Microbial Ecology

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.

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[≤] 81% Average Nucleotide Identity (ANI) to available reference genomes. Phylogenetic analyses resolved these MAGs into four well-defined clades (intraclade ANI > 99%, interclade ANI[≤] 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.

Despite their importance for individual fitness and population processes, the microbiota of many ecologically significant insects remains poorly explored. Even less is known about the interactions between microbial communities inhabiting insects and their surrounding environment. Ant infrabuccal pockets (IBPs), representing the interface between the digestive tract and the external environment, provide an opportunity to study these interactions. Here, we aimed to characterize ant-microbial interaction networks in the forest floor by profiling fungal and bacterial communities associated with the IBP of Formica polyctena ants, known as ecosystem engineers in temperate forests. We used direct microscopy, culturing, and sequencing amplicons of ITS1, ITS2, 18S rRNA marker regions to describe fungal communities, and 16S rRNA metabarcoding to characterize bacterial communities. Classical methods combined with a multi-marker amplicon sequencing allowed for a comprehensive description of the IBP microbiota. Fungal communities consistently contained representatives of 15 ecologically diverse genera, including insect-associated yeasts and primarily saprotrophic or endophytic fungi. Bacterial communities were dominated by genera previously reported from ant guts, mainly Bacilli and Alphaproteobacteria, and showed greater stability among ant colonies than fungal communities. Further studies on red wood ants IBP microbiota would enhance our understanding of their role in shaping ecological networks in forest ecosystems.

The microbiota and microbiome associated with zooplankton remains rather understudied compared to other animal groups and/or taxa. The present study aimed at investigating the whole-body bacterial microbiota of Daphnia spp. in two contrasting Greek lakes, the shallow and hypertrophic Lake Koronia vs. the deep and mesotrophic Lake Vegoritida, including both egg-bearing and non-egg-bearing individuals. In both lakes, 2,060 bacterial operational taxonomic units (OTUs) were found, with 223 of them being conditionally rare (crOTUs) with low contribution even for the dominant phyla, with L. Vegoritida having more crOTUs than L. Koronia. The individuals microbiota had inconsiderable overlap with the surrounding water microbiota in both lakes. The two lakes showed significant differences in their Daphnia -associated microbiota. L. Koronia had richer OTUs and rather homogeneous bacterial communities, with higher occupancy. Overall, no significant differences in between the microbiota of egg-bearing and non-egg-bearing Daphnia individuals in both lakes. However, regarding the most important OTUs (miOTUs), the L. Koronia miOTUs were highly overlapped between the individuals with and without eggs, with only one missing from the individuals without eggs. In L. Vegoritida the individuals without eggs had only six miOTUs and while egg-bearing individuals had nine different ones; the two lakes had no shared miOTUs., considerable differences occurred.. A total of 27 miOTUs, was found and belonged to the Pseudomonadota, unclassified Bacteria, Cyanobacteria, Bacteroidota, Bacillota and Actinomycetota. Those miOTUs, where assignment to the genus level was possible, they were related to Cyanobium, Mucilaginibacter, Flavobacterium and Staphylococcus. This study showed that lake morphotype and ecological status can exert some impact on Daphnia-associated bacterial microbiota, with more pronounced effects on egg-bearing and non-egg-bearing individuals.

Microbial diversity remains largely unexplored across environments and scales, notably because at local scales many microbial taxa exist under a dormant state. Microbial biogeography is shaped by edaphic and ecological drivers, and shifts in microbial community composition are frequently associated with host community structure and health. Nontuberculous mycobacteria represent a striking example of environmental microorganisms with opportunistic pathogenic potential. Unfortunately, data on their diversity, distribution, and ecological interactions in aquatic environments remain limited. However, understanding competition for niche space and the role of abiotic and biotic factors shaping their biogeography is crucial for predicting disease emergence and transmission. Here we aimed at i) identifying microhabitat abiotic and biotic drivers influencing their distribution, ii) assessing the predictability of their diversity and distribution across continents, and iii) examining potential exclusion or associations between pathogenic and nonpathogenic mycobacterial species. By deploying an eDNA-based metabarcoding approach from freshwater samples collected in urban and rural sites in French Guiana and Cote dIvoire, we have boosted our understanding of environmental mycobacteria ecology by highlighting the influence of habitat type, abiotic factors, and microbial interactions on mycobacterial distribution. In addition, the detection of pathogenic species further highlighted the importance of environmental reservoirs in mycobacterial disease transmission.

