multimedia: Multimodal Mediation Analysis of Microbiome Data.

Mediation analysis has emerged as a versatile tool for answering mechanistic questions in microbiome research because it provides a statistical framework for attributing treatment effects to alternative causal pathways. Using a series of linked regression models, this analysis quantifies how complementary data modalities relate to one another and respond to treatments. Despite these advances, the rigid modeling assumptions of existing software often results in users viewing mediation analysis as a black box, not something that can be inspected, critiqued, and refined. We designed the multimedia R package to make advanced mediation analysis techniques accessible to a wide audience, ensuring that all statistical components are easily interpretable and adaptable to specific problem contexts. The package provides a uniform interface to direct and indirect effect estimation, synthetic null hypothesis testing, and bootstrap confidence interval construction. We illustrate the package through two case studies. The first re-analyzes a study of the microbiome and metabolome of Inflammatory Bowel Disease patients, uncovering potential mechanistic interactions between the microbiome and disease-associated metabolites, not found in the original study. The second analyzes new data about the influence of mindfulness practice on the microbiome. The mediation analysis identifies a direct effect between a randomized mindfulness intervention and microbiome composition, highlighting shifts in taxa previously associated with depression that cannot be explained by diet or sleep behaviors alone. A gallery of examples and further documentation can be found at https://go.wisc.edu/830110.

Mapping microhabitats of lignocellulose decomposition by a microbial consortium.

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

MolPad: An R-Shiny Package for Cluster Co-Expression Analysis in Longitudinal Microbiomics.

The R-Shiny package MolPad provides an interactive dashboard for understanding the dynamics of longitudinal molecular co-expression in microbiomics. The main idea for addressing the issue is first to use a network to overview major patterns among their predictive relationships and then zoom into specific clusters of interest. It is designed with a focus-plus-context analysis strategy and automatically generates links to online curated annotations. The dashboard consists of a cluster-level network, a bar plot of taxonomic composition, a line plot of data modalities, and a table for each pathway. Further, the package includes functions that handle the data processing for creating the dashboard. This makes it beginner-friendly for users with less R programming experience. We illustrate these methods with a case study on a longitudinal metagenomics analysis of the cheese microbiome.

Bee species perform distinct foraging behaviors that are best described by different movement models.

In insect-pollinated plants, the foraging behavior of pollinators affects their pattern of movement. If distinct bee species vary in their foraging behaviors, different models may best describe their movement. In this study, we quantified and compared the fine scale movement of three bee species foraging on patches of Medicago sativa. Bee movement was described using distances and directions traveled between consecutive racemes. Bumble bees and honey bees traveled shorter distances after visiting many flowers on a raceme, while the distance traveled by leafcutting bees was independent of flower number. Transition matrices and vectors were calculated for bumble bees and honey bees to reflect their directionality of movement within foraging bouts; leafcutting bees were as likely to move in any direction. Bee species varied in their foraging behaviors, and for each bee species, we tested four movement models that differed in how distances and directions were selected, and identified the model that best explained the movement data. The fine-scale, within-patch movement of bees could not always be explained by a random movement model, and a general model of movement could not be applied to all bee species.

Genomic diversification of the specialized parasite of the fungus-growing ant symbiosis.

Fungi shape the diversity of life. Characterizing the evolution of fungi is critical to understanding symbiotic associations across kingdoms. In this study, we investigate the genomic and metabolomic diversity of the genus Escovopsis, a specialized parasite of fungus-growing ant gardens. Based on 25 high-quality draft genomes, we show that Escovopsis forms a monophyletic group arising from a mycoparasitic fungal ancestor 61.82 million years ago (Mya). Across the evolutionary history of fungus-growing ants, the dates of origin of most clades of Escovopsis correspond to the dates of origin of the fungus-growing ants whose gardens they parasitize. We reveal that genome reduction, determined by both genomic sequencing and flow cytometry, is a consistent feature across the genus Escovopsis, largely occurring in coding regions, specifically in the form of gene loss and reductions in copy numbers of genes. All functional gene categories have reduced copy numbers, but resistance and virulence genes maintain functional diversity. Biosynthetic gene clusters (BGCs) contribute to phylogenetic differences among Escovopsis spp., and sister taxa in the Hypocreaceae. The phylogenetic patterns of co-diversification among BGCs are similarly exhibited across mass spectrometry analyses of the metabolomes of Escovopsis and their sister taxa. Taken together, our results indicate that Escovopsis spp. evolved unique genomic repertoires to specialize on the fungus-growing ant-microbe symbiosis.

Natural Selection Shapes Maintenance of Orthologous sRNAs in Divergent Host-Restricted Bacterial Genomes.

