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Evaluation of Lysozyme-HCl for the Treatment of Chalkbrood Disease in Honey Bee Colonies

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Chalkbrood, caused by Ascosphaera apis (Maassen and Claussen) Olive and Spiltor, is a cosmopolitan fungal disease of honey bee larvae (Apis mellifera L.) for which there is no chemo-therapeutic control. We evaluated the efÞcacy of lysozyme-HCl, an inexpensive food-grade antimi-crobial extracted from hen egg white, for the treatment of chalkbrood disease in honey bee colonies. Our study compared three doses of lysozyme-HCl in sugar syrup (600, 3,000, and 6,000 mg) administered weekly for 3 wk among chalkbrood-inoculated colonies, colonies that were inoculated but remained untreated, and colonies neither inoculated or treated. Lysozyme-HCl at the highest dose evaluated was found to suppress development of chalkbrood disease in inoculated colonies to levels observed in uninoculated, untreated colonies, and did not adversely affect adult bee survival or brood production. Honey production was signiÞcantly negatively correlated with increased disease severity but there were no signiÞcant differences in winter survival among treatment groups. Based on our results, lysozyme-HCl appears to be a promising, safe therapeutic agent for the control of chalkbrood in honey bee colonies.
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APICULTURE AND SOCIAL INSECTS
Evaluation of Lysozyme-HCl for the Treatment of Chalkbrood Disease
in Honey Bee Colonies
A. VAN HAGA,
1,2,3
B. A. KEDDIE,
2
AND S. F. PERNAL
3
J. Econ. Entomol. 105(6): 1878Ð1889 (2012); DOI: http://dx.doi.org/10.1603/EC12227
ABSTRACT Chalkbrood, caused by Ascosphaera apis (Maassen and Claussen) Olive and Spiltor, is
a cosmopolitan fungal disease of honey bee larvae (Apis mellifera L.) for which there is no chemo-
therapeutic control. We evaluated the efÞcacy of lysozyme-HCl, an inexpensive food-grade antimi-
crobial extracted from hen egg white, for the treatment of chalkbrood disease in honey bee colonies.
Our study compared three doses of lysozyme-HCl in sugar syrup (600, 3,000, and 6,000 mg) admin-
istered weekly for 3 wk among chalkbrood-inoculated colonies, colonies that were inoculated but
remained untreated, and colonies neither inoculated or treated. Lysozyme-HCl at the highest dose
evaluated was found to suppress development of chalkbrood disease in inoculated colonies to levels
observed in uninoculated, untreated colonies, and did not adversely affect adult bee survival or brood
production. Honey production was signiÞcantly negatively correlated with increased disease severity
but there were no signiÞcant differences in winter survival among treatment groups. Based on our
results, lysozyme-HCl appears to be a promising, safe therapeutic agent for the control of chalkbrood
in honey bee colonies.
KEY WORDS Apis mellifera, honey bee, Ascosphaera apis, chalkbrood, lysozyme
Chalkbrood, a fungal disease of honey bee brood
caused by Ascosphaera apis (Maassen and Claussen)
Olive and Spiltoir (Spiltoir and Olive 1955) occurs
worldwide (Ellis and Munn 2005). IdentiÞed by the
sporulated black or nonsporulated white chalk-like
larval cadavers it produces, the spores of A. apis are
spread within and between colonies via adult worker
bees who transmit spores to honey bee larvae during
feeding (Heath 1982). Chalkbrood epizootics are vari-
able and unpredictable (BefusÐNogel et al. 1992) but
generally increase in incidence in early spring (Heath
1982). Cool temperatures, high humidity, inadequate
nutrition, stress, and genetic factors have all been
implicated as predisposing conditions in the develop-
ment of chalkbrood disease (Aronstein and Murray
2010).
Although an extensive range of available natural
compounds and fungicides has been investigated for
the treatment of chalkbrood both in the laboratory
and in the Þeld (Hornitzky 2001, Davis and Ward
2003), there is at present no registered chemothera-
peutic treatment. Widespread acceptance of a chem-
ical for the treatment of chalkbrood requires that it
must be effective, easy to use, and economical (Mena-
pace and Hale 1981). Moreover, it must not compro-
mise the safety and quality of the honey produced for
human consumption. Contamination of honey by drug
residues used in the treatment of honey bee diseases
is an important public safety issue and can affect the
import and export of hive products between countries
(McKee 2003).
Lysozyme-HCl is an inexpensive, food-grade anti-
microbial enzyme used in cheese and winemaking
processes in the European Union, United States, and
Canada to inhibit the growth of damaging gram-pos-
itive bacteria (Johnson and Larson 2005). Gram-pos-
itive bacteria with cell walls rich in peptidoglycan are
particularly susceptible to lysozyme-HCl as the main
mode of action is the hydrolysis of the §(1Ð 4) linkages
in the polymers that make up peptidoglycan causing
the cell to lyse (Strominger and Tipper 1974). In
addition, lysozyme-HCl is also capable of breaking
down chitin (Berger and Weiser 1957), a component
of most fungal cell walls (GrifÞn 1994). The antimi-
crobial activity of lysozyme-HCl is not solely depen-
dent on its enzymatic activity. Lysozyme has been
shown to cause cell death through cell membrane
disruption by distorting lipid-lipid interactions and
increasing membrane permeability (Ibrahim et al.
1996, Du¨ring et al. 1999) and because lysozyme is
highly cationic, it may be also capable of inducing
autolysis in bacteria (Ginsburg and Koren 2008). Us-
ing in vitro larval rearing assays (Van Haga 2010), it has
been shown that lysozyme-HCl is active against the
fungus A. apis, the causative agent of chalkbrood dis-
ease in honey bees, although the mode of action is
unknown.
