Genetic Codes with No Dedicated Stop Codon: Context-Dependent Translation Termination.

The prevailing view of the nuclear genetic code is that it is largely frozen and unambiguous. Flexibility in the nuclear genetic code has been demonstrated in ciliates that reassign standard stop codons to amino acids, resulting in seven variant genetic codes, including three previously undescribed ones reported here. Surprisingly, in two of these species, we find efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. How, therefore, does the translation machinery interpret a "stop" codon? We provide evidence, based on ribosomal profiling and "stop" codon depletion shortly before coding sequence ends, that mRNA 3' ends may contribute to distinguishing stop from sense in a context-dependent manner. We further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution.

Novel evolutionary insights on the interactions of the Holosporales (Alphaproteobacteria) with eukaryotic hosts from comparative genomics.

Holosporales are an alphaproteobacterial order engaging in obligate and complex associations with eukaryotes, in particular protists. The functional and evolutionary features of those interactions are still largely undisclosed. Here, we sequenced the genomes of two members of the species Bealeia paramacronuclearis (Holosporales, Holosporaceae) intracellularly associated with the ciliate protist Paramecium, which resulted in high correspondence. Consistent with the short-branched early-divergent phylogenetic position, Bealeia presents a larger functional repertoire than other Holosporaceae, comparable to those of other Holosporales families, particularly for energy metabolism and motility. Our analyses indicate that different Holosporales likely experienced at least partly autonomous genome reduction and adaptation to host interactions, for example regarding dependence on host biotin driven by multiple independent horizontal acquisitions of transporters. Among Alphaproteobacteria, this is reminiscent of the convergently evolved Rickettsiales, which however appear more diverse, possibly due to a probably more ancient origin. We identified in Bealeia and other Holosporales the plasmid-encoded putative genetic determinants of R-bodies, which may be involved in a killer trait towards symbiont-free hosts. While it is not clear whether these genes are ancestral or recently horizontally acquired, an intriguing and peculiar role of R-bodies is suggested in the evolution of the interactions of multiple Holosporales with their hosts.

"Candidatus Intestinibacterium parameciiphilum"-member of the "Candidatus Paracaedibacteraceae" family (Alphaproteobacteria, Holosporales) inhabiting the ciliated protist Paramecium.

Protists frequently host diverse bacterial symbionts, in particular those affiliated with the order Holosporales (Alphaproteobacteria). All characterised members of this bacterial lineage have been retrieved in obligate association with a wide range of eukaryotes, especially multiple protist lineages (e.g. amoebozoans, ciliates, cercozoans, euglenids, and nucleariids), as well as some metazoans (especially arthropods and related ecdysozoans). While the genus Paramecium and other ciliates have been deeply investigated for the presence of symbionts, known members of the family "Candidatus Paracaedibacteraceae" (Holosporales) are currently underrepresented in such hosts. Herein, we report the description of "Candidatus Intestinibacterium parameciiphilum" within the family "Candidatus Paracaedibacteraceae", inhabiting the cytoplasm of Paramecium biaurelia. This novel bacterium is almost twice as big as its relative "Candidatus Intestinibacterium nucleariae" from the opisthokont Nuclearia and does not present a surrounding halo. Based on phylogenetic analyses of 16S rRNA gene sequences, we identified six further potential species-level lineages within the genus. Based on the provenance of the respective samples, we investigated the environmental distribution of the representatives of "Candidatus Intestinibacterium" species. Obtained results are consistent with an obligate endosymbiotic lifestyle, with protists, in particular freshwater ones, as hosts. Thus, available data suggest that association with freshwater protists could be the ancestral condition for the members of the "Candidatus Intestinibacterium" genus.

'Candidatus Sarmatiella mevalonica' endosymbiont of the ciliate Paramecium provides insights on evolutionary plasticity among Rickettsiales.

