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Impact of Varroa destructor and deformed wing virus on emergence, cellular immunity, wing integrity and survivorship of Africanized honey bees in Mexico
Journal of Invertebrate Pathology 164 (2019) 43–48
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Impact of Varroa destructor and deformed wing virus on emergence, cellular immunity, wing integrity and survivorship of Africanized honey bees in Mexico
T
Mariana Reyes-Quintanaa, Laura G. Espinosa-Montañob, Daniel Prieto-Merlosb, Gun Koleoglua, ⁎ Tatiana Petukhovaa, Adriana Correa-Benítezb, Ernesto Guzman-Novoaa, a b
School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada Departamento de Medicina y Zootecnia en Abejas, FMVZ, UNAM, Cd. Univ., Mexico 04510, Mexico
A R T I C LE I N FO
A B S T R A C T
Keywords: Varroa destructor Deformed wing virus Africanized bees Worker emergence Hemocytes Survivorship
The ectoparasitic mite Varroa destructor is the primary health problem of honey bees (Apis mellifera) worldwide. Africanized honey bees in Brazil have demonstrated tolerance to the mite, but there is controversy about the degree of mite tolerance of Africanized bees in other countries. This study was conducted to quantify the effect of V. destructor parasitism on emergence, hemocyte concentration, wing integrity and longevity of Africanized honey bees in Mexico. Africanized bee brood were artificially infested with V. destructor mites and held in an incubator until emergence as adults and compared to non-infested controls. Deformed wing virus (DWV) presence was determined in the mites used to infest the bees. After emergence, the bees were maintained in an incubator to determine survivorship. The percentage of worker bees that emerged from parasitized cells (69%) was significantly lower than that of bees emerged from non-infested cells (96%). Newly-emerged parasitized bees had a significantly lower concentration of hemocytes in the hemolymph than non-parasitized bees. Additionally, the proportion of bees with deformed wings that emerged from V. destructor-parasitized cells was significantly higher (54%) than that of the control group (0%). The mean survival time of bees that emerged from infested and non-infested cells was 8.5 ± 0.3 and 14.4 ± 0.4 days, respectively, and the difference was significant. We conclude that V. destructor parasitism and DWV infections kill, cause deformities and inhibit cellular immunity in developing Africanized honey bees, and significantly reduce the lifespan of adult bees in Mexico. These results suggest that the tolerance of Africanized bees to V. destructor is related to adult bee mechanisms.
1. Introduction
humoral (Yang and Cox-Foster, 2005; Koleoglu et al., 2017) and cellular immune responses (Koleoglu et al., 2018), and transmit pathogenic viruses to the bees (Kevan et al., 2006; Genersch and Aubert, 2010; Anguiano-Baez et al., 2016). The deformed wing virus (DWV), in particular, causes severe morphological deformities and has been linked to winter colony mortality (Bowen-Walker et al., 1999; Dainat et al., 2012). Numerous studies have demonstrated that adult Africanized honey bees (descendants of A. m. scutellata) in the neotropics are more tolerant to the Varroa mite than their European counterparts (Moretto et al., 1991; De Jong, 1997; Guzman-Novoa et al., 1999, 2011; Medina-Flores et al., 2014), although the basis of this tolerance is still not well understood. However, despite evidence that indicates a certain degree of tolerance of Africanized bees to V. destructor mites, few studies have
Varroa destructor parasitism is the most serious health problem of western honey bee (Apis mellifera) populations worldwide. Relatively low levels of V. destructor infestation cause colonies to collapse in temperate climate countries (Rosenkranz et al., 2010) and the mite has been found to be associated with more than 85% of colony mortality cases during winter in temperate regions (Guzman-Novoa et al., 2010). Varroa mite infestations also decrease honey yields of different bee strains, including Africanized bees (Arechavaleta-Velasco and GuzmanNovoa, 2000; Medina-Flores et al., 2011; Emsen et al., 2014). These and other detrimental effects on the bees result from several types of damage caused by V. destructor. The mites feed on the hemolymph and fat tissue of brood and adult bees (Sammataro et al., 2000), inhibit their
⁎
Corresponding author. E-mail address: [email protected] (E. Guzman-Novoa).
https://doi.org/10.1016/j.jip.2019.04.009 Received 13 December 2018; Received in revised form 24 April 2019; Accepted 26 April 2019 Available online 26 April 2019 0022-2011/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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been conducted to analyze the actual damage that the mite causes in Africanized bees during their developmental stage. Several studies have documented the effects of V. destructor parasitism in developing European honey bees. For example, Bowen-Walker and Gunn (2001) found that bees emerging from V. destructor-parasitized cells weighed significantly less and had lower concentrations of protein than bees emerging from non-parasitized cells. Similar results were reported by De Jong et al. (1982b) for Africanized bees in Brazil. Bowen-Walker et al. (1999) found that only 54% of the European bees in their study emerged from cells that had been artificially parasitized with V. destructor mites. However, reports for rate of emergence from developing Africanized honey bees that are artificially parasitized with Varroa mites are lacking in the literature. Similarly, there are no reports of hemocyte concentrations in newly emerged bees parasitized by V. destructor mites. Hemocytes are cells with immune response functions that circulate in the hemolymph of insects. One of the main roles of hemocytes is to contribute to wound healing by aggregating at a wound site and releasing granular components and chemicals that form a clot (Theopold et al., 2002). Because V. destructor causes wounds in the honey bee cuticle and feeds upon its hemolymph, it would be expected that the concentration of hemocytes in the hemolymph of parasitized bees would decrease during the developmental stage of the insects. With regards to adult bee longevity, De Jong and De Jong (1983) in Brazil, found that Africanized bees emerging from Varroa-parasitized cells had a 50% shorter adult lifespan than bees emerging from nonparasitized cells. De Jong and De Jong (1983) however, did not measure the impact that Varroa parasitism has on the rate of bee emergence. Another study conducted with Africanized bees in Mexico and using similar methodology to that of De Jong and De Jong (1983), did not find differences in longevity of worker bees that were and were not parasitized by V. destructor (Romero-Vera and Otero-Colonia, 2002). This was in contrast with the results from Brazil, and suggested that the brood of Africanized bees in Mexico might be more tolerant to the mite. No other similar studies have been conducted with Africanized bees in both Mexico and Brazil to corroborate or challenge previous findings. We conducted this study to test the hypothesis that V. destructor parasitism reduces honey bee emergence and hemocyte concentration, causes deformities in wings of the bees and shortens the adult lifespan of Africanized bees in Mexico.
2.2. Effect of V. destructor parasitism on bee emergence, hemocyte concentration and occurrence of wing deformities
2. Materials and methods
2.3. Effect of V. destructor parasitism on adult bee lifespan
2.1. Sources of mites and honey bee brood
The adult bees that emerged during the previous experiment were placed in hoarding cages (12.7 × 8.5 × 14.5 cm) and were provided with two feeders, one containing 50% sucrose syrup and the other distilled water. The cages were held in an incubator (33 °C, 60% RH) to record bee mortality every 24 h until all bees in each cage had died. The experiment was repeated three times.
