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The phylum Ciliophora plays important roles in a wide range of biological studies. However, the evolutionary relationships of many groups remain unclear due to a lack of sufficient molecular data. In this study, molecular dataset was expanded with representatives from 55 orders and all major lineages. The main findings are: (1) 14 classes were recovered including one new class, Protocruziea n. cl.; (2) in addition to the two main branches, Postciliodesmatophora and Intramacronucleata, a third branch, the Mesodiniea, is identified as being basal to the other two subphyla; (3) the newly defined order Discocephalida is revealed to be a sister clade to the euplotids, strongly suggesting the separation of discocephalids from the hypotrichs; (4) the separation of mobilids from the peritrichs is not supported; (5) Loxocephalida is basal to the main scuticociliate assemblage, whereas the thigmotrichs are placed within the order Pleuronematida; (6) the monophyly of classes Phyllopharyngea, Karyorelictea, Armophorea, Prostomatea, Plagiopylea, Colpodea and Heterotrichea are confirmed; (7) ambiguous genera Askenasia , CyclotrichiumParaspathidium and Plagiocampa show close affiliation to the well known plagiopyleans; (8) validity of the subclass Rhynchostomatia is supported and (9) the systematic positions of Halteriida and Linconophoria remain unresolved and are thus regarded as incertae sedis within Spirotrichea.

Losses and gains in species diversity affect ecological stability 1 – 7 and the sustainability of ecosystem functions and services 8 – 13 . Experiments and models have revealed positive, negative and no effects of diversity on individual components of stability, such as temporal variability, resistance and resilience 2 , 3 , 6 , 11 , 12 , 14 . How these stability components covary remains poorly understood 15 . Similarly, the effects of diversity on overall ecosystem stability 16 , which is conceptually akin to ecosystem multifunctionality 17 , 18 , remain unknown. Here we studied communities of aquatic ciliates to understand how temporal variability, resistance and overall ecosystem stability responded to diversity (that is, species richness) in a large experiment involving 690 micro-ecosystems sampled 19 times over 40 days, resulting in 12,939 samplings. Species richness increased temporal stability but decreased resistance to warming. Thus, two stability components covaried negatively along the diversity gradient. Previous biodiversity manipulation studies rarely reported such negative covariation despite general predictions of the negative effects of diversity on individual stability components 3 . Integrating our findings with the ecosystem multifunctionality concept revealed hump- and U-shaped effects of diversity on overall ecosystem stability. That is, biodiversity can increase overall ecosystem stability when biodiversity is low, and decrease it when biodiversity is high, or the opposite with a U-shaped relationship. The effects of diversity on ecosystem multifunctionality would also be hump- or U-shaped if diversity had positive effects on some functions and negative effects on others. Linking the ecosystem multifunctionality concept and ecosystem stability can transform the perceived effects of diversity on ecological stability and may help to translate this science into policy-relevant information. Species richness was found to increase temporal stability but decrease resistance to warming in an experiment involving 690 micro-ecosystems consisting of 1 to 6 species of bacterivorous ciliates that were sampled over 40 days.

Lactococcus garvieae has recently been identified and listed as one of the causative agents of hyperacute hemorrhagic sepsis in fish. In intensive recirculating aquaculture systems where there are high fish densities and minimal water changes, not only will it be conducive to the growth of bacteria, but Cryptocaryon irritans as a marine protozoan fish parasite is also prone to appear. This study reports the disease status of Trachinotus ovatus in an aquaculture area in Yangjiang City, Guangdong Province. Through the diagnosis of clinical symptoms of the diseased fish, identification of specific primers, 16s rRNA sequences phylogenetic tree analysis, physiological and biochemical identification, and observation of histopathological sections, the result of the experiment is that the mass death of T . ovatus is caused by a mixture of L . garvieae and C . irritants infections. Subsequently, regression infection experiments were performed to verify Koch’s law. It was confirmed that the pathogen had strong virulence to T . ovatus . This is the first time that the co-infection of L . garvieae and C . irritans to T . ovatus was found in South China. The research results of this experiment have certain enlightenment significance for the epidemic trend of fish diseases in relevant sea areas.

