Microbial Ecology

QuestionThe large earth bumble bee (Bombus terrestris) maintains a social core gut-microbiota, similar as known from the honey bee, which plays an important role for host health and resistance. Experiments under laboratory conditions with commercial hives are limited to these vertically transmitted microbes and neglect variability by environmental influences and external acquisition of microbes. Various environmental and landscape-level factors may have an impact on the gut-microbiota of pollinating insects, with consequences for pollinator health and fitness in agroecosystems. Still, it is not fully clear whether access to a higher vs lower flower diversity will have a significant influence on the bumble bee microbiota. Here, we tested in a semi-field experiment how strongly the bumble bee microbiota changes over time when exposed to different flower diversities within outdoor flight cages. We used commercial hives to distinguish between vertically and horizontally transmitted bacteria, respectively from the nest environment or the exposed outside environment. ResultThe sequential sampling of foraging workers over a period of 35 days indicated a temporal progression of the bumble bee microbiota when exposed to outside conditions. The microbiota became not only more diverse, but changed in composition and variability over time. We observed a major increase in relative abundance of the families Lactobacillaceae, Bifidobacteriaceae and Weeksellaceae. In contrast, major core taxa like Snodgrassella and Gilliamella declined in their relative abundance over time. The genus Lactobacillus showed a high diversity and strain specific turnover, so that only specific ASVs showed an increase over time, while others had a more erratic occurrence pattern. Exposure to different flower diversities had no significant influence on the bumble bee microbiota. ConclusionThe bumble bee microbiota showed a dynamic temporal progression with distinct compositional changes and diversification over time. The exposure of bumble bees to environmental conditions, or environmental microbes, increases dissimilarity and changes the gut-community composition compared to laboratory rearing conditions. This shows the importance of environmental influences on the temporal dynamic and progression of the bumble bee microbiota. Scope statementBumble bees (Bombus terrestris) are, next to the honey bee, commercially important pollinators and widely used to enhance crop pollination service within greenhouse environments. They host a similar, but characteristic, set of core-microbiota which are of known importance for bumble bee health. Despite this, bumble bees harbor their own specific set of symbionts, which do not occur within the honey bee and seem to be more easily influenced by colonization of environmental microbes. While experiments under controlled lab-based rearing conditions often lack the influence of environmental or landscape-level drivers, field-based observation can often not resolve the influence of a single factor. One major unresolved question is which environmental factor influences the microbiota of social pollinators by environmental microbes. Especially whether monocultures (low flower diversity) are per se rather detrimental to microbiota composition compared to more balanced and diverse pollen provisions (high flower diversity). Within this article, we investigated the influence of different flower diversities as potential drivers of the bumble bee gut-microbiota under semi-field conditions. We used outdoor cages which contained a flower diversity gradient to specifically test how a low and high diversity of flower resources could influence the bumble bee microbiota over time.

BackgroundBacteria and fungi co-inhabit the soil microbiome in dynamic interactions. In the rhizosphere, fungi and bacteria have been studied to synergistically colonize soil as beneficial or as antagonists to form a pathobiome. These variations of soil bacterial community from pathogen and nonpathogen form of FOSC have been researched, however the bacterial community within the hyphosphere has yet to be studied thoroughly for direct pathogen interkingdom interactions. This study used 16S rRNA gene sequencing and a to decipher the bacteriome diversity associated with the hyphosphere of three isolates of Fusarium oxysporum f. sp. niveum race 2 (FON2) with temporal and spatial differences. ResultsOur results show a core microbiome that is shared among the three isolates regardless of the differences of spatial and temporal differences. The core hyphosphere community visualized as a ternary plot was made up 15 OTUs which were associated with all three FON2. Although a few operational taxonomic units (OTUs) were significantly correlated with a particular isolate of FON2, reported in the LDA (p<0.05), these OTUs were still present as part of the core in all isolates. Co-occurrence analysis and correlation plot identified a negative correlation among most of the microbiota which may indicate a positive correlation to the FON2 that is not tested. ConclusionsThe study indicates a core microbiota associated with FON2 regardless of the isolates temporal and spatial differences. Through our results we provide insights into the microbe-microbe dynamic of the pathogens success and its ability to recruit a core pathobiome. Our research promotes the concept of pathogens not being lone invaders but recruits from the established host microbiome to form a pathobiome.

