Termite Gut Research Articles

Metagenomics analysis of bacterial community structure from wood- and soil-feeding termites: metabolic pathways and functional structures toward the degradation of lignocellulose and recalcitrant compounds

Some essential information on gut bacterial profiles and their unique contributions to food digestion in wood-feeding termites (WFT) and soil-feeding termites (SFT) is still inadequate. The feeding type of termites is hypothesized to influence their gut bacterial composition and its functionality in degrading lignocellulose or other organic chemicals. This could potentially provide alternative approaches for the degradation of some recalcitrant environmental chemicals. Therefore, metagenomic analysis can be employed to examine the composition and functional profiles of gut bacterial symbionts in WFT and SFT. Based on the metagenomic analysis of the 16S rRNA gene sequences of gut bacterial symbionts in the WFT, Microcerotermes sp., and the SFT, Pericapritermes nitobei, the findings revealed a total of 26 major bacterial phyla, with 18 phyla commonly represented in both termites, albeit in varying abundances. Spirochaetes dominated the bacterial symbionts in Microcerotermes sp. at 55%, followed by Fibrobacters, while Firmicutes dominated the gut bacteria symbionts in P. nitobei at 95%, with Actinobacteria coming in second at 2%. Furthermore, the Shannon and phylogenetic tree diversity indices, as well as the observed operational taxonomic units and Chao 1 richness indices, were all found to be higher in the WFT than in the SFT deduced from the alpha diversity analysis. Based on the principal coordinate analysis, exhibited a significant distance dissimilarity between the gut bacterial symbionts. The results showed that the gut bacterial composition differed significantly between the WFT and SFT. Furthermore, Tax4Fun analysis evaluated bacterial functions, revealing the predominance of carbohydrate metabolism, followed by amino acid metabolism and energy metabolism in both Microcerotermes sp. and P. nitobei termites. The results implicated that bacterial symbionts inhabiting the guts of both termites were actively involved in the degradation of lignocellulose and other recalcitrant compounds.

Comparison of microbial diversity and carbohydrate-active enzymes in the hindgut of two wood-feeding termites, Globitermes sulphureus (Blattaria: Termitidae) and Coptotermes formosanus (Blattaria: Rhinotermitidae).

Wood-feeding termites have been employed as sources of novel and highly efficient lignocellulolytic enzymes due to their ability to degrade lignocellulose efficiently. As a higher wood-feeding termite, Globitermes sulphureus (Blattaria: Termitidae) plays a crucial role as a decomposer in regions such as Vietnam, Singapore, Myanmar, and Yunnan, China. However, the diversity of its gut microbiome and carbohydrate-active enzymes (CAZymes) remains unexplored. Here, we analyzed the diversity of hindgut microbial communities and CAZymes in a higher wood-feeding termite, G. sulphureus, and a lower wood-feeding termite, Coptotermes formosanus (Blattaria: Rhinotermitidae). 16S rRNA sequencing revealed that Spirochaetota, Firmicutes, and Fibrobacterota were the dominant microbiota in the hindgut of the two termite species. At the phylum level, the relative abundances of Proteobacteria and Bacteroidota were significantly greater in the hindgut of C. formosanus than in G. sulphureus. At the genus level, the relative abundances of Candidatus_Azobacteroides and Escherichia-Shigella were significantly lower in the hindgut of G. sulphureus than in C. formosanus. Metagenomic analysis revealed that glycoside hydrolases (GHs) with cellulases and hemicellulases functions were not significantly different between G. sulphureus and C. formosanus. Interestingly, the cellulases in G. sulphureus were mainly GH5_2, GH5_4, GH6, GH9, and GH45, while the hemicellulases were mainly GH11, GH8, GH10, GH11, GH26, and GH53. In C. formosanus, the cellulases were mainly GH6 and GH9, and the hemicellulases were mainly GH5_7, GH5_21, GH10, GH12, and GH53. In addition, β-glucosidase, exo-β-1,4-glucanase, and endo-β-1,4-glucanase activities did not differ significantly between the two termite species, while xylanase activity was higher in G. sulphureus than in C. formosanus. The bacteria encoding GHs in G. sulphureus were mainly Firmicutes, Fibrobacterota, and Proteobacteria, whereas Bacteroidota and Spirochaetota were the main bacteria encoding GHs in C. formosanus. Our findings characterized the microbial composition and differences in the hindgut microbiota of G. sulphureus and C. formosanus. Compared to C. formosanus, G. sulphureus is enriched in genes encoding for hemicellulase and debranching enzymes. It also highlights the rich diversity of GHs in the hindgut microbiota of G. sulphureus, including the GH5 subfamily, GH6, and GH48, with the GH6 and GH48 not previously reported in other higher termites. These results strengthen the understanding of the diversity of termite gut microbiota and CAZymes.

