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High prevalence and infection levels of Nosema ceranae in bumblebees Bombus atratus and Bombus bellicosus from Uruguay
Journal of Invertebrate Pathology 130 (2015) 165–168
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
High prevalence and infection levels of Nosema ceranae in bumblebees Bombus atratus and Bombus bellicosus from Uruguay N. Arbulo a,⇑, K. Antúnez b, S. Salvarrey c, E. Santos c, B. Branchiccela b, R. Martín-Hernández d, M. Higes d, C. Invernizzi c a
Centro Universitario Regional del Este, Universidad de la República, Rocha, Uruguay Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay Sección Etología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay d Centro Apícola Regional, Junta de Comunidades de Castilla La Mancha, 19180 Marchamalo, Spain b c
a r t i c l e
i n f o
Article history: Received 20 January 2015 Revised 16 July 2015 Accepted 31 July 2015 Available online 3 August 2015 Keywords: Microsporidia Parasites Nosemosis
a b s t r a c t Nosema ceranae is one of the most prevalent pathogens in Apis mellifera and has recently been found in multiple host species including several species of bumblebees. Prevalence and infection intensity of N. ceranae was determined in two species of native bumblebees from Uruguay. Nosema ceranae was the only microsporidia identified and mean prevalence was 72% in Bombus atratus and 63% in Bombus bellicosus, values much higher than those reported elsewhere. The presence of this pathogen in bumblebees may be threatening not only for bumblebee populations, but also to the rest of the native pollinator community and to honeybees. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Nosema ceranae is one of the most prevalent pathogens in Apis mellifera worldwide (Higes et al., 2013). Originally described in Apis cerana (Fries et al., 1996), in 2006 was found as an emergent pathogen in the European honey bee (Higes et al., 2006) and currently it is found in multiple host species (Plischuk et al., 2009; Chaimanee et al., 2011; Li et al., 2012; Graystock et al., 2013). This wide host range is of significant epidemiological and ecological concern, since some parasite can be transmitted between host individuals that share flower resources (Durrer and Schmid-Hempel, 1994). Bumblebees host a great variety of parasites (Schmid-Hempel, 2001). Until the work of Plischuk et al. (2009), Nosema bombi was the only microsporidia known to infect bumblebees (Tay et al., 2005). At present, N. ceranae has been detected in three South American (Plischuk et al., 2009), four Asian (Li et al., 2012) and seven European (Graystock et al., 2013) species. Recent studies confirmed that bumblebees can be genuinely infected by N. ceranae and may not be simply accidental carriers of its spores (Graystock et al., 2013; Fürst et al., 2014).
⇑ Corresponding author at: Centro Universitario Regional del Este – Sede Rocha, Ruta Nacional N° 9 intersección Ruta Nacional N° 15, Rocha 27000, Uruguay. E-mail address: [email protected] (N. Arbulo). http://dx.doi.org/10.1016/j.jip.2015.07.018 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.
The aim of this study was to determine the presence, prevalence and infection intensity of N. ceranae in Bombus atratus and Bombus bellicosus from Uruguay. 2. Methods 2.1. Bumblebee collection A total of 526 bumblebees, 317 B. atratus (167 females, 150 males) and 209 B. bellicosus (141 females, 68 males), were collected in April 2010 while foraging on red clover (Trifolium pratense) in three locations: Montevideo (34°500 S, 56°130 W), Canelones (34°400 S, 56°020 W) and Colonia (34°200 S, 57°410 W). Individual bees were collected directly from flowers and were stored at 20 °C until analysis. 2.2. Identification of Nosema species Abdomens of bumblebees were individually macerated with 2 ml of 95° ethanol. Macerates were centrifuged and washed to remove ethanol and pellets were resuspended in 250 ll of sterile distilled water. One hundred and fifty ll of each sample were introduced into a 96-well plate (Qiagen) with glassbeads (2 mm, Sigma) and plates were shaken for 6 min at 30 Hz. Thirty ll of ATL buffer (Qiagen 19076) and 20 ll of Proteinase K (Qiagen 19131) were added to each well and plates were incubated
