Honey Bee Genetic Stock Determines Deformed Wing Virus Symptom Severity but not Viral Load or Dissemination Following Pupal Exposure

Honey bees exposed to Varroa mites incur substantial physical damage in addition to potential exposure to vectored viruses such as Deformed wing virus (DWV) that exists as three master variants (DWV-A, DWV-B, and DWV-C) and recombinants. Although mite-resistant bees have been primarily bred to mitigate the impacts of Varroa mites, mite resistance may be associated with increased tolerance or resistance to the vectored viruses. The goal of our study is to determine if five honey bee stocks (Carniolan, Italian, Pol-Line, Russian, and Saskatraz) differ in their resistance or tolerance to DWV based on prior breeding for mite resistance. We injected white-eyed pupae with a sublethal dose (105) of DWV or exposed them to mites and then evaluated DWV levels and dissemination and morphological symptoms upon adult emergence. While we found no evidence of DWV resistance across stocks (i.e., similar rates of viral replication and dissemination), we observed that some stocks exhibited reduced symptom severity suggestive of differential tolerance. However, DWV tolerance was not consistent across mite-resistant stocks as Russian bees were most tolerant, while Pol-Line exhibited the most severe symptoms. DWV variants A and B exhibited differential dissemination patterns that interacted significantly with the treatment group but not bee stock. Furthermore, elevated DWV-B levels reduced adult emergence time, while both DWV variants were associated with symptom likelihood and severity. These data indicate that the genetic differences underlying bee resistance to Varroa mites are not necessarily correlated with DWV tolerance and may interact differentially with DWV variants, highlighting the need for further work on mechanisms of tolerance and bee stock–specific physiological interactions with pathogen variants

Potassium ion channels as a molecular target to reduce virus infection and mortality of honey bee colonies

Declines in managed honey bee populations are multifactorial but closely associated with reduced virus immunocompetence and thus, mechanisms to enhance immune function are likely to reduce viral infection rates and increase colony viability. However, gaps in knowledge regarding physiological mechanisms or ‘druggable’ target sites to enhance bee immunocompetence has prevented therapeutics development to reduce virus infection. Our data bridge this knowledge gap by identifying ATP-sensitive inward rectifier potassium ( KATP) channels as a pharmacologically tractable target for reducing virus-mediated mortality and viral replication in bees, as well as increasing an aspect of colony-level immunity. Bees infected with Israeli acute paralysis virus and provided KATP channel activators had similar mortality rates as uninfected bees. Furthermore, we show that generation of reactive oxygen species (ROS) and regulation of ROS concentrations through pharmacological activation of KATP channels can stimulate antiviral responses, highlighting a functional framework for physiological regulation of the bee immune system. Next, we tested the influence of pharmacological activation of KATP channels on infection of 6 viruses at the colony level in the field. Data strongly support that KATP channels are a field-relevant target site as colonies treated with pinacidil, a KATP channel activator, had reduced titers of seven bee-relevant viruses by up to 75-fold and reduced them to levels comparable to non-inoculated colonies. Together, these data indicate a functional linkage between KATP channels, ROS, and antiviral defense mechanisms in bees and define a toxicologically relevant pathway that can be used for novel therapeutics development to enhance bee health and colony sustainability in the field

Increased Resin Collection after Parasite Challenge: A Case of Self-Medication in Honey Bees?

The constant pressure posed by parasites has caused species throughout the animal kingdom to evolve suites of mechanisms to resist infection. Individual barriers and physiological defenses are considered the main barriers against parasites in invertebrate species. However, behavioral traits and other non-immunological defenses can also effectively reduce parasite transmission and infection intensity. In social insects, behaviors that reduce colony-level parasite loads are termed “social immunity.” One example of a behavioral defense is resin collection. Honey bees forage for plant-produced resins and incorporate them into their nest architecture. This use of resins can reduce chronic elevation of an individual bee's immune response. Since high activation of individual immunity can impose colony-level fitness costs, collection of resins may benefit both the individual and colony fitness. However the use of resins as a more direct defense against pathogens is unclear. Here we present evidence that honey bee colonies may self-medicate with plant resins in response to a fungal infection. Self-medication is generally defined as an individual responding to infection by ingesting or harvesting non-nutritive compounds or plant materials. Our results show that colonies increase resin foraging rates after a challenge with a fungal parasite (Ascophaera apis: chalkbrood or CB). Additionally, colonies experimentally enriched with resin had decreased infection intensities of this fungal parasite. If considered self-medication, this is a particularly unique example because it operates at the colony level. Most instances of self-medication involve pharmacophagy, whereby individuals change their diet in response to direct infection with a parasite. In this case with honey bees, resins are not ingested but used within the hive by adult bees exposed to fungal spores. Thus the colony, as the unit of selection, may be responding to infection through self-medication by increasing the number of individuals that forage for resin

Chalkbrood infection levels in colonies in 2008.

