Presence of Nosema ceranae associated with honeybee queen introductions

Infection, Genetics and Evolution 23 (2014) 161–168

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Presence of Nosema ceranae associated with honeybee queen introductions Irene Muñoz a, Almudena Cepero b, Maria Alice Pinto c, Raquel Martín-Hernández b,d, Mariano Higes b, Pilar De la Rúa a,⇑ a Área de Biología Animal, Dpto. de Zoología y Antropología Física, Facultad de Veterinaria, Campus de Excelencia Regional ‘‘Campus Mare Nostrum’’, Universidad de Murcia, 30100 Murcia, Spain b Centro Apícola Regional, Consejería de Agricultura, Gobierno de Castilla-La Mancha, 19180 Marchamalo, Spain c Mountain Research Centre (CIMO), Polytechnic Institute of Bragança, Campus de Sta. Apolónia, Apartado 1172, 5301-855 Bragança, Portugal d Instituto de Recursos Humanos para la Ciencia y la Tecnología (INCRECYT), Fundación Parque Científico y Tecnológico de Albacete, Spain

a r t i c l e

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Article history: Received 24 May 2013 Received in revised form 17 January 2014 Accepted 7 February 2014 Available online 22 February 2014 Keywords: Apis mellifera Nosema spp. Queen introduction Conservation

a b s t r a c t Microsporidiosis caused by Nosema species is one of the factors threatening the health of the honeybee (Apis mellifera), which is an essential element in agriculture mainly due to its pollination function. The dispersion of this pathogen may be influenced by many factors, including various aspects of beekeeping management such as introduction of queens with different origin. Herein we study the relation of the presence and distribution of Nosema spp. and the replacement of queens in honeybee populations settled on the Atlantic Canary Islands. While Nosema apis has not been detected, an increase of the presence and distribution of Nosema ceranae during the last decade has been observed in parallel with a higher frequency of foreign queens. On the other hand, a reduction of the number of N. ceranae positive colonies was observed on those islands with continued replacement of queens. We suggest that such replacement could help maintaining low rates of Nosema infection, but healthy queens native to these islands should be used in order to conserve local honeybee diversity. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Honeybees (Apis mellifera Linnaeus 1758) are essential components to modern worldwide agriculture and ecosystems providing pollination services to crops and wild plants (Klein et al., 2007; Jaffé et al., 2010; Potts et al., 2010a), but at present their health is challenged on many fronts (Moritz et al., 2010) and economic yield of crops may suffer a drastic reduction (Gallai et al., 2009). Parasites such as varroa mites (Varroa destructor), honeybee tracheal mites (Acarapis woodi), fungal (Ascosphaera spp., Nosema spp.), bacterial and viral diseases, kleptoparasites such as small hive beetles (Aethina tumida) and pesticides are dangers faced by beekeepers (vanEngelsdorp et al., 2008; Moritz et al., 2010). There is evidence for several regional declines in honeybee colonies in the US (the average winter losses were 32.6% from 2007 to 2010; vanEngelsdorp et al., 2007, 2008, vanEngelsdorp and Meixner, 2010) and Europe (25% loss of colonies between 1985 and 2005, Potts et al., 2010b). Multiple factors have been proposed as triggers for colony losses and several national programs, such as the ⇑ Corresponding author. Tel.: +34 868 884908; fax: +34 868 884906. E-mail address: [email protected] (P. De la Rúa). http://dx.doi.org/10.1016/j.meegid.2014.02.008 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.

German Honeybee Monitoring or the U.S. research team on Colony Collapse Disorder (CCD) have been initiated to collect data on honeybee mortality, to quantify this phenomenon and to try identifying the causes (Cox-Foster et al., 2007; Bacandritsos et al., 2010; Bromenshenk et al., 2010; Genersch et al., 2010; Giray et al., 2010; Higes et al., 2010a,b; Hatjina et al., 2011; Soroker et al., 2011). Microsporidiosis caused by Nosema spp. is one of the factors related with colony losses (Martín-Hernández et al., 2007; Higes et al., 2008), causing significant economic impacts to beekeepers worldwide (vanEngelsdorp et al., 2008; Giersch et al., 2009; vanEngelsdorp and Meixner, 2010; Heintz et al., 2011). Microsporidia are obligatory intracellular parasites (Martin, 2001; Corradi and Keeling, 2009) of all major animal lineages, including insects and other invertebrates (Larsson, 1986) but are also parasites of vertebrates including humans (Canning and Lom, 1986; Weber et al., 1994; Becnel and Andreadis, 1999). Currently, two microsporidian species have been described infecting the ventricular epithelium of honeybees: Nosema apis Zander (1909) and Nosema ceranae Fries et al. (1996). N. apis was first described in Europe 100 years ago, and its effects have been widely documented since then (Bailey and Ball, 1991). However, N. ceranae has been recently