We investigated the food-dependent growth and thermal response of the freshwater ciliate Colpidium kleini using numerical response (NR) experiments. This bacterivorous ciliate occurs in lotic water and the pelagial of lakes and ponds. The C. kleini strain used in this work was isolated from a small alpine lake and identified by combining detailed morphological inspections with molecular phylogeny. Specific growth rates ( r max ) were measured from 5 to 21 °C. The ciliate did not survive at 22 °C. The threshold bacterial food levels (0.3 − 2.2 × 10 6 bacterial cells mL −1 ) matched the bacterial abundance in the alpine lake from which C. kleini was isolated. The food threshold was notably lower than previously reported for C. kleini and two other Colpidium species. The threshold was similar to levels reported for oligotrich and choreotrich ciliates if expressed in terms of bacterial biomass (0.05 − 0.43 mg C L −1 ). From the NR results, we calculated physiological mortality rates at zero food concentration. The mean mortality (0.55 ± 0.17 d −1 ) of C. kleini was close to the mean estimate obtained for other planktonic ciliates that do not encyst. We used the data obtained by the NR experiments to fit a thermal performance curve (TPC). The TPC yielded a temperature optimum at 17.3 °C for C. kleini , a maximum upper thermal tolerance limit of 21.9 °C, and a thermal safety margin of 4.6 °C. We demonstrated that combining NR with TPC analysis is a powerful tool to predict better a species’ fitness in response to temperature and food.

Coral diseases have been increasingly reported over the past few decades and are a major contributor to coral decline worldwide. The Caribbean, in particular, has been noted as a hotspot for coral disease, and the aptly named white syndromes have caused the decline of the dominant reef building corals throughout their range. White band disease (WBD) has been implicated in the dramatic loss of Acropora cervicornis and Acropora palmata since the 1970s, resulting in both species being listed as critically endangered on the International Union for Conservation of Nature Red list. The causal agent of WBD remains unknown, although recent studies based on challenge experiments with filtrate from infected hosts concluded that the disease is probably caused by bacteria. Here, we report an experiment using four different antibiotic treatments, targeting different members of the disease-associated microbial community. Two antibiotics, ampicillin and paromomycin, arrested the disease completely, and by comparing with community shifts brought about by treatments that did not arrest the disease, we have identified the likely candidate causal agent or agents of WBD. Our interpretation of the experimental treatments is that one or a combination of up to three specific bacterial types, detected consistently in diseased corals but not detectable in healthy corals, are likely causal agents of WBD. In addition, a histophagous ciliate (Philaster lucinda) identical to that found consistently in association with white syndrome in Indo-Pacific acroporas was also consistently detected in all WBD samples and absent in healthy coral. Treatment with metronidazole reduced it to below detection limits, but did not arrest the disease. However, the microscopic disease signs changed, suggesting a secondary role in disease causation for this ciliate. In future studies to identify a causal agent of WBD via tests of Henle–Koch's postulates, it will be vital to experimentally control for populations of the other potential pathogens identified in this study.

The ruminal microbiome, comprising large numbers of bacteria, ciliate protozoa, archaea and fungi, responds to diet and dietary additives in a complex way. The aim of this study was to investigate the benefits of increasing the depth of the community analysis in describing and explaining responses to dietary changes. Quantitative PCR, ssu rRNA amplicon based taxa composition, diversity and co-occurrence network analyses were applied to ruminal digesta samples obtained from four multiparous Nordic Red dairy cows fitted with rumen cannulae. The cows received diets with forage:concentrate ratio either 35:65 (diet H) or 65:35 (L), supplemented or not with sunflower oil (SO) (0 or 50 g/kg diet dry matter), supplied in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments and four 35-day periods. Digesta samples were collected on days 22 and 24 and combined. QPCR provided a broad picture in which a large fall in the abundance of fungi was seen with SO in the H but not the L diet. Amplicon sequencing showed higher community diversity indices in L as compared to H diets and revealed diet specific taxa abundance changes, highlighting large differences in protozoal and fungal composition. Methanobrevibacter ruminantium and Mbb. gottschalkii dominated archaeal communities, and their abundance correlated negatively with each other. Co-occurrence network analysis provided evidence that no microbial domain played a more central role in network formation, that some minor-abundance taxa were at nodes of highest centrality, and that microbial interactions were diet specific. Networks added new dimensions to our understanding of the diet effect on rumen microbial community interactions.

