The impact of mass-flowering crops on bee pathogen dynamics.

Nearly two fifths of the Earth's land area is currently used for agriculture, substantially impacting the environment and ecosystems. Besides the direct impact through land use change, intensive agriculture can also have an indirect impact, for example by changing wildlife epidemiology. We review here the potential effects of mass-flowering crops (MFCs), which are rapidly expanding in global cropping area, on the epidemiology of known pathogens in bee pollinators. We bring together the fifty MFCs with largest global area harvested and give an overview of their pollination dependency as well as their impact on bee pollinators. When in bloom these crops provide an abundance of flowers, which can provide nutrition for bees and increase bee reproduction. After their short bloom peak, however, the fields turn into green deserts. These big changes in floral availability strongly affect the plant-pollinator network, which in turn affects the pathogen transmission network, mediated by shared flowers. We address this dual role of flowers provided by MFCs, serving as nutritional resources as well as pathogen transmission spots, and bring together the current knowledge to assess how MFCs could affect pathogen prevalence in bee pollinator communities.

Critical View on the Importance of Host Defense Strategies on Virus Distribution of Bee Viruses: What Can We Learn from SARS-CoV-2 Variants?

Bees, both wild and domesticated ones, are hosts to a plethora of viruses, with most of them infecting a wide range of bee species and genera. Although viral discovery and research on bee viruses date back over 50 years, the last decade is marked by a surge of new studies, new virus discoveries, and reports on viral transmission in and between bee species. This steep increase in research on bee viruses was mainly initiated by the global reports on honeybee colony losses and the worldwide wild bee decline, where viruses are regarded as one of the main drivers. While the knowledge gained on bee viruses has significantly progressed in a short amount of time, we believe that integration of host defense strategies and their effect on viral dynamics in the multi-host viral landscape are important aspects that are currently still missing. With the large epidemiological dataset generated over the last two years on the SARS-CoV-2 pandemic, the role of these defense mechanisms in shaping viral dynamics has become eminent. Integration of these dynamics in a multi-host system would not only greatly aid the understanding of viral dynamics as a driver of wild bee decline, but we believe bee pollinators and their viruses provide an ideal system to study the multi-host viruses and their epidemiology.

Honey bees and climate explain viral prevalence in wild bee communities on a continental scale.

Viruses are omnipresent, yet the knowledge on drivers of viral prevalence in wild host populations is often limited. Biotic factors, such as sympatric managed host species, as well as abiotic factors, such as climatic variables, are likely to impact viral prevalence. Managed and wild bees, which harbor several multi-host viruses with a mostly fecal-oral between-species transmission route, provide an excellent system with which to test for the impact of biotic and abiotic factors on viral prevalence in wild host populations. Here we show on a continental scale that the prevalence of three broad host viruses: the AKI-complex (Acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus), Deformed wing virus, and Slow bee paralysis virus in wild bee populations (bumble bees and solitary bees) is positively related to viral prevalence of sympatric honey bees as well as being impacted by climatic variables. The former highlights the need for good beekeeping practices, including Varroa destructor management to reduce honey bee viral infection and hive placement. Furthermore, we found that viral prevalence in wild bees is at its lowest at the extreme ends of both temperature and precipitation ranges. Under predicted climate change, the frequency of extremes in precipitation and temperature will continue to increase and may hence impact viral prevalence in wild bee communities.

Efficacy and biosafety assessment of neuropeptide CAPA analogues against the peach-potato aphid (Myzus persicae).

Insect CAPA neuropeptidesare considered to affect water and ion balance by mediating the physiological metabolism activities of the Malpighian tubules. In previous studies, the CAPA-PK analogue 1895 (2Abf-Suc-FGPRLamide) was reported to decrease aphid fitness when administered through microinjection or via topical application. However, a further statistically significant decrease in the fitness of aphids and an increased mortality could not be established with pairwise combinations of 1895 with other CAPA analogue. In this study, we assessed the topical application of new combinations of 1895 with five CAPA-PVK analogues on the fitness of aphids. We found that 1895 and CAPA-PVK analogue 2315 (ASG-[ß3 L]-VAFPRVamide) was statistically the most effective combination to control the peach potato aphid Myzus persicae nymphs via topical application, leading to 72% mortality. Additionally, the combination (1895+2315) was evaluated against a selection of beneficial insects, that is, a pollinator (Bombus terrestris) and three natural enemies (Chrysoperla carnea, Nasonia vitripennis, and Adalia bipunctata). We found no significant influence on food intake, weight increase, and survival for the pollinator and the three representative natural enemies. These results could facilitate to further establish and generate CAPA analogues as alternatives to broad spectrum and less friendly insecticides.