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identified on A. mellifera and now represents a major disease in colonies from temperate areas (Higes et al., 2008, 2010a; Heintz et al., 2011; Martín-Hernández et al., 2007, 2012). N. ceranae was described on Asian honeybees (Apis cerana) but has been detected in several geographically distant honeybee populations in Europe (Higes et al., 2006; Fries et al., 2006; Chauzat et al., 2007; Paxton et al., 2007; Tapaszti et al., 2009; Fries, 2010; Stevanovic et al., 2010; Whitaker et al., 2010), America (Klee et al., 2007; Calderón et al., 2008; Chen et al., 2008; Williams et al., 2008; Invernizzi et al., 2009; Guzmán-Novoa et al., 2011; Traver and Fell, 2011; Medici et al., 2012; Martínez et al., 2012), Asia (Huang et al., 2005, 2007; Chaimanee et al., 2011; Yoshiyama and Kimura, 2011), Africa (Higes et al., 2009) and Oceania (Giersch et al., 2009; Botías et al., 2012a). Infestations of either Nosema spp. result in decreased honey production and foraging activity, yielding reduced pollination productivity (Botías et al., 2012b). The presence of parasites and pathogens may be influenced by many factors, including beekeeping practices, host genetic variation or climate (Fenoy et al., 2009; Fries, 2010; Gisder et al., 2010; Botías et al., 2011; Jara et al., 2012; Martín-Hernández et al., 2009, 2012; Fontbonne et al., 2013). For example, queen replacement decreases the proportion of Nosema infected honeybees and maintains the overall infection at a level compatible with colony viability (Botías et al., 2012b), but it is also known that the distribution of subspecies such as Apis mellifera ligustica, A. m. carnica and A. m. caucasica by means of the importation of honeybee queens, favors the spread of parasites, pathogens and diseases (Mutinelli, 2011). In this sense, N. ceranae has been also detected in colonies of the worldwide distributed subspecies A. m. ligustica and A. m. carnica (Italian and Carniolan honeybees, respectively; Klee et al., 2007). From previous work, we know that introduction of honeybee queens of European origin is taking place on the Canary Islands (Muñoz and De la Rúa, 2012; Muñoz et al., 2013). Specifically, the presence at high frequency of foreign queens has been inferred from the detection of mitochondrial DNA (mtDNA) haplotypes, named C1 and C2, that characterize Italian and Carniolan honeybees, respectively (East and Central European honeybees), and M7 typical of West European honeybee populations as northern A. m. iberiensis and A. m. mellifera (Miguel et al., 2007) but also present in A. m. ligustica (Franck et al., 2000). These haplotypes are considered as foreign because through the characterization of the mitochondrial DNA chromosome (De la Rúa et al., 1998, 2001), it was confirmed that local Canarian honeybees harbor geographically limited mitochondrial haplotypes of African origin (e.g. A11, A14, A15 and A16). These haplotypes are absent in western and eastern Europe, rarely found in populations from Spain and Africa (Franck et al., 2001; De la Rúa et al., 2007; Miguel et al., 2007; Cánovas et al., 2008) and more commonly detected in Portugal (Pinto et al., 2012, 2013). While population genetic studies on Canarian honeybees are abundant (De la Rúa et al., 1998, 2001, 2002; Muñoz and De la Rúa, 2012; Muñoz et al., 2012, 2013, 2014) pathology studies are scarce and increasingly sought as colony diseases are one of the most important impediment to beekeeping activity. The islands provide an interesting scenario in which to analyze the correlation between the presence of pathogens and the level of importation of foreign honeybee queens because it is a confined environment. Here, we performed a spatial and temporal analysis of the presence and abundance of Nosema spp. on island honeybee populations from the Canarian archipelago corresponding to surveys conducted in 1998, 2008, 2010 and 2011. These data have been compared with the level of honeybee queen introductions observed on each island as inferred from mtDNA variation. We hypothesized that the islands with a higher level of foreign honeybee queens would exhibit a higher level of pathogen-positive col-