Members of the bacterial order Rickettsiales are obligatorily associated with a wide range of eukaryotic hosts. Their evolutionary trajectories, in particular concerning the origin of shared or differential traits among distant sub-lineages, are still poorly understood. Here, we characterized a novel Rickettsiales bacterium associated with the ciliate Paramecium tredecaurelia and phylogenetically related to the Rickettsia genus. Its genome encodes significant lineage-specific features, chiefly the mevalonate pathway gene repertoire, involved in isoprenoid precursor biosynthesis. Not only this pathway has never been described in Rickettsiales, it also is very rare among bacteria, though typical in eukaryotes, thus likely representing a horizontally acquired trait. The presence of these genes could enable an efficient exploitation of host-derived intermediates for isoprenoid synthesis. Moreover, we hypothesize the reversed reactions could have replaced canonical pathways for producing acetyl-CoA, essential for phospholipid biosynthesis. Additionally, we detected phylogenetically unrelated mevalonate pathway genes in metagenome-derived Rickettsiales sequences, likely indicating evolutionary convergent effects of independent horizontal gene transfer events. Accordingly, convergence, involving both gene acquisitions and losses, is highlighted as a relevant evolutionary phenomenon in Rickettsiales, possibly favoured by plasticity and comparable lifestyles, representing a potentially hidden origin of other more nuanced similarities among sub-lineages.

Incorporating mitogenome sequencing into integrative taxonomy: The multidisciplinary redescription of the ciliate Thuricola similis (Peritrichia, Vaginicolidae) provides new insights into the evolutionary relationships among Oligohymenophorea subclasses.

The evolutionary relationships among Oligohymenophorea subclasses are under debate as the phylogenomic analysis using a large dataset of nuclear coding genes is significantly different to the 18S rDNA phylogeny, and it is unfortunately not stable within and across different published studies. In addition to nuclear genes, the faster-evolving mitochondrial genes have also shown the ability to solve phylogenetic problems in many ciliated taxa. However, due to the paucity of mitochondrial data, the corresponding work is scarce, let alone the phylogenomic analysis based on mitochondrial gene dataset. In this work, we presented the characterization on Thuricola similis Bock, 1963, a loricate peritrich (Oligohymenophorea), incorporating mitogenome sequencing into integrative taxonomy. As the first mitogenome for the subclass Peritrichia, it is linear, 38,802 bp long, and contains two rRNAs, 12 tRNAs, and 43 open reading frames (ORFs). As a peculiarity, it includes a central repeated region composed of tandemly repeated A-T rich units working as a bi-transcriptional start. Moreover, taking this opportunity, the phylogenomic analyses based on a set of mitochondrial genes were also performed, revealing that T. similis, as a representative of Peritrichia subclass, branches basally to other three Oligohymenophorea subclasses, namely Hymenostomatia, Peniculia, and Scuticociliatia. Evolutionary relationships among those Oligohymenophorea subclasses were discussed, also in the light of recent phylogenomic reconstructions based on a set of nuclear genes. Besides, as a little-known species, T. similis was also redescribed and neotypified based on data from two populations collected from wastewater treatment plants (WWTPs) in Brazil and Italy, by means of integrative methods (i.e., living observation, silver staining methods, scanning and transmission electron microscopy, and 18S rDNA phylogeny). After emended diagnosis, it is characterized by: (1) the sewage habitat; (2) the lorica with a single valve and small undulations; (3) the 7-22 µm-long inner stalk; and (4) the presence of only a single postciliary microtubule on the left side of the aciliferous row in the haplokinety. Among Vaginicolidae family, our 18S rRNA gene-based phylogenetic analysis revealed that Thuricola and Cothurnia are monophyletic genera, and Vaginicola could be a polyphyletic genus.

The first case of microsporidiosis in Paramecium.

A new microsporidian species, Globosporidium paramecii gen. nov., sp. nov., from Paramecium primaurelia is described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis in Paramecium reported so far. All observed stages of the life cycle are monokaryotic. The parasites develop in the cytoplasm, at least some part of the population in endoplasmic reticulum and its derivates. Meronts divide by binary fission. Sporogonial plasmodium divides by rosette-like budding. Early sporoblasts demonstrate a well-developed exospore forming blister-like structures. Spores with distinctive spherical shape are dimorphic in size (3.7 ± 0.2 and 1.9 ± 0.2 µm). Both types of spores are characterized by a thin endospore, a short isofilar polar tube making one incomplete coil, a bipartite polaroplast, and a large posterior vacuole. Experimental infection was successful for 5 of 10 tested strains of the Paramecium aurelia species complex. All susceptible strains belong to closely related P. primaurelia and P. pentaurelia species. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship to Euplotespora binucleata (a microsporidium from the ciliate Euplotes woodruffi), to Helmichia lacustris and Mrazekia macrocyclopis, microsporidia from aquatic invertebrates.

