Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

Melissa A Y Oddie; Sandra Lanz; Bjørn Dahle; Orlando Yañez; Peter Neumann

doi:10.1371/journal.pone.0289883

. 2023 Dec 15;18(12):e0289883. doi: 10.1371/journal.pone.0289883

Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

Melissa A Y Oddie ^1,^*, Sandra Lanz ², Bjørn Dahle ¹, Orlando Yañez ², Peter Neumann ²

Editor: Stephen J Martin³

PMCID: PMC10723705 PMID: 38100484

Abstract

Western honeybee populations, Apis mellifera, in Europe have been known to survive infestations of the ectoparasitic mite, Varroa destructor, by means of natural selection. Proposed mechanisms in literature have been focused on the management of this parasite, however literature remains scare on the differences in viral ecology between colonies that have adapted to V. destructor and those that are consistently treated for it. Samples were collected from both a mite-surviving and a sympatric mite-susceptible honeybee population in Norway. The prevalence and abundances of 10 viruses, vectored by the parasite or not, were investigated in adult host workers and pupae as well as in V. destructor mites. Here we show that the mite-vectored Deformed wing virus (DWV-A) is often lower in both abundance and prevalence in the mite-surviving population in tandem with lower phoretic mite infestations compared to the mite susceptible population. However, the non-mite-vectored Black queen cell virus (BQCV), had both a higher abundance and prevalence in the mite-surviving population compared to the susceptible population. The data therefore suggest that general adaptations to virus infections may be unlikely to explain colony survival. Instead, mechanisms suppressing mite reproduction and therefore the impact seem to be more important.

Introduction

Honeybee pathogens like Deformed wing virus (DWV) and its variants, which in the past were largely benign [1], have been given novel transmission pathways through the invasive ectoparasitic mite vector Varroa destructor [2]. The mite bolsters viral titres to the point of reducing the longevity of individual bees [3]. This leads to colony weakening and collapse in many cases [3, 4]. As a result, V. destructor is one of the most serious threats to Western honeybees, both managed and wild [3–5]. Due to the limited defence mechanisms of Western honeybees, unhindered V. destructor population growth can cause numbers to reach fatal levels in late summer and autumn in temperate climates, when the bees being reared are required for winter colony hibernation [4, 6]. There is now very solid evidence that Western honeybee populations, if left untreated, have the potential to adapt to V. destructor infestations without the need for human-mediated mite control [7–9] (via large initial colony losses), and that they can develop this ability in as little as five years [10]. Previous literature has pointed to several mechanisms such as reduced post-capping period [10], potential changes in brood volatiles [11], grooming [12], brood removal [13, 14] and brood cell recapping [15]. One of the most prominent traits that has been detected in naturally surviving honeybees, regardless of the mechanisms identified, is suppressed mite reproduction (SMR) [7, 16], signified by lower average reproductive output per foundress mite. SMR has been recorded not only in Africanized bee populations surviving V. destructor [9, 17], but in the “more susceptible” European populations as well, including the Primorsky bees from Russia [18], the Gotland bees in Sweden, the Avignon bees in France [16, 19], in England, Cuba [9] and a population in the Oslo region of Norway [8].

The managed population of bees in Norway able to persist without mite treatments (mite-surviving) [8] has been used commercially since before the introduction of V. destructor into the local area approximately 30 years ago [8]. Selection efforts employed by the beekeeper managing the population included only the monitoring of docility and high honey productivity, and these traits were preserved along with the development of the mite-surviving adaptations due to natural selection (The removal of all treatment for V. destructor and subsequent loss of colonies without adaptive traits) [8]. Evidence has been gathered that suggest these bees possess mechanisms that focus on controlling the parasite directly, through SMR [7, 8, 15], however there has been no study monitoring the loads of key viruses in the system. It is possible that observed differences between the viral profiles of this population and a local control may provide more insight into the effect these “mite surviving” traits have on viruses.

The Gotland population of Varroa-surviving bees did display evidence of viral tolerance in a laboratory study, by retaining comparative levels of tested viruses but presenting a lower mortality rate [20]. If parasite suppression plays a central role in the ability of honeybees to persist through infestations, we can expect associated virus prevalence and abundance to be lower when the vector levels are also low. Levels of non-vectored viruses should remain relatively similar. Though observing similar levels of Varroa-vectored viruses between both groups will not be sufficient evidence to suggest viral tolerance, the possibility cannot be excluded, and it will provide further insight into the dynamic.

Here, we measured the abundance and prevalence of mite-vectored, non-mite-vectored and mite-associated viruses in parallel with the vector (V. destructor) infestation levels in the Norwegian mite-surviving honeybee population and compared the information to a sympatric, regularly treated control population (mite-susceptible). Viruses were measured throughout the active season in host workers. These viruses were: those known to be vectored by V. destructor: Deformed wing virus A & B, Slow bee paralysis virus, Israeli acute paralysis virus, Kashmir bee virus and Acute bee paralysis virus (DWV-A, DWV-B, SBPV, IAPV, KBV and ABPV), viruses not known to be vectored: Black queen cell virus and Lake sinai virus (BQCV and LSV), and viruses known to be associated with but not directly transmitted by V. destructor: Sacbrood virus and Chronic bee paralysis virus (SBV and CBPV). DWV-A, DWV-B and SBPV were also monitored in both developing pupae and the mites contained in the same brood cells. Colony-level V. destructor infestation levels were measured during the virus sampling period.

Materials and methods

Sample collection and Varroa destructor infestation rates

In September 2013, April and June 2014, samples were taken in Østlandet (South-eastern Norway) from local queenright A. mellifera “Buckfast” colonies known to survive V. destructor infestation without treatments for at least 16 years [21] (n = 32, 3 apiaries). Samples were also collected from colonies regularly treated against these mites with oxalic acid and/or drone brood removal (n = 69, 7 apiaries). Control colonies were managed by several beekeepers separately from the surviving colonies. All colonies were within 100km and placed in similar farmland habitat. Management practices were focused on honey production, centred around a single flowering crop (raspberry flower) and feeding schedules were designed around this, accounting for local variation in flowering times. Adult workers were collected from outer frames of the brood box. Phoretic mites were sampled using routine washing methods (~100–400 bees, [22]). Infested pupae were sampled, and their associated mites collected and stored. All samples were transported on ice to Bern, Switzerland [23] and then stored at -80°C until processing.

Sample selection and analytic approach

Pooled samples

One hundred workers and phoretic mites of the colonies sampled in April (spring) and June (summer) 2014 (n = 58; surviving = 24, susceptible = 34) were pooled and homogenized for each colony. Virus prevalence and abundance were analysed using PCR and qPCR techniques respectively.

Individual samples

Adult workers (n = 11–13 per colony) and phoretic mites (1–22 per colony) were sampled from 10 surviving and 11 susceptible colonies in September (autumn) 2013 and analysed for the presence of DWV-A. Infested honeybee pupae were collected with their associated mites from 47 colonies at eight apiaries in all seasons (29 colonies from three surviving apiaries and 18 colonies from five susceptible ones). Brood samples were taken in April and June from 15 surviving and 19 susceptible colonies. All pupae and mites preserved for viral analysis were processed by individual for DWV-A, DWV-B and SBPV.

Homogenization and RNA extraction

TN buffer (Tris 10mM, NaCl 10 mM; pH 7.6) was added to each sample (25 ml for pooled workers, 100–300 μl for pooled phoretic mites, 250 μl for individual workers and pupae and 100 μl for individual mites) and the sample was homogenized with either a Dispomix® Drive homogenizer (Medic tools) for pooled worker samples or a tissuelyser (Qiagen Retsch MM300, 1 min at 25g/s) for pooled phoretic mites and all individual samples [24]. An aliquot of 50 μl homogenate was used for RNA extraction using the NucleoSpin® RNA II kit, (Macherey-Nagel) following the manufacture’s recommendations. The total extracted RNA was diluted in 60 μl of RNase-free water.

Reverse transcription, PCR and qPCR assays

The RNA was transcribed to cDNA using M-MLV reverse transcription kit (Promega) following the manufacturer’s recommendations using a defined amount of RNA (1μg for bees and 50 ng for mites, respectively) according to fluorospectrometry (NanoDrop^TM 1000) measurements [24]. cDNAs were diluted 10-fold in nuclease-free water. With a standard qualitative PCR (0.125 My Taq^TM polymerase (Bioline), 5μl 5x buffer, 1 μl of the respective forward and reverse primers (S1 Table) and RNase-free water to complete 25 μl final volume; 2 min at 95°C, 35 cycles with 20 sec at 95°C, 20 sec at 57°C and 30 sec at 72°C, 2 min at 72°C), pooled worker and phoretic mite samples were then screened for the following viruses using routine protocols [25]): DWV-A, DWV-B, ABPV, IAPV, KBV, CBPV, SBPV, SBV, BQCV, LSV1 and LSV2. Similarly, pooled brood samples were screened for DWV-A, DWV-B, ABPV, IAPV, KBV, SBV, LSV1, LSV2, SBPV and CBPV. Positive and negative controls were used for each PCR run. Each PCR Product was analysed on 1.2% agarose gel. The agarose gel was stained with GelRed^TM and visualized by UV light. With quantitative RT-PCR (RT-qPCR; Kapa SYBR® Fast Master Mix (KAPA, Biosystems), 10 μl master mix, 3 ul of the 1:10 diluted cDNA template, 0.4 μl forward and reverse target primers (10 mM) and 6.2 μl RNase-free water; 3 min at 95°C, 40 cycles of 95°C for 3 sec and 55°C for 30 sec; melting curve: 55–95 °C with 0.5°C intervals s⁻¹) pooled worker samples, where viruses were detected with qualitative PCR, were analysed to determine virus levels (DWV-A, BQCV, LSV1, LSV2 and SBPV). Individual adult workers and phoretic mites were analysed individually for DWV-A by use of qPCR (protocol described above). Individual brood samples (pupae and brood mites) were analysed individually for the viruses detected in the PCR (DWV-A, DWV-B, and SBPV, protocol described above). Standard curves prepared from viral and β-Actin gene targets [26] were used for virus quantification and normalization, respectively. The standard curves were established by plotting the logarithm of 10-fold dilutions of purified PCR products (10⁻³ to 10⁻⁶ ng/reaction) against the corresponding Ct value as the average of two repetitions. The PCR efficiency (E = 10(−1/slope) − 1) and the linear standard equations for each target were as follows: E = 95,5%, Ct = −3.434 × +47.762, R² = 0.999 for DWV-A; E = 101,9%, Ct = −3.275× +30.881, R² = 0.998 for BQCV; E = 96,3%, Ct = −3.415 × +37.875, R² = 0.988 for LSV-1; E = 85,5%, Ct = −3.724 × +36.203, R² = 0.971 for LSV-2; E = 98,5%, Ct = −3.358 × +32.064, R² = 0.999 for SBPV and E = 110,4%, Ct = − 3.094 × + 33.163, R² = 0.994 for β-Actin. Resulting viral loads for the respective amount of RNA included in the reverse transcription reaction (1μg for bees and 50 ng for mites) were then adjusted by the various experimental dilution factors to account for the total volume of RNA per sample. A Cq cut-off value (according to the value of the negative control) was used to define the target status (positive or negative).

Sequencing

To confirm the virus identity, selected PCR-products were purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) following the manufacturer’s recommendations. The purified amplicons were commercially sequenced (Fasteris SA) and compared with reference sequences deposited in GenBank.

