Disentangling the microbial ecological factors impacting honey bee susceptibility to Paenibacillus larvae infection.

Paenibacillus larvae is a spore-forming bacterial entomopathogen and causal agent of the important honey bee larval disease, American foulbrood (AFB). Active infections by vegetative P. larvae are often deadly, highly transmissible, and incurable for colonies but, when dormant, the spore form of this pathogen can persist asymptomatically for years. Despite intensive investigation over the past century, this process has remained enigmatic. Here, we provide an up-to-date synthesis on the often overlooked microbiota factors involved in the spore-to-vegetative growth transition (corresponding with the onset of AFB disease symptoms) and offer a novel outlook on AFB pathogenesis by focusing on the 'collaborative' and 'competitive' interactions between P. larvae and other honey bee-adapted microorganisms. Furthermore, we discuss the health trade-offs associated with chronic antibiotic exposure and propose new avenues for the sustainable control of AFB via probiotic and microbiota management strategies.

Differential Selection on Caste-Associated Genes in a Subterranean Termite.

Analyzing the information-rich content of RNA can help uncover genetic events associated with social insect castes or other social polymorphisms. Here, we exploit a series of cDNA libraries previously derived from whole-body tissue of different castes as well as from three behaviourally distinct populations of the Eastern subterranean termite Reticulitermes flavipes. We found that the number (~0.5 M) of single nucleotide variants (SNVs) was roughly equal between nymph, worker and soldier caste libraries, but dN/dS (ratio of nonsynonymous to synonymous substitutions) analysis suggested that some of these variants confer a caste-specific advantage. Specifically, the dN/dS ratio was high (~4.3) for genes expressed in the defensively specialized soldier caste, relative to genes expressed by other castes (~1.7−1.8) and regardless of the North American population (Toronto, Raleigh, Boston) from which the castes were sampled. The populations, meanwhile, did show a large difference in SNV count but not in the manner expected from known demographic and behavioural differences; the highly invasive unicolonial population from Toronto was not the least diverse and did not show any other unique substitution patterns, suggesting any past bottleneck associated with invasion or with current unicoloniality has become obscured at the RNA level. Our study raises two important hypotheses relevant to termite sociobiology. First, the positive selection (dN/dS > 1) inferred for soldier-biased genes is presumably indirect and of the type mediated through kin selection, and second, the behavioural changes that accompany some social insect urban invasions (i.e., 'unicoloniality') may be detached from the loss-of-diversity expected from invasion bottlenecks.

Deteriorating microbiomes in agriculture - the unintended effects of pesticides on microbial life.

There is emerging concern regarding the unintentional and often unrecognized antimicrobial properties of "non-antimicrobial" pesticides. This includes insecticides, herbicides, and fungicides commonly used in agriculture that are known to produce broad ranging, off-target effects on beneficial wildlife, even at seemingly non-toxic low dose exposures. Notably, these obscure adverse interactions may be related to host-associated microbiome damage occurring from antimicrobial effects, rather than the presumed toxic effects of pesticides on host tissue. Here, we critically review the literature on this topic as it pertains to the rhizosphere microbiome of crop plants and gut microbiome of pollinator insects (namely managed populations of the western honey bee, Apis mellifera), since both are frequent recipients of chronic pesticide exposure. Clear linkages between pesticide mode of action and host-specific microbiome functionalities are identified in relation to potential antimicrobial risks. For example, inherent differences in nitrogen metabolism of plant- and insect-associated microbiomes may dictate whether neonicotinoid-based insecticides ultimately exert antimicrobial activities or not. Several other context-dependent scenarios are discussed. In addition to direct effects (e.g., microbicidal action of the parent compound or breakdown metabolites), pesticides may indirectly alter the trajectory of host-microbiome coevolution in honey bees via modulation of social behaviours and the insect gut-brain axis - conceivably with consequences on plant-pollinator symbiosis as well. In summary, current evidence suggests: (1) immediate action is needed by regulatory authorities in amending safety assessments for "non-antimicrobial" pesticides; and (2) that the development of host-free microbiome model systems could be useful for rapidly screening pesticides against functionally distinct microbial catalogues of interest.

No obvious transcriptome-wide signature of indirect selection in termites.

The evolution of sterile helper castes in social insects implies selection on genes that underlie variation in this nonreproductive phenotype. These focal genes confer no direct fitness and are presumed to evolve through indirect fitness effects on the helper's reproducing relatives. This separation of a gene's phenotypic effect on one caste and its fitness effect on another suggests that genes for this and other forms of reproductive altruism are buffered from selection and will thus evolve closer to the neutral rate than genes directly selected for selfish reproduction. We test this hypothesis by comparing the strength of selection at loci associated in their expression with reproductive versus sterile castes in termites. Specifically, we gather caste-biased gene expression data from four termite transcriptomes and measure the global dN/dS ratio across gene sets and phylogenetic lineages. We find that the majority of examined orthologous gene groups show patterns of nucleotide substitution that are consistent with strong purifying selection and display little evidence for distinct signatures of direct versus indirect selection in reproductive and sterile castes. For one particular species (Reticulitermes flavipes), the strength of purifying selection is relaxed in a reproductive nymph-biased gene set, which opposes the nearly neutral idea. In other species, the synonymous rate (dS) alone was often found to be the highest in the sterile worker caste, suggesting a more subtle signature of indirect selection or an altogether different relationship between caste-biased expression and rates of molecular evolution.

