Microbial Ecology

Many insect populations are experiencing thermal stress as a result of global change, making it imperative to investigate how their relationship with other organisms will be impacted by heat disturbances. Microbial symbionts, such as bacteria, have the potential to enhance or inhibit an insect's thermal tolerance. Solitary bee larvae host bacteria within their food stores ("pollen provisions"), which have been shown to benefit survival and development; however, it is unclear how heatwaves brought about by climate change will impact their relationships with these bacterial partners. In this study, we subjected blue orchard bee (Osmia lignaria) eggs and larvae to a 4-day heatwave (35 {degrees}C daytime:22 {degrees}C nighttime) or kept them at control temperatures (25 {degrees}C daytime:15 {degrees}C nighttime), then returned all bees to control temperatures for a 5-day recovery period. We assessed bacterial communities within pollen provisions and larval development stage pre-heatwave (Day 0), immediately post-heatwave (Day 4), and following the recovery period (Day 9). Bacterial community composition, diversity, and abundance were resilient to heat stress, but larval bees developed faster when subjected to a heatwave. This finding refutes the hypothesis that bacteria within pollen provisions modulate blue orchard bee responses to heat, suggesting instead that developmental effects could be more largely shaped by bee physiology or interactions with microorganisms other than bacteria.

Predatory protists are important in shaping terrestrial microbial ecosystems, but their roles in the phyllosphere, or the communities on aerial plant surfaces, are poorly understood. Previous work found that the order Colpodida dominated heterotrophic protist communities in the phyllosphere. While most protists were sporadically present, a few Colpodida variants were prevalent and abundant, indicating that these variants may represent species adapted to the phyllosphere. To identify these organisms, we cultured colpodids from field-collected tomato leaves and performed phylogenetic analysis of the 18S rRNA gene. Five of nine independent isolates matched the most prevalent Colpodida variant previously identified as leaf-enriched through amplicon sequencing, and these isolates comprised a novel clade of Paracolpoda steinii. When compared to a maize root isolate of Colpoda inflata, an abundant rhizosphere ciliate, a P. steinii isolate was similar in size and growth yield on E. coli, but grew to higher yields and formed large cyst clusters when incubated with model phyllosphere bacteria prey Erwinia and Pseudomonas. We developed and validated quantitative PCR (qPCR) methods for detection and cell abundance estimation of the P. steinii phyllosphere clade, C. inflata, and the order Colpodida in environmental samples. In inoculated greenhouse plants, qPCR-estimated protist populations matched measured inoculum levels, and protist inoculum was still detectable after five days. In an uninoculated tomato field, P. steinii was detected on all plants, with greatest abundances observed in lower leaves and after a rain event. P. steinii comprised up to 18.7% of total leaf Colpodida populations, which were estimated at up to [~]1400 organisms per gram of fresh weight. The findings demonstrate that Colpodida communities are consistently present on tomato leaves, dynamically affected by the abiotic environment, and include significant populations of P. steinii. We propose that the P. steinii isolates and qPCR tools presented can be used as a model system to investigate colonization and distribution patterns, biotic interactions, genetic adaptations, and agricultural applications of leaf predation.

Giant clams are some of the largest bivalve molluscs. They form a vital partnership with Symbiodiniaceae dinoflagellates that supply most of their energetic requirements. However, the factors that shape giant clam-associated photosymbiont communities remain unknown. Here, we profiled Symbiodiniaceae communities using ITS2 metabarcoding in eight giant clam species (Hippopus hippopus, H. porcellanus, Tridacna crocea, T. derasa, T. gigas, T. maxima, T. noae and T. squamosa) from 11 sites across the Philippine archipelago. Symbiodiniaceae community structure was shaped by an interplay between giant clam host and environment. Most giant clams were dominated by members of a single symbiont genus, with Cladocopium as the most prevalent, followed by Durusdinium and Symbiodinium. However, giant clam hosts also exhibited flexibility in their symbiotic partners that was evident across sites. Differences in giant clam-associated symbiont communities may contribute to differences in holobiont function and adaptability to variable environments. These findings deepen our understanding of giant clam-Symbiodiniaceae associations, offering a framework for predicting how giant clams may be affected by increasingly stressful reef conditions and, more importantly, informing strategies to improve mariculture and conservation practices.