onies, since imported queens may have been infected and established directly into the local apiaries without an adequate quarantine period. This event could modify the gene pool of honeybees native to these islands since foreign colonies may have greater tolerance or resistance traits as they have been exposed to these pathogens for longer time. Alternatively, if queen replacements with (preferably) local queens are performed yearly, the proportion of Nosema-infected colonies will be maintained through time, as suggested by Botías et al. (2012b). 2. Material and methods 2.1. Honeybee samples Samples were collected in honeybee colonies from the following Canary Islands: La Palma, El Hierro, La Gomera, Tenerife and Gran Canaria (Fig. 1). Beekeeping in Canary Islands shows two trends: more intensive and professional on Tenerife and Gran Canaria where the number of hives in the last twenty years ranged from 5000 to 10,000, and other more traditional on La Palma, El Hierro and La Gomera with 500–2000 colonies per island (data from the Regional Government of the Canary Islands). A total of 151 colonies (Table 1 in Supplementary Data) were sampled between spring and summer from 1998 to 2011. To avoid re-sampling error different colonies and apiaries (from different beekeepers) were sampled during the two surveys. Samples analyzed for detecting Nosema spp. included in the earlier survey of 1998 correspond to a subset of the colonies analyzed by De la Rúa et al. (2001) and those from 2008, 2010 and 2011 includes a subset of the Macaronesian samples colonies by Muñoz et al. (2013). Subsets were used due to the limited number of worker honeybees available for the adequate molecular determination of Nosema spp. From each colony, worker honeybees were taken from the inner frames in order to avoid the sampling of drifting bees (i.e. worker honeybees that could come from another colony) and stored in absolute ethanol at –20 °C until laboratory processing. 2.2. Detection of Nosema spp. Worker and drone honeybees from each colony (see Table S1 in Supplementary Data for more detailed information) were pooled and macerated in 5 ml H2O PCR grade for 2 min at high speed in a StomacherÒ 80 Biomaster (Seward, West Sussex, UK) using strainer bags (BA6040/STR, Seward). The macerate was recovered in a tube and centrifuged for 6 min at 800g. Then the supernatant was discarded and the pellet re-suspended in 1 mL of distilled water. Subsequently, 500 lL were homogenized by shaking with glass beads (2 mm of diameter, Sigma) in the TissueLyser (Qiagen) for 6 min at 30 Hz. DNA extractions were performed using BS96 DNA Tissue extraction protocol in a BioSprint workstation (Qiagen) according to manufacturer’s instructions. PCR reactions were carried out in a MastercyclerÒ ep gradient S apparatus (Eppendorf) and performed as described previously using an internal PCR control to determine the reliability of analysis (Botías et al., 2011; Martín-Hernández et al., 2012). PCRs for Nosema spp. detection were carried out in 25 lL reaction volumes with Hotsplit DNA Polymerase in gel form (BIOTOOLSÒ) containing 2.5 lL of DNA template, 200 lM total dNTP, 1 Reaction Buffer, 1 U/rxn Hotsplit DNA Polymerase, 2 mM MgCl2, 0.4 lM of each primer for Nosema spp, (321-APIS-F/R and 218-MITOC-F/R), 0.03 lM of COI-F/R primers (Martín-Hernández et al., 2007, 2012). The thermocycler program used was: 95 °C (10 min); 35 cycles of a 30 s denaturation at 95 °C, a 30 s elongation at 61.8 °C, a 45 s extension at 72 °C; and a final extension step at 72 °C for 7 min. Negative controls (for DNA extractions and PCRs) were included in every PCR

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Fig. 1. Map showing the islands sampled in the Macaronesian region.