Documenting the natural diversity of eukaryotic organisms in the nonhuman primate (NHP) gut is important for understanding the evolution of the mammalian gut microbiome, its role in digestion, health and disease, and the consequences of anthropogenic change on primate biology and conservation. Despite the ecological significance of gut-associated eukaryotes, little is known about the factors that influence their assembly and diversity in mammals. In this study, we used an 18S rRNA gene fragment metabarcoding approach to assess the eukaryotic assemblage of 62 individuals representing 16 NHP species. We find that cercopithecoids, and especially the cercopithecines, have substantially higher alpha diversity than other NHP groups. Gut-associated protists and nematodes are widespread among NHPs, consistent with their ancient association with NHP hosts. However, we do not find a consistent signal of phylosymbiosis or host-species specificity. Rather, gut eukaryotes are only weakly structured by primate phylogeny with minimal signal from diet, in contrast to previous reports of NHP gut bacteria. The results of this study indicate that gut-associated eukaryotes offer different information than gut-associated bacteria and add to our understanding of the structure of the gut microbiome.

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.

Hydrogenosomes are H2-producing mitochondrial homologs found in some anaerobic microbial eukaryotes that provide a rare intracellular niche for H2-utilizing endosymbiotic archaea. Among ciliates, anaerobic and aerobic lineages are interspersed, demonstrating that the switch to an anaerobic lifestyle with hydrogenosomes has occurred repeatedly and independently. To investigate the molecular details of this transition, we generated genomic and transcriptomic data sets from anaerobic ciliates representing three distinct lineages. Our data demonstrate that hydrogenosomes have evolved from ancestral mitochondria in each case and reveal different degrees of independent mitochondrial genome and proteome reductive evolution, including the first example of complete mitochondrial genome loss in ciliates. Intriguingly, the FeFe-hydrogenase used for generating H2 has a unique domain structure among eukaryotes and appears to have been present, potentially through a single lateral gene transfer from an unknown donor, in the common aerobic ancestor of all three lineages. The early acquisition and retention of FeFe-hydrogenase helps to explain the facility whereby mitochondrial function can be so radically modified within this diverse and ecologically important group of microbial eukaryotes.

Buxtonella sulcata is an alveolate ciliate protist, historically considered a commensal of the gastrointestinal tract of cattle. Despite the fact that its cysts are morphologically identical to those of Balantioides coli , molecular identification techniques have shed new light on its role as a pathogen. This work aimed to assess the presence of this ciliate in the population of dairy cattle on the Azorean island of Terceira by means of molecular analyses (ITS1–5.8S–ITS2 rRNA) of stool samples. A total of 116 samples were collected from adult Holstein-Friesian dairy cows, with no signs of gastrointestinal disease. A proportion of 49.1% of the samples were PCR-positive for Bu. sulcata , and 12 different genetic sequences were identified. These findings highlight the need for future research concerning the factors that influence the presence of Bu. sulcata in the gastrointestinal tract of dairy cows, the role of bovines as possible sources of infection, and the impact this ciliate may have on the health, welfare, and productivity of these animals. Buxtonella sulcata est un protiste cilié alvéolé, historiquement considéré comme un commensal du tube digestif des bovins. En dépit du fait que la morphologie de ses kystes est identique à celle de Balantioides coli , les techniques d’identification moléculaire ont apporté un nouvel éclairage sur son rôle comme pathogène. Ce travail vise à évaluer la présence de ce cilié dans la population de vaches laitières de l’île de Terceira, aux Açores, grâce à des analyses moléculaires (ARNr ITS1–5.8S–ITS2) d’échantillons de selles. Au total, 116 échantillons ont été prélevés sur des vaches laitières Holstein adultes, ne présentant aucun signe de maladie gastro-intestinale. Une proportion de 49,1 % des échantillons étaient positifs à la PCR pour Bu. sulcata et 12 séquences génétiques différentes ont été identifiées. Ces résultats soulignent la nécessité de recherches futures concernant les facteurs qui influencent la présence de Bu. sulcata dans le tractus gastro-intestinal des vaches laitières, le rôle des bovins comme sources possibles d’infection et l’impact que ce cilié peut avoir sur la santé, le bien-être et la productivité de ces animaux.