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Short Communication |
1 Institute for Bee Research, Friedrich-Engels-Str. 32, D-16540 Hohen Neuendorf, Germany
2 Faculty for Biology and Biotechnology, Ruhr University Bochum, Bochum, Germany
Correspondence
Elke Genersch
elke.genersch{at}rz.hu-berlin.de
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Ball & Allen, 1988; Cox-Foster et al., 2007; Hung et al., 1995, 1996; Shen et al., 2005b; Shimanuki et al., 1994; Yang & Cox-Foster, 2007).
The ectoparasitic mite V. destructor is an obligate parasite of the honeybee. Colonies infested by V. destructor develop the parasitic mite syndrome (Shimanuki et al., 1994) and ultimately collapse if left untreated. V. destructor has been confirmed as a vector in transmitting and activating bee virus infections and it is well established that viruses vectored by V. destructor play an important role in Varroa-induced colony collapse (Ball, 1983, 1985; Ball & Allen, 1988; Hung et al., 1995, 1996; Martin, 2001; Martin et al., 1998; Shen et al., 2005a, b; Sumpter & Martin, 2004). One of the viruses associated with mite-induced colony collapse is deformed wing virus (DWV), a plus-stranded RNA virus (Lanzi et al., 2006). Outbreaks of clinical DWV infections in honeybees have been reported to be associated with V. destructor infestation (Ball & Allen, 1988; Martin, 2001; Martin et al., 1998), while in the absence of V. destructor, DWV persists as covert infections in honeybees (Yue et al., 2007). The role of V. destructor in the transmission of DWV has been demonstrated experimentally under field conditions (Bowen-Walker et al., 1999). Furthermore, it was concluded that the number of mites within the cell was positively correlated with the amount of DWV detected in individual pupae (Shen et al., 2005b; Tentcheva et al., 2006) and the incidence of morphological deformity and death (Bowen-Walker et al., 1999; Yang & Cox-Foster, 2005). However, it was shown recently that DWV replicated in mites (Ongus et al., 2004; Yue & Genersch, 2005) and that this replication correlated with the occurrence of crippled wings (Yue & Genersch, 2005), suggesting that the transmission of DWV by V. destructor is a prerequisite for the development of crippled wings. Hence, the purpose of our study was to determine the need for V. destructor to act as a biological vector for the development of an overt DWV infection. This was achieved by correlating the levels of mite infestation of developing bees with the occurrence of crippled wings, analysing individual mites for the presence and replication of DWV, individually determining viral loads in these mites and by correlating the results with the development of clinical symptoms in bees.
Bees and mites analysed in this study were collected from eight colonies managed at the apiary of the Ruhr University Bochum, Germany. V. destructor is endemic in Germany, hence all colonies were mite-infested. Colony infestation levels ranged from 0.14 to 0.59 mites per bee for adults, and from 0.23 to 2.08 mites per cell for brood samples (Table 1). Emerging bees or bees about to emerge from the brood cells were randomly sampled from the colonies during a defined time period and evaluated for clinical symptoms of DWV-infection (crippled wings). All mites associated with the sampled bees (i.e. phoretic on the bees and/or present in the cell) were also collected (this was defined as the cell community). We collected a total of 53 asymptomatic bees, together with 135 mites, and 16 symptomatic bees, together with 60 mites (Table 1). Bees and mites were stored at –20 °C for subsequent analysis.
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and Shen et al. (2005b), we analysed the mite infestation level of the sampled bees. Around 50 % of the bees (35 of 69) had only one mite. The infestation level of the remaining bees varied between 2 and 12 mites per bee, with the highest infestation level (12 mites) observed in the case of one asymptomatic bee (Table 2). To test whether the development of crippled wings was correlated with the mite infestation level of the developing bee, the two groups (symptomatic and asymptomatic bees) were tested for homogeneity with respect to their mite infestation level using the 
2 test, with all cell communities that harboured five or more mites grouped into one category (Table 2). Statistical evaluation revealed that the mite infestation level of pupae which developed into asymptomatic bees did not differ significantly from those which developed into symptomatic bees (2=6.516, degrees of freedom=4, P=0.1638), suggesting that factors other than the individual mite infestation level determined the probability of an overt DWV infection in the parasitized developing bee.
