M-phosphate buffer for 24 h followed by

washing in the same buffer. SEM samples were dehydrated by

immersion for 15 min each in fresh solutions of 30%, 50%, 75%,

90%, and 100% acetone, and critical point dried. The dried samples

were mounted on specimen stubs using a double-sided adhesive tape

and coated with gold. Coated samples were viewed under a VEGA

LMU scanning microscope at 30 KV, measured, and photographed.

All data for statistical analysis was obtained using 120 of N. apis

and 120 of N. ceranae spores from faecal samples observed under

SEM. The length and width of each spore was calculated under

magnification of 50,000; and the distances among exospore furrows

and widths of furrows under magnification of 200,000 (Fig. 1C).

Furthermore, the differences between sculpture of the N. apis and N.

ceranae spores were observed under magnification of 5,000; 10,000;

15,000; 20,000; 25,000 and 30,000 to determine the lowest

magnification that allows the unequivocal differentiation of Nosema

species.

Statistical analysis

The SAS software (SAS Institute 2002-2003 SAS/STAT User’s Guide

Version 9.13; Cary, NC, Statistical Analysis System Institute) was used

for statistical analysis. One-way ANOVA (a group effect; i.e. N. apis

versus N. ceranae spores, was the experimental factor) was

performed for the distance between neighbour exospore furrows and

width of the furrow to compare N. apis and N. ceranae spores (Fig.

1C). Additionally, Tukey’s HSD (honestly significant difference) for

these traits in the both groups was performed. Pearson’s correlation

between the distance between the neighbour furrows and the width

of the furrow in N. apis and N. ceranae exospores was calculated. The

541

sculpture index, counted as the ratio of the distance between the

means of the furrows and the width of the furrows, was also

calculated as follows:

Where: a = distance between the means of the furrows; λ = width of

the furrow. Student's t-test significant value for the differences in the

sculpture index between N. apis and N. ceranae spores was

performed.

Results

Faecal samples

To analyse environmental spores of N. apis and N. ceranae, samples

of faeces were analysed first. Faecal samples were taken from

individual bees and were subject to duplex PCR analysis. When the

sample was infected solely by one microsporidian species, e.g. N. apis

or N. ceranae, such sample was taken to further steps of SEM

preparation. Therefore, SEM analyses, given in Tables 1- 3, are the

studies of faecal samples from individual bees infected by just one

Nosema species.

Observations of N. apis and N. ceranae spores under SEM

revealed that the spores differed in their surface structure. The spores

of N. ceranae (Fig. 1B) were more sculptured with deeper

ornamentation than those of N. apis (Fig. 1A). The spores of N. apis

had significantly larger folds than N. ceranae (Table 1), longer

distances between the furrows in the ornamentation, and wider edges

of the furrows. This was confirmed by the correlation coefficients

between the means of the furrows and the width of the furrows of N.

apis and N. ceranae exospores (Table 2). The sculpture index

indicated that the distances between N. apis furrows for the furrows

widths have a higher value than for N. ceranae (Table 3).

sculpture index = a

λ

Differentiation of N. apis and N. ceranae spores under SEM

Table 1. Differences in the sculpture of N. apis and N. ceranae

spores. a,b – differences are statistically significant for p < 0.05

(ANOVA; Tukey’s HSD test), SE – standard error, Min. – minimum trait

values, Max. – maximum trait values, CV – coefficient of variation.

Nosema spp. Mean [nm] CV

[%] SE

Min. [nm]

Max. [nm]

Distance between the means of the furrows

N. ceranae 83.62a 25 0.78 36.23 169.07

N. apis 114.54b 26 2.15 40.30 200.35

The width of the furrows

N. ceranae 28.06a 14 0.29 18.30 39.49

N. apis 35.50b 19 0.55 24.62 52.08

Table 2. Pearson’s correlation between the means of the furrows and

the width of the furrows of N. apis and N. ceranae exospores

(correlation was significant for p < 0.05, n = 240).

N. apis Distance between the means of the furrows

N. ceranae Distance between the means of the

furrows

N. apis width of the furrows

–0.22 ─

N. ceranae width of the furrows

─ +0.34

Pooled samples

In the pooled samples, the environmental spores looked exactly the

same and had the same parameters as those from the faecal samples.