1
Corresponding author: University of British Columbia, 2125 East
Mall, Vancouver, BC, Canada V6T 1Z4 (e-mail: vanhagaa@gmail.
com).
2
Department of Biological Sciences, University of Alberta, Edmon-
ton, AB, Canada T6G 2E9.
3
Agriculture and Agri-Food Canada, P.O. Box 29, Beaverlodge, AB,
Canada T0H 0C0.
Safe for both honey bee and human consumption
(Johnson and Larson 2005, Van Haga 2010), lysozyme-
HCl shows great potential as a control for chalkbrood
disease at the colony level. Its high solubility in water
and sucrose solutions (Johnson and Larson 2005) al-
lows easy integration into current management prac-
tices such as fall or spring feeding. Beekeepers rou-
tinely supplement colony diets both in the spring and
fall with sugar syrup or high fructose corn syrup to
boost colony growth. Lysozyme-HCl is also resistant
to changes in temperature and pH. As a result, it is an
extremely stable compound and will remain active
during prolonged exposure at colony temperatures of
33Ð35C (Johnson and Larson 2005). Safe, soluble, and
stable, lysozyme-HCl is an ideal candidate for colony-
level treatment of chalkbrood disease.
Although widespread, the severity of infection and
subsequent economic impact of chalkbrood on bee-
keeping operations worldwide are highly variable.
Considered by some beekeepers to be the most im-
portant brood disease encountered in their apiaries
(Jakobsons 2005), it is only a mild nuisance for others
(Ileana 2007). Even though chalkbrood is rarely lethal
to the colony, larval mortality as a result of chalkbrood
infection can have a serious impact on the buildup of
adult bee populations. It has been suggested that 100
infected larvae is equivalent to a 5% loss in potential
adult populations (Taber et al. 1975). SigniÞcant re-
ductions in the worker bee population can translate
into decreases in foraging resulting in both decreased
honey production and pollination efÞcacy. Estimates
of losses attributed to chalkbrood range from 1 to 37%
of honey yields (Heath 1982, Yakobson et al. 1991) and
up to 49% of foraging capacity (Heath 1982). Exper-
imental assessments of yield losses in honey from clo-
ver (Trifolium alexandrinum L.) in Egypt were re-
ported to be 18.4 0.7% in naturally infected colonies
and as high as 30.1 1.8% in colonies artiÞcially in-
fected with black mummies (Zaghloul et al. 2005). In
Beaverlodge, Alberta, Canada, where this study was
conducted, previous experiments did not establish a
signiÞcant relationship between chalkbrood infection
levels and honey production (Nelson and Gochnauer
1982).
In this study, therapeutic doses of lysozyme-HCl for
the treatment of chalkbrood disease in honey bee
colonies were evaluated and the impact of chalkbrood
disease on: 1) colony population, 2) honey produc-
tion, and 3) winter survival in newly established pack-
age colonies was assessed.
Materials and Methods
Colony Establishment and Management. Forty col-
onies were established 24 April 2007 at the AAFC
Beaverlodge Research Farm (5518N; 11917W).
One-kilogram packaged bees with queens (mixed
race) were imported from New Zealand and placed in
irradiated (10 kGy, Iotron Industries Canada Inc., Port
Coquitlam, BC) single brood chambers (nine frames,
full depth Langstroth supers). Each colony was
equipped with a dead bee trap, modiÞed from Illies et
al. 2002, which was attached below the front edge of
the bottom board. Bottom boards were modiÞed by
removing the rim along the rear of the colonies to
accommodate the removal of corrugated plastic sheets
(Tenplast 37 46 cm) that lay ßat beneath the brood
chamber. These sheets facilitated the counting of
chalkbrood mummies removed from the comb by
bees. Colonies were managed as single brood cham-
bers throughout the experiment. Before or during the
experiment, colonies were requeened as required
(e.g., replacing queens that had perished) with New
Zealand queens of similar age to those established with
the packages.
At the time of establishment, all colonies were
treated with Fumagilin-B (Medivet Pharmaceuticals
High River, AB, Canada) in sucrose syrup according to
label directions for package colonies (100 mg active
ingredient [AI]/colony) for the control of Nosema
apis Zander. Each colony also received a 454 g pollen
supplement patty (Global Patties, Airdrie, Alberta,
Canada). At no time in the experiment were varroa
mites (Varroa destructor Anderson and Truemann)
detected.
Colony population (adult bees, sealed and unsealed
brood cells) was assessed before start of experiment
on 11 May 2007 using a Plexiglas grid (2.5 cm
2
) and
grouped into eight strength categories (strongest to
weakest) based on adult bee and brood population
size. One colony per group was randomly selected
from within each of the eight strength categories and
assigned to each treatment group.
Colony Inoculation. Thirty-two colonies were in-
oculated on 15 May 2007 (day 1) with chalkbrood
spores according to the method outlined by Gilliam et
al. (1988). Each colony received a 113 g pollen patty
[40% (wt:wt) irradiated pollen, 35% (wt:wt) commer-
cial table grade sucrose, 5% (wt:wt) BrewerÕs yeast,
20% (wt:wt) sterile distilled water] containing chalk-
brood mummies. Mummies were collected from dis-
eased apiaries in the Beaverlodge area and homoge-
nized in 5 ml sterile distilled water using a glass/glass
tissue homogenizer (Kontes, Vineyard, NJ). For a
batch of 32 pollen patties, a combination of Þve black
and Þve white mummies (approximately double the
inoculation dose used by Gilliam et al. [1988]) to
ensure prolonged infection), were used; each pollen
patty contained 1.56 10
7
spores. Control colonies
received a similar pollen patty without the addition of
homogenized mummies. Pollen patties were placed on
the top bars in the center of the colony. The viability
and uniformity of A. apis distribution in the patty
mixture was determined by plating six pollen patty
samples collected from throughout the patty mixture
on PDY (Potato Dextrose Agar [Difco, Detroit, MI]
0.4% (wt:vol) Yeast Extract) media (Shimanuki and
Knox 2000). The plated samples were incubated at
30C for 120 h and examined for growth of A. apis.