Historically it has been difficult to study the evolution of bacterial small RNAs (sRNAs) across distantly related species. For example, identifying homologs of sRNAs is often difficult in genomes that have undergone multiple structural rearrangements. Also, some types of regulatory sRNAs evolve at rapid rates. The high degree of genomic synteny among divergent host-restricted bacterial lineages, including intracellular symbionts, is conducive to sRNA maintenance and homolog identification. In turn, symbiont genomes can provide us with novel insights into sRNA evolution. Here, we examine the sRNA expression profile of the obligate symbiont of psyllids, Carsonella ruddii, which has one of the smallest cellular genomes described. Using RNA-seq, we identified 36 and 32 antisense sRNAs (asRNAs) expressed by Carsonella from the psyllids Bactericera cockerelli (Carsonella-BC) and Diaphorina citri (Carsonella-DC), respectively. The majority of these asRNAs were associated with genes that are involved in essential amino acid biosynthetic pathways. Eleven of the asRNAs were conserved in both Carsonella lineages and the majority were maintained by selection. Notably, five of the corresponding coding sequences are also the targets of conserved asRNAs in a distantly related insect symbiont, Buchnera. We detected differential expression of two asRNAs for genes involved in arginine and leucine biosynthesis occurring between two distinct Carsonella-BC life stages. Using asRNAs identified in Carsonella, Buchnera, and Profftella which are all endosymbionts, and Escherichia coli, we determined that regions upstream of these asRNAs encode unique conserved patterns of AT/GC richness, GC skew, and sequence motifs which may be involved in asRNA regulation.

Burkholderia from Fungus Gardens of Fungus-Growing Ants Produces Antifungals That Inhibit the Specialized Parasite Escovopsis.

Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants' fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus converting the plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis. Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia and Paraburkholderia from 50% of the 52 colonies sampled, indicating that members of the family Burkholderiaceae are common inhabitants in the fungus gardens of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1 cm. Genomic assessment of fungus garden-associated Burkholderiaceae indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed that these compounds are responsible for antifungal activities that inhibit Escovopsis but, at equivalent concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of fungus-growing ant colonies. IMPORTANCE Many organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect Leucoagaricus spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, Escovopsis. The ants take multiple approaches, including weeding their fungus gardens to remove Escovopsis spores, as well as harboring Pseudonocardia spp., bacteria that produce antifungals that inhibit Escovopsis. In addition, a genus of bacteria commonly found in fungus gardens, Burkholderia, is known to produce secondary metabolites that inhibit Escovopsis spp. In this study, we isolated Burkholderia spp. from fungus-growing ants, assessed the isolates' ability to inhibit Escovopsis spp., and identified two compounds responsible for inhibition. Our findings suggest that Burkholderia spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite Escovopsis.

Continent-Scale Sampling Reveals Fine-Scale Turnover in a Beneficial Bug Symbiont.

Many members of animal-associated microbial communities, including the gut flora, are acquired from their host's environment. While many of these communities are species rich, some true bugs (Hemiptera) in the superfamilies Lygaeoidea and Coreidae allow only ingested Burkholderia to colonize and reproduce in a large portion of the midgut. We studied the spatial structuring of Burkholderia associated with a widespread omnivorous bug genus, Jalysus (Berytidae). We sampled Wickham's stilt bug, Jalysus wickhami, across the United States and performed limited sampling of its sister species, the spined stilt bug Jalysus spinosus. We asked: (1) What Burkholderia strains are hosted by Jalysus at different locations? (2) Does host insect species, host plant species, or location influence the strain these insects acquire? (3) How does Burkholderia affect the development and reproductive fitness of J. wickhami? We found: (1) Sixty-one Burkholderia strains were present across a sample of 352 individuals, but one strain dominated, accounting for almost half of all symbiont reads. Most strains were closely related to other hemipteran Burkholderia symbionts. (2) Many individuals hosted more than one strain of Burkholderia. (3) J. wickhami and J. spinosus did not differ in the strains they hosted. (4) Insects that fed on different plant species tended to host different Burkholderia, but this accounted for only 4% of the variation in strains hosted. In contrast, the location at which an insect was collected explained 27% of the variation in symbiont strains. (5) Burkholderia confers important fitness benefits to J. wickhami. In laboratory experiments, aposymbiotic (Burkholderia-free) insects developed more slowly and laid fewer eggs than symbiotic (Burkholderia-colonized) insects. (6) In the lab, nymphs sometimes acquired Burkholderia via indirect exposure to adults, indicating that horizontal symbiont transmission can occur via adult insect-mediated enrichment of Burkholderia in the local environment - a phenomenon not previously reported in bug-Burkholderia relationships. Taken together, the results suggest that for these bugs, critical nutritional requirements are outsourced to a highly diverse and spatially structured collection of Burkholderia strains acquired from the environment and, occasionally, from conspecific adults.

