Microb Ecol (2015) 70:522–533

DOI 10.1007/s00248-015-0594-7

INVERTEBRATE MICROBIOLOGY

Seasonal Variation of Honeybee Pathogens and its Association

with Pollen Diversity in Uruguay
Karina Antúnez 1 & Matilde Anido 1 & Belén Branchiccela 1 & Jorge Harriet 2 &
Juan Campa 2 & Ciro Invernizzi 3 & Estela Santos 3 & Mariano Higes 4 &
Raquel Martín-Hernández 4 & Pablo Zunino 1

Received: 30 June 2014 / Accepted: 4 March 2015 / Published online: 22 March 2015
# Springer Science+Business Media New York 2015

Abstract Honeybees are susceptible to a wide range of path-
ogens, which have been related to the occurrence of colony
loss episodes reported mainly in north hemisphere countries.
Their ability to resist those infections is compromised if they
are malnourished or exposed to pesticides. The aim of the
present study was to carry out an epidemiological study in
Uruguay, South America, in order to evaluate the dynamics
and interaction of honeybee pathogens and evaluate their as-
sociation with the presence of external stress factors such as
restricted pollen diversity and presence of agrochemicals. We
monitored 40 colonies in two apiaries over 24 months, regu-
larly quantifying colony strength, parasite and pathogen sta-
tus, and pollen diversity. Chlorinated pesticides, phosphorus,
pyrethroid, fipronil, or sulfas were not found in stored pollen
in any colony or season. Varroa destructor was widespread in
March (end of summer–beginning of autumn), decreasing af-
ter acaricide treatments. Viruses ABPV, DWV, and SBV pre-
sented a similar trend, while IAPV and KBV were not
Electronic supplementary material The online version of this article
(doi:10.1007/s00248-015-0594-7) contains supplementary material,
which is available to authorized users.
* Karina Antúnez
kantunez03@gmail.com
1
Departamento de Microbiología, Instituto de Investigaciones
Biológicas Clemente Estable, Avda. Italia 3318,
Montevideo, Uruguay
2
Sección Apicultura, Dirección de Laboratorios Veterinarios BMiguel
C. Rubino^, MGAP, Montevideo, Uruguay
3
Sección Etología, Facultad de Ciencias, Universidad de la República,
Montevideo, Uruguay
4
Centro Apícola Regional, Junta de Comunidades de Castilla La
Mancha, 19180 Marchamalo, Spain
detected. Nosema ceranae was detected along the year while
Nosema apis was detected only in one sample. Fifteen percent
of the colonies died, being associated to high V. destructor
mite load in March and high N. ceranae spore loads in Sep-
tember. Although similar results have been reported in north
hemisphere countries, this is the first study of these character-
istics in Uruguay, highlighting the regional importance. On
the other side, colonies with pollen of diverse botanical origins
showed reduced viral infection levels, suggesting that an ade-
quate nutrition is important for the development of healthy
colonies.
Keywords Apis mellifera . Colony losses . Honeybee
viruses . Varroa destructor . Nosema ceranae . Bee nutrition
Introduction
Honeybee Apis mellifera produces a wide variety of products
that have been used by humans for centuries with industrial,
medicinal, and alimentary objectives. Honey, pollen, propolis,
royal jelly, and beeswax are between those products. In addi-
tion, those insects play an essential role in the ecology of
natural environments through pollination, being essential for
the conservation of natural ecosystems and agricultural pro-
duction [1]. It has been estimated that one third of the human
diet can be traced to bee pollination. Their value for agricul-
ture has been estimated in more than $14.6 billion/year in the
USA [2].
However, due to their communal and social lifestyle, hon-
eybees are susceptible to a great variety of pathogens. Among
these, the ectoparasitic mite Varroa destructor, the
microsporidium Nosema ceranae, and different RNA viruses
are major threats for colony health [3–8]. More than 18 viruses
that affect honeybees have been described, including acute
Honeybee Pathogens and Pollen Diversity in Uruguay
bee paralysis virus (ABPV), chronic bee paralysis virus
(CBPV), black queen cell virus (BQCV), sacbrood virus
(SBV), deformed wing virus (DWV), Kashmir virus (KV),
and Israeli acute bee paralysis virus [3, 4, 9, 10].
The presence and synergistic effect of different pathogens
have been associated to large scale losses of managed colonies
in North America and Europe [5, 11–16]. The situation seems
to be different in Latin America, since although most of these
pathogens are present and widely distributed, no large-scale
colony losses have been reported, at least in the propor-
tion and extension to those observed in North America
and Europe [17].
Agricultural intensification and changes in the use of land-
scape has also been associated to colony losses [18, 19] since