Symbiosis between insects and bacteria has been established countless times. While it is well known that the symbionts originated from a variety of different bacterial taxa, it is usually difficult to determine their environmental source and a route of their acquisition by the host. In this study, we address this question using a model of Neisseriaceae symbionts in rodent lice. These bacteria established their symbiosis independently with different louse taxa (Polyplax, Hoplopleura, Neohaematopinus), most likely from the same environmental source. We first applied amplicon analysis to screen for candidate source bacterium in the louse environment, that is, three species of rodents (Microtus arvalis, Clethrionomys glareolus, and Apodemus flavicollis). The screened samples included rodent fur, skin, spleen, and ectoparasites sampled from the rodents. The amplicon analysis revealed a Neisseriaceae bacterium, closely related to the known louse symbionts. We assembled genome drafts of this environmental bacterium from all three rodent hosts. The sizes of the three drafts converged to a remarkably small size of approximately 1.4 Mbp, which is even smaller than the genomes of the related symbionts. Based on these findings, we propose a hypothetical scenario of the genome evolution during the transition of a free-living bacterium to the member of the rodent fur-associated microbiome and subsequently to the facultative and obligate louse symbionts.

Blow flies such as Lucilia sericata (Diptera: Calliphoridae) serve important ecological functions as decomposers. However, due to their close association with decaying organic matter they also play potential roles as reservoirs for pathogenic bacteria and antimicrobial resistance genes (ARGs). In this study, we characterized the bacterial communities and resistome profiles of L. sericata specimens collected from six provinces across South Korea using 16S rRNA gene metabarcoding and targeted PCR screening. The microbiome was dominated by Dysgonomonas, Vagococcus, Pseudomonas, Ignatzschineria, and Providencia with geographic variation in community structure. Flies from Chungnam exhibited the lowest microbial diversity, while samples from Jeonnam and Gyeonggi showed greater richness and evenness. Beta diversity analyses confirmed geographic structuring of bacterial communities, with semi-urban, rural locations harboring more diverse taxa. Notably, opportunistic pathogens such as Proteus mirabilis and Providencia were detected, alongside a range of ARGs (blaTEM, ermB, sul1, aac(6')-Ib-cr, cat and mecA) and integron elements (intI and intII), suggesting that L. sericata may act as a reservoir of clinically important microbes and resistance genes. ImportanceThe environment plays a significant role in shaping the microbiome of flies. Due to their motile nature and close association with decomposing matter blow flies of the species Lucilia sericata can harbor diverse bacterial communities, including potential pathogens that threaten human and animal health. Furthermore, due to their synanthropic lifestyle, this species is also exposed to bacteria that host antibiotic resistance genes (ARGs). Here we investigate the role of blowflies as reservoirs of potential pathogens and ARGs using metabarcoding. Our study revealed a diverse microbiome and resistome shaped by location and possible biotic or abiotic factors. These findings provide baseline information for wildlife surveillance and emphasize the importance of including synanthropic flies in strategies aimed at controlling the dissemination of antibiotic resistance.

Termites represent one of the most important insect groups worldwide due to their key role as plant decomposers and proxy of carbon recycling in the tropical rainforest ecosystems. Besides, high relevance in research has been given to these social insects due to a prominent role as urban pests. However, one of the most fascinating aspects of termites are their defence strategies that prevent the growth of detrimental microbiological strains on their nests. One success factor is the key role of the nest allied microbiome. Understanding how beneficial microbial strains aid termites in pathogen biocontrol strategies could provide us with an enhanced repertoire for fighting antimicrobial resistant strains or mine for genes for bioremediation purposes. We carried out a multiomics approach for dissecting the nest microbiome in a wide range of termite species, covering several feeding habits and three geographical locations at two tropical sides of the Atlantic Ocean, and an African savanna. Our experimental approach included untargeted volatile metabolomics, targeted evaluation of volatile naphthalene, taxonomical profile for bacteria and fungi through amplicon sequencing, and further dive into the genetic repertoire through a metagenomic sequencing approach. Volatile naphthalene was present in species belonging to the genera Nasutitermes and Cubitermes. We further assessed the apparent differences in terms of bacterial community structure, having found a stronger influence from feeding habits and genera, rather than the geographical location. Lastly, our metagenomic analysis revealed that the gene content provides both soil feeding genera with similar functional profiles, while the wood feeding genus shows a different one. These results seem to be independent of the geographical location, indicating that the nest functional profile is heavily influenced by the diet of the termite inhabiting, building, and maintaining the nest.

