Genome reduction and horizontal gene transfer in the evolution of Endomicrobia-rise and fall of an intracellular symbiosis with termite gut flagellates.

Bacterial endosymbionts of eukaryotic hosts typically experience massive genome reduction, but the underlying evolutionary processes are often obscured by the lack of free-living relatives. Endomicrobia, a family-level lineage of host-associated bacteria in the phylum Elusimicrobiota that comprises both free-living representatives and endosymbionts of termite gut flagellates, are an excellent model to study evolution of intracellular symbionts. We reconstructed 67 metagenome-assembled genomes (MAGs) of Endomicrobiaceae among more than 1,700 MAGs from the gut microbiota of a wide range of termites. Phylogenomic analysis confirmed a sister position of representatives from termites and ruminants, and allowed to propose eight new genera in the radiation of Endomicrobiaceae. Comparative genome analysis documented progressive genome erosion in the new genus Endomicrobiellum, which comprises all flagellate endosymbionts characterized to date. Massive gene losses were accompanied by the acquisition of new functions by horizontal gene transfer, which led to a shift from a glucose-based energy metabolism to one based on sugar phosphates. The breakdown of glycolysis and many anabolic pathways for amino acids and cofactors in several subgroups was compensated by the independent acquisition of new uptake systems, including an ATP/ADP antiporter, from other gut microbiota. The putative donors are mostly flagellate endosymbionts from other bacterial phyla, including several, hitherto unknown lineages of uncultured Alphaproteobacteria, documenting the importance of horizontal gene transfer in the convergent evolution of these intracellular symbioses. The loss of almost all biosynthetic capacities in some lineages of Endomicrobiellum suggests that their originally mutualistic relationship with flagellates is on its decline.IMPORTANCEUnicellular eukaryotes are frequently colonized by bacterial and archaeal symbionts. A prominent example are the cellulolytic gut flagellates of termites, which harbor diverse but host-specific bacterial symbionts that occur exclusively in termite guts. One of these lineages, the so-called Endomicrobia, comprises both free-living and endosymbiotic representatives, which offers the unique opportunity to study the evolutionary processes underpinning the transition from a free-living to an intracellular lifestyle. Our results revealed a progressive gene loss in energy metabolism and biosynthetic pathways, compensated by the acquisition of new functions via horizontal gene transfer from other gut bacteria, and suggest the eventual breakdown of an initially mutualistic symbiosis. Evidence for convergent evolution of unrelated endosymbionts reflects adaptations to the intracellular environment of termite gut flagellates.

Seasonal shifts in gut microbiota and cold tolerance metrics in a northern population of Reticulitermes flavipes (Blattodea: Rhinotermitidae).

Eastern subterranean termites, Reticulitermes flavipes (Kollar), are widely distributed across North America where they are exposed to a broad range of environmental conditions. However, mechanisms for overwintering are not well understood. Wisconsin is a unique location to study mechanisms of cold tolerance as it represents the northern boundary for persistent R. flavipes populations. In this study, we evaluated seasonal shifts in cold tolerance using critical thermal minimum (CTmin) and supercooling point (SCP) and examined how these measurements correlate to changes in the microbial community of the termite gut. Results showed seasonal acclimatization to cold, which is consistent with the use of behavioral freeze-avoidant mechanisms. However, these insects also demonstrated an increased susceptibility to freezing later in the season, which may be tied to changes in gut microbiota. Our results found shifts in the composition of the gut microbiome in R. flavipes between mid- to late summer and early to late fall. These differences may be suggestive of a change in metabolism to adjust to a period of reduced feeding and increased metabolic stress during overwintering. Specifically, results showed an increased abundance of Methanobrevibacter sp. (Euryarchaeota) associated with cold, which may be indicative of a metabolic shift from acetogenesis to methanogenesis associated with overwintering. Further work is needed focusing on specific contributions of certain gut microbes, particularly their role in metabolic adaptability and in providing protection from oxidative stress associated with changes in environmental conditions.

Pullenvalenes A-D: Nitric Oxide-Mediated Transcriptional Activation (NOMETA) Enables Discovery of Triterpene Aminoglycosides from Australian Termite Nest-Derived Fungi.