Genome sequencing and phylogenetic analysis of K1G4: a new Komagataeibacter strain producing bacterial cellulose from different carbon sources.

OBJECTIVE: The objective of this study was to evaluate the ability of a new Komagataeibacter xylinus strain in producing bacterial cellulose from glucose, mannitol and glycerol, and to assess the genome sequencing with special focus on bacterial cellulose related genes. RESULTS: Bacterial cellulose production during 9 days of cultivation was tested in glucose, mannitol and glycerol, respectively. Differences in the bacterial cellulose kinetic formation was observed, with a final yield of 9.47 g/L in mannitol, 8.30 g/L in glycerol and 7.57 g/L in glucose, respectively. The draft genome sequencing of K1G4 was produced, revealing a genome of 3.09 Mbp. Two structurally completed cellulose synthase operons and a third copy of the catalytic subunit of cellulose synthase were found. By using phylogenetic analysis, on the entire rRNA operon sequence, K1G4 was found to be closely related to Komagataeibacter xylinus LMG 1515T and K. xylinus K2G30. CONCLUSIONS: The different yields of bacterial cellulose produced on glucose, mannitol and glycerol can be correlated with the third copy of bcsAB operon harboured by K1G4, making it a versatile strain for industrial applications.

"Candidatus Hafkinia simulans" gen. nov., sp. nov., a Novel Holospora-Like Bacterium from the Macronucleus of the Rare Brackish Water Ciliate Frontonia salmastra (Oligohymenophorea, Ciliophora): Multidisciplinary Characterization of the New Endosymbiont and Its Host.

We characterized a novel Holospora-like bacterium (HLB) (Alphaproteobacteria, Holosporales) living in the macronucleus of the brackish water ciliate Frontonia salmastra. This bacterium was morphologically and ultrastructurally investigated, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and performed in situ hybridization experiments with a specifically-designed probe. A new taxon, "Candidatus Hafkinia simulans", was established for this HLB. The phylogeny of the family Holosporaceae based on 16S rRNA gene sequences was inferred, adding to the already available data both the sequence of the novel bacterium and those of other Holospora and HLB species recently characterized. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and placed the new endosymbiont as the sister genus of Holospora. Additionally, the host ciliate F. salmastra, recorded in Europe for the first time, was concurrently described through a multidisciplinary study. Frontonia salmastra's phylogenetic position in the subclass Peniculia and the genus Frontonia was assessed according to 18S rRNA gene sequencing. Comments on the biodiversity of this genus were added according to past and recent literature.

The Hidden World of Rickettsiales Symbionts: "Candidatus Spectririckettsia obscura," a Novel Bacterium Found in Brazilian and Indian Paramecium caudatum.

Symbioses between bacteria and eukaryotes are widespread and may have significant impact on the evolutionary history of symbiotic partners. The order Rickettsiales is a lineage of intracellular Alphaproteobacteria characterized by an obligate association with a wide range of eukaryotic hosts, including several unicellular organisms, such as ciliates and amoebas. In this work, we characterized the Rickettsiales symbionts associated with two different genotypes of the freshwater ciliate Paramecium caudatum originated from freshwater environments in distant geographical areas. Phylogenetic analyses based on 16S rRNA gene showed that the two symbionts are closely related to each other (99.4% identity), belong to the family Rickettsiaceae, but are far-related with respect to previously characterized Rickettsiales. Consequently, they were assigned to a new species of a novel genus, namely "Candidatus Spectririckettsia obscura." Screening on a database of short reads from 16S rRNA gene amplicon-based profiling studies confirmed that bacterial sequences related to the new symbiont are preferentially retrieved from freshwater environments, apparently with extremely scarce occurrence (< 0.1% positive samples). The present work provides new information on the still under-explored biodiversity of Rickettsiales, in particular those associated to ciliate host cells.

Morphology and Phylogenetic Position of Gruberia lanceolata (Gruber 1884) (Ciliophora, Heterotrichea) from Rio de Janeiro, Brazil.

