The Buzz on Honey Bees
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It’s a clear, sunny spring day, when out of the corner of your eye you notice something whiz by. But what? A moment later you are entranced, captivated by the musings of a tiny honey bee as it meanders from flower to flower. There is something enticing about honey bees that continues over and over again to draw us deeper into their world. From film to literature, stories of honey bees are abundant. In fact, my first real recollection of honey bees comes from a story that was read to me at my Nana’s house when I was younger — Berenstain Bears & the Wild Wild Honey — a fictional tale of a hungry bear and his quest for the bees’s golden treasure — honey. Story time aside, Honey Bees (Apis mellifera) are well known for their production of honey, in addition to the significant role as a keystone species in the pollination of plants and flowers.
Honey bees are critically important to agricultural crop production and to the reproduction of most flowering plant species on the planet. Yet, these essential ecosystem service providers are in decline around the world (IPBES). Increased mortality of honey bee colonies has been attributed to several factors, but is not fully understood. However, widespread herbicide and pesticide use, associated with increasing intensive agriculture, is one of the leading factors contributing to this concerning pollinator decline.
The broad-range herbicide glyphosate has long been the primary weed management system, and its utilization is growing globally in agricultural practice. Glyphosate, or perhaps better recognized as Round Up where it is the main active ingredient, is expected to be innocuous to animals, including bees, as it targets an enzyme only found in plants and microorganisms. However, bees rely on a specialized gut microbiota community that benefits growth and provides defense against pathogens. Most bee gut bacteria contain the enzyme targeted by glyphosate and are frequently exposed to the xenobiotic upon foraging by the bee (Herbert et al.). While there is variance among gut bacteria in susceptibility and tolerance to glyphosate, exposing bees to glyphosate can perturb the bee gut microbial community, affecting bee and colony health and their effectiveness as pollinators.
The Shikimate Pathway
The Shikimate Pathway links metabolism of carbohydrates to the biosynthesis of aromatic compounds, such as Phenylalanine, Tyrosine, and Tryptophan. The pathway is defined as the seven step metabolic pathway, beginning with the condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) and ending with the synthesis of chorismate, the precursor of aromatic amino acids and other secondary aromatic metabolites (Herrmann & Weaver). The Shikimate Pathway is found only in microorganisms and plants and never in animals, and is therefore an important target for herbicides, such as Round Up.
Glyphosate (N — (phosphonomethyl)glycine), the primary herbicide used globally for weed control, functions by targeting and inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme in the shikimate pathway, preventing the biosynthesis of aromatic amino acids and other secondary metabolites (Steinrücken & Amrhein). EPSPS catalyzes the reaction between PEP and Shikimate 3-Phosphate (S3P), and glyphosate is a competitive inhibitor that blocks the PEP binding site.
Due to the absence of a shikimate pathway in animals, Glyphosate is considered one of the least toxic pesticides used in agriculture (Duke & Powles). Chemical compounds that interfere with any enzyme activity in this pathway are considered “safe” for humans and other animals when handled in reasonable concentrations. In fact, glyphosate has successfully been tested in mice as a therapeutic agent against pathogenic protozoans that cause diseases such as Toxoplasmosis and Malaria (Roberts, et al.). However, there is evidence to suggest that glyphosate affects non-target organisms: for example, it has been noted to change the behavior of honeybees, reduce reproduction rates of soil-dwelling earthworms, and affect the growth of micro-algae and aquatic bacteria. Further, Glyphosate is also associated with disturbances of gut microbiota of animals living near agricultural sites, such as the gut microbial communities of honey bees.
Glyphosate Perturbs the Gut Microbial Community
To analyze the effects of Glyphosate on the gut microbial community in honey bees, Motta et al. collected adult worker bees from a single hive and treated them with either a 5 mg/L glyphosate (G-5), 10 mg/L glyphosate (G-10) or sterile sucrose syrup (control). The mentioned concentrations of Glyphosate were chosen to mimic environmental levels, which typically range between 1.4 and 7.6 mg/L, and may be encountered by bees foraging at flowering weeds (Herbert et al.). 15 bees were sampled from each group before reintroduction to the hive (day 0) and post-reintroduction to the hive (day 3), and relative and absolute abundances of gut bacteria were then assessed. At day 0, little effect of glyphosate exposure on the bee gut microbiome size was observed. At day 3, after treated bees were returned to the hive, the effects of glyphosate exposure on the bee gut microbiome were more prominent: there were observed compositional shifts in the bacterial community, as well a statistically significant reduction in the total number of gut bacteria for the G-5 treatment group, relative to control. These effects were more significant in the G-5 group compared to the G-10. The lack of significance and observed effects in the G-10 group, while unexplained, may reflect other effects of glyphosate on bees. Motta et al. recapture method failed to sample bees that died or abandoned the hive, and as bees exposed to glyphosate exhibit impaired spatial processing, therefore jeopardizing their return to the hive (Balbuena et al.), bees in the G-10 group may have been less likely to return to the hive after foraging. Consequently, recovered bees may not represent the total effect of glyphosate on treatment groups.