it directly affects the amounts and diversity of floral resources
(i.e., nectar and pollen) available for bees. As pollen is the
main source of proteins, reduction in abundance, quality, and
diversity may cause nutritional deficiencies, affecting bee
physiology, development, and social and individual
inmunocompetence [20–26]. These changes associated to pol-
len consumption exert an important influence on tolerance to
pathogens as microsporidia [27, 28], fungi [29], bacteria [30],
and viruses [31]. Besides that, pollen can be a source of anti-
microbial compounds, such as flavonoids and phenolic com-
pounds, which could inhibit the development of different
pathogens [32, 33].
In order to gain better insight into the spectrum of patho-
gens and potential threats present in Uruguayan colonies, we
monitored the presence and seasonal variation of the most
important honeybee pathogens over 2 years and evaluated
their association with the presence of external stress factors
such as pollen diversity available and presence of agrochem-
icals. This study provides a temporal characterization of hon-
eybee pathogens and offers a baseline for understanding path-
ogen threats and colony losses in Uruguay and South
America.
Material and Methods
Honeybee Colonies
Two professional apiaries located in different geographic areas
of Uruguay were used in the present study. Apiaries were
selected based on a series of requirements: performance of
general good beekeeping practices, application of adequate
treatments against V. destructor, belonging to a beekeeping
company registered in the National Register of Honeybee
Colonies of Uruguay, and records of high colony losses (about
20 %) in previous years (2006–2008, data provided by bee-
keepers). Apiary 1 was located in Colonia, in a dairy farm.
Adjacent pastures were grasses and legumes for dairy cows,
having a low pesticides pressure. Apiary 2 was located in
523
Canelones, in a similar scenario, in a cattle field with grass-
land. It was surrounded by small farms, familiar orchards, and
natural fields.
Each apiary was composed by forty colonies, local hybrids
of A. m. mellifera, A. m. scutellata, and A. m. ligustica without
previous genetic selection, located in Langstroth hives. In all
cases, queens were younger than 2 years. Colony strength was
estimated in March 2009, and according to these results, twen-
ty colonies with similar characteristics were selected. Colonies
were composed by 12.6±3.8 adult bees covered frames and
49.2±12.7 quarters of side of frames of brood. No significant
differences were detected between those parameters in apiar-
ies 1 and 2 (Mann-Whitney test, p≥0.05 in both cases). No
new queen was artificially introduced along the study.
Colony Strength Estimation
At the beginning of the study and every sampling date, colony
strength of each colony was estimated by two MGAP techni-
cians using the subjective mode, according to recommenda-
tions of Delaplane et al. [34]. Adult bee population (number of
covered frames, estimating 2200 bees per frame), brood area
(quarters of sides of frames, total 880 cm2 per side of frame),
and honey reserves (covered frames, total 880 cm2 per side of
frame) were estimated. Additional information such as the
presence of drones, queen cells, and other symptoms were
also recorded.
Samplings
Samples were collected at the end of summer–beginning of
autumn (March 11 and 16, 2009; March 23 and 24, 2010), end
of autumn–beginning of winter (June 11 and 15, 2009; June
23 and 24, 2010), end of winter–beginning of spring (October
1 and 2, 2009; September 20 and 21; 2010), and the end of
spring–beginning of summer (December 10 and 11, 2009;
December 2 and 15, 2010).
Each sample was composed of 200–300 nurse bees of at
least three unsealed brood combs for the detection of
V. destructor, 80–120 bees from peripheral combs in the brood
area for the detection of Nosema spp., 200–250 g of honey
from three different combs close to the brood area for the
detection of Paenibacillus larvae, and approximately 100 cc
of stored pollen from three different combs close to the brood
area, for the analysis of pollen botanical diversity and pres-
ence of pesticides.
Bee samples were collected, transported, and maintained in
ethanol. A subsample of nurse bees per colony (50 bees) was
transported alive to the laboratory (less than 3 h) and stored
immediately at −80 °C until analyzed, in order to avoid RNA
degradation [35].
All samplings and records were carried out by the same two
operators, from the Apiculture Section from Veterinary
524
Laboratories Division (DILAVE) of the Ministry of Livestock,
Agriculture and Fisheries [36], Uruguay. All colonies received
acaricide treatments in March, after the sampling; amitraz was
used in 2009 and flumethrin in 2010.