Additionally, two pollen patty samples were collected
from each colony if available, 3 and 10 d postinocu-
lation and plated on PDY as previously described to
conÞrm viability of A. apis spores.
December 2012 VAN HAGA ET AL.: LYSOZYME FOR TREATMENT OF CHALKBROOD IN HONEY BEES 1879
Colony Treatment. Lysozyme-HCl (inovapure 300,
Neova Technologies, Abbotsford, BC, Canada) was
mixed in 1:1 (wt:vol) sucrose syrup prepared with
table grade sucrose and applied to colonies using
frame feeders. Inoculated colonies were untreated or
given weekly applications of 600, 3,000, or 6,000 mg
lysozyme-HCl for 3 wk. The Þrst treatments for all
doses were dissolved in 2 liters of syrup, while in
subsequent treatments only 1 liter of syrup was used.
The dates of application were 15, 22, and 29 May 2007
(days 1, 8, and 15). The uninoculated and inoculated
untreated colonies were fed syrup without lysozyme-
HCl. At the fourth weekly assessment the volume of
any unconsumed syrup was measured and recorded.
In total, there were Þve treatment groups contain-
ing eight colonies each: uninoculated (uninoculated
and untreated); inoculated (inoculated and untreat-
ed); low (inoculated and treated with 600 mg 3
doses of lysozyme-HCl); medium (inoculated and
treated with 3,000 mg 3 doses of lysozyme-HCl);
high (inoculated and treated with 6,000 mg 3 doses
of lysozyme-HCl).
Colony Assessments. Disease Severity and Bee Mor-
tality. Black and white chalkbrood mummies and dead
adult bees were collected and counted from 1) dead
bee traps and 2) bottom board sheets on weekdays
from 14 May (day 0) until 28 August 2007 (day 106).
Chalkbrood mummies and bees collected on Mondays
were averaged over 3 d (Saturday, Sunday, and Mon-
day). The frames in the colony were inspected and the
numbers of black and white chalkbrood mummies in
uncapped cells were counted on days 1, 4, 8, 11, 15, 22,
29, 36, 43, 50, 57, 64, 78, 92, and 106.
Colony Strength. Colony strength was estimated on
days 5, 15, 29, 43, and 57. The areas of sealed brood,
unsealed brood, and adult bee populations were esti-
mated using a Plexiglas grid (2.5 cm
2
). Grids were
placed over all the frames in the colony and the num-
ber of squares of brood cells and worker bees were
counted on both sides of each frame. A conversion
factor of 1.5188 bees/cm
2
was used to estimate adult
bee populations (Westcott and Winston 1999) and 3.9
worker cells/cm
2
to estimate absolute numbers of
sealed and unsealed worker brood cells (Harbo 1986).
Adult bee population estimates were performed dur-
ing early morning hours before bees began to ßy.
Honey Production. Honey supers were weighed be-
fore placement on colonies and after removal to mea-
sure net honey yield. All colonies were provided one
honey super on 13 June 2007 and two additional honey
supers on 4 July 2007 after which time colonies were
provided additional honey supers as needed. Honey
was harvested from colonies on 17 July, 31 July, 14
August 2007; supers were stripped from the brood
chambers on 27 August 2007.
Stability and Persistence of Lysozyme-HCl. Stored
food samples (1.5 ml) from each colony were col-
lected from the outer edges of three brood frames to
assay for lysozyme-HCl activity 7 d after the Þrst
treatment application and then weekly for three ad-
ditional weeks. A sample from each colony was also
collected in the same manner 3 d before the start of the
experiment before treatments were applied. Samples
were stored at 5C in 1.5 ml microfuge tubes until
analysis of lysozyme-HCl concentrations by the man-
ufacturer, Neova Technologies (Abbotsford, BC, Can-
ada).
Presence of Spores. Five larvae were sampled from
each colony on days 1, 8, 15, 22, and 29. Larvae were
collected singly into sterile 1.5 ml microcentrifuge
tubes using sterile forceps and stored at 20C until
time of plating. Larvae were surface sterilized by
wrapping them in sterilized cheesecloth, dipping in
75% ethanol and immediately transferring for 60 s to
a 0.5% solution of sodium hypochlorite. Samples were
rinsed twice with sterile distilled water then immedi-
ately homogenized in 1.0 ml sterile distilled water. The
homogenate was vortexed with 15 ml YGSPA 0.01%
chloramphenicol (10 g yeast, 10 g dextrose, 13.5 g
KH
2
PO
4
, 10 g soluble starch, and 20 g agar in 1 liter
H
2
O) and poured onto plates containinga7mlsolid
media layer. Plates were incubated at 37C, 10% CO
2
for 24 h to stimulate germination and then at 37C, 0%
CO
2
to establish mycelial growth according to the
methods of Nelson and Gochnauer (1982) and An-
derson et al. (1997).
Adult nurse bees (30Ð100) were collected in 50 ml
centrifuge tubes with caps and held overnight at 4C.
The digestive tract or gut from Þve adult bees was
removed and rinsed three times with distilled water to
remove extraneous debris and microorganisms. After
rinsing, each gut was placed into a sterile 1.5 ml mi-
crocentrifuge tube and stored at 20C until time of
plating. To withdraw digestive tracts, the stinger was
grasped and gently pulled until the entire tract (honey
stomach, midgut, hindgut, and rectum) was removed
(Shimanuki and Knox 2000). After excising the
stinger, each digestive tract was homogenized in 1.0 ml
sterile distilled water and cultured according to the
methods of Anderson et al. (1997).