There were no differences in the sculpture between the spores from

one fungal species found in bees belonging to different taxa, sampled

in different seasons, from naturally and artificially infected bees, and

between the two types of bee samples (fresh and frozen). The only

differences that were observed were noticed between the sculpture of

the spores derived from two microsporidian species i.e. N. apis and.

N. ceranae. The differences allowed unequivocal differentiation of N.

apis and N. ceranae even under magnification of 15,000 x.

Intestine samples

From the naturally N. apis and N. ceranae infected colony (mix-

infection confirmed by the duplex PCR) were taken honey bees to

intestines preparation. Samples of intestines were collected from bees

with a medium level of infestation observed in a pooled sample and

estimated on 59 x 106 spores per bee.

Even a relatively mild Nosema spp. infection can be a cause of

deprivation of the physiological function of the bee alimentary tract,

due to the cover of the bee’ intestines by the layer of spores (Fig.

1D). During preparation of the SEM intestine samples, the level of

Nosema spp. infestation was counted from the pooled samples, as

mentioned above. The degree of individual bee infestation can of

course differ from the level of infection measured for the pooled

samples. For the SEM analysis, the intestines were isolated from

different bee specimens and therefore there may have been

differences in the level of Nosema spp. infection among the individual

samples. However, there were only slight differences in the cover of

intestines surfaces by Nosema spp. spores among the samples.

Generally, in all the intestines observed under SEM, spores were

present in such a great number that they formed a uniform layer on

the surface of the intestines, which could be the cause of the decline

or loss of a honey bee midgut function.

Discussion

There are a wide range of techniques for preparation of smear

samples providing better recognition of Nosema spp. (Vavra and

Larsson, 1999; Fries et al., 2013). The simplest and fastest method is

to use different observation techniques of the sample under light

microscopy. However, unequivocal identification of N. ceranae and N.

apis spores under the light microscope is not possible. Many fungal or

plant spores are shaped in a characteristic way that allows species

differentiation even in fossil samples. The same applies to the cell wall

of microsporidian spores. But the small dimensions of Nosema spores

mean that the spore sculpture can be observed only under

magnification provided by Scanning Electron Microscopy (SEM).

Ptaszyńska et al.

The microsporidian spore wall consists of two layers: the exospore

(or exosporium) and the endospore (or endosporium) (Vavra and

Larsson, 1999). The surface coat, i.e. the exosporium of Nosema

consists of three sheets and is an electron-dense glycoprotein

structure (Fries et al., 1996; Fries et al., 2006). Under SEM, the

sculpture of exosporium, which forms a thick coat around the spore,

can be observed. Observation of the internal structure of N. apis and

N. ceranae spores under Transmission Electron Microscopy (TEM)

revealed the two species can be separated on the basis of the number

of polar filament coils, i.e. N. ceranae always have fewer coils than N.

apis (Fries et al., 2006; Chen et al., 2009; Fries, 2010). In the same

TEM images, the exosporium was seen as a thick, wrinkled coat all

around the spore (Fries et al., 2006; Chen et al., 2009; Fries, 2010).

The whole surface of the exosporium can be observed under SEM

and, when compared, differences between the two Nosema species

can be seen (Fig. 1E). The exosporium sculpture of N. ceranae spores

(Fig. 1B) have deeper ornamentation than those of N. apis (Fig. 1a),

which have significantly larger folds, longer distances between the

furrows in the ornamentation, and wider edges of the furrows

(Table1).

TEM shows high quality, detailed structures and it is an excellent

tool for analysing a relatively small area of selected samples, whereas

SEM has proved to be a perfect tool for observing the details of

morphological structures and therefore it can be used to differentiate

the N. apis and N. ceranae spores on the basis of their sculpture.

Besides, SEM can be used for whole organs or surface screening in

search of spores. Intestines isolated from naturally Nosema spp.-

infected bees with a medium level of infestation were completely

covered by spores (Fig. 1D). Such a layer of spores can interfere with

the main gut function, i.e. absorption of nutrients.

