Diversity and taxonomic revision of methanogens and other archaea in the intestinal tract of terrestrial arthropods.

Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.

Mercury species in the nests and bodies of soil-feeding termites, Silvestritermes spp. (Termitidae, Syntermitinae), in French Guiana.

Mercury pollution is currently a major public health concern, given the adverse effects of mercury on wildlife and humans. Soil plays an essential role in speciation of mercury and its global cycling, while being a habitat for a wide range of terrestrial fauna. Soil fauna, primarily soil-feeding taxa that are in intimate contact with soil pollutants are key contributors in the cycling of soil mercury and might provide relevant indications about soil pollution. We studied the enrichment of various mercury species in the nests and bodies of soil-feeding termites Silvestritermes spp. in French Guiana. Soil-feeding termites are the only social insects using soil as both shelter and food and are major decomposers of organic matter in neotropical forests. Nests of S. minutus were depleted in total and mobile mercury compared to nearby soil. In contrast, they were enriched 17 times in methylmercury. The highest concentrations of methylmercury were found in body of both studied termite species, with mean bioconcentration factors of 58 for S. minutus and 179 for S. holmgreni relative to the soil. The assessment of the body distribution of methylmercury in S. minutus showed concentrations of 221 ng g-1 for the guts and even higher for the gut-free carcasses (683 ng g-1), suggesting that methylmercury is not confined to the gut where it was likely produced, but rather stored in various tissues. This enrichment in the most toxic form of Hg in termites may be of concern on termite predators and the higher levels in the food chain that may be endangered through prey-to-predator transfers and bioaccumulation. Soil-feeding termites appear to be promising candidates as bio-indicators of mercury pollution in soils of neotropical rainforest ecosystems.

Variations in the relative abundance of Wolbachia in the gut of Nasutitermes arborum across life stages and castes.

There are multiple forms of interactions between termites and bacteria. In addition to their gut microbiota, which has been intensively studied, termites host intracellular symbionts such as Wolbachia. These distinct symbioses have been so far approached independently and mostly in adult termites. We addressed the dynamics of Wolbachia and the microbiota of the eggs and gut for various life stages and castes of the wood-feeding termite, Nasutitermes arborum, using deep-sequencing of the 16S rRNA gene. Wolbachia was dominant in eggs as expected. Unexpectedly, it persisted in the gut of nearly all stages and castes, indicating a wide somatic distribution in termites. Wolbachia-related sequences clustered into few operational taxonomic units, but these were within the same genotype, acquired maternally. Wolbachia was largely dominant in DNA extracts from the guts of larvae and pre-soldiers (59.1%-99.1% of reads) where gut-resident lineages were less represented and less diverse. The reverse was true for the adult castes. This is the first study reporting the age-dependency of the relative abundance of Wolbachia in the termite gut and its negative correlation with the diversity of the microbiota. The possible mechanisms underlying this negative interaction are discussed.

Evidence from the gut microbiota of swarming alates of a vertical transmission of the bacterial symbionts in Nasutitermes arborum (Termitidae, Nasutitermitinae).

Studies on termite symbiosis have revealed that significant symbiont lineages are maintained across generations. However, most studies have focused only on the worker caste. Little is known about the gut microbiota of reproductives, the most probable vectors for transmitting these lineages to offspring. Using 16S rRNA gene-based Illumina MiSeq sequencing, we compared the gut microbiota of swarming alates of the higher termite Nasutitermes arborum with those of their nestmates from the parental colony. The OTU-based alpha diversity indices showed that the gut microbiota of the alates was at least as diverse as those of non-reproductive adults. It was largely dominated by Spirochaetes mostly of the Treponema I cluster (63.1% of reads), the same dominant taxa found in soldiers and workers of this species and in workers of closely related Nasutitermes species. The termite-specific lineages also included other representative taxa such as several clusters of Bacteroidetes and Fibrobacteres-TG3 group. The microbiota of alates was dominated by a core set of host-specific lineages (87% of reads, 77.6% of OTUs), which were always present across all castes/stages. This first comprehensive survey of the microbiota of the founding reproductives of these xylophagous higher termites shows that the bulk of the host endogenous symbionts, mostly taxa that cannot thrive outside the gut, are brought from the parent colony. The royal pair therefore seems to be a key player in the transmission of symbionts across generations and thereby in host-symbiont codiversification. The high proportion of fiber-degrading lineages in their gut suggests a wood-rich diet unlike the larval stages.