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N. Arbulo et al. / Journal of Invertebrate Pathology 130 (2015) 165–168
overnight at 56 °C. Subsequently, DNA was extracted using the BS96 DNA Tissue extraction protocol in a BioSprint apparatus (Qiagen). The presence of N. ceranae and N. apis was determined using Nosema Biotools DNA Polymerase plates containing PCR triplex (321-APIS-F/R, 218-MITOC-F/R and COI-F/R primers; MartínHernández et al., 2012) master mix. Reactions were carried out in a total volume of 50 ll and the cycling program was: 94 °C (6 min); 35 cycles of 30 s denaturation at 94 °C, 45 s annealing at 61.8 °C and 60 s extension at 72 °C; and a final extension step at 72 °C for 10 min. PCR for N. bombi detection was performed in a total volume of 25 ll using the Fast Start PCR Master mix (No. 04710452001 Roche Diagnostic), 0.4 mM of each primer, 0.2 mg ml-1 BSA, 0.1% Triton X-100 and 2.5 ll of DNA template. The cycling program was: 95 °C (10 min); 35 cycles of 30 s denaturation at 95 °C, 30 s annealing at 58.1 °C and 45 s extension at 72 °C; and a final extension step at 72 °C for 7 min. Negative controls for DNA extraction and PCR were analyzed in parallel. PCR reactions were carried out in a MastercyclerÒ ep gradient S apparatus (Eppendorf) in Centro Apícola Regional de Marchamalo (Spain). PCR products were analyzed in a QIAxcel System (Qiagen) using a QIAxcel DNA High Resolution Kit (Qiagen, No. 929002). Prevalence was calculated as the percentage of positive samples for each analyzed group. Chi square test with a 0.05 significance level was used to evaluate intraspecific differences between sexes and interspecific differences within sexes. 2.3. Spore counting Infection intensity (spores per bumblebee) was determined in positive samples. Spore count was done using a haemocytometer and a light microscope at 400. Number of spores in known volume was used to calculate spores per bumblebee. Spore detection limit was calculated at 12,500 spores per bumblebee, which corresponded to finding one spore in the total volume analyzed. Infection intensity categories were defined for the analysis: very low (<12,500 spores), low (12,500 to 5 105 spores), medium (5 105 + 1 to 25 105 spores) and high (more than 25 105 spores). The Mann–Whitney U test with a 0.05 significance level, was performed to compare infection level between the analyzed groups. 3. Results 3.1. Nosema identification and prevalence A total of 364 samples were positive for N. ceranae which was the only microsporidia species detected. Prevalence was equal or higher than 50% in different locations and sexes, with two exceptions (Fig. 1). There were intraspecific differences on prevalence between location (B. atratus: females v2 = 3.40, P = 0.182, males v2 = 20.81, P < 0.001; B. bellicosus: females v2 = 11.35, P < 0.001, males v2 = 5.40, P = 0.02) so the analysis was performed for each location separately. Prevalence in B. atratus was higher in females (Montevideo: v2 = 35.26, P < 0.001; Canelones: v2 = 7.86, P = 0.005; Colonia: v2 = 19.32, P < 0.001) while in B. bellicosus there was no difference between sexes (Canelones: v2 = 0.02, P = 0.882; Colonia: v2 = 0.03, P = 0.856) (Fig. 1). Prevalence in females was higher in B. atratus than in B. bellicosus (Canelones: v2 = 24.80, P < 0.001; Montevideo:
v2 = 6.46, P = 0.011). Regarding males, N. ceranae was more prevalent in B. atratus at Canelones (v2 = 5.36, P = 0.021) but there was no interspecific difference at Colonia (v2 = 3.46, P = 0.063). 3.2. N. ceranae infection intensity Infection intensity was determined in 293 N. ceranae positive samples. Not all positive samples (N = 364) could be microscopically analyzed due to insufficient amount of material left in 71 samples after molecular analyses. Spores were detectable in 246 samples (84%). Infection intensity reached loads as high as 44 million spores per individual. In 47 samples (16%) spores could not be detected microscopically, indicating that the number of spores was under the detection limit (12,500 spores per bumblebee). Infection intensity differed between sexes, being higher in females in both species (B. atratus: Montevideo: U = 133, P < 0.001; Canelones: U = 733.5, P < 0.001; Colonia: U = 164.5, P < 0.001. B. bellicosus: Canelones: U = 11, P < 0.001; Colonia: U = 365.5, P < 0.005) (Fig. 2). There were no interspecific differences on infection intensity between females at Canelones (U = 314.5, P = 0.209) or between males at Colonia (U = 227.5, P = 0.312). There were differences between males at Canelones, being B. atratus the most infected (U = 57.5, P < 0.001) but sample size of B. bellicosus was low. At Colonia there were differences between females with B. atratus also having higher infection intensity (U = 915, P = 0.042). 