Bacterial interactions-whether positive or negative - are crucial for the functioning of microbial communities. Though bacterial interactions are mainly expected to be negative, the sign and strength of interactions are predicted to be context dependent, with interactions typically being more positive in more stressful and nutrient-poor conditions. However, systematic studies investigating how the environment affects interactions between multiple taxa are lacking. Here, we determine if interactions between a panel of natural soil isolates change in response to the environment in which they are grown, with two different artificial media used (one simple and one complex) and a more ecologically relevant soil wash. To maximise natural variation in interactions, we collected multiple isolates from multiple sites: co-occurring (sympatric) isolates were predicted to show more negative interactions than allopatric isolates because of greater overlap in resource use. Pairwise interactions were in general negative, but more negative when grown in a complex lab-derived medium (Tryptic Soy Broth). Mutually beneficial interactions were most common in a simple resource medium (M9 minimal media) and exploitative interactions were most frequent in a soil broth. These patterns were independent of whether species originated from the same or a different site. The study supports the prediction that nutrient rich environments promote more negative interactions, and that measuring interactions of soil isolates in standard lab media is likely to misrepresent interactions occurring in natural environments.

Insects have interconnected relationships with gut microorganisms which can have impacts on behavior and survival. Understanding foundational ecological principles of the insect-associated microorganisms is important to understand how insects utilize microbes to cope with stress and different environments. Common microbiome surveys of insect microbiomes utilize 16S rRNA sequencing of metagenomic DNA. For amplicon-based surveys of insect microbiome, fragment length and 16S rRNA subunit choice may have unintended biases, and some primer combinations may under-represent genus or species richness. Contemporary solutions target sequencing of the entire 16S rRNA fragment. Here, we illustrate the benefit of full-length 16S rRNA sequencing in improving sample resolution compared to V4 which provides new insight into the gut microbiome community composition of an invasive insect. We evaluated the gut microbiome of mass-reared medfly males (Mediterranean fruit fly, Ceratitis capitata) that were collected across a nine-month sampling period. Full-length 16S rRNA PCR products of samples were prepared into a Kinnex 16S rRNA library, sequenced on a PacBio Revio system, and the resulting HiFi amplicon data was processed into amplicon sequence variants (ASVs). Our findings reveal substantial differences in bacterial compositions across different medfly cohorts when sampling the full-length amplicons which were not detectible when only considering the V4 regions. Strain-level gut microbiome variation were supported with genomes assembled from shotgun metagenomic sequencing. Our findings support that long-read sequencing of full-length 16S rRNA amplicon uncover ecologically important interactions between host and gut microbiomes and serve as a bridge between short-fragment sequencing and shotgun metagenomics.

Microbiomes typically vary between closely related host species, but how and why they diverge during host diversification remain poorly understood. Bees provide a well-established model for the role of sociality in microbiome evolution: solitary species have generalist gut bacterial symbionts, while eusocial honey bees and bumble bees have host-specific "core" gut bacterial symbionts. Curiously however, some species of stingless bees--the most diverse clade of eusocial bees--lack the core symbionts Snodgrassella and Gilliamella. Here, we tested whether group size, a fundamental trait of social organisms, explains symbiont gain and loss in stingless bees. Using specimens from a global collection, we performed 16S rRNA gene sequencing and qPCR on 187 adult workers representing 29 species and 18 genera. Maximum reported colony sizes for these species range from [~]100 to over [~]100,000 individuals. Our phylogenetic models indicate positive feedbacks between group size and the symbionts Snodgrassella and Gilliamella. We suggest that challenges faced by large groups (e.g., disease risk) may select for symbionts that supply needed functionality (e.g., pathogen protection). Our findings highlight group size as a potentially widespread driver of microbiome evolution in social hosts.

Animals from a wide taxonomic range can sense earths magnetic field, however the underlying mechanism remains one of sensory-biology greatest mysteries. One hypothesis suggests that Magnetotactic bacteria (MTB) serve as the underlying mechanism. This hypothesis predicts that MTB will be detected in animal microbiomes and might show a phylogenetic relationship with their hosts. We examined the phylogenetic relationship between various MTB species across 4,048 avian species using databases of MTB genetic presence across the tree of life and an avian phylogenetic tree. We documented 12 genera of MTB in association with 185 avian species. Three genera, Magnetospirillum, Magnetovibrio and Solidesulfovibrio, were found at relative high prevalence of positive samples (84%, 33%, 12% respectively). Further, Magnetospirillum showed a significant phylogenetic relationship with avian species in general and specifically within Psittaciformes, and Passeriformes. Our results demonstrate the power of harnessing the newly published MTB-database, with specific host-related queries. This analysis, to the best of our knowledge has never been done, and could be replicated across the animal kingdom. The relationship detected suggests an evolutionary and ecological relationship between MTB and avian hosts. These results are consistent with the symbiotic magnetic sensing hypothesis and highlights the potential role of microbiome in sensory physiology.