Uncovering the Potential of Termite Gut Microbiome for Lignocellulose Bioconversion in Anaerobic Batch Bioreactors.

Termites are xylophages, being able to digest a wide variety of lignocellulosic biomass including wood with high lignin content. This ability to feed on recalcitrant plant material is the result of complex symbiotic relationships, which involve termite-specific gut microbiomes. Therefore, these represent a potential source of microorganisms for the bioconversion of lignocellulose in bioprocesses targeting the production of carboxylates. In this study, gut microbiomes of four termite species were studied for their capacity to degrade wheat straw and produce carboxylates in controlled bioreactors. All of the gut microbiomes successfully degraded lignocellulose and up to 45% w/w of wheat straw degradation was observed, with the Nasutitermes ephratae gut-microbiome displaying the highest levels of wheat straw degradation, carboxylate production and enzymatic activity. Comparing the 16S rRNA gene diversity of the initial gut inocula to the bacterial communities in lignocellulose degradation bioreactors revealed important changes in community diversity. In particular, taxa such as Spirochaetes and Fibrobacteres that were highly abundant in the initial gut inocula were replaced by Firmicutes and Proteobacteria at the end of incubation in wheat straw bioreactors. Overall, this study demonstrates that termite-gut microbiomes constitute a reservoir of lignocellulose-degrading bacteria that can be harnessed in artificial conditions for biomass conversion processes that lead to the production of useful molecules.

Nitrous Oxide (N2O) Emissions by Termites: Does the Feeding Guild Matter?

In the tropics, termites are major players in the mineralization of organic matter leading to the production of greenhouse gases including nitrous oxide (N2O). Termites have a wide trophic diversity and their N-metabolism depends on the feeding guild. This study assessed the extent to which N2O emission levels were determined by termite feeding guild and tested the hypothesis that termite species feeding on a diet rich in N emit higher levels of N2O than those feeding on a diet low in N. An in-vitro incubation approach was used to determine the levels of N2O production in 14 termite species belonging to different feeding guilds, collected from a wide range of biomes. Fungus-growing and soil-feeding termites emit N2O. The N2O production levels varied considerably, ranging from 13.14 to 117.62 ng N2O-N d(-1) (g dry wt.)(-1) for soil-feeding species, with Cubitermes spp. having the highest production levels, and from 39.61 to 65.61 ng N2O-N d(-1) (g dry wt.)(-1) for fungus-growing species. Wood-feeding termites were net N2O consumers rather than N2O producers with a consumption ranging from 16.09 to 45.22 ng N2O-N d(-1) (g dry wt.)(-1). Incubating live termites together with their mound increased the levels of N2O production by between 6 and 13 fold for soil-feeders, with the highest increase in Capritermes capricornis, and between 14 and 34 fold for fungus-growers, with the highest increase in Macrotermes muelleri. Ammonia-oxidizing (amoA-AOB and amoA-AOA) and denitrifying (nirK, nirS, nosZ) gene markers were detected in the guts of all termite species studied. No correlation was found between the abundance of these marker genes and the levels of N2O production from different feeding guilds. Overall, these results support the hypothesis that N2O production rates were higher in termites feeding on substrates with higher N content, such as soil and fungi, compared to those feeding on N-poor wood.

Profiling the Succession of Bacterial Communities throughout the Life Stages of a Higher Termite Nasutitermes arborum (Termitidae, Nasutitermitinae) Using 16S rRNA Gene Pyrosequencing.

