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VETMIC 6303 1–10

Veterinary Microbiology xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

2 Effects of the organic acids produced by a lactic acid bacterium


3 in Apis mellifera colony development, Nosema ceranae control
4 and fumagillin efficiency
5 Q1 Matı́asMaggi a,b,*, Pedro Negri a,b, Santiago Plischuk b,c, Nicolás Szawarski a,
6 Fiorella De Piano a,d, Leonardo De Feudis a, Martı́n Eguaras a,b, Carina Audisio b,e
7 a
Laboratorio de Artrópodos, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata,
8 7600 Mar del Plata, Argentina
9 b
CONICET, Consejo Nacional de Investigaciones Cientı´ficas y Técnicas, Rivadavia 1917, C1033AJ Buenos Aires, Argentina
10 c
Centro de Estudios Parasitológicos y de Vectores (CEPAVE – CONICET CCT La Plata – UNLP), B1902CHX La Plata, Argentina
11 d
Comisión de Investigaciones Cientı´ficas (CIC), La Plata, Argentina
12 e
Instituto de Investigaciones para la Industria Quı´mica (INIQUI), Universidad Nacional de Salta, Salta, Argentina

A R T I C L E I N F O A B S T R A C T

Article history: The European honey bee Apis mellifera is known to be affected by many parasites and
Received 3 May 2013 pathogens that have great impact over the insect development. Among parasites affecting
Received in revised form 9 July 2013 bee health, Nosema ceranae is one of the main biotic factors affecting colony populations.
Accepted 29 July 2013 As honey bee populations decline, interest in pathogenic and mutualistic relationships
between bees and microorganisms has increased. The main goal of the current study was
Keywords: to assess the effect of the oral administration of the metabolites produced by Lactobacillus
Apis mellifera johnsonii CRL1647 (mainly organic acids) supplemented in syrup, on: (I) N. ceranae
Bacterial metabolites
sporulation dynamics before and after fumagillin application, and (II) performance of A.
Organic acids
mellifera colonies. Different experiments were conducted to evaluate the effects of these
Nosema ceranae control
Fumagillin
bacterial metabolites on bees: in vitro administration revealed no toxic effects against
bees. Colonies fed with the lactic acids incremented their beehive population and also the
amount of fat bodies per bee. Finally, the organic acids reduced the intensity of the
pathogen after the second application of treatment as well as enhanced the fumagillin
efficiency. This study provides important information for the development of new control
substances against nosemosis.
ß 2013 Published by Elsevier B.V.

13
14 1. Introduction affecting bee health, the parasitic mite Varroa destructor is 21
one of the main pests affecting bee colonies, and was 22
15 Apicultural economic development strongly relies on postulated to be partly responsible for worldwide colonies 23
16 the health status of honey bee colonies. The European losses observed (Levy, 2011). Another serious threat to A. 24
17 honey bee Apis mellifera is known to be affected by many mellifera is the American foulbrood caused by Paenibacillus 25
18 parasites and pathogens that have great impact over insect larvae, and also Nosemosis caused by the microsporidia 26
19 development and represent a serious threat to the Nosema apis and N. ceranae. Recently it has been proposed 27
20 ecosystem, agriculture and apiculture. Among parasites that the combined effects of all these parasites and/or 28
pathogens, along with habitat loss and pesticide poisoning, 29
could have drastic effects on honey bee colonies (Levy, 30
* Corresponding author at: Funes 3350, 7600 Mar del Plata, Argentina.
2011). 31
Tel.: +54 223 470 0898; fax: +54 223 4921711. By far, chemical insecticides are used most commonly 32
E-mail address: biomaggi@gmail.com (M. Maggi). to control parasites and pathogens affecting honey bees. 33

0378-1135/$ – see front matter ß 2013 Published by Elsevier B.V.


http://dx.doi.org/10.1016/j.vetmic.2013.07.030

Please cite this article in press as: Maggi, M., et al., Effects of the organic acids produced by a lactic acid bacterium in Apis
mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
10.1016/j.vetmic.2013.07.030
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2 M. Maggi et al. / Veterinary Microbiology xxx (2013) xxx–xxx