We report on the use of nitric oxide-mediated transcriptional activation (NOMETA) as an innovative means to detect and access new classes of microbial natural products encoded within silent biosynthetic gene clusters. A small library of termite nest- and mangrove-derived fungi and actinomyces was subjected to cultivation profiling using a miniaturized 24-well format approach (MATRIX) in the presence and absence of nitric oxide, with the resulting metabolomes subjected to comparative chemical analysis using UPLC-DAD and GNPS molecular networking. This strategy prompted study of Talaromyces sp. CMB-TN6F and Coccidiodes sp. CMB-TN39F, leading to discovery of the triterpene glycoside pullenvalenes A-D (1-4), featuring an unprecedented triterpene carbon skeleton and rare 6-O-methyl-N-acetyl-d-glucosaminyl glycoside residues. Structure elucidation of 1-4 was achieved by a combination of detailed spectroscopic analysis, chemical degradation, derivatization and synthesis, and biosynthetic considerations.

Seasonal impacts on gut microbial composition of the Eastern subterranean termite (Blattodea: Rhinotermitidae).

Termite hindguts are inhabited by symbionts that help with numerous processes, but changes in the gut microbiome due to season can potentially impact the physiology of termites. This study investigated the impact of seasonal changes on the composition of bacteria and protozoa in the termite gut. Termites were obtained monthly from May to October 2020 at a location in the central United States that typically experiences seasonal air temperatures ranging from < 0 to > 30 °C. The guts of 10 termites per biological replication were dissected and frozen within 1 day after collections. DNA was extracted from the frozen gut tissues and used for termite 16S rRNA mitochondrial gene analysis and bacterial 16S rRNA gene sequence surveys. Phylogenetic analysis of termite 16S rRNA gene sequences verified that the same colony was sampled across all time points. On processing bacterial 16S sequences, we observed alpha (observed features, Pielou's evenness, and Shannon diversity) and beta diversity (unweighted Unifrac, Bray-Curtis, and Jaccard) metrics to vary significantly across months. Based on the analysis of the composition of microbiomes with bias correction (ANCOM-BC) at the genus level, we found several significant bacterial taxa over collection months. In addition, Spearman correlation analysis demonstrated that 41 bacterial taxa were significantly correlated (positively and negatively) with average soil temperature. These results from a single termite colony suggest termite microbial communities go through seasonal changes in relative abundance related to temperature, although other seasonal effects cannot be excluded. Further investigations are required to conclusively define the consistency of microbial variation among different colonies with season.

Characterization and in-vitro plant-based control of hindgut bacteria isolated from Odontotermes obesus Rambur (Termitidae) and Heterotermes indicola Wasmann (Rhinotermitidae).

Termites cause a serious menace to wooden structures all over the world. They rely mostly on entozoic fauna residing in their hindgut for the digestion of cellulosic and hemicellulosic materials. One of the ways to control termites is through their gut symbionts. The present study was designed to characterize the hindgut bacteria isolated from Odontotermes obesus and Heterotermes indicola. Furthermore, the growth inhibitory effect of eight tropical plant extracts was investigated to find out potential control agents for these bacterial isolates. The characterization of bacteria was carried out based on their morphology, Gram staining, biochemical and amplification of 16SrRNA gene. Amplified products were sequenced to confirm their relationship with bacterial isolates from termites of other regions. The growth inhibitory effect of ethanolic leaf extracts of eight plants was evaluated in an invitro agar well diffusion method. Qualitative and quantitative phytochemical analysis of the most effective plant was carried out to learn about bioactive agents. The results confirmed the presence of five bacteria from each termite species. The Bacillus cereus, Escherichia coli, and Lysinibacillus fusiformis were common to both termites whereas Lysinibacillus xylanilyticus and Lysinibacillus macrolides were found in O. obesus only and H. indicola harbor Bacillus subtilis and Shigella sonnei in addition to common three ones. Among the plant extracts of Carica papaya, Eucalyptus camaldulensis, Osmium basilicum, Grevillea robusta, Eucalyptus globulus, Pongamia pinnata, Mentha longifolia, and Melia azedarach, the G. robusta > E. camaldulensis > O. basilicum were found to have growth inhibitory effects with increasing concentrations from 100 to 2000 µg/mL. The biodiversity of the bacterial fauna is important for the biological control of termites. Leaf extracts of these medicinal plants can be used to control termite infestation in an environment-friendly manner to save huge economic loss.

Actinomycetospora termitidis sp. nov., an insect-derived actinomycete isolated from termite (Odontotermes formosanus).

