Characterization of chitinolytic bacteria newly isolated from the termite Microcerotermes sp. and their biocontrol potential against plant pathogenic fungi.

Chitinolytic bacteria were isolated from guts and shells of the termite Microcerotermes sp. Among the nineteen morphologically different chitinolytic isolates, three isolates with highest extracellular chitinase production ratio (≥2.26) were selected. Based on molecular identification of 16S rRNA gene sequences and biochemical characterizations using API test kits and MALDI-TOF MS, these isolates were closely related to Bacillus thuringiensis (Mc_E02) and Paenibacillus species (Mc_E07 and Mc_G06). Isolate Mc_E02 exhibited the highest chitinase-specific activity (2.45 U/mg protein) at 96 h of cultivation, and the enzyme activity was optimized at pH 7.0 and 45 °C. The isolate showed highest and broad-spectrum inhibitory effect against three phytopathogenic fungi (Curvularia lunata, Colletotrichum capsici, and Fusarium oxysporum). Its 36-kDa chitinase exhibited the biomass reduction and mycelium inhibition against all fungi, with highest effects to Curvularia lunata. This research provides novel information about termite chitinolytic bacteria and their effective chitinase, with potential use as biocontrol tool.

Growth Performance, Feed Utilisation, Endogenous Digestive Enzymes, Intestinal Morphology, and Antimicrobial Effect of Pacific White Shrimp (Litopenaeus vannamei) Fed with Feed Supplemented with Pineapple Waste Crude Extract as a Functional Feed Additive.

This study used pineapple waste crude extract (PWCE) to increase the potential of Pacific white shrimp (Litopenaeus vannamei) production for food sustainability and stability. The objective was to investigate the appropriate technique to increase the yield production and quality of shrimp and decrease waste from shrimp culture. Pacific white shrimp (average body size: 0.51 g) were fed with commercial feed supplemented with PWCE at various concentrations of 0 (control), 90, 170, and 250 ppt. Shrimp were fed five times per day for 80 days. At the end of the trial, the results showed that shrimp fed with the PWCE 250 ppt supplementation provided the highest growth rate and the best feed utilisation and yield (P < 0.05). The protein content of whole shrimp in all shrimp fed with the PWCE supplementation diet was higher than that in the control group (P < 0.05). On the contrary, the variation of endogenous digestive enzymes, including protease, trypsin, and the T/C ratio, was significantly lower in shrimp fed a diet supplemented with PWCE 250 ppt (P < 0.05). While in this group, the number of microorganisms on thiosulfate-citrate-bile salt-sucrose (TCBS), blood agar, and trypticase soy agar (TSA) was lowest (P < 0.05). Furthermore, the dietary PWCE at 250 ppt increased the volume of microvilli in the hindgut of shrimp, but the supplementation at 170 ppt improved the number of F-cells in the epithelial cells of the hepatopancreas. Nevertheless, the supplementation of PWCE in the diet did not affect the water quality (P > 0.05). Therefore, pineapple waste crude extract supplementation improves both quantitative and qualitative yields and tends to reduce waste.

Improving the nutritional value and bioactivity of soybean meal in solid-state fermentation using Bacillus strains newly isolated from the gut of the termite Termes propinquus.

The present study aimed to isolate and characterize proteolytic Bacillus spp. from termite guts to test the possibility of application for improving the nutritional value and bioactivity of fermented soybean meal (FSBM). Aerobic endospore-forming bacteria were isolated from the gut of the termite Termes propinquus. Ten isolates with high levels of soy milk degradation were selected and tested for extracellular enzyme production. Among them, two isolates, Tp-5 and Tp-7, exhibited all tested hydrolytic enzyme activities (cellulase, xylanase, pectinase, amylase, protease, lipase and phytase), weak alpha hemolytic and also antagonistic activities against fish pathogenic species of Aeromonas and Streptococcus. Both phylogenetic and biochemical analyses indicated that they were closely related to Bacillus amyloliquefaciens. During solid-state fermentation of SBM, Tp-5 and Tp-7 exhibited the highest protease activity (1127.2 and 1552.4 U g-1, respectively) at 36 h, and the resulting FSBMs showed a significant increase in crude protein content and free radical-scavenging ability (P < 0.05), as well as an improvement in the composition of amino acids, metabolites and other nutrients, while indigestible materials such as fiber, lignin and hemicellulose were decreased. The potential strains, especially Tp-7, improved the nutritional value of FSBM by their strong hydrolytic and antioxidant activities, together with reducing antinutritional components.

Genome analyses of uncultured TG2/ZB3 bacteria in 'Margulisbacteria' specifically attached to ectosymbiotic spirochetes of protists in the termite gut.

We investigated the phylogenetic diversity, localisation and metabolism of an uncultured bacterial clade, Termite Group 2 (TG2), or ZB3, in the termite gut, which belongs to the candidate phylum 'Margulisbacteria'. We performed 16S rRNA amplicon sequencing analysis and detected TG2/ZB3 sequences in 40 out of 72 termite and cockroach species, which exclusively constituted a monophyletic cluster in the TG2/ZB3 clade. Fluorescence in situ hybridisation analysis in lower termites revealed that these bacteria are specifically attached to ectosymbiotic spirochetes of oxymonad gut protists. Draft genomes of four TG2/ZB3 phylotypes from a small number of bacterial cells were reconstructed, and functional genome analysis suggested that these bacteria hydrolyse and ferment cellulose/cellobiose to H2, CO2, acetate and ethanol. We also assembled a draft genome for a partner Treponema spirochete and found that it encoded genes for reductive acetogenesis from H2 and CO2. We hypothesise that the TG2/ZB3 bacteria we report here are commensal or mutualistic symbionts of the spirochetes, exploiting the spirochetes as H2 sinks. For these bacteria, we propose a novel genus, 'Candidatus Termititenax', which represents a hitherto uncharacterised class-level clade in 'Margulisbacteria'. Our findings add another layer, i.e., cellular association between bacteria, to the multi-layered symbiotic system in the termite gut.