Climatic Parameters
Information about temperature, pressure, humidity, and pre-
cipitations in both apiaries along the study was provided by
the General Direction of Meteorology (Uruguay).
Detection and Quantification of Honeybee Viruses
Ten nurse bees (kept at −80 °C) were randomly selected from
each sample and processed as described by Anido et al. [37]
and Rodríguez et al. [37]. RNA was extracted using the
QIAamp Viral RNA Mini Kit (Qiagen); DNA digestion and
reverse transcription were carried out using the QuantiTect
reverse transcription Kit (Qiagen); and detection and quanti-
fication of viruses ABPV, BQCV, DWV, KBV, IAPV, and
SBV were performed using the QuantiTec SYBR PCR Kit
(Qiagen) as previously reported [37]. Specific primers for
the detection of ABPV [38], BQCV [39], CBPV [40], DWV
[39], IAPV [41]; KBV [38], and SBV [38] were used. To
ensure the quality of RNA extraction, β-actin gene was am-
plified by qPCR using specific primers (Supporting informa-
tion, Table 1) [42].
Detection and Quantification of P. larvae
Honey samples were analyzed in order to detect the presence
of P. larvae as previously described [43, 44]. Suspected bac-
terial colonies were identified by macroscopic and microscop-
ic characterization, standard biochemical tests, and PCR as
previously described [45]. Bacterial genotyping was carried
out according to Antúnez et al. [46].
Detection and Quantification of Nosema spp
Due to the difficulty to collect forager bee samples during
winter, the presence of Nosema spp. was analyzed in bees
from peripherals combs, far from the brood area, as suggested
by Fries et al. [47].
Twenty old nurse bees were randomly selected from each
sample; total DNA was extracted using the QIAamp Viral
RNA Mini Kit (Qiagen); and the presence of N. ceranae
and/or Nosema apis was analyzed by multiplex PCR, as pre-
viously described [47, 48].
The number of Nosema sp. spores per pooled samples (80–
120 bees) was determined by phase-contrast microscopy
(magnification×400) as recommended by the OIE [49].
K. Antúnez et al.
Detection and Quantification of V. destructor
Detection and quantification of V. destructor were carried out
in bees covering the brood area (nurse bees), as recommended
by Dietemann et al. [50]. Mites were separated from bees
using ethanol 75, and infestation rates per colony and per
apiary were calculated [50].
Determination of the Botanical Origin of the Pollen
In order to determine the botanical origin of pollen samples, a
representative quantity was taken from each one, which was
subsequently homogenized in glacial acetic acid and
acetolized for observation under optical microscope. The res-
idues were mounted in gelatine-glycerine with formol [51].
The identification of pollen grains was carried out using×
1000 magnification. The microscopic preparations of the sam-
ples were quantitatively analyzed by counting at least 1200
pollen grains per slide per sample [52]. Simpson index was
calculated in order to estimate pollen diversity per colony.
Detection of Pesticides
Detection of pesticides (HCB, aldrin, dieldrin, HCH, DDT,
and their metabolites, endrin, endosulfan, diazinon, paratión,
clorpirifos, etion, endosulfan sulfate, cipermetrin, permetrin,
deltametrin, and fipronil) in pollen samples was performed by
the Chemistry Section, DILAVE-MGAP, by HPLC, according
to methods recommended by the Food Safety and Inspection
Service, USDA [53] and the Association of Official Analytical
Chemists [54]. Detection limits were as follows: chlorinated
pesticides 5–10 ppb, phosphorus 10–30 ppb, pyrethroid 10–
30 ppb, and fipronil and sulfas 10–15 ppb.
Statistical Analysis
Seasonal analysis of pathogens and parasites prevalence was
carried out taking into account the infection/infestation rate
per apiary and per season (number of infected/infested colo-
nies over the total of analyzed colonies in one apiary, in a
determined season), pathogens infection intensity per colony
and parasite infestation intensity per colony.
One set of data was generated for each apiary, and statisti-
cal analyses were independently performed. Data were ana-
lyzed in order to evaluate if they fitted parametric assump-
tions, as normal distribution analyzed by Kolmogorov-
Smirnov test and variance’s homogeneity using the Levene’s
test. In cases data fitted parametric assumptions, differences
between groups were evaluated by ANOVA and Scheffé’s
post hoc test. In other cases, Kruskal-Wallis and Mann-
Whitney post hoc tests were applied.
Then, data from both apiaries and years were joined, and
correlation tests between different parameters were performed
Honeybee Pathogens and Pollen Diversity in Uruguay
using Spearman test for quantitative variables and chi-squared
for qualitative variables. The Mann-Whitney test was used to