The mere fact of infection increases the energy demand in the

organism by the need for mounting an immunological response after

infestation, which is an energetically expensive process (Schmid-

Hempel, 2005). Generally, Nosema spp.-infected bees have a higher

energetic request connected with the infestation. Especially, foragers

with their high metabolic demand due to flight are especially prone to

an energetic stress when they are infected (Mayack and Naug, 2009).

The spore layer covering bee intestines even during medium

infestation can lead to bee malnutrition and can be the cause of

higher mortality among bees observed after Nosema spp. infection.

Although nosemosis is most likely not the reason of CCD (Engelsdorp

et al., 2009), it is still the serious bee disease connected with severe

colony depopulation.

542

Table 3. Sculpture index. ** – differences are statistically significant

(Student's t-test; df = 240; p = 0.000015).

Nosema apis Nosema ceranae

2.30** 1.81**

Probably, similar layer of spores can be formed during other

microsporidian infection located in intestines, as is the case of e.g.

Encephalitozoon intestinalis, which is the cause of chronic diarrhoea

and enteritis in AIDS patients, and can lead to the death of infected

HIV-positive patients.

Scanning Electron Microscopy (SEM) showed excellent

morphological and structural details of the microsporidian spores,

which remain gold standards for the diagnosis of human and animal

infection caused by these microorganisms. This has a great value both

as a confirmatory tool and in determining the severity of infection.

Moreover, whole glands, organs, or various surfaces can be examined

for the presence of N. apis and N. ceranae spores and possible

reservoirs or niches for storage of Nosema spores can be detected.

Acknowledgements

We sincerely thank the beekeepers who allowed us access to their

colonies and two anonymous reviewers for their constructive

comments. This research was supported by the Individual Research

Grant of Vice-Rector for Research and International Relations of UMCS

(Lublin, Poland) and with the use of equipment purchased thanks to

the financial support of the European Regional Development Fund

under the project: “Increase in R & D potential of Departments of

Chemistry, and Biology and Earth Sciences UMCS in

Lublin” (POPW.01.03.00-06-017/09-00).

References

BORSUK, G; PTASZYŃSKA, A A; OLSZEWSKI, K; PALEOLOG, J (2013)

Impact of nosemosis on the intestinal yeast flora of honey bee.

Medycyna Weterynaryjna, 69: 726–730.

BOURGEOIS, A L; RINDERER, T E; BEAMAN, L D; DANKA, R G (2010)

Genetic detection and quantification of Nosema apis and N.

ceranae in the honey bee. Journal of Invertebrate Pathology, 103:

53–58, http://dx.doi.org/10.1016/j.jip.2009.10.009

BROTHER ADAM (1983) Meine Betriebsweise. Ehrenwirt Verlag;

München, Germany.

BURGHER-MACLELLAN, K L; WILLIAMS, G R; SHUTLER, D;

MACKENZIE, K C; ROGERS, R E L (2010) Optimization of duplex

real-time PCR with melting-curve analysis for detecting the

microsporidian parasites Nosema apis and Nosema ceranae in Apis

mellifera. Canadian Entomologist, 142: 271-283,

http://dx.doi.org/10.4039/n10-010

CANTWELL, G E (1970) Standard methods for counting nosema

spores. American Bee Journal, 110: 222-223.

543

CHEN, Y-P; EVANS, J D; SMITH, I B; PETTIS, J S (2008) Nosema

ceranae is a long-present and widespread microsporidian infection

of the European honey bee (Apis mellifera) in the United States.

Journal of Invertebrate Pathology, 97: 186-188,

http://dx.doi.org/10.1016/j.jip.2007.07.010

CHEN, Y-P; EVANS, J D; MURPHY, C; GUTELL, R; ZUKER, M;

GUNDENSEN-RINDAL, D; PETTIS, J S (2009) Morphological,

molecular, and phylogenetic characterization of Nosema ceranae,

a microsporidian parasite isolated from the European honey bee,

Apis mellifera. Journal of Eukaryotic Microbiology, 56: 142–147,

http://dx.doi.org/10.1111/j.1550-7408.2008.00374.x

COPLEY, T R; JABAJI, S H (2012) Honey bee glands as possible

infection reservoirs of Nosema ceranae and Nosema apis in

naturally infected forager bees. Journal of Applied Microbiology,

112: 15-24, http://dx.doi.org/10.1111/j.1365-2672.2011.05192.x

CRAILSHEIM, K; STOLBERG, E (1989) Influence of diet, age and

colony condition upon intestinal proteolytic activity and size of the

hypopharyngeal glands in the honey bee (Apis mellifera L.).