Tropical rainforests represent one of the most diverse and productive ecosystems on Earth. High productivity is sustained by efficient and rapid cycling of nutrients through decomposing organic matter, which is for a large part made possible by symbiotic associations between plants and mycorrhizal fungi. In this association, an individual plant typically associates simultaneously with multiple fungi and the fungus associates with multiple plants, creating complex networks between fungi and plants. However, there are still very few studies that have investigated mycorrhizal fungal composition and diversity in tropical rainforest trees, particularly in Africa, and assessed the structure of the network of associations between fungi and rainforest trees. In this study, we collected root and rhizosphere soil samples from Ise Forest Reserve (Southwest Nigeria), and employed a metabarcoding approach to identify the dominant arbuscular mycorrhizal (AM) fungal taxa associating with ten co-occurring tree species and to assess variation in AM communities. Network analysis was used to elucidate the architecture of the network of associations between fungi and tree species. A total of 194 AM fungal Operational Taxonomic Units (OTUs) belonging to six families were identified, with 68% of all OTUs belonging to Glomeraceae. While AM fungal diversity did not differ between tree species, AM fungal community composition did. Network analyses showed that the network of associations was not significantly nested and showed a relatively low level of specialization (H2 = 0.43) and modularity (M = 0.44). We conclude that, although there were some differences in AM fungal community composition, the studied tree species associate with a large number of AM fungi. Similarly, most AM fungi had a large host breadth and connected most tree species to each other, thereby potentially working as interaction network hubs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/578868v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@4573ecorg.highwire.dtl.DTLVardef@1bd9974org.highwire.dtl.DTLVardef@a598ccorg.highwire.dtl.DTLVardef@1d27b76_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionSeveral Desmodium spp. are used as intercrops in push-pull pest management systems to repel insect herbivores. In addition, Desmodium suppresses the parasitic weed Striga, and diversifies the soil microbiome with negative impacts on fungi. We investigated the impact of a 2-year cropping of five Desmodium species on soil microbiome populations. MethodologyTotal DNA was obtained from root zone soil samples collected from a two-years-old common garden experiment with replicated plots of five Desmodium spp. at the international centre for insect physiology and ecology (ICIPE), Mbita, Kenya. Subsequently, 16S and ITS DNA sequencing were performed and the data was analysed by using QIIME2 and Calypso. ResultsOur findings show significant differences in composition and abundance of specific microbial taxa among the Desmodium plots and the bulk soil, with a stronger shift observed for fungal community profiles than bacteria. There was, however, no significant difference in overall diversity, richness and evenness of microbial communities among the Desmodium plots and the bulk soil. Similarly, beta diversity analysis did not reveal a significant association of variation to specific Desmodium spp. plots. Discussion and conclusionThis is the first study to compare impact and association of whole soil microbiomes to different Desmodium species. Whereas long-term Desmodium cropping clearly shifts whole microbiome communities, no significant difference in overall diversity and richness of microbial populations was observed among the studied plots. However, there was a divergence of individual taxa reflected on their increased abundance in association to specific Desmodium spp., pointing towards potential impact on ecosystem services. These findings indicate that significant shifts in whole microbial populations due to Desmodium spp. and thus potentially provision of associated ecosystem services require longer cultivation periods to solidify. Future studies should focus on techniques that monitor real-time changes in microbial populations such as RNA-seq to ascertain live and dead microbes, and thus infer ecological services.

