Microbes, including bacteria and viruses, are the main threats to the health of honey bee colonies, and cause great losses to beekeepers. Rapid and accurate diagnosis is the key to controlling and eliminating honey bee diseases, and preventing them from spreading and causing an outbreak. This review summarizes recent advances in techniques to detect honey bee diseases, including traditional methods such as polymerase chain reaction, and nextgeneration sequencing methods, and how they are applied in the diagnosis and management of such diseases. It also discusses how these methods have revolutionized disease detection, and presents the future directions in the field of clinical diagnostics.

Managed and wild bee populations contribute over $15 billion in pollination services to US agriculture, yet both are declining or becoming increasingly vulnerable to parasites and disease. The loss of healthy and diverse forage is a key driver in bee declines, so incentivizing land managers to adopt diversified bee-friendly management practices such as forage plantings and reduced pesticide use can directly increase food security, pollinator health, and farmer adaptive capacity. To better understand what might incentivize growers to adopt bee-friendly practices, we conducted a survey of California almond growers, whose orchards are entirely dependent on bee pollination and draw nearly 88% of US bee colonies each February to pollinate almond bloom. We asked 329 respondents across all major almond growing regions of CA about their adoption rate and incentives for planting cover crops, pollinator habitat, and practicing the recommended and legally required bee-friendly best management practices, as well as their interest in bee-friendly certification programs. Using a model selection framework, we evaluated which geographic, social, operational, and pollination-service related factors were predictive of bee-friendly practice adoption. We found that no single factor was a statistically significant predictor of adoption across all models, suggesting there is no silver bullet determining bee-friendly practice adoption. However, we discovered that region and concerns about future pollination services consistently emerged as important factors related to all the practices we investigated, except the adoption of legally required BMPs. These findings suggest that a regionally flexible pollinator conservation strategy focused on supporting honey bee colonies might have the highest likelihood of grower participation and adoption.

Approximately one-third of the typical human Western diet depends upon pollination for production, and honey bees (Apis mellifera) are the primary pollinators of numerous food crops, including fruits, nuts, vegetables, and oilseeds. Regional large scale losses of managed honey bee populations have increased significantly during the last decade. In particular, asymptomatic infection of honey bees with viruses and bacterial pathogens are quite common, and co-pathogenic interaction with other pathogens have led to more severe and frequent colony losses. Other multiple environmental stress factors, including agrochemical exposure, lack of quality forage, and reduced habitat, have all contributed to the considerable negative impact upon bee health. The ability to accurately diagnose diseases early could likely lead to better management and treatment strategies. While many molecular diagnostic tests such as real-time PCR and MALDI-TOF mass spectrometry have been developed to detect honey bee pathogens, they are not field-deployable and thus cannot support local apiary husbandry decision-making for disease control. Here we review the field-deployable technology termed loop-mediated isothermal amplification (LAMP) and its application to diagnose honey bee infections.

Abstract It is a widespread practice in China to keep colonies of both the western honey bee, Apis mellifera, and the eastern honey bee, Apis cerana, in close proximity. However, this practice increases opportunities for spillover of parasites and pathogens between the two host bee species, impacting spatial and temporal patterns in the occurrence and prevalence of the viruses that adversely affect bee health. We conducted a 1‐year large‐scale survey to assess the current status of viral infection in both A. mellifera and A. cerana in China. Our study focused on multiple aspects of viral infections in honey bees, including infection rate, viral load, seasonal variation, regional variation, and phylogenetic relationships of the viruses within the same species found in this study and other parts of the world. The survey showed that the black queen cell virus (BQCV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV) were common in both A. mellifera and A. cerana, and infection dynamics of BQCV, DWV, and SBV between bee species or seasons were significantly different. DWV was the most common virus in A. mellifera, and its infection rate and load in A. mellifera were higher than those in A. cerana, which reflects the high susceptibility of A. mellifera to Varroa destructor infestation. The infection rate and viral load of SBV were higher in A. cerana than in A. mellifera, indicating that SBV poses a greater threat to A. cerana than to A. mellifera. Our results also suggested that there was no geographical variation in viral dynamics in A. mellifera and A. cerana. Phylogenetic analyses of BQCV, DWV, IAPV, and SBV suggested the cross‐regional and cross‐species spread of these viruses. This study provides important insights into the complex relationships between viruses and their hosts in different seasons and regions, which will be important for developing effective disease management strategies to improve bee health.