Table 1 Percentage per island and survey of evolutionary sub-lineages and lineages, foreign queens and N. ceranae presence with confidence interval (CI, 95%) in Canarian colonies (N = number of colonies analyzed). Island

Survey

N

Sub-lineages and lineages AI

Foreign queens (%)

AIII

Presence (%)

CI (95%)

2008

9

11.11

0.00

44.44

0.00

44.44

44.44

11.11

15.0–37.0

Gran Canaria

1998 2008 2010 2011

5 10 14 19

0.00 10.00 21.43 26.32

0.00 0.00 14.29 0.00

100.00 70.00 57.14 52.63

0.00 20.00 0.00 21.05

0.00 0.00 7.14 0.00

0.00 20.00 7.14 21.05

0.00 60.00 14.29 73.68

0.0–0.0 23.0–97.0 7.0–35.0 52.0–95.0

La Gomera

1998 2008 2010

4 6 14

0.00 33.33 21.43

0.00 0.00 0.00

100.00 0.00 42.86

0.00 0.00 0.00

0.00 67.67 35.71

0.00 67.67 35.71

0.00 0.00 7.14

0.0–0.0 0.0–0.0 8.0–23.0

El Hierro

1998 2008 2011

4 7 26

0.00 0.00 0.00

0.00 0.01 0.00

100.00 57.14 61.54

0.00 0.00 0.00

0.00 42.86 38.46

0.00 42.86 38.46

0.00 0.00 30.77

0.0–0.0 0.0–0.0 12.0–50.0

La Palma

1998 2008 2010

5 12 16

80.0 41.67 37.50

0.00 0.00 0.00

20.0 41.67 56.25

0.00 0.00 0.00

0.00 16.67 6.25

0.00 16.67 6.25

0.00 75.00 68.75

0.0–0.0 46.0–104.0 43.0–94.0

151

19.87

1.32

54.97

3.97

19.87

23.84

34.44

27.0–42.0

experiment. PCR products were analyzed in a QIAxcel System (Qiagen) using a QIAxcelDNA High Resolution Kit (Qiagen, No. 929002). 2.3. Genetic determination of honeybee queen maternal origin The origin of the honeybee queens is based on the sequence variation of a mitochondrial non-coding A + T-rich region located between the tRNAleu and cox2 genes (Garnery et al., 1993). Two types of components are present in this intergenic region named P and Q (Cornuet and Garnery, 1991). The sequence P has three forms with different size based on the presence of several indels: P with 54 bp (base pairs), Po with 67 bp, or P1 with 50 bp. The sequence Q is 194–196 bp long and can be repeated up to four times within this intergenic region. The combination and number of these compo-

M

C

N. ceranae

Tenerife

Total

AII

nents is characteristic of the four evolutionary lineages of A. mellifera subspecies and the three African sub-lineages (see Fig. S1 in Supplementary Data). In this sense the subspecies from eastern and central Europe (the globally imported A. m. ligustica and A. m. carnica among others) included in the C lineage present only one Q copy and none P sequence. The combination with P and up to four Q sequences is typical of subspecies from the western Europe M lineage as A. m. mellifera and northern A. m. iberiensis, whereas African subspecies and southern A. m. iberiensis populations (A lineage) present also from one to four Q sequences but a Po sequence. The sub-lineage of the African lineage with Atlantic distribution (sub-lineage AIII), including the Macaronesian (De la Rúa et al., 1998, 2001, 2006) and the North Portuguese populations (Pinto et al., 2012, 2013), is characterized by the P1 sequence and