Combs containing brood from the source colonies were retrieved to be artificially inoculated by mites in newly capped cells. To ensure that the cells containing larvae used for the experiments had been capped recently (< 12 h), previous to retrieving the combs, mature larvae (5 d old) in uncapped cells of those combs were monitored three times daily by placing transparent acetate sheets above the combs, and the area containing open cells with mature larvae was delimited on the sheets using a marker. When the cells were capped, the combs were retrieved from the hives and used for the experiments. Between 150 and 200 newly capped larvae were subjected to one of the following two treatments. The experimental treatment consisted of cells in which two female V. destructor mites were introduced. First, the cells were painted on their outer rims with water based, nontoxic markers, to identify the treatment. Then, the cappings of these cells were opened by cutting a thin slit with a sterile blade, and the mites were introduced into the cells with a fine paintbrush. The cells were then resealed by brushing the cappings with liquid beeswax. The control treatment consisted of cells that were treated as above, but no mites were introduced. The brood cells for each treatment were covered with a wire mesh screened cage (3 mm) that was embedded manually on the comb to capture any emerging bees from the treated cells. The frames were held in screened emergence wooden cages inside an incubator at 33 °C and 60% RH until the bees emerged from their cells. The number and percentage of emerged and non-emerged bees for each treatment was determined. Additionally, the number and percentage of bees that emerged with wing deformities was determined by ocular inspection, as the bees were transferred to hoarding cages (see below). To ascertain parasitism, all bees from the infested treatment were inspected for the presence of at least one live mite. Also, subsamples of 10 newly emerged parasitized and non-parasitized bees were randomly collected and used to obtain hemolymph samples for hemocyte quantification. At least 4 µl hemolymph were obtained per bee by piercing the membrane between the second and third dorsal tergite with a #7 entomological pin (Bioquip, Dominguez, CA, USA). The hemolymph was collected with the aid of a micropipette and spread over a microscope slide. The hemocytes were stained and quantified as described by Koleoglu et al. (2018). The experiment was repeated three times.
Our study was conducted at the Environmental Educational Centre in Xochimilco, Mexico (19°15.5′N, 99° 6.3′W). Bees from several colonies maintained at the Centre were sampled to confirm their genotype (Africanized or European) through morphometric and mitochondrial DNA analyses (Nielsen et al., 1999) and to determine their V. destructor infestation levels as per De Jong et al. (1982a). Five Africanized colonies with V. destructor infestation levels > 5% were selected as sources of mites for the experiments. Another three Africanized bee colonies with low mite infestation levels (< 1%) were also selected and treated with fluvalinate strips (Apistan®) as per label instructions. These colonies were separated from the infested colonies and served as sources of mite-free brood for the experiments; examination of adult bees and brood confirmed the absence of mites. Female V. destructor mites were collected by uncapping cells containing worker bee pupae with purple eyes, and then transferring mites from parasitized cells to a Petri dish using a fine paintbrush. Subsamples of the mites were placed into sterile 2 ml microcentrifuge tubes containing RNA Later® solution (life Technologies Inc., Burlington, ON, Canada) to determine the presence of deformed wing virus (DWV), as well as haplotype. The mites in the Petri dish were used within 1 h of collection.
2.4. Identification of DWV in V. destructor mites The samples of varroa mites were analysed by reverse transcriptionPCR (RT-PCR) for DWV presence at the University of Guelph’s Honey Bee Research Centre, in Guelph, Ontario, Canada, using standard methods (de Miranda et al., 2013). Briefly, total RNA was extracted from about 50 mites per sample (Chen et al., 2000). For cDNA synthesis, 2 µg of total RNA was reverse-transcribed using Oligo (dT)18 and M-MuLV RT with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas Life Sciences; Burlington, Ontario), following the instructions of the manufacturer. The primers used to detect DWV were the forward primer of Chen et al. (2005), and the reverse primer of Guzman-Novoa et al. (2012b). All PCR reactions were done with a Mastercycler (Eppendorf, Mississauga, Ontario). Each 15 µl of reaction contained 1.5 µl of 10x PCR buffer (New England BioLabs; Pickering, Ontario), 0.5 µl 10 mM of dNTPs (Bio Basic Inc.; Markham, Ontario), 44
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1 µl of 10 µM for forward and reverse primers, 0.2 µl 5U/µl of Taq polymerase (New England BioLabs; Pickering, Ontario), 1 µl of the cDNA sample, and 7.8 µl of dd H2O. The thermocycler ran at 94 °C for 3 min, followed by 35 cycles of 30 s at 94 °C, 60 s at 58 °C and 60 s at 72 °C, and a final extension step at 72 °C for 10 min. PCR products were separated by electrophoresis in 1.1% agarose gels and stained with ethidium bromide. The amplified bands were captured in photographs using a digital camera with a Benchtop UV Transilluminator (BioDocIt™ Imaging System; Upland, CA, USA). For positive identifications, randomly selected PCR products were purified using the EZ-10 Spin Column DNA Gel Extraction Kit (Bio Basic Canada Inc.; Markham, Ontario, Canada) and sequenced at the Laboratory Services of the University of Guelph. 2.5. Mite haplotype analysis The haplotype analysis was conducted to determine if the mites were of the Korean or the Japanese haplotype because differences in mite pathogenicity have been reported for these varroa mite haplotypes (Solignac et al., 2005). Ten mites were randomly assayed per sample and assessed for mt DNA type as per Solignac et al. (2005). 2.6. Statistical analyses The data on the number of bees that emerged and did not emerge from the different treatments were subjected to contingency table analyses and Chi square values were obtained. The same statistics were used to compare treatments for bees with damaged and undamaged wings. Hemocyte counts for the two treatments were natural log (ln) transformed because they were not normally distributed and then were analysed using a student t test. Percent daily survivorship of the bees was calculated for the treatments and the data on the lifespan of the adult bees were analysed using the nonparametric Kaplan-Meier method. The estimated survival functions were compared with a nonparametric logrank test. All statistical analyses were performed with the R Statistical Program (R Development Core Team, Auckland, New Zealand) with the significance level set at P < 0.05.
Fig. 1. Comparison between a honey bee that emerged from a Varroa destructor infested cell (below) and a bee that emerged from a non-infested cell (above). A clear reduction in body size as well as deformed and undeveloped wings in the parasitized bee was observed.
bees, respectively.
3.3. Effect of V. destructor parasitism on the occurrence of wing deformities The proportion of bees with deformed wings that emerged from V. destructor-parasitized cells was significantly higher than that of the control group. All bees (100%) from the control group had normal wings, compared to 46% of the workers that emerged from parasitized cells; the other 54% had wing deformities (Fig. 1). The difference between the two groups was significant (P < 0.0001; Table 2).