Strain Odt1-22T, an insect-derived actinomycete was isolated from a termite (Odontotermes formosanus) that was collected from Chanthaburi province, Thailand. Strain Odt1-22T was aerobic, Gram-stain-positive, and produced bud-like spore chain on the substrate hypha. According to chemotaxonomic analysis, strain Odt1-22T contained meso-diaminopimelic acid in peptidoglycan and the whole-cell hydrolysates contained arabinose, galactose, glucose, and ribose. The major menaquinone was MK-8(H4). The diagnostic phospholipids were diphosphatidylglycerol, hydroxyphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. Phylogenetic analysis based on 16 S rRNA gene sequence revealed that strain Odt1-22T was identified to the genus Actinomycetospora and showed high similarity values with A. chiangmaiensis DSM 45062 T (99.24%), A. soli SF1T (99.24%) and A. corticicola 014-5 T (98.17%). The genomic size of strain Odt1-22T was 6.6 Mbp with 73.8% G + C content and 6355 coding sequences (CDSs). The genomic analysis, strain Odt1-22T and closely related species A. chiangmaiensis DSM 45062 T, A. soli SF1T and A. corticicola DSM 45772 T displayed the values of average nucleotide identity-blast (ANIb) at 83.7-84.1% and MUMmer (ANIm) at 86.6-87.0%. Moreover, the results of digital DNA-DNA hybridization values between strain Odt1-22T and related Actinomycetospora species were 45.8-50.5% that lower than the threshold value of commonly used to delineate separated species level. On the basis of phenotypic, chemotaxonomic, and genotypic data, strain Odt1-22T represented a novel species within the genus Actinomycetospora, for which the name Actinomycetospora termitidis sp. nov. is proposed. The type strain of the species is Odt1-22T (= TBRC 16192 T = NBRC 115965 T).

Involvement of a catalase gene in lignin catalysis and immune defense against pathogenic fungus in Coptotermes formosanus: a potential new target for termite control.

BACKGROUND: Detoxifying enzymes are likely involved in lignin feeding and immune defense mechanisms within termites, rendering them potential targets for biological control. However, investigations into the dual functionality of termite detoxification enzymes in vivo have not been documented. RESULTS: In this study, the complete cDNA of the catalase gene (Cfcat) derived from Coptotermes formosanus Shiraki was amplified. CFCAT comprises an open reading frame spanning 1527 bp, encoding a 508-amino acid sequence. The highest expression was observed in the epidermal tissues (including the fat body and hemolymph) followed by the foregut/salivary gland. Furthermore, we confirmed the catalase activity of the recombinant Cfcat protein. Using RNA interference (RNAi) technology, the importance of Cfcat in the lignin-feeding of C. formosanus was demonstrated, and the role of Cfcat in innate immunity was investigated. Survival assays showed that Cfcat RNAi significantly increased the susceptibility of C. formosanus to Metarhizium anisopliae. Irrespective of the infection status, Cfcat inhibition had a significant impact on multiple factors of humoral and intestinal immunity in C. formosanus. Notably, Cfcat RNAi exhibited a more pronounced immunosuppressive effect on humoral immunity than on intestinal immunity. CONCLUSION: Cfcat plays an important role in the regulation of innate immunity and lignin feeding in C. formosanus. Cfcat RNAi can weaken the immune response of termites against M. anisopliae, which may aid the biocontrol efficiency of M. anisopliae against C. formosanus. This study provides a theoretical basis and technical reference for the development of a novel biocontrol strategy targeting detoxifying enzymes of termites. © 2024 Society of Chemical Industry.

Termitidicoccus mucosus gen. nov. sp. nov. a novel Verrucomicrobiota species isolated from Reticulitermes chinensis gives insights of high adaptability of symbiotic bacteria to termite gut ecosystem.