Statistical analyses

Statistical analyses were performed using the R statistical computing software (version 4.1.2) [27] and the packages LME4 [28], gamlss [29] and nlme [30]. Phoretic mite infestation rate data were analysed using generalized linear models with a Poisson error structure, including season and population type (susceptible (treated) or surviving (untreated)) as fixed effects. Worker brood infestation rate proportions were compared using a zero-inflated beta regression model. Virus prevalence data extracted from infested pupae were analysed using generalized linear models with a binomial error structure including season, population type, pupal age, and species (mite or bee) as fixed effects. Individual worker samples were only tested for DWV-A and prevalence was analysed using a χ² test. Viral abundance data were log transformed and analysed using linear regression models with population type, brood and adult bee infestation rates, and virus as fixed effects. A post hoc analysis was included investigating the relationship of each virus to the other parameters and a Bonferroni correction was applied to account for multiple testing (α = 0.025).

Results

Mite infestation levels

Mite-surviving colonies had significantly lower phoretic mite infestation levels than susceptible colonies when all respective apiaries were pooled (χ² 0 5.88, df = 1, p = 0.017, Fig 1 and Table in S1 Table). Mite infestation levels were not significantly affected by the time of year (fall 2013, spring 2014 or summer 2014, Table in S1 Table) when all samples were considered together. Brood infestation rates were not significantly different between surviving or susceptible populations in spring and summer (Table in S1 Table); however, no data were collected on brood infestation rates in autumn (Table in S1 Table).

Fig 1 — Susceptible colonies had significantly more mites in spring and autumn (χ² 0 5.88, df = 1, p = 0.017), standard errors shown.

Virus prevalence and abundance

Pooled adult worker and mite samples

In the pooled worker and mite samples from summer 2014, the following viruses were detected: BQCV, DWV-A, LSV1, LSV2 and SBPV. Sequencing of the PCR products confirmed their identity (GenBank accession: BQCV, EF517515.1, 482 bp query length, 100% query cover, 98,55% identity; DWV-A, JF346624.1, 369 bp query length, 100% query cover, 98,37% identity; LSV2, KY465710.1, 116 bp query length, 100% query cover, 97,41% identity; SBPV, NC_014137.1, 167 bp query length, 100% query cover, 100% identity). The LSV sequence produced with the LSV1 primers was more similar to, and therefore recalled as, LSV8 (Schroeder et al. 2022, ON108639.1, 63 bp query length, 88% query cover, 89,29% identity) [31].

Virus prevalence. The surviving colonies had a significantly lower prevalence of DWV-A (χ² = 4.61, df = 1, p = 0.03, Table in S1 Table), but a higher prevalence of BQCV than the susceptible colonies (χ² = 15.03, df = 1, p<0.001, Fig 2 and Table in S1 Table). None of the other viruses showed significantly different rates between the two population types. DWV-A was generally more common in mites than in tested adult workers (χ² = 8.7, df = 1, p = 0.003, Fig 2 and Table in S1 Table) and prevalence of DWV-A did vary with season (χ² = 28.64, df = 2, p < 0.001, Table in S1 Table).

Fig 2 — Surviving (untreated) colonies had a significantly higher BQCV prevalence (χ² = 15.03, df = 1, p<0.001, Table in S1 Table), but a lower DWV-A prevalence (χ² = 6.75, df = 1, p = 0.009, Table in S1 Table), when compared to susceptible (treated) colonies. Sampled colonies n = 31 susceptible and n = 19 surviving.

Virus abundance. When comparing virus abundances between pooled worker samples from surviving and susceptible colonies, the surviving colonies had higher BQCV loads (F = 39.64, df = 1, p < 0.001, Fig 3 and Table in S1 Table), but no significant differences were detected for DWV-A, LSV1, LSV2 and SBPV (Table in S1 Table). In the pooled adult samples, DWV-A titres did increase significantly with the proportion of infested brood in a colony (F = 8.18, df = 1, p = 0.01, Table in S1 Table). Abundance of LSV1 was significantly reduced with an increase in mite brood infestation levels (F = 55.56, df = 1, p <0.001, Table in S1 Table).

Fig 3 — Medians, interquartile ranges, and maxima are shown. While there were no significant differences for most viruses (DWV-A, LSV2, SBPV) surviving colonies had significantly higher BQCV loads, compared to susceptible colonies (F = 39.64, df = 1, p < 0.001). Sampled colonies n = 19 susceptible and n = 20 surviving.

Individual adult worker samples and phoretic foundress mites

Prevalence (DWV-A only). Viral prevalence of DWV-A in individual adult worker samples was lower in surviving colonies (χ² = 33.751, df = 1, p < 0.01). However, mites showed no difference in viral prevalence between the population types (surviving or susceptible colonies, Fig 4).

Fig 4 — Fewer adult worker bees in surviving colonies tested positive for DWV-A (χ² = 33.751, df = 1, p < 0.01). Sampled colonies n = 29 surviving and n = 18 susceptible.

Individual pupae and associated mite samples

Viruses detected in brood and associated mites were DWV-A, DWV-B and SBPV, all confirmed using PCR sequencing. BQCV, LSV1 and LSV2 were not tested in individual pupae and mite samples.

Virus prevalence. Pupae sampled in surviving colonies had a lower prevalence of DWV-A (χ² = 6.75, df = 1, p = 0.009, Table in S1 Table) than susceptible colonies. Population type (surviving or susceptible) did not significantly influence the prevalence of any other tested virus (Table in S1 Table). DWV-A and SBPV varied by season (DWV-A: χ² = 28.64, df = 2, p < 0.001, SBPV: χ² = 16.44, df = 2, p < 0.001, Table in S1 Table) and DWV-B was affected by the age of the pupae (13.41, df = 1, p < 0.001, Table in S1 Table).

Virus abundance. DWV-A abundance was significantly lower in surviving pupae and mites (pupae: F = 19.78, df = 1, p < 0.001, mites: F = 11.13, df = 1, p = 0.001, Fig 5, Table in S1 Table). Abundances were highest in autumn of 2013 (F = 12.57, df = 2, p < 0.001, Table in S1 Table) and abundance varied with the age of the pupae (F = 13.2, df = 1, p < 0.001, Table in S1 Table). DWV-B and SBPV abundances did not differ significantly between population type (surviving or susceptible, Table in S1 Table).

Discussion

The data show that the mite-vectored DWV-A is lower in both abundance and prevalence in the mite-surviving honeybee population in tandem with lower phoretic mite infestations compared to the mite-susceptible population, especially at the critical period in autumn. When adult worker bees were pooled, DWV-A prevalence was lower, but not abundance, meaning that when a bee was subjected to the virus, levels of the virus were similar to the controls. The data therefore suggest that general adaptations to virus infections, though still possible, are unlikely to explain colony survival. Instead, mechanisms suppressing mite reproduction and therefore reducing vector presence seem to be more important, a hypothesis put forward by Grindrod and Martin (2021) [32].

When compared to the sympatric, susceptible colonies, phoretic mite levels and DWV-A prevalence were lower in all collected samples. DWV-A abundance was lower in individual pupae and adult worker samples but showed no significant difference in pooled adult worker samples, meaning when an adult bee was bitten by a phoretic mite, the levels of virus may often have been the same as in mite-susceptible bees. In line with this: DWV-A abundance was also lower in mites collected from surviving colony brood but not lower in mites collected off adult bees, possibly pointing to horizontal transfer, and/or the vector’s efficiency of transmission when given the chance to interact with the virus. DWV-A abundance in pooled worker samples did increase with the level of infested brood, pointing again to levels of the virus being dictated primarily by the presence of the vector. These findings fall in-line with the findings of previous studies, where viral loads in colonies displaying resistances to V. destructor were significantly lower than their susceptible counterparts, both in tested South African populations [33] and in Uruguayan populations [34].

The general pattern for mite populations in non-adapted Western honeybee colonies is a steady and exponential increase in numbers from spring to autumn [35]. Mite levels in this study were low in summer in susceptible colonies, and this may have been due to the subsequent replacement of dead colonies with new nucleus colonies when they became available in mid-June. For this reason, and likely because mite levels did not change significantly over season in surviving colonies, season was not a significant factor in dictating colony levels of V. destructor. Mite numbers measured in bee brood were not found to be different between population types; however, no brood samples were collected in autumn, the most critical period for measuring mite population loads. Previous research on the surviving population has found that mite populations are lower in autumn than in sympatric control colonies regularly treated for mites [8].

The prevalence of DWV-A in pooled phoretic mite samples was just as high in the surviving bee population as it was in the susceptible population, even though prevalence was lower in bees, i.e. mites tested positive for DWV-A in comparative frequencies between populations, while the bees in surviving colonies tested positive less frequently. This evidence was reinforced in the individual adult bee samples and phoretic mite samples. This could suggest a viral resistance in bees; however, because pooled worker samples had comparable levels of DWV-A in both populations, this seems unlikely: some workers clearly contracted comparably high abundances of the virus in surviving colonies. Observed rates in adult worker pooled samples may be an indicator of horizontal mite spread from colonies outside of the experimental apiaries (comparable abundance) and the ability of bees to reduce the number of mites and therefore the probability of being bitten by foreign mites and contracting high loads of DWV-A (lower prevalence). Horizontal transmission is an element of the system that has not received nearly enough attention in the past and requires further investigation.

In mites, DWV-A abundance was significantly lower in the surviving population when they were taken from brood cells. This is very likely a result of reduced vector infestation levels in the surviving colonies and the consequent reduction of pathogen spread.

BQCV was only tested in the pooled adult worker samples, but both prevalence and abundance were significantly higher in surviving colonies. This data combined with comparative DWV-A abundances in pooled worker bee samples suggest that an adaptation centred around a general increase in response to virus infections may be unlikely to explain colony survival. The central mechanism seems to be a control of the viral vector, V. destructor. However, traits fostering the ability to survive mite infestations may render colonies more susceptible to other threats: The cell recapping behaviour seems to be involved in suppressing mite reproduction [21] and is present in all tested mite-surviving bee populations [9, 15, 36], but opening of brood cells may result in a higher susceptibility to other pathogens, such as BQCV.

DWV-B and SBPV abundances changed with season and DWV-B levels fluctuated with pupal age but were comparable between population types. This study was performed in 2014 and 2013, and it is likely DWV-B was not present in great levels in Norway at this time, though little prior data had been collected to confirm its presence or absence and was not published. Interestingly, LSV1 and LSV2 were mostly absent in mites however little can be said about the relationship of these viruses to the mite-surviving traits observed in the bees.

In conclusion, this study provides evidence that prevalence and abundance of V. destructor-associated viruses can be lower in honeybee populations adapted to the parasite vector [8, 15, 21]. The less-frequent the vector, the lower the chance of viral transmission [32]. Viral tolerance cannot be completely excluded, as it is known that the ability to cope with V. destructor infestations is likely a product of several interacting traits [10, 37], and interactions between viruses merit more attention in literature. The higher levels of BQCV are in line with previous observations made within this population, though this is the first time quantifiable data of this pattern have been obtained. In another surviving population in Sweden, BQCV abundances were substantially lower compared to a local susceptible population [38]. This may point to different mechanisms enabling colony survival in the various surviving populations, and the independence of the selection pathways. This further suggests that adaptations to V. destructor may render colonies more vulnerable to other pathogens. Such a trade-off scenario seems likely, as higher levels of cell recapping in mite-surviving populations [8–15] increases exposure risk to the more vulnerable brood.