3. Results 3.1. Effect of V. destructor parasitism on bee emergence The proportion of bees that emerged from V. destructor-parasitized cells was significantly lower than that of the control group. The proportion of non-emerged bees (brood mortality) was 31 and 4%, for the experimental and control treatments, respectively (P < 0.0001; Table 1).
3.4. Effect of V. destructor parasitism on adult bee lifespan The lifespan of adult bees that emerged from V. destructor-parasitized cells was significantly shorter than that of the bees emerging from non-parasitized cells (χ2 = 257, df = 1, P < 0.0001). Bees from parasitized cells lived a maximum of 19 days in two of the three replicates of the experiment. Conversely, bees that emerged from nonparasitized cells lived up to 40 days (Fig. 2). The mean length of life of bees emerged from infested and non-infested cells was 8.5 ± 0.3 and 14.4 ± 0.4 days, respectively.
3.2. Effect of V. destructor parasitism on hemocyte concentration The concentration of hemocytes in the hemolymph of bees that emerged from V. destructor-parasitized cells was significantly lower than that of bees emerged from non-parasitized cells (t = −3.4, df = 58, P = 0.0011). The mean number of hemocytes/µL hemolymph was 1834 ± 129 and 1291 ± 85 for non-parasitized and parasitized Table 1 Number of emerged and non-emerged Africanized honey bee adults and proportion of emerged bees from brood cells infested and non-infested with Varroa destructor mites. Variable
Infested brood
Control group
No. emerged bees No. non-emerged bees Proportion emerged bees
385 170 0.69
568 26 0.96
Table 2 Number and proportion of Africanized honey bees that emerged with normal or deformed wings from brood cells infested and non-infested with Varroa destructor mites.
χ2
Variable
Infested brood
Control group
139.8 P < 0.0001
No. bees with normal wings No. bees with deformed wings Proportion bees with deformed wings
177 208 0.54
568 0 0.00
45
χ2
282.0 P < 0.0001
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Fig. 2. Probability of survivorship of adult worker honey bees that emerged from cells infested with Varroa destructor mites (red) and non-infested cells (green). Survival functions were estimated with the Kaplan-Meier method. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Jong et al. (1982b) showed that weight of Africanized bees emerging from cells naturally infested with Varroa mites was significantly lower than that of bees emerging from non-parasitized cells. They also observed wing damage in some newly emerged bees but reported that these physical deformities were not common. V. destructor parasitism resulted in a reduction of hemocyte concentration in the hemolymph of newly-emerged Africanized bees. This is the first report of this effect in developing bees. Koleoglu et al. (2018) previously reported that adult Africanized and European bees also lose hemocytes as a consequence of Varroa mite infestations. The implications of this loss of hemocytes are unknown but may have immunosuppressive consequences making the bees more susceptible to other pathogens. Additionally, the lower concentration of hemocytes could retard the healing of wounds caused by Varroa mites because hemocytes play an important role in clotting (Theopold et al., 2002). Clearly, this and the above cited studies are consistent with the hypothesis that the tolerance of Africanized bees to the Varroa mite does not seem to reside in mechanisms associated with their brood. The percentage of bees that emerged with deformities in their wings was significantly higher in the mite-infested bees compared to the noninfested bees (54% vs. 0%). Similar to our study using Africanized honey bees, Bowen-Walker et al. (1999) found that > 80% of European honey bees emerging from cells inoculated with two Varroa mites had deformities. Bowen-Walker et al. (1999) also detected DWV infections in the bees showing wing deformities. Similarly, in our study with Africanized bees, more than half the Varroa mite-infested bees emerged with wing damage. Bees were not analyzed for DWV presence, but subsamples of the mites used in the experiments were all positive for DWV. Our results, combined with observations of typical symptoms of overt DWV infection (Genersch and Aubert, 2010), allow us to presume that those deformities were caused by DWV infections in the bees. That 100% of bees emerging from non-infested cells did not have wing damage reinforces this assumption. Martin et al. (2012) demonstrated that mite parasitism of the brood is required for wing damage symptoms to occur in adult bees. It has also been reported that DWV replicates in the Varroa mites and when high titers of the virus are transmitted to larvae and pupae by the parasites, the emerging bees show wing damage (Bowen-Walker et al., 1999; Yue and Genersch, 2005), although there is controversy on whether replication of DWV occurs in the mites (Santillán-Galicia et al., 2008; Wilfert et al., 2016). The results of our study suggest that DWV infections were transmitted by the mites and that these infections are very damaging to Africanized bees, although perhaps relatively less so than to European bees. Hamiduzzaman et al. (2015) reported that the rate of increase of DWV infections in Africanized bees was lower than in European bees.
3.5. Identification of DWV in V. destructor mites The three V. destructor mite subsamples analysed were positive for DWV. PCR products of the reactions were sequenced (Laboratory services, University of Guelph) and the sequences (see supplementary material 1) were compared with viral sequences in GenBank (accession No. NC_004830). All sequences produced in our study showed ≥96% identity. 3.6. Mite haplotype analysis Only the Korean haplotype was detected in the mite samples. 4. Discussion We determined, in the same individual bees, the rate of emergence, hemocyte concentration, percent wing damage, and adult survivorship of Africanized honey bees parasitized with V. destructor mites or nonparasitized during the pupal stage. We showed that the health of brood and adult bees of Africanized bees in Mexico is severely affected by V. destructor parasitism. The mite was associated with more than 30% brood mortality, a 30% reduction in hemocyte concentration, and a 40% reduction in lifespan of adult honey bees. Additionally, parasitized bees were affected by wing malformations, possibly as a consequence of DWV infections transmitted by the mite. More than 50% of the bees that emerged from parasitized cells had deformities in the wings versus 0% in the bees that emerged from non-parasitized cells. It is important however, to consider that this study was conducted in the laboratory under controlled conditions and thus, it is possible that the effects observed might be less severe under field conditions. Few studies have documented the negative effects of V. destructor on bee development and longevity, particularly in Africanized honey bees. We clearly showed that populations of Africanized bees in Mexico have low tolerance to V. destructor parasitism at the brood stage; 31% of the infested brood died within their cells compared to only 4% of the control group. By comparison, Bowen-Walker et al. (1999), using a similar methodology, found that 54% of the brood of European bees artificially parasitized with between one and six mites of V. destructor, emerged (representing 46% brood mortality). Similarly, in Brazil, Mattos and Chaud-Netto (2012) inferred high mortality rates of pupae in colonies of Africanized bees that were naturally infested with V. destructor mites and reported a significant correlation between brood mortality and level of mite parasitism. However, Mattos and ChaudNetto (2012) did not observe or quantify bee emergence and did not verify the presence of mites in cells with dead pupae. Previously, De 46
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to EG. Mexico’s National Council of Science and Technology provided a scholarship to MR.