The western honeybee, Apis mellifera L., is a crop pollinator that makes royal jelly and other hive products. However, widespread concerns arise about opportunistic diseases (e.g., bacteria, fungi, or mites) or chemicals that have an effect on the health and number of colonies, as well as their activity. The relationships between the gut microbiota and its host are currently being researched extensively. The effects of Varroa destructor infection on the gut microbial community, in particular, have received little investigation. This work utilized amplicon sequencing of the bacterial and archaeal 16S rRNA genes to assess the bacterial and archaeal communities of adult bee groups (healthy and affected by Varroa designed in NG and VG, respectively) and larvae from Varroa destructor-infected hives. Our results suggest that the genus Bombella was substantially dominant in larvae, while the genera Gillamella, unidentified Lactobacillaceae, and Snodgrassella were significantly dominant in adult bees. NG and VG, on the other hand, did not differ statistically significantly. The PICRUSt study revealed a significant difference in the KEGG classifications of larvae and adult bee groups. A greater number of genes involved in cofactor and vitamin production were identified in larvae. Additionally, despite the complexity of the honeybees bacterial community, all groups exhibited a straightforward archaeal community structure. Surprisingly, methanogen was detected in low abundance in the microbiota of honeybees. In summary, larvae and adult bees infected with Varroa destructor exhibit altered gut microbiota composition and function.

Variation along environmental gradients in host-associated microbial communities is not well understood, compared to free-living microbial communities. Because elevational gradients may serve as natural proxies for climate change, understanding patterns along these gradients can inform our understanding of the threats hosts and their symbiotic microbes face in a warming world. In this study, we analysed bacterial microbiomes from pupae and adults of four Drosophila species native to Australian tropical rainforests. We sampled wild individuals at high and low elevation along two mountain gradients, to determine natural diversity patterns. Further, we sampled laboratory-reared individuals from isofemale lines established from the same localities, to see if any natural patterns are retained in the lab. In both environments, we controlled for diet to help elucidate other deterministic patterns of microbiome composition. We found small but significant differences in Drosophila bacterial community composition across elevation, with some notable taxonomic differences between different Drosophila species and sites. Further, we found that field-collected fly pupae had significantly richer microbiomes than laboratory-reared pupae. We also found similar microbiome composition in both types of provided diet, suggesting that the significant differences found among Drosophila microbiomes are the product of surrounding environments with different bacterial species pools, possibly bound to elevational differences in temperature. Our results suggest that comparative studies between lab and field specimens help reveal the true variability in microbiome communities that can exist within a single species. ImportanceBacteria form microbial communities inside most higher-level organisms, but we know little about how the microbiome varies along environmental gradients and between natural host populations and laboratory colonies. To explore such effects on insect-associated microbiomes, we studied gut microbiome in four Drosophila species over two mountain gradients in tropical Australia. We also compared these data to individuals kept in the laboratory to understand how different settings changed microbiome communities. We found that field sampled individuals had significantly higher microbiome diversity than those from the lab. In wild Drosophila populations, elevation explains a small but significant amount of the variation in their microbial communities. Our study highlights the importance of environmental bacterial sources for Drosophila microbiome composition across elevational gradients, and shows how comparative studies help reveal the true flexibility in microbiome communities that can exist within a species.

Many species of social insects introduced to regions beyond their native ranges have become highly invasive. The introduction of the eusocial European buff-tailed bumblebee, Bombus terrestris, to the island of Tasmania (Australia) [~]30 years ago is of concern due to its ecological impacts and its potential to spill over pathogens to native bees or commercially important honeybees. The health of B. terrestris is intricately connected with its gut microbiome and diet; however, environmental variables may also interact, particularly during invasion into novel environments. Using landscape-wide sampling and a metabarcoding approach to characterize the gut bacteria (16S rRNA) and diet composition from foraged pollen (ITS2: floristic diversity of pollen baskets), this study investigates how the gut microbiota of B. terrestris workers is affected by nutritional diversity ( pollenbiome) and environmental variation across diverse landscapes of its invasive range in Tasmania. Gut bacterial community composition and diversity were significantly predicted by site annual precipitation and percentage of pasture. Further, a positive interaction between site annual precipitation and site annual temperature significantly predicted gut bacterial diversity. The interaction effect of pollen diversity and average summer wind velocity was also significantly and positively related to gut bacterial diversity. Following comparison of Akaike information criterion (AIC) and sum of weights, the percentage of pasture was identified as the most strongly weighted variable, which, along with pollen diversity, had a negative impact on gut bacterial diversity. These insights help to uncover how environmental interactions affect the gut microbiome of B. terrestris in an invaded landscape with novel nutritional resources. This knowledge contributes to understanding the factors that predict the spread and persistence of invasive bumblebees.