Verrucomicrobiota is widely distributed in various habitats including insect guts. It was found to be prevalent in almost all investigated termite guts, whereas their physiological functions are not very clear. In this study we characterized the physiological and genomic properties of Verrucomicrobiota strain TSB47T isolated from Reticulitermes chinensis. The cells of strain TSB47T were Gram-stain-negative, non-motile, and non-spore-forming coccoid with one or more warts. 16S rRNA gene analysis showed that the closest relatives of strain TSB47T were Opitutaceae strain TAV1 and Ereboglobus luteus Ho45T (98.3% and 95.4% sequence similarity, respectively). Whole genome analysis revealed that there are a large number of glycoside hydrolase genes, amino acid metabolism genes, complete Mo-Fe nitrogenase and Fe-Fe nitrogenase gene clusters, as well as cbb3-type cytochrome oxidase gene in the genome of strain TSB47T. Strain TSB47T grows well under anaerobic and microaerophilic conditions with a strong tolerance to oxygen. Physiological and genomic characters of strain TSB47T indicated its high adaptability to termite gut ecosystem. Based on phenotypic and phylogenetic evidence, we suggest strain TSB47T as the type species of a novel genus in the family Opitutaceae, for which the name Termitidicoccus mucosus sp. nov. is proposed. The type strain is TSB47T (CCTCC AB2022447T; KCTC 102044T).

Taxonomic profiling of Nasutitermes takasagoensis microbiota to investigate the role of termites as vectors of bacteria linked to ironwood tree decline in Guam.

The ironwood tree (Casuarina equisetifolia, family Casuarinaceae), an indigenous agroforestry species in Guam, has been threatened by ironwood tree decline (IWTD) since 2002. Formation of bacterial ooze by the wilt pathogen from the Ralstonia solanacearum species complex and wetwood bacteria (primarily Klebsiella species) has been linked to IWTD. In addition, termite infestation of trees was statistically associated with IWTD. Termites are known carriers of a diverse microbiome. Therefore, we hypothesized that termites could be vectors of bacteria linked to IWTD. To investigate the potential role of termites as pathogen vectors, we employed next-generation 16S rRNA gene sequencing to describe the bacteria diversity of Nasutitermes takasagoensis (Family Termitidae) workers collected from 42 ironwood trees of different disease stages in Guam in association with tree-, plot-, and location-related factors. Nasutitermes takasagoensis workers account for the majority of termite infestations of ironwood trees. The bacterial phyla composition of N. takasagoensis workers was typical for wood-feeding higher termites consisting mainly of Spirochaetes and Fibrobacteres. However, Ralstonia species were not detected and Klebsiella species were rare even in termites collected from trees infected with Ralstonia and wetwood bacteria. Feeding experiments suggested that termites prefer to consume wood with low pathogen content over wood with high pathogen load. Termites were able to ingest Ralstonia but Ralstonia could not establish itself in healthy termite bodies. We concluded that N. takasagoensis workers are not vectors for Ralstonia spp. or the bacterial endophytes associated with wetwood (Klebsiella, Pantoea, Enterobacter, Citrobacter, and Erwinia) that were previously observed in IWTD-infested trees. The bacterial diversity in termite samples was significantly influenced by various factors, including Tree Health, Site Management, Plot Average Decline Severity, Proportion of Dead Trees in the Plot, Proportion of Trees with Termite Damage in the Plot, Presence of Ralstonia, and Altitude.

Emergence of putative energy parasites within Clostridia revealed by genome analysis of a novel endosymbiotic clade.

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0-1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.

Identification of Obscurolide-Type Metabolites and Antifungal Metabolites from the Termite-Associated Streptomyces neopeptinius BYF101.

Streptomyces spp. are well-known symbiotic microorganisms that produce antimicrobial metabolites against various pathogens. We isolated actinomycetes from the body surface of the termite Odontotermes formosanus and identified it as Streptomyces neopeptinius BYF101 based on 16S rRNA phylogenetic analysis. Chemical analysis of the cultures of termite-associated S. neopeptinius BYF101 via HR-MS2 and GNPS analyses enabled the isolation and identification of 20 metabolites, including the unreported obscurolide-type metabolites (1-3). The chemical structures of unreported compounds (1-3) were elucidated using HR-ESI-MS and 1D and 2D NMR analysis, and their absolute configurations were determined via chemical reactions followed by the application of competing enantioselective acylation (CEA) and computational methods for ECD and DP4+ probability calculation. The isolated compounds (1-20) were tested to determine their antifungal activity against two human fungal pathogens, Candida albicans and Cryptococcus neoformans. Among the compounds tested, indole-3-carboxylic acid (9) displayed antifungal activity against C. neoformans, with an MIC value of 12 µg/mL.

Genome-wide identification of Coptotermes formosanus immune genes and their potential roles in termite control.