Whole-Genome Sequencing And Characterization Of Two Bacillus velezensis Strains from Termitarium and A Comprehensive Comparative Genomic Analysis of Biosynthetic Gene Clusters.

The speciesBacillus velezensisis known for its biosynthetic potential and metabolic versatility in producing several secondary metabolites and promoting plant growth. In this study, we isolated twoB. velezensisstrains, WGTg-8 and WGTm-299, from the termite gut and termitarium, which exhibited antimicrobial activity against multiple clinical and phytopathogens. The whole genomes of these strains were sequenced using the Illumina platform and annotated. The genome mining of the draft genome sequences revealed 48 biological gene clusters (BGCs) responsible for synthesizing various secondary metabolites. The construction of the similarity network of the BGCs and the comparative analysis with the genetically related organisms are aided in the identification of metabolites produced by these strains. We identified biosynthetic gene clusters (BGCs) coding for macrolactin H, bacilysin, bacillibactin, amylocyclin, comX4, and LCI, found in both strains with 100% similarity. The difficidin, bacillaene, thusin_alpha, and cericidin BGCs are exclusively found in strain WGTg-8, while the colicin BGC is exclusively present in the WGTm-299 strain. The fengycin and surfactin gene clusters are present in both strains with 80% similarity. Furthermore, 28 putative NRPS BGCs, NRPS-T1PKS hybrid clusters, a T1PKS, and a bacteriocin BGC were identified with very low similarity (≤ 25%) or no similarity with known antibiotics. In addition, we found several genes coding for plant growth-promoting properties, including nitrogen metabolism, hormone synthesis, sulfur metabolism, phosphate metabolism, and a few other properties.

Termites and subsocial roaches inherited many bacterial-borne carbohydrate-active enzymes (CAZymes) from their common ancestor

Termites digest wood using Carbohydrate-Active Enzymes (CAZymes) produced by gut bacteria with whom they have cospeciated at geological timescales. Whether CAZymes were encoded in the genomes of their ancestor’s gut bacteria and transmitted to modern termites or acquired more recently from bacteria not associated with termites is unclear. We used gut metagenomes from 195 termites and one Cryptocercus, the sister group of termites, to investigate the evolution of termite gut bacterial CAZymes. We found 420 termite-specific clusters in 81 bacterial CAZyme gene trees, including 404 clusters showing strong cophylogenetic patterns with termites. Of the 420 clusters, 131 included at least one bacterial CAZyme sequence associated with Cryptocercus or Mastotermes, the sister group of all other termites. Our results suggest many bacterial CAZymes have been encoded in the genomes of termite gut bacteria since termite origin, indicating termites rely upon many bacterial CAZymes endemic to their guts to digest wood.

Unveiling lignocellulolytic potential: a genomic exploration of bacterial lineages within the termite gut

BackgroundThe microbial landscape within termite guts varies across termite families. The gut microbiota of lower termites (LT) is dominated by cellulolytic flagellates that sequester wood particles in their digestive vacuoles, whereas in the flagellate-free higher termites (HT), cellulolytic activity has been attributed to fiber-associated bacteria. However, little is known about the role of individual lineages in fiber digestion, particularly in LT.ResultsWe investigated the lignocellulolytic potential of 2223 metagenome-assembled genomes (MAGs) recovered from the gut metagenomes of 51 termite species. In the flagellate-dependent LT, cellulolytic enzymes are restricted to MAGs of Bacteroidota (Dysgonomonadaceae, Tannerellaceae, Bacteroidaceae, Azobacteroidaceae) and Spirochaetota (Breznakiellaceae) and reflect a specialization on cellodextrins, whereas their hemicellulolytic arsenal features activities on xylans and diverse heteropolymers. By contrast, the MAGs derived from flagellate-free HT possess a comprehensive arsenal of exo- and endoglucanases that resembles that of termite gut flagellates, underlining that Fibrobacterota and Spirochaetota occupy the cellulolytic niche that became vacant after the loss of the flagellates. Furthermore, we detected directly or indirectly oxygen-dependent enzymes that oxidize cellulose or modify lignin in MAGs of Pseudomonadota (Burkholderiales, Pseudomonadales) and Actinomycetota (Actinomycetales, Mycobacteriales), representing lineages located at the hindgut wall.ConclusionsThe results of this study refine our concept of symbiotic digestion of lignocellulose in termite guts, emphasizing the differential roles of specific bacterial lineages in both flagellate-dependent and flagellate-independent breakdown of cellulose and hemicelluloses, as well as a so far unappreciated role of oxygen in the depolymerization of plant fiber and lignin in the microoxic periphery during gut passage in HT.Ak1UNwnHwhcNN-Fj12kHobVideo