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also up to four Q sequences (reviewed by De la Rúa et al., 2009). There are other African sub-lineages named AI and AII that show different geographic distribution (and also haplotype frequency): AI is mainly present in south-western Iberian Peninsula (Cánovas et al., 2008), some Mediterranean islands and sub-Saharan Africa whereas AII is dominant in northern Africa but also present at low frequency in Iberia (Franck et al., 2001). Given the well-established native distribution of lineages A, M, and C and African sublineages, in this study queens were classified as foreign if they were of C or M lineage maternal ancestry and native if they were of African ancestry. While introduced African-derived queens from the mainland Iberia would be equally foreign, the maternal test implemented herein is unable to distinguish the native Macaronesian colonies from the Iberian colonies. A PCR–RFLP (restriction fragment length polymorphism) approach allows determining the combined length and sequence variation of the intergenic region, hence discriminates the three A, M and C lineages and African AI, AII, AIII sub-lineages. Furthermore, this approach has revealed two C and many A and M haplotypes within these lineages due to the combination of length and restriction patterns (Garnery et al., 1993; De la Rúa et al., 1998; Franck et al., 2001; Rortais et al., 2011; Pinto et al., 2012). Each haplotype bear by each worker and each drone is directly inherited from the queen; therefore one worker honeybee per colony was used for mtDNA identification of queens governing the colonies. Haplotype determination was achieved following standard methods (Evans et al., 2013). Briefly, DNA was extracted from a pair of legs using the ChelexÒ method (Walsh et al., 1991). The intergenic tRNAleu-cox2 region was PCR-amplified in a thermocycler PTC 100 (MJ Research) in a total volume of 12.5 lL with KapaTaq DNA Polymerase (KAPA BIOSYSTEMS), containing 2 lL of DNA template, 200 lM total dNTP, 1 Reaction Buffer, 0.5 U/rxn KapaTaq DNA Polymerase, 1.5 mM MgCl2, 0.4 lM of each primer (E2 and H2, Garnery et al., 1993). The thermocycler program used was: 94 °C (5 min); 35 cycles of a 45 s denaturation at 94 °C, a 45 s elongation at 48 °C, a 60 s extension at 62 °C; and a final extension step at 65 °C for 20 min. The size of the PCR-amplified product was determined by electrophoresis in a 1.5% agarose gel. The remaining 10 lL were digested with the restriction enzyme DraI (with recognition site 50 -TTTAAA-30 ) at 37 °C overnight. Restricted fragments were electrophoresed on 4% NuSieve agarose gels and the restriction patterns were observed with UV light and recorded for determination. Haplotypes were determined by comparison with patterns previously described.

2.4. Statistical analyses Since only N. ceranae was detected, the statistical analyses were performed to evaluate the temporal and spatial variation of this pathogen. 95% confidence intervals for the average of N. ceranae prevalence were determined by bootstrapping. The Pearson’s X2 test and one-way analysis of variance (ANOVA) with the Bonferroni post hoc method were used to compare the ‘‘presence of N. ceranae’’ between surveys, years and islands. Pearson’s correlation coefficient was calculated between the variables ‘‘evolutionary lineage or sub-lineage of the island honeybees’’, ‘‘haplotypes of the island honeybees’’ and ‘‘presence of N. ceranae’’ to explore the relationships between variables. Spearman’s correlation coefficient (rs) was calculated between the average percentage of colonies were N. ceranae was detected and average percentage of colonies with foreign queens and a linear regression analysis was performed in order to detect statistically significant linear dependence. The statistical analyses were carried out using SPSS software version 19.0.0 and the differences were considered statistically significant when a = 0.05.

3. Results We aimed at detecting and discriminating Nosema spp. in honeybee colonies from the Canary Islands sampled over a decade. While N. apis was not detected, N. ceranae was found in 52 out of the 151 colonies sampled (34.44%, Table 1). In the earlier survey from 1998, N. ceranae was not detected in any colony (although this observation is based on a reduced number of colonies from the historical sampling), whereas in the later survey (2008–2011) the microsporidium was present on every island reaching 75% of the colonies from La Palma in 2008. The average frequency of N. ceranae-positive colonies increased significantly (Pearson X2 = 13.823, P = 0.003) during 1998–2011 (Fig. 2). When the temporal change was analyzed on every Canary Island, a significant increase in the prevalence of N. ceranae during the last decade was detected on Gran Canaria (ANOVA, F = 7.745, P < 0.001) and La Palma (ANOVA, F = 5.779, P = 0.008). In relation to the evolutionary origin of the colonies 30 (19.87%) of them belongs to the C evolutionary lineage (eastern Europe), as they showed the shortest amplicon length corresponding to the presence of only one Q element. To distinguish western Europe (M) from African (A) haplotypes, the amplicon was digested with the DraI enzyme and the composition and restriction patterns (Fig. S1 in Supplementary Data) were used to define the haplotypes and corresponding evolutionary lineage (M or A) and African sublineage (AI, AII, AIII). An overall number of 14 mitochondrial haplotypes were detected (Table S1 in Supplementary Data) that grouped 23.84% of the sampled colonies into the European M and C lineages and 76.16% into the three African sub-lineages. Specifically, Gran Canaria was the only island where the three lineages A, C and M co-occurred, while in the remaining islands both A and C lineages were observed. The African sublineage with Atlantic distribution was the most frequent and widespread sublineage detected in every year and island. We considered that colonies showing either M or C haplotypes correspond to honeybee queens recently introduced on the Canary Islands. The C lineage was not observed in 1998 but was detected in every later survey whereas the M lineage was only detected in 2008 and 2011 (Fig. 3). Overall, a significant increase of colonies (Pearson X2 = 11.015, P = 0.012) with introduced queens was detected between the earlier (1998) and the later (2008–2011) surveys. Temporal analysis revealed a significant relationship between evolutionary lineage and presence of N. ceranae in 2011 (Pearson X2 = 8.243, P = 0.041). An increase of N. ceranae-positive colonies belonging to the African sub-lineage AIII was detected that year, since 40.91% of the 22 positive colonies for N. ceranae showed haplotypes characteristic to belonging to this sub-lineage. The percentage of foreign queens and N. ceranae-positive colonies in the

