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The prevalence of Acarapis woodi in Spanish honey bee (Apis mellifera) colonies
Experimental Parasitology 132 (2012) 530–536
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The prevalence of Acarapis woodi in Spanish honey bee (Apis mellifera) colonies Encarna Garrido-Bailón a, Carolina Bartolomé b, Lourdes Prieto c, Cristina Botías a, Amparo Martínez-Salvador d, Aránzazu Meana e, Raquel Martín-Hernández a,f, Mariano Higes a,⇑ a
Bee Pathology Laboratory, Centro Apícola Regional (CAR), Junta de Comunidades de Castilla La Mancha, 19180 Marchamalo, Spain Departamento de Anatomía Patolóxica e Ciencias Forenses, Grupo de Medicina Xenómica, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain c Instituto Universitario de Investigación en Ciencias Policiales (IUICP), Comisaría General de Policía Científica, DNA Laboratory, Madrid, Spain d Epidemiology Consultant, C/Puente la Reina, 28050 Madrid, Spain e Animal Health Department, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain f Instituto de Recursos Humanos para la Ciencia y la Tecnología (INCRECYT). Fundación Parque Científico y Tecnológico de Albacete, Spain b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" A new polymerase chain reaction
(PCR) was developed to identify Acarapis woodi. " A great prevalence was detected in Spain during the 2006 and 2007 years. " Series doubts about the current classification of Acarapis species have arised.
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 25 July 2012 Accepted 28 August 2012 Available online 10 September 2012 Keywords: Acarapis woodi Apis mellifera PCR Colony-loss Temperate area
a b s t r a c t Acarapis woodi is an internal obligate parasite of the respiratory system of honey bees which provokes significant economic losses in many geographical areas. The main aim of this study was assess the A. woodi role in the ‘‘higher honey bee colony losses phenomenon’’ in Spain. Therefore, a new polymerase chain reaction (PCR) was developed to amplify the mitochondrial cytochrome oxidase I gene (COI) and so the actual prevalence of A. woodi in Spanish honey bee colonies in 2006 and 2007 was determined as part of a wider survey. The results revealed a greater prevalence than expected in most of the geographical areas studied where has been generally underestimated One problem encountered in this study was to distinguish between A. woodi and other species (Acarapis dorsalis and Acarapis externus) at the molecular level. Furthermore, the patterns of genetic divergence across sequences raised serious doubts about the current classification of these organisms. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
⇑ Corresponding author. Address: Bee Pathology Laboratory, Centro Apícola Regional, Camino San Martín s/n, 19180 Marchamalo, Spain. Fax: +34 949 250 176. E-mail addresses: [email protected] (E. Garrido-Bailón), [email protected] (C. Bartolomé), [email protected] (L. Prieto), [email protected] (C. Botías), [email protected] (A. Martínez-Salvador), [email protected] (A. Meana), [email protected] (R. Martín-Hernández), [email protected] (M. Higes). 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2012.08.018
Acarapisosis is a disease of the adult honey bee (Apis mellifera) caused by the microscopic tracheal tarsonemid mite, Acarapis woodi. This parasite was first identified on the Isle of Wight (England) in 1919 and since then, it has been associated with high levels of bee mortality and poor winter survival (McMullan and Brown, 2009; Otis and Scott-Dupree, 1992; Villa and Rinderer, 2008a,b). A. woodi is an internal obligate parasite of the respiratory system,
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which lives and reproduces primarily in the large prothoracic trachea of the bee, although it can also be found in the head, and in thoracic and abdominal air sacs (Giordani, 1965). This organism feeds on the haemolymph of its host and it is a vector of several honey bee viruses (Bailey, 1975, 1985, 1999; Collison, 2001; Komeili and Ambrose, 1991). The pathogenic effects of A. woodi on individual bees depend on the number of parasites within the tracheae and they can be attributed to both mechanical injury and to physiological disorders resulting from the obstruction of the air ducts, lesions in the trachea walls and the depletion of haemolymph. Increased parasitic load causes the tracheal walls, which are normally white and translucent, to become opaque and discolored, with blotchy black areas that are thought to be due to melanin crusts (Giordani, 1964). This parasite has been reported worldwide (Matheson, 1993), except for Sweden Norway, Denmark, New Zealand, Australia and the state of Hawaii (Sammataro et al., 2000), and it is recognized as the cause of significant economic losses. However, little is known regarding its actual prevalence and impact on honey bee colonies in Mediterranean countries. The traditional diagnostic methods are very time consuming and they are based on direct visualization of A. woodi or its lesions in the tracheas (OIE, 2008). Other methods as enzyme-linked immunosorbent assays (ELISA) (Fichter, 1988; (Grant et al., 1993; Ragsdale and Furgala, 1987; Ragsdale and Kjer, 1989) or methods based on the visualization of guanine under ultraviolet light, the main end product of nitrogen metabolism in mites (Mozes-Koch and Gerson, 1997), are little used in routine diagnostic. Recently, new molecular diagnostic techniques were developed to identify A. woodi using a nested PCR to amplify the COI gene (Evans et al., 2007) or different set of primers to amplify the same gene (Kojima et al., 2011). The aim of the present study was to determine the prevalence of A. woodi in honey bee colonies in professional apiaries in Spain in 2006 and 2007, using a PCR protocol that detects A. woodi in a single step. In addition, as new information on A. woodi and related mites, such as Acarapis externus or Acarapis dorsalis, has recently become available at GenBank, we analyzed genetic distances to determine whether the sequences deposited in GenBank were phylogenetically accurate. The results of these studies have important implications not only for our understanding of the prevalence of this mite and its influence on colony losses but also, for the phylogenetic classification of this genus.
2. Methods 2.1. New primers design The mitochondrial COI gene was selected for PCR amplification from A. woodi. All the A. woodi COI sequences available as of March 2010 in GenBank (http://www.ncbi.nlm.nih.gov/sites/entrez?db= taxonomy) were compiled (EU190886, FJ603294.1, FJ603296.1 and GQ916565.1), although sequence FJ603295.1 was excluded due to its short length. A multiple alignment of the four available sequences was carried out using ClustalW (http://www.ebi.ac.uk/ clustalw/), which allowed us to identify polymorphic points and to avoid these when designing the primers. Primers were selected visually, ensuring that the resulting amplicon was short enough to allow amplification in adverse conditions (e.g. poorly preserved samples). The selected primers were: forward primer (AcarFor) 50 -CGGGCCCGAGCTTATTTTACTGCTG-30 ; reverse primer (AcarRev) 50 -GCGCCTGTCAATCCACCTACAGAAA-30 . The CG tails added to primers are shown in bold and they are underlined). The expected size of the amplicon was 162 bp. Primer suitability (G + C content and melting temperature) was evaluated using the IDT OligoAnalyzer software (http://www.idtd-
531
na.com/analyzer/Applications/OligAnalyzer), and G or GC tails were added to the 50 end of each primer to standardize the melting temperatures of the primer sets. Potential primer interactions (hairpin, homodimer, and heterodimer structures for the two primers) were tested using the AutoDimer program (http:// www.cstl.nist.gov/div831/strbase//AutoDimerHomepage/AutoDimerProgramHomepage.htm). Species specificity was determined by conducting a search for nearly exact matches using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Once the specific annealing of primers was verified, PCR amplification and sequencing of three samples were performed. A gradient PCR (58 ± 5 °C) was performed to empirically determine the annealing temperatures of the primer pairs. 2.2. Sample collection Determining the prevalence of A. woodi was part of a wider survey designed to study ‘‘higher honey bee colony losses phenomenon’’ in Spain, involving the study of many different pathogens. The number of colonies to be sampled was calculated in relation to the number of apiaries registered in 2003 (source: Spanish Ministry of Agriculture), with an expected prevalence of honey bee depopulation and losses of 40% (CAR, unpublished data), a precision rate of 10% and a confidence level of 95%. The number of samples was subsequently distributed in proportion to the number of apiaries in each region in which colonies were selected randomly (Fig. 1). This cross-sectional study was carried out between spring 2006 and autumn 2007 and it involved a total of 1943 adult bee samples distributed as follows: 630 honey bee colonies in spring 2006; 458 in autumn 2006; 526 in spring 2007; and 329 in autumn 2007. All samples were submitted to the Bee Pathology Laboratory (CAR) by beekeepers or veterinary services in charge of the beekeeping associations’ apiaries. Bee samples were stored aseptically at 20 °C prior to testing. The parasitized honey bee samples used as controls were sent to CAR by Dr. McMullan (Ireland) and Dr. José Villa (USA). Each apiary was geo-referred and they were linked with the bioclimate belt described by Rivas-Martínez (1987) in the phytogeographical regions of the Iberian Peninsula as previously described (Martín-Hernández et al., 2012) in relation to climatic and altitudinal variables and vegetation attributes of the territory (five belts in Mediterranean region: thermo-,meso-, supra-, oroand cryoromediterranean, from lower to higher altitude; and four belts in the Eurosiberian region: colline, montane, subalpine and alpine). The distribution of A. woodi was related to the agroclimatic information obtained and treated with Geographical Information Systems (GIS, v. 9.0). Pathogens distribution and proportions found were compared through Pearson Chi2 (v2). 2.3. DNA extraction The thorax and head of over 100 house honey bees were crushed for 4 min at high velocity in 50 ml of MilliQ water using a Stomacher machine (Stomacher 80 Biomaster – Seward) and plastic bags equipped with a filter (to retain the honey bee exoskeleton). The macerates were centrifuged for 6 min at 800g, the supernatant was discarded and the pellets were resuspended in 1 ml of distilled water (PCR grade). An aliquot of each resuspended pellet (150 ll) was placed in a 96-well plate (Qiagen) to which glass beads had been added (2 mm diameter, Sigma). At least one blank well with water alone was included for every 20 samples as a negative control of extraction. Positive and negative control samples were included in the plates.