Ciliates of the genus Gruberia are poorly studied. Consequently, most species lack detailed morphological descriptions, and all gene sequences in GenBank are not classified at the species level. In this study, a detailed morphological description of a population of G. lanceolata from Brazil is presented, based on live and protargol-stained organisms. We also present the 18S rRNA gene sequence and the phylogenetic position of this species. The primary characteristics of G. lanceolata from the Maricá Lagoon are as follows: an elongate fusiform body 280-870 × 40-160 µm in size; rosy cortical granules; a peristome occupying approximately 1/3-1/2 of body length; an adoral zone comprising 115-330 membranelles; a paroral membrane in 35-50 fragments; and a moniliform macronucleus with 11-16 nodules. Based on our observations and data from pertinent literature, we suggest G. beninensis to be a junior synonym of G. lanceolata.

Strengths and Biases of High-Throughput Sequencing Data in the Characterization of Freshwater Ciliate Microbiomes.

Molecular surveys of eukaryotic microbial communities employing high-throughput sequencing (HTS) techniques are rapidly supplanting traditional morphological approaches due to their larger data output and reduced bench work time. Here, we directly compare morphological and Illumina data obtained from the same samples, in an effort to characterize ciliate faunas from sediments in freshwater environments. We show how in silico processing affects the final outcome of our HTS analysis, providing evidence that quality filtering protocols strongly impact the number of predicted taxa, but not downstream conclusions such as biogeography patterns. We determine the abundance distribution of ciliates, showing that a small fraction of abundant taxa dominates read counts. At the same time, we advance reasons to believe that biases affecting HTS abundances may be significant enough to blur part of the underlying biological picture. We confirmed that the HTS approach detects many more taxa than morphological inspections, and highlight how the difference varies among taxonomic groups. Finally, we hypothesize that the two datasets actually correspond to different conceptions of "diversity," and consequently that neither is entirely superior to the other when investigating environmental protists.

Disentangling the Taxonomy of Rickettsiales and Description of Two Novel Symbionts ("Candidatus Bealeia paramacronuclearis" and "Candidatus Fokinia cryptica") Sharing the Cytoplasm of the Ciliate Protist Paramecium biaurelia.

In the past 10 years, the number of endosymbionts described within the bacterial order Rickettsiales has constantly grown. Since 2006, 18 novel Rickettsiales genera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts from Paramecium collected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium "Candidatus Bealeia paramacronuclearis" occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, "Candidatus Fokinia cryptica," whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that "Candidatus Bealeia paramacronuclearis" clusters with the so-called "basal" Rickettsiales, and "Candidatus Fokinia cryptica" belongs to "Candidatus Midichloriaceae." We obtained tree topologies showing a separation of Rickettsiales into at least two groups: one represented by the families Rickettsiaceae, Anaplasmataceae, and "Candidatus Midichloriaceae" (RAM clade), and the other represented by "basal Rickettsiales," including "Candidatus Bealeia paramacronuclearis." Therefore, and in accordance with recent publications, we propose to limit the order Rickettsiales to the RAM clade and to raise "basal Rickettsiales" to an independent order, Holosporales ord. nov., inside Alphaproteobacteria, which presently includes four family-level clades. Additionally, we define the family "Candidatus Hepatincolaceae" and redefine the family Holosporaceae IMPORTANCE: In this paper, we provide the characterization of two novel bacterial symbionts inhabiting the same Paramecium host (Ciliophora, Alveolata). Both symbionts belong to "traditional" Rickettsiales, one representing a new species of the genus "Candidatus Fokinia" ("Candidatus Midichloriaceae"), and the other representing a new genus of a "basal" Rickettsiales According to newly characterized sequences and to a critical revision of recent literature, we propose a taxonomic reorganization of "traditional" Rickettsiales that we split into two orders: Rickettsiales sensu stricto and Holosporales ord. nov. This work represents a critical revision, including new records of a group of symbionts frequently occurring in protists and whose biodiversity is still largely underestimated.

A House for Two--Double Bacterial Infection in Euplotes woodruffi Sq1 (Ciliophora, Euplotia) Sampled in Southeastern Brazil.