We also showed that adult Africanized bees in Mexico have significantly shorter lifespans if they are parasitized by V. destructor during the brood stage compared with non-parasitized bees. This result agrees with that of De Jong and De Jong (1983) for naturally parasitized Africanized bees in Brazil. In contrast, another study conducted in Mexico with Africanized bees did not find differences in longevity between adult bees emerging from mite parasitized and non-parasitized cells (Romero-Vera and Otero-Colina, 2002). One possible explanation is that Romero-Vera and Otero-Colina (2002) used bees that were infested with tracheal mites (Acarapis woodi) in their non-Varroa control group, and given that Acarapis woodi also affects the health of honey bees in similar ways to Varroa, the effects caused by each mite could not be separated and may have confounded the results. The Varroa mites were of the Korean haplotype, as were mites recovered in two large regions of Mexico in another study (AnguianoBaez et al., 2016). The Korean haplotype is considered the most pathogenic of the V. destructor haplotypes (Solignac et al., 2005), possibly explaining some of the damaging effects on the health of Africanized bees we observed. However, because DWV is also highly pathogenic to honey bees (McMenamin and Genersch, 2015), it is possible that the reduction of the adult lifespan observed in this study was at least partially a consequence of DWV infections. Yang and Cox-Foster (2007) and Dainat et al. (2012) reported that DWV infections significantly reduce the lifespan of honey bees; future studies that assess the effects of single and multiple pathogens on the health of Africanized bees should quantify the relative effect of DWV infections and V. destructor parasitism on bee longevity. Our results indicate that V. destructor parasitism and DWV infections are highly detrimental to the health of Africanized bees in Mexico and could negatively impact colonies. A significantly lower rate of adult bee emergence, many with deformities, inhibited immune cellular responses and shorter livespans, would result in weaker colonies that cannot secure and store adequate food resources; a situation that is less productive for the beekeeper and has a higher risk of colony collapse compared with healthy colonies. Our results also suggest that the tolerance of Africanized bees to V. destructor infestations, which has been demonstrated in numerous studies (Moretto et al., 1991; De Jong, 1997, Guzman-Novoa et al., 1999, 2011; Medina-Flores et al., 2014), does not seem to rely on mechanisms associated with their brood, for example, suppressed mite reproduction. It is possible that the tolerance of Africanized bees to these mites resides more in mechanisms associated with and performed by adult bees, for example, hygienic and grooming behavior (Boecking and Spivak, 1999; Guerra et al., 2000; ArechavaletaVelasco and Guzman-Novoa, 2001; Guzman-Novoa et al., 2012a). In conclusion, we demonstrated that the development, cellular immunity, wing integrity and lifespan of Africanized honey bees in Mexico, are adversely affected by V. destructor parasitism and DWV infections. Our results do not suggest that Africanized bees in Mexico are more tolerant to V. destructor than Africanized bees in Brazil or European bees elsewhere for the parameters measured, nor that the tolerance of Africanized bees to V. destructor resides in brood-related mechanisms. Comparative studies between Africanized bees from different neotropical regions, as well as between these bees and European bees, are necessary not only to assess the relative damage caused by Varroa mite infestations and viral infections, but to quantify the amount of damage that these parasites cause in different genotypes of bees, and the specific implications resulting from these effects.
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Guzman-Novoa, E., Emsen, B., Unger, P., Espinosa-Montaño, L.G., Petukhova, T., 2012a. Genotypic variability and relationships between mite infestation levels, mite damage, grooming intensity, and removal of Varroa destructor mites in selected strains of worker honey bees (Apis mellifera L.). J. Invertebr. Pathol. 110, 314–320. Guzman-Novoa, E., Hamiduzzaman, M.M., Espinosa-Montaño, L.G., Correa-Benitez, A., Ponce-Vazquez, R., 2012b. First detection of four viruses in honey bee (Apis mellifera) workers with and without deformed wings and Varroa destructor in Mexico. J. Apic. Res. 51, 342–346. https://doi.org/10.3896/IBRA.1.51.4.08. Hamiduzzaman, M.M., Guzman-Novoa, E., Goodwin, P.H., Reyes-Quintana, M., Koleoglu,
Acknowledgments We are grateful to Nicolas Mejia and Margarita Flores from the Environmental Educational Centre in Xochimilco, Mexico for their assistance. We are also grateful to Mollah Hamiduzzaman for performing the DWV analyses in varroa mite samples. This study was partially funded by a National University of Mexico grant to AC and by a grant from the Natural Sciences and Engineering Research Council of Canada 47
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Identification of Africanized honey bees (Hymenoptera: Apidae) incorporating morphometrics and an improved PCR mitotyping procedure. Ann. Entomol. Soc. Am. 92, 167–174. Romero-Vera, C., Otero-Colina, G., 2002. Effect of single and successive infestation of Varroa destructor and Acarapis woodi on the longevity of worker honey bees Apis mellifera. Am. Bee J. 142, 54–57. Rosenkranz, P., Aumeier, P., Ziegelmann, B., 2010. Biology and control of Varroa destructor. J. Invertebr. Pathol. 103, 96–119. Sammataro, D., Gerson, U., Needham, G., 2000. Parasitic mites of honey bees: life history, implications and impact. Annu. Rev. Entomol. 45, 519–548. Santillán-Galicia, M.T., Carzaniga, R., Ball, B.V., Alderson, P.G., 2008. Immunolocalization of deformed wing virus particles within the mite Varroa destructor. J. Gen. Virol. 89, 1685–1689. Solignac, M., Cournet, J.M., Vautrin, D., Le Conte, Y., Anderson, D., Evans, J., Cros-Arteli, S., Navajas, M., 2005. The invasive Korean and Japan types of Varroa destructor, ectoparasitic mites of the Western honeybee (Apis mellifera), are two partially isolated clones. Proc. R. Soc. B 272, 411–419. Theopold, U., Li, D., Fabbri, M., Schefer, C., Schmidt, O., 2002. The coagulation of insect hemolymph. Cell. Mol. Life Sci. 59, 363–372. Wilfert, L., Long, G., Leggett, H.C., Schmid-Hempel, P., Butlin, R., Martin, S.J.M., Boots, M., 2016. Deformed wing virus is a recent global epidemic in honeybees driven by Varroa mites. Science 351, 594–597. Yang, X., Cox-Foster, D.L., 2005. Impact of an ectoparasite on the immunity and pathology of an invertebrate: evidence for host immunosuppression and viral amplification. Proc. Natl. Acad. Sci. USA 102, 7470–7475. Yang, X., Cox-Foster, D.L., 2007. Effects of parasitization by Varroa destructor on survivorship and physiological traits of Apis mellifera in correlation with viral incidence microbial challenge. Parasitol. 134, 405–412. Yue, C., Genersch, E., 2005. RT-PCR analysis of deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor). J. Gen. Virol. 86, 3419–3424.