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Fig. 1. Distribution of the prevalence of A. woodi in Spain, according to the bioclimatic belts described by Rivas-Martínez, 1987: Montane (C), Colline (D), supramediterranean (G), mesomediterranean (H), termomediterranean (I).
The plates were shaken in a TissueLyser machine (Qiagen) and 30 ll of ATL buffer (Cat. No. 19076, Qiagen) and 20 ll of Proteinase K (Cat. No. 19131, Qiagen) were added to each well and incubated overnight at 56 °C. DNA was subsequently extracted using a BS96 DNA Tissue extraction protocol in a BioSprint Workstation (Qiagen) and the plates were frozen at 20 °C until use. 2.4. Polymerase Chain Reaction PCR amplification was performed with a Mastercycler ep gradient S (Eppendorf), under the following conditions: Fifty micro litter reaction cocktail containing 25 ll of Fast Star PCR Master, (Cat. No. 04 710 452 001, Roche Diagnostic), 0.3 lM of each primer, 0.2 mg/ ml bovine serum albumin, 0.1% Triton X-100 and 5 ll of the DNA template. The thermocycler program was set as follows: 95 °C (10 min); 35 cycles of a 30 s denaturalization at 95 °C, a 30 s elongation at 61.8 °C and a 45 s extension at 72 °C; and a final extension step at 72 °C for 7 min. For each PCR, templates corresponding to positive (reference Acarapis spp., extracts as template) and negative (H2O as template) controls were run along with DNA extracted from the isolates. PCR amplicons were analyzed in QIAxcel System (Qiagen), using a QIAxcel DNA Screening Kit (Cat. No. 929004, Qiagen), analyzing the negative controls (both extraction and PCR negatives) in parallel to detect possible contamination.
2.5. Sequence analysis All PCR positive products were initially purified with Qiaquick PCR Purification Kit (Cat. No. 28104 Qiagen) in a QiaCube machine (Qiagen) and they were later sequenced in both directions in an Applied Biosystem Genetic Analyzer 3730 automated sequencer. The sequence data obtained was checked visually using Chromas 1.43 software and it was then compared with the sequences deposited in GenBank using BLAST. 2.6. Sequence divergence and phylogenetic analysis of Acarapis COI data in GenBank All overlapping Acarapis COI sequences available in GenBank as on December 2011 were edited and aligned using Bioedit (http:// www.mbio.ncsu.edu/BioEdit/bioedit.html). In order to analyze the maximum number of sites, three A. woodi (AB38409.1, EU190886.1, FJ603295.1) and one A. externus (AB638410.1) sequences were excluded from the final alignment due to their short length. To estimate the synonymous nucleotide divergence between sequences (KS) we applied the Nei–Gojobori method with Jukes-Cantor correction (Nei and Gojobori, 1986) implemented in MEGA4 (Tamura et al., 2007). A neighbor-joining tree was generated for synonymous sites with the same software using the Nei–Gojobori method (Nei and Gojobori, 1986), with Jukes-Cantor
533
E. Garrido-Bailón et al. / Experimental Parasitology 132 (2012) 530–536 Table 1 Prevalence and confidence interval (95%) of Acarapis woodi in Spain in the transverse study relative to the 1943 samples. A. woodi (%) Year
n
Annual Prevalence
CI 95%
2006
1089
13
10,99–15,09
2007
854
15,5
12,97–17,94
Spring Autumn Spring Autumn
Seasonal Prevalence
CI 95%
11 15,9 12,7 19,8
8,4–13,5 12,5–19,4 9,8–15,7 15,3–24,2
Table 2 Distribution of A. woodi in Spain according to the bioclimatic belts (Rivas-Martínez, 1987). A. woodi Bioclimatic belt
Climatic characteristics
n
Positive
Prevalence (%)
v2
p
Mesomediterranean (H)
T 17 to 13 °C, m 4 to 1 °C, M 14 to 9 °C, IT 350 to 210, H: X–IV. Semi-arid to hyper-humid T 19 to 17 °C, m 10 to 4 °C, M 18 to 14 °C, IT 470 to 350, H: XII–II. Arid to humid T > 12 °C, m>2 °C, M > 10 °C, It > 240, H: XI–IV. Sub-humid to Hyper-Humid T 12 to 6 °C, m 2 to -4 °C, M 10 to 3 °C, IT 240 to 50, H: IX–VI. Sub-humid to Hyper-Humid T 13to 8 °C, m 1 to 4 °C, M 9 to 2 °C, IT 210 to 60, H: IX–VI. Semi-arid to Hyper-humid
338
27
8
–
–
173
21
12,1
2,4a
169
22
13
228
35
15,4
Termomediterranean (I) Colline (D) Montane (C) Supramediterranean(G)
263
47
17,9
v2
p
0,125a
–
–
3,3a
0,068a
0,06b
0,8054b
7,5a
0,006a
–
–
13,
a
a
0,000
0,58
c
0,4454c
Bold indicates statistical significance. T = annual average temperature, m = average of minim temperature on the colder month. M = average of maximum temperatures on the colder month. IT: Termic index = (T + m + M) 10. H: months (in Roman numbers) when frost is statistically probable to happen. Ombroclimate classification according to rainfall: Arid < 200 mm, Semi-arid 200–350 mm, Dry 350–600 mm, Sub-humid 600–1000 mm, Humid 1000–1600 mm, Hyper-humid > 1600 mm. a Prevalence of A. woodi on each bioclimatic belt was compared with the lowest level (Mesomediterranean H). b Prevalence of A. woodi on Colline (D) was compared with Termomediterranean (I). c Prevalence in Supramediterranean (G) belt compared with Montane (C).
correction for multiple hits. A bootstrap test (2000 replicates) was performed to assess the reliability of the resulting phylogenetic tree. 3. Results 3.1. PCR reproducibility, sensitivity and specificity In this work, a new PCR protocol was developed on COI gene. In temperature gradient PCR tests, the best amplification was obtained at 59 °C. Indeed, this was the highest temperature at which amplicons could be clearly seen and consequently, the risk of nonspecific amplification was minimized. Several primer concentrations (from 0.2 to 0.6 lM) were also assessed and the best results were obtained at 0.3 lM. All positive controls yielded amplicons of the expected size (162 bp) and sequences. Reproducibility was assessed by repeating the entire process (from DNA extraction to PCR amplicons analyzed in QIAxcel System, Qiagen) in 10 samples 5 times. The same results were obtained with each sample, demonstrating the reliability of the method. In addition, infected bees (positive controls) from different countries (Ireland and USA) were analyzed with our protocol, and the expected amplicons and A. woodi COI sequences were obtained in all cases. 3.2. A. woodi prevalence in Spanish samples collected in 2006 and 2007 A total of 274 house worker bee samples out of 1943 tested positive for A. woodi infection in 2006 (13%) and 2007 (15.5%) and no significant differences were found between the two sampled years (v2 = 2,31; p = 0,13). When the seasonal average prevalence of A. woodis was assessed, the Autumn prevalence in each year (Table 1) was significantly high when compared with spring of the corre-
sponding year (Spring/Autumn 2006 v2 = 5,86, p = 0,015; Spring/ Autumn 2007 v2 = 7,57, p = 0,006), and no differences statistical significant were found between spring or autumn of both years (Spring 2006/2007, v2 = 0,92; p = 0,337; Autumn 2006/2007, v2 = 1,93; p = 0,165). A. woodi was detected at least once in all the regions included in the study (Fig. 1). The prevalence was significantly higher in the montane and supramediterranean belts (Table 2), corresponding to the colder regions the in Spain, where the longest periods when frost are possible. However, A. woodi as well was detected in hotter areas of the country where the climate tends to be drier and hotter than in northern areas. All the amplicons generated were sent for sequencing and the sequences obtained matched the A. woodi COI sequence. Indeed, two example sequences were submitted to GenBank (accession numbers HM213853 and HM213854). No other Acarapis spp., were detected in these samples.
