Small RNA-mediated genome rearrangement pathways in ciliates.

Most eukaryotes employ a combination of transcriptional and post-transcriptional silencing mechanisms to suppress transposons, yet ciliates employ a more extreme approach. They separate germline and somatic functions into distinct nuclei, enabling the elimination of transposons from the active somatic genome through diverse small RNA-mediated genome rearrangement pathways during sexual processes.

Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.

Trade-offs of lipid remodeling in a marine predator-prey interaction in response to phosphorus limitation.

Phosphorus (P) is a key nutrient limiting bacterial growth and primary production in the oceans. Unsurprisingly, marine microbes have evolved sophisticated strategies to adapt to P limitation, one of which involves the remodeling of membrane lipids by replacing phospholipids with non-P-containing surrogate lipids. This strategy is adopted by both cosmopolitan marine phytoplankton and heterotrophic bacteria and serves to reduce the cellular P quota. However, little, if anything, is known of the biological consequences of lipid remodeling. Here, using the marine bacterium Phaeobacter sp. MED193 and the ciliate Uronema marinum as a model, we sought to assess the effect of remodeling on bacteria-protist interactions. We discovered an important trade-off between either escape from ingestion or resistance to digestion. Thus, Phaeobacter grown under P-replete conditions was readily ingested by Uronema, but not easily digested, supporting only limited predator growth. In contrast, following membrane lipid remodeling in response to P depletion, Phaeobacter was less likely to be captured by Uronema, thanks to the reduced expression of mannosylated glycoconjugates. However, once ingested, membrane-remodeled cells were unable to prevent phagosome acidification, became more susceptible to digestion, and, as such, allowed rapid growth of the ciliate predator. This trade-off between adapting to a P-limited environment and susceptibility to protist grazing suggests the more efficient removal of low-P prey that potentially has important implications for the functioning of the marine microbial food web in terms of trophic energy transfer and nutrient export efficiency.

Identification of novel, functional, long noncoding RNAs involved in programmed, large-scale genome rearrangements.

Noncoding RNAs (ncRNAs) make up to ∼98% percent of the transcriptome of a given organism. In recent years, one relatively new class of ncRNAs, long noncoding RNAs (lncRNAs), were shown to be more than mere by-products of gene expression and regulation. The unicellular eukaryote Paramecium tetraurelia is a member of the ciliate phylum, an extremely heterogeneous group of organisms found in most bodies of water across the globe. A hallmark of ciliate genetics is nuclear dimorphism and programmed elimination of transposons and transposon-derived DNA elements, the latter of which is essential for the maintenance of the somatic genome. Paramecium and ciliates in general harbor a plethora of different ncRNA species, some of which drive the process of large-scale genome rearrangements, including DNA elimination, during sexual development. Here, we identify and validate the first known functional lncRNAs in ciliates to date. Using deep-sequencing and subsequent bioinformatic processing and experimental validation, we show that Paramecium expresses at least 15 lncRNAs. These candidates were predicted by a highly conservative pipeline, and informatic analyses hint at differential expression during development. Depletion of two lncRNAs, lnc1 and lnc15, resulted in clear phenotypes, decreased survival, morphological impairment, and a global effect on DNA elimination.

Comparative genomic analysis of symbiotic and free-living Fluviibacter phosphoraccumulans strains provides insights into the evolutionary origins of obligate Euplotes-bacterial endosymbioses.