Lethal disruption of the bacterial gut community in Eastern subterranean termite caused by boric acid.

The Eastern subterranean termite, Reticulitermes flavipes (Kollar) (Blattodea: Rhinotermitidae), is a significant pest, causing extensive damage to structures that amount to substantial economic losses. Boric acid is widely used for wood preservation due to its stability and broad-spectrum insecticidal properties, yet its impact on termite gut microbiomes and the implications of such effects remain understudied. Our study evaluates the dose-dependent mortality of R. flavipes upon being provided boric acid treated filter papers and investigates the resulting dysbiosis within the termite gut microbiome. Consistent with reports from other insects, mortality increased in a dose-dependent manner, with the highest boric acid concentration (203.7 µg/cm2 of filter paper) significantly reducing termite survival. 16S rRNA gene sequencing of the gut bacterial microbiome revealed notable shifts in composition, indicating boric acid-induced dysbiosis. Aside from an overall decrease in bacterial diversity, the relative abundance of some symbionts essential for termite nutrition decreased in response to higher boric acid concentrations, while several opportunistic pathogens increased. Our findings extend the understanding of boric acid's mode of action in termites, emphasizing its ability to significantly modulate the bacterial symbiont community, which can have dire effects on termite biology. Considering its ability to protect wood from further termite consumption, our study supports the continued use of boric acid and related compounds for termite-resistant treatments for wood.

Screening of aerobic cellulolytic bacteria from the gut of termite for cellulase production

Screening of aerobic cellulolytic bacteria from the gut of termite for cellulase production

Shaping the tripartite symbiosis: are termite microbiome functions directed by the environmentally acquired fungal cultivar?

Microbiome assembly critically impacts the ability of hosts to access beneficial symbiont functions. Fungus-farming termites have co-evolved with a fungal cultivar as a primary food source and complex gut microbiomes, which collectively perform complementary degradation of plant biomass. A large subset of the bacterial community residing within termite guts are inherited (vertically transmitted) from parental colonies, while the fungal symbiont is, in most termite species, acquired from the environment (horizontally transmitted). It has remained unknown how the gut microbiota sustains incipient colonies prior to the acquisition of the fungal cultivar, and how, if at all, bacterial contributions are modulated by fungus garden establishment. Here, we test the latter by determining the composition and predicted functions of the gut microbiome using metabarcoding and shotgun metagenomics, respectively. We focus our functional predictions on bacterial carbohydrate-active enzyme and nitrogen cycling genes and verify compositional patterns of the former through enzyme activity assays. Our findings reveal that the vast majority of microbial functions are encoded in the inherited microbiome, and that the establishment of fungal gardens incurs only minor modulations of predicted bacterial capacities for carbohydrate and nitrogen metabolism. While we cannot rule out that other symbiont functions are gained post-fungus garden establishment, our findings suggest that fungus-farming termite hosts are equipped with a near-complete set of gut microbiome functions at the earliest stages of colony life. This inherited, incipient bacterial microbiome likely contributes to the high extent of functional specificity and coevolution observed between termite hosts, gut microbiomes, and the fungal cultivar.