In terms of infectious diseases caused by a variety of microorganisms, the ability to promptly and accurately identify the causative agents is the first step on the path to all types of effective management of such infections. Among the various factors that are affecting global bee health, viruses have often been linked to honey bee colony losses and they pose a serious threat to the fraction of agriculture that depends on the service of pollinators. Over the past few decades, PCR-based molecular methods have provided powerful tools for rapid, specific, and sensitive detection and the quantification of difficult-to-grow pathogenic microorganisms such as viruses in honey bees. However, PCR-based methods require nucleic acid extraction and purification, which can be quite laborious and time-consuming and they involve the use of organic solvents and chaotropic agents like phenol and chloroform which are volatile and highly toxic. In response, we developed a novel and non-sacrificial method for detecting viral infections in honey bees. As little as 1 μl of hemolymph was collected from adult workers, larvae, and queens of bee colonies by puncturing the soft inter-tergal integument between the second and third dorsal tergum with a fine glass capillary. The hemolymph was then diluted and subjected to RT-PCR analysis directly. The puncture wound caused by the glass capillary was found to heal automatically and rapidly without any trouble and the lifespan of the experimental workers remained unaffected. Using this method, we detected multiple viruses including Deformed wing virus (DWV), Black queen cell virus (BQCV), and Sacbrood virus (SBV) in infected bees. Furthermore, expressed transcripts that indicate the induction of innate immune response to the virus infections were also detected in the hemolymph of infected bees. The simplicity and cost-effectiveness of this innovative approach will allow it to be a valuable, time-saving, safer, and more environmentally friendly contribution to bee disease management programs.