Marine non-cyanobacterial diazotrophs (NCDs) are recognized as globally distributed, however, few representatives have been isolated in pure cultures. As a result, understanding the physiology, growth rate, substrate preference and dinitrogen (N2) fixation capabilities proves difficult. Thalassolituus haligoni. sp. nov., BB40 was isolated from a fjord-like inlet within Kjipuktuk (Halifax), Nova Scotia. The fully sequenced genome displayed all necessary genes required for N2 fixation, and various carbon uptake pathways. The gram-negative flagellated rod shape bacterium displayed significantly higher growth rates in medium amended with nitrate (NO3-) or ammonia (NH3), compared to dissolved N2, as the sole nitrogen source. Biological N2 fixation rates were detectable across all conditions, measuring a range from 9.34 x 10-6 to 1.4 x 10-1 fmol N cell-1 day-1. Growth of the isolate was successful between 4 {degrees}C up to 35 {degrees}C, with a Topt of 20 {degrees}C for N2, and between 27 - 30 {degrees}C for fixed nitrogen (NO3- and NH3). The closest relatives to T. haligoni, were found to be the uncultured Arc-gamma-03 (99% average nucleotide identity (ANI)) and Oceanobacter antarcticus (81% ANI). T. haligoni also displays versatile capabilities for growth on various carbon, and nitrogen sources, and antibiotics. Collectively this study provides an in-depth physiological assessment of an Oceanospirillales diazotrophic species which we presently have limited knowledge of.

AbstractSoil microbes have sophisticated mechanisms to detect and respond to short pulses of C inputs, often involving changes in gene-expression. We studied gene transcription in a soil microbial community before, and 8, 24, and 48h after glucose addition (0.7 mg C g-1 dry soil) to understand how microbes react to periods of substrate excess and subsequent starvation. The relative transcript abundance of genes associated with energy metabolism and biosynthesis of amino acids, lipids, nucleotides, and cell wall components increased 8h after glucose addition. By 24 and 48h, the abundances of these transcripts reversed. Transcript abundance for genes associated with degradation of lipids, nucleotides, and (hetero)cyclic hydrocarbons decreased at 8h, but increased 24 and 48h after glucose addition. Simultaneously with a rise in transcripts for energy production and biosynthesis at 8h, transcription of regulatory genes for the exponential growth phase and ribosome assembly and maturation increased. In contrast, at 24 and 48h, transcript abundance for genes associated with ribosomal hibernation, sporulation, and regulation of the stationary phase increased, while transcripts for regulators for the exponential phase, and ribosome activation decreased. Based on changes in transcript abundance of phosphoenolpyruvate carboxylase and pyruvate carboxylase, it appeared that 8h after glucose addition glycolytic activity was high, however, gluconeogenesis returned at 24 and 48h. High levels of transcripts for nrtC-ntrB indicated N limitation 8 and 24h after glucose addition. Transcripts associated with Type VI Secretion Systems increased 24 and 48h after start of the experiment, suggesting a short lag between primary consumers and predatory bacteria. These results illustrate how metatranscriptome analysis can be used to study the ecophysiology of soil microbes providing details on the timing of exponential and stationary phase processes, coordination between anabolism and catabolism, and emerging nutrient limitations in natural soil communities. Research HighlightsO_LIWe studied gene transcription of a soil microbial community after glucose addition C_LIO_LITranscript abundances for biosynthesis and energy production initially increased, while those for degradation decreased C_LIO_LITranscripts of regulators and sporulation genes indicated start of stationary phase at 24h C_LIO_LINitrogen limitation induced transcription of nitrogen stress genes C_LI

