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.

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.

Insights into the Metabolomic Capacity of Podaxis and Isolation of Podaxisterols A-D, Ergosterol Derivatives Carrying Nitrosyl Cyanide-Derived Modifications.

Cultures of a termite-associated and a free-living member of the fungal genus Podaxis, revived from spores maintained in century-old herbarium collections, were analyzed for their insecticidal and antimicrobial effects. Their secondary metabolomes were explored to uncover possible adaptive mechanisms of termite association, and dereplication of LC-HRMS/MS data sets led to the isolation of podaxisterols A-D (1-4), modified ergosterol derivatives that result from a Diels-Alder reaction with endogenous nitrosyl cyanide. Chemical structures were determined based on HRMS/MS and NMR analyses as well as X-ray crystallography. The putative origin of the endogenous fungal nitrosyl cyanide and ergosterol derivatives is discussed based on results obtained from stable isotope experiments and in silico analysis. Our "omics"-driven analysis of this underexplored yet worldwide distributed fungal genus builds a foundation for studies on a potential metabolic adaptations to diverse lifestyles.

Termite-engineered microbial communities of termite nest structures: a new dimension to the extended phenotype.

Termites are a prototypical example of the 'extended phenotype' given their ability to shape their environments by constructing complex nesting structures and cultivating fungus gardens. Such engineered structures provide termites with stable, protected habitats, and nutritious food sources, respectively. Recent studies have suggested that these termite-engineered structures harbour Actinobacteria-dominated microbial communities. In this review, we describe the composition, activities, and consequences of microbial communities associated with termite mounds, other nests, and fungus gardens. Culture-dependent and culture-independent studies indicate that these structures each harbour specialized microbial communities distinct from those in termite guts and surrounding soils. Termites select microbial communities in these structures through various means: opportunistic recruitment from surrounding soils; controlling physicochemical properties of nesting structures; excreting hydrogen, methane, and other gases as bacterial energy sources; and pretreating lignocellulose to facilitate fungal cultivation in gardens. These engineered communities potentially benefit termites by producing antimicrobial compounds, facilitating lignocellulose digestion, and enhancing energetic efficiency of the termite 'metaorganism'. Moreover, mound-associated communities have been shown to be globally significant in controlling emissions of methane and enhancing agricultural fertility. Altogether, these considerations suggest that the microbiomes selected by some animals extend much beyond their bodies, providing a new dimension to the 'extended phenotype'.

GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.

Trichoderma-Derived Pentapeptides from the Infected Nest Mycobiome of the Subterranean Termite Coptotermes testaceus.

Termites live in a dynamic environment where colony health is strongly influenced by surrounding microbes. However, little is known about the mycobiomes of lower termites and their nests, and how these change in response to disease. Here we compared the individual and nest mycobiomes of a healthy subterranean termite colony (Coptotermes testaceus) to one infected and ultimately eradicated by a fungal pathogen. We identified Trichoderma species in the materials of both nests, but they were also abundant in the infected termites. Methanolic extracts of Trichoderma sp. FHG000531, isolated from the infected nest, were screened for secondary metabolites by UHPLC-HR MS/MS-guided molecular networking. We identified many bioactive compounds with potential roles in the eradication of the infected colony, as well as a cluster of six unknown peptides. The novel peptide FE011 was isolated and characterized by NMR spectroscopy. The function of this novel peptide family as well as the role of Trichoderma species in dying termite colonies therefore requires further investigation.

Exploring the region-wise diversity and functions of symbiotic bacteria in the gut system of wood-feeding termite, Coptotermes formosanus, toward the degradation of cellulose, hemicellulose, and organic dyes.