3.3. Suitability of the GenBank COI sequences The analysis of the patterns of genetic differentiation across sequences raised serious doubts about the current classification of these organisms (Table 3). In some cases the level of synonymous divergence (Ks) between sequences attributed to the same species was greater than that observed between sequences assigned to different species. For example, the KS values for two A. dorsalis (GQ916568.1 and GQ916567.1) and two A. externus (HQ243442.1 and GQ916566.1) COI sequences were 23% and 30%, respectively. These values were greater than the synonymous divergence between GQ916566.1 (A. externus) and any of the three A. woodi sequences (22%). Indeed, KS values as large as those observed here (in the order of 10% or greater) strongly suggest that these sequences belong to five different lineages.
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Table 3 Synonymous divergence between Acarapis sequences in GenBank calculated using the Nei–Gojobori method with Jukes-Cantor correction.
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
0.00 0.00 0.17 0.25 0.23 0.15 0.15 0.25 0.25 0.15 0.15 0.25 0.25 0.25 0.25 0.22
0.00 0.17 0.25 0.23 0.15 0.15 0.25 0.25 0.15 0.15 0.25 0.25 0.25 0.25 0.22
0.17 0.25 0.23 0.15 0.15 0.25 0.25 0.15 0.15 0.25 0.25 0.25 0.25 0.22
0.21 0.23 0.01 0.01 0.21 0.21 0.01 0.01 0.25 0.25 0.25 0.25 0.22
0.01 0.23 0.23 0.00 0.00 0.23 0.23 0.08 0.08 0.08 0.08 0.23
0.21 0.21 0.01 0.01 0.21 0.21 0.09 0.09 0.09 0.09 0.22
0.00 0.23 0.23 0.00 0.00 0.27 0.27 0.27 0.27 0.20
0.23 0.23 0.00 0.00 0.27 0.27 0.27 0.27 0.20
0.00 0.23 0.23 0.08 0.08 0.08 0.08 0.23
0.23 0.23 0.08 0.08 0.08 0.08 0.23
0.00 0.27 0.27 0.27 0.27 0.20
0.27 0.27 0.27 0.27 0.20
0.00 0.00 0.00 0.30
0.00 0.00 0.30
0.00 0.30
0.30
A. woodi: [1] FJ603294.1, [2] FJ603296.1, [3] GQ916565.1, [4] GQ916567.1; A. dorsalis. [5] HQ243438.1, [6] GQ916568.1, [7] HQ243439.1, [8] HQ243437.1, [9] HQ243436.1, [10] HQ243435.1, [11] HQ243434.1, [12] HQ243433.1, [13] HQ243442.1; A. externus: [14] HQ243441.1, [15] HQ243440.1, [16] FJ603293.1 and [17] GQ916566.1.
In contrast to the conflicting classification of the A. dorsalis and A. externus datasets, A. woodi sequences (FJ603294.1, FJ603296.1 and GQ916565.1) in GenBank were reliably grouped, showed no divergence (KS = 0.0) and were identical to those obtained in this study (data not shown). To illustrate this finding, we constructed a phylogenetic tree (Fig. 2) that reflects both the consistency of the A. woodi clade, showing identical sequences, and the incongruent names given to the remaining species. Two very divergent groups of ‘‘A. externus’’ and ‘‘A. dorsalis’’ could be distinguished, indicating the existence of at least four different lineages represented by four clades (HQ243433.1, GQ916566.1, HQ243435.1 and FJ603293.1). Although GQ916567.1 and GQ916568.1 differ slightly from the other sequences of the HQ243433.1 and HQ243435.1 clades, respectively, the synonymous pairwise divergence (KS = 1.3%) between each one of them and their respective groups is compatible with these sequences belonging to these clusters. However, the possibility of being distinct entities cannot be excluded. 4. Discussion This is the first extensive study of the prevalence of A. woodi in Spain, and the only such study in a European country in the last decade that uses an accurate and sensitive method that can be easily applied in the laboratory setting without the need for specialized or expensive equipment. In this first study, we do not determine the percentage of parasitized worker bees in each positive colony due to the design of the study and the number of positives samples found, much higher than expected. This is of great importance to assess the mite impact on the colony and the diagnostic method developed in this work we will do so in future studies of this parasite. The most common method to detect A. woodi has been to visualize mites in the tracheal trunk under a microscope following dissection of individual honeybees (Giordani, 1974). Several modifications to this classic technique have been proposed over the years, such as the incubation of discs cut from the thorax in 10% KOH (Shimanuki and Knox, 1991) or lactic acid, staining with dyes like methylene blue (Peng and Nasr, 1985) and thiazol blue tetrazolium to distinguish live from dead mites (Liu, 1995), the flotation of mites (Camazine, 1985), large sampling trials using ELISA (Fichter, 1988; Grant et al., 1993; Ragsdale and Furgala, 1987; Ragsdale and Kjer, 1989), or detection of the presence of guanine (Mozes-Koch and Gerson, 1997). However the molecular tech-
niques are often more sensitive, especially in cases of low parasite loads. One of the challenges in the present study was to distinguish A. woodi from other species of the same genus which also infect honey bees using a molecular approach. In February 2012, there were six COI sequences of A. woodi deposited in GenBank-without including ours- (AB638409.1, EU190886.1, FJ603294.1, FJ603295.1, FJ603296.1, GQ916565.1), six of A. externus (AB638410.1, FJ603293.1; GQ916566.1; HQ243440.1 to HQ243442.1) and nine of A. dorsalis (GQ916567.1, GQ916568.1, HQ243433.1 to HQ243439.1). There were four additional COI sequences of A. woodi (AB634837.1, AB634838.1, HQ162656.1, HQ162657.1), one of A. dorsalis (HQ162658.1) and five of A. externus (AB634839.1, HQ162659.1 to HQ162662.1), that did not overlap with the previous ones and were discarded from further analysis. Sequencing of all the amplified products confirmed the infection by A. woodi, and these products were identical to all the COI sequences found in GenBank from this organism. There were no polymorphisms (KS = 0.0 among A. woodi COI sequences), and indeed, this is the only species of this genus that has been clearly and correctly identified by several authors (Delmiglio et al. unpublished; Evans et al., 2007; Kojima et al., 2011). By contrast, the genetic distances between sequences attributed to A. externus (GQ916566.1, HQ243440.1-HQ243442.1and FJ603293.1, Delmiglio et al. unpublished data and Ward et al. unpublished data) and A. dorsalis (GQ916567.1, GQ916568.1, HQ243433.1–HQ243439.1, Delmiglio et al. unpublished data and Ward et al. unpublished data), respectively, were greater than those observed between sequences assigned to different species. This indicates that the nomenclature of these organisms is incompatible with their current classification. As such, it is necessary to further clarify this issue and to establish the true origin of these sequences, which almost certainly belong to at least four different species. The method described here is very accurate and sensitive. The short size of our amplicon allows A. woodi to be detected even in samples that are not perfectly preserved (e.g. those that contain degraded DNA) and the limit of detection is close to that obtained using nested PCR. We found that the prevalence of A. woodi in honey bee colonies was higher than expected in all Spanish geographical areas, including areas with climatic conditions not considered suitable for the development of A. woodi. Moreover, the sequencing of all the products amplified from our samples confirmed infection by A. woodi alone, with no other mites detected in any of the samples studied.
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Fig. 2. Neighbor-joining tree (synonymous sites) of Acarapis sequences deposited in GenBank, generated with the Nei–Gojobori method with Jukes-Cantor correction. The numbers on the branches indicate the bootstrap values.