relate quantitative with qualitative variables.
Finally, colonies were divided into three groups according
to the percentage of adult bee population loss along 1 year,
being group 1 completely depopulated colonies, group 2 col-
onies that reduced their adult population, and group 3 colonies
that remain stable or increased their adult population. Infec-
tion levels of different pathogens in different seasons of the
year were compared by Kruskal-Wallis test. In all cases, P
values under 0.05 were considered significant.
Results
Colony Strength and Symptoms
Adult honeybee population and brood area were high in sum-
mer (from December to March), with averages of 28,800±
9450 bees and 6700±4300 cm2 of brood area and decreased
to winter with averages of 9700±4100 bees and 570±619 cm2
of brood area, in agreement with their biological cycle. Honey
reserves also followed this trend, increasing in summer
months (December to March) and decreasing in winter
months (June to September) (Supporting information, Fig. 1).
Although at the beginning of the study all colonies looked
healthy, different disease symptoms were evidenced by oper-
ators along the study. The main symptom was an uneven
brood pattern (sporadic cases in each colony), being present
in about 10 % of total analyzed colonies. European foulbrood
symptoms were detected in 1 % of the colonies, while Amer-
ican foulbrood symptoms or chalkbrood disease were not
identified in any of the colonies at any of the sampling dates.
Queen cells were evidenced in 8 % of the total analyzed col-
onies, while drone layer workers were evidenced in 2.5 % of
the colonies.
Climatic Parameters
Climatic conditions (temperature, pressure, and relative hu-
midity) in both locations were similar between them along
the study and were in agreement with previously described
conditions for those areas, as informed by General Direction
of Meteorology (Uruguay) (Supporting information, Fig. 2).
Precipitations were stronger in 2010 in both apiaries compared
with 2009.
Viral Prevalence and Infection Intensity
Honeybee viruses ABPV, BQCV, DWV, and SBV were suc-
cessfully detected by real-time RT-PCR, obtaining unique
melting peaks for each amplified product, coinciding with
525
those previously determined in our laboratory (Anido et al.
[37]; 37). KBV or IAPV were not detected.
At the beginning of the experiment, March 2009, BQCV,
ABPV, DWV, and SBV were detected in most of the colonies
in both apiaries (Supporting information, Fig. 3). In the case of
BQCV, prevalence and viral infection intensities were high
along the whole study, with an average of 84 and
82 % of infected colonies per season in apiaries 1 and
2, respectively (Fig. 1).
On the other side, DWV and ABPV presented high preva-
lence rates and viral loads in March 2009 in both apiaries
(average 95 % of infected colonies per virus in both apiaries),
decreasing over the course of both years. Sacbrood virus also
followed this trend although infection pattern varied among
local conditions. Correlation analysis between viral infection
intensities showed positive and significant associations, al-
though in most cases were weak to moderate (Table 1).
V. destructor Prevalence and Infestation Intensity
In March 2009, most colonies showed detectable levels of
varroa mites (95 and 99 % for apiaries 1 and 2, respectively,
Fig. 2). Amitraz was immediately applied to control the mite,
which was evidenced in the reduction of infestation observed
in June. Colonies with detectable levels of mites and total mite
infestation intensity increased steadily along the year. In
March 2010 another treatment was applied (flumethrin),
1 week before the sampling in case of apiary 1 and after the
sampling in apiary 2. Mite loads were positively correlated
with ABPV, DWV, and SBV viral infection intensities, with
weak–moderate values (Table 1).
N. apis and N. ceranae Prevalence and Infection Intensity
Prevalence of colonies with detectable levels of Nosema spp.
was low (around 10 %) in March, peaking during winter and
spring (from June to December) (Fig. 3). Infection intensity
per colony and per season were highly variable (Fig. 3) and
were negatively and weakly correlated to DWV and SBV
2009 viral infection intensities (Table 1). N. ceranae was by
far the predominant Nosema sp. found, while N. apis
was only detected in one sample along the whole study
period (March 2009).
P. larvae Prevalence
Clinical signs of American foulbrood were not observed in
any colony at any inspection date. However, P. larvae spores
were detected in honey collected from two colonies from api-
ary 1 (in March and June of 2009) and seven colonies from
apiary 2 (one in March 2009, one in December 2009, four in
June 2010, one in September 2010). All isolates were
526 K. Antúnez et al.