Fig. 2. Mean values of N. ceranae-positive Canarian honeybee colonies per sampled year.

I. Muñoz et al. / Infection, Genetics and Evolution 23 (2014) 161–168

Fig. 3. Percentage of African sub-lineages (AI, AII and AIII) and foreign lineages (Western M and Eastern C evolutionary lineages) in the Canarian honeybee colonies per year. N = number of sampled colonies.

present survey (years 2008, 2010 and 2011) showed that islands with a higher percentage of colonies with foreign queens had significantly lower N. ceranae presence (rs = –0.711, P = 0.021) and the regression analysis showed that the presence of N. ceranae is negatively associated with the abundance of foreign queens (R2 = 0.469, Fig. 4).

4. Discussion This study provides the first temporal and spatial description of the presence of Nosema spp. on the Canary Islands. In this region, during the last decade, N. apis has not been detected in the analyzed colonies, in contrast with N. ceranae which has being increas-

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ingly dispersed and observed in recent years. These data on the increased frequency of N. ceranae-positive colonies agree with its known worldwide distribution and tendency (Huang et al., 2005, 2007; Higes et al., 2006, 2009; Fries et al., 2006; Chauzat et al., 2007; Klee et al., 2007; Paxton et al., 2007; Calderón et al., 2008; Chen et al., 2008; Williams et al., 2008; Giersch et al., 2009; Invernizzi et al., 2009; Tapaszti et al., 2009; van Engelsdorp et al., 2009; Fries, 2010; Stevanovic et al., 2010; Whitaker et al., 2010; Chaimanee et al., 2011; Guzmán-Novoa et al., 2011; Traver and Fell, 2011; Yoshiyama and Kimura, 2011; Medici et al., 2012; Botías et al., 2012a; Martínez et al., 2012). Seemingly, N. ceranae was accidently introduced to the Canarian islands by imported honeybees queens and it was established during the last decade. In contrast, N. apis has not been introduced yet or alternatively its spreading appears to be limited. Differences in the distribution of both Nosema spp. are consistent with the preferences for different climatic conditions observed in other studies (Fenoy et al., 2009; Fries, 2010; Gisder et al., 2010; Botías et al., 2011; Martín-Hernández et al., 2009, 2012). While N. apis infections have been associated with colder climates, N. ceranae appears to dominate in warmer climates (Fries, 2010; Gisder et al., 2010), which could determine the higher biotic potential of N. ceranae at different temperatures (Fenoy et al., 2009; Martín-Hernández et al., 2007, 2009). In the Canarian region, the tropical climate (Fernández-Palacios et al., 2011) seems to favor the distribution of N. ceranae since the microsporidium has dispersed on every island. The presence of both Western (M) and Eastern (C) European evolutionary lineages is evidence for the introduction of foreign queens into the Canary Islands (De la Rúa et al., 2001, 2002; Muñoz and De la Rúa, 2012; Muñoz et al., 2013). In the current study a significant increase of foreign queens has been detected during the time period analyzed. These honeybee queens have different geographic origin as depicted from the distinct haplotypes detected.

Fig. 4. Correlation of N. ceranae presence with the percentage of foreign queen in 2008–2011 survey. Name codes correspond to: Tenerife-2008 (TF08), Gran Canaria-2008 (GC08), Gran Canaria-2010 (GC10), Gran Canaria-2011 (GC11), La Gomera-2008 (GO08), La Gomera-2010 (GO10), El Hierro-2008 (HI08), El Hierro-2011 (HI11), La Palma2008 (LP08) and La Palma-2010 (LP10).