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), suggesting that viral replication is a key factor. To corroborate these previous findings, we analysed a total of 195 mites, with 135 mites collected from 53 healthy bees and 60 mites collected from 16 symptomatic bees (Table 1
). Total RNA was extracted from mites and bees as described previously (Yue & Genersch, 2005), using the RNeasy kit and following the manufacturer's protocols (Qiagen). Eluted RNA (50 µl) was stored at –70 °C for subsequent analysis. One-step RT-PCR was performed according to standard protocols (One-step-RT-PCR kit; Qiagen) as described previously (Yue & Genersch, 2005), using the primer pair F1/B1 (Genersch, 2005). Viral replication was analysed via a modified two-step RT-PCR with an elevated reaction temperature (Laskus et al., 1998) and tagged primers (Craggs et al., 2001) to allow the specific detection of plus- and minus-strand DWV RNA as described previously (Yue & Genersch, 2005). In rare cases, RNA extracted from a mite collected from an asymptomatic bee gave an extremely faint signal for the negative-strand RNA as opposed to the strong signals always obtained from RNA extracted from mites collected from symptomatic bees (Fig. 1a). To check for the specificity of these hardly visible bands, the strand-specific RT-PCR was supplemented with an additional control (Celle et al., 2008) performed by analysing cDNAs obtained with random hexamer primers (F15/Tag and B23/Tag; Yue & Genersch, 2005) for the PCR analysis; this normally gave a negative result. In rare cases, these controls resulted in an extremely faint band (Fig. 1b), proving that such bands are false positives. At the same time, a control PCR using DWV-specific primers F15/B23 (Genersch, 2005) was performed on these random cDNAs, giving a strong positive result (Fig. 1b).
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). In seven of 53 cell communities collected from the cells of pupae which developed into asymptomatic bees, we found DWV-negative as well as DWV-positive mites. In nine cell communities, only DWV-negative mites were found and DWV-positive mites only were found in 37 cell communities. In contrast, all mites collected from symptomatic bees were DWV-positive and all of them also tested strongly positive for the replicative strand of viral RNA (Fig. 1a). These results again substantiated and strongly supported the correlation between viral replication in mites and the development of crippled wings (Yue & Genersch, 2005).
Viral load in pupae or bees and the development of crippled wings have been a topic of several studies and it has been speculated that the amount of DWV transmitted by infesting mites is crucial for the development of crippled wings, especially since symptomatic bees are characterized by the presence of a high virus titre (Chen et al., 2005; Shen et al., 2005b; Tentcheva et al., 2006). However, our previous results implicated a correlation between viral replication in mites and crippled wings (Yue & Genersch, 2005). To show that these two hypotheses are not mutually exclusive but rather support each other, we quantified the viral load in the collected mites. This was achieved by using quantitative real-time RT-PCR (qRT-PCR) using the QuantiTect reverse transcription kit (Qiagen) for the reverse transcription reaction and the QuantiTect SYBR green PCR kit (Qiagen) and a Chromo4 real-time PCR thermal cycler (Bio-Rad) for the PCR amplification, performed according to the manufacturer's instructions. The qRT-PCR analysis was performed with the following cycling conditions: 15 min at 95 °C followed by 38 cycles of 1 min at 94 °C, 1 min at 54.3 °C and 1 min at 72 °C. Plate reads were performed after each annealing and extension step. After a final elongation step for 10 min at 72 °C, a melting curve analysis was performed with a range of 70 to 95 °C as a control for the specificity of the amplified product. In addition, PCR products (5 µl per reaction) were analysed on a 1 % agarose gel. The DNA bands were stained with ethidium bromide and visualized by UV light. An external DNA standard was generated by amplifying a viral genomic fragment of 1520 bp via RT-PCR, using the primers Fstd (5'-GGACCATCCTTCCAGTCTACGAT-3') and Bstd (5'-CTGTAGGTTGTGCTCCTGATGAAGA-3'); this contained the 354 bp fragment that was amplified by the primer pair F1/B1 (Genersch, 2005) for quantification. A triplicate dilution series of this external DNA standard, covering the range of 101 to 107 molecules, was included with every qRT-PCR run to give a standard curve which had a reaction efficiency of 91.6–97.8 % and a linear detection range from 101 to 107 molecules (r2 between 0.966 and 0.999), allowing the absolute quantification of the DWV genome equivalents per mite.
