Trypanosome Infection Disturbs the Gut Microbiota of Bumble Bee Hosts

Host-microbe interactions are ubiquitous with both positive and negative outcomes for host health and fitness. While pathogens abound, there has been an increased focus beneficial host-microbial community associations, such as native gut microbiota that can aid in disease resistance, nutrient sequestration, and detoxification. However, while beneficial microbes can protect against infection, there is a limited appreciation for how pathogen infection may disrupt healthy gut microbiota structure and function. Crithidia bombi is a common trypanosomatid gut parasite of bumble bees with fitness-relevant effects on individuals and colonies. The native bumble bee gut microbiota has been shown to increase protection against Crithidia, and an association has been found between infection occurrence and host gut microbiota structure in nature. However, we hypothesize that the association between gut microbiota structure and infection could also emerge if infection disrupts the healthy gut community, especially early during the gut community establishment. Using experimental inoculations, we investigate if Crithidia infection alters the bumble bee gut microbiota community. Bombus impatiens workers were exposed early in microbiota colonization or post-establishment to one of three C. bombi strains or left naïve. Subsequently, guts were dissected to quantify total bacterial load, numbers of key bumble bee gut bacterial symbionts, and C. bombi. We find that infection by Crithidia leads to a reduction in total gut bacteria, and when it occurs early during microbiota colonization, Lactobacillus spp. and Gilliamella spp. numbers are reduced and increased, respectively. This provides evidence for an infection-mediated disruption of the gut microbiota balance, which could explain microbiota structure and infection associations in the field. Furthermore, healthy microbiota perturbation is another way that pathogens may detrimentally affect hosts, and may underlie some previously described infection effects. This adds to our understanding of the multi-dimensional and directionality of effects between host organisms and their parasitic and beneficial microbes

Studies on the novel effects of feeding non-thermally treated honeybee gathered pollen on the colony stability and outputs of commercially-reared bumble bees ( Bombus terrestris) for pollination services

Pollination is a vital resource, carried out mainly by animals, which without could threaten food security on a global scale. Of the agricultural crops that are produced globally, animal pollination is responsible for 40%. Among animal pollinators, insects such as the honey and bumblebee are considered some of the most important for pollination of foods such as almonds, apples, strawberries, plums and blueberries. Commercial colonies of both are sold species are sold every year, which includes the global importation to over 50 countries of one million bumblebee colonies for the purpose of pollination. This practice of using commercially produced bee colonies for pollination is worth an estimated $14 billion to the US economy and$15 billion to the European economy per annum. Despite the largescale use of commercial colonies, wild bees are still seen as major contributors to the pollination of crops and flowering plants. However, their survival is undertreat, due to a multitude of factors, such as climate change, pesticides, habitat lose and disease with the latter being considered as some of the major drivers. One of the major concerns with disease spread has been brought about by the introduction of commercial colonies. Commercial colonies are marketed as pathogen free, however many studies have found that this is not always the case. Disease carrying bees have been known to forage large distances and then disperse pathogens on flowers, which in turn act as reservoirs. Separate studies in both Canada and Ireland found that certain bee parasites were more prevalent in wild bees the closer they were to commercial greenhouses and that this lessened the further away the bees were captured. Wild bees who then forage on these flowers then pick up the pathogens and bring them back to their nests, contaminating the hives, a practice known as pathogen spillover. Furthermore evidence has also being found that suggests not only are commercial bees a source of contamination, but so is the pollen that they are fed on. Commercial pollen is collected by honeybees and fed to both commercial honey and bumblebees colonies. Studies have found that it is often contaminated with parasites such as Crithidia bombi and Nosema species as well as some viruses. This suggests that commercial pollen could be a major driver in the spread of pathogens from commercial colonies to wild colonies and could be responsible.ye

Fecal transplant allows transmission of the gut microbiota in honey bees.

The study of the fecal microbiota is crucial for unraveling the pathways through which gut symbionts are acquired and transmitted. While stable gut microbial communities are essential for honey bee health, their modes of acquisition and transmission are yet to be confirmed. The gut of honey bees is colonized by symbiotic bacteria within 5 days after emergence from their wax cells as adults. Few studies have suggested that bees could be colonized in part via contact with fecal matter in the hive. However, the composition of the fecal microbiota is still unknown. It is particularly unclear whether all bacterial species can be found viable in the feces and can therefore be transmitted to newborn nestmates. Using 16S rRNA gene amplicon sequencing, we revealed that the composition of the honey bee fecal microbiota is strikingly similar to the microbiota of entire guts. We found that fecal transplantation resulted in gut microbial communities similar to those obtained from feeding gut homogenates. Our study shows that fecal sampling and transplantation are viable tools for the non-invasive analysis of bacterial community composition and host-microbe interactions. It also implies that contact of young bees with fecal matter in the hive is a plausible route for gut microbiota acquisition. Honey bees are crucial pollinators for many crops and wildflowers. They are also powerful models for studying microbiome-host interactions. However, current methods rely on gut tissue disruption to analyze microbiota composition and use gut homogenates to inoculate microbiota-deprived bees. Here, we provide two new and non-invasive approaches that will open doors to longitudinal studies: fecal sampling and transplantation. Furthermore, our findings provide insights into gut microbiota transmission in social insects by showing that ingestion of fecal matter can result in gut microbiota acquisition

Interplay between gut symbionts and behavioral variation in social insects.