4. Discussion We presented the detection of N. ceranae in native B. atratus and B. bellicosus from Uruguay. Since N. ceranae in A. mellifera is widely distributed in Uruguay since the nineties (Invernizzi et al., 2009; Anido et al., in press), bumblebees might have acquired spores through their close contact with honey bees like Plischuk et al. (2009) suggested for Argentina. Mean prevalence values of N. ceranae found in B. atratus and B. bellicosus (72% and 63%, respectively) are much higher than those reported in other countries for the same (Plischuk et al., 2009) or other bumblebee species (Li et al., 2012; Graystock et al., 2013; Fürst et al., 2014). The high prevalence could reflect the rapid spread of a recent invasion of N. ceranae to Uruguayan bumblebees. Nevertheless, high prevalence does not necessarily mean that an epizootic is occurring (Cordes et al., 2012) and alternatively, B. atratus and B. bellicosus may be natural reservoirs of the pathogen supporting high populations of N. ceranae. The location-specific differences found on prevalence indicate that not all populations may be equally affected as has already been suggested for other parasites in different bumblebee species (Cordes et al., 2012). The same pathogen (e.g. N. bombi or Crithidia spp.) could generate infection with different characteristics (prevalence or intensity) in different bumblebee species (Cordes et al., 2012). Here, interspecific differences were found in prevalence of N. ceranae mainly in females and there were also some interspecific differences on spore loads. On the other hand, species infection intensity was lower for males in both bumblebee species. Sex effect at field conditions have been reported for various parasites in several bumblebee species although there appears to be no general pattern (review in Ruiz-Gonzalez and Brown (2006), Murray et al. (2013)). The lower infection intensity found in males may be due to higher resistance that prevents infection growing further or to highly infected males
N. Arbulo et al. / Journal of Invertebrate Pathology 130 (2015) 165–168
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Fig. 1. Percentage of N. ceranae positive samples analyzed through PCR (prevalence) in Bombus atratus and B. bellicosus females (F) and males (M) from Montevideo, Canelones and Colonia. Numbers inside bars are sample size. Significance level of Chi square tests are show between bars of compared female/male pairs: ⁄: P < 0.05, ⁄⁄⁄: P < 0.001, ns: non-significant.
Fig. 2. Relative frequencies distribution of N. ceranae infection intensity in females (F) and males (M) of Bombus atratus and B. bellicosus in Montevideo, Canelones and Colonia. Numbers inside bars are sample size. 293 N. ceranae positive samples were included in this analysis because not all positive samples (N = 364) could be microscopically analyzed. Categories were defined as follows: very low (<12,500 spores), low (12,500 to 5 105 spores), medium (5 105 + 1 to 25 105 spores) and high (>25 105 spores). Significance level of Mann–Whitney U tests are show between bars of compared female/male pairs: ⁄⁄: P < 0.005, ⁄⁄⁄: P < 0.001, ns: non-significant.
dying early and so being under represented in the samples. Alternatively, differences between sexes could be due to different chances of getting infected associated to sex distinctive behavior (Murray et al., 2013): males leaving the nest permanently few days after emergence versus nest centered females that may be more exposed to parasite contaminated material. Data about N. ceranae infection effects on B. terrestris are alarming since it appears to be a highly virulent pathogen rapidly reducing bumblebees’ survival (Graystock et al., 2013). The presence of such a threatening pathogen in Uruguayan bumblebees may detrimental not only for bumblebee populations, but also to the rest of the native pollinator community and to honeybees.