34 This application is usually performed when symptoms of 2. Materials and methods 95


35 the different diseases are observed in bee colonies, and
36 sustaining these strategies over time could lead to 2.1. Biological material 96
37 pesticide residues on commercial bee products and
In vitro experiments and laboratory measurements were 97
38 resistance phenomena in bee pests (Maggi et al., 2011).
conducted in the Arthropods Laboratory of the National 98
39 In the last few years, there was increasing evidence of
University of Mar del Plata. Field trials were carried out in 99
40 different ways to ensure bee health and indirectly, parasite
an experimental apiary located in Santa Paula farm (National 100
41 tolerance. As honey bee populations decline, interest in
Rout 226, 10 km south of Mar del Plata, Argentina). 101
42 pathogenic and mutualistic relationships between bees
Experiments were performed between February and Sep- 102
43 and their pathogens has increased. Microorganisms
tember 2012. Beehive inspections were performed at midday. 103
44 associated with A. mellifera have received special attention
45 as a new option for integrated pest management (IPM). In 2.2. Bacterial metabolites 104
46 this example, strains of bacteria and their products have
47 become an interesting field for experimentation as they are The antimicrobial compounds synthesized by L. john- 105
48 commonly isolated from the hive environment and bees’ sonii CRL1647 were recovered in the cell-free supernatants 106
49 digestive tracts (Audisio et al., 2011). Their potential (CFS) as explained elsewhere (Audisio et al., 2011). Briefly, 107
50 effects on bees have been evaluated in three different this lactic acid bacterium was grown during 24 h in MRS 108
51 ways. First, bacteria strains have been selected and studied broth (MRS, Britania, Argentina) at 37 8C under micro- 109
52 as probiotic supplements for bee consumption, testing for aerophilic conditions. Then, CFS was recovered by cen- 110
53 effects on colony development. In these studies the trifuging (10,000  g, 15 min, 10 8C), filtering (0.22 mm 111
54 administration of Lactobacillus and Bacillus strains helped mesh), and kept at 4 8C until used. 112
55 the development of bee colonies by enhancing the brood
56 and also honey yield (Sabaté et al., 2012). Second, bacteria 2.3. Organic acids quantification by HPLC 113
57 (or their metabolites) were tested as alternative control
58 methods for bee parasites. Sabaté et al. (2012) have Concentration of all organic acids produced by L. 114
59 documented a negative impact against N. ceranae and V. johnsonii CRL1647 was determined by HPLC according to 115
60 destructor when bacterial administration was applied on Audisio et al. (2011). The CFS from a MRS culture was 116
61 beehives in field conditions. Also, Porrini et al. (2010) have deproteinized and filter-sterilized (0.45 mm) before the 117
62 reported that particular surfactins can alter spore HPLC analyses. The sample amount injected was 20 ml. The 118
63 structure. Third, bacterial strains were studied as activa- column temperature was 55 8C and the flow rate of the 119
64 Q2 tors of immune-competence in bees. Evans and Lopez H2SO4 10 mM mobile phase was 0.6 ml/min. Detection was 120
65 (2007) have demonstrated activation of antibacterial carried out by determining the refraction index using a 121
66 peptide expression when bees were fed bacteria. These 2142LKB Differential Refractometer. The chromatography 122
67 authors also proposed that nonpathogenic bacteria could column (Rezec Organic Acid, Phenomenex) had a diameter 123
68 be used as a probiotic supply to enhance honey bee of 7.8 mm and a length of 300 mm. Peakprofiles, integra- 124
69 humoral immunity. tion, and quantification were obtained with a CR601 125
70 Nosema ceranae is an emergent parasite of A. mellifera Shimadzu chromatopac integrator (Shimadzu Corporation, 126
71 apparently more virulent than N. apis. Currently, N. Analytical Instrument Division, Kyoto, Japan). All tests 127
72 ceranae infections seem to be correlated with declining were carried out in duplicate. 128
73 populations of honey bees in Spain (Higes et al., 2008).
74 Taking into account that the major commercial medica- 2.4. Assay I: toxicity effects of bacterial metabolite 129
75 tion available for Nosema control is based on the antibiotic against honey bees 130
76 fumagillin (Williams et al., 2008) and that this drug is not
Before this experiment, bees remained without food for 131
77 legal in most countries, alternative management strate-
4 h to produce starvation. Then, each bee was confined 132
78 gies should be developed to confront this microparasite.
inside a plastic tube (length: 3 cm; diameter: 1 cm) and fed 133
79 Moreover, recent reports have provided controversial
by mean of a micropipette tip (200 ml) with different doses 134
80 results about fumagillin efficacy (Williams et al., 2008)
of pure metabolite (20; 30; 40 and 60 ml per bee) (Fig. 1). 135
81 and also about its undesirable effects related to residues
Each treatment was replicated 20 times. Syrup 2:1 136
82 generated in honey (Lopez et al., 2008). In this way, much
(sugar:water) and sterile MRS broth was used as control 137
83 effort in the last few years has focused on alternatives to
treatments. Once the metabolite dose was consumed, 138
84 control this bee disease, most of them involving alter-
candy (mixture of powdered sugar and water) was placed 139
85 natives to chemical substances. Even though great
inside each tube to provide food resource to the bees. 140
86 progress has been achieved and extensive efforts are
Individual bee mortality per treatment was recorded at 24, 141
87 being made in the study of these compounds properties,
48 and 72 h. Bees were kept under incubator conditions 142
88 more research is still needed.
during toxicity experiments (33  0.79 8C; 60  3.3% HR). 143
89 The goal of the current study is to assess the effects of
90 the oral administration of the metabolites produced by 2.5. Assay II: effects of metabolite administration on 144
91 Lactobacillus johnsonii CRL1647, mainly identified as colony development 145
92 organic acids (Audisio et al., 2011), on: (I) N. ceranae
93 sporulation dynamics before and after fumagillin applica- Field research was carried out with a local A. mellifera 146
94 tion, and (II) performance of A. mellifera colonies. ecotype (A. mellifera mellifera  A. mellifera ligustica) 147