Exploring the potential of insect gut symbionts for polyethylene biodegradation

The global issue of synthetic plastic pollution is escalating, necessitating innovative bioremediation approaches. Specific insect species have developed symbiotic associations with intestinal microorganisms that possess the ability to degrade resistant plant materials, such as lignocellulose. This is the first study to our knowledge to isolate, characterize, and compare microbial isolates (yeast and bacteria) extracted from the guts of wood-feeding termites (WFT) and lesser waxworms (LWW). The objective was to determine their efficacy in decomposing recalcitrant pollutants, such as low-density polyethylene (LDPE). The isolates belonged to numerous taxonomic groups, as determined by molecular identification; these groups included Bacillus, Citrobacter, Cellulosimicrobium, Rhodococcus, Pseudomonas, Candida, and others. A number of isolates had the potential to degrade LDPE. After 45 days of incubation, the polymer sheets lost 19.8 % of their weight for Bacillus cereus (LDPE-DB2) and 15.4 % of their weight for Candida guilliermondii (LDPE-DY6). Additionally, LDPE degraded by LDPE-DY6 showed a tensile strength loss of 50.3 % compared to that degraded by LDPE-DB2 (58.3 %). During this time, robust biofilm formation was observed on the surfaces of the polymers. Extracellular enzymes such as cutinases, aryl alcohol oxidases, and peroxidases, secreted by specific yeast and bacterial isolates, contribute to the degradation of plastic and recalcitrant biopolymers. Notably, the isolates from LWW demonstrated a greater advantage in this regard than those from WFT. Bacillus cereus, Pseudomonas aeruginosa, Candida guilliermondii, and Sterigmatomyces halophilus were found to have the highest activities in degrading LDPE among the studied WFT and LWW gut-derived symbionts. This study paves the way for a new opportunity to enhance polyethylene degradation by insect gut bacteria and yeasts that may facilitate biotechnological applications for plastic waste remediation.

Termite gut microbiome being a rich source of potential lignocellulolytic bacterial strains

A study was carried out to identify prospective cellulose degrading bacteria isolated from termite gut collected from various agriculture sites. Eight of the bacterial isolates displayed a very distinct zone of clearing on CMC (Carboxylmethyl cellulose) agar media after staining with 1% Congo red, indicating that they may have cellulose degrading ability. On a CMC agar plate, CRDB6 had the largest colony diameter (3.08 cm) as well as clear zone diameter (0.96 cm), however CRDB 1 has maximum cellulolytic activity (1.45 cm) followed by CRDB5 and CRDB6 (1.33 cm and 1.28 cm) respectively. CRDB3, CRDB4 and CRDB9 showed Gram negative reaction among crop residue degrading bacterial isolates, while the remainder showed Gram positive reaction. The morphological characteristics of the bacteria isolates, such as colony shape, elevation, margin, colour and opacity, differed. Isolates CRDB6 produced highest β-glucosidase activity (12.66 m L-1) followed by CRDB1 (12.20 mg m L-1) and CRDB5 (10.21 m L-1) in Luria broth culture. Indole acetic acid production (with tryptophan) was greater in CRDB1 (389.33 mg/ml), CRDB6 (356.9 m L-1) and CRDB3 (315.96 m L-1) while as P-solubilization potential was peaked in CRDB6 (2.41 m L-1) followed by CRDB4 (1.76 m L-1) and CRDB3. Overall, isolate CRDB6 exhibited the highest cellulolytic activity as compared to rest of the bacterial isolates. These results will help in preparing bacterial consortia for the management of crop residues.

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.

Selection of Potential Bacteria in Termite Nest and Gut for Sustainable Agriculture

In this study, bacteria with the best abilities in cellulose degradation, siderophore production, phosphate solubility, and Pythium parasitica inhibition were selected from termite nests and guts. The isolate BTNASP 5-2, BTPK 5-3 and BTNA 5-1 from termite guts exhibited highest in siderophore production index (SPI) (4.16 ± 0.21), phosphate solubilizing index (PSI) (2.10 ± 0.14) and percentage inhibition of radial growth (PIRG) (67.07 ± 4.02 %), respectively. The BGNACMC 4-3 isolated from termite nest gave the highest cellulolytic index (CI) of 5.17 ± 0.24. Bacterial classification was performed using 16s rRNA gene sequencing. The isolates BTPK 5-3, BGNACMC 4-3 and BTNA 5-1 were found closely related to Bacillus cereus, whereas the bacterial isolate BTNASP 5-2 was closely related to Bacillus subtilis. It is also suggested that the Bacillus cereus exhibited a variety of biological activities, denoting the highest cellulase, phosphate-solubilizing and antifungal activities, while Bacillus subtilis produced only a siderophore. The results obtained suggest that the bacteria selected will be used to develop bio-compost to promote plant growth, leading to sustainable farming. HIGHLIGHTS Bacillus cereus exhibited a variety of biological activities, denoting the highest cellulase, phosphate-solubilizing, and antifungal activities Bacillus subtilis produced a siderophore The highest cellulose-degrading bacteria ( cereus BGNACMC4-3) found in termite mound nest GRAPHICAL ABSTRACT

Lignin Degradation by Klebsiella aerogenes TL3 under Anaerobic Conditions.