Winter Survival. Colonies were assessed for sur-
vival (presence of queen and brood) the following
spring on 14 May 2008. Frames in the colony were
inspected and number of black and white chalkbrood
mummies in uncapped cells counted. Colony popu-
lation (unsealed brood, sealed brood, and adult bees)
was estimated as previously described.
Statistical Analysis. Colonies were excluded from
the experiment when chronic queenlessness or dis-
eases other than chalkbrood resulted in less than one
frame of healthy unsealed brood at any time before
over-wintering. The numbers of colonies included in
the Þnal analysis for each of the treatment groups
were: uninoculated (7); inoculated (6); low (8); me-
dium (5); and high (7).
Differences among treatment groups for the num-
ber of average mummies (total, black, and white)
collected daily from traps, average weekly frame
mummy counts, adult mortality, honey yield, accu-
mulation of lysozyme-HCl in stored food and differ-
ences in the number of white and black mummies
collected were compared using one-way analysis of
variance (ANOVA) and a posteriori comparisons
1880 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 6
(TukeyÐKramer honestly signiÞcant difference [HSD];
0.05).
Winter survival between treatment groups was
compared using a
2
test (
0.05). Differences in
colony strength (adult bee population, sealed, and
unsealed brood cells) among over-wintered colonies
were compared using ANOVA and a posteriori com-
parisons (TukeyÐKramer HSD;
0.05).
The relationship between disease severity and
honey yield was modeled using linear regression.
Honey yield was transformed using a Log
10
transfor-
mation to stabilize the variance and normalize the
data.
The effect of time on disease severity (square root
transformed to stabilize variances in the sample data),
adult bee mortality, adult bee populations, sealed, and
unsealed brood cells between treatment groups was
compared using repeated measures ANOVA. The
HuynhÐFeldt correction was used, as assumptions of
sphericity were not met. Contrast analysis was used to
compare the differences within treatment groups for
disease severity. All analyses were performed using
JMP (7.01, SAS Institute, Gary, NC).
Results
Disease Severity and Adult Bee Mortality. The
mean number of total chalkbrood mummies collected
daily from the traps and bottom boards (Fig. 1) dif-
fered signiÞcantly between the low and inoculated
treatment groups and the uninoculated, medium, and
high treatment groups (F112.5413; df 4, 3526; P
0.0001). The inoculated and low treatment groups
produced between 12.5 and 13.7 mummies daily, over
16 times more than the uninoculated and high treat-
ment groups and four times more than the medium
treatment group. SigniÞcantly higher numbers of
black mummies were collected (F102.48; df 4,
3526; P0.0001) from colonies in the inoculated
treatment group than the low treatment group but
signiÞcantly higher numbers of white mummies were
collected (F87.75511; df 4, 3526; P0.0001) from
the low treatment colonies than the inoculated treat-
ment group. Both the low and inoculated treatment
groups produced signiÞcantly more black and white
mummies than the uninoculated, medium, and high
treatment groups. Numbers of both black and white
mummies collected daily did not differ signiÞcantly
between the uninoculated, medium, and high treat-
ments and ranged between 0.38Ð1.8 and 0.32Ð1.04
mummies, respectively.
Similar to the daily collections, the total numbers of
chalkbrood mummies counted in the brood frames
during the weekly colony inspections (F14.2929;
df 4, 489; P0.0001) were signiÞcantly higher in the
inoculated and low treatment group brood frames
than in the uninoculated, medium, and high treatment
groups (Fig. 2). On average, 34Ð37 mummies per week
were counted in the inoculated and low treatment brood
frames compared with the 3Ð8 mummies in the brood
frames in the other treatment groups. Similar to the total
chalkbrood counts, the numbers of both black and white
mummies counted separately in the uninoculated, me-
dium, and high treatment groups were similar and sig-
niÞcantly lower than the inoculated and low treatment
groups (F
Black Mummies
12.34, df 4, 489, P0.0001;
F
White Mummies
9.63, df 4, 489, P0.0001). The
numbers of white mummies in the inoculated and low
Fig. 1. The mean daily number of mummies (total, black, and white) collected on weekdays from traps and bottom boards
for each treatment group from 14 May to 28 August 2007; collections on Monday were averaged over Saturday, Sunday, and
Monday. The numbers of total, black, and white mummies collected were analyzed separately. Treatments with different
letters are signiÞcantly different at
0.05 (TukeyÐKramer HSD). For each category different letter styles were used to
indicate signiÞcance: total mummy production (gray, uppercase, italicized letters, A); black mummy production (black,
lowercase, Greek letters,
); white mummy production (black, lowercase letters, a).
December 2012 VAN HAGA ET AL.: LYSOZYME FOR TREATMENT OF CHALKBROOD IN HONEY BEES 1881
treatment groups averaged 17.8 Ð20.3 per week and were
higher than the number of black mummies counted. In
comparison, the uninoculated, medium, and high treat-
ment groups had extremely low levels of mummies in the
brood frames ranging from 1.5 to 2.9 black mummies and
1.2Ð5.0 white mummies per week.
Numerical differences in the number of black mum-
mies visible in the brood frames compared with the
number of visible white mummies was not signiÞcant
among treatment groups (F0.35; df 4, 489; P
0.84) over the entire duration of the experiment (F
0.7615; df 1, 986; P0.3831) but the differences in
the number of black mummies compared with the
number of white mummies collected daily from the
traps and bottom boards were signiÞcant (F88.3566;
df 1, 7060; P0.0001) (Fig. 3). In the daily counts
signiÞcantly more black than white mummies, 7.4
more per day, were collected from the inoculated
colonies compared with the other treatment groups
(F65.85; df 4, 3526; P0.0001).