Ticks are obligate hematophagous arthropods and feed exclusively on blood. As blood is nutrient-poor, ticks rely on bacterial endosymbionts to synthesise nutrients, yet the diversity and functional roles of these symbionts in Australian ticks remain largely uncharacterised. This is critical to address as these ticks are of high medical importance in Australia. In this study, shotgun metagenomic sequencing was performed on Bothriocroton concolor, Bothriocroton hydrosauri, Haemaphysalis longicornis and Ixodes holocyclus, enabling the recovery of six complete or partial metagenome-assembled genomes (MAGs). These comprised Coxiella-like endosymbionts (CLE), a facultative Rickettsia symbiont, and two Midichloria mitochondrii strains (Ixholo1 and Ixholo2). Functional annotation of these taxon-specific symbionts revealed the absence of virulence factors and the presence of B-vitamin and/or heme biosynthesis genes, indicative of nutritional mutualism, which is essential for tick hematophagy. The CLEs additionally harbour genes of the shikimate pathway, which modulate blood feeding in ticks by regulating serotonin biosynthesis. Furthermore, functional annotation and pangenomic analysis of Midichloria spp. found evidence that the genus may encompass multiple species, as well as the retention of genes potentially associated with an intramitochondrial lifestyle in M. mitochondrii Ixholo2. Tick microbiomes are dominated by non-pathogenic microorganisms, which are often overshadowed by pathogens. These include the endosymbionts, which can influence host biology and pathogen transmission, and are fundamental for the development of diagnostic tools and taxon-specific tick biocontrols.

Antimicrobial resistance (AMR) is one of the greatest challenges within the One Health continuum. Exploring transmission routes between health domains and determining their driving forces are key priorities for future research. This effort can be effectively supported by landscape epidemiology, a field of science that integrates methods from landscape ecology and epidemiology to unravel the complex interdependencies behind disease transmission. This exploratory study aimed to demonstrate that landscape diversity and the degree of hemeroby (anthropogenic impact) correlate with the composition of bacterial communities and their AMR profiles. To test this hypothesis, submandibular lymph nodes from Cervidae and Suidae were collected to detect Staphylococcus and Mammaliicoccus bacteria and characterise their AMR features using selective culture and the VITEK 2 Compact automated system. As a result, the bacterial community in the more natural landscape was more diverse, characterised by the dominance of Mammaliicoccus sciuri and pan-susceptible isolates of Staphylococcus hyicus, and it displayed a low-level, heterogeneous AMR profile. Within the more hemerobic landscape, the bacterial community was characterised by the dominance of Staphylococcus epidermidis, a human-adapted species, and the AMR profile showed signs of higher antimicrobial pressure from both public health and veterinary origins. Although this study was based on only two study sites and was therefore not suitable for drawing definite conclusions, the findings suggest that human impact manifests itself in both bacterial and AMR profiles. A high prevalence of mammaliicocci and a heterogeneous AMR profile appeared to be indicators of naturalness. Conversely, the dominance of a human-adapted bacterial species and the accumulation of AMR features characteristic of medical environments likely indicate higher degrees of hemeroby.

Knowledge of the family Lactobacillaceae rests largely on isolates from foods and a few repeatedly sampled hosts. Reference databases give an unrecognised sequence the name of its nearest relative, so a lineage with no entry of its own is renamed rather than flagged. Here we classify the family across the public plant and invertebrate amplicon record to locate the hosts carrying undescribed lineages. Across 3,344 independent 16S rRNA gene amplicon studies, every sequence cluster was tested against a type-strain reference and placed at the deepest rank it supports. Of 204,813 classified clusters, 20,516 were named to species and 127,906 to genus, while 4,741 matched no described species. Six published datasets whose authors could name their Lactobacillaceae only as "Lactobacillus", or not at all, are reclassified here. Genera described from one habitat occur far beyond it, three bee-associated genera occurring on Rosaceae and Brassicaceae at several times their rate on wind-pollinated grasses, and we found no published Bombilactobacillus record from a plant. Undescribed lineages concentrate in the least-cultured hosts, reaching 86.1% and 77.4% of studies in the stingless bees Melipona and Tetragonula, and are most divergent in the turtle ant Cephalotes and, among plants, in tea, Camellia. An independent genome-resolved survey of pot honey converges on the same two genera. The primary descriptions of forty-eight species from these hosts specify a supplemented medium, so the hosts carrying undescribed lineages also indicate how to culture them.