Fig. 1 Seasonal variation of viral infection intensity per colony of ABPV, BQCV, DWV, and SBV (relative quantification) in apiaries 1 and 2, during
2009 and 2010. Apiaries 1 and 2 were independently analyzed. Different letters indicate the presence of significant differences (p≤0.05)

classified as genotype ERIC-I BOXA. Presence of P. larvae
was not statistically correlated to the presence of other
pathogens.
Pollen Diversity and Pathogen Infection Intensities
Availability of pollen from diverse botanical origins (estimat-
ed through the Simpson index) varied along the year, being
significantly higher in summer (December) compared to win-
ter months (June, z=3.99, p<0.001, and September, z=4.66,
p<0.001, respectively). Botanical species identified also var-
ied along the study. Main species found per season, distribu-
tion in the apiary, and abundance per colony are shown in
Supporting information, Table 2.
Diversity of stored pollen present in the colonies was neg-
atively correlated to viral infection levels by different viruses,
in the same month and in the following ones (Table 2). Cor-
relations were weak to strong, depending on the month and the
virus. Strongest correlations were found between pollen diver-
sity in March and BQCV and SBV in September, pollen
Honeybee Pathogens and Pollen Diversity in Uruguay 527

Table 1 Spearman correlations between pest and pathogens loads strongly negatively correlated with viral infection intensities
Valid N Spearman R p level of ABPV, DWV, and SBV in the same and following months
(Table 3). As an example, abundance of those species in
ABPV and BQCV 292 0.137 0.019 March and June was strongly and negatively correlated with
ABPV and DWV 292 0.412 <0.001 ABPV, DWV, and SBV in the same months.
ABPV and SBV 292 0.348 <0.001 Pollen diversity was not correlated with adult bee popula-
BQCV and DWV 293 0.154 0.008 tion, brood area, or honey production in the same or following
BQCV and SBV 293 0.224 <0.001 months (p>0.05 in all cases).
DWV and SBV 293 0.334 <0.001
ABPV and V. destructor 286 0.150 0.011 Pesticides
DWV and V. destructor 287 0.273 <0.001
SBV and V. destructor 287 0.315 <0.001 No residues of chlorinated pesticides, phosphorus, pyrethroid,
DWV and N. ceranae 290 −0.175 0.003 or fipronil were detected in any pollen sample during the
SBV and N. ceranae 290 −0.196 0.001 study.
Two apiaries and years were analyzed together. Only statistically signif-
icant correlations are shown (p≤0.05). Moderate to high correlations are Depopulation of Colonies
underlined
As previously presented, at the beginning of the study, all
diversity in June and DWV from June to September, and colonies presented similar colony strength. When colonies
pollen diversity in September and N. ceranae during the same were monitored over the time, three groups were clearly dis-
month and BQCV in December. tinguished. Group 1 was composed of 12 completely
Even more, when analyses per botanical species were per- depopulated colonies (15 %), 10 died during winter–begin-
formed, we found that abundance of Baccharis trimera, ning of spring 2009 and 2 during winter–beginning of spring
Baccharis spicata, and Trifolium repens was moderate to 2010. Among these, five colonies were queenless, five