A closer examination of the nontransformed daily
average of total mummies collected in each treatment
Fig. 2. The mean number of visible chalkbrood mummies (black and white) in brood frames counted weekly for each
treatment group from 14 May to 28 August 2007. The numbers of total, black, and white mummies counted were analyzed
separately. Treatments with different letters are signiÞcantly different at
0.05 (TukeyÐKramer HSD). For each category
different letter styles were used to indicate signiÞcance: total mummy production (gray, uppercase, italicized letters, A); black
mummy production (black, lowercase, Greek letters,
); white mummy production (black, lowercase letters, a).
Fig. 3. The mean number of black and white chalkbrood mummies collected daily from the traps and bottom boards or
counted weekly in brood frames for all colonies in the experiment (n33) from 14 May to 28 August 2007. Daily and weekly
counts were analyzed separately by ANOVA. Letters denote a signiÞcant difference (
0.05) in the number of black
mummies compared with the number of white mummies counted within each category.
1882 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 6
group over the period of 14 May through 28 August
2007 reveals some general trends (Fig. 4). Five days
postinoculation chalkbrood mummies appeared in the
traps and bottom boards of all inoculated treatment
groups and peaked in number 9 d after inoculation.
Mummy count decreased almost immediately and 12 d
postinoculation, the medium and high dose treatment
groups were producing extremely low levels of mum-
mies until late July when an increase and ßuctuation
in mummy counts was recorded for the medium treat-
ment group. Mummy counts in the inoculated and low
treatment colonies also decreased 12 d postinocula-
tion but mummy levels ßuctuated in both treatment
groups for the remainder of the experiment. The peak
in mummy production at the end of July, 10 wk pos-
tinoculation, seen in the medium treatment group was
also seen in the low and inoculated treatment groups.
The uninoculated colonies produced minimal chalk-
brood mummies for the entirety of the experiment but
5 wk postinoculation small increases in mummy count
began and occurred sporadically for the remainder of
the experiment. Although repeated measures analysis
of daily mummy counts (square root transformed
data) over the duration of the experiment (106 d)
shows a marginally nonsigniÞcant treatment effect
(F2.63; df 4,28; P0.0552), it does reveal a
signiÞcant time effect (F6.43; df 7.12, 199.43; P
0.0001) and signiÞcant time by treatment interaction
(F1.57; df 28.48, 199.43; P0.0406). Posthoc
comparisons reveal a signiÞcant difference in the ef-
fect of the treatment over time (F3.05; df 7.12,
199.43; P0.0043) when the uninoculated, medium,
and high dose treatment groups are contrasted with
the inoculated and low treatment groups.
Within each treatment group the cumulative num-
ber of mummies collected daily varied among colo-
nies. Regardless of scale, there were one or two col-
onies in each treatment group that produced at least
two or three times more mummies than the other
colonies. In each of the inoculated and low treatment
groups there were colonies that produced 4,000
mummies over the duration of the experiment, 10
times the maximum amount produced by any colony
in the uninoculated or high treatment groups.
There were no signiÞcant differences in the mean
adult bee mortality among treatment groups (F1.65;
Fig. 4. Mean number of chalkbrood mummies collected daily from traps and bottom boards for each treatment group
from 14 May through 28 August 2007. Pollen patties (113 g) containing homogenized chalkbrood mummies were applied to
all inoculated treatment groups (Inoculated, Low, Medium, High) and clean patties to the uninoculated treatment group
(Uninoculated) simultaneously with Þrst treatment of lysozyme-HCl on 15 May 2007. All treatment groups received three
weekly treatments of 50% (wt:vol) sucrose syrup containing 0 (Uninoculated, Inoculated), 600 (Low), 3,000 (Medium), or
6,000 mg (High) of lysozyme-HCl.
December 2012 VAN HAGA ET AL.: LYSOZYME FOR TREATMENT OF CHALKBROOD IN HONEY BEES 1883
df 4, 3526; P0.1597). Adult bee mortality ranged
from 8.7 bees/d in the inoculated treatment group to
10.5 bees/d in the uninoculated colonies. Repeated
measure analysis of adult bee mortality over time (14
May through 28 August 2007) did not show a signif-
icant treatment effect (F0.5639; df 4, 28; P
0.6908) or time by treatment interaction (F0.8136;
df 68.885, 482.2; P0.8547). There was a signiÞcant
time effect (F20.4678; df 17.221, 482.2; P0.0001)
corresponding to increased adult bee mortality across
all treatment groups on the day after colony inspec-
tions and assessments.
Colony Strength. Repeated measures analysis on
number of adult bees, number of sealed brood cells,
and number of unsealed brood cells counted every 2
wk from the start of the experiment until 10 July 2007
showed a signiÞcant effect of time for all three mea-
sures of the population (F
Adult
160.27, df 3.68,
102.91, P0.0001; F
Sealed
56.78, df 3.87, 108.31, P
0.0001; F
Unsealed
35.89, df 2.82, 78.87, P0.0001).
In general, all measures of population increased in
number over time except adult bee populations, which
increased until 26 June and decreased 10 July. There
was no signiÞcant treatment effect (F
Adult
0.4732,
df 4,28, P0.7550; F
Sealed
0.0350, df 4, 28, P
0.9975; F
Unsealed
0.2296, df 4, 28, P0.9195) or
time by treatment interaction for adult bee, sealed
brood, or unsealed brood numbers (F
Adult
0.7467,
df 14.70, 102.91, P0.7292; F
Sealed
0.8458, df
15.47, 108.31, P0.6283; F
Unsealed
1.10, df 11.27,
78.87, P0.3738).