The presence of suppressed mite reproduction in all recorded mite-surviving Western honeybee populations so far [7, 8, 39] suggests that this is the most successful natural strategy enabling survival of infested host colonies, though care must be taken to account for local challenges and it is always better to focus on the adaptive potential of regional stocks [40, 41]. Viral tolerance cannot be discounted however, and we must also consider the adaptive potential of mites, future studies on V. destructor-survivability in honeybees may benefit from a focus on the bees’ ability to regulate the parasite and not endure the mites and associated viral infections.

Supporting information

S1 Table. Model output for the full analysis.

Parameters include: the population type (treated or untreated), “Season” is the time of year the samples were taken (Spring, Summer or Autumn), “Species”, whether the organism screened was a honeybee or a V. destructor mite and “Brood/Worker infestation” The V. destructor infestation rate sampled per colony in either brood or adult worker bees.

(PDF)

Click here for additional data file.^{(161.8KB, pdf)}

S2 Table. Primers used for the qualitative and quantitative detection of bee viruses.

(PDF)

Click here for additional data file.^{(161.5KB, pdf)}

S1 File. Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors.

(DOCX)

Click here for additional data file.^{(12.8KB, docx)}

S1 Data

(XLSX)

Click here for additional data file.^{(10.7KB, xlsx)}

S2 Data

(XLSX)

Click here for additional data file.^{(17.2KB, xlsx)}

S3 Data

(XLSX)

Click here for additional data file.^{(14.3KB, xlsx)}

S4 Data

(XLSX)

Click here for additional data file.^{(27.6KB, xlsx)}

S5 Data

(XLSX)

Click here for additional data file.^{(22.2KB, xlsx)}

S6 Data

(XLSX)

Click here for additional data file.^{(90.3KB, xlsx)}

Acknowledgments

We are grateful to the cooperating beekeepers that allowed us to collect samples from their honeybee colonies.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Financial support was granted to Peter Neumann and Bjørn Dahle by the Vinetum foundation and by the Research Council of Norway (grant. Nb 207694) respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Martin SJ, Highfield AC, Brettell L, Villalobos EM, Budge GE, Powell M, et al. Global Honey Bee Viral Landscape Altered by a Parasitic Mite. Science. 2012;336: 1304–1306. doi: 10.1126/science.1220941 [DOI] [PubMed] [Google Scholar]
2.Anderson DL, Trueman JWH. Varroa jacobsoni (Acari: Varroidae) is more than one species. Exp Appl Acarol. 2000;24: 165–189. doi: 10.1023/A:1006456720416 [DOI] [PubMed] [Google Scholar]
3.Dainat B, Evans JD, Chen YP, Gauthier L, Neumann P. Dead or Alive: Deformed Wing Virus and Varroa destructor Reduce the Life Span of Winter Honeybees. Appl Environ Microbiol. 2012;78: 981–987. doi: 10.1128/AEM.06537-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Rosenkranz P, Aumeier P, Ziegelmann B. Biology and control of Varroa destructor. J Invert Path. 2010;103: S96–S119. doi: 10.1016/j.jip.2009.07.016 [DOI] [PubMed] [Google Scholar]
5.Conte YL, Ellis M, Ritter W. Varroa mites and honey bee health: can Varroa explain part of the colony losses? Apidologie. 2010;41: 353–363. doi: 10.1051/apido/2010017 [DOI] [Google Scholar]
6.Dietemann V, Pflugfelder J, Anderson D, Charrière J-D, Chejanovsky N, Dainat B, et al. Varroa destructor: research avenues towards sustainable control. J Apic Res. 2012;51: 125–132. doi: 10.3896/IBRA.1.51.1.15 [DOI] [Google Scholar]
7.Locke B. Natural Varroa mite-surviving Apis mellifera honeybee populations. Apidologie. 2016;47: 467–482. doi: 10.1007/s13592-015-0412-8 [DOI] [Google Scholar]
8.Oddie MAY, Dahle B, Neumann P. Norwegian honey bees surviving Varroa destructor mite infestations by means of natural selection. PeerJ. 2017;5: e3956. doi: 10.7717/peerj.3956 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Luis AR, Grindrod I, Webb G, Piñeiro AP, Martin SJ. Recapping and mite removal behaviour in Cuba: home to the world’s largest population of Varroa-resistant European honeybees. Sci Rep. 2022;12: 15597. doi: 10.1038/s41598-022-19871-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Oddie MAY, Dahle B, Neumann P. Reduced Postcapping Period in Honey Bees Surviving Varroa destructor by Means of Natural Selection. Insects. 2018;9: 149. doi: 10.3390/insects9040149 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Mondet F, Kim SH, Miranda JR de, Beslay D, Conte YL, Mercer AR. Specific Cues Associated With Honey Bee Social Defence against Varroa destructor Infested Brood. Sci Rep. 2016;6: 1–8. doi: 10.1038/srep25444 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Guzman-Novoa E, Emsen B, Unger P, Espinosa-Montaño LG, Petukhova T. 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 Invert Path. 2012;110: 314–320. doi: 10.1016/j.jip.2012.03.020 [DOI] [PubMed] [Google Scholar]
13.Harris JW, Danka RG, Villa JD. Honey Bees (Hymenoptera: Apidae) with the Trait of Varroa Sensitive Hygiene Remove Brood with All Reproductive Stages of Varroa Mites (Mesostigmata: Varroidae). Ann Entomol Soc Am. 2010;103: 146–152. doi: 10.1603/AN09138 [DOI] [Google Scholar]
14.Danka RG, Harris JW, Villa JD. Expression of Varroa Sensitive Hygiene (VSH) in Commercial VSH Honey Bees (Hymenoptera: Apidae). J Econ Entomol. 2011;104: 745–749. doi: 10.1603/ec10401 [DOI] [PubMed] [Google Scholar]
15.Oddie M, Büchler R, Dahle B, Kovacic M, Conte YL, Locke B, et al. Rapid parallel evolution overcomes global honey bee parasite. Sci Rep. 2018;8: 1–9. doi: 10.1038/s41598-018-26001-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Locke B, Conte YL, Crauser D, Fries I. Host adaptations reduce the reproductive success of Varroa destructor in two distinct European honey bee populations. Ecol Evol. 2012;2: 1144–1150. doi: 10.1002/ece3.248 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Camazine S. Differential Reproduction of the Mite, Varroa jacobsoni (Mesostigmata: Varroidae), on Africanized and European Honey Bees (Hymenoptera: Apidae). Ann Entomol Soc Am. 1986;79: 801–803. doi: 10.1093/aesa/79.5.801 [DOI] [Google Scholar]
18.de Guzman LI, Rinderer TE, Frake AM. Comparative reproduction of Varroa destructor in different types of Russian and Italian honey bee combs. Exp Appl Acarol. 2008;44: 227–238. doi: 10.1007/s10493-008-9142-1 [DOI] [PubMed] [Google Scholar]
19.Locke B, Fries I. Characteristics of honey bee colonies (Apis mellifera) in Sweden surviving Varroa destructor infestation. Apidologie. 2011;42: 533–542. doi: 10.1007/s13592-011-0029-5 [DOI] [Google Scholar]
20.Thaduri S, Stephan JG, de Miranda JR, Locke B. Disentangling host-parasite-pathogen interactions in a varroa-resistant honeybee population reveals virus tolerance as an independent, naturally adapted survival mechanism. Sci Rep. 2019;9: 6221. doi: 10.1038/s41598-019-42741-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Oddie MAY, Burke A, Dahle B, Le Conte Y, Mondet F, Locke B. Reproductive success of the parasitic mite (Varroa destructor) is lower in honeybee colonies that target infested cells with recapping. Sci Rep. 2021;11: 9133. doi: 10.1038/s41598-021-88592-y [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Dietemann V, Nazzi F, Martin SJ, Anderson DL, Locke B, Delaplane KS, et al. Standard methods for varroa research. J Apic Res. 2013;52: 1–54. doi: 10.3896/IBRA.1.52.1.09 [DOI] [Google Scholar]
23.Dainat B, Evans JD, Chen YP, Neumann P. Sampling and RNA quality for diagnosis of honey bee viruses using quantitative PCR. Journal of Virological Methods. 2011;174: 150–152. doi: 10.1016/j.jviromet.2011.03.029 [DOI] [PubMed] [Google Scholar]
24.Evans JD, Schwarz RS, Chen YP, Budge G, Cornman RS, De la Rua P, et al. Standard methods for molecular research in Apis mellifera. In: Dietemann V, Ellis JD, Neumann P, editors. The COLOSS BEEBOOK, Volume I: standard methods for Apis mellifera research J Apic Res. 2013. [Google Scholar]
25.de Miranda JR, Bailey L, Ball BV, Blanchard P, Budge GE, Chejanovsky N, et al. Standard methods for virus research in Apis mellifera. Journal of Apicultural Research. 2013;52: 1–56. doi: 10.3896/IBRA.1.52.4.22 [DOI] [Google Scholar]
26.Lourenço AP, Mackert A, Cristino A dos S, Simões ZLP. Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie. 2008;39: 372–385. [Google Scholar]
27.R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria; 2013. Available: http://www.r-project.org/index.html [Google Scholar]
28.Bates D, Mächler M, Bolker B, Walker S. Fitting Linear Mixed-Effects Models using lme4. arXiv:14065823 [stat]. 2014. [cited 4 May 2020]. Available: http://arxiv.org/abs/1406.5823 [Google Scholar]
29.Rigby RA, Stasinopoulos DM. Generalized additive models for location, scale and shape,(with discussion). Applied Statistics. 2005;54: 507–554. [Google Scholar]
30.Pinheiro J, Bates D, DebRoy S, Sarkar D, R Core Team. nlme: Linear and Nonlinear Mixed Effects Models. 2022. Available: https://CRAN.R-project.org/package=nlme [Google Scholar]
31.Schroeder DC, Stone JL, Weeks A, Jacobs M, DaSilva J, Butts S, et al. Two Distinct Genomic Lineages of Sinaivirus Detected in Guyanese Africanized Honey Bees. Microbiology Resource Announcements. 2022;11: e00512–22. doi: 10.1128/mra.00512-22 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Grindrod I, Martin SJ. Parallel evolution of Varroa resistance in honey bees: a common mechanism across continents? Proceedings of the Royal Society B: Biological Sciences. 2021;288: 20211375. doi: 10.1098/rspb.2021.1375 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.de Souza FS, Allsopp MH, Martin SJ. Deformed wing virus prevalence and load in honeybees in South Africa. Arch Virol. 2021;166: 237–241. doi: 10.1007/s00705-020-04863-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Mendoza Y, Tomasco IH, Antúnez K, Castelli L, Branchiccela B, Santos E, et al. Unraveling Honey Bee–Varroa destructor Interaction: Multiple Factors Involved in Differential Resistance between Two Uruguayan Populations. Veterinary Sciences 2020;7: 116. doi: 10.3390/vetsci7030116 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Traynor KS, Mondet F, de Miranda JR, Techer M, Kowallik V, Oddie MAY, et al. Varroa destructor: A Complex Parasite, Crippling Honey Bees Worldwide. Trends in Parasitology. 2020;36: 592–606. doi: 10.1016/j.pt.2020.04.004 [DOI] [PubMed] [Google Scholar]
36.Martin SJ, Hawkins GP, Brettell LE, Reece N, Correia-Oliveira ME, Allsopp MH. Varroa destructor reproduction and cell re-capping in mite-resistant Apis mellifera populations. Apidologie. 2020;51: 369–381. doi: 10.1007/s13592-019-00721-9 [DOI] [Google Scholar]
37.Mondet F, Beaurepaire A, McAfee A, Locke B, Alaux C, Blanchard S, et al. Honey bee survival mechanisms against the parasite Varroa destructor: a systematic review of phenotypic and genomic research efforts. International Journal for Parasitology. 2020;50: 433–447. doi: 10.1016/j.ijpara.2020.03.005 [DOI] [PubMed] [Google Scholar]
38.Locke B, Forsgren E, Miranda JR de. Increased Tolerance and Resistance to Virus Infections: A Possible Factor in the Survival of Varroa destructor-Resistant Honey Bees (Apis mellifera). PLOS ONE. 2014;9: e99998. doi: 10.1371/journal.pone.0099998 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Conte YL, Vaublanc G de, Crauser D, Jeanne F, Rousselle J-C, Bécard J-M. Honey bee colonies that have survived Varroa destructor. Apidologie. 2007;38: 566–572. doi: 10.1051/apido:2007040 [DOI] [Google Scholar]
40.Rinderer TE, Harris JW, Hunt GJ, De Guzman LI. Breeding for resistance to Varroa destructor in North America. Apidologie. 2010;41: 409–424. doi: 10.1051/apido/2010015 [DOI] [Google Scholar]
41.Berg S, Fuchs S, Koeniger N, Rinderer TE. Preliminary results on the comparison of Primorski honey bees. Apidologie. 2004;35: 552–554. [Google Scholar]