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In this sense, C1 is more frequently bear by A. m. ligustica honeybees from the Italian Peninsula, and C2 has been observed in the Balkan Peninsula (Muñoz et al., 2009) where the Carniolan honeybee (A. m. carnica) is present. The M haplotypes (M4, M4’ and M7) are typical of West European honeybee populations, as northern A. m. iberiensis (Cánovas et al., 2008) and A. m. mellifera (Rortais et al., 2011), which range from northern Spain to Scandinavia, and of A. m. ligustica as a result of the hybrid origin of this subspecies (Franck et al., 2000). The practice of importing queens with different origins is not only detrimental for the conservation of the local ecotypes (De la Rúa et al., 2009) but it may also contribute to the spread of pathogens (Mutinelli, 2011), as there are often inadequate sanitary measures to control the health of the imported honeybees. Indeed, the introduction of foreign queens in the Canarian Islands may have led to the introduction of N. ceranae, since in the earlier survey N. ceranae infected colonies and foreign introduced queens were not detected in the islands included in this study. It should be noted that introduced honeybees were detected on the Canaries (up to 15% of the colonies, Muñoz et al., 2013) but due to the reduced number of honeybees per colony available for an adequate Nosema detection, only a subset of the original sampling has been included in the present study. Another result of this study is the dispersion of N. ceranae throughout the Canary Islands during the last decade and the change in the pattern of distribution on the basis of the maternal origin of the colonies. N. ceranae has been observed in honeybees belonging to every detected lineage and sub-lineage (except AII) and its presence increased in introduced M and C honeybees and remains stable in colonies belonging to AI and AIII African sub-lineages during the last years. This could be related with the lack of host-specificity of N. ceranae, which has been found in a wide range of hosts in relation to A. mellifera evolutionary lineages (Jara et al., 2012; Fontbonne et al., 2013), Apis species (Botías et al., 2012a; Martín-Hernández et al., 2012) and bumblebees in South America (Plischuk et al., 2009), China (Li et al., 2012) and Europe (Evison et al., 2012), but whether local honeybees are more susceptible than imported due to less contact time with this pathogen remains to be studied. On La Palma, the high presence of N. ceranae infected colonies may be related to the distribution of queens reared in a particular mating area and distributed among those beekeepers involved in the established honeybee conservation program (Muñoz and De la Rúa, 2012). If those queens included in the program were infected by N. ceranae, then the pathogen could have spilt over other colonies on the island and also to other islands (as La Gomera and El Hierro) where beekeepers are preferentially using queens bred on La Palma. Therefore, not only the evolutionary origin should be determined but also pathological analyses should be done in the colonies involved in the conservation program to avoid dissemination of foreign queens and this and other pathogens. Relatively low values of N. ceranae infection were observed on Tenerife, an island where there is intensive beekeeping with many professional beekeepers yearly replacing queens with others of local and/or foreign origin and (probably) using chemical treatments, and La Gomera, where beekeepers have introduced queens from different origins (either from other Canary Islands or Europe) since 2008. Indeed, the removal of the honeybee queen and subsequent replacement with a younger queen decreases the proportion of Nosema-infected forager and house honeybees (Botías et al., 2012b). Although in our study the extant presence of N. ceranae was lower on those islands with higher proportion of foreign queens, it must be taken into account the importance of maintaining the Canarian diversity and protecting local ecotypes in order to cope with future environmental changes. The Canarian islands harbor honeybee populations or ecotypes which show genetic differences between island populations and also with other European and African continental honeybee populations (De la Rúa et al., 2001; Muñoz et al.,