Journal of Insect Physiology, 35: 595–602.

CZEKOŃSKA, K (2001) Quality of honey bee queens commercially

available in southern Poland. Journal of Apicultural Science, 45:

5-11.

DADE, H A (2009) Anatomy and dissection of the honey bee (revised

Ed.). International Bee Research Association; Cardiff, UK. 196 pp.

ISBN 0-86098-214-9

DESEYN, J; BILLEN, J (2005) Age-dependent morphology and

ultrastructure of the hypopharyngeal gland of Apis mellifera

workers (Hymenoptera, Apidae). Apidologie, 36: 49–57,

http://dx.doi.org/10.1051/apido:2004068

VAN ENGELSDORP, D; EVANS, J D; SAEGERMAN, C; MULLIN, C;

HAUBRUGE, E; NGUYEN, B K; FRAZIER, M; FRAZIER, J; COX-

FOSTER, D; CHEN, Y-P; UNDERWOOD, R; TARPY, D R; PETTIS, J

S (2009) Colony Collapse Disorder: a descriptive study. PLoS ONE,

4: E6481, http://dx.doi.org/10.1371/journal.pone.0006481

FORSGREN, E; FRIES, I (2010) Comparative virulence of Nosema

ceranae and Nosema apis in individual European honey bees.

Veterinary Parasitology, 170: 212-217,

http://dx.doi.org/10.1016/j.vetpar.2010.02.010.

FRIES, I (1988) Infectivity and multiplication of Nosema apis Z. in the

ventriculus of the honey bee. Apidologie, 19: 319–328,

http://dx.doi.org/10.1051/apido:19880310

FRIES, I (2010) Nosema ceranae in European honey bees (Apis

mellifera). Journal of Invertebrate Pathology, 103: S73–S79,

http://dx.doi.org/10.1016/j.jip.2009.06.017

Differentiation of N. apis and N. ceranae spores under SEM

FRIES, I; FENG, F; SILVA, A D; SLEMENDA, S B; PIENIAZEK, N J

(1996) Nosema ceranae n. sp. (Microspora, Nosematidae),

morphological and molecular characterization of a microsporidian

parasite of the Asian honey bee Apis cerana (Hymenoptera,

Apidae). European Journal of Protistology, 32: 356–365.

http://dx.doi.org/10.1016/S0932-4739(96)80059-9

FRIES, I; MARTÍN, R; MEANA, A; GARCÍA-PALENCIA, P; HIGES, M

(2006) Natural infections of Nosema ceranae in European honey

bees. Journal of Apicultural Research, 45: 230–233.

FRIES, I; CHAUZAT, M-P; CHEN, Y-P; DOUBLET, V; GENERSCH, E;

GISDER, S; HIGES, M; MCMAHON, D P; MARTÍN-HERNÁNDEZ, R;

NATSOPOULOU, M; PAXTON, R J; TANNER, G; WEBSTER, T C;

WILLIAMS, G R (2013) Standard methods for nosema research. In

V Dietemann; J D Ellis, P Neumann (Eds) The COLOSS BEEBOOK:

Volume II: Standard methods for Apis mellifera pest and pathogen

research. Journal of Apicultural Research 52(1):

http://dx.doi.org/10.3896/IBRA.1.52.1.14

DE GRAAF, D; JACOBS, F J (1991) Tissue specificity of Nosema apis.

Journal of Invertebrate Pathology, 58: 277–278.