PLoS One. 2023 Dec 15;18(12):e0289883. doi: 10.1371/journal.pone.0289883.r001

Author response to previous submission

15 Dec 2022

Attachment

Submitted filename: Plos one review_Rebuttal_2022_Lanz-Oddie.docx

Click here for additional data file.^{(24.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0289883.r002

Decision Letter 0

Olav Rueppell

18 Jan 2023

PONE-D-22-33692Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectorsPLOS ONE

Dear Dr. Oddie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Specifically, reviewer 2 was very critical and has explained several important issues. They are concerned about the rigor of your methods and terminology and also raised the possibility that this manuscript was previously reviewed and then submitted here without sufficiently addressing previous reviews. This would be a waste of everybody's time and therefore I would urge you to careful pay attention to the criticisms of that reviewer if you chose to revise and resubmit.

Please submit your revised manuscript by Mar 04 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Olav Rueppell

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper by Oddie et al. focuses on the analysis of viral infections (prevalence and viral loads) in Norwegian honey bee colonies that naturally survived infestation by Varroa destructor. They found lower V. destructor infestation levels in surviving colonies compared to Varroa susceptible colonies, and detected BQCV, DWV-A, LSV1, LSV2 and SBPV in all the colonies. The prevalence and viral load of Deformed Wing Virus (DWV-A) was lower in the mite surviving colonies in contrast to BQCV. Since the former virus is vectored by the mite they concluded that their findings suggests that lower mite levels may account for reduced prevalence and titers DWV-A rather than other mechanism of virus resistance in the surviving colonies

The study is interesting, well performed and described and it contributes to our knowledge on virus-resistance observed in V. destructor naturally surviving honey bee colonies. The conclusion is sound and it paves the way for further investigation of these mechanisms at the colony and individual bee levels.

The statement at lines 79-81 that indicates that the IKA viruses (IAPV, KBV and ABPV) and SBPV are not vectored by V. destructor should be changed since these viruses can be vectored by the mite as stated in Yañez et al. 2020, Bee Viruses: Routes of Infection in Hymenoptera.

Reviewer #2: Editors,

The manuscript entitled, “Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors” by Oddie et. al describes differences in virus abundance between a reported samples obtained from colonies that are reportedly mite-resistant (i.e., survivor honey bee stock) compared to samples obtained from conventionally managed colonies. This is an interesting topic, but as currently presented it was too difficult to interpret the data to ensure accuracy, and the text requires additional work prior to publication.

Points to clarify or address before publication include:

1. Figures – In general, virus abundance data be better presented as box and whisker graphs that include and data point for all samples (i.e. rather than bar charts), so that readers can get a better understanding of the values and actual variability in the data.

2. Abstract Line 21 / Introduction – While the citations in the introduction provide insight into mechanisms / behaviors associated with mite resistance and indicate that these adaptations can occur within 5 years (which seems short, if selection is acting at the colony level). It would be good for the authors to elaborate on this topic in regard to selection at the individual and/or colony level, etc. and/or revise the first line in their abstract and other place in the text. The researchers should be in other examples of hosts that have developed resistant to parasites over a similar relative time period (i.e., discuss typically ‘generation time’ for a honey bee colony, which is likely 1-2 years+ , and the number of years of mite exposure in Norway, etc.) in order to better describe how the mite-resistance described in this manuscript is/may be the result of natural selection.

3. Abstract Line 22 - The Abstract should be changed since if the colonies were selected from mite resistance, that may result in lower viral load since mites are amplifying vectors of several honey bee viruses and likely passive vectors of many others – but that does not mean that the honey bees have developed resistance to the viruses. While the Abstract gets to this point it begins by stating “It is possible that resistance to the viruses spread by this parasite may contribute to colony survival,. . .” which is misleading/ confusing.

4. Abstract – “The damaging variant of DWV, (DWV-A) .. “ is incorrect, both DWV-A and DWV-B can be detrimental to honey bees, as well as recombinant viruses.

5. The authors need to include a list of primers used in this study, including the primers used to distinguish DWV-A vs. DWV-B. Were the viruses sequence? Were DWV-recombinants assessed? Without this information, some of the DWV abundance in this study may not be accounted for, and therefore the conclusions would only be based on some of the DWV. Regional differences in RNA viruses, which have required new primers for detection and quantification may be needed (e.g., see Daughenbaugh et al Viruses 2021 doi: 10.3390/v13020291, Moore et al. J. Gen. Virol. 2010 doi: 10.1099/vir.0.025965-0, and others).

6. The Virus nomenclature needs to be revised throughout the manuscript, Lines 77-80+, to match ICTV guidelines. Virus nomenclature suggestion based on ICTV: , “black queen cell virus” and “deformed wing virus”, etc. be written in lower case, when referred to in-general (i.e., not a specific strain or species) should be written in lower case and not-italicized.

See: https://talk.ictvonline.org/information/w/faq/386/how-to-write-virus-species-and-other-taxa-names

Considering “deformed wing virus-A” is a virus name, “A virus name should never be italicized, even when it includes the name of a host species or genus, and should be written in lower case. The first letters of words in a virus name, including the first word, should only begin with a capital when these words are proper nouns (including host genus names but not virus genus names) or start a sentence.” Alternatively, for specific virus species, the authors could use “Deformed wing virus-A” (i.e., first letter capitalized, and italicized word), but this is rarely used.

7. Figures, were all colonies assessed on the same date? The supplemental information is good, but would be improved by inclusion of sample date (not just season) or at least including the exact sample dates in the methods. Since the virus abundance in any colony changes over time.

8. The use of the word “titres” is inaccurate and should be revised throughout the manuscript.

Virus titer or viral titre = titre typically refers to the concentration of infectious viral particles

The authors present “viral abundance” or “viral load” data in the form of RNA or cDNA copies per bee (which would be better presented as copies/ xxx ng RNA), therefore the text should be revised.

9. Virus abundance data is a major component of this study – yet it is unclear how this was determined in this study, and the reporting on figures, etc. requires major modification.

Specifically –

(a) Figure 3 – Log copies per colony is not the correct axis label.

It should be log(10) copies per 1000 ng total RNA (if they scaled results to represent total RT reaction).

The reaction volumes for RT and qPCR reactions should be included in the methods section and it should be clearly stated if Line 139 “3 ul cDNA” is really “3 ul of the 1:10 cDNA” – since, as written it is impossible to compare the virus copy number reported in this paper to other studies.

The researchers do not know the copy number per colony, since the assessment is done per x ul of a cDNA reaction – it represents some amount of RNA sample obtained from a subset of bees sampled from one colony.

(b) Lines 146 – 152, it is unclear how virus standards were made since simple dilutions of an unknown stock would not work to calculate “virus copy numbers” .

What did the authors use as a virus standard?

The standard data should be included and graphed to evaluate copy number and the qPCR efficiency.

PCR efficiency typically expressed as a percentage

(i.e., 10^(1/slope “3.32) – 1 = 1 or 100%

I am not sure how the authors calculated efficiency.

(c) The qPCR standard curves used to calculate virus abundance per qPCR well, and the math required to make that number representative of a given amount or RNA need to be clearly presented in this manuscript.

10. Virus abundance in individual colonies varies over time within a colony and the variation between individual colonies with the same apiary and/or region can be high.

The sample size in this study, which was n=10 mite-tolerant colonies, and n=11 conventionally managed colonies for virus abundance data was low. This sample sizes should clearly stated in the figures / figure legends The methods section reports higher numbers of both colonies in other sections making it difficult to see that the virus data came from a subset of the total number of colonies described.

The authors should revise the text describing putative virus resistance vs. tolerance since with a total of 21 colonies (i.e., 10 or 11 colonies per group) it would be difficult to try to assess the potential of virus resistance vs. tolerance.

For pupae and mite assessment it seems, 29 samples were obtained from mite resistant colonies located in three apiaries and 18 conventionally managed mite-susceptible colonies from five apiaries.

Since sample sizes vary for each figure, they must be included in the figures and figure legends, they should be included in every figure or figure legend.

11. Discussion – Although the virus levels reported in honey bees in this study were lower in the “mite-resistant stock” were lower than the “conventionally managed susceptible stock” the number of mites were also lower – so the conclusion stated at the beginning of the discussion may not be accurate (i.e., Line 252), lower mite numbers may be associated with lower virus numbers.

12. As written the discussion is difficult to read, and included big overarching conclusions based on the data obtained from a relatively few number of colonies – that may or may not be exhibiting “mite-resistance” phenotype given that the levels of mite in the brood are similar in the different stocks (although they are missing the autumn time point).

There may be other areas to comment on in the discussion, but since the virus data was difficult to review/interpret it is not possible to fully evaluate the discussion.

Minor points to clarify or address before publication include:

1. Fig. 2 – y-axis does not need decimal places (e.g., 10%, instead of 10.00%)

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Nor Chejanovsky

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Dec 15;18(12):e0289883. doi: 10.1371/journal.pone.0289883.r003

Author response to Decision Letter 0

24 Mar 2023

Response: We firstly thank the reviewer very much for their time and effort. This correction has been made and the paper added to citations.

Reviewer #2: Editors,

Points to clarify or address before publication include:

Response: We thank the reviewer for thorough work and have endeavoured to address all points made:

We have added the points to the box and whisker plots as advised.

Response: We thank the reviewer for highlighting these evolutionary aspects. There is consensus that European (-derived) populations of western honeybees, Apis mellifera, can survive infestations of Varroa destructor by means of natural selection This has been reviewed in detail by Locke 2016 and confirmed since, e.g. Oddie et al 2017 among many others. Clearly, selection at both individual bee and colony level are important in this regard as is the rather long generation time of honeybees. However, this has been dealt with in detail elsewhere (Locke 2016). So, here we just mention this briefly, cite the respective review accordingly, and prefer instead to focus on the possible role of the ability to cope with virus infections contributing to colony survival.