Please cite this article in press as: Maggi, M., et al., Effects of the organic acids produced by a lactic acid bacterium in Apis
mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
10.1016/j.vetmic.2013.07.030
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M. Maggi et al. / Veterinary Microbiology xxx (2013) xxx–xxx 3

Fig. 1. Systemic administration of the bacterial metabolite in vitro conditions. Each tube was embedded into a styrofoam base. One single worker bee per
tube was introduced and fed by mean of a micropipette tip (200 ml) with different doses of pure metabolite (20; 30; 40 and 60 ml per bee). Each treatment
was replicated 20 times. Syrup 2:1 (sugar:water) and sterile MRS broth was used as control treatments. Once the metabolite dose was consumed, candy was
placed inside each tube to provide food resource to the bees. Individual bee mortality per treatment was recorded at 24, 48 and 72 h. Bees were kept at
33  0.79 8C; 60  3.3% HR.

148 (Abrahamovich et al., 2007), kept in standard Langstroth inspected and afterword the first treatment application 177
149 hives. Prior to these studies, colonies had received parasitic was performed. Any change was compared with the 178
150 control treatment against V. destructor (Flumevar1, two untreated control hives. All other conditions (weather, 179
151 strips per hive with a total of 1 g of flumethrine). Hives nourishment and supervision) were identical. The para- 180
152 were naturally infected with N. ceranae. Molecular meters to quantify the general state of the colonies during 181
153 diagnoses based on samples collected in Santa Paula the evaluation were as follows: number of combs fully 182
154 apiary were performed and recently published (Genebank covered with bees, open and sealed brood areas (following 183
155 AN FJ425736; Medici et al., 2012). In addition, N. ceranae the methodology of Branco et al., 1999) and quantity of 184
156 was the only microsporidium found in the southeast of honey and pollen (estimated as box surface covered with 185
157 Buenos Aires province (Medici et al., 2012). Colonies used honey or pollen respectively). A final quantification of 186
158 in these assays were comprised of 15 nuclei prepared with colony development was performed on September 15th 187
159 sister queens (obtained from a commercial bee house), one (end of winter). Here, we established the number of frames 188
160 non operculated brood comb, two operculated brood covered with adult bees as an estimator of the strength of 189
161 combs plus the bees present in these, and finally, one frame each colony. 190
162 full of honey. Bacterial metabolite administration was
163 carried out by a Doolittle feeder. Five colonies (Group 2.6. Assay III: effects of metabolite administration on fat 191
164 A = LM) received 50 ml of pure metabolite + 200 ml of bodies of workers bees 192
165 syrup 2:1 (sugar:water). Five colonies (Group B, control
166 1 = CM) received 50 ml of culture sterile media + 200 ml of Just before the first treatment application ( control, 193
167 syrup 2:1, and another five colonies (Group C, control T0) and the same day of the third and fifth ones (prior 194
168 2 = syrup) received 250 ml of syrup 2:1. Treatments were to its application: T2 and T4 respectively), a sample 195
169 provided respectively to each group (A, B and C), five times of 50 nurse bees per colony was collected from the 196
170 at intervals of five days (time-points T0, T1, T2, T3 and T4) brood nest (Table 1). Bees were frozen until laboratory 197
171 (Table 1). assays. 198
172 The progress of the hives treated with the bacterial To measure fat bodies the protocol of Wilson-Rich et al. 199
173 metabolite was monitored prior to each treatment (2008) was followed. Adult abdomens were severed from 200
174 application (time-points T0, T1, T2, T3 and T4) and also thoraces and dried for three days at room temperature. 201
175 one week after the fifth application (designated as time- Abdomens were weighed and washed in ethyl ether for 202
176 point T5) (Table 1). As an example: at T0, the colonies were 24 h to dissolve fat. Larvae and pupae were not included in 203

Please cite this article in press as: Maggi, M., et al., Effects of the organic acids produced by a lactic acid bacterium in Apis
mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
10.1016/j.vetmic.2013.07.030
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4 M. Maggi et al. / Veterinary Microbiology xxx (2013) xxx–xxx

Table 1
Variables analyzed for each sampling time-point.

T0 T1 T2 T3 T4 T5 T6 T7

Sampling of colony development x x x x x x – –


N. ceranae sampling x – x – x x x x
Fat bodies x – x – x – – –
Fumagillin application – – – – x x x –
Metabolite application x x x x x – – –