Glyphosate Exposure Increases Susceptibility to Opportunistic Pathogens
To determine whether perturbation of glyphosate on gut microbial communities affects host health, Motta et al. analyzed the susceptibility of glyphosate treated bees to an opportunistic bacterial pathogen. Bees with a conventional gut microbioata and bees lacking a gut microbiota were challenged with the opportunistic bacterial pathogen Serratia marcescens kz19. Control groups not challenged with Serratia were additionally included to ensure that increased mortality was attributable to the effects of glyphosate on the gut microbiota or to direct effects of glyphosate on the bees. As seen in Figure 2., in bees challenged with Serratia, mortality rates were statistically, significantly greater compared to the controls. Further, bees exposed to glyphosate, but not challenged with Serratia, showed survival rates much higher than in the Serratia-challenged groups (Fig.2), reinforcing that glyphosate is not a direct cause of mortality in bees. Rather, glyphosate reduces the protective effect of the gut microbiota against opportunistic pathogens, therefore increasing the host susceptibility to such pathogens, such as Serratia.
EPSPS: a Sensitive and Insensitive Type
As might be predicted, not all bacterial strains share the same susceptibility to Glyphosate. There is a level of variance that exists among gut bacteria in susceptibility and tolerance to the xenobiotic. Bacterial EPSPS exists in two main classes, which corresponds to two distinct phylogenetic clusters that differ in sensitivity to glyphosate. Class I is naturally sensitive, showing a significant effect of glyphosate, whereas class II is insensitive and shows no response to glyphosate (Cao et al.). To identify the EPSPS types present in the bee gut microbiota, Motta et al. constructed a phylogenetic tree using the EPSPS protein of bacterial strains isolated from honey bees, in addition to other representative organisms, such as bumble bees, bacteria, and plants. (Fig. 3). EPSPS sequences from S. alvi, G. apicola, F. perrara, Bifidobacterium, and Apibacter adventoris are clustered with those from other organisms containing a class I EPSPS, and therefore these bacteria are predicted to be sensitive to glyphosate and demonstrate reduced tolerance. Contrarily, sequences from B. apis, Parasaccharibacter apium and Lactobacillus Firm-4 clustered with other bacteria containing a class II EPSPS, and are therefore expected to show no response to glyphosate exposure and a higher resilience, as can be seen in Figure 3.
Not all Class 1 EPSPS ensue a lower resilience to Glyphosate. In fact, S. alvi strains wkB2 and wkB298, despite containing a class I EPSPS, grow as well in
the presence of glyphosate as in its absence, with no initial delay in growth. In order to understand the mechanism that prevents some gut microbial strains from growing in the presence of glyphosate, Motta et al. complemented E. coli ΔaroA with aroA genes cloned from bee gut bacterial strains as well as with the E. coli K12 aroA, which is recognized to be sensitive to glyphosate. The aroA locus encodes EPSPS and has been identified to contribute to Glyphosate resistance (Comai et al.). Transformants carrying the aroA gene from S. alvi, G. apicola, and B. apis were able to grow at a similar rate to the transformant carrying the aroA gene from E. coli. The addition of glyphosate resulted in a delay in growth for transformants carrying the aroA gene. Although S. alvi strains wkB2 and wkB298 were resistant to glyphosate, their aroA versions
were sensitive to glyphosate. Consequently, these bacterial strains are likely to employ a novel mechanism of glyphosate resistance: Motta et al. hypothesizes that these bee gut microbes, as well as others, may have evolved alternative glyphosate resistance mechanisms over a course of history of exposure to the xenobiotic. The presence of an unknown mechanism conferring resistance to glyphosate invites the opportunities of future studies to identify this mechanism, as well as determine the evolutionary origin of resistance.
Honey Bees: A Keystone Species
Exposure of bees to glyphosate, as demonstrated, can perturb their beneficial gut microbiota, ensuing ramifications for both individual and colonial bee health and their effectiveness as pollinators. Honey bees are commonly recognized as a keystone species for their significant role in pollination. Defined by Chapin et al., a keystone species is a species that is ecologically distinct from others in its ecosystem and has a much greater impact on said ecosystem than would be expected given its body size. Biologists have known for a while that honey bees are widespread and abundant, but in his recent study, Hung as identified in quantitative terms that they are currently the most successful pollinators in the world.