Quantification of viral genome equivalents in mites that were collected from symptomatic bees and, therefore, contained replicating virus, had a viral load between 2x1010 and 1x1012 genome equivalents. In contrast, mites from infested pupae that developed into asymptomatic bees contained between 5x104 and 1x108 DWV genome equivalents. Statistical evaluation of these data by one-way analysis of variance (ANOVA; general linear model) (degrees of freedom=67, F=117.19, P=0.0) revealed that the differences in viral load between mites collected from symptomatic or asymptomatic bees were highly significant (confidence interval=0.05). Fisher's least significant difference multiple comparison test (degrees of freedom=67, MSE=1.793118x1022, critical value=1.9960) also showed that a virus titre in mites of 1010 to 1012 strongly correlated with viral replication, suggesting that such a high virus titre can only be achieved through replication in mites.
An accepted view is that the transmission of DWV to pupae through V. destructor is a prerequisite for the development of crippled wings (Ball & Allen, 1988; Bowen-Walker et al., 1999; Shen et al., 2005b; Yue & Genersch, 2005). The exact pathogenic mechanisms in the triangular relationship between honeybees as hosts, V. destructor as a virus vector and DWV as a pathogen are poorly understood.
Two explanatory approaches can be found in the literature. One explanation is that V. destructor acts as a virus vector but more importantly, it induces an immunosuppression in the host, thereby activating covert virus infections (Shen et al., 2005b; Yang & Cox-Foster, 2005, 2007). Accordingly, the degree of mite infestation of a single pupa was reported to be positively correlated with the probability of crippled wings (Bowen-Walker et al., 1999) or severity of the symptoms (Yang, 2004; Yang & Cox-Foster, 2005). The results presented in this study did not confirm these correlations. No significant difference in mite infestation level could be demonstrated between bees emerging with malformed or normal wings.
The other explanation is that V. destructor not only acts as a mechanical vector of DWV but also has the potential to act as a biological vector by supporting DWV replication prior to transmission. We previously showed that this replication correlates to the development of crippled wings (Yue & Genersch, 2005) and we have duplicated these results here. The correlation between DWV replication in mites and the development of wing deformity (and conversely, the correlation between absence of replication in mites and absence of clinical symptoms in bees) suggested that, although DWV transmission by V. destructor is a prerequisite for the development of crippled wings, a crucial factor is the replication of DWV in the mite prior to transmission. We now provide correlative evidence that this replication produces a high DWV titre in these mites (1010–1012 genome equivalents per mite), which is at least two orders of magnitude higher than the viral titre in mites collected from asymptomatic bees. These results suggested that it is necessary for the DWV titre in the infecting mites to reach a certain threshold level (achieved through replication in these mites) for infested, and hence infected, bees to emerge with crippled wings.
This raises the question of why a certain DWV titre may be crucial for the formation of crippled wings. One possible explanation is that whether DWV will cause clinical symptoms in the adult bee is determined by the number of virus particles transmitted by the mite that circulate in the pupal haemolymph and infect all possible tissues. This explanation is supported by earlier studies suggesting that the level of DWV present in the bees determines whether they hatch as symptomatic, non-viable bees, or emerge as asymptomatic bees (Bowen-Walker et al., 1999; Chen et al., 2005).
Another explanation comes from the quasi-species concept for RNA viruses, which proposes that RNA viruses tend to form mutant swarms due to their high failure rate during replication (Domingo, 2002; Eigen, 1996; Holmes & Moya, 2002). Assuming that only certain DWV mutants are able to cause crippled wings in the developing bee, the likelihood of such a mutant will be positively correlated with the replication rate in the vector and the host and, therefore, with the viral population size that finally infects the pupa. Hence, efficient DWV replication that results in a high virus titre in mites, as shown in our study, may allow the generation of a certain DWV mutant already present in the vector V. destructor that is able to cause clinical symptoms of DWV infection in the bees. Accordingly, it may not be the number of viral particles transmitted by the mite that are essential for the development of crippled wings, but rather the presence of certain mutants among the vector-transmitted viruses.
Based on our results, we now propose the following model for the pathogenesis of an overt DWV infection in mite-infested pupae. DWV transmission to pupae by V. destructor is the prerequisite for the development of crippled wings in the adult bee. DWV replication in mites prior to viral transmission is then necessary to produce a viral titre above a certain threshold (more than 1010 viral genome equivalents per mite). Following this, either the large number of viral particles vectored by the mite and/or the existence of a certain viral mutant in the transmitted viral population will cause the development of crippled wings as clinical symptoms of an overt DWV infection.
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ACKNOWLEDGEMENTS |
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Received 15 July 2008;
accepted 13 October 2008.
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