Response: We are afraid that the reviewer may have misinterpreted part of the matter: The surviving colonies investigated in this study were not selected for any mite resistance whatsoever. Instead, natural selection has enabled colony survival which may result from resistance / tolerance to mite infestations and our virus infections.

However, the reviewer is correct in another point: our main hypothesis is that viruses which are vectored by this mite should correlate with vector infestations levels and those not vectored by this mite should not. If the ability to cope with virus infections is an important factor contributing to colony survival, then virus levels should in general be low regardless of vector abundance and independent of a virus being vectored or not. On the other hand given that virus levels positively correlate with vector abundance and are equally high or even higher in non-mite-vectored viruses compared to mite-vectored ones, than the ability to generally cope with virus infections seems to be less relevant for colony survival than the ability to supress mite reproduction. Our data do indicate the latter. We have now rewritten the abstract to make this clearer to the readership.

The reviewer is also absolutely correct about the terminology. From our data, it cannot be concluded about resistance / tolerance whatsoever. Instead, we stick to "mite-surviving" as a well known colony phenotype regardless of the actual underlying mechanism.

4. Abstract – “The damaging variant of DWV, (DWV-A) .. “ is incorrect, both DWV-A and DWV-B can be detrimental to honey bees, as well as recombinant viruses.

Response: We have changed the terminology here to reflect the reviewer’s comment.

Response: The paper now has a primer table in the supplementary files. More detailed information about the virus sequences were added to the text.

See: https://talk.ictvonline.org/information/w/faq/386/how-to-write-virus-species-and-other-taxa-names

Response: The virus nomenclature has now been carefully considered throughout the entire MS.

Response: We thank the reviewer for highlighting this important point. Yes, indeed, seasonal fluctuations in virus levels are well-known. Therefore, we have of course taken this into account and sampled both mite-surviving and mite susceptible colonies in the same months. This has now been added.

8. The use of the word “titres” is inaccurate and should be revised throughout the manuscript.

Virus titer or viral titre = titre typically refers to the concentration of infectious viral particles

Response: The entire text has been revised accordingly.

9. Virus abundance data is a major component of this study – yet it is unclear how this was determined in this study, and the reporting on figures, etc. requires major modification.

Specifically –

(a) Figure 3 – Log copies per colony is not the correct axis label.

It should be log(10) copies per 1000 ng total RNA (if they scaled results to represent total RT reaction).

(b) Lines 146 – 152, it is unclear how virus standards were made since simple dilutions of an unknown stock would not work to calculate “virus copy numbers” .

What did the authors use as a virus standard?

The standard data should be included and graphed to evaluate copy number and the qPCR efficiency.

PCR efficiency typically expressed as a percentage

(i.e., 10^(1/slope “3.32) – 1 = 1 or 100%

I am not sure how the authors calculated efficiency.

Response: We have addressed all of these concerns accordingly. In particular, the axis label on figure 3 has been changed to “Log copies per pooled sample”. More detailed information about the qPCR efficiencies and the standard curves were added to the text.

10. Virus abundance in individual colonies varies over time within a colony and the variation between individual colonies with the same apiary and/or region can be high.

For pupae and mite assessment it seems, 29 samples were obtained from mite resistant colonies located in three apiaries and 18 conventionally managed mite-susceptible colonies from five apiaries.

Since sample sizes vary for each figure, they must be included in the figures and figure legends, they should be included in every figure or figure legend.

Response: We agree with the sample size issue and are now much more careful in our data interpretation. Nevertheless, we are convinced that our data are solid. Again, we also agree with the virus resistance vs. tolerance issue (see above) and have removed this from the entire text. Finally, sample sizes have been added in the figure caption for each figure.

Response: Again, we fully agree. The wording has been changed. Yes indeed, in our data low mite numbers correlate with low levels of the mite-vectored DWV-A thereby nicely confirming our hypothesis. We have now reworded accordingly.

There may be other areas to comment on in the discussion, but since the virus data was difficult to review/interpret it is not possible to fully evaluate the discussion.

Response: The mite survival of the colonies used for this experiment has been confirmed earlier and is referenced extensively in the introduction and discussion. In any case, we have substantially revised the discussion to make it clearer and more concise. In particular, we are much more careful now with our conclusions based on the sample size matter raised above.

Minor points to clarify or address before publication include:

1. Fig. 2 – y-axis does not need decimal places (e.g., 10%, instead of 10.00%)

Response: This has been addressed.

Attachment

Submitted filename: Oddie etal2023_PlosOne_response to reviewers_MO4_yao_PN.docx

Click here for additional data file.^{(27.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0289883.r004

Decision Letter 1

Olav Rueppell

11 Apr 2023

PONE-D-22-33692R1Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectorsPLOS ONE

Dear Dr. Oddie,

Thank you for submitting your manuscript to PLOS ONE. As you can see below, I sent the manuscript out to one of the previous reviewers who was very critical before but is now almost satisfied. Therefore, we invite you to submit a revised version of the manuscript that takes their remaining concerns into account. One issue that was not directly stated in the review but was brought to my attention was your assertion in line 45. While I (and the reviewer) agree that the potential exists, I would suggest to caution readers that this potential may not be realized. Encouraging "Darwinian beekeeping" may be your goal, but I think it is only fair to put in more cautious language for beekeepers that may well read an open-access article and gamble their livelihood on that potential. The survivor populations exist, but they are not necessarily the rule (which makes them particularly interesting).Also, please check for spelling and other minor mistakes throughout. Some mistakes are not caught by automatic spell-checkers (e.g., line 65: "be" instead of "me").

Please submit your revised manuscript by May 26 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Olav Rueppell

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Reviewer #2:

The manuscript entitled, “Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors” by Oddie et. al was significantly improved by the review process.

Points to clarify or address before publication include:

1. While I understand that the abstract “European honeybee populations,

Apis mellifera, “ is indicating “Apis mellifera” populations in Europe, since some papers still define Apis mellifera as the European honey bee (as opposed to the Western honey bee) – the authors should revise the abstract to make sure this is clear – since PONE has a general readership.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

PLoS One. 2023 Dec 15;18(12):e0289883. doi: 10.1371/journal.pone.0289883.r005

Author response to Decision Letter 1

13 Apr 2023

Response to reviewer

Reviewer #2 comments:

The manuscript entitled, “Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors” by Oddie et. al was significantly improved by the review process.

Points to clarify or address before publication include:

1. While I understand that the abstract “European honeybee populations,

Response:

The term in question meant to be very general and is meant to include all populations of Apis mellifera, as it is known that A. m. scutellata is very capable of persisting through V. destructor infestations. However, since this paper is wholly about a population actually bred in Europe, we have made this distinction.

Attachment

Submitted filename: Oddie-etal2023-Response to reviewer.docx

Click here for additional data file.^{(12.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0289883.r006

Decision Letter 2

Olav Rueppell

9 May 2023

PONE-D-22-33692R2

Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

PLOS ONE

Dear Dr. Oddie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 23 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Olav Rueppell

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments:

Thank you for re-submitting your manuscript to PLOS ONE. As indicated in our previous correspondence, I felt obligated to it out for additional review and I was fortunately able to quickly secure two additional reviewers. As you can see, both have additional comments and concerns. While I do not think that the demand of reviewer #3 for additional experiments is warranted, I do think that both reviewers raise many important points. Most importantly, they confirm my concern about the language that could mislead beekeepers and cause them to stop treating for Varroa. I quote an excerpt from their confidential comments to the editor in this case: "The most important point, however, is that the wording concerning the evolution of mite tolerance must be adjusted (first sentence of the abstract and l. 45-47 in the Introduction)." Reviewer #4 also indicates a number of other necessary changes (for example Table 1 now seems to be missing from the manuscript?). If you are prepared to perform the necessary revisions, please follow the directions below. If you are not willing to do so, please just indicate this in an email to save the work of another submission because I will be forced to reject the manuscript in that case.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: The paper by Oddie et al. analyzes viral infections (prevalence and viral loads) in Norwegian honey bee colonies that apparently survived infestation by Varroa destructor naturally. However, as a reviewer, I find the provided information insufficient to verify the authors' statement. The term "naturally survival" is not explicitly defined, and there is no information on the management system or procedures involved in the study. The lack of fundamental information, such as the size and strength of the colonies at the beginning of the experiment, feeding schedules, and honey collection, makes it difficult to evaluate the article's argument thoroughly.

The researchers found lower V. destructor infestation levels in surviving colonies compared to Varroa-susceptible colonies, which confirms the results from Dr. Oddie's previous work. The researchers detected BQCV, DWV-A, LSV1, LSV2, and SBPV in all the colonies. The prevalence and viral load of Deformed Wing Virus (DWV-A) was lower in the mite-surviving colonies, in contrast to BQCV. The authors concluded that their findings suggest that reduced mite levels may account for the lower prevalence and titers of DWV-A, rather than other mechanisms of virus resistance in the surviving colonies.

The study is interesting, but caution should be exercised when interpreting the results. In honey bee research conducted in the field, it can be challenging to obtain reliable control groups. Thus, it is uncertain what diseases or conditions were initially present in the colonies at the beginning of the experiment, which could interfere with the intended measurements.

Although the authors measured some pathogens, it is clear that unmeasured pathogens interfere with the replication of another pathogen, making it difficult to draw firm conclusions.

My suggestion is to include the missing information management information and perhaps perform an additional experiment by injecting bees with purified viruses and measuring replication efficiency in these two honey bee populations.

This experiment could provide the authors with a more robust indication of resistance selection mechanisms than those proposed in this manuscript.

Without more information, any conclusion regarding the mechanisms by which these "naturally" surviving honey bees survive varroa mite infestations is pure conjecture.

Reviewer #4: In this study, the authors hypothesized that in honey bee populations that survive V. destructor infestations without treatment, that is, that are "mite survivors," the bees may also have increased tolerance or resistance to viral infections vectored by the mite. The results presented here from analyzing honey bee colonies originating from the "mite-surviving" population and from a "normal", hence, mite susceptible population over three successive seasons in 2013/2014 are interesting, the paper is nicely written, however, some amendments and corrections are still necessary:

l. 16-17: The wording needs to be amended. This reads as if Western honey bees in general are known to survive V. destructor infestation. But this is not the case. It is true that there are reports and research on a few populations that survive mite infestation without treatment, e.g. in France, Sweden, Norway. But - to the best of my knowledge - these "adapted" bees lost their "adaptation" once transferred to other environments/regions of Europe.

l. 19-20: This is a very simplistic view on virus infections and host tolerance. Host induced reduction of viral titers may be one possibility but sustaining higher viral titers may be another one and selection of less virulent virus variants may be yet another one.

Please reword the sentence and make clear that you only test one of several different possibilities here.

l. 25-26: What about DWV-B? That is the relevant, clearly mite associated DWV-variant. Why are you concentrating on the less virulent variant DWV-A? Please explain and justify your choice of variant.

l. 37: Please replace the term "recombinants" by the term "variants", because recombinants are only a subclass of the different variants within the DWV quasispecies.

l. 38: The wording is wrong: It is NOT the virus that developed new transmission routes but a new transmission route for DWV has opened up through the mite.

l. 45-47: The wording is misleading: Actually, small adapted populations have been reported from several European regions and have been analyzed for many years by now to identify the mechanisms behind these so far rather rare evolutionary events. Although these populations are worth to be studied, the sentence, as it reads right now, implies that every honey bee population would become "adapted" if left untreated. However, that such a development (evolution) would automatically occur within 5 years, if the colonies would be left untreated, has never been shown. Moreover, bee colonies from these allegedly mite-tolerant populations were no longer able to survive mite infestation without treatment when brought to other regions in Europe. The question still remains whether it is the bee or the mite or both that adapt to "lack of treatment".