Honey Production. Although mean honey yield did
not differ signiÞcantly among treatment groups (F
0.2703; df 4,28; P0.8946), there was a mean
increase in honey yield as the amount of lysozyme-
HCl applied to the colonies increased (Fig. 5). Honey
yields ranged from 99.5 kg in the inoculated untreated
treatment group to 124.2 kg in the inoculated high
dose treatment group. Nevertheless, there was a sig-
niÞcant (F16.03; df 1,31; P0.0004) and corre-
lated (R
2
0.34) relationship between the total mum-
mies collected from traps and bottom boards and
honey yield. As the number of mummies collected
increased, honey yield decreased (Fig. 6).
Stability and Persistence of Lysozyme-HCl. Ly-
sozyme-HCl activity was detected in all treatment
groups given lysozyme-HCl (Fig. 7) and not found in
the treatment groups given only sugar syrup. The
amount of lysozyme-HCl detected in the stored food
samples increased over time in a dose-dependent
manner. Repeated measures analysis on the amount of
lysozyme-HCl detected over time showed a signiÞcant
treatment effect (F45.88; df 4,28; P0.0001),
time effect (F5.81; df 2.36,66; P0.003), and
treatment by time interaction (F3.63; df 9.43,66;
P0.0008). Posthoc comparisons revealed a signiÞ-
cant difference in the effect of treatment over time
when the high dose treatment group was contrasted
with both of the medium and low dose groups (F
9.07; df 2.36,66; P0.0002).
Presence of Spores. Spores were present in all treat-
ment groups in either the larvae or adult bee digestive
tracts over all dates sampled.
Winter Survival. There were no signiÞcant differ-
ences in the percentage of colonies surviving the win-
ter among treatment groups (
2
6.18; df 4,33; P
0.1502); winter survival ranged from 57% in the uni-
noculated treatment group to 100% in the inoculated
and medium dose treatment groups. Survival in the
low and high treatment groups was 88 and 86%, re-
spectively.
Fig. 5. Mean honey production (kilogram) per colony compared among treatment groups. Honey was collected for the
duration of the trial (14 May through 28 August 2007). Treatments were not signiÞcantly different at
0.05 (TukeyÐKramer
HSD).
1884 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 6
At the time of inspection, 14 May 2008, there were
no signiÞcant differences in the adult bee populations
(F0.4643; df 4, 23; P0.7612), number of sealed
brood cells (F0.4497; df 4, 23; P0.7715), or
unsealed brood cells (F0.5731; df 4, 23; P
0.6849). There were no signiÞcant differences among
treatment groups in the number of total (F1.088;
df 4, 23; P0.3857), black (F0.8952; df 4, 23;
P0.4828), or white (F1.279; df 4,23; P0.3071)
chalkbrood mummies visible in the brood frames
when inspected on 14 May 2008 (Fig. 8). However, in
the inoculated and low treatment groups total mummy
counts visible in the brood frame averaged between
9.5 and 26.7, higher than the other treatment groups
that averaged 0.5Ð1.8 chalkbrood mummies per col-
ony.
Discussion
Lysozyme-HCl in the medium (3,000 mg 3 doses)
and highest (6,000 mg 3 doses) doses tested was
capable of suppressing mummy production in artiÞ-
cially inoculated colonies to levels similar to that of
uninoculated untreated colonies. Mean daily mummy
production was 15 times lower in the inoculated high
dose treatment group than in the inoculated untreated
colonies. Although all inoculated treatment groups
saw a dramatic increase in mean mummy production
5 d postinoculation and a sharp decrease 1 wk later, it
was only the highest dose treatment group that sup-
pressed mummy production until the end of the sum-
mer. Long-term suppression in the high dose treat-
ment group may be the result of the signiÞcantly
Fig. 6. The relationship between total mummies collected daily from traps and bottom boards and honey yield (kilogram).
Each data point represents an individual colony (n33). Data presented in the Þgure is untransformed.
Fig. 7. The mean amount (parts per million, ppm) of lysozyme-HCl detected in the stored food collected from the outer
edges of three brood frames of each colony compared among treatment groups that were administered three dosages of
lysozyme-HCl. Colonies were sampled before the Þrst treatment was applied and then weekly for 4 wk afterwards.
December 2012 VAN HAGA ET AL.: LYSOZYME FOR TREATMENT OF CHALKBROOD IN HONEY BEES 1885
higher levels of lysozyme-HCl built up in the stored
food cells surrounding the brood comb. The lowest
dose (600 mg 3 doses) of lysozyme-HCl assessed
was ineffective and produced high numbers of chalk-
brood mummies in artiÞcially inoculated colonies
throughout the entirety of the experiment.
Mummy numbers in the uninoculated untreated
colonies were low throughout the experiment but did
show slight increases in July and August. Drifting, the
movement of adult bees from one colony into another,
could be one explanation of how A. apis spores were
transmitted into uninoculated colonies. Similarly,
spores could have been transferred at forage sites
common to the apiary (Heath 1982). However, spores
were detected at the start of the experiment in the
larvae and worker bee guts before inoculation indi-
cating a preexisting chalkbrood infection. It is com-
mon for low levels of A. apis spores to be detected in
colonies asymptomatic for chalkbrood disease (Gil-
liam 1986). The source of A. apis spores in the colonies
preinoculation was likely the package bees imported
from New Zealand as the equipment the bees were
established in was disinfected by irradiation.