The reports about prevalence of tracheal mites in Spain are scarce. In a study performed between 1974 and 1979, years previous to the entry of Varroa destructor in the Iberian Peninsula, the prevalence of A. woodi on clinical samples was estimated in an 18.4% and it was even considered as an endemic pathogen in same areas (Gómez Pajuelo and Fernández Arroyo, 1979). In a study performed in the South of Spain some years later (1990–1991), when V. destructor had spread around the country, A. woodi was reported in four out of 35 (11%) of colonies studied but only in the 2.5% of samples analyzed (Orantes and García-Fernández, 1997). The lower prevalence in this study was linked to the acaricide treatments against varroa for more than 10 years, and for that reason in last decades tracheal mites were considered almost to be disappeared in Spain (Centro Apícola Regional, data not shown). Despite these previous studies were performed using direct observation of the parasite and they are undoubtedly not as sensitive as molecular detection, we can see that prevalence in same moments of our survey (e.g. spring 2007) is close to values previous to the entry of Varroa in Spain. A Greek study using a similar method reported that the acarapisosis problem was self-correcting under their experimental conditions, and a long-term decrease in the incidence of A. woodi from 1986 to 1995 was reported (Bacandritsos and Saitanis, 2004). In agreement with previous data (Ruijter and Eijnde, 1997), these authors confirmed the higher prevalence of the parasite during autumn months, and a lower prevalence during hot and dry periods (summer), a pattern that was exacerbated in conditions of high temperature and low humidity. Similar results were found in our study were the prevalence in autumn was higher than in spring. In our study, although the higher prevalence was found in colder regions, interestingly, we detected A. woodi DNA in samples from warmer climatic belts of Spain, where the climate is traditionally characterized by high temperatures and short rainy periods. Parasitism of honey bees by tracheal mites continues to produce problems for beekeepers (Villa and Rinderer, 2008a,b). Susceptible colonies are often weakened or killed when tracheal mite populations augment (De Guzmán et al., 2006), and increased winter mortality has been associated with high levels of tracheal mite infestation in the autumn (Furgala et al., 1989). There are no reliable clinical signs to diagnose acarapisosis since the signs of infection are not specific and bees behave in much the same way as
those affected by other diseases or disorders (OIE, 2008), crawling in the front of the hive and climbing blades of grass, unable to fly. Dysentery may occur (OIE, 2008), causing beekeepers to confuse these symptoms with those of nosemosis due to Nosema apis. Although clinical outbreaks of acarapisosis were not frequent in any of the areas sampled here, the detection of a higher prevalence of mites than expected (regardless of the higher sensitivity of the PCR) indicates that parasites apparently remain undetected by classical microscopy methods. The National Spanish Control Program for varroosis has enhanced Acarapis control through the regular application of miticides to honey bee colonies. Although no reliable studies of the efficacy of miticide treatment exist, it appears that these treatments are sufficiently effective to prevent clinical outbreaks, particularly since the prevalence we report never exceeds 30%, considered to be the level at which colony losses occur (Bailey, 1981). The presence of A. woodi in colonies should not be underestimated, especially when the percentage of parasitized bees is high (Villa, personal communication). Indeed, honey bee colonies parasitized with Varroa destructor and A. woodi sustain considerably greater mortality during winter months than uninfected colonies, or those with just one mite species (Downey and Winston, 2001). This scenario may also be applicable to colonies parasitized with other pathogens such as Nosema ceranae, which is highly prevalent in Spain (Higes et al., 2010; Martín-Hernández et al., 2007; Orantes and García-Fernández, 1997), and that requires notification of the OIE. Compared with the well-known effects of pathogens such as V. destrutor or Nosema spp., A. woodi has been somewhat neglected, as witnessed by the few research projects into Acarapis spp., that receive funding and the limited publication of new scientific data in this field. The development of new tools to enhance our understanding of this prevalent parasite will help to define the interactions that occur between pathogens, as well as aiding the identification of epidemiological factors influencing its prevalence and determine the consequences of climate change on A. woodi.
5. Conclusions Our results show that the prevalence A. woodi in our country is comparable with that recorded before the massive application of
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acaricides for control of the varroosis. This result suggests a profound reflection on the reasons that have caused this situation and their direct influence on the health status of colonies of bees in many areas of a Mediterranean country. Since the prevalence of this pathogen has been generally underestimated, further studies should be conducted to determine if these levels are cause for concern The patterns of genetic differentiation and phylogenetic analysis of Acarapis sequences deposited in Genbank seriously question the species assignment of these organisms, except in the case of A. woodi, that is concordant in all studies. Acknowledgments The authors wish to thank the Spanish Beekeeper Association for supplying the samples, and Dr. McMullan and Dr. Villa for the control parasitized honey bee samples. Dr. Villa also made a constructive criticism of this work. This study was supported by funding from the Junta de Comunidades de Castilla-La Mancha (Consejería de Agricultura and Consejería de Educación), INIA (RTA2005-152 and RTA 2008-00020-C02-01, FEDER funding) and INCRECYT, European Social Funds. We would like to thank to Virginia Albendea, Carmen Abascal, Carmen Rogerio and Teresa Corrales for their technical support. References Bacandritsos, N.K., Saitanis, C.J., 2004. A field study on the long-term incidence of Acarapis woodi in Greece. J. Apic. Res. 43, 21–26. Bailey, L., 1975. Recent research on honeybee viruses. Bee World 56, 55–64. Bailey, L., 1981. Honey Bee Pathology. Academic Press, London, UK. Bailey, L., 1985. Acarapis woodi: a modern appraisal. Bee World 66, 99–104. Bailey, L., 1999. The century of Acarapis woodi. Am. Bee J. 139, 541–542. Camazine, S., 1985. Tracheal flotation: a rapid method for the detection of honey bee acarine disease. Am. Bee J. 125, 104–105. Collison, C.H., 2001. The pathological effects of the tracheal mite on its host. In: Webster, T.C., Delaplane, K.S. (Eds.), Mites of the Honeybee. Dadant and Sons, Hamilton, Illinois, pp. 57–71. De Guzmán, L.I., Rinderer, T.E., Bigalk, M., Tubbs, H., Bernard, S.J., 2006. Russian honey bee (Hymenoptera: Apidae) colonies: Acarapis woodi (Acari: Tarsonemidae) infestations and overwintering survival. J. Econ. Entomol. 98, 1796–1801. Downey, D.L., Winston, M.L., 2001. Honey bee colony mortality and productivity with single and dual infestations of parasitic mite species. Apidologie 32, 567– 575. Evans, J.D., Pettis, J.S., Smith, I.B., 2007. A diagnostic genetic test for the honey bee tracheal mite. J. Apic. Res. 46, 195–197. Fichter, B.L., 1988. ELISA detection of Acarapis woodi. In: Needham, G.R., Page, R.E., Jr., Delfinado-Baker, M., Bowman, C.E. (Eds.), Africanized Honey Bees and Bee Mites. Ellis Horwood, Chichester, UK, pp. 526–529. Furgala, B., Duff, S.R., Aboulfaraj, S., Ragsdale, D.W., Hyser, R.A., 1989. Some effects of the honey bee tracheal mite (Acarapis woodi) on non-migratory honey bee colonies in east central Minnesota. Am. Bee J. 129, 195–197. Giordani, G., 1965. Laboratory research on Acarapis woodi Rennie, the causative agent of acarine disease of honeybees (Apis mellifera L.) Note 2. Bull. Apic. 6, 185–203. Giordani, G., 1974. Méthodes de diagnostic des maladies des abelles adultes. Dianostic de l´acariose. Bull. Apic., 17 (In French).