In recent years, the use of microbes to control termites has attracted increasing attention. It was found that pathogenic bacteria, nematodes, and fungi effectively control termites under laboratory conditions. However, their effects have not been replicated in the field, and one reason for this is the complex immune defense mechanisms of termites, which are mainly regulated by immune genes. Therefore, altering the expression of immune genes may have a positive influence on the biocontrol efficacy of termites. Coptotermes formosanus Shiraki is one of the most economically important termite pests worldwide. Currently, the large-scale identification of immune genes in C. formosanus is primarily based on cDNA library or transcriptome data rather than at the genomic level. In this study, we identified the immune genes of C. formosanus according to genome-wide analysis. In addition, our transcriptome analysis showed that immune genes were significantly downregulated when C. formosanus was exposed to the fungus Metarhizium anisopliae or nematodes. Finally, we found that injecting dsRNA to inhibit three immune genes (CfPGRP-SC1, CfSCRB3, and CfHemocytin), which recognize infectious microbes, significantly increased the lethal effect of M. anisopliae on termites. These immune genes show great potential for C. formosanus management based on RNAi. These results also increase the number of known immune genes in C. formosanus which will provide a more comprehensive insight into the molecular basis of immunity in termites.

Impact of Wood Age on Termite Microbial Assemblages.

The decomposition of wood and detritus is challenging to most macroscopic organisms due to the recalcitrant nature of lignocellulose. Moreover, woody plants often protect themselves by synthesizing toxic or nocent compounds which infuse their tissues. Termites are essential wood decomposers in warmer terrestrial ecosystems and, as such, they have to cope with high concentrations of plant toxins in wood. In this paper, we evaluated the influence of wood age on the gut microbial (bacterial and fungal) communities associated with the termites Reticulitermes flavipes (Rhinotermitidae) (Kollar, 1837) and Microcerotermes biroi (Termitidae) (Desneux, 1905). We confirmed that the secondary metabolite concentration decreased with wood age. We identified a core microbial consortium maintained in the gut of R. flavipes and M. biroi and found that its diversity and composition were not altered by the wood age. Therefore, the concentration of secondary metabolites had no effect on the termite gut microbiome. We also found that both termite feeding activities and wood age affect the wood microbiome. Whether the increasing relative abundance of microbes with termite activities is beneficial to the termites is unknown and remains to be investigated. IMPORTANCE Termites can feed on wood thanks to their association with their gut microbes. However, the current understanding of termites as holobiont is limited. To our knowledge, no studies comprehensively reveal the influence of wood age on the termite-associated microbial assemblage. The wood of many tree species contains high concentrations of plant toxins that can vary with their age and may influence microbes. Here, we studied the impact of Norway spruce wood of varying ages and terpene concentrations on the microbial communities associated with the termites Reticulitermes flavipes (Rhinotermitidae) and Microcerotermes biroi (Termitidae). We performed a bacterial 16S rRNA and fungal ITS2 metabarcoding study to reveal the microbial communities associated with R. flavipes and M. biroi and their impact on shaping the wood microbiome. We noted that a stable core microbiome in the termites was unaltered by the feeding substrate, while termite activities influenced the wood microbiome, suggesting that plant secondary metabolites have negligible effects on the termite gut microbiome. Hence, our study shed new insights into the termite-associated microbial assemblage under the influence of varying amounts of terpene content in wood and provides a groundwork for future investigations for developing symbiont-mediated termite control measures.

A genetic linkage map and improved genome assembly of the termite symbiont Termitomyces cryptogamus.

BACKGROUND: The termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect. METHODS: To explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle. RESULTS: Our linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi. CONCLUSIONS: The apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate.

Genome-scale community modeling for deciphering the inter-microbial metabolic interactions in fungus-farming termite gut microbiome.