Acknowledgments Authors would like to thank to Facultad de Agronomía, Familia Sánchez-Santos and INIA – La Estanzuela for allowing bumblebee collection at their facilities. Also thank to the staff of the Apiculture Section at INIA and Bruno Invernizzi for field assistance. The authors wish to thank two anonymous reviewers for their valuable comments and suggestions to improve the quality of the manuscript. N. Arbulo was partially supported by the Unidad de Apoyo Académico de la Comisión Coordinadora del Interior and the Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) of the Universidad de la República.
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References Anido, M., Branchiccela, B., Castelli, L., Harriet, J., Campá, J., Zunino, P., Antúnez, K., in press. Prevalence and distribution of honey bee pathogens in Uruguay. J. Apcultural Res. Chaimanee, V., Chen, Y., Pettis, J.S., Cornman, R.S., Chantawannakul, P., 2011. Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand. J. Invertebr. Pathol. 107, 229–233. Cordes, N., Huang, W.-F., Strange, J.P., Cameron, S.A., Griswold, T.L., Lozier, J.D., Solter, L.F., 2012. Interspecific geographic distribution and variation of the pathogens Nosema bombi and Crithidia species in United States bumble bee populations. J. Invertebr. Pathol. 109, 209–216. Durrer, S., Schmid-Hempel, P., 1994. Shared use of flowers leads to horizontal pathogen transmission. Proc. Biol. Sci. R. Soc. 258, 299–302. Fries, I., Feng, F., da Silva, A., Slemend, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356–365. Fürst, M.A., McMahon, D.P., Osborne, J.L., Paxton, R.J., Brown, M.J.F., 2014. Disease associations between honeybees and bumblebees as a threat to wild pollinators. Nature 506, 364–366. Graystock, P., Yates, K., Darevill, B., Goulson, D., Hughes, W.O.H., 2013. Emerging dangers: deadly effects of an emergent parasite in a new pollinator host. J. Invertebr. Pathol. 114, 114–119. Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honey bees in Europe. J. Invertebr. Pathol. 92, 93–95. Higes, M., Meana, A., Bartolomé, C., Botías, C., Martín-Hernández, R., 2013. Nosema ceranae (Microsporidia), a controversial 21st century honey bee pathogen. Environ. Microbiol. Rep. 5 (1), 17–29.
Invernizzi, C., Abud, C., Tomasco, I., Harriet, J., Ramallo, G., Campá, J., Katz, H., Gardiol, G., Mendoza, Y., 2009. Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay. J. Invertebr. Pathol. 101, 150–153. Li, J., Chen, W., Wu, J., Peng, W., An, J., Schmid-Hempel, P., Schmid-Hempel, R., 2012. Diversity of Nosema associated with bumblebees (Bombus spp.) from China. Int. J. Parasitol. 42 (1), 49–61. Martín-Hernández, R., Botías, C., Garrido Bailón, E., Martínez-Salvador, A., Prieto, L., Meana, A., Higes, M., 2012. Microsporidia infecting Apis mellifera: coexistence or competition. Is Nosema ceranae replacing Nosema apis? Environ. Microbiol. 14 (8), 2127–2138. Murray, T.E., Coffey, M.F., Kehoe, E., Horgan, F.G., 2013. Pathogen prevalence in commercially reared bumble bees and evidence of spillover in conspecific populations. Biol. Cons. 159, 269–276. Plischuk, S., Martín-Hernández, R., Prieto, L., Lucía, M., Botías, C., Meana, A., Abrahamovich, A.H., Lange, C., Higes, M., 2009. South American native bumblebees (Hymenoptera: Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera). Environ. Microbiol. Rep. 1 (2), 131–135. Ruiz-Gonzalez, M.X., Brown, M.J.F., 2006. Males vs workers: testing the assumptions of the haploid susceptibility hypothesis in bumblebees. Behav. Ecol. Sociobiol. 60, 501–509. Schmid-Hempel, P., 2001. On the evolutionary ecology of host-parasite interactions: addressing the question with regard to bumblebees and their parasites. Naturwissenschaften 88, 147–158. Tay, W.T., O’Mahony, E.M., Paxton, R.J., 2005. Complete rRNA gene sequences reveal that the microsporidium Nosema bombi infects diverse bumblebee (Bombus spp.) hosts and contains multiple polymorphic sites. J. Eukaryot. Microbiol. 52 (6), 505–513.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
High prevalence and infection levels of Nosema ceranae in bumblebees Bombus atratus and Bombus bellicosus from Uruguay N. Arbulo a,⇑, K. Antúnez b, S. Salvarrey c, E. Santos c, B. Branchiccela b, R. Martín-Hernández d, M. Higes d, C. Invernizzi c a