Giordani, G., 1964. Rechercher au laboratoire sur Acarapis woodi (Rebbue), agent de l´acariose des abeilles (Apis mellifera L.) Note 4.. Bull. Apic. 7, 43–60 (In French). Gómez Pajuelo, A., Fernández Arroyo, M.P., 1979. Enfermedades de las abejas en España. XXVII Congreso Internacional de Apicultura. Apimondia, Bucarest, pp. 357–361. Grant, G., Nelson, D., Olsen, P., Rice, W.A., 1993. The ELISA detection of tracheal mites in whole honey bee samples. Am. Bee J. 133, 652–655. Higes, M., Martín-Hernández, R., Meana, A., 2010. Nosema ceranae in Europe: an emergent type C nosemosis. Apidologie 41, 375–392. Kojima, Y., Yoshiyama, M., Kimura, K., Kadowaki, T., 2011. PCR-based detection of a tracheal mite of the honey bee Acarapis woodi. J. Invert. Pathol. 108, 135–137. Komeili, A.B., Ambrose, J.T., 1991. Electron microscope studies of tracheae and flight muscles of noninfested, Acarapis woodi infested, crawling honeybees (Apis mellifera). Am. Bee J. 131, 253–257. Liu, T.P., 1995. A rapid differential staining technique for live and dead tracheal mites. Canadian Beekeeping 18, 155. Martín-Hernández, R., Meana, A., Prieto, L., Martínez-Salvador, A., Garrido-Bailón, E., Higes, M., 2007. Outcome of colonization of Apis mellifera by Nosema ceranae. Appl. Environ. Microbiol. 73, 6331–6338. 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, 2127–2138. Matheson, A., 1993. World bee health report. Bee World 74, 176–212. McMullan, J.B., Brown, M.J.F., 2009. A qualitative model of mortality in honey bee (Apis mellifera) colonies infested with tracheal mites (Acarapis woodi). Exp. Appl. Acarol. 47, 225–234. Mozes-Koch, R., Gerson, U., 1997. Guanine visualization: a new method for diagnosing tracheal mite infestation of honey bees. Apidologie 28, 3–9. Nei, M., Gojobori, T., 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3, 418–426. Office International des Epizooties: 2008 Manual of Standards for Diagnostic Test and Vaccines. URL:. Orantes, F.J., García-Fernández, P., 1997. A scientific note on the current low levels of honey bee tracheal mite in southern Spain. Apidologie 28, 149–150. Otis, G.W., Scott-Dupree, C.D., 1992. Effects of Acarapis woodi on overwintered colonies of honey bees (Hymenoptera: Apidae) in New York. J. Econ. Entomol. 85, 40–46. Peng, Y.S., Nasr, M.E., 1985. Detection of honey bee tracheal mites (Acarapis woodi) by simple staining techniques. J. Invert. Pathol. 46, 325–331. Ragsdale, D., Furgala, B., 1987. A serological approach to the detection of Acarapis woodi parasitism in honey bees using an enzyme-linked immunosorbent assay. Apidologie 18, 1–10. Ragsdale, D., Kjer, K.M., 1989. Diagnosis of tracheal mite (Acarapis woodi Rennie) parasitism of honey bees using a monoclonal based enzyme-linked immunosorbent assay. Am. Bee J. 129, 550–553. Rivas-Martínez, S., 1987. Memoria del mapa de series de Vegetación de España. Serie Técnica. Ministerio de Agricultura, Pesca y Alimentación I.C.O.N.A., Madrid. URL: . Sammataro, D., Gerson, U., Needham, G., 2000. Parasitic mites of honey bees: life history, implications and impact. Annu. Rev. Entomol. 45, 519–548. Shimanuki, H., Knox, D.A., 1991. Diagnosis of honey bee diseases. US Department of Agriculture, Agriculture Handbook. No. AH-690, p. 53. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA 4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Villa, J.D., Rinderer, T., 2008a. Inheritance of resistance to Acarapis woodi (Acari: Tarsonemidae) in crosses between selected resistant russian and selected susceptible US honey bees (Hymenoptera: Apidae). J. Econ. Entomol. 101, 1756– 1759. Villa, J.D., Rinderer, T.E., 2008b. Inheritance of resistance to Acarapis woodi (Acari: Tarsonemidae) in crosses between selected resistant Russian and selected susceptible US honey bees (Hymenorptera: Apidae). J. Econ. Entomol. 101, 1726–1759.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
The prevalence of Acarapis woodi in Spanish honey bee (Apis mellifera) colonies Encarna Garrido-Bailón a, Carolina Bartolomé b, Lourdes Prieto c, Cristina Botías a, Amparo Martínez-Salvador d, Aránzazu Meana e, Raquel Martín-Hernández a,f, Mariano Higes a,⇑ a
Bee Pathology Laboratory, Centro Apícola Regional (CAR), Junta de Comunidades de Castilla La Mancha, 19180 Marchamalo, Spain Departamento de Anatomía Patolóxica e Ciencias Forenses, Grupo de Medicina Xenómica, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain c Instituto Universitario de Investigación en Ciencias Policiales (IUICP), Comisaría General de Policía Científica, DNA Laboratory, Madrid, Spain d Epidemiology Consultant, C/Puente la Reina, 28050 Madrid, Spain e Animal Health Department, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain f Instituto de Recursos Humanos para la Ciencia y la Tecnología (INCRECYT). Fundación Parque Científico y Tecnológico de Albacete, Spain b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" A new polymerase chain reaction
(PCR) was developed to identify Acarapis woodi. " A great prevalence was detected in Spain during the 2006 and 2007 years. " Series doubts about the current classification of Acarapis species have arised.
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 25 July 2012 Accepted 28 August 2012 Available online 10 September 2012 Keywords: Acarapis woodi Apis mellifera PCR Colony-loss Temperate area
a b s t r a c t Acarapis woodi is an internal obligate parasite of the respiratory system of honey bees which provokes significant economic losses in many geographical areas. The main aim of this study was assess the A. woodi role in the ‘‘higher honey bee colony losses phenomenon’’ in Spain. Therefore, a new polymerase chain reaction (PCR) was developed to amplify the mitochondrial cytochrome oxidase I gene (COI) and so the actual prevalence of A. woodi in Spanish honey bee colonies in 2006 and 2007 was determined as part of a wider survey. The results revealed a greater prevalence than expected in most of the geographical areas studied where has been generally underestimated One problem encountered in this study was to distinguish between A. woodi and other species (Acarapis dorsalis and Acarapis externus) at the molecular level. Furthermore, the patterns of genetic divergence across sequences raised serious doubts about the current classification of these organisms. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
⇑ Corresponding author. Address: Bee Pathology Laboratory, Centro Apícola Regional, Camino San Martín s/n, 19180 Marchamalo, Spain. Fax: +34 949 250 176. E-mail addresses: [email protected] (E. Garrido-Bailón), [email protected] (C. Bartolomé), [email protected] (L. Prieto), [email protected] (C. Botías), [email protected] (A. Martínez-Salvador), [email protected] (A. Meana), [email protected] (R. Martín-Hernández), [email protected] (M. Higes). 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2012.08.018
Acarapisosis is a disease of the adult honey bee (Apis mellifera) caused by the microscopic tracheal tarsonemid mite, Acarapis woodi. This parasite was first identified on the Isle of Wight (England) in 1919 and since then, it has been associated with high levels of bee mortality and poor winter survival (McMullan and Brown, 2009; Otis and Scott-Dupree, 1992; Villa and Rinderer, 2008a,b). A. woodi is an internal obligate parasite of the respiratory system,
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which lives and reproduces primarily in the large prothoracic trachea of the bee, although it can also be found in the head, and in thoracic and abdominal air sacs (Giordani, 1965). This organism feeds on the haemolymph of its host and it is a vector of several honey bee viruses (Bailey, 1975, 1985, 1999; Collison, 2001; Komeili and Ambrose, 1991). The pathogenic effects of A. woodi on individual bees depend on the number of parasites within the tracheae and they can be attributed to both mechanical injury and to physiological disorders resulting from the obstruction of the air ducts, lesions in the trachea walls and the depletion of haemolymph. Increased parasitic load causes the tracheal walls, which are normally white and translucent, to become opaque and discolored, with blotchy black areas that are thought to be due to melanin crusts (Giordani, 1964). This parasite has been reported worldwide (Matheson, 1993), except for Sweden Norway, Denmark, New Zealand, Australia and the state of Hawaii (Sammataro et al., 2000), and it is recognized as the cause of significant economic losses. However, little is known regarding its actual prevalence and impact on honey bee colonies in Mediterranean countries. The traditional diagnostic methods are very time consuming and they are based on direct visualization of A. woodi or its lesions in the tracheas (OIE, 2008). Other methods as enzyme-linked immunosorbent assays (ELISA) (Fichter, 1988; (Grant et al., 1993; Ragsdale and Furgala, 1987; Ragsdale and Kjer, 1989) or methods based on the visualization of guanine under ultraviolet light, the main end product of nitrogen metabolism in mites (Mozes-Koch and Gerson, 1997), are little used in routine diagnostic. Recently, new molecular diagnostic techniques were developed to identify A. woodi using a nested PCR to amplify the COI gene (Evans et al., 2007) or different set of primers to amplify the same gene (Kojima et al., 2011). The aim of the present study was to determine the prevalence of A. woodi in honey bee colonies in professional apiaries in Spain in 2006 and 2007, using a PCR protocol that detects A. woodi in a single step. In addition, as new information on A. woodi and related mites, such as Acarapis externus or Acarapis dorsalis, has recently become available at GenBank, we analyzed genetic distances to determine whether the sequences deposited in GenBank were phylogenetically accurate. The results of these studies have important implications not only for our understanding of the prevalence of this mite and its influence on colony losses but also, for the phylogenetic classification of this genus.