Fig. 2 Seasonal variation of

V. destructor in apiaries 1 and 2,
during 2009 and 2010. Infection
rate per apiary per season and
mite infestation intensity per
colony are shown. Apiaries 1 and
2 were independently analyzed.
Different letters indicate the
presence of significant differences
(p≤0.05)
528 K. Antúnez et al.

Fig. 3 Seasonal variation of

N. ceranae in apiaries 1 and 2,
during 2009 and 2010. Infection
rate per apiary per season and
infection intensity per colony are
shown. Apiaries 1 and 2 were
independently analyzed. Different
letters indicate the presence of
significant differences (p≤0.05)

showed starvation symptoms while in the rest two colonies, Group 2 was composed of colonies which suffered reduc-
no special symptoms were observed. tion of adult bee population (29 %) from March to December
(from 8 to 83 % of the initial population), and group 3 was
Table 2 Spearman correlations between pollen diversity (estimated composed by colonies which populations remained stable or
trough Simpson index) and pathogens infection levels increased along the experiment (56 %).
Valid N Spearman R p value Depopulated colonies (group 1) showed a significantly
higher level of V. destructor in March and a higher level of
Pollen diversity in March N. ceranae spores in September, than healthy colonies (group
BQCV in March 78 −0.214 0.060 3; Fig. 4, z=2.46, p=0.04, and z=2.64, p=0.02, respectively).
SBV in March 78 −0.232 0.041 Pollen diversity in different seasons was not related to colo-
ABPV in June 77 −0.261 0.022 nies depopulation (p>0.05 in all cases).
BQCV in September 72 −0.479 <0.001
SBV in September 72 −0.433 <0.001
SBV in December 62 −0.404 0.001
Pollen diversity in June
Discussion
DWV in June 37 −0.503 0.001
In the present manuscript, we present the first long-term epi-
SBV in June 37 −0.311 0.061
demiological study of the most important honeybee pathogens
ABPV in September 32 −0.360 0.043
in Uruguay, in colonies from two professional apiaries located
BQCV in September 32 −0.384 0.030
in Colonia and Canelones and evaluated their association with
DWV in September 32 −0.497 0.004
pollen diversity of colonies. Apiaries were located in dairy
ABPV in December 27 −0.624 0.001
farms or cattle ranch, with low pressure of pesticides or agro-
Pollen diversity in September
chemicals, fact that was confirmed by the absence of residues
N. ceranae in September 46 −0.418 0.004
of chlorinated pesticides, phosphorus, pyrethroid, and fipronil
BQCV in December 41 −0.565 <0.001
in stored pollen. Climatic conditions (temperature and humid-
DWV in December 41 0.360 0.021
ity) at both apiaries areas were mild, although a severe drought
Two apiaries and years were analyzed together. Only statistically signif- affected the country in 2009, directly affecting pollen diversity
icant correlations are shown. Moderate to high correlations are underlined in both apiaries.
Honeybee Pathogens and Pollen Diversity in Uruguay 529

