Symbiotic relationships between eukaryotes and prokaryotes played pivotal roles in the evolution of life and drove the emergence of specialized symbiotic structures in animals, plants and fungi. The host-evolved symbiotic structures of microbial eukaryotes - the vast majority of such hosts in nature - remain largely unstudied. Here we describe highly structured symbiosomes within three free-living anaerobic protists (Anaeramoeba spp.). We dissect this symbiosis using complete genome sequencing and transcriptomics of host and symbiont cells coupled with fluorescence in situ hybridization, and 3D reconstruction using focused-ion-beam scanning electron microscopy. The emergence of the symbiosome is underpinned by expansion of gene families encoding regulators of membrane trafficking and phagosomal maturation and extensive bacteria-to-eukaryote lateral transfer. The symbionts reside deep within a symbiosomal membrane network that enables metabolic syntrophy by precisely positioning sulfate-reducing bacteria alongside host hydrogenosomes. Importantly, the symbionts maintain connections to the Anaeramoeba plasma membrane, blurring traditional boundaries between ecto- and endosymbiosis.

• MeSH
• anaerobióza MeSH
• Eukaryota genetika   metabolismus   MeSH
• fylogeneze MeSH
• hybridizace in situ fluorescenční MeSH
• mikroskopie elektronová rastrovací MeSH
• přenos genů horizontální MeSH
• symbióza * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• Endomicrobiaceae, Parabasalia, convergent evolution, endosymbionts, lateral gene transfer, termites,
• MeSH
• Bacteria * genetika   klasifikace   MeSH
• fylogeneze * MeSH
• genom bakteriální * MeSH
• Isoptera * mikrobiologie   parazitologie   MeSH
• metagenom MeSH
• molekulární evoluce MeSH
• přenos genů horizontální * MeSH
• střevní mikroflóra * MeSH
• symbióza * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

• Klíčová slova
• biodiversity, diptera, heteroptera, host specificity, monoxenous trypanosomatids, multiple infections, nanopore sequencing, phylogeny, systematics,
• MeSH
• Diptera genetika   MeSH
• fylogeneze * MeSH
• Hemiptera parazitologie   genetika   MeSH
• koinfekce * parazitologie   MeSH
• protozoální DNA genetika   analýza   MeSH
• RNA ribozomální 18S * genetika   analýza   MeSH
• Trypanosomatina * genetika   klasifikace   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Kuba epidemiologie   MeSH
• Názvy látek
• protozoální DNA MeSH
• RNA ribozomální 18S * MeSH

The association between anaerobic ciliates and methanogenic archaea has been recognized for over a century. Nevertheless, knowledge of these associations is limited to a few ciliate species, and so the identification of patterns of host-symbiont specificity has been largely speculative. In this study, we integrated microscopy and genetic identification to survey the methanogenic symbionts of 32 free-living anaerobic ciliate species, mainly from the order Metopida. Based on Sanger and Illumina sequencing of the 16S rRNA gene, our results show that a single methanogenic symbiont population, belonging to Methanobacterium, Methanoregula, or Methanocorpusculum, is dominant in each host strain. Moreover, the host's taxonomy (genus and above) and environment (i.e. endobiotic, marine/brackish, or freshwater) are linked with the methanogen identity at the genus level, demonstrating a strong specificity and fidelity in the association. We also established cultures containing artificially co-occurring anaerobic ciliate species harboring different methanogenic symbionts. This revealed that the host-methanogen relationship is stable over short timescales in cultures without evidence of methanogenic symbiont exchanges, although our intraspecific survey indicated that metopids also tend to replace their methanogens over longer evolutionary timescales. Therefore, anaerobic ciliates have adapted a mixed transmission mode to maintain and replace their methanogenic symbionts, allowing them to thrive in oxygen-depleted environments.

• Klíčová slova
• anaerobiosis, archaea, endosymbionts, methane, symbiosis, syntrophy, transmission mode,
• MeSH
• anaerobióza MeSH
• Ciliophora * klasifikace   genetika   fyziologie   MeSH
• DNA archebakterií genetika   MeSH
• ekosystém * MeSH
• fylogeneze * MeSH
• methan * metabolismus   MeSH
• RNA ribozomální 16S * genetika   MeSH
• sekvenční analýza DNA MeSH
• symbióza * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA archebakterií MeSH
• methan * MeSH
• RNA ribozomální 16S * MeSH

Ciliates are a diverse group of protists known for their ability to establish various partnerships and thrive in a wide variety of oxygen-depleted environments. Most anaerobic ciliates harbor methanogens, one of the few known archaea living intracellularly. These methanogens increase the metabolic efficiency of host fermentation via syntrophic use of host end-product in methanogenesis. Despite the ubiquity of these symbioses in anoxic habitats, patterns of symbiont specificity and fidelity are not well known. We surveyed two unrelated, commonly found groups of anaerobic ciliates, the Plagiopylea and Metopida, isolated from anoxic marine sediments. We sequenced host 18S rRNA and symbiont 16S rRNA marker genes as well as the symbiont internal transcribed spacer region from our cultured ciliates to identify hosts and their associated methanogenic symbionts. We found that marine ciliates from both of these co-occurring, divergent groups harbor closely related yet distinct intracellular archaea within the Methanocorpusculum genus. The symbionts appear to be stable at the host species level, but at higher taxonomic levels, there is evidence that symbiont replacements have occurred. Gaining insight into this unique association will deepen our understanding of the complex transmission modes of marine microbial symbionts, and the mutualistic microbial interactions occurring across domains of life.