Symbioses are widespread and highly successful but vulnerable to the stress sensitivity of either symbiont. In some symbioses, turnover of symbionts has been shown to confer resilience to stressors. While similar mechanisms have been proposed for lichen symbioses, direct evidence for rapid adaptive symbiont turnover has not been shown. We tested the photobiont community composition and physiological responses of the foliose lichen symbiosis Flavoparmelia caperata-Trebouxia to urbanization-induced stress in a transplant experiment. We found evidence for significant compositional change in the photobiont community along an urbanization gradient (measured as vegetation cover), reflecting a turnover in dominance of Trebouxia OTUs from A46 to I05 in more urbanized transplant sites. This change in symbiont composition is associated with a greater physiological tolerance for urbanization, consistent with the hypothesized adaptive role of photobiont turnover. These findings support rapid photobiont turnover as a potential adaptive response to environmental change in lichen symbioses.

Background and AimsAbiotic stress is a major constraint for citrus production in Mediterranean environments, where water deficit and salinity frequently occur. This is particularly relevant for perennial crops, like citrus, where limited options for stress avoidance exist. Rootstocks are extensively employed to enhance stress resilience; however, their influence on the root microbiome under abiotic stress remains largely unexplored. Here, we investigated the effects of water stress and salinity on the diversity, composition, and interactions of bacterial and fungal communities in two citrus rootstocks with reported contrasting phenotypes, such as Bitters, which has been described as exhibiting a promising tolerance to both water and salt stress, and Carrizo, which is generally reported to be highly sensitive to these conditions. MethodsThe distinct rootstocks have been subjected to either water stress or salt stress and compared with the non-stressed rootstocks. At the end of stress period, they were profiled and then integrated with recorded plant morphological (i.e. root volume), physiological (water potential, abscisic acid, chlorophyll and chlorophyll content meter) and biochemical measurements (abscisic acid and catalase). In parallel, we used a high-throughput amplicon sequencing to profile bacterial and fungal communities inhabiting the rhizosphere and endorhizosphere microhabitats of the rootstocks in both stresses and in non-treated conditions. Finally, we used correlations and multivariate analysis to determine relationships between plant performance and microbiome putatively underpinning stress adaptation and tolerance. ResultsAcross all treatments, microbial community composition was primarily shaped by microhabitat, with clear differentiation between rhizosphere and endorhizosphere. Abiotic stress significantly restructured microbial communities, particularly in the rhizosphere, while the endorhizosphere exhibited stronger genotype-dependent patterns. Bacterial communities showed pronounced stress-driven enrichments of taxa belonging to the main phyla (such as Proteobacteria, Actinobacteriota and Bacteroidota), with selective recruitment of taxa putatively associated with stress adaptation, whereas the response of fungal taxa (more represented by Ascomycota, Basidiomycota and Glomeromycota phyla) was less consistent and mainly microhabitat-driven. Notably, the two rootstocks exhibited distinct physiological strategies, with Bitters by increased proline accumulation and root volume and Carrizo characterized by enhanced ABA and catalase. ConclusionsOur findings showed Bitters outperform Carrizo in terms of tolerance to both water and salinity stress. In both rootstocks, specific bacterial taxa such as high abundant core or rare members, were associated with distinct phenotypic parameters, highlighting the importance of integrating plant and microbiome perspectives for improving stress resilience in citrus.

