Despite an increased understanding of functions in sponge microbiomes, the interactions among the symbionts and between symbionts and host are not well characterized. Here we reconstructed the metabolic interactions within the sponge Cymbastela concentrica microbiome in the context of functional features of symbiotic diatoms and the host. Three genome bins (CcPhy, CcNi and CcThau) were recovered from metagenomic data of C. concentrica, belonging to the proteobacterial family Phyllobacteriaceae, the Nitrospira genus and the thaumarchaeal order Nitrosopumilales. Gene expression was estimated by mapping C. concentrica metatranscriptomic reads. Our analyses indicated that CcPhy is heterotrophic, while CcNi and CcThau are chemolithoautotrophs. CcPhy expressed many transporters for the acquisition of dissolved organic compounds, likely available through the sponge's filtration activity and symbiotic carbon fixation. Coupled nitrification by CcThau and CcNi was reconstructed, supported by the observed close proximity of the cells in fluorescence in situ hybridization. CcPhy facultative anaerobic respiration and assimilation by diatoms may consume the resulting nitrate. Transcriptional analysis of diatom and sponge functions indicated that these organisms are likely sources of organic compounds, for example, creatine/creatinine and dissolved organic carbon, for other members of the symbiosis. Our results suggest that organic nitrogen compounds, for example, creatine, creatinine, urea and cyanate, fuel the nitrogen cycle within the sponge. This study provides an unprecedented view of the metabolic interactions within sponge-microbe symbiosis, bridging the gap between cell-and community-level knowledge.

Background Kissing bugs (Triatominae) are blood-feeding insects best known as the vectors of Trypanosoma cruzi, the causative agent of Chagas’ disease. Considering the high epidemiological relevance of these vectors, their biology and bacterial symbiosis remains surprisingly understudied. While previous investigations revealed generally low individual complexity but high among-individual variability of the triatomine microbiomes, any consistent microbiome determinants have not yet been identified across multiple Triatominae species. Methods To obtain a more comprehensive view of triatomine microbiomes, we investigated the host-microbiome relationship of five Triatoma species sampled from white-throated woodrat (Neotoma albigula) nests in multiple locations across the USA. We applied optimised 16S rRNA gene metabarcoding with a novel 18S rRNA gene blocking primer to a set of 170 T. cruzi-negative individuals across all six instars. Results Triatomine gut microbiome composition is strongly influenced by three principal factors: ontogeny, species identity, and the environment. The microbiomes are characterised by significant loss in bacterial diversity throughout ontogenetic development. First instars possess the highest bacterial diversity while adult microbiomes are routinely dominated by a single taxon. Primarily, the bacterial genus Dietzia dominates late-stage nymphs and adults of T. rubida, T. protracta, and T. lecticularia but is not present in the phylogenetically more distant T. gerstaeckeri and T. sanguisuga. Species-specific microbiome composition, particularly pronounced in early instars, is further modulated by locality-specific effects. In addition, pathogenic bacteria of the genus Bartonella, acquired from the vertebrate hosts, are an abundant component of Triatoma microbiomes. Conclusion Our study is the first to demonstrate deterministic patterns in microbiome composition among all life stages and multiple Triatoma species. We hypothesise that triatomine microbiome assemblages are produced by species- and life stage-dependent uptake of environmental bacteria and multiple indirect transmission strategies that promote bacterial transfer between individuals. Altogether, our study highlights the complexity of Triatominae symbiosis with bacteria and warrant further investigation to understand microbiome function in these important vectors.

Despite the development of several cultivation methods, the rate of discovery of microorganisms that are yet-to-be cultivated outpaces the rate of isolating and cultivating novel species in the laboratory. Furthermore, no current cultivation technique is capable of selectively isolating and cultivating specific bacterial taxa or phylogenetic groups independently of morphological or physiological properties. Here, we developed a new method to isolate living bacteria solely based on their 16S rRNA gene sequence. We showed that bacteria can survive a modified version of the standard fluorescence in situ hybridization (FISH) procedure, in which fixation is omitted and other factors, such as centrifugation and buffers, are optimized. We also demonstrated that labelled DNA probes can be introduced into living bacterial cells by means of chemical transformation and that specific hybridization occurs. This new method, which we call live-FISH, was then combined with fluorescence-activated cell sorting (FACS) to sort specific taxonomic groups of bacteria from a mock and natural bacterial communities and subsequently culture them. Live-FISH represents the first attempt to systematically optimize conditions known to affect cell viability during FISH and then to sort bacterial cells surviving the procedure. No sophisticated probe design is required, making live-FISH a straightforward method to be potentially used in combination with other single-cell techniques and for the isolation and cultivation of new microorganisms.

BackgroundKissing bugs (Triatominae) are blood-feeding insects best known as the vectors of Trypanosoma cruzi, the causative agent of Chagas' disease. Considering the high epidemiological relevance of these vectors, their biology and bacterial symbiosis remains surprisingly understudied. While previous investigations revealed generally low individual complexity but high among-individual variability of the triatomine microbiomes, any consistent microbiome determinants have not yet been identified across multiple Triatominae species. Methods To obtain a more comprehensive view of triatomine microbiomes, we investigated the host-microbiome relationship of five Triatoma species sampled from white-throated woodrat (Neotoma albigula) nests in multiple locations across the USA. We applied optimized 16S rRNA gene metabarcoding with a novel 18S rRNA gene blocking primer to a set of 170 T. cruzi negative individuals across all six instars. Results Triatomine gut microbiome composition is strongly influenced by three principal factors: ontogeny, species identity, and the environment. The microbiomes are characterised by significant loss in bacterial diversity throughout ontogenetic development. First instars possess the highest bacterial diversity while adult microbiomes are routinely dominated by a single taxon. Primarily, the bacterial genus Dietzia dominates late-stage nymphs and adults of T. rubida, T. protracta, and T. lecticularia, but is not present in the phylogenetically more distant T. gerstaeckeri and T. sanguisuga. Species-specific microbiome composition, particularly pronounced in early instars, is further modulated by locality-specific effects. In addition, pathogenic bacteria of the genus Bartonella, acquired from the vertebrate hosts, are an abundant component of Triatoma microbiomes. ConclusionOur study is the first to demonstrate deterministic patterns in microbiome composition among all life stages and multiple Triatoma species. We hypothesize that triatomine microbiome assemblages are produced by species-and life stage-dependent uptake of environmental bacteria and multiple indirect transmission strategies that promote bacterial transfer between individuals. Altogether, our study highlights the complexity of Triatominae symbiosis with bacteria and warrant further investigation to understand microbiome function in these important vectors.