HIGES, M; MARTÍN, R; MEANA, A (2006) Nosema ceranae, a new

microsporidian parasite in honey bees in Europe. Journal of

Invertebrate Pathology, 92: 93–95,

http://dx.doi.org/10.1016/j.jip.2006.02.005

HIGES, M; GARCIA-PALENCIA, P; MARTIN-HERNANDEZ, R; MEANA, A

(2007) Experimental infection of Apis mellifera honey bees with

Nosema ceranae (Microsporidia). Journal of Invertebrate

Pathology, 94: 211-217.

http://dx.doi.org/10.1016/j.jip2006.11.001

HIGES, M; MARTÍN-HERNÁNDEZ, R; MEANA, A (2010) Nosema

ceranae in Europe, an emergent type C nosemosis. Apidologie,

41: 375-392, http://dx.doi.org/10.1051/apido/2010019

HORNITZKY, M (2008) Nosema disease – Literature review and three

surveys of beekeepers – Part 2. Rural Industries Research and

Development Corporation. Pub. No. 08/006, 2008. RIRDC;

Australia.

HUANG, W F; JIANG, J H; WANG C H (2005) Nosema ceranae

infection in Apis mellifera. In Proceedings 38th Annual Meeting of

Society for Invertebrate Pathology, Anchorage, Alaska, USA.

HUANG, W F; SOLTER, L F (2013) Comparative development and

tissue tropism of Nosema apis and Nosema ceranae. Journal of

Invertebrate Pathology, 113: 35-41.

http://dx.doi.org/doi:10.1016/j.jip.2013.01.001

Ptaszyńska et al.

LIU, F; WANG, Q; DAI, P L; WU Y, Y; SONG, H K; ZHOU, T (2008)

Natural stripe of microsporidia of honey bee in China. Chinese

Bulletin of Entomology, 45: 963–966.

MALONE, L A; TODD, J H; BURGESS, E P J; CHRISTELLER, J T (2004)

Development of hypopharyngeal glands in adult honey bees fed

with a Bt toxin, a biotin binding protein and a protease inhibitor.

Apidologie, 35: 655–664,

http://dx.doi.org/10.1051/apido:2004063

MARTÍN-HERNÁNDEZ, R; MEANA, A; PRIETO, L; MARTÍNEZ

SALVADOR, A; GARRIDO-BAILÓN, E; HIGES, M (2007) Outcome

of colonization of Apis mellifera by Nosema ceranae. Applied and

Environmental Microbiology, 73: 6331–6338,

http://dx.doi.org/10.1128/AEM.00270-07

MAYACK, C; NAUG, D (2009) Energetic stress in the honey bee Apis

mellifera from Nosema ceranae infection. Journal of Invertebrate

Pathology, 100: 185–188,

http://dx.doi.org/10.1016/j.jip.2008.12.001

PAXTON, R J; KLEE, J; KORPELA, S; FRIES, I (2007) Nosema ceranae

has infected Apis mellifera in Europe since at least 1998 and may

be more virulent than Nosema apis. Apidologie, 38: 558–565,

http://dx.doi.org/10.1051/apido:2007037

PTASZYŃSKA, A A; BORSUK, G; ANUSIEWICZ, M; MUŁENKO, W

(2012) Location of Nosema spp. spores within body of honey bee.

Medycyna Weterynaryjna, 68: 618-621.

PTASZYŃSKA, A A; BORSUK, G; WOŹNIAKOWSKI, G; GNAT, S;

MAŁEK, W (2014) Loop-mediated isothermal amplification (LAMP)

assays for rapid detection and differentiation of Nosema apis and

N. ceranae in honey bees. FEMS Microbiology Letters, 357: 40–

48, http://dx.doi.org/10.1111/1574-6968.12521

SAS/STAT (date) Users Guide release 9.13. Statistical Analysis System

Institute; Cary, NC, USA.

SCHMID-HEMPEL, P (2005) Evolutionary ecology of insect immune

defences. Annual Review of Entomology, 50: 529-551.

http://dx.doi.org/10.1146/annurev.ento.50.071803.130420

VAVRA, J; LARSSON, R (1999) Structure of the microsporidia. In M

Wittner; L M Weiss (Eds). The microsporidia and microsporidiosis.

ASM Press; Washington DC; USA. pp. 7-84.

ZANDER, E (1909) Tierische Parasiten als Krankenheitserreger bei der

Biene. Münchener Bienenzeitung, 31: 196–204.

544