Lactobacillus spp. attenuate antibiotic-induced immune and microbiota dysregulation in honey bees.

Widespread antibiotic usage in apiculture contributes substantially to the global dissemination of antimicrobial resistance and has the potential to negatively influence bacterial symbionts of honey bees (Apis mellifera). Here, we show that routine antibiotic administration with oxytetracycline selectively increased tetB (efflux pump resistance gene) abundance in the gut microbiota of adult workers while concurrently depleting several key symbionts known to regulate immune function and nutrient metabolism such as Frischella perrera and Lactobacillus Firm-5 strains. These microbial changes were functionally characterized by decreased capped brood counts (marker of hive nutritional status and productivity) and reduced antimicrobial capacity of adult hemolymph (indicator of immune competence). Importantly, combination therapy with three immunostimulatory Lactobacillus strains could mitigate antibiotic-associated microbiota dysbiosis and immune deficits in adult workers, as well as maximize the intended benefit of oxytetracycline by suppressing larval pathogen loads to near-undetectable levels. We conclude that microbial-based therapeutics may offer a simple but effective solution to reduce honey bee disease burden, environmental xenobiotic exposure, and spread of antimicrobial resistance.

Gene-regulatory context of honey bee worker sterility.

The highly organized societies of the Western honey bee Apis mellifera feature a highly reproductive queen at the center of attention and a large cohort of daughters that suppress their own reproduction to help rear more sisters, some of whom become queens themselves. This reproductive altruism is peculiar because in theory it evolves via indirect selection on genes for altruism that are expressed in the sterile workers but not in the reproductive queens. In this study we attempt to situate lists of genes previously implicated in queenright worker sterility into a broader regulatory framework. To do so we use a model bee brain transcriptional regulatory network as a template to infer how sets of genes responsive to ovary-suppressing queen pheromone are functionally interconnected over the model's topology. We predict that genes jointly involved in the regulation of worker sterility should be tightly networked, relative to genes whose functions are unrelated to each other. We find that sets of mapped genes - ranging in size from 17 to 250 - are well dispersed across the network's substructural scaffolds, suggesting that ovary de-activation involves genes that reside within more than one transcriptional regulatory module. For some sets, however, this dispersion is biased into certain areas of the network's substructure. Our analysis identifies the regions enriched for sterility genes and likewise identifies local hub genes that are presumably critical to subnetwork function. Our work offers a glimpse into the gene regulatory context of honey bee worker sterility and uses this context to identify new candidate gene targets for functional analysis. Finally, to the extent that any sterility-related modules identified here have evolved via selection for worker altruism, we can assume that this selection was indirect and of the type specifically invoked by inclusive fitness theory.

Novel probiotic approach to counter Paenibacillus larvae infection in honey bees.

American foulbrood (AFB) is a highly virulent disease afflicting honey bees (Apis mellifera). The causative organism, Paenibacillus larvae, attacks honey bee brood and renders entire hives dysfunctional during active disease states, but more commonly resides in hives asymptomatically as inactive spores that elude even vigilant beekeepers. The mechanism of this pathogenic transition is not fully understood, and no cure exists for AFB. Here, we evaluated how hive supplementation with probiotic lactobacilli (delivered through a nutrient patty; BioPatty) affected colony resistance towards a naturally occurring AFB outbreak. Results demonstrated a significantly lower pathogen load and proteolytic activity of honey bee larvae from BioPatty-treated hives. Interestingly, a distinctive shift in the microbiota composition of adult nurse bees occurred irrespective of treatment group during the monitoring period, but only vehicle-supplemented nurse bees exhibited higher P. larvae loads. In vitro experiments utilizing laboratory-reared honey bee larvae showed Lactobacillus plantarum Lp39, Lactobacillus rhamnosus GR-1, and Lactobacillus kunkeei BR-1 (contained in the BioPatty) could reduce pathogen load, upregulate expression of key immune genes, and improve survival during P. larvae infection. These findings suggest the usage of a lactobacilli-containing hive supplement, which is practical and affordable for beekeepers, may be effective for reducing enzootic pathogen-related hive losses.

From gene list to gene network: Recognizing functional connections that regulate behavioral traits.

The study of social breeding systems is often gene focused, and the field of insect sociobiology has been successful at assimilating tools and techniques from molecular biology. One common output from sociogenomic studies is a gene list. Gene lists are readily generated from microarray, RNA sequencing, or other molecular screens that typically aim to prioritize genes based on the differences in their expression. Gene lists, however, are often unsatisfying because the information they provide is simply tabular and does not explain how genes interact with each other, or how genetic interactions change in real time under social or environmental circumstances. Here, we promote a view that is relatively common to molecular systems biology, where gene lists are converted into gene networks that better describe the functional connections that regulate behavioral traits. We present a narrative related to honeybee worker sterility to show how network analysis can be used to reprioritize candidate genes based on connectivity rather than their freestanding expression values. Networks can also reveal multigene modules, motifs, clusters or other system-wide properties that might not be apparent from an ab initio list. We argue that because network analyses are not restricted to "genes" as nodes, their implementation can potentially connect multiple levels of biological organization into a single, progressively complex study system.