Although predominantly black mummies were col-
lected from the dead bee traps and bottom boards,
similar numbers of black and white mummies were
counted in the weekly frame inspections. Addition-
ally, the number of black to white mummies collected
daily was signiÞcantly higher in the inoculated un-
treated and low dose colonies compared with the
other treatment groups. The color variation in chalk-
brood mummies is because of the presence of spore
cysts that are brown to black when mature; mummies
remain white if the fungus does not mate and produce
spores (Gilliam et al. 1988). Diseased larvae left to
sporulate and harden into black mummies contain as
many as 1 10
8
A. apis spores (Nelson and Gochnauer
1982), and if not removed provide a constant source
of spores for reinfection. The higher level of black
mummies and spores in the colony feeds back into and
intensiÞes the natural disease cycle, and may cause the
ßuctuating levels of mummies seen in the inoculated
untreated and low dose treatment groups throughout
the experiment. The natural infection cycle may have
been interrupted as the chalkbrood mummies that
were collected daily were removed and discarded.
Mummies left on the bottom boards and at the en-
trance of colonies provide a reservoir of spores that
left uncollected could have increased disease severity
in infected colonies or increased transmission of
chalkbrood disease in uninoculated colonies.
Because the frame inspections involved counting
only what was visible, chalkbrood mummies in capped
cells were not counted. The differences between the
daily collections and weekly frame counts may mean
that once detected and uncapped, black mummies are
removed faster than white mummies. The detection
and removal of diseased larvae or hygienic behavior is
a heritable colony-level trait important in disease re-
sistance (Rothenbuhler 1964) and studies have shown
that colonies that exhibit this behavior are more re-
sistant to chalkbrood (Gilliam et al. 1983, Spivak and
Reuter 1998). Hygienic behavior is a quantitative trait
inßuenced by multiple loci (Lapidge et al. 2002) and
individual worker bees within a colony have different
response thresholds to stimuli that trigger the uncap-
ping of the brood cell and the removal of the larva
(Oxley et al. 2010). Olfactory sensitivity to chalkbrood
Fig. 8. The average number of visible chalkbrood mummies (black and white) in brood frames counted at the end of
the experiment (14 May 2008) for each treatment group. Total, black, and white mummies from each treatment group were
analyzed separately. Treatments with different letters are signiÞcantly different at
0.05 (TukeyÐKramer HSD). For each
category different letter styles were used to indicate signiÞcance: total mummy production (gray, uppercase, italicized letters,
A); black mummy production (black, lowercase, Greek letters,
); white mummy production (black, lowercase letters, a).
For total (A), black (
), and white (a) mummy counts.
1886 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 6
odors is higher in honey bees bred for hygienic be-
havior than in bees from nonhygienic lines (Master-
man et al. 2001, Gramacho and Spivak 2003) and re-
cently, it was shown that the volatile compound
phenethyl acetate isolated from larvae infected with
A. apis (presporulation) was capable of inducing hy-
gienic behavior in the Þeld (Swanson et al. 2009). If
infected larvae are removed before A. apis sporulates,
the disease cycle is interrupted. However, it is not
known if black sporulated mummies have other vola-
tiles not found in infected nonsporulated larvae or if
they have a greater quantity of the volatile phenethyl
acetate. The colonies in this experiment were not
assayed for hygienic behavior and it was not known
which colonies were naturally resistant to chalkbrood
disease. Although the queens were all imported from
the same source, the genetic background and relat-
edness of the queens used in the Þeld trial was un-
known. However, it was noted that within all treat-
ment groups, one or two colonies appeared to be more
susceptible to chalkbrood disease than the others re-
gardless of treatment that may be a result of genetic
variability in the population.
Genetic variation in honey production among col-
onies is also well documented (Guzma´nÐNovoa and
Gary 1993) and may be one reason why there was high
variability in honey production within treatment
groups. There were no signiÞcant differences among
treatment groups in mean honey production even
though the inoculated high dose treatment group pro-
duced on average 24 kg more than the inoculated
untreated group and surprisingly, 18 kg more than the
uninoculated untreated colonies. As the dose of ly-
sozyme-HCl applied to the colony increased, so did
mean honey production. It may be that lysozyme-HCl
is having a positive effect on colony disease suppres-
sion and production unrelated to chalkbrood suppres-
sion. Although not signiÞcant, from a management
perspective an 18Ð24 kg increase in mean honey yield
per colony is economically important and reßects a
$60Ð90 increase per colony at current honey values
(U.S. Department of Agriculture [USDA] 2012) es-
pecially when the cost of the highest treatment ad-
ministered (6,000 mg 3 doses lysozyme-HCl) is less
than $0.50.
Although there were no signiÞcant differences in
mean honey production among treatment groups
there was a moderately correlated signiÞcant negative
relationship between total mummies collected and
honey production. As chalkbrood disease increases in
severity, increased larval death should translate into
fewer adult worker bees and overall lower colony
productivity but the consequences of larval death may
not be immediate or straightforward. The honey bee
colony can be considered a superorganism where in-
dividual actions are determined by colony needs (See-
ley 1989). Division of labor in honey bee colonies is
age-related and different age castes task specialize.
However, worker bees demonstrate great ßexibility in
performing age-related tasks important to colony Þt-
ness throughout their lifetime (Winston 1987). For-
aging is generally the Þnal task a worker bee performs
in her lifetime but in the absence of an older aged
cohort, younger bees will become precocious forag-
ers. Conversely, in the absence of young worker bees,
older aged workers will switch to brood tending and
other tasks typically performed by younger bees (See-
ley 1989). The ßexibility of the worker bee population
is one factor that contributes to colony resilience
when dealing with disturbance. Combined with the
ability of the queen bee to lay 1,000 Ð2,000 eggs per day
(Bodenheimer 1937) it may mean that the impact of
chalkbrood disease is minimal unless severe and pro-
longed. Additionally, if there is a lag between larval
death and adult worker bee reduction, it may be that
in the Peace River region where the honey ßow is brief
but intense (Pankiw 1968), that chalkbrood disease
severity will not be a good predictor of honey pro-
duction. This is illustrated by the comparison of two
colonies within the same treatment group; one colony
that produced 2,548 mummies over the entire exper-
iment yielded 38 kg more honey than a colony that
produced only 84 chalkbrood mummies.