G., Correa-Benitez, A., Petukhova, T., 2015. Differential responses of Africanized and European honey bees (Apis mellifera) to viral replication following mechanical transmission or Varroa destructor parasitism. J. Invertebr. Pathol. 126, 12–20. https:// doi.org/10.1016/j.jip.2014.12.004. Kevan, P.G., Hannan, M.A., Ostiguy, N., Guzman-Novoa, E., 2006. A summary of the varroa-virus disease complex in honey bees. Am. Bee J. 146, 694–697. Koleoglu, G., Goodwin, P.H., Reyes-Quintana, M., Hamiduzzaman, M.M., Guzman-Novoa, E., 2017. Effect of Varroa destructor, wounding and varroa homogenate on gene expression in brood and adult honey bees. PLoS ONE 12 (1), e0169669. https://doi. org/10.1371/journal.pone.0169669. Koleoglu, G., Goodwin, P.H., Reyes-Quintana, M., Hamiduzzaman, M.M., Guzman-Novoa, E., 2018. Varroa destructor parasitism reduces hemocyte concentrations and prophenol oxidase gene expression in bees from two populations. Parasitol. Res. 117, 1175–1183. https://doi.org/10.1007/s00436-018-5796-8. Martin, S.J., Highfield, A.C., Brettell, L., Villalobos, E.M., Budge, G.E., Powell, M., Nikaido, S., Schroeder, D.C., 2012. Global honey bee viral landscape altered by a parasitic mite. Science 336, 1304–1306. Mattos, I.M., Chaud-Netto, J., 2012. Analysis of mortality in Africanized honey bee colonies with high levels of infestation by Varroa destructor. Sociobiol. 59, 1–12. McMenamin, A.J., Genersch, E., 2015. Honey bee colony losses and associated viruses. Curr. Opin. Insect Sci. 8, 121–129. Medina-Flores, C., Guzman-Novoa, E., Aréchiga-Flores, C.F., Aguilera-Soto, J.I., GutiérezPiña, F.J., 2011. Effect of Varroa destructor infestations on honey yields of Apis mellifera colonies in Mexico’s semi-arid high plateau. Rev. Mex. Cienc. Pecu. 2, 313–317. Medina-Flores, C.A., Guzman-Novoa, E., Hamiduzzaman, M.M., Aréchiga-Flores, C.F., López-Carlos, A., 2014. Africanized honey bees (Apis mellifera) have low infestation levels of the mite Varroa destructor in different ecological regions in Mexico. Gen. Mol. Res. 13, 7282–7293. Moretto, G., Goncalves, L.S., De Jong, D., Bichuette, M.Z., 1991. The effects of climate and bee race on Varroa jacobsoni Oud. infestations in Brazil. Apidologie 22, 197–203. Nielsen, D.I., Ebert, P.R., Hunt, G.J., Guzman-Novoa, E., Kinee, S.A., Page Jr, R.E., 1999.
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Impact of Varroa destructor and deformed wing virus on emergence, cellular immunity, wing integrity and survivorship of Africanized honey bees in Mexico
T
Mariana Reyes-Quintanaa, Laura G. Espinosa-Montañob, Daniel Prieto-Merlosb, Gun Koleoglua, ⁎ Tatiana Petukhovaa, Adriana Correa-Benítezb, Ernesto Guzman-Novoaa, a b
School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada Departamento de Medicina y Zootecnia en Abejas, FMVZ, UNAM, Cd. Univ., Mexico 04510, Mexico
A R T I C LE I N FO
A B S T R A C T
Keywords: Varroa destructor Deformed wing virus Africanized bees Worker emergence Hemocytes Survivorship
The ectoparasitic mite Varroa destructor is the primary health problem of honey bees (Apis mellifera) worldwide. Africanized honey bees in Brazil have demonstrated tolerance to the mite, but there is controversy about the degree of mite tolerance of Africanized bees in other countries. This study was conducted to quantify the effect of V. destructor parasitism on emergence, hemocyte concentration, wing integrity and longevity of Africanized honey bees in Mexico. Africanized bee brood were artificially infested with V. destructor mites and held in an incubator until emergence as adults and compared to non-infested controls. Deformed wing virus (DWV) presence was determined in the mites used to infest the bees. After emergence, the bees were maintained in an incubator to determine survivorship. The percentage of worker bees that emerged from parasitized cells (69%) was significantly lower than that of bees emerged from non-infested cells (96%). Newly-emerged parasitized bees had a significantly lower concentration of hemocytes in the hemolymph than non-parasitized bees. Additionally, the proportion of bees with deformed wings that emerged from V. destructor-parasitized cells was significantly higher (54%) than that of the control group (0%). The mean survival time of bees that emerged from infested and non-infested cells was 8.5 ± 0.3 and 14.4 ± 0.4 days, respectively, and the difference was significant. We conclude that V. destructor parasitism and DWV infections kill, cause deformities and inhibit cellular immunity in developing Africanized honey bees, and significantly reduce the lifespan of adult bees in Mexico. These results suggest that the tolerance of Africanized bees to V. destructor is related to adult bee mechanisms.
1. Introduction
humoral (Yang and Cox-Foster, 2005; Koleoglu et al., 2017) and cellular immune responses (Koleoglu et al., 2018), and transmit pathogenic viruses to the bees (Kevan et al., 2006; Genersch and Aubert, 2010; Anguiano-Baez et al., 2016). The deformed wing virus (DWV), in particular, causes severe morphological deformities and has been linked to winter colony mortality (Bowen-Walker et al., 1999; Dainat et al., 2012). Numerous studies have demonstrated that adult Africanized honey bees (descendants of A. m. scutellata) in the neotropics are more tolerant to the Varroa mite than their European counterparts (Moretto et al., 1991; De Jong, 1997; Guzman-Novoa et al., 1999, 2011; Medina-Flores et al., 2014), although the basis of this tolerance is still not well understood. However, despite evidence that indicates a certain degree of tolerance of Africanized bees to V. destructor mites, few studies have
Varroa destructor parasitism is the most serious health problem of western honey bee (Apis mellifera) populations worldwide. Relatively low levels of V. destructor infestation cause colonies to collapse in temperate climate countries (Rosenkranz et al., 2010) and the mite has been found to be associated with more than 85% of colony mortality cases during winter in temperate regions (Guzman-Novoa et al., 2010). Varroa mite infestations also decrease honey yields of different bee strains, including Africanized bees (Arechavaleta-Velasco and GuzmanNovoa, 2000; Medina-Flores et al., 2011; Emsen et al., 2014). These and other detrimental effects on the bees result from several types of damage caused by V. destructor. The mites feed on the hemolymph and fat tissue of brood and adult bees (Sammataro et al., 2000), inhibit their
⁎
Corresponding author. E-mail address: [email protected] (E. Guzman-Novoa).