Centro Universitario Regional del Este, Universidad de la República, Rocha, Uruguay Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay Sección Etología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay d Centro Apícola Regional, Junta de Comunidades de Castilla La Mancha, 19180 Marchamalo, Spain b c
a r t i c l e
i n f o
Article history: Received 20 January 2015 Revised 16 July 2015 Accepted 31 July 2015 Available online 3 August 2015 Keywords: Microsporidia Parasites Nosemosis
a b s t r a c t Nosema ceranae is one of the most prevalent pathogens in Apis mellifera and has recently been found in multiple host species including several species of bumblebees. Prevalence and infection intensity of N. ceranae was determined in two species of native bumblebees from Uruguay. Nosema ceranae was the only microsporidia identified and mean prevalence was 72% in Bombus atratus and 63% in Bombus bellicosus, values much higher than those reported elsewhere. The presence of this pathogen in bumblebees may be threatening not only for bumblebee populations, but also to the rest of the native pollinator community and to honeybees. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Nosema ceranae is one of the most prevalent pathogens in Apis mellifera worldwide (Higes et al., 2013). Originally described in Apis cerana (Fries et al., 1996), in 2006 was found as an emergent pathogen in the European honey bee (Higes et al., 2006) and currently it is found in multiple host species (Plischuk et al., 2009; Chaimanee et al., 2011; Li et al., 2012; Graystock et al., 2013). This wide host range is of significant epidemiological and ecological concern, since some parasite can be transmitted between host individuals that share flower resources (Durrer and Schmid-Hempel, 1994). Bumblebees host a great variety of parasites (Schmid-Hempel, 2001). Until the work of Plischuk et al. (2009), Nosema bombi was the only microsporidia known to infect bumblebees (Tay et al., 2005). At present, N. ceranae has been detected in three South American (Plischuk et al., 2009), four Asian (Li et al., 2012) and seven European (Graystock et al., 2013) species. Recent studies confirmed that bumblebees can be genuinely infected by N. ceranae and may not be simply accidental carriers of its spores (Graystock et al., 2013; Fürst et al., 2014).
⇑ Corresponding author at: Centro Universitario Regional del Este – Sede Rocha, Ruta Nacional N° 9 intersección Ruta Nacional N° 15, Rocha 27000, Uruguay. E-mail address: [email protected] (N. Arbulo). http://dx.doi.org/10.1016/j.jip.2015.07.018 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.
The aim of this study was to determine the presence, prevalence and infection intensity of N. ceranae in Bombus atratus and Bombus bellicosus from Uruguay. 2. Methods 2.1. Bumblebee collection A total of 526 bumblebees, 317 B. atratus (167 females, 150 males) and 209 B. bellicosus (141 females, 68 males), were collected in April 2010 while foraging on red clover (Trifolium pratense) in three locations: Montevideo (34°500 S, 56°130 W), Canelones (34°400 S, 56°020 W) and Colonia (34°200 S, 57°410 W). Individual bees were collected directly from flowers and were stored at 20 °C until analysis. 2.2. Identification of Nosema species Abdomens of bumblebees were individually macerated with 2 ml of 95° ethanol. Macerates were centrifuged and washed to remove ethanol and pellets were resuspended in 250 ll of sterile distilled water. One hundred and fifty ll of each sample were introduced into a 96-well plate (Qiagen) with glassbeads (2 mm, Sigma) and plates were shaken for 6 min at 30 Hz. Thirty ll of ATL buffer (Qiagen 19076) and 20 ll of Proteinase K (Qiagen 19131) were added to each well and plates were incubated