204 this assay because of their lack of hardened cuticle. 2.8. Statistics 253
205 Abdomens were then dried for three days and weighed
206 again. The fat body was calculated as the percent change in Two-way ANOVA was performed to analyze the effects 254
207 abdominal weight after the ethyl ether wash. of treatments and time on colony development and fat 255
bodies. Comparison of the average values was carried out 256
208 2.7. Assay IV: effects of the bacterial metabolite on Nosema using Tukey test (p < 0.05). A non-parametric test was 257
209 sporulation and fumagillin efficacy and efficiency performed to analyze N. ceranae prevalence and intensity 258
among treatments and time of sampling (Kruskal–Wallis 259
210 To assess the effect of the L. johnsonii CRL1647 test). Comparison of the average values was carried out 260
211 metabolite administration on the development of N. using Student–Nueman–Keuls test (p < 0.05). 261
212 ceranae, samples of bees from each colony were taken:
213 (a) prior the first treatment application (control, time- 3. Results 262
214 point T0); (b) the same day the third treatment application,
215 prior to apply it (time-point T2) and (c) the same day of the 3.1. Bacterial metabolites quantification by HPLC analyses 263
216 fifth treatment application, prior to apply it (time-point T4)
217 (Table 1). The concentration in the CFS of the organic acids 264
218 To evaluate the effect of the metabolite on fumagillin produced by L. johnsonii CRL1647 is reported in Table 2. 265
219 efficacy and efficiency, the same day of the fifth Three acids were detected: lactic acid, phenyl-lactic acid 266
220 treatment application (time-point T4), colonies also were and acetic acid. 267
221 treated with 2% fumagillin (Nosemix-B1 Solemar SA),
222 according to label instructions. Each colony received 3.2. Assay I: toxicity effects of bacterial metabolite 268
223 6 mg fumagillin per bee-covered comb, three times at against honey bees 269
224 intervals of seven days (time-points T4, T5 and T6). Bee
225 samples were taken from each colony after each The in vitro administration of the CFS, containing 270
226 fumagillin application, and also one week after the last mainly organic acids, did not produce bee mortality during 271
227 drug application (time-points T5, T6 and T7 respectively) treatment, even in high doses and after 72 h of exposure 272
228 (3 times) (Table 1). (60 ml of bacterial metabolite produced 10% mortality). 273
229 Sampling was carried out always at midday, since Table 3 depicts bee mortality (in percentage) for the doses 274
230 Meana et al. (2010) reported that bees collected for tested. Both control treatments reached only 10% bee 275
231 measuring Nosema spore loads should be collected at the mortality after 72 h. 276
232 same time of day to be comparable. The entrance of every
233 hive was closed with foam rubber so that foraging bees 3.3. Assay II: effects of metabolite administration on 277
234 piled up and a representative sample group of more than colony development 278
235 50 individuals could be collected and put into a flask with
236 ethanol 70%. One sample group from each colony was Adult bee populations at the end of the test increased in 279
237 taken at each time-point ‘‘T’’ as indicated above (T0, T2, T4, the different treatments by (I) 39.5% when the bacterial L. 280
238 T5, T6, and T7) (see also Table 1, Figs. 5 and 6). In the
239 laboratory, the abdomens of 50 bees from each sample Table 2
240 group were individually homogenized in 2 ml of double Organic acid production by Lactobacillus johnsonii CRL1647.
241 distilled water and checked for the presence of Nosema Lactic acid Phenyl-lactic Acetic acid
242 spores under a compound microscope (400 and 1000) (mM) acid (mM) (mM)
243 Q3 to obtain infection levels (percentage of infected bees per CRL1647 128.1 0.3 38.0
244 hive, colony level, adapted from Smart and Sheppard,
245 2011). Later on, ten of these positive homogenates were
Table 3
246 randomly selected; spore amounts were quantified using
Honey bee mortality (%) after 24, 48 and 72 h for different doses (ml) of
247 an improved Neubauer haemocytometer, yielding the organic acids isolated from Lactobacillus johnsonii CRL1647.
248 number of mature spores per bee (intensity, individual
0 ml 20 ml 30 ml 40 ml 60 ml
249 level). In sample groups without ten positive cases, all
250 positives were quantified by haemocytometer. Table 1 24 h 0 0 0 0 0
48 h 10 0 4 0 10
251 summarizes the sampling time-points (from T0 to T7) and
72 h 10 0 10 0 10
252 all the variables analyzed.

Please cite this article in press as: Maggi, M., et al., Effects of the organic acids produced by a lactic acid bacterium in Apis
mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
10.1016/j.vetmic.2013.07.030
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M. Maggi et al. / Veterinary Microbiology xxx (2013) xxx–xxx 5

Fig. 2. Evolution of different beehive parameters during the field trial. (A) Depicts the progress of adult bee population. (B–E) Do the same for total combs
with sealed brood per colony, total combs with open brood per colony, total combs with honey per colony and total combs with pollen per colony
respectively. At time-point 0, all parameters were recorded from the hive. Then, first application of the three treatments was performed and repeated four
more times every five days (time-points T1, T2, T3 and T4). LM = group of colonies which received five applications of 50 ml of bacterial metabolite plus
200 ml of syrup 2:1 (sugar:water); CM = group of colonies which received five applications of 50 ml of culture media plus 200 ml of syrup 2:1; syrup = group
of colonies which received five applications of 250 of syrup 2:1.