https://doi.org/10.1016/j.jip.2019.04.009 Received 13 December 2018; Received in revised form 24 April 2019; Accepted 26 April 2019 Available online 26 April 2019 0022-2011/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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been conducted to analyze the actual damage that the mite causes in Africanized bees during their developmental stage. Several studies have documented the effects of V. destructor parasitism in developing European honey bees. For example, Bowen-Walker and Gunn (2001) found that bees emerging from V. destructor-parasitized cells weighed significantly less and had lower concentrations of protein than bees emerging from non-parasitized cells. Similar results were reported by De Jong et al. (1982b) for Africanized bees in Brazil. Bowen-Walker et al. (1999) found that only 54% of the European bees in their study emerged from cells that had been artificially parasitized with V. destructor mites. However, reports for rate of emergence from developing Africanized honey bees that are artificially parasitized with Varroa mites are lacking in the literature. Similarly, there are no reports of hemocyte concentrations in newly emerged bees parasitized by V. destructor mites. Hemocytes are cells with immune response functions that circulate in the hemolymph of insects. One of the main roles of hemocytes is to contribute to wound healing by aggregating at a wound site and releasing granular components and chemicals that form a clot (Theopold et al., 2002). Because V. destructor causes wounds in the honey bee cuticle and feeds upon its hemolymph, it would be expected that the concentration of hemocytes in the hemolymph of parasitized bees would decrease during the developmental stage of the insects. With regards to adult bee longevity, De Jong and De Jong (1983) in Brazil, found that Africanized bees emerging from Varroa-parasitized cells had a 50% shorter adult lifespan than bees emerging from nonparasitized cells. De Jong and De Jong (1983) however, did not measure the impact that Varroa parasitism has on the rate of bee emergence. Another study conducted with Africanized bees in Mexico and using similar methodology to that of De Jong and De Jong (1983), did not find differences in longevity of worker bees that were and were not parasitized by V. destructor (Romero-Vera and Otero-Colonia, 2002). This was in contrast with the results from Brazil, and suggested that the brood of Africanized bees in Mexico might be more tolerant to the mite. No other similar studies have been conducted with Africanized bees in both Mexico and Brazil to corroborate or challenge previous findings. We conducted this study to test the hypothesis that V. destructor parasitism reduces honey bee emergence and hemocyte concentration, causes deformities in wings of the bees and shortens the adult lifespan of Africanized bees in Mexico.
2.2. Effect of V. destructor parasitism on bee emergence, hemocyte concentration and occurrence of wing deformities
2. Materials and methods
2.3. Effect of V. destructor parasitism on adult bee lifespan
2.1. Sources of mites and honey bee brood
The adult bees that emerged during the previous experiment were placed in hoarding cages (12.7 × 8.5 × 14.5 cm) and were provided with two feeders, one containing 50% sucrose syrup and the other distilled water. The cages were held in an incubator (33 °C, 60% RH) to record bee mortality every 24 h until all bees in each cage had died. The experiment was repeated three times.
Combs containing brood from the source colonies were retrieved to be artificially inoculated by mites in newly capped cells. To ensure that the cells containing larvae used for the experiments had been capped recently (< 12 h), previous to retrieving the combs, mature larvae (5 d old) in uncapped cells of those combs were monitored three times daily by placing transparent acetate sheets above the combs, and the area containing open cells with mature larvae was delimited on the sheets using a marker. When the cells were capped, the combs were retrieved from the hives and used for the experiments. Between 150 and 200 newly capped larvae were subjected to one of the following two treatments. The experimental treatment consisted of cells in which two female V. destructor mites were introduced. First, the cells were painted on their outer rims with water based, nontoxic markers, to identify the treatment. Then, the cappings of these cells were opened by cutting a thin slit with a sterile blade, and the mites were introduced into the cells with a fine paintbrush. The cells were then resealed by brushing the cappings with liquid beeswax. The control treatment consisted of cells that were treated as above, but no mites were introduced. The brood cells for each treatment were covered with a wire mesh screened cage (3 mm) that was embedded manually on the comb to capture any emerging bees from the treated cells. The frames were held in screened emergence wooden cages inside an incubator at 33 °C and 60% RH until the bees emerged from their cells. The number and percentage of emerged and non-emerged bees for each treatment was determined. Additionally, the number and percentage of bees that emerged with wing deformities was determined by ocular inspection, as the bees were transferred to hoarding cages (see below). To ascertain parasitism, all bees from the infested treatment were inspected for the presence of at least one live mite. Also, subsamples of 10 newly emerged parasitized and non-parasitized bees were randomly collected and used to obtain hemolymph samples for hemocyte quantification. At least 4 µl hemolymph were obtained per bee by piercing the membrane between the second and third dorsal tergite with a #7 entomological pin (Bioquip, Dominguez, CA, USA). The hemolymph was collected with the aid of a micropipette and spread over a microscope slide. The hemocytes were stained and quantified as described by Koleoglu et al. (2018). The experiment was repeated three times.
Our study was conducted at the Environmental Educational Centre in Xochimilco, Mexico (19°15.5′N, 99° 6.3′W). Bees from several colonies maintained at the Centre were sampled to confirm their genotype (Africanized or European) through morphometric and mitochondrial DNA analyses (Nielsen et al., 1999) and to determine their V. destructor infestation levels as per De Jong et al. (1982a). Five Africanized colonies with V. destructor infestation levels > 5% were selected as sources of mites for the experiments. Another three Africanized bee colonies with low mite infestation levels (< 1%) were also selected and treated with fluvalinate strips (Apistan®) as per label instructions. These colonies were separated from the infested colonies and served as sources of mite-free brood for the experiments; examination of adult bees and brood confirmed the absence of mites. Female V. destructor mites were collected by uncapping cells containing worker bee pupae with purple eyes, and then transferring mites from parasitized cells to a Petri dish using a fine paintbrush. Subsamples of the mites were placed into sterile 2 ml microcentrifuge tubes containing RNA Later® solution (life Technologies Inc., Burlington, ON, Canada) to determine the presence of deformed wing virus (DWV), as well as haplotype. The mites in the Petri dish were used within 1 h of collection.