Nelson and Gochnauer (1982) hypothesized that in
years of high honey production, infection levels re-
quired to cause economic loss must be higher than in
years with low honey production. Honey yields in the
province of Alberta and speciÞcally the Peace region
of northern Alberta where this study was conducted
have been historically high. In the last decade (2001Ð
2011) Alberta has produced 40% of Canadian honey
yields (Statistics Canada 2002Ð2005, 2006, 2007Ð2011)
and the Peace region alone produced 11% of total
Canadian honey production between 2007Ð2010 (Al-
berta Agriculture and Rural Development 2007Ð
2010). The mean honey yield per colony in the Peace
region during that time period was 65 kg per colony,
10 kg more than the overall Albertan average, and 12
kg more than the average Canadian yield. It may be
that in northern Alberta where honey production is
high, chalkbrood disease will have minimal economic
impact.
There were no signiÞcant differences in adult bee
populations, sealed or unsealed brood cells between
treatment groups at any of the dates measured. The
buildup of adult bee populations peaked 26 June
(14,500 bees) coinciding with the start of major
nectar ßow in the Peace River region (Pankiw 1968)
and decreased on 10 July. A previous study on package
bee colonies in Manitoba (Nelson and Jay 1972)
showed similar numbers of adult bees on 21 June 1969
and 22 June 1970 (15,000Ð20,000 bees) but the pop-
ulations continued to increase throughout the summer
and did not experience the decline observed in this
experiment. Package colonies in the Canadian Prairies
take 110 d postestablishment to reach maximum pop-
ulation levels of 45,000Ð50,000 bees (Nelson and Jay
1982). Population measurements ended 10 July before
colonies reached maximum population levels and the
effects of chalkbrood disease on colony strength for
the entire season was not assessed. However, colony
populations were assessed one year later on 14 May
2008 and there were no signiÞcant differences among
treatment groups. Additionally, winter survival of col-
December 2012 VAN HAGA ET AL.: LYSOZYME FOR TREATMENT OF CHALKBROOD IN HONEY BEES 1887
onies did not differ signiÞcantly among treatment
groups and although the mean number of chalkbrood
mummies counted in the brood frames of the colonies
were higher in the inoculated untreated and low dose
treatment groups, it was not signiÞcant.
Trends in the pathology of the disease in the colony
mirrored the results of Taber (1986) who used the
same method of artiÞcial inoculation and observed
emergence of chalkbrood mummies 3 d postinocula-
tion and complete removal by 3 wk in chalkbrood-
susceptible colonies. Although there was a decrease in
the number of mummies collected in this experiment
2 wk postinoculation, unlike Taber (1986) and Gilliam
et al. (1988), some colonies never fully recovered. The
use of chalkbrood mummies as an inoculant in Þeld
studies is problematic as mummies are not a pure
source of A. apis spores and can contain other molds,
yeast, and bacteria (Johnson et al. 2005). The response
of the colony to inoculation by homogenized mum-
mies may not only be to chalkbrood infection but
other microbes as well. It has been shown that inoc-
ulation of colonies with chalkbrood mummies is more
infective than by A. apis spores alone (Jakobsons
2005). However, it is unlikely that colonies under
natural conditions would encounter uncontaminated
A. apis spores. In this experiment, the pathology of the
disease may have also been affected by the presence
of preexisting A. apis spores from another source.
Heterogeneity in the biochemistry and virulence of
different A. apis strains has been reported (Gilliam and
Lorenz 1993, Jakobsons 2005) and it is not known if
different strains also vary in their susceptibility to
lysozyme-HCl.
The impact of chalkbrood disease on package col-
onies is variable and individual colony response to
artiÞcial inoculation with A. apis spores is inßuenced
by both environmental and genetic factors. Despite
the highly variable response to chalkbrood infection,
lysozyme-HCl at the medium and highest doses tested
signiÞcantly reduced disease severity to levels similar
to that of uninoculated colonies. The highest dose
(6,000 mg 3 doses) evaluated suppressed mummy
production for the entire summer. Inexpensive and
easily integrated into established colony management
practices, lysozyme-HCl at the highest dose evaluated
is an effective control for chalkbrood disease.
Acknowledgments
The authors thank John Zhang and Neova Technologies
Inc. for supplying the lysozyme-HCl and performing detec-
tions of lysozyme-HCl in the stored food samples. Special
thanks are owed to Andony Melathopoulos, Wendy Walter,
Peter Mills, and Sterling Smith for their technical assistance.
We also thank Devon Coupland, Robert Annett, and Brandon
Smith, our summer students, for their contributions to Þeld
experiments. Funding was provided by Neova Technologies
Inc., the Canadian Bee Research Fund, the Alberta Beekeep-
ersÕ Commission, Bee Maid Honey, and the Matching Invest-
ment Initiative of AAFC.
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Received 5 June 2012; accepted 31 August 2012.
December 2012 VAN HAGA ET AL.: LYSOZYME FOR TREATMENT OF CHALKBROOD IN HONEY BEES 1889
... At present, there is no treatment licensed to control it. A main issue is to avoid the possible presence of drug residues in the hive products; for this reason, different natural products have been screened [169]. ...
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