Endosymbiosis is a widespread and important phenomenon requiring diverse model systems. Ciliates are a widespread group of protists that often form symbioses with diverse microorganisms. Endosymbioses between the ciliate Euplotes and heritable bacterial symbionts are common in nature, and four essential symbionts were described: Polynucleobacter necessarius, "Candidatus Protistobacter heckmanni," "Ca. Devosia symbiotica," and "Ca. Devosia euplotis." Among them, only the genus Polynucleobacter comprises very close free-living and symbiotic representatives, which makes it an excellent model for investigating symbiont replacements and recent symbioses. In this article, we characterized a novel endosymbiont inhabiting the cytoplasm of Euplotes octocarinatus and found that it is a close relative of the free-living bacterium Fluviibacter phosphoraccumulans (Betaproteobacteria and Rhodocyclales). We present the complete genome sequence and annotation of the symbiotic Fluviibacter. Comparative analyses indicate that the genome of symbiotic Fluviibacter is small in size and rich in pseudogenes when compared with free-living strains, which seems to fit the prediction for recently established endosymbionts undergoing genome erosion. Further comparative analysis revealed reduced metabolic capacities in symbiotic Fluviibacter, which implies that the symbiont relies on the host Euplotes for carbon sources, organic nitrogen and sulfur, and some cofactors. We also estimated substitution rates between symbiotic and free-living Fluviibacter pairs for 233 genes; the results showed that symbiotic Fluviibacter displays higher dN/dS mean value than free-living relatives, which suggested that genetic drift is the main driving force behind molecular evolution in endosymbionts. IMPORTANCE: In the long history of symbiosis research, most studies focused mainly on organelles or bacteria within multicellular hosts. The single-celled protists receive little attention despite harboring an immense diversity of symbiotic associations with bacteria and archaea. One subgroup of the ciliate Euplotes species is strictly dependent on essential symbionts for survival and has emerged as a valuable model for understanding symbiont replacements and recent symbioses. However, almost all of our knowledge about the evolution and functions of Euplotes symbioses comes from the Euplotes-Polynucleobacter system. In this article, we report a novel essential symbiont, which also has very close free-living relatives. Genome analysis indicated that it is a recently established endosymbiont undergoing genome erosion and relies on the Euplotes host for many essential molecules. Our results provide support for the notion that essential symbionts of the ciliate Euplotes evolve from free-living progenitors in the natural water environment.

Interactions between Balantidium ctenopharyngodoni and microbiota reveal its low pathogenicity in the hindgut of grass carp.

BACKGROUND: Hosts, parasites, and microbiota interact with each other, forming a complex ecosystem. Alterations to the microbial structure have been observed in various enteric parasitic infections (e.g. parasitic protists and helminths). Interestingly, some parasites are associated with healthy gut microbiota linked to the intestinal eubiosis state. So the changes in bacteria and metabolites induced by parasite infection may offer benefits to the host, including protection from other parasitesand promotion of intestinal health. The only ciliate known to inhabit the hindgut of grass carp, Balantidium ctenopharyngodoni, does not cause obvious damage to the intestinal mucosa. To date, its impact on intestinal microbiota composition remains unknown. In this study, we investigated the microbial composition in the hindgut of grass carp infected with B. ctenopharyngodoni, as well as the changes of metabolites in intestinal contents resulting from infection. RESULTS: Colonization by B. ctenopharyngodoni was associated with an increase in bacterial diversity, a higher relative abundance of Clostridium, and a lower abundance of Enterobacteriaceae. The family Aeromonadaceae and the genus Citrobacter had significantly lower relative abundance in infected fish. Additionally, grass carp infected with B. ctenopharyngodoni exhibited a significant increase in creatine content in the hindgut. This suggested that the presence of B. ctenopharyngodoni may improve intestinal health through changes in microbiota and metabolites. CONCLUSIONS: We found that grass carp infected with B. ctenopharyngodoni exhibit a healthy microbiota with an increased bacterial diversity. The results suggested that B. ctenopharyngodoni reshaped the composition of hindgut microbiota similarly to other protists with low pathogenicity. The shifts in the microbiota and metabolites during the colonization and proliferation of B. ctenopharyngodoni indicated that it may provide positive effects in the hindgut of grass carp.

Northern coral triangle coral ciliates diseases and disease prevention: A first record.