Blood-feeding insects typically depend on obligate intracellular bacterial symbionts that provide essential B vitamins absent from vertebrate blood. In contrast, kissing bugs (Triatominae) have long been considered atypical because they rely primarily on extracellular gut-associated bacteria. Recent reports of the genus Symbiopectobacterium in Rhodnius species raise questions about the diversity and evolution of symbiosis in these insects. Here, we investigate the distribution, genome evolution, and tissue localization of Symbiopectobacterium in the genus Rhodnius. Using comparative metagenomics, phylogenomics, fluorescence in situ hybridization, transmission electron microscopy, and hemolymph screening, we characterize a Symbiopectobacterium genome from Rhodnius prolixus and assess its occurrence across publicly available datasets representing multiple Rhodnius species. The R. prolixus strain possesses a large, highly dynamic genome enriched in mobile genetic elements, pseudogenes, and remnants of secretion systems, while retaining biosynthetic pathways for several B vitamins. Comparative analysis revealed variation in genome reduction among Rhodnius-associated strains, suggesting ongoing and potentially independent transitions toward host-restricted symbiosis. Localization analyses detected Symbiopectobacterium intracellularly within posterior midgut epithelial cells and occasionally in the hemolymph, consistent with a facultative intracellular lifestyle. However, no bacteriomes or stable intracellular structures were observed. Together, these findings indicate that Symbiopectobacterium represents an intermediate stage in the transition from environmentally associated bacteria to obligate intracellular mutualists in Triatominae.

Forest-associated Saccharomycotina occur at low relative abundance, limiting inference about their diversity and dynamics. We sampled leaf litter weekly for 47 weeks across a subtropical forest in northern Taiwan. Enrichment, isolation and ITS sequencing recovered 687 isolates, including 613 Saccharomycotina representing 56 described species and 77 putatively novel operational taxonomic units. Rarefaction indicated unsampled culturable diversity. Among litter traps, community dissimilarity was high and dominated by taxon replacement, but neither topography nor geographic distance was associated with composition, and turnover matched randomised expectations. Richness peaked during warm, wet periods and declined in winter, and minimum temperature showed the strongest statistical association. Composition was associated with maximum temperature, minimum relative humidity, precipitation and solar radiation. Selected isolates' thermal optima covaried with collection-week temperatures, and two October Magnusiomyces magnusii isolates had higher optima than four winter isolates. Together, these findings reveal substantial culturable diversity and seasonal community restructuring consistent with temperature-related filtering.

Gut microbiome responses to anthropogenic disturbance vary across wildlife species, even within similarly disturbed landscapes. What drives this variation is unclear: whether it reflects anthropogenic exposure itself or broader ecological differences among hosts. We tested this using three macaque species with contrasting ecology, Bonnet, Rhesus, and Lion-tailed macaques, sampled across 12 sites in southern India spanning contrasting anthropogenic exposure, using 16S rRNA gene sequencing (n = 127) and shotgun metagenomics on a subset of samples. The two synurbanized species exhibited a similar magnitude of microbiome restructuring but differed in the taxa underlying these changes; no differentially abundant amplicon sequence variants were shared across all three species, indicating that shared anthropogenic exposure did not produce uniform microbial responses across hosts. The specialist Lion-tailed macaque showed a more extensive response, characterized by reduced diversity and phylogenetically structured compositional change. The Bonnet macaque showed greater microbial similarity with the Rhesus macaque than with the Lion-tailed macaque during sympatric co-occurrence. Despite taxonomic divergence, functional pathway architecture was broadly conserved across species and habitats, with selective shifts in pathways including vitamin B6 biosynthesis and fermentation. Together, these findings show that microbiome responses to anthropogenic environments are jointly shaped by ecological context and host ecology, with host differences in diet, habitat use, and ecological history influencing the magnitude and nature of microbial restructuring. These findings show that taxonomic diversity and functional potential respond as partially decoupled axes under anthropogenic pressure, with implications for assessing microbiome resilience across ecologically heterogeneous wildlife.

The reintroduction of extinct or endangered species to restore ecosystem function is an essential aspect of rewilding. The Wilder Blean Project at West Blean and Thornden Woods in Canterbury, UK, is committed to rewilding natural processes and enhancing biodiversity in one of England's oldest and largest areas of ancient woodland. The introduction of European bison (Bison bonasus) is an important part of the project. However, how the reintroduction of large herbivores influences local biodiversity and ecosystem functions during the early stages of rewilding remains poorly understood. Soil samples were collected from the same sampling sites before and two years after bison were reintroduced and profiled by metagenomic sequencing using Oxford Nanopore Technologies sequencing platforms. The results showed that the alpha diversity of soil organisms did not change significantly before and after the introduction of European bison, while beta diversity showed modest shifts in community composition. The relative abundance of some nitrogen-fixing and photosynthetic microbial genera showed declines in the 2024 Bison Area, while the mycorrhizal fungus genus Rhizophagus was significantly less abundant than in the 2024 Control Area. Despite relatively stable taxonomic diversity, functional composition differed significantly between the 2022 and 2024 Bison areas and among the 2024 rewilding treatments, revealing a decoupling between taxonomic diversity and functional composition. Amino acid synthesis pathways and carbon metabolism pathways were significantly enriched. These findings highlight the potential of long-read Oxford Nanopore metagenomics to reveal functional shifts that may not be apparent from taxonomic diversity alone. Although these early-stage responses cannot yet predict long-term rewilding trajectories, continued longitudinal monitoring integrating microbial, soil physicochemical, and ecosystem-level measurements will be essential to determine the persistence and ecological significance of these functional shifts.