Previous surveys of the gut microbiota of termites have been limited to the worker caste. Termite gut microbiota has been well documented over the last decades and consists mainly of lineages specific to the gut microbiome which are maintained across generations. Despite this intimate relationship, little is known of how symbionts are transmitted to each generation of the host, especially in higher termites where proctodeal feeding has never been reported. The bacterial succession across life stages of the wood-feeding higher termite Nasutitermes arborum was characterized by 16S rRNA gene deep sequencing. The microbial community in the eggs, mainly affiliated to Proteobacteria and Actinobacteria, was markedly different from the communities in the following developmental stages. In the first instar and last instar larvae and worker caste termites, Proteobacteria and Actinobacteria were less abundant than Firmicutes, Bacteroidetes, Spirochaetes, Fibrobacteres and the candidate phylum TG3 from the last instar larvae. Most of the representatives of these phyla (except Firmicutes) were identified as termite-gut specific lineages, although their relative abundances differed. The most salient difference between last instar larvae and worker caste termites was the very high proportion of Spirochaetes, most of which were affiliated to the Treponema Ic, Ia and If subclusters, in workers. The results suggest that termite symbionts are not transmitted from mother to offspring but become established by a gradual process allowing the offspring to have access to the bulk of the microbiota prior to the emergence of workers, and, therefore, presumably through social exchanges with nursing workers.

Characterization of N2O emission and associated bacterial communities from the gut of wood-feeding termite Nasutitermes voeltzkowi.

Xylophagous termites rely on nitrogen deficient foodstuff with a low C/N ratio. Most research work has focused on nitrogen fixation in termites highlighting important inflow and assimilation of atmospheric nitrogen into their bodies fundamentally geared up by their intestinal microbial symbionts. Most of termite body nitrogen is of atmospheric origin, and microbially aided nitrification is the principal source of this nitrogen acquisition, but contrarily, the information regarding potent denitrification process is very scarce and poorly known, although the termite gut is considered to carry all favorable criteria necessary for microbial denitrification. Therefore, in this study, it is hypothesized that whether nitrification and denitrification processes coexist in intestinal milieu of xylophagous termites or not, and if yes, then is there any link between the denitrification product, i.e., N2O and nitrogen content of the food substrate, and moreover where these bacterial communities are found along the length of termite gut. To answer these questions, we measured in vivo N2O emission by Nasutitermes voeltzkowi (Nasutitermitinae) maintained on different substrates with varying C/N ratio, and also, molecular techniques were applied to study the diversity (DGGE) and density (qPCR) of bacterial communities in anterior and posterior gut portions. Rersults revealed that xylophagous termites emit feeble amount of N2O and molecular studies confirmed this finding by illustrating the presence of an ample density of N2O-reductase (nosZ) gene in the intestinal tract of these termites. Furthermore, intestinal bacterial communities of these termites were found more dense and diverse in posterior than anterior portion of the gut.

Increased lead availability and enzyme activities in root-adhering soil of Lantana camara during phytoextraction in the presence of earthworms.

Earthworms are known to increase availability of heavy metals in soils and also play an important role in maintaining the structure and quality of soil. The introduction of earthworms into soils contaminated with metals in the presence of a potential hyperaccumulator has been suggested as an aid for phytoremediation processes. The present study was conducted to evaluate: (i) the effects of earthworms on lead availability in artificially contaminated soil at 500 and 1000 mg kg(-1) Pb in the presence of Lantana camara, a hyperaccumulator, (ii) the effects of earthworms and lead on soil properties such as pH, cation exchange capacity (CEC), organic matter (OM), total and available N, P and K and (iii) soil enzyme activities. Earthworms increased the bioavailable Pb in root-adhering soil by a factor of 2 to 3 in the contaminated soils at concentrations of 500 to 1000 mg Pb kg(-1), respectively. In lead contaminated soils, the presence of earthworms led to a significant decrease in soil pH by about 0.2 but increased CEC by 17% and OM by more than 30%. Earthworm activities also increased the activities of N-acetylglucosamidase, ß-glucosidase, cellulase, xylanase, alkaline and acid phosphatase, urease and fluorescein diacetate assay (FDA). These results indicate that the ecological context for phytoremediation should be broadened by considering plant-soil-earthworm interactions as they influence both plant health and absorption of heavy metals. They also showed that the enzyme activities monitored could serve as useful proxies for phytoremediation capability and, more generally, for soil quality as a whole.

Effect of earthworms on plant Lantana camara Pb-uptake and on bacterial communities in root-adhering soil.