Honey bees, originally native to Northern Africa, the Middle East and Southern Europe, represent a cosmopolitan species: having become naturalized in ecosystems across the globe as a result of anthropogenic transport, these bees are able to thrive all around the world. Honey bees are the most widely distributed pollinator, and they’re abundant in many systems. They are able to visit a large diversity of plant species — most whose flowers their bodies fit into — and they ere active for long seasons, sometimes even year-round in select ecosystems (Hung et al.). While the role of honey bees in agricultural is well understood, with honey bees ranking as the most frequent single species of pollinator for crops worldwide (Calderone), still little is understood about the homey bees role in a natural ecosystem.
To analyze the importance and the dominant numbers of the western honey bee (Apis mellifera) despite the great diversity of other bee species encountered in naturally occurring landscapes globally, Hung et al. exploited a recent trend in pollination research — the documentation of community-level, plant –pollinator interaction networks, additionally known as pollination networks. Quantitative pollination network studies document the identity and frequency of each type of pollinator visiting each plant species within a locality (Memmott). Honey bees were recorded in 89 percent of the pollination networks in the honey bee’s native range and in 61 percent of pollination networks in non-native regions, locals where honey bees have been introduced by humans. This translates to approximately one out of eight interactions between a non-agricultural plant and a pollinator being carried out by the honey bee (Hung et al.).
The honey bee’s global importance is further underscored when considering that it is but only one of tens of thousands of pollinating species in the world, including wasps, flies, beetles, butterflies, moths and other bee species. In fact; in San Diego, CA, where there exist established, non-native honey bee colonies, honey bees are responsible for over 75 percent of pollinator visits to native plants, representing the highest honey bee dominance in the introduced range of the honey bee (Wong). This is in lieu of the fact that there are more than 650 other species of native bees in San Diego County, as well as many other native pollinating insects — wasps, beetles, butterflies, etc.
Even in the presence of a highly abundant species that pollinates many plant species (ie. honey bees), there is still a need for healthy populations of other pollinators in order to fulfill the adequate pollination requirements for entire plant communities. In natural habitats where honey bees are present, they nevertheless fail to visit approximately half (49%) of all animal-pollinated plant species (Hung et al.), indicating that despite all the pollinating that honey bees do, they are hardly alone: a significant portion of many flowering plant taxa and assemblages remain dependent on non-A. mellifera visitors for pollination. Consequently, it is absolutely crucial to continue conservation efforts of non-honey-bee pollinators worldwide, even in places dominated by honey bees.
Closing Remarks
Buzz about the significance of honey bees pollination services in both agricultural and natural ecosystems and concerns about the implications of their decline are widespread. As increased mortality rates in honey bee colonies are on the rise paralleling an increase in agricultural practices, it is important to distinguish between the health of feral honey bee populations in natural ecosystems and the health of managed honey bee colonies in agricultural systems. It is important to not confuse the health of managed honey bees with the conservation status of the bee as honey bees appear to be thriving in many natural ecosystems.
As in many animals, honey bees are dependent on their gut microbial community for a vast variety of functions, from food processing to defense against pathogens. Perturbations to these communities have the potential to lead to negative consequences for host fitness, as exampled by Glyphosates’ effects on the bee gut microbiota, from altered composition to increased susceptibility of foreign opportunistic pathogen invasion.
While some species of bacterial strains in the bee gut can tolerate high concentrations of glyphosate due to the presence of a class II EPSPS
enzyme, others are sensitive due to the presence of a class I
EPSPS. However, some bacterial strains with a class I EPSPS may confer resistance to Glyphosate through yet another unknown mechanism. Since bee gut symbionts affect bee development, nutrition, and defense against natural enemies, perturbations of these gut communities may be a factor making bees more susceptible to environmental stressors including poor nutrition and pathogens.
Despite recent increases in the mortality of managed honey bee colonies, these increases are found to be unrelated to the proportion of honey bee visits in natural habitats worldwide (Hung et al.). Agents responsible for increased mortality in managed colonies, such as the herbicide Glyphosate, can affect wild and feral honey bee colonies, however ongoing research suggests that unmanaged, natural populations of honey bees may be better able to cope with parasites and pathogens compared to managed populations (Loftus et al.). As an abundant, super-generalist pollinator, A. mellifera may influence the fitness and behaviour of competing pollinators, alter plant reproduction, and facilitate the spread of non-native weeds and pathogens. Given the ecological importance of honey bees, changes in the species distribution and abundance may impact the evolution of co-occurring animal-pollinated
plants and pollinators.
Animal-mediated pollination represents a vital ecosystem service (Ashman et al.), and quantification of and insight into the pollination services provided by the cosmopolitan, generalist A. mellifera will provide insight into the functioning of many terrestrial ecosystems. Further studies are necessitated to investigate how honey bees, and potential changes in its range and population size, will shape the ecology, evolution and conservation of plants, pollinators and their interactions in natural habitats on local and global scales.
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