The gut microbiome of eusocial corbiculate bees, which include honeybees, bumblebees, and stingless bees, consists of anciently associated, host-specific bacteria that are vital for bee health. Two symbionts, Snodgrassella and Gilliamella, are ubiquitous in honeybees and bumblebees. However, their presence varies in the stingless bee clade (Meliponini), a group with pantropical distribution. They are absent or rare in the diverse genus Melipona, indicating a shift in microbiota composition in this lineage. To identify the main members of the Melipona microbiota, we combined newly collected and published data from field-collected individuals of several species. Additionally, we identified the localization of the dominant microbiota members within the gut regions of Melipona quadrifasciata anthidioides. The dominant microbiota of Melipona species includes members of the genera Bifidobacterium, Lactobacillus, Apilactobacillus, Floricoccus, and Bombella. Among these, Apilactobacillus and Bombella dominate in the crop, whereas Apilactobacillus and other members of the Lactobacillaceae dominate the ventriculus. The ileum lacks Snodgrassella or Gilliamella but contains a putative new symbiont close to Floricoccus, as well as strains of Bifidobacterium, Lactobacillaceae (including Apilactobacillus), and Bombella. The rectum is dominated by Bifidobacterium and Lactobacillus. In summary, the Melipona microbiota is compositionally distinct but shows spatial organization paralleling that of other eusocial corbiculate bees.

1.Honeybees (Apis Mellifera) perform an essential role in the ecosystem and economy through pollination of insect-pollinated plants, but their population is declining. Many causes of honeybees decline are likely to be influenced by the microbiome which is thought to play an important role in bees and is particularly susceptible to infection and pesticides. However, there has been no systematic review or meta-analysis on honeybee microbiome data. Therefore, we conducted the first systematic meta-analysis of 16S-rRNA data to address this gap in the literature. Four studies were in a usable format - accounting for 336 honeybees worth of data - the largest such dataset to the best of our knowledge. We analysed these datasets in QIIME2 and visualised the results in R-studio. For the first time, we conducted a multi-study evaluation of the core and rare bee microbiome and confirmed previous compositional microbiome data. We established that Snodgrassella, Lactobacillus, Bifidobacterium, Fructobacillus and Saccaribacter form part of the core microbiome and identify 251 rare bacterial genera. Additional components of the core microbiome were likely obscured by incomplete classification. Future studies should refine and add to our existing dataset to produce a more conclusive and high-resolution portrait of the honeybee microbiome. Furthermore, we emphasise the need for an actively curated dataset and enforcement of data sharing standards. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/473299v1_fig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1d9c414org.highwire.dtl.DTLVardef@1d82d2forg.highwire.dtl.DTLVardef@17e6aa1org.highwire.dtl.DTLVardef@8aa415_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1C_FLOATNO Graphical abstract. Made by the author in Biorender.com. C_FIG

Nematodes are widely abundant soil metazoa and often referred to as indicators of soil health. While recent advances in next-generation sequencing technologies have accelerated research in microbial ecology, the ecology of nematodes remains poorly elucidated, partly due to the lack of reliable and validated sequencing strategies. Objectives of the present study were (i) to compare commonly used primer sets and to identify the most suitable primer set for metabarcoding of nematodes; (ii) to establish and validate a high-throughput sequencing strategy for nematodes using Illumina paired-end sequencing. In this study, we tested four primer sets for amplicon sequencing: JB3/JB5 (mitochondrial, I3-M11 partition); SSU_04F/SSU_22R (18S rRNA, V1-V2 region); Nemf/18Sr2b (18S rRNA, V6-V8 region) from earlier studies; and MMSF/MMSR (18S rRNA, V4-V5 region), a newly developed primer set from this study. In order to test the primer sets, we used 22 samples of individual nematode species, 20 mock communities, 20 soil samples, 20 spiked soil samples (mock communities in soil), and 4 root/rhizosphere soil samples. We successfully amplified the target regions (I3-M11 partition of the COI gene; V1-V2, V4-V8 region of 18S rRNA gene) from these 86 DNA samples with the four different primer combinations and sequenced the amplicons on an Illumina MiSeq sequencing platform. We found that the MMSF/MMSR and Nemf/18Sr2b were efficient in detecting nematode compared to JB and SSU primer sets based on annotation of sequence reads at genus and in some cases at species level. Therefore, these primer sets are suggested for studies of nematode communities in agricultural environments.