<p>The data is from all colonies used in 2008 regardless of use in analyses of foraging rates (n = 5 resin-poor unchallenged colonies; n = 6 each for resin-poor challenged, resin-rich unchallenged and resin-rich challenged colonies). Resin-rich unchallenged colonies positive for CB-infection either had persistent low levels of infection (1 colony) or low-levels of infection at only one time point (2 colonies). Letters indicate significant differences in the total number of mummies based on two-way ANOVA.</p

Change in resin foraging rates before and after challenge.

<p>Data were analyzed using ANOVA for each series with p-values reported within each graph. A) 2008 (n = 7 unchallenged, n = 10 challenged); B) 2009 (n = 8 for unchallenged, chalkbrood, and American foulbrood; n = 9 for <i>Metarhizium</i>; C) 2010 (n = 6 unchallenged, n = 7 challenged); D) Data combined for unchallenged and chalkbrood challenge treatments over the three years of study.</p

Change in pollen foraging rates before and after challenge.

<p>Data were analyzed using ANOVA for each series with p-values reported within each graph. A) 2008 (n = 7 unchallenged, n = 10 challenged); B) 2009 (n = 8 for unchallenged, chalkbrood, and American foulbrood; n = 9 for <i>Metarhizium</i>); C) 2010 (n = 6 unchallenged, n = 7 challenged); D) Data combined for unchallenged and chalkbrood challenge treatments over the three years of study.</p

Potassium ion channels as a molecular target to reduce virus infection and mortality of honey bee colonies

Abstract Declines in managed honey bee populations are multifactorial but closely associated with reduced virus immunocompetence and thus, mechanisms to enhance immune function are likely to reduce viral infection rates and increase colony viability. However, gaps in knowledge regarding physiological mechanisms or ‘druggable’ target sites to enhance bee immunocompetence has prevented therapeutics development to reduce virus infection. Our data bridge this knowledge gap by identifying ATP-sensitive inward rectifier potassium (KATP) channels as a pharmacologically tractable target for reducing virus-mediated mortality and viral replication in bees, as well as increasing an aspect of colony-level immunity. Bees infected with Israeli acute paralysis virus and provided KATP channel activators had similar mortality rates as uninfected bees. Furthermore, we show that generation of reactive oxygen species (ROS) and regulation of ROS concentrations through pharmacological activation of KATP channels can stimulate antiviral responses, highlighting a functional framework for physiological regulation of the bee immune system. Next, we tested the influence of pharmacological activation of KATP channels on infection of 6 viruses at the colony level in the field. Data strongly support that KATP channels are a field-relevant target site as colonies treated with pinacidil, a KATP channel activator, had reduced titers of seven bee-relevant viruses by up to 75-fold and reduced them to levels comparable to non-inoculated colonies. Together, these data indicate a functional linkage between KATP channels, ROS, and antiviral defense mechanisms in bees and define a toxicologically relevant pathway that can be used for novel therapeutics development to enhance bee health and colony sustainability in the field

Honey Bee Genetic Stock Determines Deformed Wing Virus Symptom Severity but not Viral Load or Dissemination Following Pupal Exposure