281 johnsonii CRL1647 metabolite (LM) was provided (from 3.4. Assay III: effects of metabolite administration on fat 305
282 4.3  0.44 combs covered with bees at the beginning of the bodies of worker bees 306
283 field trial to 6  0.97 at the end); (II) 26.0% when culture
284 media (CM) was provided (from 4.6  0.65 combs covered Metabolite administration/delivery (LM) on beehives 307
285 with bees at the beginning of the field trial to 5.8  0.67 at the induced a significant increase in fat body production 308
286 end) and (III) 12.0% when colonies received syrup alone compared with culture media (CM) and syrup adminis- 309
287 (syrup) (from 4.1  0.54 combs covered with bees at the tration: after T2, worker bees exposed to LM increased their 310
288 beginning of the field trial to 4.5  0.7) (Fig. 2). Considering fat bodies stores from 2.48 to 5.76 mg (Fig. 4 and Table 5). 311
289 statistical analyses, significant differences were observed in After four applications of LM, a pronounced decrease was 312
290 the adult bee population per colony among treatments and observed: fat bodies decreased from 5.76 (T2) to 1.06 mg 313
291 time (p < 0.05, Table 4). Brood population also varied through per bee at the end of the field trial (T4). In the control 314
292 the field trial. A continuous reduction of sealed brood for LM groups, the CM treatment did not produce significant 315
293 treatment could be observed after the third dose application
294 (T2) explaining the increase of adult bees on beehives (from
Table 4
295 2  0.5 combs with sealed brood at the beginning of the field
Statistical output for the two way ANOVA conducted to test the
296 trial to 1.6  0.4 at the end) (Fig. 2, p < 0.05 and Table 4). differences among treatments on colony performance.
297 Pollen stores were not affected by treatments (p > 0.05).
298 However, statistical differences were detected in honey Colony variable Treatment Time

299 storage for the three groups over time (p < 0.05, Table 4) but p value F value p value F value
300 no differences were detected among treatments at the end of Adult bees <0.001 16.6 <0.001 4.63
301 the experiment (p > 0.05). After winter, clear differences Sealed brood 0.04 3.15 <0.001 4.34
302 were observed for colony strength among treatments. One of Open brood 0.97 0.02 0.01 3.29
Pollen 0.57 0.88 0.37 1.08
303 the five colonies from the control group died during the
Honey 0.88 0.12 <0.001 4.24
304 winter. These results are represented in Fig. 3.

Please cite this article in press as: Maggi, M., et al., Effects of the organic acids produced by a lactic acid bacterium in Apis
mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
10.1016/j.vetmic.2013.07.030
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6 M. Maggi et al. / Veterinary Microbiology xxx (2013) xxx–xxx

Table 5
Mean values of fat bodies (mg) per honey bees and standard deviation
(between brackets) measured over time for each treatment. Different
letters indicate statistical differences (two way ANOVA, p < 0.05) inside
time and treatment and also between them. T0 represents the time before
the first treatment application (control) and show the basal status of the
mean fat body/bee for each group of colonies. T2 represents the status of
the mean fat body/bee after two treatment application. T4 represents the
status of the mean fat boy/bee after four treatment application.
LM = group of colonies which received five applications of 50 ml of
bacterial metabolite plus 200 ml of syrup 2:1 (sugar and water);
CM = group of colonies which received five applications of 50 ml of
culture media plus 200 ml of syrup 2:1; syrup = group of colonies which
received five applications of 250 of syrup 2:1.

Treatment T0 T2 T4

LM 2.48 (1.14) a 5.76 (2.9) b 1.06 (0.32) a


MC 2.06 (0.6) a 3.17 (2.45) a 2.4 (1.73) a
Syrup 2.58 (1.21) a 3.23 (0.78) a 5.74 (2.76) b

Fig. 3. Boxplot for the mean number of combs covered with adult bees
after winter for each group of colonies involved on the field trial.
the parasitosis, no improvement on its final efficacy due 329
the administration of the bacterial metabolite was 330
316 changes in fat bodies per bee over time (p > 0.05). detected: all groups reached levels of Nosema prevalence 331
317 However, an increase in fat body production was observed close to 0% (Fig. 5 and Table 6). 332
318 in workers sampled from colonies fed only with syrup:
319 from 2.58 at T0 to 5.74 mg per bee at T4. 3.5.2. Nosemosis at individual level 333
Those colonies which were fed with bacterial metabo- 334
320 3.5. Assay IV: effects of the bacterial metabolite on Nosema lites showed a decrease in the average number of spores 335
321 sporulation per bee: after the fourth application of the organic acids, 336
Nosema intensity decreased from 5.52 to 2.61  106 spores 337
322 3.5.1. Nosemosis at colony level per bee (Fig. 6 and Table 7). Moreover, both control group 338
323 Prevalence of N. ceranae over time was similar for the colonies showed an increase in the intensity of N. ceranae 339
324 three groups of colonies involved in the field trial. Infection after T0: from 1.74 to 4.28  106 spores per bee in the CM 340
325 levels ranged from 16.4 to 25.6% at the beginning of the group, and from 3.14 to 4.65 spores per bee in the syrup 341
326 trial. By the end of the treatment applications, these values group (Fig. 6 and Table 7). In this way, the administration of 342
327 decreased to 0.4–1.2% (Fig. 5 and Table 6). Although the
328 fumagillin application was successful in the control of

