A multifactorial approach to honeybees health: from multiple causes to consequences and possible actions

In the last decades, large-scale losses of honey bee (Apis mellifera L.) colonies have been recorded all over the world. After years of intense investigation, no specific causal agent for the widespread colony losses has been found but rather a multifactorial origin has been proposed for this syndrome. Biotic and abiotic factors contribute to this phenomenon, but several studies indicate that the ectoparasitic mite Varroa destructor and the Deformed Wing Virus (DWV) play an important role. Recent research has shown that DWV infections at low viral levels are asymptomatic because the honey bee immune competence is able to contain virus replication. As soon as any stress factor interferes with this equilibrium, competing for metabolic resources or negatively acting on immunity, uncontrolled viral replication is promoted, resulting in the transition from a benign covert infection to a devastating overt disease. Xenobiotics, abiotic stressors, malnutrition and other factors can further contribute to complicate the situation. The aim of this thesis was to investigate, at the individual level, how different stress factors and nutrition interact to influence the survival of honey bees. To this aim, we subdivided the study in two phases; in the first one, we assessed how several stress factors as well as pollen influence honey bee health. In a second phase of this study, we investigated how some of these stressors act in combination with the others and in combination with nutrition. We selected seven factors that are possibly implicated in the multifactorial syndrome related to colony losses: pollen, as a natural supply of amino acids and lipids for the honey bee; hydroxymethylfurfural (HMF), a toxic compound contained in additional sugars syrups; acidity, which normally characterizes the sugar syrups mentioned above; nicotine, which is a toxic alkaloid that bees can encounter in the environment and with a mode of action similar to that of neonicotinoid insecticides; a temperature 2-3 degrees below that normally found within the hive; V. destructor, the most dangerous ectoparasite of honey bees and Deformed wing virus (DWV), a key pathogen of honey bees. We confirmed the beneficial effect of pollen nutrition on honey bees as negatively affected by parasites and pathogens. To gain insight into the mechanisms underlying these effects, we interfered with the energetic pathway of mite infested bees using rapamycin, a chemical inhibitor of mTOR, a protein complex that regulates cell growth and other key cellular processes. We observed that rapamycin, just like pollen, decreases DWV load in a manner that could be related to the stimulation of autophagy. More in general, we noted the important role played by nutrition in the interactions between honey bee and other stressors. We also documented for the first time a kind of physiological anorexia triggered by V. destructor infestation, which leads to a reduced energy availability that influences the capacity of honey bees to cope with other stressors. Moreover, we shed light on the possible detrimental side effects of supplementary nutrition administered to bees by beekeepers with homemade sugar syrups. In fact, acidity and HMF that are normally present in such syrups are toxic for honey bees. Lastly, with this work we showed how unpredictable it can be the relationship between stress factors. Since an analytic study of all the factors that can affect honey bee health is unimaginable, the attention should be focused on the metabolic process accounting for the observed interactions in order to develop one or several models that could help to predict the outcome of such interactions

Neonicotinoid Clothianidin reduces honey bee immune response and contributes to Varroa mite proliferation

The neonicotinoid Clothianidin has a negative impact on NF-\u3baB signaling and on immune responses controlled by this transcription factor, which can boost the proliferation of honey bee parasites and pathogens. This effect has been well documented for the replication of deformed wing virus (DWV) induced by Clothianidin in honey bees bearing an asymptomatic infection. Here, we conduct infestation experiments of treated bees to show that the immune-suppression exerted by Clothianidin is associated with an enhanced fertility of the parasitic mite Varroa destructor, as a possible consequence of a higher feeding efficiency. A conceptual model is proposed to describe the synergistic interactions among different stress agents acting on honey bees

Neonicotinoid Clothianidin reduces honey bee immune response and contributes to Varroa mite proliferation

The neonicotinoid Clothianidin has a negative impact on NF-κB signaling and on immune responses controlled by this transcription factor, which can boost the proliferation of honey bee parasites and pathogens. This effect has been well documented for the replication of deformed wing virus (DWV) induced by Clothianidin in honey bees bearing an asymptomatic infection. Here, we conduct infestation experiments of treated bees to show that the immune-suppression exerted by Clothianidin is associated with an enhanced fertility of the parasitic mite Varroa destructor, as a possible consequence of a higher feeding efficiency. A conceptual model is proposed to describe the synergistic interactions among different stress agents acting on honey bees

A deeper understanding of system interactions can explain contradictory field results on pesticide impact on honey bees