Social insects exhibit a high degree of intraspecific behavioral variation. Moreover, they often harbor specialized microbial communities in their gut. Recent studies suggest that these two characteristics of social insects are interlinked: insect behavioral phenotypes affect their gut microbiota composition, partly through exposure to different environments and diet, and in return, the gut microbiota has been shown to influence insect behavior. Here, we discuss the bidirectional relationship existing between intraspecific variation in gut microbiota composition and behavioral phenotypes in social insects

Effects of glyphosate and glyphosate-based herbicide on learning and memory of the buff-tailed bumblebee (Bombus terrestris)

Abstract The decline of insect pollinators is a global concern, and the use of pesticides has been identified as a potential cause for it. Glyphosate-based herbicides (GBHs) are the world's most used pesticides, but until recent years they have been claimed to be safe for non-target organisms, such as pollinators. Using controlled arena experiments, we investigated the effects on the learning and long-term memory of buff-tailed bumblebees, Bombus terrestris (L.) (Hymenoptera: Apidae), of a single field-realistic dose of glyphosate, both in its pure form and as a commercial herbicide (Roundup Gold) containing the active ingredient (a.i.) glyphosate and other co-formulants. We found that glyphosate in its pure form caused deterioration in the learning ability of bumblebees in a 10-color discrimination experiment; the glyphosate-treated bees discriminated colors over 10% worse than the untreated control bees. However, the commercially used GBH (Roundup Gold) had no observable effect on the learning ability of the bumblebees, despite the fact that this herbicide contained the same amount of glyphosate as its pure form. These findings shed light on the potential risks associated with agrochemicals previously considered safe for pollinators and emphasize the need for comprehensive risk assessments of pesticides, including evaluations of cognitive functions in pollinators. Therefore, we propose that future pesticide testing should incorporate broader assessments to ensure the safety of pollinators in agricultural landscapes.Abstract The decline of insect pollinators is a global concern, and the use of pesticides has been identified as a potential cause for it. Glyphosate-based herbicides (GBHs) are the world's most used pesticides, but until recent years they have been claimed to be safe for non-target organisms, such as pollinators. Using controlled arena experiments, we investigated the effects on the learning and long-term memory of buff-tailed bumblebees, Bombus terrestris (L.) (Hymenoptera: Apidae), of a single field-realistic dose of glyphosate, both in its pure form and as a commercial herbicide (Roundup Gold) containing the active ingredient (a.i.) glyphosate and other co-formulants. We found that glyphosate in its pure form caused deterioration in the learning ability of bumblebees in a 10-color discrimination experiment; the glyphosate-treated bees discriminated colors over 10% worse than the untreated control bees. However, the commercially used GBH (Roundup Gold) had no observable effect on the learning ability of the bumblebees, despite the fact that this herbicide contained the same amount of glyphosate as its pure form. These findings shed light on the potential risks associated with agrochemicals previously considered safe for pollinators and emphasize the need for comprehensive risk assessments of pesticides, including evaluations of cognitive functions in pollinators. Therefore, we propose that future pesticide testing should incorporate broader assessments to ensure the safety of pollinators in agricultural landscapes

Gut Symbiont Viability in Honey Bees Exposed to Agrochemical Stressors

The honey bee gut microbiome is essential for protecting this pollinator against abiotic and biotic stressors, including the prevention of harmful gut parasites and pathogens. Previous studies have not only demonstrated a linkage of bee gut dysbiosis to increased immunodeficiencies and pathogen sensitivities, but also report the maladaptation of the gut microbiome in bees exposed to agricultural and apicultural chemistries. There are few techniques available that allow for a simple and reliable analysis of the relative proportions of live and dead gut microbes in bees exposed to these chemistries. Previous techniques for measuring gut symbiont dysbiosis are temporally limited by the digestion and excretion of non-viable, double-stranded DNA (dsDNA) from the host. Here, I will report a propidium monoazide (PMA)-based qPCR technique to quantify the antibiotic- and fungicide-mediated dysbiosis of the bee gut microbiome. Bees fed the antibiotics oxytetracycline and tylosin exhibited a 78% and 82% reduction, respectively, of gut bacteria abundance when compared to untreated bees. Similarly, gut microbes in bees fed chlorothalonil and Fumagilin-B were reduced by 44% and 68%, respectively, compared to untreated bees. These data demonstrate the bee microbiome to be depauperated within 24 h of exposure to agricultural and apicultural chemistries. These data support previous evidence that agrochemical exposures may increase pathogenicity of bee pathogens and gut parasites because of the critical role gut microbiomes play in aiding the host immune system. Fungicides, such as chlorothalonil, are not regulated to the extent of other pesticides and are sprayed during the high activity periods of pollinators when incidental exposure are more likely to occur. This PMA-based qPCR approach, coupled with DNA sequencing, is a useful technology that can rapidly identify changes in abundance and diversity of bee gut symbionts after fungicide or antibiotic exposures. In turn, a PMA-based qPCR approach can assist in the discovery of abiotic and biotic stressors of bee gut symbionts, which is an important step towards reducing the loss of a managed agricultural pollinator. Advisor: Troy D. Anderso