The avocado (Persea americana Mill.) is a widely demanded fruit worldwide. While its palatable taste is one of the reasons it is favoured so, the fleshy interior contains a wide range of vitamins, minerals and other health boosting components. Not only this, but the pulp and seed are also valued in the oil-processing, pharmaceutical and cosmetic industries. The demand for avocados is thus extensive, and production of the fruit has increased five-fold over the last 35 years to meet this demand. Despite centuries of selection and hybridisation, avocado trees are still characterised by a low-yielding nature. The oil-rich flesh, alternate bearing, competition between vegetative and reproductive flushes, and disease proliferation all contribute to low yields in commercial orchards. There is another factor, widely investigated and yet poorly understood, that further reduces yields in avocados: sub-optimal pollination. When cultivated in its native region of central Mexico, many endemic insect species facilitate pollination between the synchronous, dichogamous avocado flowers. As avocado cultivation expands outside its native region, honey bee hives are installed in orchards to improve pollination. However, more attractive blooms of other angiosperms (citrus, for example) can cause a mass honey bee exodus out of the avocado orchards. This results in reduced pollination and consequently, fruit set and yield are seriously compromised. This dissertation aimed to explore the notion of low pollination rates in avocados. Factors inherent to the avocado’s physiology, such as floral morphology and scent were considered in conjunction with external influences, including cold stress and nutrient availability. Furthermore, practical management strategies to combat low pollination in avocado orchards were surveyed. Simple methods were used to manually count honey bee activity amongst the canopies of a range of cultivars. Honey bees appeared to favour ‘B-type’ cultivars, such as P. americana ‘Fuerte’ and ‘Zutano’. Visitations to flowers open in the female and male phases showed honey bees are more partial to functionally male flowers. This may be due to the dual reward of pollen in addition to nectar, as well as the grander floral display when the flowers are open in the male phase. Floral volatile exudation also plays an important role in pollinator attraction. Solid-phase microextraction (SPME) and thermal desorption with comprehensive gas chromatography with time-of-flight mass spectrometry (GC x GC-TOFMS) showed that depending on the avocado flowers’ cultivar and sexual phase, distinct floral volatiles are released. Flowers sampled in the actively male phase generally released a higher concentration of volatiles, which may contribute to them being preferred by honey bees. In addition to this, linalool (a volatile known for its honey bee attracting properties) was detected in very little amounts in the avocado flowers. This may explain why flowers that exude higher concentrations of linalool (like citrus, for example) are more attractive to honey bees. With these findings in mind, practical methods to encourage honey bee foraging amongst avocado flowers were investigated. Flowering lavender (Lavandula stoechas) and lemongrass (Cymbopogon citratus) essential oils were used to attract honey bees to an avocado orchard in bloom. Honey bee activity was then monitored over a period of time during the flowering season. The lemongrass lures proved effective in increasing honey bee activity, while the lavender plants increased fruit set in trees experiencing an ‘off’ production year. This highlights the importance of promoting pollination in orchards suffering from reduced flowering sites during ‘off’ years. Therefore, this study pioneered possible solutions that can be introduced into commercial avocado orchards to mitigate the problem of low pollination by honey bee vectors. However, these methods will not be effective if the pollen itself is compromised. As avocado production expands into sub-tropical and temperate regions the trees experience more cold stress. A novel pollen counting methodology was used in conjunction with light microscopy to evaluate the effects of cold temperature on pollen development at flower anthesis. There were many problems observed with the anthers and pollen, including absent and empty compartments, and malformed pollen grains. P. americana ‘Fuerte’ anthers appeared to be more adept at maintaining pollen health during cold periods. The number of pollen counted in flowers sampled during the actively female and male phases was also different, suggesting the overnight closure between the phases is sensitive to cold temperature. Considering the avocado trees used in this study are regularly exposed to temperatures below the minimum required for successful meiosis in tropical plants, it is reasonable to suggest these trees will suffer from inadequate pollen production and development. These findings highlight the need for further investigation into how pollen development in avocado trees is affected by cold stress, especially when the trees are cultivated in cooler climates. Lastly, nutrient analysis of floral material (a factor poorly understood in its relation to avocado production) was analysed using nitric acid (HNO₃) digestions and inductively coupled plasma (ICP) analytical methods. The results showed how flowers accumulate varying amounts of potassium, boron, phosphorous, and calcium based on their cultivar, sexual phase, and sampling location. Furthermore, there was a differential accumulation of nutrients between the reproductive and vegetative growth points. These findings show how the critical reproductive sites have a greater ‘pull’ for nutrients. This was the first known attempt to investigate avocado flower nutrition and consider the impact thereof on pollination. While the direct role of many of these nutrients on pollen development in avocados is not well known, other reports claim these nutrients can protect against the effects of cold stress, improve overall pollen health, and even repel honey bees when present in high concentrations. Ultimately, this dissertation highlighted the importance of applying holistic investigative methods to explore the notion of low pollination in avocados. This study found that many factors, including honey bee preferences for certain flowers based on sexual phase and scent, in addition to external factors such as cold stress and nutrient availability, may play interconnected roles that limit pollination and fruit set in avocados. Furthermore, the possibility of improving pollination in avocado orchards by using attractive measures was highlighted. Therefore, this study was successful in evaluating the causes for sub-optimal pollination in avocados and potential solutions thereof. However, as is the case with scientific investigation, this study also uncovered additional facets that require deeper understanding. Future research should thus focus on long-term studies in commercial orchards to evaluate the roles played by alternate bearing, drought, and insecticide sprays on pollinator activity. In addition, the influence of cold stress and nutrient deficiencies on specific physiological processes such as pollen production must be investigated. This will also require the development of floral nutrient statuses, which can be achieved during long-term studies.