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overnight at 56 °C. Subsequently, DNA was extracted using the BS96 DNA Tissue extraction protocol in a BioSprint apparatus (Qiagen). The presence of N. ceranae and N. apis was determined using Nosema Biotools DNA Polymerase plates containing PCR triplex (321-APIS-F/R, 218-MITOC-F/R and COI-F/R primers; MartínHernández et al., 2012) master mix. Reactions were carried out in a total volume of 50 ll and the cycling program was: 94 °C (6 min); 35 cycles of 30 s denaturation at 94 °C, 45 s annealing at 61.8 °C and 60 s extension at 72 °C; and a final extension step at 72 °C for 10 min. PCR for N. bombi detection was performed in a total volume of 25 ll using the Fast Start PCR Master mix (No. 04710452001 Roche Diagnostic), 0.4 mM of each primer, 0.2 mg ml-1 BSA, 0.1% Triton X-100 and 2.5 ll of DNA template. The cycling program was: 95 °C (10 min); 35 cycles of 30 s denaturation at 95 °C, 30 s annealing at 58.1 °C and 45 s extension at 72 °C; and a final extension step at 72 °C for 7 min. Negative controls for DNA extraction and PCR were analyzed in parallel. PCR reactions were carried out in a MastercyclerÒ ep gradient S apparatus (Eppendorf) in Centro Apícola Regional de Marchamalo (Spain). PCR products were analyzed in a QIAxcel System (Qiagen) using a QIAxcel DNA High Resolution Kit (Qiagen, No. 929002). Prevalence was calculated as the percentage of positive samples for each analyzed group. Chi square test with a 0.05 significance level was used to evaluate intraspecific differences between sexes and interspecific differences within sexes. 2.3. Spore counting Infection intensity (spores per bumblebee) was determined in positive samples. Spore count was done using a haemocytometer and a light microscope at 400. Number of spores in known volume was used to calculate spores per bumblebee. Spore detection limit was calculated at 12,500 spores per bumblebee, which corresponded to finding one spore in the total volume analyzed. Infection intensity categories were defined for the analysis: very low (<12,500 spores), low (12,500 to 5 105 spores), medium (5 105 + 1 to 25 105 spores) and high (more than 25 105 spores). The Mann–Whitney U test with a 0.05 significance level, was performed to compare infection level between the analyzed groups. 3. Results 3.1. Nosema identification and prevalence A total of 364 samples were positive for N. ceranae which was the only microsporidia species detected. Prevalence was equal or higher than 50% in different locations and sexes, with two exceptions (Fig. 1). There were intraspecific differences on prevalence between location (B. atratus: females v2 = 3.40, P = 0.182, males v2 = 20.81, P < 0.001; B. bellicosus: females v2 = 11.35, P < 0.001, males v2 = 5.40, P = 0.02) so the analysis was performed for each location separately. Prevalence in B. atratus was higher in females (Montevideo: v2 = 35.26, P < 0.001; Canelones: v2 = 7.86, P = 0.005; Colonia: v2 = 19.32, P < 0.001) while in B. bellicosus there was no difference between sexes (Canelones: v2 = 0.02, P = 0.882; Colonia: v2 = 0.03, P = 0.856) (Fig. 1). Prevalence in females was higher in B. atratus than in B. bellicosus (Canelones: v2 = 24.80, P < 0.001; Montevideo:
v2 = 6.46, P = 0.011). Regarding males, N. ceranae was more prevalent in B. atratus at Canelones (v2 = 5.36, P = 0.021) but there was no interspecific difference at Colonia (v2 = 3.46, P = 0.063). 3.2. N. ceranae infection intensity Infection intensity was determined in 293 N. ceranae positive samples. Not all positive samples (N = 364) could be microscopically analyzed due to insufficient amount of material left in 71 samples after molecular analyses. Spores were detectable in 246 samples (84%). Infection intensity reached loads as high as 44 million spores per individual. In 47 samples (16%) spores could not be detected microscopically, indicating that the number of spores was under the detection limit (12,500 spores per bumblebee). Infection intensity differed between sexes, being higher in females in both species (B. atratus: Montevideo: U = 133, P < 0.001; Canelones: U = 733.5, P < 0.001; Colonia: U = 164.5, P < 0.001. B. bellicosus: Canelones: U = 11, P < 0.001; Colonia: U = 365.5, P < 0.005) (Fig. 2). There were no interspecific differences on infection intensity between females at Canelones (U = 314.5, P = 0.209) or between males at Colonia (U = 227.5, P = 0.312). There were differences between males at Canelones, being B. atratus the most infected (U = 57.5, P < 0.001) but sample size of B. bellicosus was low. At Colonia there were differences between females with B. atratus also having higher infection intensity (U = 915, P = 0.042). 