2.4. Identification of DWV in V. destructor mites The samples of varroa mites were analysed by reverse transcriptionPCR (RT-PCR) for DWV presence at the University of Guelph’s Honey Bee Research Centre, in Guelph, Ontario, Canada, using standard methods (de Miranda et al., 2013). Briefly, total RNA was extracted from about 50 mites per sample (Chen et al., 2000). For cDNA synthesis, 2 µg of total RNA was reverse-transcribed using Oligo (dT)18 and M-MuLV RT with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas Life Sciences; Burlington, Ontario), following the instructions of the manufacturer. The primers used to detect DWV were the forward primer of Chen et al. (2005), and the reverse primer of Guzman-Novoa et al. (2012b). All PCR reactions were done with a Mastercycler (Eppendorf, Mississauga, Ontario). Each 15 µl of reaction contained 1.5 µl of 10x PCR buffer (New England BioLabs; Pickering, Ontario), 0.5 µl 10 mM of dNTPs (Bio Basic Inc.; Markham, Ontario), 44
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1 µl of 10 µM for forward and reverse primers, 0.2 µl 5U/µl of Taq polymerase (New England BioLabs; Pickering, Ontario), 1 µl of the cDNA sample, and 7.8 µl of dd H2O. The thermocycler ran at 94 °C for 3 min, followed by 35 cycles of 30 s at 94 °C, 60 s at 58 °C and 60 s at 72 °C, and a final extension step at 72 °C for 10 min. PCR products were separated by electrophoresis in 1.1% agarose gels and stained with ethidium bromide. The amplified bands were captured in photographs using a digital camera with a Benchtop UV Transilluminator (BioDocIt™ Imaging System; Upland, CA, USA). For positive identifications, randomly selected PCR products were purified using the EZ-10 Spin Column DNA Gel Extraction Kit (Bio Basic Canada Inc.; Markham, Ontario, Canada) and sequenced at the Laboratory Services of the University of Guelph. 2.5. Mite haplotype analysis The haplotype analysis was conducted to determine if the mites were of the Korean or the Japanese haplotype because differences in mite pathogenicity have been reported for these varroa mite haplotypes (Solignac et al., 2005). Ten mites were randomly assayed per sample and assessed for mt DNA type as per Solignac et al. (2005). 2.6. Statistical analyses The data on the number of bees that emerged and did not emerge from the different treatments were subjected to contingency table analyses and Chi square values were obtained. The same statistics were used to compare treatments for bees with damaged and undamaged wings. Hemocyte counts for the two treatments were natural log (ln) transformed because they were not normally distributed and then were analysed using a student t test. Percent daily survivorship of the bees was calculated for the treatments and the data on the lifespan of the adult bees were analysed using the nonparametric Kaplan-Meier method. The estimated survival functions were compared with a nonparametric logrank test. All statistical analyses were performed with the R Statistical Program (R Development Core Team, Auckland, New Zealand) with the significance level set at P < 0.05.
Fig. 1. Comparison between a honey bee that emerged from a Varroa destructor infested cell (below) and a bee that emerged from a non-infested cell (above). A clear reduction in body size as well as deformed and undeveloped wings in the parasitized bee was observed.
bees, respectively.
3.3. Effect of V. destructor parasitism on the occurrence of wing deformities The proportion of bees with deformed wings that emerged from V. destructor-parasitized cells was significantly higher than that of the control group. All bees (100%) from the control group had normal wings, compared to 46% of the workers that emerged from parasitized cells; the other 54% had wing deformities (Fig. 1). The difference between the two groups was significant (P < 0.0001; Table 2).
3. Results 3.1. Effect of V. destructor parasitism on bee emergence The proportion of bees that emerged from V. destructor-parasitized cells was significantly lower than that of the control group. The proportion of non-emerged bees (brood mortality) was 31 and 4%, for the experimental and control treatments, respectively (P < 0.0001; Table 1).
3.4. Effect of V. destructor parasitism on adult bee lifespan The lifespan of adult bees that emerged from V. destructor-parasitized cells was significantly shorter than that of the bees emerging from non-parasitized cells (χ2 = 257, df = 1, P < 0.0001). Bees from parasitized cells lived a maximum of 19 days in two of the three replicates of the experiment. Conversely, bees that emerged from nonparasitized cells lived up to 40 days (Fig. 2). The mean length of life of bees emerged from infested and non-infested cells was 8.5 ± 0.3 and 14.4 ± 0.4 days, respectively.
3.2. Effect of V. destructor parasitism on hemocyte concentration The concentration of hemocytes in the hemolymph of bees that emerged from V. destructor-parasitized cells was significantly lower than that of bees emerged from non-parasitized cells (t = −3.4, df = 58, P = 0.0011). The mean number of hemocytes/µL hemolymph was 1834 ± 129 and 1291 ± 85 for non-parasitized and parasitized Table 1 Number of emerged and non-emerged Africanized honey bee adults and proportion of emerged bees from brood cells infested and non-infested with Varroa destructor mites. Variable
Infested brood
Control group
No. emerged bees No. non-emerged bees Proportion emerged bees
385 170 0.69
568 26 0.96
Table 2 Number and proportion of Africanized honey bees that emerged with normal or deformed wings from brood cells infested and non-infested with Varroa destructor mites.
χ2
Variable
Infested brood
Control group
139.8 P < 0.0001
No. bees with normal wings No. bees with deformed wings Proportion bees with deformed wings
177 208 0.54
568 0 0.00
45
χ2
282.0 P < 0.0001
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Fig. 2. Probability of survivorship of adult worker honey bees that emerged from cells infested with Varroa destructor mites (red) and non-infested cells (green). Survival functions were estimated with the Kaplan-Meier method. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Jong et al. (1982b) showed that weight of Africanized bees emerging from cells naturally infested with Varroa mites was significantly lower than that of bees emerging from non-parasitized cells. They also observed wing damage in some newly emerged bees but reported that these physical deformities were not common. V. destructor parasitism resulted in a reduction of hemocyte concentration in the hemolymph of newly-emerged Africanized bees. This is the first report of this effect in developing bees. Koleoglu et al. (2018) previously reported that adult Africanized and European bees also lose hemocytes as a consequence of Varroa mite infestations. The implications of this loss of hemocytes are unknown but may have immunosuppressive consequences making the bees more susceptible to other pathogens. Additionally, the lower concentration of hemocytes could retard the healing of wounds caused by Varroa mites because hemocytes play an important role in clotting (Theopold et al., 2002). Clearly, this and the above cited studies are consistent with the hypothesis that the tolerance of Africanized bees to the Varroa mite does not seem to reside in mechanisms associated with their brood. The percentage of bees that emerged with deformities in their wings was significantly higher in the mite-infested bees compared to the noninfested bees (54% vs. 0%). Similar to our study using Africanized honey bees, Bowen-Walker et al. (1999) found that > 80% of European honey bees emerging from cells inoculated with two Varroa mites had deformities. Bowen-Walker et al. (1999) also detected DWV infections in the bees showing wing deformities. Similarly, in our study with Africanized bees, more than half the Varroa mite-infested bees emerged with wing damage. Bees were not analyzed for DWV presence, but subsamples of the mites used in the experiments were all positive for DWV. Our results, combined with observations of typical symptoms of overt DWV infection (Genersch and Aubert, 2010), allow us to presume that those deformities were caused by DWV infections in the bees. That 100% of bees emerging from non-infested cells did not have wing damage reinforces this assumption. Martin et al. (2012) demonstrated that mite parasitism of the brood is required for wing damage symptoms to occur in adult bees. It has also been reported that DWV replicates in the Varroa mites and when high titers of the virus are transmitted to larvae and pupae by the parasites, the emerging bees show wing damage (Bowen-Walker et al., 1999; Yue and Genersch, 2005), although there is controversy on whether replication of DWV occurs in the mites (Santillán-Galicia et al., 2008; Wilfert et al., 2016). The results of our study suggest that DWV infections were transmitted by the mites and that these infections are very damaging to Africanized bees, although perhaps relatively less so than to European bees. Hamiduzzaman et al. (2015) reported that the rate of increase of DWV infections in Africanized bees was lower than in European bees.