Table 3 Spearman correlations

between botanical species Valid N Spearman R p value
identified in stored pollen and
pathogens infection levels B. spicata in March ABPV in March 67 −0.425 <0.001
DWV in March 67 −0.509 <0.001
SBV in March 67 −0.459 <0.001
ABPV in June 67 −0.314 0.010
DWV in June 67 −0.444 <0.001
SBV in June 67 −0.270 0.027
ABPV in September 67 −0.306 0.012
BQCVS in September 67 −0.294 0.016
DWV in September 67 −0.313 0.010
B. spicata in June DWV in June 36 −0.430 0.009
B. trimera in March ABPV in March 67 −0.348 0.004
DWV in March 67 −0.410 0.001
SBV in March 67 −0.488 <0.001
ABPV in June 67 −0.268 0.028
DWV in June 67 −0.348 0.004
SBV in June 67 −0.264 0.031
DWV in September 67 −0.281 0.021
B. trimera in June ABPV in June 36 −0.377 0.023
DWV in June 36 −0.572 <0.001
SBV in June 36 −0.355 0.034
BQCV in September 36 −0.599 <0.001
T. pratense in March ABPV in March 67 −0.272 0.026
DWV in March 67 −0.306 0.012
T. pratense in June ABPV in June 36 −0.412 0.012
DWV in June 36 −0.618 <0.001
T. repens in March ABPV in March 67 −0.518 <0.001
DWV in March 67 −0.574 <0.001
SBV in March 67 −0.524 <0.001
ABPV in June 67 −0.370 0.002
DWV in June 67 −0.549 <0.001
SBV in June 67 −0.311 0.010
ABPV in September 67 −0.354 0.003
BQCVS in September 67 −0.360 0.003
DWV in September 67 −0.250 0.041
T. repens in June ABPV in June 36 −0.376 0.024
DWV in June 36 −0.511 0.001
BQCV in September 36 −0.440 0.007
ABPV in December 36 −0.343 0.040
T. repens in September ABPV in September 47 −0.337 0.021
DWV in September 47 −0.343 0.018

Two apiaries and years were analyzed together. Only statistically significant correlations are shown (p≤0.05).
Moderate to high correlations are underlined