Phylogenetic Diversity and Single-Cell Genome Analysis of "Melainabacteria", a Non-Photosynthetic Cyanobacterial Group, in the Termite Gut.

Termite guts harbor diverse yet-uncultured bacteria, including a non-photosynthetic cyanobacterial group, the class "Melainabacteria". We herein reported the phylogenetic diversity of "Melainabacteria" in the guts of diverse termites and conducted a single-cell genome analysis of a melainabacterium obtained from the gut of the termite Termes propinquus. We performed amplicon sequencing of 16S rRNA genes from the guts of 60 termite and eight cockroach species, and detected melainabacterial sequences in 48 out of the 68 insect species, albeit with low abundances (0.02-1.90%). Most of the melainabacterial sequences obtained were assigned to the order "Gastranaerophilales" and appeared to form clusters unique to termites and cockroaches. A single-cell genome of a melainabacterium, designated phylotype Tpq-Mel-01, was obtained using a fluorescence-activated cell sorter and whole genome amplification. The genome shared basic features with other melainabacterial genomes previously reconstructed from the metagenomes of human and koala feces. The bacterium had a small genome (~1.6 Mb) and possessed fermentative pathways possibly using sugars and chitobiose as carbon and energy sources, while the pathways for photosynthesis and carbon fixation were not found. The genome contained genes for flagellar components and chemotaxis; therefore, the bacterium is likely motile. A fluorescence in situ hybridization analysis showed that the cells of Tpq-Mel-01 and/or its close relatives are short rods with the dimensions of 1.1±0.2 µm by 0.5±0.1 µm; for these bacteria, we propose the novel species, "Candidatus Gastranaerophilus termiticola". Our results provide fundamental information on "Melainabacteria" in the termite gut and expand our knowledge on this underrepresented, non-photosynthetic cyanobacterial group.

Microorganisms with novel dissimilatory (bi)sulfite reductase genes are widespread and part of the core microbiota in low-sulfate peatlands.

Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented "core" members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions.

A 'rare biosphere' microorganism contributes to sulfate reduction in a peatland.

Methane emission from peatlands contributes substantially to global warming but is significantly reduced by sulfate reduction, which is fuelled by globally increasing aerial sulfur pollution. However, the biology behind sulfate reduction in terrestrial ecosystems is not well understood and the key players for this process as well as their abundance remained unidentified. Comparative 16S rRNA gene stable isotope probing (SIP) in the presence and absence of sulfate indicated that a Desulfosporosinus species, which constitutes only 0.006% of the total microbial community 16S rRNA genes, is an important sulfate reducer in a long-term experimental peatland field site. Parallel SIP using dsrAB (encoding subunit A and B of the dissimilatory (bi)sulfite reductase) identified no additional sulfate reducers under the conditions tested. For the identified Desulfosporosinus species a high cell-specific sulfate reduction rate of up to 341 fmol SO4²â» cell⁻¹ day⁻¹ was estimated. Thus, the small Desulfosporosinus population has the potential to reduce sulfate in situ at a rate of 4.0-36.8 nmol (g soil w. wt.)⁻¹ day⁻¹, sufficient to account for a considerable part of sulfate reduction in the peat soil. Modeling of sulfate diffusion to such highly active cells identified no limitation in sulfate supply even at bulk concentrations as low as 10 µM. Collectively, these data show that the identified Desulfosporosinus species, despite being a member of the 'rare biosphere', contributes to an important biogeochemical process that diverts the carbon flow in peatlands from methane to CO2 and, thus, alters their contribution to global warming.

Phylogenetic diversity, localization, and cell morphologies of members of the candidate phylum TG3 and a subphylum in the phylum Fibrobacteres, recently discovered bacterial groups dominant in termite guts.

Recently we discovered two novel, deeply branching lineages in the domain Bacteria from termite guts by PCR-based analyses of 16S rRNA (Y. Hongoh, P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, C. Vongkaluang, N. Noparatnaraporn, and T. Kudo, Appl. Environ. Microbiol. 71:6590-6599, 2005). Here, we report on the specific detection of these bacteria, the candidate phylum TG3 (Termite Group 3) and a subphylum in the phylum Fibrobacteres, by fluorescence in situ hybridization in the guts of the wood-feeding termites Microcerotermes sp. and Nasutitermes takasagoensis. Both bacterial groups were detected almost exclusively from the luminal fluid of the dilated portion in the hindgut. Each accounted for approximately 10% of the total prokaryotic cells, constituting the second-most dominant groups in the whole-gut microbiota. The detected cells of both groups were in undulate or vibroid forms and apparently resembled small spirochetes. The cell sizes were 0.2 to 0.4 by 1.3 to 6.0 microm and 0.2 to 0.3 by 1.3 to 4.9 microm in the TG3 and Fibrobacteres, respectively. Using PCR screenings with specific primers, we found that both groups are distributed among various termites. The obtained clones formed monophyletic clusters that were delineated by the host genus rather than by the geographic distance, implying a robust association between these bacteria and host termites. TG3 clones were also obtained from a cockroach gut, lake sediment, rice paddy soil, and deep-sea sediments. Our results suggest that the TG3 and Fibrobacteres bacteria are autochthonous gut symbionts of various termites and that the TG3 members are also widely distributed among various other environments.

Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host.

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic "termite clusters" comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.