A 2000-Year-Old Bacillus stercoris Strain Sheds Light on the Evolution of Cyclic Antimicrobial Lipopeptide Synthesis.

Some bacteria (notably the genera Bacillus and Clostridium) have the capacity to form endospores that can survive for millions of years in isolated habitats. The genomes of such ancient bacteria provide unique opportunities to understand bacterial evolution and metabolic capabilities over longer time scales. Herein, we sequenced the genome of a 2000-year-old bacterial strain (Mal05) isolated from intact apple seeds recovered during archaeological excavations of a Roman villa in Italy. Phylogenomic analyses revealed that this strain belongs to the species Bacillus stercoris and that it is placed in an early-branching position compared to most other strains of this species. Similar to other Bacillus species, B. stercoris Mal05 had been previously shown to possess antifungal activity. Its genome encodes all the genes necessary for the biosynthesis of fengycin and surfactin, two cyclic lipopeptides known to play a role in the competition of Bacilli with other microorganisms due to their antimicrobial activity. Comparative genomics and analyses of selective pressure demonstrate that these genes are present in all sequenced B. stercoris strains, despite the fact that they are not under strong purifying selection. Hence, these genes may not be essential for the fitness of these bacteria, but they can still provide a competitive advantage against other microorganisms present in the same environment.

Exploring the expressiveness of abstract metabolic networks.

Metabolism is characterised by chemical reactions linked to each other, creating a complex network structure. The whole metabolic network is divided into pathways of chemical reactions, such that every pathway is a metabolic function. A simplified representation of metabolism, which we call an abstract metabolic network, is a graph in which metabolic pathways are nodes and there is an edge between two nodes if their corresponding pathways share one or more compounds. The abstract metabolic network of a given organism results in a small network that requires low computational power to be analysed and makes it a suitable model to perform a large-scale comparison of organisms' metabolism. To explore the potentials and limits of such a basic representation, we considered a comprehensive set of KEGG organisms, represented through their abstract metabolic network. We performed pairwise comparisons using graph kernel methods and analyse the results through exploratory data analysis and machine learning techniques. The results show that abstract metabolic networks discriminate macro evolutionary events, indicating that they are expressive enough to capture key steps in metabolism evolution.

Complete Genome Sequence of Rhynchophorus ferrugineus Endocytobiont "Candidatus Nardonella dryophthoridicola" Strain NardRF.

We report the complete genome sequence and annotation of "Candidatus Nardonella dryophthoridicola" strain NardRF, obtained by sequencing its host bacteriome, Rhynchophorus ferrugineus, using Oxford Nanopore technology.

Endosymbiosis morphological reorganization during metamorphosis diverges in weevils.

Virtually all animals associate with beneficial symbiotic bacteria. Whether and how these associations are modulated across a host's lifecycle is an important question in disentangling animal-bacteria interactions. We recently reported a case of complete morphological reorganization of symbiosis during metamorphosis of the cereal weevil, Sitophilus oryzae. In this model, the bacteriome, a specialized organ that houses the intracellular bacterium Sodalis pierantonius, undergoes a two-phase remodeling program synchronously driven by host and endosymbiont, resulting in a localization shift and the formation of multiple new bacteriomes. Here, we provide comparative data in a closely-related coleopteran, the red palm weevil Rhynchophorus ferrugineus, which is associated with the ancestral endosymbiont Nardonella. Using cell imaging experiments, we show that the red pal weevil bacteriome remains unchanged during metamorphosis, hence contrasting with what we reported in the cereal weevil S. oryzae. These findings highlight the complexity and divergence of host-symbiont interactions and their intertwining with host development, even in closely-related species. Abbreviations: DAPI: 4',6-diamidino-2-phenylindole; FISH: Fluorescence in situ hybridization; T3SS: Type III secretion system.

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae).

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.

Evidence for a conserved microbiota across the different developmental stages of Plodia interpunctella.