Changes in Aphid Host Plant Diet Influence the Small-RNA Expression Profiles of Its Obligate Nutritional Symbiont, Buchnera.

Plants are a difficult food resource to use, and herbivorous insects have evolved a variety of mechanisms that allow them to fully exploit this poor nutritional resource. One such mechanism is the maintenance of bacterial symbionts that aid in host plant feeding and development. The majority of these intracellular symbionts have highly eroded genomes that lack many key regulatory genes; consequently, it is unclear if these symbionts can respond to changes in the insect's diet to facilitate host plant use. There is emerging evidence that symbionts with highly eroded genomes express small RNAs (sRNAs), some of which potentially regulate gene expression. In this study, we sought to determine if the reduced genome of the nutritional symbiont (Buchnera) in the pea aphid responds to changes in the aphid's host plant diet. Using transcriptome sequencing (RNA-seq), Buchnera sRNA expression profiles were characterized within two Buchnera life stages when pea aphids fed on either alfalfa or fava bean. Overall, this study demonstrates that Buchnera sRNA expression changes not only with life stage but also with changes in aphid host plant diet. Of the 321 sRNAs characterized in this study, 47% were previously identified and 22% showed evidence of conservation in two or more Buchnera taxa. Functionally, 13 differentially expressed sRNAs were predicted to target genes related to pathways involved in essential amino acid biosynthesis. Overall, results from this study reveal that host plant diet influences the expression of conserved and lineage-specific sRNAs in Buchnera and that these sRNAs display distinct host plant-specific expression profiles among biological replicates.IMPORTANCE In general, the genomes of intracellular bacterial symbionts are reduced compared to those of free-living relatives and lack many key regulatory genes. Many of these reduced genomes belong to obligate mutualists of insects that feed on a diet that is deficient in essential nutrients, such as essential amino acids. It is unclear if these symbionts respond with their host to changes in insect diet, because of their reduced regulatory capacity. Emerging evidence suggests that these symbionts express small RNAs (sRNAs) that regulate gene expression at the posttranscriptional level. Therefore, in this study, we sought to determine if the reduced genome of the nutritional symbiont Buchnera in the pea aphid responds to changes in the aphid's host plant diet. This study demonstrates for the first time that Buchnera sRNAs, some conserved in two or more Buchnera lineages, are differentially expressed when aphids feed on different plant species and potentially target genes within essential amino acid biosynthesis pathways.

It's a small, small world: unravelling the role and evolution of small RNAs in organelle and endosymbiont genomes.

Organelles and host-restricted bacterial symbionts are characterized by having highly reduced genomes that lack many key regulatory genes and elements. Thus, it has been hypothesized that the eukaryotic nuclear genome is primarily responsible for regulating these symbioses. However, with the discovery of organelle- and symbiont-expressed small RNAs (sRNAs) there is emerging evidence that these sRNAs may play a role in gene regulation as well. Here, we compare the diversity of organelle and bacterial symbiont sRNAs recently identified using genome-enabled '-omic' technologies and discuss their potential role in gene regulation. We also discuss how the genome architecture of small genomes may influence the evolution of these sRNAs and their potential function. Additionally, these new studies suggest that some sRNAs are conserved within organelle and symbiont taxa and respond to changes in the environment and/or their hosts. In summary, these results suggest that organelle and symbiont sRNAs may play a role in gene regulation in addition to nuclear-encoded host mechanisms.

Efficacy of In Vivo Electroporation on the Delivery of Molecular Agents into Aphid (Hemiptera: Aphididae) Ovarioles.

While the wealth of genomic data presently available is increasing rapidly, the advancement of functional genomics technologies for the large majority of these organisms has lagged behind. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 system is an emerging gene-editing technology derived from a bacterial adaptive immune system that has proven highly effective in multiple model systems. Here, the CRISPR/Cas9 system was delivered into the ovarioles of the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphididae), with a new delivery method utilizing in vivo electroporation. To validate gene-editing, a target sequence within the marker tor pigment gene was chosen, and gene-editing was predicted to result in white pigmentation in the offspring of treated adult aphids. Adult aphids (10-d old) were injected with the tor single guide RNA and Cas9 complex and subsequently subjected to electroporation. Adult aphids were given 4 d to produce viviparous offspring. After offspring developed for 6 d, DNA was extracted and sequenced to validate if CRISPR/Cas9-directed gene editing occurred. A survival rate over 70% was found in treated adult aphids. A distinct white pigmentation was found in 2.5% of aphids; however, gene-editing within the target sequence was not found in any of the individuals screened. Presence of white aphids without gene-editing suggests other mechanisms may have influenced pigmentation. High survival rates in experimental treatments demonstrate the robustness of this new technique, and further refinement of this technique may prove it as an effective functional genomics tool for viviparous insects and/or gene editing at a somatic level.