2. Methods 2.1. New primers design The mitochondrial COI gene was selected for PCR amplification from A. woodi. All the A. woodi COI sequences available as of March 2010 in GenBank (http://www.ncbi.nlm.nih.gov/sites/entrez?db= taxonomy) were compiled (EU190886, FJ603294.1, FJ603296.1 and GQ916565.1), although sequence FJ603295.1 was excluded due to its short length. A multiple alignment of the four available sequences was carried out using ClustalW (http://www.ebi.ac.uk/ clustalw/), which allowed us to identify polymorphic points and to avoid these when designing the primers. Primers were selected visually, ensuring that the resulting amplicon was short enough to allow amplification in adverse conditions (e.g. poorly preserved samples). The selected primers were: forward primer (AcarFor) 50 -CGGGCCCGAGCTTATTTTACTGCTG-30 ; reverse primer (AcarRev) 50 -GCGCCTGTCAATCCACCTACAGAAA-30 . The CG tails added to primers are shown in bold and they are underlined). The expected size of the amplicon was 162 bp. Primer suitability (G + C content and melting temperature) was evaluated using the IDT OligoAnalyzer software (http://www.idtd-
531
na.com/analyzer/Applications/OligAnalyzer), and G or GC tails were added to the 50 end of each primer to standardize the melting temperatures of the primer sets. Potential primer interactions (hairpin, homodimer, and heterodimer structures for the two primers) were tested using the AutoDimer program (http:// www.cstl.nist.gov/div831/strbase//AutoDimerHomepage/AutoDimerProgramHomepage.htm). Species specificity was determined by conducting a search for nearly exact matches using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Once the specific annealing of primers was verified, PCR amplification and sequencing of three samples were performed. A gradient PCR (58 ± 5 °C) was performed to empirically determine the annealing temperatures of the primer pairs. 2.2. Sample collection Determining the prevalence of A. woodi was part of a wider survey designed to study ‘‘higher honey bee colony losses phenomenon’’ in Spain, involving the study of many different pathogens. The number of colonies to be sampled was calculated in relation to the number of apiaries registered in 2003 (source: Spanish Ministry of Agriculture), with an expected prevalence of honey bee depopulation and losses of 40% (CAR, unpublished data), a precision rate of 10% and a confidence level of 95%. The number of samples was subsequently distributed in proportion to the number of apiaries in each region in which colonies were selected randomly (Fig. 1). This cross-sectional study was carried out between spring 2006 and autumn 2007 and it involved a total of 1943 adult bee samples distributed as follows: 630 honey bee colonies in spring 2006; 458 in autumn 2006; 526 in spring 2007; and 329 in autumn 2007. All samples were submitted to the Bee Pathology Laboratory (CAR) by beekeepers or veterinary services in charge of the beekeeping associations’ apiaries. Bee samples were stored aseptically at 20 °C prior to testing. The parasitized honey bee samples used as controls were sent to CAR by Dr. McMullan (Ireland) and Dr. José Villa (USA). Each apiary was geo-referred and they were linked with the bioclimate belt described by Rivas-Martínez (1987) in the phytogeographical regions of the Iberian Peninsula as previously described (Martín-Hernández et al., 2012) in relation to climatic and altitudinal variables and vegetation attributes of the territory (five belts in Mediterranean region: thermo-,meso-, supra-, oroand cryoromediterranean, from lower to higher altitude; and four belts in the Eurosiberian region: colline, montane, subalpine and alpine). The distribution of A. woodi was related to the agroclimatic information obtained and treated with Geographical Information Systems (GIS, v. 9.0). Pathogens distribution and proportions found were compared through Pearson Chi2 (v2). 2.3. DNA extraction The thorax and head of over 100 house honey bees were crushed for 4 min at high velocity in 50 ml of MilliQ water using a Stomacher machine (Stomacher 80 Biomaster – Seward) and plastic bags equipped with a filter (to retain the honey bee exoskeleton). The macerates were centrifuged for 6 min at 800g, the supernatant was discarded and the pellets were resuspended in 1 ml of distilled water (PCR grade). An aliquot of each resuspended pellet (150 ll) was placed in a 96-well plate (Qiagen) to which glass beads had been added (2 mm diameter, Sigma). At least one blank well with water alone was included for every 20 samples as a negative control of extraction. Positive and negative control samples were included in the plates.
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Fig. 1. Distribution of the prevalence of A. woodi in Spain, according to the bioclimatic belts described by Rivas-Martínez, 1987: Montane (C), Colline (D), supramediterranean (G), mesomediterranean (H), termomediterranean (I).
The plates were shaken in a TissueLyser machine (Qiagen) and 30 ll of ATL buffer (Cat. No. 19076, Qiagen) and 20 ll of Proteinase K (Cat. No. 19131, Qiagen) were added to each well and incubated overnight at 56 °C. DNA was subsequently extracted using a BS96 DNA Tissue extraction protocol in a BioSprint Workstation (Qiagen) and the plates were frozen at 20 °C until use. 2.4. Polymerase Chain Reaction PCR amplification was performed with a Mastercycler ep gradient S (Eppendorf), under the following conditions: Fifty micro litter reaction cocktail containing 25 ll of Fast Star PCR Master, (Cat. No. 04 710 452 001, Roche Diagnostic), 0.3 lM of each primer, 0.2 mg/ ml bovine serum albumin, 0.1% Triton X-100 and 5 ll of the DNA template. The thermocycler program was set as follows: 95 °C (10 min); 35 cycles of a 30 s denaturalization at 95 °C, a 30 s elongation at 61.8 °C and a 45 s extension at 72 °C; and a final extension step at 72 °C for 7 min. For each PCR, templates corresponding to positive (reference Acarapis spp., extracts as template) and negative (H2O as template) controls were run along with DNA extracted from the isolates. PCR amplicons were analyzed in QIAxcel System (Qiagen), using a QIAxcel DNA Screening Kit (Cat. No. 929004, Qiagen), analyzing the negative controls (both extraction and PCR negatives) in parallel to detect possible contamination.
2.5. Sequence analysis All PCR positive products were initially purified with Qiaquick PCR Purification Kit (Cat. No. 28104 Qiagen) in a QiaCube machine (Qiagen) and they were later sequenced in both directions in an Applied Biosystem Genetic Analyzer 3730 automated sequencer. The sequence data obtained was checked visually using Chromas 1.43 software and it was then compared with the sequences deposited in GenBank using BLAST. 2.6. Sequence divergence and phylogenetic analysis of Acarapis COI data in GenBank All overlapping Acarapis COI sequences available in GenBank as on December 2011 were edited and aligned using Bioedit (http:// www.mbio.ncsu.edu/BioEdit/bioedit.html). In order to analyze the maximum number of sites, three A. woodi (AB38409.1, EU190886.1, FJ603295.1) and one A. externus (AB638410.1) sequences were excluded from the final alignment due to their short length. To estimate the synonymous nucleotide divergence between sequences (KS) we applied the Nei–Gojobori method with Jukes-Cantor correction (Nei and Gojobori, 1986) implemented in MEGA4 (Tamura et al., 2007). A neighbor-joining tree was generated for synonymous sites with the same software using the Nei–Gojobori method (Nei and Gojobori, 1986), with Jukes-Cantor
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E. Garrido-Bailón et al. / Experimental Parasitology 132 (2012) 530–536 Table 1 Prevalence and confidence interval (95%) of Acarapis woodi in Spain in the transverse study relative to the 1943 samples. A. woodi (%) Year
n
Annual Prevalence
CI 95%
2006
1089
13
10,99–15,09
2007
854
15,5
12,97–17,94
Spring Autumn Spring Autumn
Seasonal Prevalence
CI 95%
11 15,9 12,7 19,8
8,4–13,5 12,5–19,4 9,8–15,7 15,3–24,2
Table 2 Distribution of A. woodi in Spain according to the bioclimatic belts (Rivas-Martínez, 1987). A. woodi Bioclimatic belt
Climatic characteristics
n
Positive
Prevalence (%)
v2
p
Mesomediterranean (H)
T 17 to 13 °C, m 4 to 1 °C, M 14 to 9 °C, IT 350 to 210, H: X–IV. Semi-arid to hyper-humid T 19 to 17 °C, m 10 to 4 °C, M 18 to 14 °C, IT 470 to 350, H: XII–II. Arid to humid T > 12 °C, m>2 °C, M > 10 °C, It > 240, H: XI–IV. Sub-humid to Hyper-Humid T 12 to 6 °C, m 2 to -4 °C, M 10 to 3 °C, IT 240 to 50, H: IX–VI. Sub-humid to Hyper-Humid T 13to 8 °C, m 1 to 4 °C, M 9 to 2 °C, IT 210 to 60, H: IX–VI. Semi-arid to Hyper-humid
338
27
8
–
–
173
21
12,1
2,4a
169
22
13
228
35
15,4
Termomediterranean (I) Colline (D) Montane (C) Supramediterranean(G)
263
47
17,9
v2
p
0,125a
–
–
3,3a
0,068a
0,06b
0,8054b
7,5a
0,006a
–
–
13,
a
a
0,000
0,58
c
0,4454c
Bold indicates statistical significance. T = annual average temperature, m = average of minim temperature on the colder month. M = average of maximum temperatures on the colder month. IT: Termic index = (T + m + M) 10. H: months (in Roman numbers) when frost is statistically probable to happen. Ombroclimate classification according to rainfall: Arid < 200 mm, Semi-arid 200–350 mm, Dry 350–600 mm, Sub-humid 600–1000 mm, Humid 1000–1600 mm, Hyper-humid > 1600 mm. a Prevalence of A. woodi on each bioclimatic belt was compared with the lowest level (Mesomediterranean H). b Prevalence of A. woodi on Colline (D) was compared with Termomediterranean (I). c Prevalence in Supramediterranean (G) belt compared with Montane (C).