Several ciliated protists form symbiotic associations with a diversity of microorganisms, leading to drastic impact on their ecology and evolution. In this work, two Euplotes spp. sampled in Rio de Janeiro, Brazil, were identified based on morphological and molecular features as Euplotes woodruffi strain Sq1 and E. encysticus strain Sq2 and investigated for the presence of endosymbionts. While E. woodruffi Sq1 stably hosts two bacterial populations, namely Polynucleobacter necessarius (Betaproteobacteria) and a new member of the family "Candidatus Midichloriaceae" (Alphaproteobacteria, Rickettsiales), here described as "Candidatus Bandiella woodruffii," branching with a broad host range bacterial group found in association with cnidarians, sponges, euglenoids, and some arthropods; in E. encysticus Sq2 no symbiotic bacterium could be detected. The dispersion ability of this novel bacterium was tested by co-incubating E. woodruffi Sq1 with three different ciliate species. Among the tested strains "Ca. B. woodruffii" could only be detected in association with E. encysticus Sq2 with a prevalence of 20 % after 1 week and 40 % after 2 weeks, maintaining this level for up to 6 months. Nevertheless, this apparent in vitro association was abolished when E. woodruffi Sq1 donor was removed from the microcosm, suggesting that this bacterium has the capacity for at least a short-term survival outside its natural host and the aptitude to ephemerally interact with other organisms. Together, these findings strongly suggest the need for more detailed investigations to evaluate the host range for "Ca. B. woodruffii" and any possible pathogenic effect of this bacterium on other organisms including humans.

Evaluation of Enrichment Protocols for Bacterial Endosymbionts of Ciliates by Real-Time PCR.

Large-scale studies on obligate bacterial endosymbionts may frequently require preliminary purification and enrichment protocols, which are often elaborate to set up and to evaluate, especially if the host organism is a protist. The purpose of this study was to develop a real-time PCR-based strategy and employ it for assessing two of such enrichment protocols for Holospora caryophila, hosted by the ciliate Paramecium. Four SSU rRNA gene-targeted real-time PCR assays were designed, which allowed to compare the amount of H. caryophila to other organisms, namely the host, its food bacterium (Raoultella planticola), and free-living bacteria present in the culture medium. By the use of the real-time PCR assays in combination, it was possible to conclude that the "cell fractionation" protocol was quite successful in the enrichment of the symbiont, while the "Percoll gradient" protocol will need further refinements to be fully repeatable. The proposed approach has the potential to facilitate and encourage future studies on the yet underexplored field of bacterial endosymbionts of ciliates and other protists. It can also find valuable applications for experimental questions other than those tested, such as fast and precise assessment of symbiont abundance in natural populations and comparison among multiple coexisting symbionts.

Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria.

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.

Microbial Consortium Associated with the Antarctic Marine Ciliate Euplotes focardii: An Investigation from Genomic Sequences.

We report the characterization of the bacterial consortium associated to Euplotes focardii, a strictly psychrophilic marine ciliate that was maintained in laboratory cultures at 4 °C after its first isolation from Terra Nova Bay, in Antarctica. By Illumina genome analyser, we obtained 11,179 contigs of potential prokaryotic origin and classified them according to the NCBI's prokaryotic attributes table. The majority of these sequences correspond to either Bacteroidetes (16 %) or Proteobacteria (78 %). The latter were dominated by gamma- (39 %, including sequences related to the pathogenic genus Francisella), and alpha-proteobacterial (30 %) sequences. Analysis of the Pfam domain family and Gene Ontology term variation revealed that the most frequent terms that appear unique to this consortium correspond to proteins involved in "transmembrane transporter activity" and "oxidoreductase activity". Furthermore, we identified genes that encode for enzymes involved in the catabolism of complex substance for energy reserves. We also characterized members of the transposase and integrase superfamilies, whose role in bacterial evolution is well documented, as well as putative antifreeze proteins. Antibiotic treatments of E. focardii cultures delayed the cell division of the ciliate. To conclude, our results indicate that this consortium is largely represented by bacteria derived from the original Antarctic sample and may contribute to the survival of E. focardii in laboratory condition. Furthermore, our results suggest that these bacteria may have a more general role in E. focardii survival in its natural cold and oxidative environment.

Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton.

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as "bacterial microtubules" (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening.

Molecular phylogenetic position and description of a new genus and species of freshwater Chaetonotidae (Gastrotricha: Chaetonotida: Paucitubulatina), and the annotation of its mitochondrial genome.