PremiseThe soils in lowland tropics are teeming with microbial life which can impact plant community structure and diversity through plant-soil feedbacks. While bacteria and fungi have been the focus of most studies in the tropics, the oomycetes may have an outsized effect on seed and seedling health and survival, given their affinity for environments with increased precipitation and temperature. MethodsWe assessed the diversity and pathogenicity of oomycete species present in a lowland tropical forest in Panama. We used both a culture dependent leaf-baiting assay and culture independent soil DNA metabarcoding methods to quantify zoospore abundance and species diversity. A subset of the isolates from the baiting assay were used to evaluate pathogenicity and aggressiveness on seedlings of three tree species. Key resultsOomycetes are ubiquitous and common members of the soil microbial community in lowland tropical forests and zoospore abundance was far greater compared to similar studies from temperate and mediterranean forests. We also observed variation in oomycete species ability to infect host plants. Species of Pythium were more aggressive, while species of Phytopythium caused less disease but were more diverse and commonly isolated from the soil. Finally, we found that individual hosts accumulate a distinct oomycete community and was the only factor that had an effect community structure. ConclusionsCollectively, these finding demonstrate that oomycetes are ubiquitous, host-generalist pathogens and saprophytes, that have the potential to impact seed and seedling survival in lowland tropical forests

Understanding the biotic drivers of diversity is a major goal of microbial ecology. One approach towards tackling this issue is to interrogate relatively simple communities that are easy to observe and perturb. Figs (syconia) of the genus Ficus represent such a system. Here, we describe the microbial communities of Ficus septica figs, which are associated with the nematode Caenorhabditis inopinata (the sister species of the C. elegans genetic model system). In 2019, 38 Ficus septica figs (across 12 plants in Taiwan) were dissected, and metadata such as foundress wasp number and nematode occupancy were collected for each fig. Suspensions derived from interior fig material and fig surface washes were prepared for 16S microbial metabarcoding. Over 3,000 OTUs were detected, and microbial communities were dominated by members of Proteobacteria, Bacteroidota, and Actinobacteriota. Although microbial communities of fig exteriors and interiors can be distinguished, levels of microbial alpha diversity were comparable across these areas of the fig. Nematodes likewise had no detectable impact on microbial alpha diversity, although nematodes were associated with a modest change in microbial community composition. A handful of OTUs (associated with the genera Kosokonia, Ochobactrum, and Stenotrophomonas) revealed potential differential abundance among figs varying in nematode occupancy. Additionally, foundress wasp number was negatively correlated with microbial alpha diversity. These findings set the stage for future studies that directly test the role of nematode and wasp occupancy on microbial communities, as well as investigations that probe nematode-microbe interactions through laboratory experiments. Taken together, these results constitute a fundamental step in characterizing the natural microbial communities of figs and Caenorhabditis nematodes. ImportanceUnraveling why different species live in different places is a longstanding open question in ecology, and it is clear that interspecific interactions among species are a major contributor to species distributions. Ficus figs are a useful system for ecological studies because they are relatively simple microcosms where characterizing animal community composition of multiple samples is straightforward. Additionally, Caenorhabditis inopinata, a close relative of the C. elegans genetic model system, thrives in Ficus septica figs. Here, we tie 16S microbial metabarcoding to nematode and wasp occupancy data to understand the causes of bacterial community composition in F. septica figs. We found that microbial composition, but not total diversity, varies among fig surface and interiors. Likewise, we found that nematode occupancy impacts microbial composition but not alpha diversity. Moreover, we show that as the number of foundress wasps increases, the microbial alpha diversity decreases. Finally, we identified OTUs that are potentially associated with nematode occupancy. Taken together, these results represent a key step in describing a microcommunity wherein ecological genetic hypotheses can be tested, as well as one that can potentially reveal the roles of uncharacterized genes in established model systems.