Diversity and composition of lepidopteran microbiotas are poorly investigated, especially across the different developmental stages. To improve this knowledge, we characterize the microbiota among different developmental stages of the Indian meal moth, Plodia interpunctella, which is considered one of the major pest of commodities worldwide. Using culture-independent approach based on Illumina 16S rRNA gene sequencing we characterized the microbiota of four developmental stages: eggs, first-, and last-instar larvae, and adult. A total of 1022 bacterial OTUs were obtained, showing a quite diversified microbiota associated to all the analyzed stages. The microbiotas associated with P. interpunctella resulted almost constant throughout the developmental stages, with approximately 77% of bacterial OTUs belonging to the phylum of Proteobacteria. The dominant bacterial genus is represented by Burkholderia (∼64%), followed by Propionibacterium, Delftia, Pseudomonas, and Stenotrophomonas. A core bacterial community, composed of 139 OTUs, was detected in all the developmental stages, among which 112 OTUs were assigned to the genus Burkholderia. A phylogenetic reconstruction, based on the 16S rRNA, revealed that our Burkholderia OTUs clustered with Burkholderia cepacia complex, in the same group of those isolated from the hemipterans Gossyparia spuria and Acanthococcus aceris. The functional profiling, predicted on the base of the bacterial 16S rRNA, indicates differences in the metabolic pathways related to metabolism of amino acids between preimaginal and adult stages. We can hypothesize that bacteria may support the insect host during preimaginal stages.

The Cost of Metabolic Interactions in Symbioses between Insects and Bacteria with Reduced Genomes.

Various intracellular bacterial symbionts that provide their host with essential nutrients have much-reduced genomes, attributed largely to genomic decay and relaxed selection. To obtain quantitative estimates of the metabolic function of these bacteria, we reconstructed genome- and transcriptome-informed metabolic models of three xylem-feeding insects that bear two bacterial symbionts with complementary metabolic functions: a primary symbiont, Sulcia, that has codiversified with the insects, and a coprimary symbiont of distinct taxonomic origin and with different degrees of genome reduction in each insect species (Hodgkinia in a cicada, Baumannia in a sharpshooter, and Sodalis in a spittlebug). Our simulations reveal extensive bidirectional flux of multiple metabolites between each symbiont and the host, but near-complete metabolic segregation (i.e., near absence of metabolic cross-feeding) between the two symbionts, a likely mode of host control over symbiont metabolism. Genome reduction of the symbionts is associated with an increased number of host metabolic inputs to the symbiont and also reduced metabolic cost to the host. In particular, Sulcia and Hodgkinia with genomes of ≤0.3 Mb are calculated to recycle ∼30 to 80% of host-derived nitrogen to essential amino acids returned to the host, while Baumannia and Sodalis with genomes of ≥0.6 Mb recycle 10 to 15% of host nitrogen. We hypothesize that genome reduction of symbionts may be driven by selection for increased host control and reduced host costs, as well as by the stochastic process of genomic decay and relaxed selection.IMPORTANCE Current understanding of many animal-microbial symbioses involving unculturable bacterial symbionts with much-reduced genomes derives almost entirely from nonquantitative inferences from genome data. To overcome this limitation, we reconstructed multipartner metabolic models that quantify both the metabolic fluxes within and between three xylem-feeding insects and their bacterial symbionts. This revealed near-complete metabolic segregation between cooccurring bacterial symbionts, despite extensive metabolite exchange between each symbiont and the host, suggestive of strict host controls over the metabolism of its symbionts. We extended the model analysis to investigate metabolic costs. The positive relationship between symbiont genome size and the metabolic cost incurred by the host points to fitness benefits to the host of bearing symbionts with small genomes. The multicompartment metabolic models developed here can be applied to other symbioses that are not readily tractable to experimental approaches.

New Insights into the Microbiota of Moth Pests.

In recent years, next generation sequencing (NGS) technologies have helped to improve our understanding of the bacterial communities associated with insects, shedding light on their wide taxonomic and functional diversity. To date, little is known about the microbiota of lepidopterans, which includes some of the most damaging agricultural and forest pests worldwide. Studying their microbiota could help us better understand their ecology and offer insights into developing new pest control strategies. In this paper, we review the literature pertaining to the microbiota of lepidopterans with a focus on pests, and highlight potential recurrent patterns regarding microbiota structure and composition.

A genomic investigation of ecological differentiation between free-living and Drosophila-associated bacteria.

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome-scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.

Differentiating current and past PCB and PCDD/F sources: The role of a large contaminated soil site in an industrialized city area.