correction for multiple hits. A bootstrap test (2000 replicates) was performed to assess the reliability of the resulting phylogenetic tree. 3. Results 3.1. PCR reproducibility, sensitivity and specificity In this work, a new PCR protocol was developed on COI gene. In temperature gradient PCR tests, the best amplification was obtained at 59 °C. Indeed, this was the highest temperature at which amplicons could be clearly seen and consequently, the risk of nonspecific amplification was minimized. Several primer concentrations (from 0.2 to 0.6 lM) were also assessed and the best results were obtained at 0.3 lM. All positive controls yielded amplicons of the expected size (162 bp) and sequences. Reproducibility was assessed by repeating the entire process (from DNA extraction to PCR amplicons analyzed in QIAxcel System, Qiagen) in 10 samples 5 times. The same results were obtained with each sample, demonstrating the reliability of the method. In addition, infected bees (positive controls) from different countries (Ireland and USA) were analyzed with our protocol, and the expected amplicons and A. woodi COI sequences were obtained in all cases. 3.2. A. woodi prevalence in Spanish samples collected in 2006 and 2007 A total of 274 house worker bee samples out of 1943 tested positive for A. woodi infection in 2006 (13%) and 2007 (15.5%) and no significant differences were found between the two sampled years (v2 = 2,31; p = 0,13). When the seasonal average prevalence of A. woodis was assessed, the Autumn prevalence in each year (Table 1) was significantly high when compared with spring of the corre-
sponding year (Spring/Autumn 2006 v2 = 5,86, p = 0,015; Spring/ Autumn 2007 v2 = 7,57, p = 0,006), and no differences statistical significant were found between spring or autumn of both years (Spring 2006/2007, v2 = 0,92; p = 0,337; Autumn 2006/2007, v2 = 1,93; p = 0,165). A. woodi was detected at least once in all the regions included in the study (Fig. 1). The prevalence was significantly higher in the montane and supramediterranean belts (Table 2), corresponding to the colder regions the in Spain, where the longest periods when frost are possible. However, A. woodi as well was detected in hotter areas of the country where the climate tends to be drier and hotter than in northern areas. All the amplicons generated were sent for sequencing and the sequences obtained matched the A. woodi COI sequence. Indeed, two example sequences were submitted to GenBank (accession numbers HM213853 and HM213854). No other Acarapis spp., were detected in these samples.
3.3. Suitability of the GenBank COI sequences The analysis of the patterns of genetic differentiation across sequences raised serious doubts about the current classification of these organisms (Table 3). In some cases the level of synonymous divergence (Ks) between sequences attributed to the same species was greater than that observed between sequences assigned to different species. For example, the KS values for two A. dorsalis (GQ916568.1 and GQ916567.1) and two A. externus (HQ243442.1 and GQ916566.1) COI sequences were 23% and 30%, respectively. These values were greater than the synonymous divergence between GQ916566.1 (A. externus) and any of the three A. woodi sequences (22%). Indeed, KS values as large as those observed here (in the order of 10% or greater) strongly suggest that these sequences belong to five different lineages.
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Table 3 Synonymous divergence between Acarapis sequences in GenBank calculated using the Nei–Gojobori method with Jukes-Cantor correction.
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
0.00 0.00 0.17 0.25 0.23 0.15 0.15 0.25 0.25 0.15 0.15 0.25 0.25 0.25 0.25 0.22
0.00 0.17 0.25 0.23 0.15 0.15 0.25 0.25 0.15 0.15 0.25 0.25 0.25 0.25 0.22
0.17 0.25 0.23 0.15 0.15 0.25 0.25 0.15 0.15 0.25 0.25 0.25 0.25 0.22
0.21 0.23 0.01 0.01 0.21 0.21 0.01 0.01 0.25 0.25 0.25 0.25 0.22
0.01 0.23 0.23 0.00 0.00 0.23 0.23 0.08 0.08 0.08 0.08 0.23
0.21 0.21 0.01 0.01 0.21 0.21 0.09 0.09 0.09 0.09 0.22
0.00 0.23 0.23 0.00 0.00 0.27 0.27 0.27 0.27 0.20
0.23 0.23 0.00 0.00 0.27 0.27 0.27 0.27 0.20
0.00 0.23 0.23 0.08 0.08 0.08 0.08 0.23
0.23 0.23 0.08 0.08 0.08 0.08 0.23
0.00 0.27 0.27 0.27 0.27 0.20
0.27 0.27 0.27 0.27 0.20
0.00 0.00 0.00 0.30
0.00 0.00 0.30
0.00 0.30
0.30
A. woodi: [1] FJ603294.1, [2] FJ603296.1, [3] GQ916565.1, [4] GQ916567.1; A. dorsalis. [5] HQ243438.1, [6] GQ916568.1, [7] HQ243439.1, [8] HQ243437.1, [9] HQ243436.1, [10] HQ243435.1, [11] HQ243434.1, [12] HQ243433.1, [13] HQ243442.1; A. externus: [14] HQ243441.1, [15] HQ243440.1, [16] FJ603293.1 and [17] GQ916566.1.
In contrast to the conflicting classification of the A. dorsalis and A. externus datasets, A. woodi sequences (FJ603294.1, FJ603296.1 and GQ916565.1) in GenBank were reliably grouped, showed no divergence (KS = 0.0) and were identical to those obtained in this study (data not shown). To illustrate this finding, we constructed a phylogenetic tree (Fig. 2) that reflects both the consistency of the A. woodi clade, showing identical sequences, and the incongruent names given to the remaining species. Two very divergent groups of ‘‘A. externus’’ and ‘‘A. dorsalis’’ could be distinguished, indicating the existence of at least four different lineages represented by four clades (HQ243433.1, GQ916566.1, HQ243435.1 and FJ603293.1). Although GQ916567.1 and GQ916568.1 differ slightly from the other sequences of the HQ243433.1 and HQ243435.1 clades, respectively, the synonymous pairwise divergence (KS = 1.3%) between each one of them and their respective groups is compatible with these sequences belonging to these clusters. However, the possibility of being distinct entities cannot be excluded. 4. Discussion This is the first extensive study of the prevalence of A. woodi in Spain, and the only such study in a European country in the last decade that uses an accurate and sensitive method that can be easily applied in the laboratory setting without the need for specialized or expensive equipment. In this first study, we do not determine the percentage of parasitized worker bees in each positive colony due to the design of the study and the number of positives samples found, much higher than expected. This is of great importance to assess the mite impact on the colony and the diagnostic method developed in this work we will do so in future studies of this parasite. The most common method to detect A. woodi has been to visualize mites in the tracheal trunk under a microscope following dissection of individual honeybees (Giordani, 1974). Several modifications to this classic technique have been proposed over the years, such as the incubation of discs cut from the thorax in 10% KOH (Shimanuki and Knox, 1991) or lactic acid, staining with dyes like methylene blue (Peng and Nasr, 1985) and thiazol blue tetrazolium to distinguish live from dead mites (Liu, 1995), the flotation of mites (Camazine, 1985), large sampling trials using ELISA (Fichter, 1988; Grant et al., 1993; Ragsdale and Furgala, 1987; Ragsdale and Kjer, 1989), or detection of the presence of guanine (Mozes-Koch and Gerson, 1997). However the molecular tech-
niques are often more sensitive, especially in cases of low parasite loads. One of the challenges in the present study was to distinguish A. woodi from other species of the same genus which also infect honey bees using a molecular approach. In February 2012, there were six COI sequences of A. woodi deposited in GenBank-without including ours- (AB638409.1, EU190886.1, FJ603294.1, FJ603295.1, FJ603296.1, GQ916565.1), six of A. externus (AB638410.1, FJ603293.1; GQ916566.1; HQ243440.1 to HQ243442.1) and nine of A. dorsalis (GQ916567.1, GQ916568.1, HQ243433.1 to HQ243439.1). There were four additional COI sequences of A. woodi (AB634837.1, AB634838.1, HQ162656.1, HQ162657.1), one of A. dorsalis (HQ162658.1) and five of A. externus (AB634839.1, HQ162659.1 to HQ162662.1), that did not overlap with the previous ones and were discarded from further analysis. Sequencing of all the amplified products confirmed the infection by A. woodi, and these products were identical to all the COI sequences found in GenBank from this organism. There were no polymorphisms (KS = 0.0 among A. woodi COI sequences), and indeed, this is the only species of this genus that has been clearly and correctly identified by several authors (Delmiglio et al. unpublished; Evans et al., 2007; Kojima et al., 2011). By contrast, the genetic distances between sequences attributed to A. externus (GQ916566.1, HQ243440.1-HQ243442.1and FJ603293.1, Delmiglio et al. unpublished data and Ward et al. unpublished data) and A. dorsalis (GQ916567.1, GQ916568.1, HQ243433.1–HQ243439.1, Delmiglio et al. unpublished data and Ward et al. unpublished data), respectively, were greater than those observed between sequences assigned to different species. This indicates that the nomenclature of these organisms is incompatible with their current classification. As such, it is necessary to further clarify this issue and to establish the true origin of these sequences, which almost certainly belong to at least four different species. The method described here is very accurate and sensitive. The short size of our amplicon allows A. woodi to be detected even in samples that are not perfectly preserved (e.g. those that contain degraded DNA) and the limit of detection is close to that obtained using nested PCR. We found that the prevalence of A. woodi in honey bee colonies was higher than expected in all Spanish geographical areas, including areas with climatic conditions not considered suitable for the development of A. woodi. Moreover, the sequencing of all the products amplified from our samples confirmed infection by A. woodi alone, with no other mites detected in any of the samples studied.
