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Cells of Cryptocaryon irritans at different life history stages were studied using both light and electron microscopy. The characteristics of several organelles were revealed for the first time at the ultrastructural level. It was confirmed that the cytostome of trophonts, protomonts and theronts was surrounded by cilium–palp triplets rather than ciliary triplets. The nematodesmata underlying the circumoral dikinetids were single bundles, whereas these were always paired in Prorodontids. Toxicysts were present in late-stage tomonts and theronts, but were absent in trophonts and protomonts. We posited that toxicysts might play a role in infection and invasion of host-fish tissue by theronts. The adoral brosse was unlike that of any other family of the class Prostomatea based on its location and morphology. Membranous folds were present in trophonts, protomonts and theronts. These folds were longer and more highly developed in C. irritans than in exclusively free-living prostome ciliates suggesting that they might be linked to parasitism in C. irritans. Trophonts, protomonts and theronts had multiple contractile vacuoles. The basic ultrastructure of the contractile vacuole of C. irritans was similar to that of other kinetofragminophoran ciliates. They might play different roles in different stages of the life cycle since their ultrastructure varied among trophonts, protomonts and theronts.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

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.

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.

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.

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.

Prochilodus lineatus is a freshwater fish species found in South America. It is common in aquaculture, but few studies regarding diseases of this fish have been performed. This study presents data of the occurrence of Trichodina heterodentata Duncan, 1977, as well as the pathological alterations detected by light and scanning electron microscopy (SEM). Twenty 20-day-old larvae were harvested from an earth pond and examined. Larvae showed erratic swimming on the pond edges and some had a whitish tegument. Larval smears were either impregnated with silver nitrate or stained with Giemsa stain to observe the taxonomic features of the ciliates. Five larvae were fixed in formalin solution for histopathological analysis, and another five specimens were fixed in glutaraldehyde for SEM. All larvae were diagnosed with a severe infestation by trichodinid T. heterodentata. Histological sections showed discrete hyperplasia of the gill filaments with subepithelial oedema of the secondary lamellae. In the SEM, suction areas were observed on the skin, gill and eye; corrosion and ulceration of the fins were associated with the bacterial presence of cocci on the lesions. This is the first report of T. heterodentata in P. lineatus that is responsible for an acute disease that culminates in larval mortality.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 μm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus) , prasinophytes (chiefly Ostreococcus) , fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Cryptocaryoniasis, caused by Cryptocaryon irritans , is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila , a culturable ciliate, to develop a mimetic C. irritans . Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans , to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis , allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using “mimetic parasites”.

Mitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis 1 , 2 . Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution 3 . As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation 4 . Here we describe ‘ Candidatus Azoamicus ciliaticola’, which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. ‘ Candidatus A. ciliaticola’ contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron–sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. ‘ Candidatus A. ciliaticola’ and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria. ‘ Candidatus Azoamicus ciliaticola’ transfers energy to its ciliate host in the form of ATP and enables this host to breathe nitrate, demonstrating that eukaryotes with remnant mitochondria can secondarily acquire energy-providing endosymbionts.