Towards Integrated Pest and Pollinator Management in Intensive Pear Cultivation: A Case Study from Belgium.

Recently, the concept of Integrated Pest Management (IPM) was further extended into Integrated Pest and Pollinator Management (IPPM). Implementation of IPPM strategies entails the combination of actions for pest and pollinator management providing complementary or synergistic benefits for yield and/or quality of the harvest. The aim of this study was to examine IPPM elements (i.e., mixed hedgerow, nesting boxes for mason bees, Osmia spp.) and demonstrate their impact in the practical context of modern commercial fruit cultivation in a 4-year case study in an intensive 'Conference' pear orchard. The outcomes of visual observations during transect walks and molecular analysis of pollen collected by mason bees, showed the importance of additional floral resources for the presence of mason bees and other pollinating insects in the orchard environment. Pear quality assessments indicated that insect-mediated pollination had a significant positive impact, with a tendency for higher quality pears in the close vicinity of Osmia nesting boxes. However, despite the fact that pear pollen was also detected in Osmia spp. nest cells, the amount and frequency of pear pollen collection for their nest built-up turned out to be rather low. In the same intensive pear orchard studied for pollination effects, we simultaneously demonstrate the impact of a mixed hedgerow to enhance integrated pest control.

More is less: mass-flowering fruit tree crops dilute parasite transmission between bees.

Parasites influence wild bee population dynamics and are regarded as one of the main drivers of wild bee decline. Most of these parasites are mainly transmitted between bee species via the use of shared floral resources. Disturbance of the plant-pollinator network at a location can hence disturb the transmission of these parasites. Expansion and intensification of agriculture, another major driver of wild bee decline, often disturbs local plant-pollinator networks by altering the availability and diversity of floral resources. Mass-flowering crops are an extreme example as they provide an abundance of floral resources for a short period of time, substantially altering the present plant-pollinator network. This likely has repercussions on parasite transmission in the pollinator community. Using the bloom of mass-flowering crops we tested the hypothesis that an increase in floral resources can dilute parasite transmission in the pollinator community. To test this, we analysed the presence of parasites in the pollen of the brood cell provisions of Osmia spp., collected from trap nests placed in apple and sweet cherry orchards. We collected pollen at several time intervals during and after mass bloom, and found that pollen collected during mass bloom had significantly lower parasite prevalence compared with pollen collected after mass bloom. Furthermore, using pollen barcoding data we found that the presence of MFCs in pollen was a good predictor for lower parasite prevalence. Taken together, our results indicate that an increase in flower availability can reduce parasite transmission between bees.

Managed bumble bees acquire parasites from their foraging environment: A case study on parasite spillback.

The use of commercially reared bumble bees in agricultural environments has been recognized as a potential threat to wild pollinators due to competition, genetic contamination, and most notably, disease transmission. Higher parasite prevalence near greenhouses where managed bumble bees are used has been linked to parasite spillover from managed to wild bees. However, pathogen transmission is not unidirectional, and can also flow from wild to managed bees. These newly infected managed bees can subsequently re-infect (other) wild bees, in a process known as spillback, which is an alternative explanation for the increased parasite prevalence near greenhouses. Reducing parasite prevalence in managed bees is key to controlling host-parasite dynamics in cases of spillover; in spillback, producing managed bees that are resilient to infection is important. Here we establish that the managed bumble bee Bombus terrestris can acquire parasites from their foraging environment, which is the major infection route for Apicystis spp. and Crithidia spp., but not for Nosema spp.. Managed B. terrestris were found to have a higher prevalence of Crithdia and a higher load of Apicystis than local wild conspecifics, showing that for these parasites, spillback is a possible risk scenario.

Network Centrality as an Indicator for Pollinator Parasite Transmission via Flowers.

Parasites are important actors within ecosystems. However, a key aspect to unraveling parasite epidemiology is understanding transmission. The bee pollinator community harbors several multihost parasites, which have been shown to be able to spread between species via flowers. Hence the plant-pollinator network can provide valuable information on the transmission of these parasites between species. Although several controlled experiments have shown that flowers function as a transmission hub for parasites, the link with the plant-pollinator network has rarely been addressed in the field. Here, one can hypothesize that the most central flowers in the network are more likely to enable parasite transmission between species. In this study, we test this hypothesis in three local plant-pollinator networks and show that the centrality of a plant in a weighted plant-pollinator network is a good predictor of the presence of multihost pollinator parasites on the plant's flowers.