The wood-feeding termite Coptotermes formosanus represents a unique and impressive system for lignocellulose degradation. The highly efficient digestion of lignocellulose is achieved through symbiosis with gut symbionts like bacteria. Despite extensive research during the last three decades, diversity of bacterial symbionts residing in individual gut regions of the termite and their associated functions is still lacking. To this end, cellulose, xylan, and dye-decolorization bacteria residing in foregut, midgut, and hindgut regions of C. formosanus were enlisted by using enrichment and culture-dependent molecular methods. A total of 87 bacterial strains were successfully isolated from different gut regions of C. formosanus which belonged to 27 different species of 10 genera, majorly affiliated with Proteobacteria (80%) and Firmicutes (18.3%). Among the gut regions, 37.9% of the total bacterial isolates were observed in the hindgut that demonstrated predominance of cellulolytic bacteria (47.6%). The majority of the xylanolytic and dye-decolorization bacteria (50%) were obtained from the foregut and midgut, respectively. Actinobacteria represented by Dietza sp. was observed in the hindgut only. Based on species richness, the highest diversity was observed in midgut and hindgut regions each of which harbored seven unique bacterial species. The members of Enterobacter, Klebsiella, and Pseudomonas were common among the gut regions. The lignocellulolytic activities of the selected potential bacteria signpost their assistance to the host for lignocellulose digestion. The overall results indicate that C. formosanus harbors diverse communities of lignocellulolytic bacteria in different regions of the gut system. These observations will significantly advance our understanding of the termite-bacteria symbiosis and their microbial ecology uniquely existed in different gut regions of C. formosanus, which may further shed a light on its potential values at termite-modeled biotechnology.

Antiparasitic Ovalicin Derivatives from Pseudallescheria boydii, a Mutualistic Fungus of French Guiana Termites.

Social insects are in mutualism with microorganisms, contributing to their resistance against infectious diseases. The fungus Pseudallescheria boydii SNB-CN85 isolated from termites produces ovalicin derivatives resulting from the esterification of the less hindered site of the ovalicin epoxide by long-chain fatty acids. Their structures were elucidated using spectroscopic analysis and semisynthesis from ovalicin. For ovalicin, these compounds displayed antiprotozoal activities against Plasmodium falciparum and Trypanosoma brucei, with IC50 values of 19.8 and 1.1 µM, respectively, for the most active compound, i.e., ovalicin linoleate. In parallel, metabolomic profiling of a collection of P. boydii strains associated with termites made it possible to highlight this class of compounds together with tyroscherin derivatives in all strains. Finally, the complete genome of P. boydii strains was obtained by sequencing, and the cluster of potential ovalicin and ovalicin biosynthesis genes was annotated. Through these metabolomic and genomic analyses, a new ovalicin derivative named boyden C, in which the 6-membered ring of ovalicin was opened by oxidative cleavage, was isolated and structurally characterized.

Simultaneous Single-Cell Genome and Transcriptome Sequencing of Termite Hindgut Protists Reveals Metabolic and Evolutionary Traits of Their Endosymbionts.

Some of the protist species which colonize the hindguts of wood-feeding Reticulitermes termites are associated with endosymbiotic bacteria belonging to the genus Endomicrobium. In this study, we focused on the endosymbionts of three protist species from Reticulitermes flavipes, as follows: Pyrsonympha vertens, Trichonympha agilis, and Dinenympha species II. Since these protist hosts represented members of different taxa which colonize separate niches within the hindguts of their termite hosts, we investigated if these differences translated to differential gene content and expression in their endosymbionts. Following assembly and comparative genome and transcriptome analyses, we discovered that these endosymbionts differed with respect to some possible niche-specific traits, such as carbon metabolism. Our analyses suggest that species-specific genes related to carbon metabolism were acquired by horizontal gene transfer (HGT) and may have come from taxa which are common in the termite hind gut. In addition, our analyses suggested that these endosymbionts contain and express genes related to natural transformation (competence) and recombination. Taken together, the presence of genes acquired by HGT and a putative competence pathway suggest that these endosymbionts are not cut off from gene flow and that competence may be a mechanism by which members of Endomicrobium can acquire new traits. IMPORTANCE The composition and structure of wood, which contains cellulose, hemicellulose, and lignin, prevent most organisms from using this common food source. Termites are a rare exception among animals, and they rely on a complex microbiota housed in their hindguts to use wood as a source of food. The lower termite, Reticulitermes flavipes, houses a variety of protists and prokaryotes that are the key players in the disassembly of lignocellulose. Here, we describe the genomes and the gene expression profiles of five Endomicrobium endosymbionts living inside three different protist species from R. flavipes. Data from these genomes suggest that these Endomicrobium species have different mechanisms for using carbon. In addition, they harbor genes that may be used to import DNA from their environment. This process of DNA uptake may contribute to the high levels of horizontal gene transfer noted previously in Endomicrobium species.