Cities and contaminated areas can be primary or secondary sources of polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and other chemicals, into air and soil and can influence the regional level of some of these pollutants. In a contaminated site, the evaluation of such emissions can be crucial in the choice of the remediation technology to be adopted. In the city of Brescia (Northern Italy), more than 100 ha of agricultural areas were contaminated with PCBs, PCDD/Fs and heavy metals, originating from the activities of a former PCB factory. In order to evaluate the current emissions of PCBs and PCDD/Fs from the contaminated site, in a location where other current sources are present, we compared measured and predicted air concentrations, resulting from chemical volatilization from soils as well as fingerprints of Brescia soils and of soils contaminated by specific sources. The results confirm that the contaminated area is still a current and important secondary source of PCBs to the air, and to a lesser extent of PCDFs (especially the more volatile), but not for PCDDs. PCBs in soils have fingerprints similar to highly chlorinated mixtures, indicating contamination by these mixtures and/or a long weathering process. PCB 209 is also present at important levels. PCDD fingerprints in soil cannot be related to current emission sources, while PCDFs are compatible to industrial and municipal waste incineration, although weathering and/or natural attenuation may have played a role in modifying such soil fingerprints. Finally, we combined chemical and microbiological analyses to provide an integrated approach to evaluate soil fingerprints and their variation in a wider perspective, which accounts for the mutual effects between contamination and soil microbiota, a pivotal hint for addressing in situ bioremediation activities.

Bacterial diversity shift determined by different diets in the gut of the spotted wing fly Drosophila suzukii is primarily reflected on acetic acid bacteria.

The pivotal role of diet in shaping gut microbiota has been evaluated in different animal models, including insects. Drosophila flies harbour an inconstant microbiota among which acetic acid bacteria (AAB) are important components. Here, we investigated the bacterial and AAB components of the invasive pest Drosophila suzukii microbiota, by studying the same insect population separately grown on fruit-based or non-fruit artificial diet. AAB were highly prevalent in the gut under both diets (90 and 92% infection rates with fruits and artificial diet respectively). Fluorescent in situ hybridization and recolonization experiments with green fluorescent protein (Gfp)-labelled strains showed AAB capability to massively colonize insect gut. High-throughput sequencing on 16S rRNA gene indicated that the bacterial microbiota of guts fed with the two diets clustered separately. By excluding AAB-related OTUs from the analysis, insect bacterial communities did not cluster separately according to the diet, suggesting that diet-based diversification of the community is primarily reflected on the AAB component of the community. Diet influenced also AAB alpha-diversity, with separate OTU distributions based on diets. High prevalence, localization and massive recolonization, together with AAB clustering behaviour in relation to diet, suggest an AAB role in the D. suzukii gut response to diet modification.

Hydrocarbon pollutants shape bacterial community assembly of harbor sediments.

Petroleum pollution results in co-contamination by different classes of molecules, entailing the occurrence of marine sediments difficult to remediate, as in the case of the Ancona harbor (Mediterranean Sea, Italy). Autochthonous bioaugmentation (ABA), by exploiting the indigenous microbes of the environment to be treated, could represent a successful bioremediation strategy. In this perspective we aimed to i) identify the main drivers of the bacterial communities' richness in the sediments, ii) establish enrichment cultures with different hydrocarbon pollutants evaluating their effects on the bacterial communities' composition, and iii) obtain a collection of hydrocarbon degrading bacteria potentially exploitable in ABA. The correlation between the selection of different specialized bacterial populations and the type of pollutants was demonstrated by culture-independent analyses, and by establishing a collection of bacteria with different hydrocarbon degradation traits. Our observations indicate that pollution dictates the diversity of sediment bacterial communities and shapes the ABA potential in harbor sediments.

Draft Genome Sequence of the Hydrocarbon-Degrading Bacterium Alcanivorax dieselolei KS-293 Isolated from Surface Seawater in the Eastern Mediterranean Sea.

We report here the draft genome sequence of Alcanivorax dieselolei KS-293, a hydrocarbonoclastic bacterium isolated from the Mediterranean Sea, by supplying diesel oil as the sole carbon source. This strain contains multiple putative genes associated with hydrocarbon degradation pathways and that are highly similar to those described in A. dieselolei type strain B5.

Plant-mediated interspecific horizontal transmission of an intracellular symbiont in insects.

Intracellular reproductive manipulators, such as Candidatus Cardinium and Wolbachia are vertically transmitted to progeny but rarely show co-speciation with the host. In sap-feeding insects, plant tissues have been proposed as alternative horizontal routes of interspecific transmission, but experimental evidence is limited. Here we report results from experiments that show that Cardinium is horizontally transmitted between different phloem sap-feeding insect species through plants. Quantitative PCR and in situ hybridization experiments indicated that the leafhopper Scaphoideus titanus releases Cardinium from its salivary glands during feeding on both artificial media and grapevine leaves. Successional time-course feeding experiments with S. titanus initially fed sugar solutions or small areas of grapevine leaves followed by feeding by the phytoplasma vector Macrosteles quadripunctulatus or the grapevine feeder Empoasca vitis revealed that the symbionts were transmitted to both species. Explaining interspecific horizontal transmission through plants improves our understanding of how symbionts spread, their lifestyle and the symbiont-host intermixed evolutionary pattern.