A sRNA in a reduced mutualistic symbiont genome regulates its own gene expression.

Similar to other nutritional endosymbionts that are obligate for host survival, the mutualistic aphid endosymbiont, Buchnera, has a highly reduced genome with few regulatory elements. Until recently, it was thought that aphid hosts were primarily responsible for regulating their symbiotic relationship. However, we recently revealed that Buchnera displays differential protein regulation, but not mRNA expression. We also identified a number of conserved small RNAs (sRNAs) that are expressed among Buchnera taxa. In this study, we investigate whether differential protein regulation in Buchnera is the result of post-transcriptional gene regulation via sRNAs. We characterize the sRNA profile of two Buchnera life stages: (i) when Buchnera is transitioning from an extracellular proliferating state in aphid embryos and (ii) when Buchnera is in an intracellular nonproliferating state in aphid bacteriocytes (specialized symbiont cells). Overall, we identified 90 differentially expressed sRNAs, 97% of which were upregulated in aphid embryos. Of these sRNAs, the majority were predicted to be involved in the regulation of various metabolic processes, including arginine biosynthesis. Using a heterologous dual expression vector, we reveal for the first time that a Buchnera antisense sRNA can post-transcriptionally interact with its cognate Buchnera coding sequence, carB, a gene involved in arginine biosynthesis. These results corroborate our in vivo RNAseq and proteomic data, where the candidate antisense sRNA carB and the protein CarB are significantly upregulated in aphid embryos. Overall, we demonstrate that Buchnera may regulate gene expression independently from its host by utilizing sRNAs.

Novel Insights into Insect-Microbe Interactions-Role of Epigenomics and Small RNAs.

It has become increasingly clear that microbes form close associations with the vast majority of animal species, especially insects. In fact, an array of diverse microbes is known to form shared metabolic pathways with their insect hosts. A growing area of research in insect-microbe interactions, notably for hemipteran insects and their mutualistic symbionts, is to elucidate the regulation of this inter-domain metabolism. This review examines two new emerging mechanisms of gene regulation and their importance in host-microbe interactions. Specifically, we highlight how the incipient areas of research on regulatory "dark matter" such as epigenomics and small RNAs, can play a pivotal role in the evolution of both insect and microbe gene regulation. We then propose specific models of how these dynamic forms of gene regulation can influence insect-symbiont-plant interactions. Future studies in this area of research will give us a systematic understanding of how these symbiotic microbes and animals reciprocally respond to and regulate their shared metabolic processes.

The role of pollinators in maintaining variation in flower colour in the Rocky Mountain columbine, Aquilegia coerulea.

BACKGROUND AND AIMS: Flower colour varies within and among populations of the Rocky Mountain columbine, Aquilegia coerulea, in conjunction with the abundance of its two major pollinators, hawkmoths and bumble-bees. This study seeks to understand whether the choice of flower colour by these major pollinators can help explain the variation in flower colour observed in A. coerulea populations. METHODS: Dual choice assays and experimental arrays of blue and white flowers were used to determine the preference of hawkmoths and bumble-bees for flower colour. A test was made to determine whether a differential preference for flower colour, with bumble-bees preferring blue and hawkmoths white flowers, could explain the variation in flower colour. Whether a single pollinator could maintain a flower colour polymorphism was examined by testing to see if preference for a flower colour varied between day and dusk for hawkmoths and whether bumble-bees preferred novel or rare flower colour morphs. KEY RESULTS: Hawkmoths preferred blue flowers under both day and dusk light conditions. Naïve bumble-bees preferred blue flowers but quickly learned to forage randomly on the two colour morphs when similar rewards were presented in the flowers. Bees quickly learned to associate a flower colour with a pollen reward. Prior experience affected the choice of flower colour by bees, but they did not preferentially visit novel flower colours or rare or common colour morphs. CONCLUSIONS: Differences in flower colour preference between the two major pollinators could not explain the variation in flower colour observed in A. coerulea. The preference of hawkmoths for flower colour did not change between day and dusk, and bumble-bees did not prefer a novel or a rare flower colour morph. The data therefore suggest that factors other than pollinators may be more likely to affect the flower colour variation observed in A. coerulea.