Lignin, the largest non-carbohydrate component of lignocellulosic biomass, is also a recalcitrant component of the plant cell wall. While the aerobic degradation mechanism of lignin has been well-documented, the anaerobic degradation mechanism is still largely elusive. In this work, a versatile facultative anaerobic lignin-degrading bacterium, Klebsiella aerogenes TL3, was isolated from a termite gut, and was found to metabolize a variety of carbon sources and produce a single kind or multiple kinds of acids. The percent degradation of alkali lignin reached 14.8% under anaerobic conditions, and could reach 17.4% in the presence of glucose within 72 h. Based on the results of infrared spectroscopy and 2D nuclear magnetic resonance analysis, it can be inferred that the anaerobic degradation of lignin may undergo the cleavage of the C-O bond (β-O-4), as well as the C-C bond (β-5 and β-β), and involve the oxidation of the side chain, demethylation, and the destruction of the aromatic ring skeleton. Although the anaerobic degradation of lignin by TL3 was slightly weaker than that under aerobic conditions, it could be further enhanced by adding glucose as an electron donor. These results may shed new light on the mechanisms of anaerobic lignin degradation.

Biodegradation of aniline blue dye by salt-tolerant Bacillus thuringiensis DHC4 isolated from soil-feeding termite guts

The existence of aniline blue in aquatic environments poses a large threat to aquatic organisms. Microbial degradation of aniline blue has been frequently used in remediation because of its effectiveness, low cost, and environmental friendliness. However, the mechanisms by which the dye can be biodegraded are not well understood. In this study, Bacillus thuringiensis DHC4 was isolated from the gut of a soil-feeding termite, and its ability to degrade aniline blue was investigated. The results showed that DHC4 was highly resistant to both aniline blue and salt, with the decolorization rate reaching 78% and 67% after 96 h of incubation at initial dye concentrations of 1 g/L and 7% salinity, respectively. Aniline blue degradation products were identified based on UV–vis, FTIR, and GC–MS analyses, and genome analysis confirmed that DHC4 contained many potential dye-degrading genes. Transcriptome analysis revealed significant up-regulation of methyltransferase, nitroreductase, arylamine N-acetyltransferase and oxidoreductase genes on exposure to aniline blue, indicating their active role in its biodegradation. A new degradation pathway of aniline blue by DHC4 is proposed, based on analysis of metabolites and differentially expressed genes. This mechanism involves cleavage of aniline blue by oxidoreductases into mono- and diphenyl ring molecules, followed by further methylation and alkylation through methyltransferases. The phytotoxicity results showed that aniline blue was degraded by DHC4 into less toxic metabolites. These results provide new insights into the mechanism of biodegradation of aniline blue by microorganisms.

Transmission dynamics of symbiotic protist communities in the termite gut: association with host adult eclosion and dispersal.

The fidelity of vertical transmission is a critical factor in maintaining mutualistic associations with microorganisms. The obligate mutualism between termites and intestinal protist communities has been maintained for over 130 million years, suggesting the faithful transmission of diverse protist species across host generations. Although a severe bottleneck can occur when alates disperse with gut protists, how protist communities are maintained during this process remains largely unknown. In this study, we examined the dynamics of intestinal protist communities during adult eclosion and alate dispersal in the termite Reticulitermes speratus. We found that the protist community structure in last-instar nymphs differed significantly from that in workers and persisted intact during adult eclosion, whereas all protists disappeared from the gut during moults between worker stages. The number of protists in nymphs and alates was substantially lower than in workers, whereas the proportion of protist species exhibiting low abundance in workers was higher in nymphs and alates. Using a simulation-based approach, we demonstrate that such changes in the protist community composition of nymphs and alates improve the transmission efficiency of whole protist species communities. This study thus provides novel insights into how termites have maintained mutualistic relationships with diverse gut microbiota for generations.

