Synergistic parasite-pathogen interactions mediated by host immunity can drive the collapse of honeybee colonies

Abstract

The health of the honeybee and, indirectly, global crop production are threatened by several biotic and abiotic factors, which play a poorly defined role in the induction of widespread colony losses. Recent descriptive studies suggest that colony losses are often related to the interaction between pathogens and other stress factors, including parasites. Through an integrated analysis of the population and molecular changes associated with the collapse of honeybee colonies infested by the parasitic mite Varroa destructor, we show that this parasite can de-stabilise the within-host dynamics of Deformed wing virus (DWV), transforming a cryptic and vertically transmitted virus into a rapidly replicating killer, which attains lethal levels late in the season. The de-stabilisation of DWV infection is associated with an immunosuppression syndrome, characterized by a strong down-regulation of the transcription factor NF-κB. The centrality of NF-κB in host responses to a range of environmental challenges suggests that this transcription factor can act as a common currency underlying colony collapse that may be triggered by different causes. Our results offer an integrated account for the multifactorial origin of honeybee losses and a new framework for assessing, and possibly mitigating, the impact of environmental challenges on honeybee health.

Figure 1. Seasonal dynamics of bees in colonies with low and high levels of mite infestation.

(A) Estimated bee numbers recorded in each hive in October, when a sudden decrease of bee population was observed in highly infested colonies. (B) Bee mortality over time. The error bars indicate the standard deviation; mean values significantly different are denoted with asterisks (*P≤0.05; **P≤0.01). Bee population in highly infested colonies reached minimum levels in October, because of a marked increase of bee mortality.

Figure 2. Mites and DWV in low and highly infested colonies.

(A) Number of mites per 1,000 bees. (B) Seasonal prevalence of Deformed wing virus (DWV) in low and highly infested colonies. (C) Number of DWV genome copies in infected honeybees, collected in September and October from low and highly infested colonies. The error bars indicate the standard deviation; mean values significantly different are denoted with asterisks (*P≤0.05; **P≤0.01). Mite population steadily increased along the season in untreated colonies; DWV prevalence approached 100% at the end of the season both in low and highly infested colonies, but the number of genome copies was much higher in highly infested colonies.

Figure 3. Varroa infestation and DWV genome copies in infested bees and the effect of viral load on bee survival.

(A) Number of DWV genome copies in honeybees larvae artificially infested with different numbers of V. destructor mites, for different time intervals; the error bars indicate the standard error. (B) Survival of honeybees larvae injected with two different dilutions (1∶1,000 and 1∶100,000) of a whole body lysate of bees with deformed wings (DW) and of bees with normal wings as control (NW). Infestation by the Varroa mite caused increasing number of DWV genome copies in infected bees, this significantly affected bee longevity.

Figure 4. Dorsal expression in virus free and virus infected bees.

Dorsal copies in virus free and virus infected honeybee larvae, either infested or not with one Varroa mite, 12 days after cell sealing; the error bars indicate the standard deviation. Average viral load in infected bee larvae, uninfested or infested by the Varroa mite, was 2.40E+10 and 3.22E+12, respectively. Dorsal expression was significantly reduced in virus infected bees compared to virus free bees, while Varroa infestation did not affect gene expression.

Figure 5. Effect of the down-regulation of the transcription factor dorsal-1A by RNAi on DWV replication in bees.

(A) Dorsal-1A transcript level in bees fed for different times with a sucrose/protein solution, containing dsRNA of honeybee dorsal-1A (dsRNA Dorsal) or dsRNA of Green Fluorescent Protein (dsRNA GFP) as a control. (B) Deformed wing virus genome copies in bees treated as above. The error bars indicate the standard deviation. The significant rate (H = 7.00, df = 1: P = 0.008) of silencing of the target gene triggered a significant increase (H = 9.61, df = 1: P = 0.002) of viral replication.

Figure 6. Accelerating or ‘threshold’ immuno-suppression by DWV can create bistable DWV dynamics.

The stable (solid line) and unstable (dotted line) equilibrium level of DWV (arbitrary scale) are calculated from equations S4, S5, and plotted as a function of increasing levels of immune depletion (y). Below the dotted line, the virus can be efficiently regulated by the immune-system to some intermediate (potentially cryptic) density, represented by the solid line. Above the dotted line (and for high y, any point to right of intersection with solid line), the virus cannot be efficiently regulated and a viral explosion ensues. Any factor such as mite feeding that depletes the immune system (increasing y) will first cause a gradual increase in copy number, V (moving right along the solid line), and then at a defined point (intersection of solid and dotted lines), a viral explosion will ensue. Parameters are x = 0.09 (y>x ensures that the virus can invade from rare) and z = 0.4.

Figure 7. Schematic diagram of within-host viral copy number (V) and immune currency (I) dynamics.

The bold lines represent dynamical processes captured explicitly in equations S4, S5. In this model, the viral population dynamics are governed by two antagonistic processes, replication and control (by the immune system). The immune dynamics are in turn governed by three processes; maintenance (increasing immune stocks), stressors (depleting immune stocks) and a specific impact of virally-mediated immune modification (ranging from excitatory to suppressive). The dotted lines represent processes that are external to the model: 1) over-growth of the virus directly leads to increased bee mortality and collapse of the colony (Figures 1 and 2); 2) despite impending collapse within a focal colony, the virus can escape its host via horizontal transmission facilitated by its mite symbiont , ; 3) the mite may gain further advantages from its association with an immuno-suppressive virus, as the suppression will further release immunological control of mite feeding; 4) the mite can affect honeybee survival .