• Klíčová slova
• Metopida, Plagiopylea, anaerobic protists, anoxic sediments, archaea, methanogens, symbiosis, syntrophy,
• MeSH
• anaerobióza MeSH
• Ciliophora * klasifikace   genetika   fyziologie   MeSH
• DNA archebakterií genetika   chemie   MeSH
• fylogeneze * MeSH
• geologické sedimenty * mikrobiologie   MeSH
• mořská voda mikrobiologie   parazitologie   MeSH
• RNA ribozomální 16S * genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• sekvenční analýza DNA MeSH
• symbióza * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA archebakterií MeSH
• RNA ribozomální 16S * MeSH
• RNA ribozomální 18S MeSH

Gene transfer agents (GTAs) are virus-like structures that package and transfer prokaryotic DNA from donor to recipient prokaryotic cells. Here, we describe widespread GTA gene clusters in the highly reduced genomes of bacterial endosymbionts from microbial eukaryotes (protists). Homologs of the GTA capsid and portal complexes were initially found to be present in several highly reduced alphaproteobacterial endosymbionts of diplonemid protists (Rickettsiales and Rhodospirillales). Evidence of GTA expression was found in polyA-enriched metatranscriptomes of the diplonemid hosts and their endosymbionts, but due to biases in the polyA-enrichment methods, levels of GTA expression could not be determined. Examining the genomes of closely related bacteria revealed that the pattern of retained GTA head/capsid complexes with missing tail components was common across Rickettsiales and Holosporaceae (Rhodospirillales), all obligate symbionts with a wide variety of eukaryotic hosts. A dN/dS analysis of Rickettsiales and Holosporaceae symbionts revealed that purifying selection is likely the main driver of GTA evolution in symbionts, suggesting they remain functional, but the ecological function of GTAs in bacterial symbionts is unknown. In particular, it is unclear how increasing horizontal gene transfer in small, largely clonal endosymbiont populations can explain GTA retention, and, therefore, the structures may have been repurposed in endosymbionts for host interactions. Either way, their widespread retention and conservation in endosymbionts of diverse eukaryotes suggests an important role in symbiosis.

• Klíčová slova
• Holosporaceae, Rickettsiales, endosymbiosis, evolution, gene transfer agent, protist,
• MeSH
• Bacteria genetika   MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• přenos genů horizontální MeSH
• symbióza genetika   MeSH
• viry * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The symbiont-associated (SA) environmental package is a new extension to the minimum information about any (x) sequence (MIxS) standards, established by the Parasite Microbiome Project (PMP) consortium, in collaboration with the Genomics Standard Consortium. The SA was built upon the host-associated MIxS standard, but reflects the nestedness of symbiont-associated microbiota within and across host-symbiont-microbe interactions. This package is designed to facilitate the collection and reporting of a broad range of metadata information that apply to symbionts such as life history traits, association with one or multiple host organisms, or the nature of host-symbiont interactions along the mutualism-parasitism continuum. To better reflect the inherent nestedness of all biological systems, we present a novel feature that allows users to co-localize samples, to nest a package within another package, and to identify replicates. Adoption of the MIxS-SA and of the new terms will facilitate reports of complex sampling design from a myriad of environments.

• Publikační typ
• časopisecké články MeSH

Eukaryotic organelles supposedly evolved from their bacterial ancestors because of their benefits to host cells. However, organelles are quite often retained, even when the beneficial metabolic pathway is lost, due to something other than the original beneficial function. The organellar function essential for cell survival is, in the end, the result of organellar evolution, particularly losses of redundant metabolic pathways present in both the host and endosymbiont, followed by a gradual distribution of metabolic functions between the organelle and host. Such biological division of metabolic labor leads to mutual dependence of the endosymbiont and host. Changing environmental conditions, such as the gradual shift of an organism from aerobic to anaerobic conditions or light to dark, can make the original benefit useless. Therefore, it can be challenging to deduce the original beneficial function, if there is any, underlying organellar acquisition. However, it is also possible that the organelle is retained because it simply resists being eliminated or digested untill it becomes indispensable.

• Klíčová slova
• benefit, endosymbiosis, essential function, mitochondrion, organelle, plastid,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• Klíčová slova
• ecology, genomics, holobiont concept, metabolism, prokaryotic symbionts, protists,
• Publikační typ
• úvodníky MeSH