While the suppression of toxic cyanobacteria by aquatic plants has long been recognized, few studies have clearly differentiated between the allelopathic effects of the plant itself and the inhibitory influence of its associated microbiome. This study aimed to clarify the primary inhibitory agent by pre-culturing Myriophyllum spicatum (Eurasian watermilfoil) under three conditions: (1) BG11 medium, (2) live Microcystis aeruginosa KW culture, and (3) a Microcystis-symbiotic microbiome (excluding Microcystis cells). After a 7-day pre-culture, Myriophyllum shoots were transferred to fresh Microcystis culture. The Myriophyllum pre-cultured in Microcystis culture exhibited rapid inhibition against Microcystis (84% within day 1), whereas the Myriophyllum pre-cultured in BG11 medium showed delayed responses (89% by day 7). In contrast, inhibition remained below 50% in the Myriophyllum pre-cultured with the Microcystis-symbiotic microbiome. Notably, plant-derived soluble compounds exhibited weak inhibitory effects, whereas the microbiome showed stronger inhibitory activity, indicating that the plant-associated microbiome plays a more dominant role than the plant itself. Exposure to Microcystis triggered significant shifts in plant-symbiotic microbial community composition, leading to rapid enhancement of inhibitory activity in the Myriophyllum microbiome. Microbial community analysis identified 28 bacterial taxa closely associated with the inhibitory response, including strains involved in organic matter degradation, adhesion, biofilm formation, and predatory behavior. Meta-transcriptomic analysis further confirmed increased expression of genes related to bacterial adhesion, biofilm formation, and carbohydrate metabolism following Microcystis exposure, highlighting functional adaptations linked to cyanobacterial suppression. These findings underline the role of microbiome-mediated cyanobactericidal mechanisms, providing new insights into a nature-based solution for mitigating Microcystis-dominated harmful algal blooms.

Heritable bacterial symbionts are pervasive in terrestrial arthropods, often imposing reproductive manipulations to promote their own spread within host populations. Co-infections are common, potentially allowing symbiont co-infectors to hitchhike through a host population. However, adverse thermal conditions can disrupt these communities, particularly when co-infectors vary in their thermal sensitivity. We used a multi-generation experiment to test whether warm (29 {degrees}C) conditions disrupted spread of heritable symbionts through uninfected populations of the spider, Mermessus fradeorum. We tested two common infection combinations: a single infection with a cytoplasmic incompatibility (CI) inducing Rickettsiella or a feminizing co-infection that included a feminizing Wolbachia, the same Rickettsiella, and up to three apparent hitchhikers (two additional Wolbachia strains and Tisiphia). We initiated replicate populations with 1/3 of one infection type and 2/3 uninfected spiders, evaluating population infection rate over 5 spider generations under different temperature regimes. Under cool (21{degrees}C) conditions, Wolbachia feminization drove co-infection to 88% and Rickettsiella CI drove single infection to 83% of host populations. Vertical transmission for all symbionts was high (97-99%) and hitchhiking symbionts also spread effectively. Under warm conditions, feminization and CI efficacy were reduced, and symbionts suffered variably reduced vertical transmission. Warm conditions ultimately destroyed the co-infecting symbiont consortium and impeded symbiont spread. On its own, though, Rickettsiella was still able to increase, despite reduced strength of CI. We hypothesize that contrasting tensions between feminizing spread of the symbiont consortium versus environmentally driven loss of function and transmission may explain observed patterns of mixed infections in field populations of this spider.

Rising ocean temperatures disrupt previously stable coral-microbe interactions, leading to widespread coral mortality and threatening reef ecosystems worldwide. Growing evidence demonstrates the coral microbiome, including protists, plays a critical role in the host response to thermal stress. Specifically, corallicolids (Phylum: Apicomplexa) are positively correlated with thermal stress mortality in soft corals. This study investigates changes in the eukaryotic microbiome of the stony coral, Pocillopora damicornis, across an experimental thermal stress event. Using anti-metazoan 18S rRNA gene metabarcoding, protist communities were assessed at four time-points during experimental thermal stress. Outside of the Symbiodiniaceae, a prominent shift in microbiome composition during thermal stress was observed, most notably a significant increase and dominance in Corallicolida abundance in heat-stressed corals, while other protists declined substantially. Increased corallicolid abundance concurrent with bleaching suggests an overlooked compounding stressor beyond the loss of algal symbionts during heat stress. These results contrast with previous research on Pocillopora microbiomes showing prokaryotic community stability throughout stress, and support the hypothesis that thermal stress may alter the coral-corallicolid relationship, potentially shifting corallicolids from a commensal to a parasitic role, and synergistically contributing to coral mortality during and after heat stress. This work provides critical insight into the role of protists in marine holobionts, supports their inclusion in future microbiome studies, and informs strategies to improve coral resilience under climate change.

Microbiome transplantation, used to treat human disease, can enhance thermal and pathogen resilience in bleaching-susceptible corals via coral microbiome transplantation (CMT), though success is donor- and recipient-dependent. In this study, less thermally tolerant Pachyseris speciosa fragments were exposed to ciprofloxacin or an antibiotic cocktail for 24 h, then received CMT from the thermally tolerant Acropora millepora from Singapore's turbid reef system. Alpha diversity increased only in antibiotic-treated, CMT fragments, demonstrating that antibiotic-induced dysbiosis enhanced bacterial uptake. Coral microbiome assemblage shifted significantly at 1 and 10 d, regardless of antibiotic treatment or Acropora inoculum. Antibiotic-induced dysbiosis did not enhance uptake of donor's core ASVs (e.g., Endozoicomonas spp.). However, early uptake favoured potential pathogens like Vibrio spp., while longer inoculation allowed for uptake of unculturable environmental taxa. Our approach of using antibiotic pretreatment followed by whole microbiome transplant parallels human faecal microbiota transplantation to restore gut health.

Background: Microplastics have been detected in many freshwater ecosystems, including stagnant waters where mosquito larvae develop. Larvae are therefore exposed to microplastic pollution, which may affect their life history traits or microbiota along with immune gene expression. However, these effects have never been tested in mosquitoes, despite their major public health importance as vectors of numerous pathogens. Method: We exposed mosquito larvae, from hatching to adult emergence, to four concentrations of polyethylene microplastics (MPs): 0, 60, 200, and 600 MPs/mL. Fourth-instar larvae and newly emerged adult females were collected for each treatment. To investigate the effects of MPs on gene expression and bacterial microbiota, RNA sequencing and 16S metabarcoding approaches were performed on three biological replicates for each developmental stage. Results: Microplastic exposure induced a non-monotonic dose-dependent transcriptomic response. In larvae, only a limited number of genes were differentially expressed (five to eight per concentration), with immune-related genes downregulated at both low and high concentrations. In adult females, the intermediate concentration (200 MPs/mL) elicited the strongest response with 14 differentially expressed genes (DEGs). Regarding the microbiota, microplastic exposure reduced bacterial diversity in larvae, with the lowest diversity observed at the highest concentration. However, no significant changes were detected in the microbiota of adult females. Conclusion: Overall, this study shows that adult females are affected by larval exposure to MPs (i.e. differential expression in immune-related genes) and warrants further studies in this field. This includes: 1) investigating further the effects of MPs on mosquitoes' populations (e.g. through multi-generational studies), 2) gaining more environmentally relevant knowledge on MP effects (i.e. using MPs with a biofilm and/or adsorbed pollutants) and 3) focusing on the effects of MPs on mosquitoes' vectorial capacities.