The objective of this study was to re-analyse the molecular phylogeny and/or the morphology of all species, which have been attributed to the so-far mono-generic fungal family Ambisporaceae. The genus Ambispora has been well-known for its spore bi-morphy described even from single spore clusters. Triple-walled spores are differentiated on sporiferous saccules, while mono-walled spores are formed on simple subtending hyphae. New phylogenetic analyses reveal dissimilarities of [≥]10% in partial nrDNA gene of three different stable phylogenetic clades and thus suggest the division of Ambispora into three genera, which simultaneously request for advanced morphological separations. These advances are primarily based on the more diverse spore wall composition of the ambisporoid-acaulosporoid morph rather than on the rather simple-glomoid morph. While all known species of the triple-walled morph have an evanescent to semi-permanent outer spore wall, i) Am. fennica, Am. brasiliensis, Am. gerdemannii and Am. nicolsonii have a smooth, permanent central spore wall (Am. fennica clade, A), ii) the central wall of Am. appendicula, Am. callosa, Am. leptoticha and Am. jimgerdemannii is alveolate (Am. appendicula clade, B), and iii) the central wall of Am. granatensis is smooth, but easily degraded, thus rather short-lived and not permanent but evanescent (Am. granatensis clade, C). In conclusion, species of the Am. fennica clade represent the genus Ambispora, while species of the Am. appendicula clade represent the new genus Appendiculaspora, and the mono-specific Am. granatensis clade represents the new genus Ephemerapareta. Species of an additional morph, with triple-walled spores, but apparently formed on subtending hyphae, and having a diagnostic reticulate, football-like middle wall, are here separated from the revised genus Ambispora based solely on morphological analyses, since molecular identification analyses so far failed and remained merely unknown. This later morph and genus is based on the type species Pelotaspora reticulata comb. nov, and on P. austrolatina sp. nov. Concomitant molecular phylogenetic and morphological analyses are needed to attribute not only Pelotaspora spp., but also those species, for which hitherto only the ambisporoid-glomoid morph has been observed correctly within the family Ambisporaceae. Without molecular analyses, such species with glomoid but unknown ambisporoid-acaulosporoid morph have to be retained within Ambispora.

O_LIPhyllosphere microbiomes are subject to microbial import from various sources and undergo substantial changes during phenological changes of plants. However, these processes are still poorly understood for forest canopies. We propose that phenology-driven changes in host properties, and rainwater-mediated, within-canopy transport shape the phyllosphere microbiome in temperate forests. Leaves and throughfall samples were collected from oak, ash and linden trees at top, mid, and bottom canopy positions at the Leipzig canopy crane facility (Germany) at time points representing early, mid and late phenological stages. Bacterial community composition was assessed by 16S rRNA gene amplicon sequencing. C_LIO_LIPhenological stages explained 19% of phyllosphere bacterial community variation, followed by tree species identity (12%) and canopy position (2%). Later phenological stages exhibited more homogeneous and functionally redundant phyllosphere communities along with a strong decline of plant pathogens and increasing potential for microbially mediated biocontrol mechanisms. Throughfall transported up to 1011 bacterial cells per litre with maximum bacterial fluxes at the canopy top. C_LIO_LIOur findings demonstrate that in temperate forests, phenology-driven effects on the phyllosphere microbiome are far more important than tree species specific effects. Extent and selectivity of throughfall-mediated mobilization may play a crucial role for the spatial heterogeneity of microbial communities in tree crowns. C_LI

BackgroundBuilt environment microbiome studies have identified numerous factors that shape indoor microbiomes, yet the reproducibility of these findings across buildings, timepoints, and research groups remains unclear. Differences in sequencing protocols, sampling design, and environments pose major challenges for cross-study comparisons, particularly in low-biomass environments where technical variation can obscure biological signal. To address this gap, we constructed a simple ontology which groups samples into one of three categories: hand, hand-associated surfaces, and floor then applied it to four publicly available 16S rRNA gene datasets: a hospital, university dormitory, Air Force dormitory, and private residential houses. ResultsWe identified strong and reproducible separation between floors and surfaces with frequent human contact. We found that floors consistently harbored soil-associated taxa, including KD4-96, 67-14, Skermanella, and Sphingobacterium, whereas hands and hand-associated surfaces were enriched with skin-associated genera, such as Lawsonella and Cutibacterium. Within studies, these results were generally consistent across timepoints. Across studies, mixed-model PERMANOVA analysis revealed significant clustering by sample type, with modest effects of study, suggesting that biological signal outweighed differences in laboratory or sequencing methods. Leave-one-study-out random forest models achieved high AUCs for hand vs. floor comparisons (0.865 to 0.921), moderate AUCs for hand-associated vs. floor comparisons, and weaker performance for hand vs. hand-associated comparisons. Application of the batch-correction method DEBIAS-M did not improve effect sizes or classification performance, indicating that reproducible structure was already discernible without batch adjustment. ConclusionsDespite substantial temporal and environmental heterogeneity among studies, we found that the built environment microbiome has a reproducible bacterial signal. There was consistent enrichment of soil-derived taxa on floors and human-associated taxa on hands and hand-associated surfaces suggesting a stable microbiome despite differences in building type, occupancy, and methodology. These findings establish an important foundation for future studies, suggesting cross-study comparability, the accuracy of ecological inference, and the ability to support the development of predictive applications in indoor microbiome research.