Fig. 5. Mean prevalence of Nosema ceranae (%) over time for the three
groups of colonies involved on the field trial. T0: time before the first
treatment application (control). Basal status of the mean prevalence of N.
Fig. 4. Variation of fat body per bee among treatments and time. T0: time ceranae for each group of colonies. T2 and T4: status of the mean
before the first treatment application (control). Basal status of the mean prevalence of N. ceranae after two and four treatment applications
fat body/bee for each group of colonies. T2: status of the mean fat body/ respectively. T5, T6 and T7: effects of the first, second and third fumagillin
bee after two treatment applications. T4: status of the mean fat body/bee application against N. ceranae prevalence. LM = group of colonies which
after four treatment applications. LM = group of colonies which received received five applications of 50 ml of bacterial metabolite plus 200 ml of
five applications of 50 ml of bacterial metabolite plus 200 ml of syrup 2:1 syrup 2:1 (sugar:water); CM = group of colonies which received five
(sugar:water); CM = group of colonies which received five applications of applications of 50 ml of culture media plus 200 ml of syrup 2:1;
50 ml of culture media plus 200 ml of syrup 2:1; syrup = group of colonies syrup = group of colonies which received five applications of 250 of
which received five applications of 250 of syrup 2:1. syrup 2:1.

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Table 6
Mean prevalence of Nosema ceranae (%) and standard deviations (between brackets) over time for the three groups of colonies involved on the field trial.
Different letters indicate statistical differences (Kruskal–Wallis test, p < 0.05) inside time and treatment and also among them. LM = group of colonies which
received five applications of 50 ml of bacterial metabolite plus 200 ml of syrup 2:1 (sugar and water); CM = group of colonies which received five
applications of 50 ml of culture media plus 200 ml of syrup 2:1; syrup = group of colonies which received five applications of 250 of syrup 2:1.

Treatment T0 T2 T4 T5 T6 T7

LM 24 (5.8) a 26.6 (6.2) a 33.2 (8.1) a 21.2 (11.1) a 3.4 (3.4) c 1.2 (1.1) c
CM 25.6 (7.5) a 24 (11) a 30.8 (14.6) a 10.4 (5.5) b 0.8 (1.1) c 0.4 (0.9) c
Syrup 16.4 (7.9) b 10.8 (7.7) b 34 (25.2) a 17.2 (7.3) b 2.4 (1.7) c 1.2 (1.8) c

Table 7
Mean intensity of Nosema ceranae and standard deviations (between brackets) over time for the three groups of colonies involved on the field trial. Different
letters indicate statistical differences (Kruskal–Wallis test, p < 0.05) inside time and treatment and also between them. Values were expressed in millions of
spores per bee. LM = group of colonies which received five applications of 50 ml of bacterial metabolite plus 200 ml of syrup 2:1 (sugar and water);
CM = group of colonies which received five applications of 50 ml of culture media plus 200 ml of syrup 2:1; syrup = group of colonies which received five
applications of 250 of syrup 2:1.

Treatment T0 T2 T4 T5 T6 T7

LM 5.52 (5.3) a 2.8 (2.1) b 2.61 (1.7) b 2.51(1.5) b 2.21(1.1) b 2.77 (3.7) b
CM 1.74 (1.15) b 4.08 (2.9) a 4.28 (2.3) a 3.39 (2.6) a 4.43 (1.5) a 3.85(3.1) a
Syrup 3.14 (3.2) a 5.4 (4.1) a 4.65 (4.2) a 2.84 (2.7) b 6.13 (4.1) a 4.45 (2.3) a

343 the bacterial metabolite improved the final efficiency of 4. Discussion 352
344 fumagillin when single infected bees were analyzed. Fig. 6
345 and Table 7 depicts these effects: those colonies which Thus far, most studies on honey bee microbiota have 353
346 received the administrations of the bacterial metabolite focused on disease-causing microorganisms and colony 354
347 maintained lower levels of N. ceranae intensity during declines while much less emphasis has been given to non- 355
348 fumagillin application and afterwards (from 2.51 at T5 pathogenic microorganisms and their potential health 356
349 to 2.77  106 spores per bee at T7). However, colonies benefits for individual bees or whole colonies. Intestinal 357
350 belonging to the control groups experienced a reinfesta- microbiota of most organisms plays a crucial role in 358
351 tion (from 2.84 at T5 to 4.45  106 spores per bee at T7). nutrient assimilation and immune function, and accumu- 359
lating evidence for a worldwide association between A. 360
mellifera and a core set of bacterial phylotypes does exist 361
(Martinson et al., 2011). A growing awareness about the 362
importance of the intestinal microbiota composition for 363
honey bee health and growth has been reported (Olofsson Q4 364
and Vásquez, 2008; Martinson et al., 2011). Moreover, bees 365
themselves are constantly exposed to pesticides, which in 366
turn may impair beneficial in-hive microbes (Yoder et al., 367
2012). Because specific bacteria are consistently associated 368
with A. mellifera, it has been suggested that these bacteria 369
are beneficial mutualists (Martinson et al., 2011). 370
Honey bee gut microbiota appears to be relatively 371
simple and consistent across individuals, compared to gut 372
microbiota of other insects (Moran et al., 2012). Currently, 373
knowledge about the benefits of Apis bacteria on bee health 374
is scarce, with the exception of some lactic acid bacteria 375
(LAB) effects. These bacteria might decrease the infection 376
rate of A. mellifera larvae exposed to Paenibacillus larvae 377
(Forsgren et al., 2010). In addition, LAB have been isolated 378
Fig. 6. Mean intensity of Nosema ceranae (spores/infected bee) over time from the crop of A. mellifera, freshly collected pollen and 379
for the three groups of colonies involved on the field trial. T0: time before
freshly fermented bee bread, which is the protein source 380
first treatment application (control). Basal status of the mean intensity of
N. ceranae for each group of colonies. T2 and T4: status of the mean intensity for larval stages (Vásquez and Olofsson, 2009). Fermenta- 381
of N. ceranae after two and four treatment applications respectively. T5, T6 tive properties of LAB could aid in the conversion of nectar 382
and T7:effects of the first, second and third fumagillin application against N. to honey as well as in the conversion of pollen to bee bread 383
ceranae intensity. LM = group of colonies which received five applications of and its protection from spoilage (Olofsson and Vásquez, 384
50 ml of bacterial metabolite plus 200 ml of syrup 2:1 (sugar:water);
CM = group of colonies which received five applications of 50 ml of culture
2008; Vásquez and Olofsson, 2009). Bacteria associated 385
media plus 200 ml of syrup 2:1; syrup = group of colonies which received with bumble bees can also benefit their hosts against 386
five applications of 250 of syrup 2:1. parasites (Koch and Schmid-Hempel, 2012). 387