Bee Viruses: Routes of Infection in Hymenoptera.

Numerous studies have recently reported on the discovery of bee viruses in different arthropod species and their possible transmission routes, vastly increasing our understanding of these viruses and their distribution. Here, we review the current literature on the recent advances in understanding the transmission of viruses, both on the presence of bee viruses in Apis and non-Apis bee species and on the discovery of previously unknown bee viruses. The natural transmission of bee viruses will be discussed among different bee species and other insects. Finally, the research potential of in vivo (host organisms) and in vitro (cell lines) serial passages of bee viruses is discussed, from the perspective of the host-virus landscape changes and potential transmission routes for emerging bee virus infections.

Diversity and Global Distribution of Viruses of the Western Honey Bee, Apis mellifera.

In the past centuries, viruses have benefited from globalization to spread across the globe, infecting new host species and populations. A growing number of viruses have been documented in the western honey bee, Apis mellifera. Several of these contribute significantly to honey bee colony losses. This review synthetizes the knowledge of the diversity and distribution of honey-bee-infecting viruses, including recent data from high-throughput sequencing (HTS). After presenting the diversity of viruses and their corresponding symptoms, we surveyed the scientific literature for the prevalence of these pathogens across the globe. The geographical distribution shows that the most prevalent viruses (deformed wing virus, sacbrood virus, black queen cell virus and acute paralysis complex) are also the most widely distributed. We discuss the ecological drivers that influence the distribution of these pathogens in worldwide honey bee populations. Besides the natural transmission routes and the resulting temporal dynamics, global trade contributes to their dissemination. As recent evidence shows that these viruses are often multihost pathogens, their spread is a risk for both the beekeeping industry and the pollination services provided by managed and wild pollinators.

Double-stranded RNA reduces growth rates of the gut parasite Crithidia mellificae.

Parasites of managed bees can disrupt the colony success of the host, but also influence local bee-parasite dynamics, which is regarded as a threat for wild bees. Therapeutic measures have been suggested to improve the health of managed bees, for instance, exploiting the bees' RNA interference (RNAi) pathway to treat against viral pathogens. Gut trypanosomes are an important group of bee parasites in at least two common managed bee species, i.e., managed Apis mellifera and reared Bombus terrestris. In several trypanosomes, RNAi activity is present, while in other associated genes of RNAi, such as Dicer-like (DCL) and Argonaute (AGO), it is lost. Up to date, the ability to exploit the RNAi of gut trypanosomes of bees has remained unexplored. Here, we screened parasite genomes of two honey bee protozoa (Crithidia mellificae and Lotmaria passim) and two bumble bee protozoa (Crithidia bombi and Crithidia expoeki) for the presence of DCL and AGO proteins. For C. mellificae, we constructed a double-stranded RNA (dsRNA) targeting kinetoplastid membrane protein-11 (KMP-11) to test the RNAi potential to kill this parasite. Transfection with KMP-11 dsRNA, but also adding it to the growth medium resulted in small growth reduction of the trypanosome C. mellificae, thereby showing the limited potential to apply dsRNA therapeutics to control trypanosome infection in managed honey bee species. Within bumble bees, there seems to be no application potentials against C. bombi, as we could only retrieve non-functional DCL- and AGO-related genes within the genome of this bumble bee parasite.

Establishment of wildflower fields in poor quality landscapes enhances micro-parasite prevalence in wild bumble bees.

The current worldwide pollinator decline is caused by the interplay of different drivers. Several strategies have been undertaken to counteract or halt this decline, one of which is the implementation of wildflower fields. These supplementary flowers provide extra food resources and have proven their success in increasing pollinator biodiversity and abundance. Yet such landscape alterations could also alter the host-pathogen dynamics of pollinators, which could affect the populations. In this study, we investigated the influence of sown wildflower fields on the prevalence of micro-parasites and viruses in the wild bumble bee Bombus pascuorum, one of the most abundant bumble bee species in Europe and the Netherlands. We found that the effect of sown wildflower fields on micro-parasite prevalence is affected by the composition of the surrounding landscape and the size of the flower field. The prevalence of micro-parasites increases with increasing size of sown wildflower fields in landscapes with few semi-natural landscape elements. This effect was not observed in landscapes with a high amount of semi-natural landscape elements. We elaborate on two mechanisms which can support these findings: (1) "transmission hot spots" within the altered flower-networks, which could negatively impact hosts experiencing an increased exposure; (2) improved tolerance of the hosts, withstanding higher parasite populations.