Insight into the synergistic effects of surface microstructures and wall peristalsis on glucose production in a bionic microreactor

The enzymatic hydrolysis stage is regarded as a key step in the production of biofuels derived from lignocellulosic biomass, but the elevated production expenses and limited conversion efficiency are still not favorable for practical applications. In this study, inspired by the efficient degradation of cellulose in the termite gut, a cellulase-loaded flexible microreactor model with bionic surface structures was first proposed for the production of fermentable glucose. In such a system, the fold-like structures can provide more enzyme loading sites, and villus-like structures can not only increase enzyme loading but also improve radial transfer in the microreactor. These two structures synergized well and resulted in a 58.5% increase in glucose production compared to the control. Applying peristalsis can further enhance mass transfer by the formation of backflow and vortex, and reducing the peristaltic period is more effective than increasing the peristaltic amplitude for glucose production enhancement. The glucose concentration obtained by the synergistic enhancement of peristalsis and surface structure was 2.3 times of the control. Moreover, for reactions with higher intrinsic reaction rates, the system is in a mass transfer-limited state and peristaltic enhancement is more effective. These results give insight into the design of the bionic flexible microreactor for efficient glucose production.

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.

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.

In vitro biological control of Pyrrhoderma noxium using volatile compounds produced by termite gut-associated streptomycetes.

Pyrrhoderma noxium is a plant pathogen that causes economic losses in agricultural and forestry industries, including significant destruction to amenity trees within the city of Brisbane in Australia. Use of chemical control agents are restricted in public areas, there is therefore an urgent need to investigate biological control approaches. Members of the phylum Actinomycetota, commonly known as actinomycetes, are known for their industrially important secondary metabolites including antifungal agents. They have proven to be ideal candidates to produce environmentally friendly compounds including the volatile organic compounds (VOCs) which can be used as biofumigants. Different Streptomyces species (n=15) previously isolated from the guts of termites and stored in the University of the Sunshine Coast'sMicrobial Library were tested for their antifungal VOCs against Pyrrhoderma noxium. Fourteen of them were found to display inhibition (39.39-100%) to the mycelial development of the pathogen. Strongest antifungal activity displaying isolates USC-592, USC-595, USC-6910 and USC-6928 against the pathogen were selected for further investigations. Their VOCs were also found to have plant growth promotional activity observed for Arabidopsis thaliana with an increase of root length (22-36%) and shoot length (26-57%). The chlorophyll content of the test plant had a slight increase of 11.8% as well. Identified VOCs included geosmin, 2-methylisoborneol, 2-methylbutyrate, methylene cyclopentane, β-pinene, dimethyl disulfide, ethyl isovalerate, methoxyphenyl-oxime and α-pinene. Additionally, all 15 Streptomyces isolates were found to produce siderophores and indole acetic acid as well as the enzyme chitinase which is known to break down the fungal cell wall. Findings indicate that termite gut-associated streptomycetes might be used to control Pyrrhoderma noxium by utilizing their wide range of inhibitory mechanisms.

Development and evaluation of agro-waste biodegradation formulation

In the study, twenty strains of cellulose degrading microbes (Pseudomonas spp., Bacillus spp., Aspergillus spp. etc.) were isolated from the gut of termites and earthworms collected from different locations. The cellulolytic activity of the isolates of Aspergillus spp. were determined based on the formation of clear zone on CMC (Carboxymethyl cellulose) media. The strain C18 of Aspergillus flavus isolated from the gut of termite produced the largest zone with maximum cellulolytic index of 7.64. All the purified strains were subjected to in vitro cellulase activity through DNS (3,5-dinitrosalicylic acid) method which showed that strain C18 of Aspergillus flavus produced the maximum amount of glucose (0.507 mg ML ) and the -1 cellulase enzyme ( IU ML ) was recorded as 0.187 IU ML . Out of twenty microbial strains, ten best -1 -1 microbial strains were selected and subjected to in vitro FPA (filter paper assay). Aspergillus flavus strain C18 was found most effective with maximum glucose (1.059 mg ml ) and cellulase enzyme (0.196 -1 IUML ) production after 9 days of inoculation and therefore the consortium was made from two most -1 effective Aspergillus flavus strains (C12 and C18) which were found best effective in degradation of wheat straw substrate i.e., 61 per cent degradation of initial substrate weight. After evaluation of each strain, effective strains (C12 and C18) were used to prepare consortium formulations for commercial use in the field. These cellulolytic microbes have significant role in agro-waste management by degradation without harming the land and environment.. KEYWORDS :Agro-waste, Aspergillus flavus, biodegradation, cellulolytic microbes, formulation