Therefore, I request that the authors rephrase this sentence in a way that it better reflects the current knowledge and does not leave the impression that stopping mite treatment for five years will solve the mite problem and result in mite surviving honey bees all over Europe.

l. 54-55: Especially the Primorsky bees and the Gotland bees were tested for survival without Varroa treatment in Germany and they were no longer able to survive mite infestation without treatment. This has been published and should not be ignored because it is a very interesting aspect regarding the evolution of mite tolerance in honey bee populations.

l. 78: SBV and CBPV are known to be vectored by V. destructor, please read the literature.

That the vector capacity of the mite has not yet been shown for the other viruses mentioned does not mean that the mite is not able to vector them. There are just too few comprehensive studies on viruses in mites. Please correct and rephrase.

l. 172: It would be better to use the same terminology in the text and in the table, i.e. either worker infestation rate or phoretic mite infestation levels. Please correct.

l. 174: Will the supporting information (raw data in VirusVarroa.zip) also be available to the reader? If so, please refer here to this information but remove the names of the beekeepers in the file.

l. 182: (i) Please indicate whether mean +/- SD or +/- SEM is shown. (ii) The number of the colonies differs considerably between spring, summer, autumn. Why? Did colonies die? Did all "surviving" colonies really survive the winter season and how many of the "susceptible" colonies did not survive the winter season? (iii) Acc to MatMeth, the samples were collected in Sept 2013, April 2014 and June 2014. I assume that spring is April 2014, summer is June 2014 and autumn is Sept 2013. The proper order of the seasons in the figure then were autumn, spring, summer. Please rearrange and add the year. It will then become clear that information on winter mortality must exist because the colonies were observed over winter. Please provide this information. It is absolutely necessary for interpreting the data. Are the “surviving” colonies really surviving, are the susceptible colonies really more susceptible (more of them collapsing over winter) than the “surviving” colonies? Plesase add the requested information and consider it in the interpretation of your data.

l. 194: I assume that all colonies that were sampled in April and June 2014 survived the winter 2013/2014. Hence, strictly speaking, all colonies were "surviving" colonies". If this is not the case, please specify and differentiate between truely surviving colonies and replaced colonies in both groups.

I think it is better to keep writing mite-surviving and mite-susceptible colonies because otherwise the impression is given that the other colonies have not survived - but they had, because they were sampled!? Or didn't they? Then please specify.

Please amend the terminology throughout the manuscript.

l. 202, test stat (X2) 15.30: In Table 1, the value 15.03 is given. Please correct. Please explain "(from qpcr negative results)" given in the Table.

l. 203, test stat (X2) 6.75: In Table 1, the value 4.61 is given. Please correct.

l. 203, p value 0.009: In Table 1, the value 0,03 is given. Please correct.

l. 204: Acc to MatMeth, for virus prevalence pooled samples were collected in April and June 2014. Please provide this information somewhere in the legend of the Fig and explain/justify why you combined the results from two sampling time points.

l. 209, figure 4: It must be Fig. 3. Please correct.

l. 217: Please stick to one terminology! For instance, either write “mite-surviving colonies and mite-susceptible colonies” or “colonies from the mite-surviving population and the mite-susceptible population”, but don't switch between susceptible, mite-susceptible, treated etc. colonies.

This is especially relevant if you use a term here (treated) that cannot be found in the Figure or elsewhere.

l. 223: I cannot find results for individual adult workers in Table 1, only for (i) individual worker brood, (ii) pooled adult worker, and (iii) individual viral loads pupae: Likewise searching for X2 33.751 did not yield a result. Please correct.

l. 225: Please be consistent: If the legend of the axis says "wandering mites" don't use "phoretic mites" in the figure legend.

l. 238: For pupae, I can only find data for individual viral loads but not for prevalence in Table 1. Please correct.

l. 240: In Table 1 the term "viral load" is used, here it is "virus abundance". Please be consistent and rather take viral load throughout.

l. 246: Fig. 5 is about prevalence and abundance, not only prevalence. “Abundance” is better named viral load. Please correct.

l. 246: Fig. 5 is supposed to show surviving pupae and associated mites (see l. 241), not adult workers and phoretic mites. Please correct.

l. 247: Fig 5 is a box plot showing more than just means. Please describe correctly what is shown and specify the box and error bars (SD? SEM?).

l. 249: Here the use of "surviving" colonies is especially problematic. Since these data are from autumn, the use of the term surviving implies that the data of surviving colonies from both groups (mite survivjng and mite susceptible) are shown here, but I assume that surviving actually means “mite surviving”. Please correct.

l. 256: Please provide data on what happened over winter. Did the described differences had any influence on the mortality rate? Did the highly infested and virus infected colonies all die or did they survive? This information is lacking but highly relevant. Please add this information.

l. 259: Please also mention and properly discuss the possibility that the experimental design (pools of entire bees were analyzed, only DWV-A was analyzed instead of DWV-B or even better both variants) was not optimal for answering the question.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: No

**********

PLoS One. 2023 Dec 15;18(12):e0289883. doi: 10.1371/journal.pone.0289883.r007

Author response to Decision Letter 2

20 Jun 2023

Although the authors measured some pathogens, it is clear that unmeasured pathogens interfere with the replication of another pathogen, making it difficult to draw firm conclusions.

This experiment could provide the authors with a more robust indication of resistance selection mechanisms than those proposed in this manuscript.

Without more information, any conclusion regarding the mechanisms by which these "naturally" surviving honey bees survive varroa mite infestations is pure conjecture.

Response:

We thank the reviewer for the time and effort they placed in reviewing our paper.

Thew reviewer’s argument is well-founded, viral loads shift over seasons and time. We have softened the language around the findings and removed the hypothesis on viral tolerance focusing instead on the expected pattern in viral loads for parasite suppression.

Our observational studies were performed over the course of an entire year, so we do believe the most valuable shifts were captured in the data. We have provided further clarification as to the management and origins of the colonies, however the timing of management is defined by area, as temperature is dictated very much by the geography of the region and can separate seasonal patterns by as much as two weeks in as little a distance as 50km. The difficulty of standardizing management to collect meaningful data is an ever-present problem in field observations and should not be a deterrent to presenting observed patterns.

There are undoubtedly other pathogens affecting this system, however, the same can be said for all virus studies published, it is impossible to account for all interactions in a single study, and we feel this does not subtract from the legitimacy of the observational information presented in this paper.

The ability of this population to mitigate Varroa infestation levels is well-described in sourced papers and is not a subject of debate in this study.

We acknowledge that a manipulative experiment would support the findings but is not necessary in this case, as this is an observational study examining viral loads at the population level between two stocks with the fundamental difference of being adapted to V. destructor or not.

Response: Firstly, we thank the reviewer for the time and effort they placed in reviewing our paper.

We have amended the abstract to clarify that we mean some populations and not all Western honeybees.

We are aware of a difference in experience between North American and European beekeeping in terms of Varroa destructor resistance, however the reviewer has received only a part of the picture presented by current literature:

The claim that adapted bees “lose” their adaptation is untrue and unrealistic in the context of evolution. The changes in environment and Varroa density provide challenges that can reduce the effectiveness of traits that have been adapted for a specific environment, therefore it has always been recommended to focus on breeding resistance in locally-adapted bees. We have restated this now in the paper (line 330-332).

There are many cases of untreated Western honeybees persisting in the face of Varroa, one of the latest published cases being the entire country of Cuba (Luis et al. 2022), where genetics studies revealed an almost exclusively European origin of the bees.

The argument of the loss of adaptation when bees are moved also falls drastically outside the scope of this paper, as there is no mention or encouragement of moving bees in any part of this paper.

Please reword the sentence and make clear that you only test one of several different possibilities here.

Response: We have clarified that we mean to observe viral loads based on the known differences between these populations, we agree that this study is not sufficient to measure all possibilities of viral tolerance, we have also reworded the hypotheses to clarify our focus on the parasite suppression and its effect on viral loads.

l. 25-26: What about DWV-B? That is the relevant, clearly mite associated DWV-variant. Why are you concentrating on the less virulent variant DWV-A? Please explain and justify your choice of variant.

Response: DWV-B was discussed and tested for (line125), but when the data was collected it was not found in most of the samples. The reviewer must consider the spread of DWV-B and the timing of the study, coupled with the fact that Norway is a very closed country for honeybee imports and it is very possible this variant was not present in the population at the time.

l. 37: Please replace the term "recombinants" by the term "variants", because recombinants are only a subclass of the different variants within the DWV quasispecies.

Response: This has been amended.

l. 38: The wording is wrong: It is NOT the virus that developed new transmission routes but a new transmission route for DWV has opened up through the mite.

Response: This has been amended.

Response: We disagree with the reviewer’s claims here. There is a very abundant body of literature that provides evidence for the ability of Western honeybees to adapt to Varroa and we have cited it. We acknowledge the fundamental differences in experience between North American and European beekeeping operations, but this should not be a factor that dictates the interpretation of previous peer-reviewed evidence. Our wording was cautious, and “would develop” was not used, but “can” indicating the possibility but not a definite outcome. It has been observed to occur in as little as 5 years, this is in the literature cited. To the argument that honeybees “lose their adaptation when moved”, we refer to the argument and cited papers above.

We believe the language is sufficiently cautious given the evidence we have presented, there are no claims that stopping mite treatments will result in resistant bees indefinitely, and we will not be changing the wording here.

Response: Here the reviewer likely refers to a paper written in 2004 by Berg et al. referenced below, however there was another study published on this same stock moved to the United States which showed a retention of the trait and the subsequent creation of the Russian Honeybee Breeder’s Association in the US (Rinderer et al. 2010), also referenced below.

We have cited both these papers and insist that Varroa resistance is a practical and realistic strategy as long as biology and local environments are considered.

Berg S., Fuchs S., Koeniger N., Rinderer T.E. (2004) Preliminary results on the comparison of Primorski honey bees, Apidologie 35, 552–554.

Rinderer TE, Harris JW, Hunt GJ, De Guzman LI: (2010) Breeding for resistance to Varroa destructor in North America. Apidologie 41:409-424 http://dx.doi.org/10.1051/apido/2010015.

We restate that the argument of moving bees does not apply to this paper, resistant bees have been shown to keep their resistance consistently when managed in the areas where they were bred. This has been documented in scientific literature in at least 8 independent cases across the globe.

l. 78: SBV and CBPV are known to be vectored by V. destructor, please read the literature.

Response: The reviewer cannot claim that these viruses are vectored by Varroa, only associations have been shown in previous literature and these associations are not consistent. According to a paper published in 2022 (O’Shea-Wheller) CBPV can be associated with Varroa but viral transmission pathways have been “elusive”, citing two others: Seitz et al. (2019) and Ribière et al (2010), the same associations were found for SBV (Shen et al. 2005). We have edited the text to describe this association. If the reviewer insists on both viruses being actively vectored by Varroa, we ask that they provide literature where this has been shown, otherwise we must adhere to the consensus provided by the scientific literature found.

l. 172: It would be better to use the same terminology in the text and in the table, i.e. either worker infestation rate or phoretic mite infestation levels. Please correct.