4. Discussion We presented the detection of N. ceranae in native B. atratus and B. bellicosus from Uruguay. Since N. ceranae in A. mellifera is widely distributed in Uruguay since the nineties (Invernizzi et al., 2009; Anido et al., in press), bumblebees might have acquired spores through their close contact with honey bees like Plischuk et al. (2009) suggested for Argentina. Mean prevalence values of N. ceranae found in B. atratus and B. bellicosus (72% and 63%, respectively) are much higher than those reported in other countries for the same (Plischuk et al., 2009) or other bumblebee species (Li et al., 2012; Graystock et al., 2013; Fürst et al., 2014). The high prevalence could reflect the rapid spread of a recent invasion of N. ceranae to Uruguayan bumblebees. Nevertheless, high prevalence does not necessarily mean that an epizootic is occurring (Cordes et al., 2012) and alternatively, B. atratus and B. bellicosus may be natural reservoirs of the pathogen supporting high populations of N. ceranae. The location-specific differences found on prevalence indicate that not all populations may be equally affected as has already been suggested for other parasites in different bumblebee species (Cordes et al., 2012). The same pathogen (e.g. N. bombi or Crithidia spp.) could generate infection with different characteristics (prevalence or intensity) in different bumblebee species (Cordes et al., 2012). Here, interspecific differences were found in prevalence of N. ceranae mainly in females and there were also some interspecific differences on spore loads. On the other hand, species infection intensity was lower for males in both bumblebee species. Sex effect at field conditions have been reported for various parasites in several bumblebee species although there appears to be no general pattern (review in Ruiz-Gonzalez and Brown (2006), Murray et al. (2013)). The lower infection intensity found in males may be due to higher resistance that prevents infection growing further or to highly infected males
N. Arbulo et al. / Journal of Invertebrate Pathology 130 (2015) 165–168
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Fig. 1. Percentage of N. ceranae positive samples analyzed through PCR (prevalence) in Bombus atratus and B. bellicosus females (F) and males (M) from Montevideo, Canelones and Colonia. Numbers inside bars are sample size. Significance level of Chi square tests are show between bars of compared female/male pairs: ⁄: P < 0.05, ⁄⁄⁄: P < 0.001, ns: non-significant.
Fig. 2. Relative frequencies distribution of N. ceranae infection intensity in females (F) and males (M) of Bombus atratus and B. bellicosus in Montevideo, Canelones and Colonia. Numbers inside bars are sample size. 293 N. ceranae positive samples were included in this analysis because not all positive samples (N = 364) could be microscopically analyzed. Categories were defined as follows: very low (<12,500 spores), low (12,500 to 5 105 spores), medium (5 105 + 1 to 25 105 spores) and high (>25 105 spores). Significance level of Mann–Whitney U tests are show between bars of compared female/male pairs: ⁄⁄: P < 0.005, ⁄⁄⁄: P < 0.001, ns: non-significant.
dying early and so being under represented in the samples. Alternatively, differences between sexes could be due to different chances of getting infected associated to sex distinctive behavior (Murray et al., 2013): males leaving the nest permanently few days after emergence versus nest centered females that may be more exposed to parasite contaminated material. Data about N. ceranae infection effects on B. terrestris are alarming since it appears to be a highly virulent pathogen rapidly reducing bumblebees’ survival (Graystock et al., 2013). The presence of such a threatening pathogen in Uruguayan bumblebees may detrimental not only for bumblebee populations, but also to the rest of the native pollinator community and to honeybees.
Acknowledgments Authors would like to thank to Facultad de Agronomía, Familia Sánchez-Santos and INIA – La Estanzuela for allowing bumblebee collection at their facilities. Also thank to the staff of the Apiculture Section at INIA and Bruno Invernizzi for field assistance. The authors wish to thank two anonymous reviewers for their valuable comments and suggestions to improve the quality of the manuscript. N. Arbulo was partially supported by the Unidad de Apoyo Académico de la Comisión Coordinadora del Interior and the Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) of the Universidad de la República.
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