Seasonal dynamics of honeybee population, brood,
and honey reserves along the study was in accordance
with the expected biological cycle of bees, rising in
spring and summer and declining in autumn and winter.
V. destructor cycle was linked to honeybee cycle, in-
creasing its infestation intensity along summer to
autumn, when high amount of brood is available for
reproduction [7]. Infestation levels abruptly decreased
in autumn, after the application of miticides. Infection
intensities of RNA viruses ABPV, BQCV, DWV, and
SBV were associated to V. destructor, although in some
cases, associations were weak.
530
Fig. 4 Classification of colonies in groups according to the percentage of
adult bee losses. a Three groups of colonies were clearly distinguished:
completely depopulated along 1 year (1), suffered reduction of adult bee
The strong impact of the mite on colonies development and
survival and its association with honeybee viruses has been
widely documented [7, 8, 15, 55]. Moreover, it has been pro-
posed that global spread of V. destructor has contributed to the
selection of highly pathogenic DWV variants [56].
In Uruguay, those pathogens had previously been detected,
being widely spread [57, 58]. Beekeepers must apply acari-
cides at the beginning of the fall, to avoid colony losses asso-
ciated to V. destructor. On the other side, KBV and IAPV,
pathogens potentially associated to colony losses [3, 59, 60]
have not been detected.
In the case of microsporidia, N. ceranae was detected along
the study in both apiaries, according to previous findings in
Uruguay [61], while N. apis was only detected in one sample.
The negative correlation detected between N. ceranae and
DWV may be the result of antagonistic effects, as suggested
by Costa et al. [62]. Since both pathogens multiply in the
digestive tract [4, 28, 63, 64], they may be competing for host
cells. Weak correlation values found in the present work may
be related to the analysis of the complete bee, instead of
analysis of bee midgut, where correlation is stronger
[62].
In the case of P. larvae, no clinical signs of American
foulbrood were detected in colonies and the prevalence of this
bacterium in honey was extremely low, coinciding with pre-
vious results where a national prevalence of 2 % was estimat-
ed. P. larvae genotype ERIC-I BOXA was detected, coincid-
ing with circulating genotypes in the country [65]. This path-
ogen was first detected in Uruguay in 1999, and 2 years later,
it had spread all over the country, reaching a 51 % prevalence
in honey [43]. The progress in the understanding of the dis-
ease has led to an improvement of laboratory diagnostic tech-
niques, epidemiological studies, and treatment strategies,
which have reduced the detrimental effects of the disease
[9]. However, the epidemiologic surveillance should not be
neglected, in order to avoid the entrance of other highly path-
ogenic genotypes.
At the beginning of the present study, all colonies presented
similar colony strengths. However, along the 2-year study,
K. Antúnez et al.
population (2), and was stable or increased the adult bee population (3). b
Infestation intensity of V. destructor in March in the three groups. c
Infection intensity of N. ceranae in September in the three groups
15 % of the colonies died, probably associated to the presence
of V. destructor and N. ceranae. Both agents significantly
affect honeybee health and have been previously related to
colony losses [5, 9, 12, 16, 66]. On the other side, van
Engelsdorp et al. [67] demonstrated that colonies with evi-
dence of queen replacement or queen failure exhibited a
higher relative risk of colony death over the short term. Al-
though we were not able to calculate this risk since queens
were not marked at the beginning of the study, queen failure
was observed in 42 % of the dead colonies.
Besides honeybee pathogens, types, abundance, and diver-
sity of pollen available for bees, nutrition strongly affects hon-
eybee health [20–23, 25–27, 29–31]. DeGrandi-Hoffman
et al. [31] reported that DWV infection levels were lower in
bees fed with pollen compared to those fed with sugar syrup
and Invernizzi et al. [68] reported that colonies fed with pollen
from diverse botanical origins presented lower levels of
N. ceranae spores compared to colonies fed only with one
pollen species. In the present manuscript, we show evidence
that feeding on pollen of diverse botanical origin is correlated
to low infection intensities by different pathogens (ABPV,
BQCV, DWV, SBV, and N. ceranae). Even more, we found
that some pollen species (B. trimera, B. spicata, T. repens, and
T. pratense) were negatively correlated to of infection intensi-
ties of different viruses. In this regard, biological properties of
these species have already been described. B. trimera
has shown antihelmintic activity [69] and anti-inflamma-
tory, antioxidant, and anticancer properties, probably re-
lated to the presence of phenolic compounds [70].
B. spicata has shown antiviral and trypanocidal activity
[71, 72], while T. pratense has also shown antiviral
activity related to the presence of quercetin and other
phenolic compounds [73, 74].
The present findings suggest that an adequate nutrition
based on pollen from diverse botanical origin is important
for defense against pathogens. The mechanisms underlying
this finding can be related to the alteration of individual and
social immunocompetence and physiology, as suggested in
previous works [21, 27, 75].
Honeybee Pathogens and Pollen Diversity in Uruguay
Finally, although in the present study no residues of pesti-
cides were detected, residues of imidacloprid, fipronil, endo-
sulfan, coumaphos, cypermethrin, ethion, and chlorpyrifos
had previously been detected in Uruguayan colonies, indicat-
ing that the problem is present [76]. Those residues may be
acting synergistically with pathogens, affecting honeybee
health and development.
In conclusion, presence of N. ceranae, V. destructor, and
queen failure are related to colony losses in Uruguay. An
adequate nutrition based on pollen from diverse botanical
was correlated to a reduction in pathogens intensities, suggest-
ing its importance for the development of healthy honeybee
colonies.
Acknowledgments This work was financed by the Instituto
Nacional de Investigación Agropecuaria (INIA), grant INIA-FPTA 258.
Authors thank Dennis van Engelsdorp for the help with manuscript
organization.
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