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Fig. 2. Neighbor-joining tree (synonymous sites) of Acarapis sequences deposited in GenBank, generated with the Nei–Gojobori method with Jukes-Cantor correction. The numbers on the branches indicate the bootstrap values.
The reports about prevalence of tracheal mites in Spain are scarce. In a study performed between 1974 and 1979, years previous to the entry of Varroa destructor in the Iberian Peninsula, the prevalence of A. woodi on clinical samples was estimated in an 18.4% and it was even considered as an endemic pathogen in same areas (Gómez Pajuelo and Fernández Arroyo, 1979). In a study performed in the South of Spain some years later (1990–1991), when V. destructor had spread around the country, A. woodi was reported in four out of 35 (11%) of colonies studied but only in the 2.5% of samples analyzed (Orantes and García-Fernández, 1997). The lower prevalence in this study was linked to the acaricide treatments against varroa for more than 10 years, and for that reason in last decades tracheal mites were considered almost to be disappeared in Spain (Centro Apícola Regional, data not shown). Despite these previous studies were performed using direct observation of the parasite and they are undoubtedly not as sensitive as molecular detection, we can see that prevalence in same moments of our survey (e.g. spring 2007) is close to values previous to the entry of Varroa in Spain. A Greek study using a similar method reported that the acarapisosis problem was self-correcting under their experimental conditions, and a long-term decrease in the incidence of A. woodi from 1986 to 1995 was reported (Bacandritsos and Saitanis, 2004). In agreement with previous data (Ruijter and Eijnde, 1997), these authors confirmed the higher prevalence of the parasite during autumn months, and a lower prevalence during hot and dry periods (summer), a pattern that was exacerbated in conditions of high temperature and low humidity. Similar results were found in our study were the prevalence in autumn was higher than in spring. In our study, although the higher prevalence was found in colder regions, interestingly, we detected A. woodi DNA in samples from warmer climatic belts of Spain, where the climate is traditionally characterized by high temperatures and short rainy periods. Parasitism of honey bees by tracheal mites continues to produce problems for beekeepers (Villa and Rinderer, 2008a,b). Susceptible colonies are often weakened or killed when tracheal mite populations augment (De Guzmán et al., 2006), and increased winter mortality has been associated with high levels of tracheal mite infestation in the autumn (Furgala et al., 1989). There are no reliable clinical signs to diagnose acarapisosis since the signs of infection are not specific and bees behave in much the same way as
those affected by other diseases or disorders (OIE, 2008), crawling in the front of the hive and climbing blades of grass, unable to fly. Dysentery may occur (OIE, 2008), causing beekeepers to confuse these symptoms with those of nosemosis due to Nosema apis. Although clinical outbreaks of acarapisosis were not frequent in any of the areas sampled here, the detection of a higher prevalence of mites than expected (regardless of the higher sensitivity of the PCR) indicates that parasites apparently remain undetected by classical microscopy methods. The National Spanish Control Program for varroosis has enhanced Acarapis control through the regular application of miticides to honey bee colonies. Although no reliable studies of the efficacy of miticide treatment exist, it appears that these treatments are sufficiently effective to prevent clinical outbreaks, particularly since the prevalence we report never exceeds 30%, considered to be the level at which colony losses occur (Bailey, 1981). The presence of A. woodi in colonies should not be underestimated, especially when the percentage of parasitized bees is high (Villa, personal communication). Indeed, honey bee colonies parasitized with Varroa destructor and A. woodi sustain considerably greater mortality during winter months than uninfected colonies, or those with just one mite species (Downey and Winston, 2001). This scenario may also be applicable to colonies parasitized with other pathogens such as Nosema ceranae, which is highly prevalent in Spain (Higes et al., 2010; Martín-Hernández et al., 2007; Orantes and García-Fernández, 1997), and that requires notification of the OIE. Compared with the well-known effects of pathogens such as V. destrutor or Nosema spp., A. woodi has been somewhat neglected, as witnessed by the few research projects into Acarapis spp., that receive funding and the limited publication of new scientific data in this field. The development of new tools to enhance our understanding of this prevalent parasite will help to define the interactions that occur between pathogens, as well as aiding the identification of epidemiological factors influencing its prevalence and determine the consequences of climate change on A. woodi.
5. Conclusions Our results show that the prevalence A. woodi in our country is comparable with that recorded before the massive application of
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acaricides for control of the varroosis. This result suggests a profound reflection on the reasons that have caused this situation and their direct influence on the health status of colonies of bees in many areas of a Mediterranean country. Since the prevalence of this pathogen has been generally underestimated, further studies should be conducted to determine if these levels are cause for concern The patterns of genetic differentiation and phylogenetic analysis of Acarapis sequences deposited in Genbank seriously question the species assignment of these organisms, except in the case of A. woodi, that is concordant in all studies. Acknowledgments The authors wish to thank the Spanish Beekeeper Association for supplying the samples, and Dr. McMullan and Dr. Villa for the control parasitized honey bee samples. Dr. Villa also made a constructive criticism of this work. This study was supported by funding from the Junta de Comunidades de Castilla-La Mancha (Consejería de Agricultura and Consejería de Educación), INIA (RTA2005-152 and RTA 2008-00020-C02-01, FEDER funding) and INCRECYT, European Social Funds. We would like to thank to Virginia Albendea, Carmen Abascal, Carmen Rogerio and Teresa Corrales for their technical support. References Bacandritsos, N.K., Saitanis, C.J., 2004. A field study on the long-term incidence of Acarapis woodi in Greece. J. Apic. Res. 43, 21–26. Bailey, L., 1975. Recent research on honeybee viruses. Bee World 56, 55–64. Bailey, L., 1981. Honey Bee Pathology. Academic Press, London, UK. Bailey, L., 1985. Acarapis woodi: a modern appraisal. Bee World 66, 99–104. Bailey, L., 1999. The century of Acarapis woodi. Am. Bee J. 139, 541–542. Camazine, S., 1985. Tracheal flotation: a rapid method for the detection of honey bee acarine disease. Am. Bee J. 125, 104–105. Collison, C.H., 2001. The pathological effects of the tracheal mite on its host. In: Webster, T.C., Delaplane, K.S. (Eds.), Mites of the Honeybee. Dadant and Sons, Hamilton, Illinois, pp. 57–71. De Guzmán, L.I., Rinderer, T.E., Bigalk, M., Tubbs, H., Bernard, S.J., 2006. Russian honey bee (Hymenoptera: Apidae) colonies: Acarapis woodi (Acari: Tarsonemidae) infestations and overwintering survival. J. Econ. Entomol. 98, 1796–1801. Downey, D.L., Winston, M.L., 2001. Honey bee colony mortality and productivity with single and dual infestations of parasitic mite species. Apidologie 32, 567– 575. Evans, J.D., Pettis, J.S., Smith, I.B., 2007. A diagnostic genetic test for the honey bee tracheal mite. J. Apic. Res. 46, 195–197. Fichter, B.L., 1988. ELISA detection of Acarapis woodi. In: Needham, G.R., Page, R.E., Jr., Delfinado-Baker, M., Bowman, C.E. (Eds.), Africanized Honey Bees and Bee Mites. Ellis Horwood, Chichester, UK, pp. 526–529. Furgala, B., Duff, S.R., Aboulfaraj, S., Ragsdale, D.W., Hyser, R.A., 1989. Some effects of the honey bee tracheal mite (Acarapis woodi) on non-migratory honey bee colonies in east central Minnesota. Am. Bee J. 129, 195–197. Giordani, G., 1965. Laboratory research on Acarapis woodi Rennie, the causative agent of acarine disease of honeybees (Apis mellifera L.) Note 2. Bull. Apic. 6, 185–203. Giordani, G., 1974. Méthodes de diagnostic des maladies des abelles adultes. Dianostic de l´acariose. Bull. Apic., 17 (In French).
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