The present study aimed to assess the potential abilities of Lantana camara, an invasive plant species for phytoremediation in the presence of earthworm Pontoscolex corethrurus. Effects of earthworm on growth and lead (Pb) uptake by L. camara plant were studied in soil artificially contaminated at 500 or 1000mg of Pb kg(-1) soil. This species has a promising value for phytoremediation because it can uptake as much as 10% of 1000mgkg(-1) of Pb per year. Moreover, the presence of earthworms enhanced plant biomass by about 1.5-2 times and increased the uptake of lead by about 2-3 times. In the presence of earthworm, L. camara was thus able to uptake up 20% of Pb presence in the soil, corresponding to remediation time of 5 years if all organs are removed. As soil microorganisms are known to mediate many interactions between earthworms and plants, we documented the effect of earthworms on the bacterial community of root-adhering soil of L. camara. Cultivable bacterial biomass of root-adhering soil increased in the presence of earthworms. Similar trend was observed on bacterial metabolic activities. The increase of lead concentrations from 500 to 1000mgkg(-1) did not have any significant effect either on plant growth or on bacterial biomass and global activities but affected the structure and functional diversity of the bacterial community. These results showed that we should broaden the ecological context of phytoremediation by considering plant/microbial community/earthworm interactions that influence the absorption of heavy metals.

Molecular diversity and host specificity of termite-associated Xylaria.

Studies have revealed that some Xylaria species were closely associated with fungus-growing termite nests. However this relationship rarely had been investigated and the host specificity of termite-associated Xylaria was not yet clearly established. Eighteen Xylaria rDNA-ITS sequences were obtained from fungus combs belonging to 11 Macrotermitinae species from eight regions. Low diversity was found between isolates, and nine sequences were retrieved. Termite-associated Xylaria were shown to be monophyletic, with three main clades, all including strains from various termite hosts and geographical localities. This new molecular study shows no species specificity with respect to fungus-growing termites, which suggests that there might be substrate specialization.

Occurrence of fungi in combs of fungus-growing termites (Isoptera: Termitidae, Macrotermitinae).

Fungus-growing termites cultivate their mutualistic basidiomycete Termitomyces species on a substrate called a fungal comb. Here, the Suicide Polymerase Endonuclease Restriction (SuPER) method was adapted for the first time to a fungal study to determine the entire fungal community of fungal combs and to test whether fungi other than the symbiotic cultivar interact with termite hosts. Our molecular analyses show that although active combs are dominated by Termitomyces fungi isolated with direct Polymerase Endonuclease Restriction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE), they can also harbor some filamentous fungi and yeasts only revealed by SuPER PCR-DGGE. This is the first molecular evidence of the presence of non-Termitomyces species in active combs. However, because there is no evidence for a species-specific relationship between these fungi and termites, they are mere transient guests with no specialization in the symbiosis. It is however surprising to notice that termite-associated Xylaria strains were not isolated from active combs even though they are frequently retrieved when nests are abandoned by termites. This finding highlights the implication of fungus-growing termites in the regulation of fungi occurring within the combs and also suggests that they might not have any particular evolutionary-based association with Xylaria species.

Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods.

The use of denaturing gradient gel electrophoresis (DGGE) and traditional culture-depending methods for examining the bacterial community of traditional cassava starch fermentation were investigated. It appeared that DGGE profiles of total DNA of cassava dough exhibited 10 distinguishable bands. In contrast, DGGE fingerprints of bacteria recovered from enrichment cultures of fermented dough gave variable profiles containing fewer bands. Bands corresponding to five bacterial species detected by direct PCR-DGGE of total DNA from of cassava dough were also observed in DGGE patterns of enrichment cultures. Eighteen strains were isolated from cultures selected on the basis of their DGGE banding patterns. Assessment of bacterial identification by 16S rDNA sequence similarity revealed that band comigration implied sequence identity. Comparison of 16S rDNA sequences of excised DGGE bands and recovered pure culture isolates with those in GENBANK and the RDP databases revealed that representative bacteria of fermented cassava dough were Lactobacillus and Pediococcus species as well as species of Clostridium, Propionibacterium and Bacillus. Some Lactobacillus species detected in dough samples by sequence analysis of DGGE bands were not recovered in any of the five culture media and conditions used. On the other hand, some species recovered as pure cultures from enrichments were not detected by direct DGGE analysis of total bacterial DNA from cassava dough. Our results provide evidence of the necessity to combine both culture-dependent and culture-independent methods for better description of microbial communities in indigenous cassava starch fermentations.