Myrmecocystus honeypot ants rely on specialized workers, repletes, to store dissolved carbohydrates in their crops long term. The repletes store this liquid, which does not spoil in their crops, for many months at a time. When resources are scarce, repletes redistribute the stored nutrients to their colony members via trophallaxis. While we suspect that the gut microbiome of honeypot ants may aid in spoilage prevention, before we can investigate this, we must first characterize it. Here, we used 16S rRNA gene sequencing to determine the microbial community composition across six Myrmecocystus honeypot ant species, sampling multiple colonies, castes, and organs. We found that microbiome community composition was strongly shaped by species, with variation between colonies in M. arenarius, M. depilis, and M. mexicanus. Organ level differences were observed in the crop and midgut in M. mexicanus. Caste differences were observed in M. flaviceps and M. mexicanus. Replete crops of M. mexicanus and M. depilis were enriched in Fructilactobacillus, other lactic acid bacteria, and acetic acid bacteria, whereas halophiles were more prominent in the gut of species such as M. flaviceps and M. wheeleri. In this study we demonstrate that Myrmecocystus ants host species-specific gut microbiomes and identify an association between lactic acid bacteria, acetic acid bacteria, and halophiles within replete crops. While much work remains in understanding the roles of the microbes in the symbiosis with their host ants, the dominance of these particular taxonomic groups suggests an association with a high sugar environment and a potential microbial role in preventing spoilage of the crop contents.

Understanding bumble bee gut health is imperative as these vital pollinators are subjected to pathogenic infections and thermal stress from climate change. The gut microbiome serves as an indicator for health and fitness and indicates the types of stress. To investigate the combined effects of thermal stress and pathogenic infection on bee guts, we performed a two-by-two crossed design where Bombus impatiens workers were subjected to hot conditions, pathogenic infection, or both. Incubation groups were given 2-weeks of stress conditions, with infected bees initially inoculated with Crithidia bombi, a common bee gut parasite. We measured body size, quantified the infection intensity of C. bombi using qPCR, and defined the composition of the gut microbiome using full-length 16S rRNA gene amplicon sequencing on an Oxford Nanopore Technologies Mk1D. While the core gut microbiome thrived with genera such as Bombilactobacillus and Snodgrassila which were not impacted by treatment; there were notable changes in other key organisms. Asaia bogorensis spiked in control temperature infected organisms, while species of Lactobacillus were overtaken in hot temperatures by significant increases in Apilactobacillus kunkeei. Species such as Citrobacter freundii dominated in hot infected bees suggesting an increased immunocompromised state from the combined stressors impacts on bee gut health. Our novel combined effects from thermal stress and pathogenic infection strengthen existing literature and provide new directions on how to quantify the health-state of wild bees based on their gut microbiome composition. These insights enable us to better understand how bees will be further impacted in changing landscapes. ImportanceWe find significant changes in bees gut microbiome especially with an increased abundance of lactic acid bacteria. These lactic acid bacteria are often specialized: based on infection status, temperature, and the combined effects. These insights are vital to researchers, especially those studying wild bee gut health where they have an uncontrolled system and need to make assumptions about bee stress based on fitness and microbiome. Our detailed outline of relevant species will provide wild bee researchers with a baseline to determine thermal stress and recent infection status based on microbiome communities. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/687019v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1528ae6org.highwire.dtl.DTLVardef@1d100dorg.highwire.dtl.DTLVardef@8ea77org.highwire.dtl.DTLVardef@159620a_HPS_FORMAT_FIGEXP M_FIG C_FIG Overview of the experimental design where bees were either given control temperatures or thermal stress and/or an infection by Crithidia bombi. The bees microcolonies were incubated for 2-weeks before sacrificing the bees, extracting DNA, and performing downstream analysis. Full length 16S rRNA gene amplicon sequencing was performed on an Oxford Nanopore MK1D sequencer while Crithidia bombi infections were quantified using quantitative polymerase chain reaction.