Response: This has been amended.

l. 174: Will the supporting information (raw data in VirusVarroa.zip) also be available to the reader? If so, please refer here to this information but remove the names of the beekeepers in the file.

Response: All data is uploaded and available as per the journal’s requirements. The names of the beekeepers and locations have been changed to provide anonymity.

Response: We have now indicated that the standard errors are shown.

The number of colonies differ due to reasons including loss, accessibility and beekeeper permissions, some colonies are moved onto heather in the local migratory practices. Winter loss data was not collected and falls outside the scope of the paper, because, as stated above, the fact that these colonies can survive mite infestations has already been published in peer-reviewed work multiple times and is not a subject for debate, nor an argument made by the data presented, it is taken as a well-studied fact. Please refer to the numerous studies cited in this work.

Please amend the terminology throughout the manuscript.

Response: Please refer to above comment, this has been addressed.

l. 202, test stat (X2) 15.30: In Table 1, the value 15.03 is given. Please correct. Please explain "(from qpcr negative results)" given in the Table.

Response: This has been amended. Prevalence taken from the absence or presence of virus in the qpcr analysis, binary presence absence tests were not performed, only the quantitative pcrs but this is sufficient to show the presence or absence of a virus.

l. 203, test stat (X2) 6.75: In Table 1, the value 4.61 is given. Please correct.

Response: This has been amended.

l. 203, p value 0.009: In Table 1, the value 0,03 is given. Please correct.

Response: This has been amended.

Response: This has been amended. The graph depicts prevalence for viruses sampled in June which are most relevant. As season did not affect most viruses, this was decided as the most concise way of conveying the important results. We have clarified the season in the figure caption.

l. 209, figure 4: It must be Fig. 3. Please correct.

Response: This has been amended.

This is especially relevant if you use a term here (treated) that cannot be found in the Figure or elsewhere.

Response: Changes have been made to reinforce “surviving” and “susceptible” as the central terminology, however, sometimes these terms are accompanied with “untreated” and “treated” to emphasize the fact that the colonies that have resistance to the parasites are not given any treatment for varroa. This is an important distinction and we press that it should remain.

Response: These data were analysed using a χ2 test and not a glm. Therefore table 1 should not have been referenced. The text has been amended in methods and results.

l. 225: Please be consistent: If the legend of the axis says "wandering mites" don't use "phoretic mites" in the figure legend.

Response: There is no mention of “wandering” mites in text or in figures or tables. Phoretic is used for mites on adult worker bees and the term “associated” is used for mites found in brood to indicate that the mite and brood samples were paired.

l. 238: For pupae, I can only find data for individual viral loads but not for prevalence in Table 1. Please correct.

Response: Models 3a-c display the data for individual pupae and mite sample prevalence. These models have been set lower in the table to reflect the order of presentation in text and are now models 5a-c.

l. 240: In Table 1 the term "viral load" is used, here it is "virus abundance". Please be consistent and rather take viral load throughout.

Response: Previous reviews had the term changed to “abundance”, we have now made this consistent throughout the manuscript and supporting documentation.

l. 246: Fig. 5 is about prevalence and abundance, not only prevalence. “Abundance” is better named viral load. Please correct.

Response: Figure 5 is strictly about abundance, the text has been corrected, please see above comment in regards to chosen terminology.

l. 246: Fig. 5 is supposed to show surviving pupae and associated mites (see l. 241), not adult workers and phoretic mites. Please correct.

Response: This has been amended.

l. 247: Fig 5 is a box plot showing more than just means. Please describe correctly what is shown and specify the box and error bars (SD? SEM?).

Response: This has been amended.

Response: This has been addressed in previous comments: Winter loss data was not collected and falls outside the scope of the paper, because, as stated above, the fact that these colonies can survive mite infestations has already been published in peer-reviewed work multiple times and is not a subject for debate, nor an argument made by the data presented.

Response: This has been addressed in previous comments.

Attachment

Submitted filename: Oddie-etal2023-VirusVarroa_comments_2.2_clean.docx

Click here for additional data file.^{(26.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0289883.r008

Decision Letter 3

Stephen J Martin

29 Jun 2023

PONE-D-22-33692R3Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectorsPLOS ONE

Dear Dr. Oddie,

Please submit your revised manuscript by Aug 13 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Stephen J. Martin

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments:

I can see that this ms has been reviewed by 4 people at various times and 3 suggest minor revisions. The final and latest one suggested major revision which I am satisfied you have addressed fully in your changes to the final versions and in the response letter. As the new editor for the ms I have carefully read the most recent version and suggest the following minor changes before final acceptance as I am not requesting any more reviews so these are the final changes.

Line 39 remove full stop after ‘many cases’

Line 81 reword (BQCV and LSV),

Line 104 Change 100 to ‘One hundred’

Line 272, maybe add 'as suggested by Grindrod and Martin (2021)'?.

Line 327 remove full stop before refs

Check refs for Latin names and put in italics.

Both these previous studies found lower DWV viral loads in resistant populations so worth adding into your discussion as it strengthens your findings:

de Souza FS, Allsopp M, Martin SJ (2020) Deformed wing virus prevalence and load in honeybees in South Africa. Arch Virol (166): 237–241.

Mendoza Y et al., (2020) Unravelling Honey Bee–Varroa destructor Interaction: Multiple Factors Involved in Differential Resistance between Two Uruguayan Populations. Vet Sci (7):116.

Also, Grindrod and Martin (2021) states in the discussion that “a reduced mite burden also reduces the number of viral vectors” and you come to the same conclusion so maybe worth citing this ref in that context to support your ideas.

Grindrod I, Martin SJ (2021) Parallel Evolution of Varroa Resistance in Honey Bees; a common mechanism across continents? Proc. R. Soc. B 288: 20211375.

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2023 Dec 15;18(12):e0289883. doi: 10.1371/journal.pone.0289883.r009

Author response to Decision Letter 3

20 Jul 2023

Journal Requirements:

Author comment: To our knowledge, none of the references cited have been retracted, species names have been italicized as per the editor’s request.

Additional Editor Comments:

Response: We are very happy with the editor reassignment and thank you very graciously for the feedback.

Line 39 remove full stop after ‘many cases’

Line 81 reword (BQCV and LSV),

Line 104 Change 100 to ‘One hundred’

Line 272, maybe add 'as suggested by Grindrod and Martin (2021)'?.

Line 327 remove full stop before refs

Check refs for Latin names and put in italics.

Both these previous studies found lower DWV viral loads in resistant populations so worth adding into your discussion as it strengthens your findings:

de Souza FS, Allsopp M, Martin SJ (2020) Deformed wing virus prevalence and load in honeybees in South Africa. Arch Virol (166): 237–241.

Mendoza Y et al., (2020) Unravelling Honey Bee–Varroa destructor Interaction: Multiple Factors Involved in Differential Resistance between Two Uruguayan Populations. Vet Sci (7):116.

Grindrod I, Martin SJ (2021) Parallel Evolution of Varroa Resistance in Honey Bees; a common mechanism across continents? Proc. R. Soc. B 288: 20211375.

Response: All changes have been made precisely as the editor has specified, new references have been cited and discussed in text and all minor edits have been made (line numbers match those specified)

Attachment

Submitted filename: Oddie-etal2023-VirusVarroa_comments_2.3_clean.docx

Click here for additional data file.^{(13.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0289883.r010

Decision Letter 4

Stephen J Martin

28 Jul 2023

Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

PONE-D-22-33692R4

Dear Dr. Oddie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stephen J. Martin

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

A nice study

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0289883.r011

Acceptance letter

Stephen J Martin

1 Sep 2023

PONE-D-22-33692R4

Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

Dear Dr. Oddie:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Stephen J. Martin

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Model output for the full analysis.

(PDF)

Click here for additional data file.^{(161.8KB, pdf)}

S2 Table. Primers used for the qualitative and quantitative detection of bee viruses.

(PDF)

Click here for additional data file.^{(161.5KB, pdf)}

S1 File. Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors.

(DOCX)

Click here for additional data file.^{(12.8KB, docx)}

S1 Data

(XLSX)

Click here for additional data file.^{(10.7KB, xlsx)}

S2 Data

(XLSX)

Click here for additional data file.^{(17.2KB, xlsx)}

S3 Data

(XLSX)

Click here for additional data file.^{(14.3KB, xlsx)}

S4 Data

(XLSX)

Click here for additional data file.^{(27.6KB, xlsx)}

S5 Data

(XLSX)

Click here for additional data file.^{(22.2KB, xlsx)}

S6 Data

(XLSX)

Click here for additional data file.^{(90.3KB, xlsx)}

Attachment

Submitted filename: Plos one review_Rebuttal_2022_Lanz-Oddie.docx

Click here for additional data file.^{(24.7KB, docx)}

Attachment

Submitted filename: Oddie etal2023_PlosOne_response to reviewers_MO4_yao_PN.docx

Click here for additional data file.^{(27.8KB, docx)}

Attachment

Submitted filename: Oddie-etal2023-Response to reviewer.docx

Click here for additional data file.^{(12.8KB, docx)}

Attachment

Submitted filename: Oddie-etal2023-VirusVarroa_comments_2.2_clean.docx

Click here for additional data file.^{(26.2KB, docx)}

Attachment

Submitted filename: Oddie-etal2023-VirusVarroa_comments_2.3_clean.docx

Click here for additional data file.^{(13.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0289883.ref001] 1.Martin SJ, Highfield AC, Brettell L, Villalobos EM, Budge GE, Powell M, et al. Global Honey Bee Viral Landscape Altered by a Parasitic Mite. Science. 2012;336: 1304–1306. doi: 10.1126/science.1220941 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref002] 2.Anderson DL, Trueman JWH. Varroa jacobsoni (Acari: Varroidae) is more than one species. Exp Appl Acarol. 2000;24: 165–189. doi: 10.1023/A:1006456720416 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref003] 3.Dainat B, Evans JD, Chen YP, Gauthier L, Neumann P. Dead or Alive: Deformed Wing Virus and Varroa destructor Reduce the Life Span of Winter Honeybees. Appl Environ Microbiol. 2012;78: 981–987. doi: 10.1128/AEM.06537-11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref004] 4.Rosenkranz P, Aumeier P, Ziegelmann B. Biology and control of Varroa destructor. J Invert Path. 2010;103: S96–S119. doi: 10.1016/j.jip.2009.07.016 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref005] 5.Conte YL, Ellis M, Ritter W. Varroa mites and honey bee health: can Varroa explain part of the colony losses? Apidologie. 2010;41: 353–363. doi: 10.1051/apido/2010017 [DOI] [Google Scholar]

[pone.0289883.ref006] 6.Dietemann V, Pflugfelder J, Anderson D, Charrière J-D, Chejanovsky N, Dainat B, et al. Varroa destructor: research avenues towards sustainable control. J Apic Res. 2012;51: 125–132. doi: 10.3896/IBRA.1.51.1.15 [DOI] [Google Scholar]

[pone.0289883.ref007] 7.Locke B. Natural Varroa mite-surviving Apis mellifera honeybee populations. Apidologie. 2016;47: 467–482. doi: 10.1007/s13592-015-0412-8 [DOI] [Google Scholar]