Stressful conditions reveal decrease in size, modification of shape but relatively stable asymmetry in bumblebee wings.

Human activities can generate a wide variety of direct and indirect effects on animals, which can manifest as environmental and genetic stressors. Several phenotypic markers have been proposed as indicators of these stressful conditions but have displayed contrasting results, depending, among others, on the phenotypic trait measured. Knowing the worldwide decline of multiple bumblebee species, it is important to understand these stressors and link them with the drivers of decline. We assessed the impact of several stressors (i.e. natural toxin-, parasite-, thermic- and inbreeding- stress) on both wing shape and size and their variability as well as their directional and fluctuating asymmetries. The total data set includes 650 individuals of Bombus terrestris (Hymenoptera: Apidae). Overall wing size and shape were affected by all the tested stressors. Except for the sinigrin (e.g. glucosinolate) stress, each stress implies a decrease of wing size. Size variance was affected by several stressors, contrary to shape variance that was affected by none of them. Although wing size directional and fluctuating asymmetries were significantly affected by sinigrin, parasites and high temperatures, neither directional nor fluctuating shape asymmetry was significantly affected by any tested stressor. Parasites and high temperatures led to the strongest phenotype modifications. Overall size and shape were the most sensitive morphological traits, which contrasts with the common view that fluctuating asymmetry is the major phenotypic marker of stress.

Infection with the multi-host micro-parasite Apicystis bombi (Apicomplexa: Neogregarinorida) decreases survival of the solitary bee Osmia bicornis.

The micro-parasites present in the Apidae pollinator community are mostly multi-host pathogens. To study the impact of these multi-host pathogens on the Apidae pollinator community, as a driver of wild bee decline, pathological studies are needed for different hosts. Yet data on the pathogenesis of these pathogens for different genera and species is scarce or lacking, especially for solitary bee species. In this study, we monitored the effect of the multi-host micro-parasite Apicystis bombi on the lifespan of Osmia bicornis, a common solitary bee. Our results show that A. bombi infection significantly reduces O. bicornis survival.

Interaction effects of different drivers of wild bee decline and their influence on host-pathogen dynamics.

Wild bee decline is a multi-factorial problem, yet it is crucial to understand the impact of a single driver. Hereto the interaction effects of wild bee decline with multiple natural and anthropogenic stressors need to be clear. This is also true for the driver 'pathogens', as stressor induced disturbances of natural host-pathogen dynamics can unbalance settled virulence equilibria. Invasive species, bee domestication, habitat loss, climate changes and insecticides are recognized drivers of wild bee decline, but all influence host-pathogen dynamics as well. Many wild bee pathogens have multiple hosts, which relaxes the host-density limitation of virulence evolution. In conclusion, disturbances of bee-pathogen dynamics can be compared to a game of Russian roulette.

Systemic Israeli acute paralysis virus (IAPV) infection in bumblebees (Bombus terrestris) through feeding and injection.

Israeli acute paralysis virus (IAPV) can cause a systemic infection, resulting in mortality in both Apis and Bombus spp. bees. However, little is known about the virus infection dynamics within bee tissues. Here, we established systemic IAPV infections in reared bumblebee Bombus terrestris workers through feeding and injection and investigated the mortality, tissue tropism and viral localization. Injection of approximately 500 IAPV (IAPVinj stock) particles resulted in acute infection, viral loads within tissues that were relatively stable from bee to bee, and a distinctive tissue tropism, making this method suitable for studying systemic IAPV infection in bumblebees. Feeding with approximately 1 × 106 particles of the same virus stock did not result in systemic infection. A high-concentration stock of IAPV (IAPVfed stock) allowed us to feed bumblebees with approximately 1 × 109 viral particles, which induced both chronic and acute infection. We also observed a higher variability in viral titers within tissues and less clear tissue tropism during systemic infection, making feeding with IAPVfed stock less optimal for studying IAPV systemic infection. Strikingly, both infection methods and stocks with different viral loads gave a similar viral localization pattern in the brain and midgut of bumblebees with an acute infection. The implications of these findings in the study of the local immunity in bees and barriers to oral transmission are discussed. Our data provide useful information on the establishment of a systemic viral infection in bees.