Bacteria of the genus Achromatium are known for their large cell sizes and intracellular calcium carbonate deposits. Achromatium inhabit freshwater, brackish, and marine sediments where they accumulate to high abundances at the oxic-anoxic interface. These bacteria alter their vertical position in the sediment along with daily fluctuations in oxygen concentrations. Yet, the mechanism behind their migration in the sediment remains unknown. In this study, we used chemotaxis assays and time-lapse microphotography to analyze the motility and chemotactic behavior of Achromatium oxaliferum. Microscopic observations revealed that rolling and gliding were the main forms of locomotion exhibited by Achromatium. In absence of any stimulant, the movement appeared to be mostly random and changes in direction frequently occurred. Chemotaxis assays showed a negative chemotaxis of Achromatium to oxygen, sulfide, and nitrate, as evidenced by the change from undirected to directed locomotion against the respective chemical gradient. For periods of more than 1 hour, Achromatium cells moved continuously towards regions of low concentration. We further investigated whether the genetic repertoire of Achromatium corresponds to our observations. Based on lab experiments and bioinformatic analyses we conclude that Achomatium motility is propelled by type IV pili guided by a plethora of chemo- and photoreceptors. We conclude that Achromatium uses negative chemo- and phototaxis to confine their distribution in aquatic sediments between opposing oxygen and sulfide gradients. This allows Achromatium to dynamically adjust its position in redox gradients, and thus is likely to have a major contribution to its success in the global colonization of diverse aquatic sediments.

The Hawaiian islands are among the most geologically and volcanically active places on Earth. While the Hawaiian Archipelago is known for its animal and plant diversity, much less is known about microbial diversity in the areas diverse habitats. In this study, we focused on steam vent associated biofilms found on the most volcanically active island of Hawai{square}i, also known as the Big Island. From 46 samples from various biofilms and associated features around fumaroles emitting water steam, we generated amplicon and metagenomic sequences. Amplicon data showed that Chloroflexota and Cyanobacteriota are the numerically dominant phyla in these biofilm communities. We constructed 363 non-redundant medium to high-quality metagenome-assembled genomes (MAGs) that are at least 70% complete and with less than 5% contamination. Ten MAGs belong in the domain Archaea, and 353 belong in the domain Bacteria. This dataset could provide valuable insights into microbial diversity and ecology around volcanic features in Hawai i and elsewhere.

Microbial residents of ectothermic hosts are exposed to variations in temperature that have the potential to impact their physiology and the host-microbe symbiotic relationship. In this experimental warming study, laboratory populations of American cockroaches (Periplaneta americana) were kept at a baseline low room temperature of 20-22{degrees}C or a high temperature of 30{degrees}C for two weeks. We quantified bacterial load and performed high-throughput 16S rRNA gene sequencing to assess the hindgut microbiomes response to a near 10{degrees}C shift in environmental temperature. We report modest impacts of temperature on cockroach gut microbiome composition. The high temperature treatment induced increases in the relative abundance of Proteobacteria and Euryarchaeota phyla as well as the Lactobacillaceae and Enterococcaceae families. We also observed increased interindividual variability. There were no significant differences in the dominant Bacteroidota or Firmicutes phyla and no significant losses or reductions in taxa or bacterial load, respectively. This suggests that the gut community of American cockroaches is largely resilient to prolonged increases in temperature and has implications for the cockroach to withstand the impacts of climate change. ImportanceInsects, as with most animals, often harbor microbial symbionts that play an essential role in host health and nutrition. As insects are ectotherms, these microbial symbionts are subject to the same temperature fluctuations as their hosts, potentially impacting host temperature responses. Here, we demonstrate that the American cockroach (Periplaneta americana) gut microbiome exhibits only modest changes following an [~]10{degrees}C increase in environmental temperature. This contrasts with studies in other insects, whose microbiota were highly responsive to temperature variation. This work illustrates that the microbiota of insects may vary in their sensitivity to long-term temperature shifts, providing a more comprehensive understanding of potential variability in insect responses to climate change.