Specialized microbial communities in the fungus-farming termite gut and fungal comb microbiome help maintain host nutrition through interactive biochemical activities of complex carbohydrate degradation. Numerous research studies have been focused on identifying the microbial species in the termite gut and fungal comb microbiota, but the community-wide metabolic interaction patterns remain obscure. The inter-microbial metabolic interactions in the community environment are essential for executing biochemical processes like complex carbohydrate degradation and maintaining the host's physicochemical homeostasis. Recent progress in high-throughput sequencing techniques and mathematical modeling provides suitable platforms for constructing multispecies genome-scale community metabolic models that can render sound knowledge about microbial metabolic interaction patterns. Here, we have implemented the genome-scale metabolic modeling strategy to map the relationship between genes, proteins, and reactions of 12 key bacterial species from fungal cultivating termite gut and fungal comb microbiota. The resulting individual genome-scale metabolic models (GEMs) have been analyzed using flux balance analysis (FBA) to optimize the metabolic flux distribution pattern. Further, these individual GEMs have been integrated into genome-scale community metabolic models where a heuristics-based computational procedure has been employed to track the inter-microbial metabolic interactions. Two separate genome-scale community metabolic models were reconstructed for the O. badius gut and fungal comb microbiome. Analysis of the community models showed up to ∼167% increased flux range in lignocellulose degradation, amino acid biosynthesis, and nucleotide metabolism pathways. The inter-microbial metabolic exchange of amino acids, SCFAs, and small sugars was also upregulated in the multispecies community for maximum biomass formation. The flux variability analysis (FVA) has also been performed to calculate the feasible flux range of metabolic reactions. Furthermore, based on the calculated metabolic flux values, newly defined parameters, i.e., pairwise metabolic assistance (PMA) and community metabolic assistance (CMA) showed that the microbial species are getting up to 15% higher metabolic benefits in the multispecies community compared to pairwise growth. Assessment of the inter-microbial metabolic interaction patterns through pairwise growth support index (PGSI) indicated an increased mutualistic interaction in the termite gut environment compared to the fungal comb. Thus, this genome-scale community modeling study provides a systematic methodology to understand the inter-microbial interaction patterns with several newly defined parameters like PMA, CMA, and PGSI. The microbial metabolic assistance and interaction patterns derived from this computational approach will enhance the understanding of combinatorial microbial activities and may help develop effective synergistic microcosms to utilize complex plant polymers.

Adaptations of Pseudoxylaria towards a comb-associated lifestyle in fungus-farming termite colonies.

Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria (Xylariaceae), named Pseudoxylaria, whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended ("sit and wait" strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces, and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity.

OfGNBP silencing enhances the toxicity of Serratia marcescens Bizio (SM1) to Odontotermes formosanus (Shiraki).

The immunity of insects plays a vital role in their survival. Our experiments found that lipopolysaccharide (LPS) and glucono-δ-lactone (GDL) could influence the virulence of Serratia marcescens Bizio (SM1) to Odontotermes formosanus (Shiraki) by affecting the immunity. Gram-negative binding proteins (GNBPs) are an important pattern recognition proteins that play a crucial role in the innate immune system. Therefore, two OfGNBPs were cloned in O. formosanus. The expression of OfGNBPs was significantly changed by LPS,SM1 and GDL, not prick. In addition, the immune-related gene expression, the phenoloxidase activity and antibacterial activity of donor termites and recipient termites were significantly induced by SM1. Furthermore, the knockdown of OfGNBP by RNA interference reduced not only individual immunity but also social immunity in O. formosanus, which increased the virulence of SM1 to O. formosanus. Importantly, dsOfGNBP alone also had good control effect on O. formosanus. In summary, we concluded that dsOfGNBPs are important termite immunosuppressants.

Tripartite Symbiotic Digestion of Lignocellulose in the Digestive System of a Fungus-Growing Termite.

Fungus-growing termites are efficient in degrading and digesting plant substrates, achieved through the engagement of symbiotic gut microbiota and lignocellulolytic Termitomyces fungi cultivated for protein-rich food. Insights into where specific plant biomass components are targeted during the decomposition process are sparse. In this study, we performed several analytical approaches on the fate of plant biomass components and did amplicon sequencing of the 16S rRNA gene to investigate the lignocellulose digestion in the symbiotic system of the fungus-growing termite Odontotermes formosanus (Shiraki) and to compare bacterial communities across the different stages in the degradation process. We observed a gradual reduction of lignocellulose components throughout the process. Our findings support that the digestive tract of young workers initiates the degradation of lignocellulose but leaves most of the lignin, hemicellulose, and cellulose, which enters the fresh fungus comb, where decomposition primarily occurs. We found a high diversity and quantity of monomeric sugars in older parts of the fungus comb, indicating that the decomposition of lignocellulose enriches the old comb with sugars that can be utilized by Termitomyces and termite workers. Amplicon sequencing of the 16S rRNA gene showed clear differences in community composition associated with the different stages of plant biomass decomposition which could work synergistically with Termitomyces to shape the digestion process. IMPORTANCE Fungus-farming termites have a mutualist association with fungi of the genus Termitomyces and gut microbiota to support the nearly complete decomposition of lignocellulose to gain access to nutrients. This elaborate strategy of plant biomass digestion makes them ecologically successful dominant decomposers in (sub)tropical Old World ecosystems. We employed acid detergent fiber analysis, high-performance anion-exchange chromatography (HPAEC), high-performance liquid chromatography (HPLC), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), pyrolysis gas chromatography-mass spectrometry (Py-GC-MS), and amplicon sequencing of the 16S rRNA gene to examine which lignocellulose components were digested and which bacteria were abundant throughout the decomposition process. Our findings suggest that although the first gut passage initiates lignocellulose digestion, the most prominent decomposition occurs within the fungus comb. Moreover, distinct bacterial communities were associated with different stages of decomposition, potentially contributing to the breakdown of particular plant components.