Bacterial population and biodegradation potential in chronically crude oil-contaminated marine sediments are strongly linked to temperature.

Two of the largest crude oil-polluted areas in the world are the semi-enclosed Mediterranean and Red Seas, but the effect of chronic pollution remains incompletely understood on a large scale. We compared the influence of environmental and geographical constraints and anthropogenic forces (hydrocarbon input) on bacterial communities in eight geographically separated oil-polluted sites along the coastlines of the Mediterranean and Red Seas. The differences in community compositions and their biodegradation potential were primarily associated (P < 0.05) with both temperature and chemical diversity. Furthermore, we observed a link between temperature and chemical and biological diversity that was stronger in chronically polluted sites than in pristine ones where accidental oil spills occurred. We propose that low temperature increases bacterial richness while decreasing catabolic diversity and that chronic pollution promotes catabolic diversification. Our results further suggest that the bacterial populations in chronically polluted sites may respond more promptly in degrading petroleum after accidental oil spills.

Mutual exclusion of Asaia and Wolbachia in the reproductive organs of mosquito vectors.

BACKGROUND: Wolbachia is a group of intracellular maternally inherited bacteria infecting a high number of arthropod species. Their presence in different mosquito species has been largely described, but Aedes aegypti, the main vector of Dengue virus, has never been found naturally infected by Wolbachia. Similarly, malaria vectors and other anophelines are normally negative to Wolbachia, with the exception of an African population where these bacteria have recently been detected. Asaia is an acetic acid bacterium stably associated with several mosquito species, found as a dominant microorganism of the mosquito microbiota. Asaia has been described in gut, salivary glands and in reproductive organs of adult mosquitoes in Ae. aegypti and in anophelines. It has recently been shown that Asaia may impede vertical transmission of Wolbachia in Anopheles mosquitoes. Here we present an experimental study, aimed at determining whether there is a negative interference between Asaia and Wolbachia, for the gonad niche in mosquitoes. METHODS: Different methods (PCR and qPCR, monoclonal antibody staining and FISH) have been used to address the question of the co-localization and the relative presence/abundance of the two symbionts. PCR and qPCR were performed to qualitatively and quantitatively verify the distribution of Asaia and Wolbachia in different mosquito species/organs. Monoclonal antibody staining and FISH were performed to localize the symbionts in different mosquito species. RESULTS: Here we provide evidence that, in Anopheles and in other mosquitoes, there is a reciprocal negative interference between Asaia and Wolbachia symbionts, in terms of the colonization of the gonads. In particular, we have shown that in some mosquito species the presence of one of the symbionts prevented the establishment of the second, while in other systems the symbionts were co-localized, although at reduced densities. CONCLUSIONS: A mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes.

Effects of the diet on the microbiota of the red palm weevil (Coleoptera: Dryophthoridae).

Rhynchophorus ferrugineus, also known as the red palm weevil, is regarded as the major pest of palm trees. Although studies of the microbiota associated with this species have been performed in recent years, little attention has been dedicated to the influence of the diet in shaping the host bacterial community. Here, we investigated the influence of food sources (i.e. palm tissues vs apple based substrate) on the microbial diversity associated with RPW, which was compared with the microbiota associated with wild individuals of the sister species Rhynchophorus vulneratus. The bacterial characterization was performed using a culture independent approach, i.e. the 16S rRNA pyrotag, and a culture dependent approach for a subset of the samples, in order to obtain bacterial isolates from RPW tissues. The bacterial community appeared significantly influenced by diet. Proteobacteria resulted to be the most abundant clade and was present in all the specimens of the three examined weevil groups. Within Proteobacteria, Enterobacteriaceae were identified in all the organs analysed, including hemolymph and reproductive organs. The apple-fed RPWs and the wild R. vulneratus showed a second dominant taxon within Firmicutes that was scarcely present in the microbiota associated with palm-fed RPWs. A comparative analysis on the bacteria associated with the palm tissues highlighted that 12 bacterial genera out of the 13 identified in the plant tissues were also present in weevils, thus indicating that palm tissues may present a source for bacterial acquisition.