AO_SCPLOWBSTRACTC_SCPLOWOrnithodoros phacochoerus are nidicolous soft ticks of the Ornithodoros moubata complex of species known to be vectors of the African swine fever (ASF) virus. These Ornithodoros ticks depend on endosymbionts to produce essential nutrients necessary for their development. However, endosymbionts are only a part of the complex microbiota hosted by the tick. This microbiota often includes primary or secondary endosymbionts, commensal species from the environment, and, most of the time, some pathogens. The present study was performed to understand the organization and spatial distribution of the microbiota of O. phacochoerus. One of the objectives was to investigate if the pathogen of interest (ASF virus) is involved in the organization of the microbiota through pathogen-induced dysbiosis or other interactions. For this purpose, 704 O. phacochoerus ticks were collected from two conservation areas in Mozambique. Sequencing was performed targeting the V3-V4 region of the 16S rRNA gene, and the resulting dataset was processed using FROGS to characterize the bacterial microbiota hosted by the ticks. The results indicate that the microbiota of Ornithodoros phacochoerus contains very low bacterial diversity, with one primary endosymbiont (Francisella-like endosymbiont), one potential secondary endosymbiont (Rickettsiella), and very few environmental or pathogenic bacterial species. We found a clear spatial structure of the microbiota, with ticks from the same sampling site showing similar patterns. On the contrary, no association with the infectious status for African swine fever virus was detected, suggesting that this pathogen does not shape Ornithodoros microbial communities. Our results on tick - microbiota - pathogen - environment interactions in nidicolous soft ticks, showed patterns that differ from most hard tick studies.

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota. The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales. Cells of strains AB-alg1T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1T (JCM 35393T=UQM 41574T) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae. ImportanceThe presented isolates are the first isolated representatives of an environmental family of Opitutales, part of the core microbiome of alkaline soda lakes. These bacteria feed on polysaccharides. We present the key enzymatic machinery for the polysaccharide breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example in washing powders and biomass waste recycling.

Macroalgae serve as critical habitat-formers and primary producers in coastal ecosystems, functioning across near-subtidal and intertidal zones in three distinct states: substrate-attached, free-floating (drift), and beach-cast. While substrate-attached macroalgae are susceptible to infectious diseases with significant ecological implications, diseases affecting drift macroalgal communities remain virtually unstudied. Here, we investigated bleaching disease - one of the most common macroalgal afflictions - in the drift rhodophyte Halymenia floresii from the Gulf of Mexico. Using 16S rRNA gene high-throughput sequencing and scanning electron microscopy, we characterized the bacterial community structure and composition associated with the free-floating healthy, bleached and degrading H. floresii to understand how bacterial partners respond to host health status. Principal Coordinate Analysis based on UniFrac distance revealed distinct clustering of bacterial communities according to host health condition. Shannon diversity indices showed distinct patterns ranging from 1.14 - 3.15 for healthy, bleached, and degrading samples, while Simpson indices ranged from 0.62 to 0.91, reflecting substantial variation in community evenness. In healthy samples, Cyanobacteria (17 - 52%) and Pseudomonadota (previously, Proteobacteria) (41 - 81%) dominated, and the bleached samples were characterized by elevated Bacteroidota (formerly, Bacteroidetes) (5 - 35%) and Pseudomonadota (41 - 88%). Notably, Novosphingobium (25 - 49%) dominated healthy hosts while showing lower abundance in degrading (13 - 17%) and bleached (18 - 22%) specimens. Conversely, Reinekea emerged as a dominant genus (22.5%) specifically in bleached samples, suggesting a potential role in disease pathogenesis. Microbial network analysis using NetCoMi revealed three distinct bacterial clusters corresponding to health states: a healthy-associated cluster dominated by Novosphingobium and uncultured Cyanobacterial with predominantly positive associations, and two disease-associated clusters enriched in opportunistic genera including Reinekea, Vibrio, Colwellia, and Alteromonas, indicating network reorganization from cooperative to exploitative interactions. This study provides the first descriptive assessment of microbiome transitions associated with bleaching disease in a drift macroalga and highlights the importance of considering free-floating macroalgal diseases and their potential impacts on coastal ecosystem health.