First Neotropical record of the association between brown sclerotium-forming fungi and termite eggs in a nest of Coptotermes gestroi (Blattaria, Isoptera, Rhinotermitidae).

Insects and fungi are abundant in many environments, in which facultative and/or obligate associations involving these groups have been established during evolution. In termites, mutualism with fungi is well reported for some termite lineages (e.g., Macrotermitinae). Within some subterranean termite species (Rhinotermitidae), egg-mimicking fungi, also referred to as "termite "balls", are often harbored inside the nest, mixed to the egg piles. Such interaction seems to be advantageous for both partners since the fungi are protected inside the nest while they may serve as an additional food source and also provide cellulases which may be incorporated into the termite digestive process. Although such mutualism has been reported for seven species of Reticulitermes and Coptotermes formosanus, all the samplings were restricted to temperate regions. Here, we provide the first Neotropical record of this termite-fungus association, and the first report for Coptotermes gestroi. The morphological characters of the "termite balls" observed in a C. gestroi nest resemble those already reported for Reticulitermes spp. and the congeneric species C. formosanus. They include a color ranging from light to dark brown, spherical shape, and a reduced diameter (0.23-0.34 mm). Our findings provide new insights into the geographical distribution of the association between termites and sclerotium-forming fungi. Future genetic analyses will be valuable aiming to identify the egg-mimicking fungi associated with C. gestroi and shed light on the evolution of this fascinating symbiosis.

Bacterial species metabolic interaction network for deciphering the lignocellulolytic system in fungal cultivating termite gut microbiota.

Fungus-cultivating termite Odontotermes badius developed a mutualistic association with Termitomyces fungi for the plant material decomposition and providing a food source for the host survival. The mutualistic relationship sifted the microbiome composition of the termite gut and Termitomyces fungal comb. Symbiotic bacterial communities in the O. badius gut and fungal comb have been studied extensively to identify abundant bacteria and their lignocellulose degradation capabilities. Despite several metagenomic studies, the species-wide metabolic interaction patterns of bacterial communities in termite gut and fungal comb remains unclear. The bacterial species metabolic interaction network (BSMIN) has been constructed with 230 bacteria identified from the O. badius gut and fungal comb microbiota. The network portrayed the metabolic map of the entire microbiota and highlighted several inter-species biochemical interactions like cross-feeding, metabolic interdependency, and competition. Further, the reconstruction and analysis of the bacterial influence network (BIN) quantified the positive and negative pairwise influences in the termite gut and fungal comb microbial communities. Several key macromolecule degraders and fermentative microbial entities have been identified by analyzing the BIN. The mechanistic interplay between these influential microbial groups and the crucial glycoside hydrolases (GH) enzymes produced by the macromolecule degraders execute the community-wide functionality of lignocellulose degradation and subsequent fermentation. The metabolic interaction pattern between the nine influential microbial species has been determined by considering them growing in a synthetic microbial community. Competition (30%), parasitism (47%), and mutualism (17%) were predicted to be the major mode of metabolic interaction in this synthetic microbial community. Further, the antagonistic metabolic effect was found to be very high in the metabolic-deprived condition, which may disrupt the community functionality. Thus, metabolic interactions of the crucial bacterial species and their GH enzyme cocktail identified from the O. badius gut and fungal comb microbiota may provide essential knowledge for developing a synthetic microcosm with efficient lignocellulolytic machinery.