Chaetonotidae is the most diversified family of the entire phylum Gastrotricha; it comprises ~430 species distributed across 16 genera. The current classification, established mainly on morphological traits, has been challenged in recent years by phylogenetic studies, indicating that the cuticular ornamentations used to discriminate among species may be misleading when used to identify groupings, which has been the practice until now. Therefore, a consensus is developing toward implementing novel approaches to better define species identity and affiliation at a higher taxonomic ranking. Using an integrative morphological and molecular approach, including annotation of the mitogenome, we report on some freshwater gastrotrichs characterised by a mixture of two types of cuticular scales diagnostic of the genera Aspidiophorus and Heterolepidoderma . Our specimens' overall anatomical characteristics find no correspondence in the taxa of these two genera, calling for their affiliation to a new species. Phylogenetic analyses based on the sequence of the ribosomal RNA genes of 96 taxa consistently found the new species unrelated to Aspidiophorus or Heterolepidoderma but allied with Chaetonotus aff. subtilis, as a subset of a larger clade, including mostly planktonic species. Morphological uniqueness and position along the non-monophyletic Chaetonotidae branch advocate erecting a new genus to accommodate the current specimens; consequently, the name Litigonotus ghinii gen. nov., sp. nov. is proposed. The complete mitochondrial genome of the new taxon resulted in a single circular molecule 14,384 bp long, including 13 protein-coding genes, 17 tRNA genes and 2 rRNAs genes, showing a perfect synteny and collinearity with the only other gastrotrich mitogenome available, a possible hint of a high level of conservation in the mitochondria of Chaetonotidae. ZooBank: urn:lsid:zoobank.org:pub:9803F659-306F-4EC3-A73B-8C704069F24A.

Host association and intracellularity evolved multiple times independently in the Rickettsiales.

The order Rickettsiales (Alphaproteobacteria) encompasses multiple diverse lineages of host-associated bacteria, including pathogens, reproductive manipulators, and mutualists. Here, in order to understand how intracellularity and host association originated in this order, and whether they are ancestral or convergently evolved characteristics, we built a large and phylogenetically-balanced dataset that includes de novo sequenced genomes and a selection of published genomic and metagenomic assemblies. We perform detailed functional reconstructions that clearly indicates "late" and parallel evolution of obligate host-association in different Rickettsiales lineages. According to the depicted scenario, multiple independent horizontal acquisitions of transporters led to the progressive loss of biosynthesis of nucleotides, amino acids and other metabolites, producing distinct conditions of host-dependence. Each clade experienced a different pattern of evolution of the ancestral arsenal of interaction apparatuses, including development of specialised effectors involved in the lineage-specific mechanisms of host cell adhesion and/or invasion.

Marine copepod culture as a potential source of bioplastic-degrading microbiome: The case of poly(butylene succinate-co-adipate).

The poly(butylene succinate-co-adipate) (PBSA) is emerging as environmentally sustainable polyester for applications in marine environment. In this work the capacity of microbiome associated with marine plankton culture to degrade PBSA, was tested. A taxonomic and functional characterization of the microbiome associated with the copepod Acartia tonsa, reared in controlled conditions, was analysed by 16S rDNA metabarcoding, in newly-formed adult stages and after 7 d of incubation. A predictive functional metagenomic profile was inferred for hydrolytic activities involved in bioplastic degradation with a particular focus on PBSA. The copepod-microbiome was also characterized in newly-formed carcasses of A. tonsa, and after 7 and 33 d of incubation in the plankton culture medium. Copepod-microbiome showed hydrolytic activities at all developmental stages of the alive copepods and their carcasses, however, the evenness of the hydrolytic bacterial community significantly increased with the time of incubation in carcasses. Microbial genera, never described in association with copepods: Devosia, Kordia, Lentibacter, Methylotenera, Rheinheimera, Marinagarivorans, Paraglaciecola, Pseudophaeobacter, Gaiella, Streptomyces and Kribbella sps., were retrieved. Kribbella sp. showed carboxylesterase activity and Streptomyces sp. showed carboxylesterase, triacylglycerol lipase and cutinase activities, that might be involved in PBSA degradation. A culturomic approach, adopted to isolate bacterial specimen from carcasses, led to the isolation of the bacterial strain, Vibrio sp. 01 tested for the capacity to promote the hydrolysis of the ester bonds. Granules of PBSA, incubated 82 d at 20 °C with Vibrio sp. 01, were characterized by scanning electron microscopy, infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry, showing fractures compared to the control sample, and hydrolysis of ester bonds. These preliminary results are encouraging for further investigation on the ability of the microbiome associated with plankton to biodegrade polyesters, such as PBSA, and increasing knowledge on microorganisms involved in bioplastic degradation in marine environment.