Spanning the Symbiotic Spectrum: From Bees and Microbes to Art and Science

The following dissertation covers several case studies in symbiosis. Chapter 1 investigates the evolution of pathogenicity of Ascosphaera fungi. Chapter 2 uncovers the microbial composition of the gut of stingless bees across diet types (pollinivorous, facultative necrophagy and obligate necrophagy). Chapter 3 investigates the function of the microbes in the guts of stingless bees across the same three diet types. Chapter 4 extends the concept of symbiosis to art-science collaborations and discusses the impact for science communication. Overall, these four chapters illustrate the importance of flexibility in symbiotic relationships

Searching for the gut microbial contributing factors to social behavior in rodent models of autism spectrum disorder

Social impairment is one of the major symptoms in multiple psychiatric disorders, including autism spectrum disorder (ASD). Accumulated studies indicate a crucial role for the gut microbiota in social development, but these mechanisms remain unclear. This review focuses on two strategies adopted to elucidate the complicated relationship between gut bacteria and host social behavior. In a top-down approach, researchers have attempted to correlate behavioral abnormalities with altered gut microbial profiles in rodent models of ASD, including BTBR mice, maternal immune activation (MIA), maternal valproic acid (VPA) and maternal high-fat diet (MHFD) offspring. In a bottom-up approach, researchers use germ-free (GF) animals, antibiotics, probiotics or pathogens to manipulate the intestinal environment and ascertain effects on social behavior. The combination of both approaches will hopefully pinpoint specific bacterial communities that control host social behavior. Further discussion of how brain development and circuitry is impacted by depletion of gut microbiota is also included. The converging evidence strongly suggests that gut microbes affect host social behavior through the alteration of brain neural circuits. Investigation of intestinal microbiota and host social behavior will unveil any bidirectional communication between the gut and brain and provide alternative therapeutic targets for ASD

Evaluating wild and commercial populations of Bombus terrestris ssp. audax (Harris, 1780): from genotype to phenotype.

Bees, including bumblebees, are highly valued for the pollination services they provide to natural ecosystems and agricultural crops. However, many bee species are facing declines, likely a result of habitat loss, pesticide use and climate change. Additionally, the use of imported commercial bumblebee colonies for crop pollination poses several risks to wild pollinators, including competition, hybridisation and pathogen spillover. A stock-take is needed of wild bees on both genetic and functional levels to identify vulnerable populations, detect local adaptations and to prevent further pollinator losses. We examine wild Irish B. terrestris ssp. audax on genomic, proteomic, and behavioural levels with reference to British and commercial populations to deepen our understanding of the selective processes acting on wild and domesticated bumblebee populations. We find that wild Irish and British populations of B. t. audax are distinctive on genomic levels and exhibit differential signatures of selection. We also find putative evidence for genetic distinctions between wild and commercial populations. A genomic examination of canonical immune genes in wild, Irish bumblebees highlighted several genes undergoing positive, purifying and possibly balancing selection, possibly reflecting their functional diversity and indicating recent adaptation. We uncover distinctions in the proteomes of wild and commercial lineages of lab-reared worker bee fat bodies and brains, as well as in the proteomic responses of these organs to pesticide exposure and infection. Finally, distinctions in the growth dynamics of wild and commercial lineages of B. t. audax colonies were identified alongside differences in the bacterial and fungal gut microbiomes of lab-reared wild and commercial workers. Overall, the findings of this thesis provide novel insights into the genetic, physiological, and behavioural distinctions between wild and domesticated populations of B. t. audax which will likely have major implications for how we conserve valuable genetic resources and manage commercial bumblebee imports

Variation of gut microbiota composition in a honey bee breeding population: exploring potential links with docility and honey production

The current global decline of bee populations is of great concern due to their crucial role as pollinators and for the conservation of biodiversity. Today the survival of bees is increasingly dependent on beekeeping practices. In this context, the present study explores the composition of honey bee gut microbiota, its changes in time and its potential relationship with two key traits of interest to beekeepers: docility and honey yield. In this study, 77 colonies, belonging to a breeding population selected for these phenotypes, were sampled three times over a 5-month period, leading to a total of 190 samples. Results showed that Apis mellifera, differently from other insects, hosts a specialised gut microbial community composed of five ever-present bacterial taxa. However, the proportional abundance of these bacterial taxa undergoes significant seasonal shifts, reflecting seasonal changes in diet. Moreover, the association between the composition of the honey bee microbiota and honey production was identified. In conclusion, this study offers insights into the composition and the seasonal dynamics of honey bee gut microbiota