[pone.0289883.ref008] 8.Oddie MAY, Dahle B, Neumann P. Norwegian honey bees surviving Varroa destructor mite infestations by means of natural selection. PeerJ. 2017;5: e3956. doi: 10.7717/peerj.3956 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref009] 9.Luis AR, Grindrod I, Webb G, Piñeiro AP, Martin SJ. Recapping and mite removal behaviour in Cuba: home to the world’s largest population of Varroa-resistant European honeybees. Sci Rep. 2022;12: 15597. doi: 10.1038/s41598-022-19871-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref010] 10.Oddie MAY, Dahle B, Neumann P. Reduced Postcapping Period in Honey Bees Surviving Varroa destructor by Means of Natural Selection. Insects. 2018;9: 149. doi: 10.3390/insects9040149 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref011] 11.Mondet F, Kim SH, Miranda JR de, Beslay D, Conte YL, Mercer AR. Specific Cues Associated With Honey Bee Social Defence against Varroa destructor Infested Brood. Sci Rep. 2016;6: 1–8. doi: 10.1038/srep25444 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref012] 12.Guzman-Novoa E, Emsen B, Unger P, Espinosa-Montaño LG, Petukhova T. 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 Invert Path. 2012;110: 314–320. doi: 10.1016/j.jip.2012.03.020 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref013] 13.Harris JW, Danka RG, Villa JD. Honey Bees (Hymenoptera: Apidae) with the Trait of Varroa Sensitive Hygiene Remove Brood with All Reproductive Stages of Varroa Mites (Mesostigmata: Varroidae). Ann Entomol Soc Am. 2010;103: 146–152. doi: 10.1603/AN09138 [DOI] [Google Scholar]

[pone.0289883.ref014] 14.Danka RG, Harris JW, Villa JD. Expression of Varroa Sensitive Hygiene (VSH) in Commercial VSH Honey Bees (Hymenoptera: Apidae). J Econ Entomol. 2011;104: 745–749. doi: 10.1603/ec10401 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref015] 15.Oddie M, Büchler R, Dahle B, Kovacic M, Conte YL, Locke B, et al. Rapid parallel evolution overcomes global honey bee parasite. Sci Rep. 2018;8: 1–9. doi: 10.1038/s41598-018-26001-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref016] 16.Locke B, Conte YL, Crauser D, Fries I. Host adaptations reduce the reproductive success of Varroa destructor in two distinct European honey bee populations. Ecol Evol. 2012;2: 1144–1150. doi: 10.1002/ece3.248 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref017] 17.Camazine S. Differential Reproduction of the Mite, Varroa jacobsoni (Mesostigmata: Varroidae), on Africanized and European Honey Bees (Hymenoptera: Apidae). Ann Entomol Soc Am. 1986;79: 801–803. doi: 10.1093/aesa/79.5.801 [DOI] [Google Scholar]

[pone.0289883.ref018] 18.de Guzman LI, Rinderer TE, Frake AM. Comparative reproduction of Varroa destructor in different types of Russian and Italian honey bee combs. Exp Appl Acarol. 2008;44: 227–238. doi: 10.1007/s10493-008-9142-1 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref019] 19.Locke B, Fries I. Characteristics of honey bee colonies (Apis mellifera) in Sweden surviving Varroa destructor infestation. Apidologie. 2011;42: 533–542. doi: 10.1007/s13592-011-0029-5 [DOI] [Google Scholar]

[pone.0289883.ref020] 20.Thaduri S, Stephan JG, de Miranda JR, Locke B. Disentangling host-parasite-pathogen interactions in a varroa-resistant honeybee population reveals virus tolerance as an independent, naturally adapted survival mechanism. Sci Rep. 2019;9: 6221. doi: 10.1038/s41598-019-42741-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref021] 21.Oddie MAY, Burke A, Dahle B, Le Conte Y, Mondet F, Locke B. Reproductive success of the parasitic mite (Varroa destructor) is lower in honeybee colonies that target infested cells with recapping. Sci Rep. 2021;11: 9133. doi: 10.1038/s41598-021-88592-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref022] 22.Dietemann V, Nazzi F, Martin SJ, Anderson DL, Locke B, Delaplane KS, et al. Standard methods for varroa research. J Apic Res. 2013;52: 1–54. doi: 10.3896/IBRA.1.52.1.09 [DOI] [Google Scholar]

[pone.0289883.ref023] 23.Dainat B, Evans JD, Chen YP, Neumann P. Sampling and RNA quality for diagnosis of honey bee viruses using quantitative PCR. Journal of Virological Methods. 2011;174: 150–152. doi: 10.1016/j.jviromet.2011.03.029 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref024] 24.Evans JD, Schwarz RS, Chen YP, Budge G, Cornman RS, De la Rua P, et al. Standard methods for molecular research in Apis mellifera. In: Dietemann V, Ellis JD, Neumann P, editors. The COLOSS BEEBOOK, Volume I: standard methods for Apis mellifera research J Apic Res. 2013. [Google Scholar]

[pone.0289883.ref025] 25.de Miranda JR, Bailey L, Ball BV, Blanchard P, Budge GE, Chejanovsky N, et al. Standard methods for virus research in Apis mellifera. Journal of Apicultural Research. 2013;52: 1–56. doi: 10.3896/IBRA.1.52.4.22 [DOI] [Google Scholar]

[pone.0289883.ref026] 26.Lourenço AP, Mackert A, Cristino A dos S, Simões ZLP. Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie. 2008;39: 372–385. [Google Scholar]

[pone.0289883.ref027] 27.R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria; 2013. Available: http://www.r-project.org/index.html [Google Scholar]

[pone.0289883.ref028] 28.Bates D, Mächler M, Bolker B, Walker S. Fitting Linear Mixed-Effects Models using lme4. arXiv:14065823 [stat]. 2014. [cited 4 May 2020]. Available: http://arxiv.org/abs/1406.5823 [Google Scholar]

[pone.0289883.ref029] 29.Rigby RA, Stasinopoulos DM. Generalized additive models for location, scale and shape,(with discussion). Applied Statistics. 2005;54: 507–554. [Google Scholar]

[pone.0289883.ref030] 30.Pinheiro J, Bates D, DebRoy S, Sarkar D, R Core Team. nlme: Linear and Nonlinear Mixed Effects Models. 2022. Available: https://CRAN.R-project.org/package=nlme [Google Scholar]

[pone.0289883.ref031] 31.Schroeder DC, Stone JL, Weeks A, Jacobs M, DaSilva J, Butts S, et al. Two Distinct Genomic Lineages of Sinaivirus Detected in Guyanese Africanized Honey Bees. Microbiology Resource Announcements. 2022;11: e00512–22. doi: 10.1128/mra.00512-22 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref032] 32.Grindrod I, Martin SJ. Parallel evolution of Varroa resistance in honey bees: a common mechanism across continents? Proceedings of the Royal Society B: Biological Sciences. 2021;288: 20211375. doi: 10.1098/rspb.2021.1375 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref033] 33.de Souza FS, Allsopp MH, Martin SJ. Deformed wing virus prevalence and load in honeybees in South Africa. Arch Virol. 2021;166: 237–241. doi: 10.1007/s00705-020-04863-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref034] 34.Mendoza Y, Tomasco IH, Antúnez K, Castelli L, Branchiccela B, Santos E, et al. Unraveling Honey Bee–Varroa destructor Interaction: Multiple Factors Involved in Differential Resistance between Two Uruguayan Populations. Veterinary Sciences 2020;7: 116. doi: 10.3390/vetsci7030116 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref035] 35.Traynor KS, Mondet F, de Miranda JR, Techer M, Kowallik V, Oddie MAY, et al. Varroa destructor: A Complex Parasite, Crippling Honey Bees Worldwide. Trends in Parasitology. 2020;36: 592–606. doi: 10.1016/j.pt.2020.04.004 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref036] 36.Martin SJ, Hawkins GP, Brettell LE, Reece N, Correia-Oliveira ME, Allsopp MH. Varroa destructor reproduction and cell re-capping in mite-resistant Apis mellifera populations. Apidologie. 2020;51: 369–381. doi: 10.1007/s13592-019-00721-9 [DOI] [Google Scholar]

[pone.0289883.ref037] 37.Mondet F, Beaurepaire A, McAfee A, Locke B, Alaux C, Blanchard S, et al. Honey bee survival mechanisms against the parasite Varroa destructor: a systematic review of phenotypic and genomic research efforts. International Journal for Parasitology. 2020;50: 433–447. doi: 10.1016/j.ijpara.2020.03.005 [DOI] [PubMed] [Google Scholar]

[pone.0289883.ref038] 38.Locke B, Forsgren E, Miranda JR de. Increased Tolerance and Resistance to Virus Infections: A Possible Factor in the Survival of Varroa destructor-Resistant Honey Bees (Apis mellifera). PLOS ONE. 2014;9: e99998. doi: 10.1371/journal.pone.0099998 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0289883.ref039] 39.Conte YL, Vaublanc G de, Crauser D, Jeanne F, Rousselle J-C, Bécard J-M. Honey bee colonies that have survived Varroa destructor. Apidologie. 2007;38: 566–572. doi: 10.1051/apido:2007040 [DOI] [Google Scholar]

[pone.0289883.ref040] 40.Rinderer TE, Harris JW, Hunt GJ, De Guzman LI. Breeding for resistance to Varroa destructor in North America. Apidologie. 2010;41: 409–424. doi: 10.1051/apido/2010015 [DOI] [Google Scholar]

[pone.0289883.ref041] 41.Berg S, Fuchs S, Koeniger N, Rinderer TE. Preliminary results on the comparison of Primorski honey bees. Apidologie. 2004;35: 552–554. [Google Scholar]

PERMALINK

Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

Melissa A Y Oddie

Sandra Lanz

Bjørn Dahle

Orlando Yañez

Peter Neumann

Roles

Abstract

Introduction

Materials and methods

Sample collection and Varroa destructor infestation rates

Sample selection and analytic approach

Pooled samples

Individual samples

Homogenization and RNA extraction

Reverse transcription, PCR and qPCR assays

Sequencing

Statistical analyses

Results

Mite infestation levels

Fig 1. Number of mites, Varroa destructor, per 100 adult workers per colony in both mite-surviving and mite-susceptible host colonies, Apis mellifera, in spring, summer and autumn.

Virus prevalence and abundance

Pooled adult worker and mite samples

Fig 2. Colony-level prevalence of the detected viruses in pooled adult worker samples in June 2014.

Fig 3. Virus abundances in pooled adult worker samples of summer 2014 from surviving and susceptible Apis mellifera host colonies.

Individual adult worker samples and phoretic foundress mites

Fig 4. Proportion of DWV-A-positive workers and phoretic mites collected from mite washes in spring, summer, and autumn.

Individual pupae and associated mite samples

Fig 5. Abundance of DWV-A in worker pupae and associated mites in autumn 2013 from surviving (untreated) and susceptible (treated) colonies.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Author response to previous submission

Decision Letter 0

Olav Rueppell

Roles

Author response to Decision Letter 0

Decision Letter 1

Olav Rueppell

Roles

Author response to Decision Letter 1

Decision Letter 2

Olav Rueppell

Roles

Author response to Decision Letter 2

Decision Letter 3

Stephen J Martin

Roles

Author response to Decision Letter 3

Decision Letter 4

Stephen J Martin

Roles

Acceptance letter

Stephen J Martin

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases