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Axenic dietary restriction (ADR) is highly effective in extending lifespan of C. elegans but its effects on healthspan improvement is less well characterized. Using transmission electron microscopy, morphometric analyses, and functional assays, we found ADR can preserve tissue ultrastructure, including the cuticle, epidermis, and intestinal lumen, and reduce age-associated pathologies like gonad degeneration, uterine tumor clusters, pharyngeal deterioration, and intestinal atrophy. However, there was no notable improvement in behavioral and functional metrics. Our results underscore that lifespan extension through ADR does not inherently translate to broad healthspan improvements.

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Nearly 50 years after the ground-breaking isolation of the primary Comptonia peregrina microsymbiont under axenic conditions, efforts to isolate a substantial number of Protofrankia and Frankia strains continue with enduring challenges and complexities. This study aimed to streamline genomic insights through comparative and predictive tools to extract traits crucial for isolating specific Frankia in axenic conditions. Pangenome analysis unveiled significant genetic diversity, suggesting untapped potential for cultivation strategies. Shared metabolic strategies in cellular components, central metabolic pathways, and resource acquisition traits offered promising avenues for cultivation. Ecological trait extraction indicated that most uncultured strains exhibit no apparent barriers to axenic growth. Despite ongoing challenges, potential caveats, and errors that could bias predictive analyses, this study provides a nuanced perspective. It highlights potential breakthroughs and guides refined cultivation strategies for these yet-uncultured strains. We advocate for tailored media formulations enriched with simple carbon sources in aerobic environments, with atmospheric nitrogen optionally sufficient to minimize contamination risks. Temperature adjustments should align with strain preferences-28-29°C for Frankia and 32-35°C for Protofrankia-while maintaining an alkaline pH. Given potential extended incubation periods (predicted doubling times ranging from 3.26 to 9.60 days, possibly up to 21.98 days), patience and rigorous contamination monitoring are crucial for optimizing cultivation conditions.

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Most terrestrial plants are associated with symbiotic Glomeromycotina fungi, commonly known as arbuscular mycorrhizal (AM) fungi. AM fungi increase plant biomass in phosphate-depleted conditions by allocating mineral nutrients to the host; therefore, host roots actively exude various specialized metabolites and orchestrate symbiotic partners. The hyphal branching activity induced by strigolactones (SLs), a category of plant hormones, was previously discovered using an in vitro assay system. For this bioassay, AM fungi of the Gigaspora genus (Gigasporaeae) are commonly used due to their linear hyphal elongation and because the simple branching pattern is convenient for microscopic observation. However, many researchers have also used Glomeraceae fungi, such as Rhizophagus species, as the symbiotic partner of host plants, although they often exhibit a complex hyphal branching pattern. Here, we describe a method to produce and quantify the hyphal branches of the popular model AM fungus Rhizophagus irregularis. In this system, R. irregularis spores are sandwiched between gels, and chemicals of interest are diffused from the surface of the gel to the germinating spores. This method enables the positive effect of a synthetic SL on R. irregularis hyphal branching to be reproduced. This method could thus be useful to quantify the physiological effects of synthesized chemicals or plant-derived specialized metabolites on R. irregularis. Key features • Development of an in vitro hyphal branching assay using germinating spores of Rhizophagus irregularis. • This in vitro assay system builds upon a method developed by Kameoka et al. [1] but modified to make it more applicable to hydrophilic compounds. • Optimized for R. irregularis to count the hyphal branches. • This bioassay requires at least 12 days to be done.

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Identifying the nutritional requirements and growth conditions of microorganisms is crucial for determining their applicability in industry and understanding their role in clinical ecology. Predatory bacteria such as Bdellovibrio bacteriovorus have emerged as promising tools for combating infections by human bacterial pathogens due to their natural killing features. Bdellovibrio's lifecycle occurs inside prey cells, using the cytoplasm as a source of nutrients and energy. However, this lifecycle supposes a challenge when determining the specific uptake of metabolites from the prey to complete the growth inside cells, a process that has not been completely elucidated. Here, following a model-based approach, we illuminate the ability of B. bacteriovorus to replicate DNA, increase biomass, and generate adenosine triphosphate (ATP) in an amino acid-based rich media in the absence of prey, keeping intact its predatory capacity. In this culture, we determined the main carbon sources used and their preference, being glutamate, serine, aspartate, isoleucine, and threonine. This study offers new insights into the role of predatory bacteria in natural environments and establishes the basis for developing new Bdellovibrio applications using appropriate metabolic and physiological methodologies. KEY POINTS: • Amino acids support axenic lifestyle of Bdellovibrio bacteriovorus. • B. bacteriovorus preserves its predatory ability when growing in the absence of prey.

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Many organisms harbor heritable bacterial symbionts that offer context-specific benefits to their hosts. In some of these symbioses, symbionts live inside host cells as endosymbionts. Studying the biology of endosymbiosis is challenging because it is hard to independently cultivate hosts and endosymbionts. A recent study, using a simple defined growth medium at ambient temperature, established an axenic culture of the pea aphid's heritable bacterial endosymbiont, Candidatus Fukatsuia symbiotica (G. P. Maeda, M. K. Kelly, A. Sundar, and N. A. Moran, mBio 15:e03253-23, 2024, https://doi.org/10.1128/mbio.03253-23). Notably, the monoculture was capable of host recolonization, was stably transmitted, and returned similar host phenotypes to those observed in native infections. This advance in uncoupling the cultivation of an endosymbiont and its host opens avenues for genetic manipulation of the endosymbiont that will facilitate hypothesis-driven work to explore the mechanisms of host-endosymbiont biology and potentially facilitate the development of symbiont-mediated practical-application biotechnologies.

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Bryophytes are rich sources of diverse secondary metabolites with a wide range of biological activities, including anti-inflammatory, antitumor and antimicrobial effects. The aim of this study was to investigate the chemical composition of extracts from two different genotypes (Serbian and Hungarian) of the axenic moss Atrichum undulatum and evaluate the immunomodulatory potential of the prepared extracts in vitro. Both genotypes of moss samples were cultivated in vitro and subsequently extracted in a Soxhlet apparatus with methanol or ethyl acetate. The highest concentration of total phenolic compounds was found in the methanolic extract of the Serbian genotype (54.25 mg GAE/g extract), while the ethyl acetate extract of the Hungarian genotype showed the highest concentration of phenolic acids (163.20 mg CAE/extract), flavonoids (35.57 mg QE/extract), and flavonols (2.25 mg QE/extract). The extracts showed anti-neuroinflammatory properties by reducing the production of reactive oxygen species, nitric oxide, and tumor necrosis factor alpha by lipopolysaccharide-stimulated microglial cells. Moreover, they mitigated the cytotoxic effects of the pro-inflammatory mediators produced by activated microglia on neurons. The data obtained suggest that extracts from A. undulatum moss have promising anti-neuroinflammatory and neuroprotective properties, making them interesting candidates for further research to combat neuroinflammation.

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BACKGROUND: The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). RESULTS: Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. CONCLUSION: Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.

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BACKGROUND: Environmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains. RESULTS: We applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen. CONCLUSIONS: Our study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.

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Blastocystis sp. is a prevalent protistan parasite found globally in the gastrointestinal tract of humans and various animals. This review aims to elucidate the advancements in research on axenic isolation techniques for Blastocystis sp. and their diverse applications. Axenic isolation, involving the culture and isolation of Blastocystis sp. free from any other organisms, necessitates the application of specific media and a series of axenic treatment methods. These methods encompass antibiotic treatment, monoclonal culture, differential centrifugation, density gradient separation, micromanipulation and the combined use of culture media. Critical factors influencing axenic isolation effectiveness include medium composition, culture temperature, medium characteristics, antibiotic type and dosage and the subtype (ST) of Blastocystis sp. Applications of axenic isolation encompass exploring pathogenicity, karyotype and ST analysis, immunoassay, characterization of surface chemical structure and lipid composition and understanding drug treatment effects. This review serves as a valuable reference for clinicians and scientists in selecting appropriate axenic isolation methods.

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Our previous studies have shown that CyanoHAB LPS (lipopolysaccharides) and LPS from cyanobacterial cultures induce pro-inflammatory effects on intestinal epithelial and immune cells in vitro. To expand our understanding, we investigated their impact on human keratinocytes, which are targeted during water recreational activities. LPS samples were isolated from CyanoHAB biomasses dominated by Microcystis, Aphanizomenon, Planktothrix, and Dolichospermum, or from axenic cultures of these genera. We identified two CyanoHAB biomasses containing a high proportion of Gram-negative bacteria, including potentially pathogenic genera. These biomasses showed the highest induction of interleukin (IL) 8, IL-6, C-C motif chemokine ligand (CCL) 2 (also known as MCP-1), and CCL20 production by HaCaT cells. Interestingly, all CyanoHAB-derived LPS and LPS from axenic cultures (except for Microcystis) accelerated cell proliferation and migration. Our findings highlight the role of G- bacteria composition and LPS structural disparities in influencing these effects, with implications for skin health during recreational activities.

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Plants harbor complex and highly diverse fungal endophyte communities (FECs), making it difficult to evaluate the functional role of individual taxa, subsets of the community, or the FEC as a whole. To reduce the complexity of this system, we aimed to produce fungi-null wheat (Triticum aestivum) plants. To this end, we treated seeds with heat and fungicides and generated plants from rescued embryos and callus tissue. A culture-based approach and reverse transcription PCR analysis were negative, indicating that all treatments produced plants apparently free of fungi. However, the analysis of DNA using digital droplet PCR and next-generation sequencing revealed that tissues from all treatments retained low levels but diversity-rich FECs. While the FECs varied in composition across treatments and tissues, they all included core taxa of the mycobiome. The reduced fungal biomass, along with the changes in FEC composition, negatively affected plant development, supporting a FEC contribution to proper plant development and fitness. Our discovery that a large part of the FEC cannot be separated from plants and can be transmitted through seeds and tissue culture calls for reevaluation of particular microbiome paradigms, such as core taxa concepts, transmission modes, and functional species.IMPORTANCEThe native microbiome in a given plant must be considered when evaluating the effect of a single taxon or synthetic community. The pre-existing microbiome can interact with artificially added microbial cargo, which affects the final outcome. Such issues can be at least partially solved by the use of endophyte-free plants, which provide a clean background that should be useful in determining the effect of a single taxon, taxa combinations, or the entire microbiome on plant performance. Previous reports regarded plants as endophyte-free or axenic by the lack of fungal growth on culture media or the generation of plants from tissue cultures. We showed here that while fungi could not be isolated from fungicide-treated or tissue culture-regenerated plants, nevertheless, all plants contained rich fungal endophyte communities; namely, it was impossible to create fungi-free wheat plants. Our results call for rethinking fundamental microbiome-related concepts, such as core taxa, transmission mode, and functional species.

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We report the draft genome sequence of strain B0820 of the cyanobacterium Tychonema bourrellyi isolated from the epilimnion of Lake Garda and assembled from a metagenome of a non-axenic culture. The strain analyzed was shown to produce anatoxin-a, a potent neurotoxin that can cause fatal intoxication in exposed organisms.

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In this study, we describe the generation of two new species of axenic mosquito, Aedes albopictus and Aedes triseriatus. Along with Aedes aegypti, axenic larvae of these three species were exposed to an environmental water source to document the assembly of the microbiome in a common garden experiment. Additionally, the larvae were reared either individually or combinatorially with the other species to characterize the effects of co-rearing on the composition of the microbiome. We found that the microbiome of the larvae was composed of a relatively low-diversity collection of bacteria from the colonizing water. The abundance of bacteria in the water was a poor predictor of their abundance in the larvae, suggesting the larval microbiome is made up of a subset of relatively rare aquatic bacteria. We found 11 bacterial 16S rRNA gene amplicon sequence variants (ASVs) that were conserved among ≥90% of the mosquitoes sampled, including 2 found in 100% of the larvae, pointing to a conserved core of bacteria capable of colonizing all three species of mosquito. Yet, the abundance of these ASVs varied widely between larvae, suggesting individuals harbored largely unique microbiome structures, even if they overlapped in membership. Finally, larvae reared in a tripartite mix of the host-species consistently showed a convergence in the structure of their microbiome, indicating that multi-species interactions between hosts potentially lead to shifts in the composition of their respective microbiomes. IMPORTANCE This study is the first report of the axenic (free of external microbes) rearing of two species of mosquito, Aedes albopictus and Aedes triseriatus. Our previous report of axenic Aedes aegypti brings the number of axenic species to three. We designed a method to perform a common garden experiment to characterize the bacteria the three species of axenic larvae assemble from their surroundings. Furthermore, species could be reared in isolation or in multi-species combinations to assess how host-species interactions influence the composition of the microbiome. We found all three species recruited a common core of bacteria from their rearing water, with a large contingent of rare and sporadically detected bacteria. Finally, we also show that co-rearing of mosquito larvae leads to a coalescence in the composition of their microbiome, indicating that host-species interactions potentially influence the composition of the microbiome.

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Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.

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To attend to the growing world demand for mushrooms, it is interesting to increase the system's productivity, improve quality and reduce production costs. This study aimed to optimize the production and quality of fruiting bodies of the edible and medicinal mushroom Lentinula edodes (shiitake), in agroresidues substrate using appropriate strain and spawn formulation. The evaluation was conducted using two strains under seven different spawn formulations (Control [C]: Sorghum grain + 2.5% CaCO3; (2) C + 2.5% sawdust; (T3) C + 5% sawdust; (T4) C + 2.5% peat; (T5) C + 5% peat; (T6) C + 1.25% sawdust + 1.25% peat; (T7) C + 2.5% sawdust + 2.5% peat) that were inoculated into the blocks at a proportion of 2% (w/w). The substrate was formulated with 63% rice straw, 20% sawdust, 15% wheat bran, and 2% CaCO3 and sterilized. The incubation period was 87 days. Two flushes were obtained. Adding small aliquots of peat and sawdust to the inoculum gave significantly higher morphological results than the control in all variables analyzed. The days required for the first harvest ranged from 87 to 94 days. The average weight of basidiomes ranged from 6.38 to 28.75 g. The productivity data show superior results for the treatments in which the spawn was supplemented with sawdust and peat. Enhanced bioconversion with supplemented spawn shows promises for yield and composition improvement, crucial for commercial viability. It can be concluded that shiitake production using agroresidues such as straw can be increased using a suitable strain/spawn for optimal production.

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Pneumocystis jirovecii, a fungus causing severe Pneumocystis pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with P. jirovecii and a small number of experiments involving animal-derived Pneumocystis species have been published to date. However, P. jirovecii culture conditions may differ significantly from those of animal-derived Pneumocystis, as there are major genotypic and phenotypic differences between them. Establishing a well-performing P. jirovecii cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for Pneumocystis jirovecii. To identify promising approaches for cultivation, a literature survey encompassing animal-derived Pneumocystis cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for P. jirovecii culture. This allowed us to develop a medium that produced a 42.6-fold increase in P. jirovecii qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing Pneumocystis clusters in the final medium, DMEM-O3. P. jirovecii doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated P. jirovecii under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.

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Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.

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Due to high growth rate, outstanding abiotic stress tolerance, and rich value-added substances, Chrysotila roscoffensis, belonging to the phylum of Haptophyta, can be considered as a versatile resource for industrial exploitation of bioactive compounds. However, the application potential of C. roscoffensis has drawn attention until just recently, and the understanding related to the biological properties of this species is still scarce. For example, the sensitivities of C. roscoffensis to antibiotics, which is essential for the verification of heterotrophic capacity and the establishment of efficient genetic manipulation system is still unavailable. Aiming to provide fundamental information for future exploitation, the sensitivities of C. roscoffensis to nine types of antibiotics were tested in this study. The results demonstrated that C. roscoffensis exhibited relatively high resistances to ampicillin, kanamycin, streptomycin, gentamicin, and geneticin, while was sensitive to bleomycin, hygromycin B, paromomycin, and chloramphenicol. Using the former five types of antibiotics, a bacteria removal strategy was established tentatively. Finally, the axenicity of treated C. roscoffensis was confirmed based on a multi-strategy method including solid plate, 16S rDNA amplification, and nuclear acid staining. This report can provide valuable information for the development of optimal selection markers, which are meaningful for more extensive transgenic studies in C. roscoffensis. Moreover, our study also paves the way for the establishment of heterotrophic/mixotrophic cultivation modes of C. roscoffensis.

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Myxogastrea is a group of eukaryotic microorganisms included in Amoebozoa. Its life cycle includes two trophic stages: plasmodia and myxamoeflagellates. However, only about 102 species have their complete life cycle known in literature and only about 18 species have their plasmodial axenic culture accomplished in laboratory conditions. The research presented herein involved culturing of Physarum galbeum on the water agar medium. The events that transpired during its life cycle including spore germination, plasmodia formation, and sporocarp development were documented especially the subglobose or discoid sporotheca and the stalk formation. The spores germinated by the V-shape split method to release a single protoplasm. Yellow-green pigmented phaneroplasmodia developed into sporocarps by subhypothallic type. The present article gives details of the sporocarp development of P. galbeum and its plasmodial axenic culture on solid and liquid mediums.

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