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8 M. Maggi et al. / Veterinary Microbiology xxx (2013) xxx–xxx

388 In this work, we have reported another type of LAB pollinators’ health. Proteins, carbohydrates, minerals, 449
389 benefit in A. mellifera colonies. This benefit was measured lipids, and vitamins are supplied primarily by nectar, 450
390 indirectly by the administration of organic acids in bee pollen and water. When these resources are depleted, bees 451
391 hives produced by the strain L. johnsonii CRL1647. This must use proteins and lipids from their own tissues to 452
392 bacterium was previously isolated from the bee gut and produce larval food and survive for a short period of time. 453
393 selected mainly by its organic acids synthesis (Audisio Therefore, stronger colonies could enhance survival, 454
394 et al., 2011). The results of this study indicate firstly that moreover when poor nutrition is explicitly identified as 455
395 feeding bees high doses of these metabolites is not lethal a probable contributing factor in recent colony losses 456
396 after 72 h of exposure. Control mortality did not exceed (vanEngelsdorp et al., 2010). 457
397 10%, thus complying with the trials standards set forth by With regard to the bacterial metabolite and its impact 458
398 OECD (OECD, No. 213 and No. 214 (1998), OEPP/EPPO on Nosema levels, two types of monitoring of the parasite 459
399 Guideline No. 170). Additionally, treating the colonies with were selected: prevalence (colony level) and intensity 460
400 the bacterial metabolite increased population levels. Similar (individual level). Mean rate of infected honey bees 461
401 results were found by Audisio and Benitez-Arhendts (2011), (prevalence) seems to be the best reliable method to 462
402 using L. johnsonii CRL1647 bacterial cells. In addition, those evaluate the health status of a colony (Higes et al., 2008; 463
403 colonies fed with the bacterial metabolite survived better Meana et al., 2010). Our results showed that metabolite 464
404 over winter than colonies in the control group (one colony supplementation did not modify the dynamics of the 465
405 fed only with syrup died during winter and the others disease at the colonial level. The initial prevalence of 466
406 showed decreased populations compared to the treat- nosemosis in the colonies was comparable at the start of 467
407 ment group LM). Although it was known that particular the experiment and, even with some minor variations in T2, 468
408 strains of L. johnsonii can produce different antimicrobial the colony infections were similar and did not show 469
409 peptides (as a result of its own metabolism), the strain statistical differences in both pre- and post-fumagillin 470
410 CRL1647 used in this survey was selected on the basis of application. This fact could be related to the limited 471
411 its acidificant power (138 mM of lactic acid) due to their timeframe of the experiment. A long-term (e.g.: seasonal) 472
412 synthesis of organics acids (Audisio et al., 2011). metabolite supplementation may give appreciable results 473
413 Microbial diversity and by products may provide at this level. However, when nosemosis development was 474
414 colonization resistance to pathogens (Dillon and Charnley, analyzed individually, differences between treatments 475
415 2002) and may be of extraordinary relevance to honey bee were observed. A mean spore count per bee is used as 476
416 health, given that they have a greatly reduced immune an indicator of success of the pathogen in the individual 477
417 system relative to other model insects (Evans et al., 2006). host (Smart and Sheppard, 2011). A higher spore load 478
418 Hence, colonies fed with LM could reach larger sizes than affects the insect, causing a suite of metabolic changes 479
419 other treatment groups as a consequence of a better health (Bailey, 1981). Infected bees have lower levels of protein, 480
420 status induced by the organic acids. Moreover, we have resulting in a reduced hypopharengeal gland (Malone and 481
421 reported that fat bodies have also significantly increased Gatehouse, 1998), as well as altered fatty acid composition 482
422 after two applications of bacterial metabolites, where in the hemolymph (Roberts, 1968). Also, N. ceranae 483
423 organic acids were the main components, when compared shortens bee life span causing greater colony mortality 484
424 with control groups. Fat bodies play a major role in the life in winter (Nitschmann, 1957). Here, we have showed that 485
425 of insects, being involved in multiple metabolic functions. application of our bacterial metabolite alone has strongly 486
426 Among the most important functions are: (a) to store and reduced the spore loads per bee. Additionally, when 487
427 release energy in response to energy demands of the insect coupled with fumagillin, the amounts of spores per bee 488
428 and (b) to produce several antimicrobial peptides, acting decreased during application of the metabolite, resulting 489
429 similar to a vertebrate liver (Lavine and Strand, 2002). in an improvement in the final efficiency of the drug: those 490
430 These two functions are closely related and could explain colonies which did not receive the metabolite (groups CM 491
431 the better survival and the bigger population size of those and syrup) had regularly increased amounts of spores in 492
432 colonies fed with the bacterial metabolite: a bee with more the individuals examined. Hence, organic acids produced 493
433 fat bodies implies a healthier bee. In A. mellifera colonies, by a L. johnsonii strain could: (a) contribute as an organic 494
434 nutritional deficiencies that affect the immune response tool to reduce individual loads of Nosema without 495
435 can accelerate the spread of disease among nest mates, fumagillin application and (b) keep controlled the reinfes- 496
436 increasing pathogen levels and reducing adult longevity tation when antibiotic control is used, improving its 497
437 and survival (Mayack and Naug, 2009). What began as a efficacy. The significance of our findings is more evident 498
438 nutritional deficiency could quickly develop into colony considering that: (a) fumagillin is prohibited in some 499
439 loss due to an infectious disease. By this pathway, nutrition countries, (b) fumagillin has been the topic of controversy 500
440 is a key factor in resistance to pathogens (Rowley and regarding its efficiency in Nosema control (Willians et al., 501
441 Powell, 2007). A recent study on A. mellifera argues that 2011) and (c) fumagillin use could result in honey residues 502
442 poor nutrition depresses the immune system and conse- (Lopez et al., 2008). These results could be of high impact 503
443 quently could drive colony loss (vanEngelsdorp et al., for IPM of nosemosis. 504
444 2010). In the same way, Allaux et al. (2010) found that bees Our investigations were not sufficient to explain which 505
445 fed with diets rich in protein modify both individual and mechanisms are involved on Nosema reduction when 506
446 social immune competencies. These authors suggest a link bacterial metabolite supplementation on colonies is 507
447 between nutrition and immunity in bees, underlining the applied. However, different hypotheses could be postu- 508
448 fundamental role of the availability of resources for lated: (a) Organic acids synthesized by L. johnsonii CRL1647 509