Pot honey, the honey produced by stingless bees, is valued for its antimicrobial capacity, which may be influenced by its microbial content. While Lactobacillaceae species are commonly associated with honeybees and honey microbiomes, most studies have focused on Apis mellifera, leaving pot honey microbial diversity largely unexplored. We present the first pot honey shotgun metagenomic analysis from bee species Melipona beecheii and Scaptotrigona mexicana. We reconstructed 24 metagenome-assembled genomes (MAGs), 15 of which lacked close matches to any described species, showing[&le;] 81% Average Nucleotide Identity (ANI) to available reference genomes. Phylogenetic analyses resolved these MAGs into four well-defined clades (intraclade ANI > 99%, interclade ANI[&le;] 81%), consistent with four novel species within the family Lactobacillaceae. GTDB-Tk classification placed MAG clades 1 and 2 closest to Nicoliella, and clades 3 and 4 closest to Acetilactobacillus. We validated the presence of these lineages in honey by sequencing three isolates that clustered within MAG clade 2. Aminoacid similarity (AAI/cAAI) indicates the presence of two genus-level lineages: one occupying a transitional genomic space near Nicoliella, and a second representing an undescribed genus. The genomic similarity of our MAGs and isolates to those from pot honey or larval food in Malaysia, Brazil, and Australia suggests these taxa are closely associated with stingless bees and may contribute to honey properties. By reducing the genomic underrepresentation of evolutionarily divergent sister clades related to Nicoliella and Acetilactobacillus, our genome-resolved analyses reveal a globally distributed, phylogenetically cohesive Lactobacillaceae species complex dominating pot honey.

The basidiomycete Serpula lacrymans is responsible for timber destruction in houses. Basidiomycetes are known to harbor a diverse but poorly understood microbial community of bacteria, archaea, yeasts, and filamentous fungi in their fruiting bodies. In this study, we used amplicon-sequencing to analyze the abundance and composition of prokaryotic communities associated with fruiting bodies of S. lacrymans and compared them to communities of surrounding material to access the background community structure. Our findings indicate that bacterial genera cluster depended on sample type, and that the main driver for microbial diversity is specimen, followed by sample origin. The most abundant bacterial phylum identified in the fruiting bodies was Pseudomonadota, followed by Actinomycetota and Bacteroidota. The prokaryote community of the mycelium was dominated by Actinomycetota, Halobacterota, and Pseudomonadota. Actinomycetota was the most abundant phylum in both environment samples (infested timber and underground scree), followed by Bacillota in wood and Pseudomonadota in underground scree. Nocardioides, Pseudomonas, Pseudonochardia, Streptomyces and Rubrobacter spp. were among others found to comprise the core microbiome of S. lacrymans basidiocarps. This research contributes to the understanding of the holobiont S. lacrymans and gives hints to potential bacterial phyla important for its development and life style. Highlights- The prokaryote communities associated with S. lacrymans mycelia and fruiting bodies as well as wood and non-woody substrate form distinct clusters. - Across all samples 30% of OTUs were shared (core microbiome) while the number of unique OTUs was small. - Fruiting bodies (n= 8) of S. lacrymans shared a core set of 365 OTUs, dominated by Actinobacteriodota (44%), Pseudomonadota (28%), and Acidobacteriodota (9%). - Tissue/sample type is the main factor influencing diversity, followed by sample origin.

Recent research has elucidated many factors which play a role in the development and composition of human microbiomes. In this study we briefly examine the microbiomes of saliva and fecal samples from 71 indigenous individuals, and chicha samples from 28 single family households in a remote community in the Ecuadorian Amazon. Fecal and saliva samples were collected at two separate time points whereas chicha samples were collected at four time points, once each day of the fermentation process. In total 324 samples were collected: 113 saliva, 108 chicha, and 103 fecal. Microbial composition and diversity were assessed using shotgun metagenome sequence data. Chicha samples were found to be nearly entirely composed of the order Lactobacillales, accounting for 90.1% of the relative abundance. Saliva samples also contained a high relative abundance of Lactobacillales (31.9%) as well as being composed of Neisseriales (12.8%), Actinomycineae (8.7%), Bacteroidales (7.0%), Clostridiales (6.8%), Micrococcineae (6.5%), and Pasteurellales (6.0%). Fecal samples were largely composed of the three orders Clostridiales (33.7%), Bacteroidales (21.9%), and Bifidobacteriales (16.5%). Comparison of -diversity, as calculated by Shannons Diversity Index, in mothers and their offspring showed no significant difference between the two groups in either fecal or saliva samples. Comparison of {beta}-diversity in fecal and saliva samples, as calculated by the Bray-Curtis Dissimilarity measure, within household units and between differing households showed that members of the same household were significantly less dissimilar to each other than to members of other households in the community. Average microbiome composition for individuals within fecal and saliva samples was assessed to determine the impact of an individuals household on the composition of their microbiome. Household was determined to have a significant impact on both fecal and oral microbiome compositions.