Modulation of fecal Clostridiales bacteria and butyrate by probiotic intervention with Lactobacillus paracasei DG varies among healthy adults.

BACKGROUND: The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE: This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS: High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS: Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations <25 mmol/kg of wet feces, the probiotic contributed to a 329 ± 255% (mean ± SD) increment in butyrate concomitantly with an ∼55% decrease in Ruminococcus (P = 0.016) and a 150% increase in an abundantly represented unclassified Bacteroidales genus (P = 0.05). CONCLUSIONS: The intake of L. paracasei DG increased the Blautia:Coprococcus ratio, which, according to the literature, can potentially confer a health benefit on the host. The probiotic impact on the microbiota and on short-chain fatty acids, however, seems to strictly depend on the initial characteristics of the intestinal microbial ecosystem. In particular, fecal butyrate concentrations could represent an important biomarker for identifying subjects who may benefit from probiotic treatment. This trial was registered at www.controlled-trials.com/isrctn as ISRCTN56945491.

A new strain of Wolbachia in an alpine population of the viviparous Oreina cacaliae (Coleoptera: Chrysomelidae).

Microbial symbionts played a central role in insect evolution. Oreina cacaliae (Schrank, 1785) (Coleoptera: Chrysomelidae) is a rare example of a viviparous insect, able to feed on toxic plants and sequester toxic compounds. In the current study, the microbiota associated with O. cacaliae was characterized using a culture-independent approach, targeting the 16S rRNA bacterial gene. The obtained 16S rRNA gene sequences were analyzed and identified at different taxonomic levels. Wolbachia was the dominant bacterium, both in male and female (100 and 91.9%, respectively) individuals; the detected Wolbachia was described as a new sequence type based on multilocus sequence typing (Wolbachia ST375 Ocac_A_wVdO). After phylogenetic analyses, Wolbachia ST375 Ocac_A_wVdO was attributed to the supergroup A. Immunofluorescence assays and electron microscopy confirmed the presence of Wolbachia within O. cacaliae oocytes, confirming its transovarial transmission in this species. Representatives of six species of Oreina were tested for the presence of Wolbachia through specific polymerase chain reaction, and a dendrogram was generated for these species based on coxI gene sequences. The Wolbachia harbored by different species of Oreina were characterized by multilocus sequence typing. Five out of the six examined Oreina species were positive for Wolbachia, with four of these harboring the same sequence type.

Geosphere-biosphere interactions in bio-activity volcanic lakes: evidences from Hule and Rìo Cuarto (Costa Rica).

Hule and Río Cuarto are maar lakes located 11 and 18 km N of Poás volcano along a 27 km long fracture zone, in the Central Volcanic Range of Costa Rica. Both lakes are characterized by a stable thermic and chemical stratification and recently they were affected by fish killing events likely related to the uprising of deep anoxic waters to the surface caused by rollover phenomena. The vertical profiles of temperature, pH, redox potential, chemical and isotopic compositions of water and dissolved gases, as well as prokaryotic diversity estimated by DNA fingerprinting and massive 16S rRNA pyrosequencing along the water column of the two lakes, have highlighted that different bio-geochemical processes occur in these meromictic lakes. Although the two lakes host different bacterial and archaeal phylogenetic groups, water and gas chemistry in both lakes is controlled by the same prokaryotic functions, especially regarding the CO2-CH4 cycle. Addition of hydrothermal CO2 through the bottom of the lakes plays a fundamental priming role in developing a stable water stratification and fuelling anoxic bacterial and archaeal populations. Methanogens and methane oxidizers as well as autotrophic and heterotrophic aerobic bacteria responsible of organic carbon recycling resulted to be stratified with depth and strictly related to the chemical-physical conditions and availability of free oxygen, affecting both the CO2 and CH4 chemical concentrations and their isotopic compositions along the water column. Hule and Río Cuarto lakes were demonstrated to contain a CO2 (CH4, N2)-rich gas reservoir mainly controlled by the interactions occurring between geosphere and biosphere. Thus, we introduced the term of bio-activity volcanic lakes to distinguish these lakes, which have analogues worldwide (e.g. Kivu: D.R.C.-Rwanda; Albano, Monticchio and Averno: Italy; Pavin: France) from volcanic lakes only characterized by geogenic CO2 reservoir such as Nyos and Monoun (Cameroon).