Most cockroaches and the termite Mastotermes darwiniensis are associated with Blattabacterium, an ancient obligate endosymbiont that participates in the nitrogen metabolism of its host. Blattabacterium has been vertically transmitted since it was acquired by the common ancestor of cockroaches and termites and was reportedly lost twice, once in the cockroach genus Nocticola and once in all termites except Mastotermes darwiniensis. Here, we acquired cockroach specimens spanning most of the cockroach phylogenetic tree to study Blattabacterium using shotgun sequencing. We found no traces of Blattabacterium in 64 specimens from ten independent lineages of cockroaches across three families: Blattellidae, Pseudophyllodromiidae, and Anaplectidae. The absence of Blattabacterium was confirmed with three PCR amplifications targeting the 16S and 23S ribosomal genes with primers specific to Blattabacterium. Notably, cockroaches lacking Blattabacterium were often infected by Rickettsia and Wolbachia, many of which were related to the mutualistic Wolbachia strain of Cimex lectularius, the common bed bug. These results indicate that cockroaches from Blattellidae, Pseudophyllodromiidae and Anaplectidae have lost their ancestral Blattabacterium endosymbiont at least ten times independently, with many of these losses possibly facilitated and compensated by new associations with mutualistic Wolbachia strains that may help provision the host with B vitamins.

Alkaline and calcareous soils can induce iron (Fe) limitation in plants, yet the responses of root-associated microbial communities to beneficial rhizobacteria under these conditions remain poorly understood in cotton. Here, we investigated the effects of Variovorax paradoxus on plant performance, Fe nutrition, and root microbiome dynamics in cotton exposed to bicarbonate-induced Fe limitation. In this study, V. paradoxus inoculation under bicarbonate-induced Fe limitation significantly improved photosynthetic parameters, growth parameters, and tissue Fe status. Interestingly, V. paradoxus partially suppressed the Fe-deficiency-induced increase in root ferric-chelate reductase activity without further increasing rhizosphere siderophore activity. This response suggests that improved Fe availability reduced the demand for maximal activation of the intrinsic Strategy I response. Despite improved plant health, V. paradoxus reduced root C levels, suggesting altered belowground carbon utilization associated with bacterial inoculation and stress conditions. Split-root experiments further showed that inoculating both root compartments showed substantially greater recovery than unilateral inoculation, indicating that broader root exposure to V. paradoxus enhanced the beneficial response. Although bacterial alpha diversity remained unchanged, V. paradoxus significantly altered bacterial community composition and enriched Cellvibrio together with the fungal taxa Funneliformis and Dominikia under Fe limitation. Exploratory analysis identified the plant-beneficial fungal hubs Funneliformis and Serendipita in the V. paradoxus-treated community under indirect Fe deficiency, along with the core genera Pseudomonas, Hydrogenophaga, and Funneliformis and the indicator taxa Shinella and Aquabispora. Spearman correlation analysis further associated Streptomyces with root Fe accumulation and biomass, while Epicoccum and Sordariales were positively associated with siderophore production in cotton exposed to bicarbonate-induced Fe limitation and inoculated with V. paradoxus. These findings demonstrate the potential of V. paradoxus and identify candidate microbial partners for microbiome-informed biofertilizers to improve Fe nutrition in cotton grown in calcareous soils.

Host genetic markers often fail to resolve regional origins in highly connected or panmictic marine species. The Japanese jack mackerel, Trachurus japonicus, is a commercially important fishery species around Japan that shows little or no detectable population structure. Here, we used nationwide multi-omics profiling to compare host genomic variation and gut microbiome composition in wild T. japonicus collected from coastal regions across Japan. We generated MIG-seq data for 43 individuals and 16S rRNA gene profiles for 24 individuals; after quality filtering, 19 individuals remained for matched host-microbiome comparison. Genome-wide host SNP analyses showed weak or absent geographic population structure, consistent with previous evidence of panmixia in Japanese waters. In contrast, gut microbiome composition showed geographic structuring based on Bray-Curtis dissimilarity and PERMANOVA, and this pattern was not explained by proximity to river mouths or host-related variables. Locality- or individual-associated bacterial lineages contributed to the observed differences in the microbiome, while chloroplast-associated and Cyanobacteria-assigned ASVs suggested recent dietary or environmental input. These results indicate that gut microbiome can show regional biological variation not apparent from host genetic markers alone. Our study provides a proof-of-concept example of integrating host genomics and gut microbiome profiling to evaluate regional characteristics and origins in highly connected marine animals.