3.5. Identification of DWV in V. destructor mites The three V. destructor mite subsamples analysed were positive for DWV. PCR products of the reactions were sequenced (Laboratory services, University of Guelph) and the sequences (see supplementary material 1) were compared with viral sequences in GenBank (accession No. NC_004830). All sequences produced in our study showed ≥96% identity. 3.6. Mite haplotype analysis Only the Korean haplotype was detected in the mite samples. 4. Discussion We determined, in the same individual bees, the rate of emergence, hemocyte concentration, percent wing damage, and adult survivorship of Africanized honey bees parasitized with V. destructor mites or nonparasitized during the pupal stage. We showed that the health of brood and adult bees of Africanized bees in Mexico is severely affected by V. destructor parasitism. The mite was associated with more than 30% brood mortality, a 30% reduction in hemocyte concentration, and a 40% reduction in lifespan of adult honey bees. Additionally, parasitized bees were affected by wing malformations, possibly as a consequence of DWV infections transmitted by the mite. More than 50% of the bees that emerged from parasitized cells had deformities in the wings versus 0% in the bees that emerged from non-parasitized cells. It is important however, to consider that this study was conducted in the laboratory under controlled conditions and thus, it is possible that the effects observed might be less severe under field conditions. Few studies have documented the negative effects of V. destructor on bee development and longevity, particularly in Africanized honey bees. We clearly showed that populations of Africanized bees in Mexico have low tolerance to V. destructor parasitism at the brood stage; 31% of the infested brood died within their cells compared to only 4% of the control group. By comparison, Bowen-Walker et al. (1999), using a similar methodology, found that 54% of the brood of European bees artificially parasitized with between one and six mites of V. destructor, emerged (representing 46% brood mortality). Similarly, in Brazil, Mattos and Chaud-Netto (2012) inferred high mortality rates of pupae in colonies of Africanized bees that were naturally infested with V. destructor mites and reported a significant correlation between brood mortality and level of mite parasitism. However, Mattos and ChaudNetto (2012) did not observe or quantify bee emergence and did not verify the presence of mites in cells with dead pupae. Previously, De 46
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to EG. Mexico’s National Council of Science and Technology provided a scholarship to MR.
We also showed that adult Africanized bees in Mexico have significantly shorter lifespans if they are parasitized by V. destructor during the brood stage compared with non-parasitized bees. This result agrees with that of De Jong and De Jong (1983) for naturally parasitized Africanized bees in Brazil. In contrast, another study conducted in Mexico with Africanized bees did not find differences in longevity between adult bees emerging from mite parasitized and non-parasitized cells (Romero-Vera and Otero-Colina, 2002). One possible explanation is that Romero-Vera and Otero-Colina (2002) used bees that were infested with tracheal mites (Acarapis woodi) in their non-Varroa control group, and given that Acarapis woodi also affects the health of honey bees in similar ways to Varroa, the effects caused by each mite could not be separated and may have confounded the results. The Varroa mites were of the Korean haplotype, as were mites recovered in two large regions of Mexico in another study (AnguianoBaez et al., 2016). The Korean haplotype is considered the most pathogenic of the V. destructor haplotypes (Solignac et al., 2005), possibly explaining some of the damaging effects on the health of Africanized bees we observed. However, because DWV is also highly pathogenic to honey bees (McMenamin and Genersch, 2015), it is possible that the reduction of the adult lifespan observed in this study was at least partially a consequence of DWV infections. Yang and Cox-Foster (2007) and Dainat et al. (2012) reported that DWV infections significantly reduce the lifespan of honey bees; future studies that assess the effects of single and multiple pathogens on the health of Africanized bees should quantify the relative effect of DWV infections and V. destructor parasitism on bee longevity. Our results indicate that V. destructor parasitism and DWV infections are highly detrimental to the health of Africanized bees in Mexico and could negatively impact colonies. A significantly lower rate of adult bee emergence, many with deformities, inhibited immune cellular responses and shorter livespans, would result in weaker colonies that cannot secure and store adequate food resources; a situation that is less productive for the beekeeper and has a higher risk of colony collapse compared with healthy colonies. Our results also suggest that the tolerance of Africanized bees to V. destructor infestations, which has been demonstrated in numerous studies (Moretto et al., 1991; De Jong, 1997, Guzman-Novoa et al., 1999, 2011; Medina-Flores et al., 2014), does not seem to rely on mechanisms associated with their brood, for example, suppressed mite reproduction. It is possible that the tolerance of Africanized bees to these mites resides more in mechanisms associated with and performed by adult bees, for example, hygienic and grooming behavior (Boecking and Spivak, 1999; Guerra et al., 2000; ArechavaletaVelasco and Guzman-Novoa, 2001; Guzman-Novoa et al., 2012a). In conclusion, we demonstrated that the development, cellular immunity, wing integrity and lifespan of Africanized honey bees in Mexico, are adversely affected by V. destructor parasitism and DWV infections. Our results do not suggest that Africanized bees in Mexico are more tolerant to V. destructor than Africanized bees in Brazil or European bees elsewhere for the parameters measured, nor that the tolerance of Africanized bees to V. destructor resides in brood-related mechanisms. Comparative studies between Africanized bees from different neotropical regions, as well as between these bees and European bees, are necessary not only to assess the relative damage caused by Varroa mite infestations and viral infections, but to quantify the amount of damage that these parasites cause in different genotypes of bees, and the specific implications resulting from these effects.
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Guzman-Novoa, E., Emsen, B., Unger, P., Espinosa-Montaño, L.G., Petukhova, T., 2012a. Genotypic variability and relationships between mite infestation levels, mite damage, grooming intensity, and removal of Varroa destructor mites in selected strains of worker honey bees (Apis mellifera L.). J. Invertebr. Pathol. 110, 314–320. Guzman-Novoa, E., Hamiduzzaman, M.M., Espinosa-Montaño, L.G., Correa-Benitez, A., Ponce-Vazquez, R., 2012b. First detection of four viruses in honey bee (Apis mellifera) workers with and without deformed wings and Varroa destructor in Mexico. J. Apic. Res. 51, 342–346. https://doi.org/10.3896/IBRA.1.51.4.08. Hamiduzzaman, M.M., Guzman-Novoa, E., Goodwin, P.H., Reyes-Quintana, M., Koleoglu,
Acknowledgments We are grateful to Nicolas Mejia and Margarita Flores from the Environmental Educational Centre in Xochimilco, Mexico for their assistance. We are also grateful to Mollah Hamiduzzaman for performing the DWV analyses in varroa mite samples. This study was partially funded by a National University of Mexico grant to AC and by a grant from the Natural Sciences and Engineering Research Council of Canada 47
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