2014). Therefore, the yearly replacement of queens with local genetic material and the use of good beekeeping practices will not only help to maintain low rates of Nosema infection and other pathogens, but will also contribute to the preservation and conservation of the local honeybee ecotypes in the Canary Islands. Acknowledgments The authors wish to thank O. Sánchez, A.I. Asensio, V. Albendea, C. Rogerio, T. Corrales and C. Abascal for their technical support and to numerous beekeepers who kindly provided the honeybee samples. Financial support was provided by Fundación Séneca (project 11961/PI/09) to P. De la Rúa, by Fundação para a Ciência e Tecnología and COMPETE/QREN/EU (project PTDC/BIA-BEC/099640/ 2008) to M.A. Pinto, and by Junta de Comunidades de Castilla-La Mancha (Consejería de Agricultura and Consejería de Educación) and INIA-FEDER (RTA 2008-00020-C02-01 and RTA 2009-000105C02-01) to R. Martín-Hernández and M. Higes. I. Muñoz was supported by the Ministry of Education, Culture and Sports. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2014.02. 008. References Bacandritsos, N., Granato, A., Budge, G., Papanastasiou, I., Roinioti, E., Caldon, M., Falcaro, C., Gallina, A., Mutinelli, F., 2010. Sudden deaths and colony population decline in Greek honey bee colonies. J. Invertebr. Pathol. 105, 335–340. Bailey, L., Ball, B.V., 1991. Honey Bee Pathology, second ed. Academic Press, London, 208p. Becnel, J.J., Andreadis, T.G., 1999. Microsporidia in insects. In: Wittner, M., Weiss, L.M. (Eds.), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC, pp. 447–501. Botías, C., Martín-Hernández, R., Garrido-Bailón, E., González-Porto, A., MartínezSalvador, A., De la Rúa, P., Meana, A., Higes, M., 2011. The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade. Res. Vet. Sci. 93, 150–155. Botías, C., Anderson, D.L., Meana, A., Garrido-Bailón, E., Martín-Hernández, R., Higes, M., 2012a. Further evidence of an oriental origin for Nosema ceranae (microsporidia: nosematidae). J. Invertebr. Pathol. 110, 108–113. Botías, C., Martín-Hernández, R., Días, J., García-Palencia, P., Matabuena, M., Juarranz, A., Barrios, L., Meana, A., Nanetti, A., Higes, M., 2012b. The effect of induced queen replacement on Nosema spp. infection in honey bee (Apis mellifera iberiensis) colonies. Environ. Microbiol. 14, 845–859. Bromenshenk, J.J., Henderson, C.B., Wick, C.H., Stanford, M.F., Zulich, A.W., Jabbour, R.E., Deshpande, S.V., McCubbin, P.E., Seccomb, R.A., Welch, P.M., Williams, T., Firth, D.R., Skowronski, E., Lehmann, M.M., Bilimoria, S.L., Gress, J., Wanner, K.W., Cramer Jr., R.A., 2010. Iridovirus and microsporidian linked to honey bee colony decline. PLoS ONE 5, e13181. Calderón, R.A., Sanchez, L.A., Yañez, O., Fallas, N., 2008. Presence of Nosema ceranae in Africanized honey bee colonies in Costa Rica. J. Apic. Res. 47, 328–329. Canning, E.U., Lom, J., 1986. The microsporidia of vertebrates, Academic Press, New York, NY, pp. 1–16. Cánovas, F., De la Rúa, P., Serrano, J., Galián, J., 2008. Geographic patterns of mitochondrial DNA variation in Apis mellifera iberiensis (hymenoptera: apidae). J. Zool. Syst. Evol. Res. 46, 24–30. Chaimanee, V., Chen, Y., Pettis, J.S., Scott, C.R., Chantawannakul, P., 2011. Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand. J. Invertebr. Pathol. 107, 229–233. Chauzat, M.P., Higes, M., Martín-Hernández, R., Meana, A., Cougoule, N., Faucon, J.P., 2007. Presence of Nosema ceranae in French honey bee colonies. J. Apic. Res. 46, 127–128. Chen, Y., Evans, J.D., Smith, I.B., Pettis, J.S., 2008. Nosema ceranae is a long-present and wide-spread microsporidian infection of the European honey bee (Apis mellifera) in the United States. J. Invertebr. Pathol. 97, 186–188. Cornuet, J.M., Garnery, L., 1991. Phylogenetic relationship in the genus Apis inferred from mitochondrial DNA sequence data. Apidologie 22, 627–642. Corradi, N., Keeling, P.J., 2009. Microsporidia: a journey through radical taxonomical revisions. Fungal Biol. Rev. 23, 1–8. Cox-Foster, D.L., Conlan, S., Holmes, E.C., Palacios, G., Evans, J.D., Moran, N.A., Quan, P.L., Briese, T., Hornig, M., Geiser, D.M., Martinson, V., vanEngelsdorp, D., Kalkstein, A.L., Drysdale, A., Hui, J., Zhai, J., Cui, L., Hutchison, S.K., Simons, J.F., Egholm, M., Pettis, J.S., Lipkin, W.I., 2007. Metagenomic survey of microbes in honey bee colony collapse disorder. Science 318, 283–287.

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