While there is widespread concern regarding the impact of pesticides on honey bees, well-replicated field experiments, to date, have failed to provide clear insights on pesticide effects. Here, we adopt a systems biology approach to gain insights into the web of interactions amongst the factors influencing honey bee health. We put the focus on the properties of the system that depend upon its architecture and not on the strength, often unknown, of each single interaction. Then we test in vivo, on caged honey bees, the predictions derived from this modelling analysis. We show that the impact of toxic compounds on honey bee health can be shaped by the concurrent stressors affecting bees. We demonstrate that the immune-suppressive capacity of the widespread pathogen of bees, deformed wing virus, can introduce a critical positive feed-back loop in the system causing bistability, i.e., two stable equilibria. Therefore, honey bees under similar initial conditions can experience different consequences when exposed to the same stressor, including prolonged survival or premature death. The latter can generate an increased vulnerability of the hive to dwindling and collapse. Our conclusions reconcile contrasting field-testing outcomes and have important implications for the application of field studies to complex systems

Haemolymph removal by Varroa mite destabilizes the dynamical interaction between immune effectors and virus in bees, as predicted by Volterra's model

The association between the deformed wing virus and the parasitic mite Varroa destructor has been identified as a major cause of worldwide honeybee colony losses. The mite acts as a vector of the viral pathogen and can trigger its replication in infected bees. However, the mechanistic details underlying this tripartite interaction are still poorly defined, and, particularly, the causes of viral proliferation in mite-infested bees. Here, we develop and test a novel hypothesis that mite feeding destabilizes viral immune control through the removal of both virus and immune effectors, triggering uncontrolled viral replication. Our hypothesis is grounded on the predator-prey theory developed by Volterra, which predicts prey proliferation when both predators and preys are constantly removed from the system. Consistent with this hypothesis, we show that the experimental removal of increasing volumes of haemolymph from individual bees results in increasing viral densities. By contrast, we do not find consistent support for alternative proposed mechanisms of viral expansion via mite immune suppression or within-host viral evolution. Our results suggest that haemolymph removal plays an important role in the enhanced pathogen virulence observed in the presence of feeding Varroa mites. Overall, these results provide a new model for the mechanisms driving pathogen-parasite interactions in bees, which ultimately underpin honeybee health decline and colony losses

Honeybees use various criteria to select the site for performing the waggle dances on the comb

After returning to the hive, successful honeybee foragers dance on the surface of the comb, where they interact with dance followers. It has been shown that bees establish a specific site for their waggle dances that is likely marked with chemical signals. By recording the site where dances take place on the comb in a single-frame observation hive, we investigated the relative importance of three different criteria for the selection of the dance floor by bees, including the distance from the hive entrance, the cell filling, and the chemical marking by bees and found that all these criteria play a role, albeit their importance does not seem to be equal

Possible side effects of sugar supplementary nutrition on honey bee health

Food shortage, along with biotic stressors, contributes to winter honey bee colony losses. In autumn, to support honey bee colonies and prepare them for the winter season, beekeepers can supply homemade syrups which could contain compounds with possible negative side effects. In this study, we investigated the toxicity of one of those compounds (e.g., hydroxymethylfurfural, HMF) at doses consistent with literature data both to healthy bees and bees challenged with their most important parasite (i.e., Varroa destructor ). To strengthen available data on HMF concentration in sugar syrups, we also investigated HMF formation in homemade 2:1 inverted sugar syrup, considering, in particular, the influence of temperature or boiling time on different homemade sugar syrups according to their acidity. Finally, we studied the effects of the acidity of sugar syrups on honeybee survival, and tested whether or not sucrose inversion through acidification is really necessary.We show that doses of HMF similar to those reported as sublethal in the literature appear to be non-toxic even tomite infested bees.However, the amount of HMF that can be found in homemade syrups, which increases with temperature and acidity, can be much higher and can cause significant bee mortality. Moreover, we highlighted the detrimental effect of syrups acidity on honeybee survival, suggesting that the addition of lemon or any other acidifying substance to invert the sucrose could be harmful and not necessary. Our results suggest a responsible approach to homemade colony nutrition

Spectral separability of bark beetle infestation stages: A single-tree time-series analysis using Planet imagery

Bark beetles cause severe damage to European forests leading to impacts on many sectors, from the environmental to the economical. Timely mapping of the different stages of an attack is very important. Remote sensing has been widely used to map bark beetle damage using both airborne and satellite data. Newly available satellite multispectral data with a daily revisit time and high spatial resolution has the potential to monitor an attack in all its phases. This study explores the spectral separability of bark beetle infestation stages using the Planet imagery at individual tree level. Multi-temporal spectral analysis of 78 trees in different stages of a spruce bark beetle attack was carried out. Bands and vegetation indexes derived from 42 multispectral images were compared to eleven field surveys over a time span of approximately four months. The spectral separability analysis was done considering three criteria exploring: 1) the significance of the differences, 2) the magnitude of the differences and 3) the separability in a supervised classification context. The field surveys reported different effects depending on the season of the bark beetle attack - spring vs. summer. Spectral bands and indexes extracted from trees in the healthy and red-stage were significantly different. Trees in the green-attack stage at the end of the summer showed a statistically significant difference from healthy trees. The separability measured with a supervised classifier showed that it is possible to separate healthy, green-attack and red-stage trees with high accuracy values (kappa accuracy above 0.9)