Honey bees exposed to Varroa mites incur substantial physical damage in addition to potential exposure to vectored viruses such as Deformed wing virus (DWV) that exists as three master variants (DWV-A, DWV-B, and DWV-C) and recombinants. Although mite-resistant bees have been primarily bred to mitigate the impacts of Varroa mites, mite resistance may be associated with increased tolerance or resistance to the vectored viruses. The goal of our study is to determine if five honey bee stocks (Carniolan, Italian, Pol-Line, Russian, and Saskatraz) differ in their resistance or tolerance to DWV based on prior breeding for mite resistance. We injected white-eyed pupae with a sublethal dose (105) of DWV or exposed them to mites and then evaluated DWV levels and dissemination and morphological symptoms upon adult emergence. While we found no evidence of DWV resistance across stocks (i.e., similar rates of viral replication and dissemination), we observed that some stocks exhibited reduced symptom severity suggestive of differential tolerance. However, DWV tolerance was not consistent across mite-resistant stocks as Russian bees were most tolerant, while Pol-Line exhibited the most severe symptoms. DWV variants A and B exhibited differential dissemination patterns that interacted significantly with the treatment group but not bee stock. Furthermore, elevated DWV-B levels reduced adult emergence time, while both DWV variants were associated with symptom likelihood and severity. These data indicate that the genetic differences underlying bee resistance to Varroa mites are not necessarily correlated with DWV tolerance and may interact differentially with DWV variants, highlighting the need for further work on mechanisms of tolerance and bee stock–specific physiological interactions with pathogen variants.</jats:p

Thinking inside the box: Restoring the propolis envelope facilitates honey bee social immunity.

When wild honey bee colonies (Apis mellifera) nest in hollow tree cavities, they coat the rough cavity walls with a continuous layer of propolis, a substance comprised primarily of plant resins. Studies have shown that the resulting "propolis envelope" leads to both individual- and colony-level health benefits. Unfortunately, the smooth wooden boxes most commonly used in beekeeping do little to stimulate propolis collection. As a result, most managed bees live in hives that are propolis-poor. In this study, we assessed different surface texture treatments (rough wood boxes, boxes outfitted with propolis traps, and standard, smooth wood boxes) in terms of their ability to stimulate propolis collection, and we examined the effect of propolis on colony health, pathogen loads, immune gene expression, bacterial gene expression, survivorship, and honey production in both stationary and migratory beekeeping contexts. We found that rough wood boxes are the most effective box type for stimulating propolis deposition. Although the use of rough wood boxes did not improve colony survivorship overall, Melissococcus plutonius detections via gene expression were significantly lower in rough wood boxes, and viral loads for multiple viruses tended to decrease as propolis deposition increased. By the end of year one, honey bee populations in migratory rough box colonies were also significantly larger than those in migratory control colonies. The use of rough wood boxes did correspond with decreased honey production in year one migratory colonies but had no effect during year two. Finally, in both stationary and migratory operations, propolis deposition was correlated with a seasonal decrease and/or stabilization in the expression of multiple immune and bacterial genes, suggesting that propolis-rich environments contribute to hive homeostasis. These findings provide support for the practical implementation of rough box hives as a means to enhance propolis collection and colony health in multiple beekeeping contexts

DataSheet1_Honey Bee Genetic Stock Determines Deformed Wing Virus Symptom Severity but not Viral Load or Dissemination Following Pupal Exposure.docx

Honey bees exposed to Varroa mites incur substantial physical damage in addition to potential exposure to vectored viruses such as Deformed wing virus (DWV) that exists as three master variants (DWV-A, DWV-B, and DWV-C) and recombinants. Although mite-resistant bees have been primarily bred to mitigate the impacts of Varroa mites, mite resistance may be associated with increased tolerance or resistance to the vectored viruses. The goal of our study is to determine if five honey bee stocks (Carniolan, Italian, Pol-Line, Russian, and Saskatraz) differ in their resistance or tolerance to DWV based on prior breeding for mite resistance. We injected white-eyed pupae with a sublethal dose (105) of DWV or exposed them to mites and then evaluated DWV levels and dissemination and morphological symptoms upon adult emergence. While we found no evidence of DWV resistance across stocks (i.e., similar rates of viral replication and dissemination), we observed that some stocks exhibited reduced symptom severity suggestive of differential tolerance. However, DWV tolerance was not consistent across mite-resistant stocks as Russian bees were most tolerant, while Pol-Line exhibited the most severe symptoms. DWV variants A and B exhibited differential dissemination patterns that interacted significantly with the treatment group but not bee stock. Furthermore, elevated DWV-B levels reduced adult emergence time, while both DWV variants were associated with symptom likelihood and severity. These data indicate that the genetic differences underlying bee resistance to Varroa mites are not necessarily correlated with DWV tolerance and may interact differentially with DWV variants, highlighting the need for further work on mechanisms of tolerance and bee stock–specific physiological interactions with pathogen variants.</p