Absence of Leishmaniinae and Nosematidae in stingless bees.

Bee pollination is an indispensable component of global food production and plays a crucial role in sustainable agriculture. The worldwide decline of bee populations, including wild pollinators, poses a threat to this system. However, most studies to date are situated in temperate regions where Apini and Bombini are very abundant pollinators. Tropical and subtropical regions where stingless bees (Apidae: Meliponini) are generally very common, are often overlooked. These bees also face pressure due to deforestation and agricultural intensification as well as the growing use and spread of exotic pollinators as Apis mellifera and Bombus species. The loss or decline of this important bee tribe would have a large impact on their provided ecosystem services, in both wild and agricultural landscapes. The importance of pollinator diseases, which can contribute to decline, has not been investigated so far in this bee tribe. Here we report on the first large pathogen screening of Meliponini species in southern Brazil. Remarkably we observed that there was an absence of Leishmaniinae and Nosematidae, and a very low occurrence of Apicystis bombi. Our data on disease prevalence in both understudied areas and species, can greatly improve our knowledge on the distribution of pathogens among bee species.

Large pathogen screening reveals first report of Megaselia scalaris (Diptera: Phoridae) parasitizing Apis mellifera intermissa (Hymenoptera: Apidae).

As it is most likely that global warming will also lead to a shift in pollinator-habitats northwards, the study of southern species becomes more and more important. Pathogen screenings in subspecies of Apis mellifera capable of withstanding higher temperatures, provide an insight into future pathogen host interactions. Screenings in different climate regions also provide a global perspective on the prevalence of certain pathogens. In this project, we performed a pathogen screening in Apis mellifera intermissa, a native subspecies of Algeria in northern Africa. Colonies were sampled from different areas in the region of Annaba over a period of two years. Several pathogens were detected, among them Apicystis bombi, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae, Lake Sinai Virus, Sacbrood Virus and Deformed Wing Virus (DWV). Our screening also revealed a phoroid fly, Megaselia scalaris, parasitizing honey bee colonies, which we report here for the first time. In addition, we found DWV to be present in the adult flies and replicating virus in the larval stages of the fly, which could indicate that M. scalaris acts as a vector of DWV.

The Effect of Oral Administration of dsRNA on Viral Replication and Mortality in Bombus terrestris.

Israeli acute paralysis virus (IAPV), a single-stranded RNA virus, has a worldwide distribution and affects honeybees as well as other important pollinators. IAPV infection in honeybees has been successfully repressed by exploiting the RNA interference (RNAi) pathway of the insect's innate immune response with virus-specific double stranded RNA (dsRNA). Here we investigated the effect of IAPV infection in the bumblebee Bombus terrestris and its tissue tropism. B. terrestris is a common pollinator of wild flowers in Europe and is used for biological pollination in agriculture. Infection experiments demonstrated a similar pathology and tissue tropism in bumblebees as reported for honeybees. The effect of oral administration of virus-specific dsRNA was examined and resulted in an effective silencing of the virus, irrespective of the length. Interestingly, we observed that non-specific dsRNA was also efficient against IAPV. However further study is needed to clarify the precise mechanism behind this effect. Finally we believe that our data are indicative of the possibility to use dsRNA for a broad range viral protection in bumblebees.

The immune response of the small interfering RNA pathway in the defense against bee viruses.

Most bee viruses are RNA viruses belonging to two major families of Dicistroviridae and Iflaviridae. During viral infection, virus-derived double stranded RNAs activate a major host innate immune pathway, namely the small interfering RNAs pathway (siRNA pathway), which degrades the viral RNA or the viral genome. This results in 21-22 nucleotide-long virus-derived siRNAs (vsiRNAs). Recent studies showed that vsiRNAs, matching to viruses from the family of Dicistroviridae and Iflaviridae, were generated in infected bees. Moreover, higher virus titers in honeybees also resulted in higher amounts of vsiRNAs, demonstrating that the siRNA response is proportional to the intensity of viral infection. Intriguingly, non-specific dsRNA could also trigger an immune response, leading to the restriction of the viral infection, however this mechanism is still unclear. Other findings demonstrated that bees can be protected through introducing virus specific-dsRNA to activate the siRNA response against the target virus. The latter is highlighting a new strategy to tackle bee viruses.