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mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
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558 The authors have no conflict of interest to declare. Roberts, M.D., 1968. Fatty acids in honey bees (Apis mellifera) infected 631
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559 Acknowledgments quasi-specific, or nonspecific? J. Immunol. 179, 7209–7214. 634
Sabaté, D.C., Cruz, M.S., Benı́tez-Ahrendts, M.R., Audisio, M.C., 2012. 635
560 We would like to thank Dr. Martı́n Porrini, for his Beneficial effects of Bacillus subtilis subsp. subtilis Mori2, a honey- 636
associated strain, on honeybee colony performance. Probiotics Anti- 637
561 advices on research design, as well as Neil Sanscrainte microb. Prot. 4, 39–46. 638
562 (USDA-ARS-CMAVE, Gainesville, FL) for his assistance in Smart, M., Sheppard, M., 2011. Nosema ceranae in age cohorts of the 639
563 the wording of the manuscript. The authors also acknowl- western honey bee (Apis mellifera). J. Invert. Pathol. 109, 148–151. 640
vanEngelsdorp, D., Hayes, J., Underwood, R., Pettis, J., 2010. A survey of 641
564 edge UNMDP and CONICET. This research was supported honey bee colony losses in the United States, fall 2008 to spring 2009. 642
565 by a grant of ANPCyT, PICT 2011 to M. M. J. Apic. Res. 49, 7–14. 643

Please cite this article in press as: Maggi, M., et al., Effects of the organic acids produced by a lactic acid bacterium in Apis
mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
10.1016/j.vetmic.2013.07.030
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647 Symbionts as major modulators of insect health: lactic acid bacteria Yoder, J.A., Hedges, B.Z., Heydinger, D.J., Sammataro, D., DeGrandi-Hoff- 655
648 and honeybees. PLoS ONE 7 (3) e33188. man, G., 2012. Differences among fungicides targeting beneficial 656
649 Williams, G.R., Sampson, M.A., Shutler, D., Rogers, R., 2008. Does fuma- fungi associated with honey bee colonies. In: Sammataro, Yoder, 657
650 gillin control the recently detected invasive parasite Nosema ceranae (Eds.), Honey Bee Colony Health: Challenges and Sustainable Solu- 658
651 in western honeybees (Apis mellifera)? J. Invert. Pathol. 99, 342–344. tions. CRC Press, pp. 181–192. Q5 659

Please cite this article in press as: Maggi, M., et al., Effects of the organic acids produced by a lactic acid bacterium in Apis
mellifera colony development, Nosema ceranae control and fumagillin efficiency. Vet. Microbiol. (2013), http://dx.doi.org/
10.1016/j.vetmic.2013.07.030

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