This study is the first to report ciliate infection on soft corals in the Northern Coral Triangle. Infected Briareum violacea will undergo tissue ulceration and death within a short period of time. This ciliate was identified as Scuticociliatia sp. through 18S rRNA gene identification. In this study, the body length of the ciliate was approximately 80-85 µm before parasitizing the B. violacea. After being parasitizing, the body length was approximately 200-250 µm, and the body width was 50 µm. Body size increased three times after parasitism. According to observations, ciliates will first parasitize the coral endoderm in the early stage of infection, and no ciliates were found in the ectoderm. Preliminary judgment suggests that it may invade the coral endoderm through the mouth for parasitism. After parasitism, the ciliate eats the coral tissue and zooxanthellae. The antioxidant enzymes SOD, CAT, and MDA of infected corals were significantly increased, which also means that the corals are in a stress response. Ciliates will eat the zooxanthellae in the coral body, resulting in a significant reduction in the number of zooxanthellae and chlorophyll a. To effectively prevent and treat this disease, Combretum indicum extract was used in this study. It is a tropical plant commonly used medicinally to treat roundworms, pinworms and parasitic diseases. The results showed that at a concentration of 1500-2500 ppm, Combretum indicum extract can be used to treat ciliates and can applied via medicinal bath therapy for long periods without causing coral stress reactions. The results of this study regarding coral disease prevention are in line with SDG 14 and promote the practical application of coral reef ecological sustainability and large-scale coral aquaculture.

Evaluation of in vitro treatments against the causative agent of Diadema antillarum scuticociliatosis (DaSc).

In the 1980s, a mass die-off of the long-spined sea urchin Diadema antillarum occurred on Florida and Caribbean coral reefs. D. antillarum populations largely did not recover, and in 2022, remaining populations experienced another mass mortality event. A ciliate most similar to Philaster apodigitiformis was identified as the causative agent of the 2022 event, which was named D. antillarum scuticociliatosis (DaSc). Here, we investigated possible treatments for this pathogen. We tested the efficacy of 10 compounds at final concentrations of 100, 50, 25, 12.5, 6.25, and 3.13 µM, or a 10-fold serial dilution series, against ciliates cultured from an infected D. antillarum specimen. Of the tested compounds, 8 induced 100% ciliate mortality at some dose after 24 h. The most effective (defined as those requiring the lowest dose to induce 100% ciliate mortality) were quinacrine and tomatine (both effective at 12.5 µM), followed by furaltadone and plumbagin (25 µM), bithionol sulfoxide and 2'4' dihydroxychalcone (50 µM), and oxyclozanide and carnidazole (100 µM). Toltrazuril and a commercially available anticiliate product containing naphthoquinones were not effective at any dose tested. Shortened (15 min) time trials were performed using ciliate cultures reared in natural seawater to better reflect natural environmental conditions, and revealed that 2 of the compounds (quinacrine and tomatine) induced 100% ciliate mortality at 100 µM, with tomatine also effective at 50 µM. This study identified several treatments effective against the causative agent of DaSc in vitro, but their toxicity and utility in vivo remain unknown.

Culture techniques for ciliate protozoa from the rumen: Recent advances and persistent challenges.

Ciliate protozoa are key members of the microbial community of the rumen. Their study is important to the health and productivity of cattle, which are their hosts. However, there have been persistent challenges in culturing this microbial group in the laboratory. This review will sum up recent advances along with these persistent challenges. Protozoa have been maintained in three types of cultures (ex vivo, in vitro batch, in vitro continuous). Ex vivo cultures are prepared readily from rumen contents by washing away contaminating cells (e.g., bacteria). They have been useful in making basic observations of metabolism, such as which types of fermentation products protozoa form. However, these cultures can be maintained for only short periods (minutes or hours). In vitro batch and in vitro continuous cultures can be used in longer experiments (weeks or longer). However, it is not currently possible to maintain protozoa in these cultures unless bacteria are also present. We conclude the review with a protocol for preparing ex vivo cultures of protozoa. Our protocol has been standardized and used successfully across animal diets, users, and institutions. We anticipate this review will prepare others to culture rumen ciliate protozoa and reach new insights into this important microbial group.

The energy metabolism of Balantidium polyvacuolum inhabiting the hindgut of Xenocypris davidi.

Anaerobic parasitic ciliates are a specialized group of ciliates that are adapted to anoxic and oxygen-depleted habitats. Among them, Balantidium polyvacuolum, which inhabits the hindgut of Xenocyprinae fishes, has received very limited scientific attention, so the molecular mechanism of its adaptation to the digestive tract microenvironment is still unclear. In this study, transmission electron microscopy (TEM) and single-cell transcriptome analysis were used to uncover the metabolism of B. polyvacuolum. Starch granules, endosymbiotic bacteria, and multiple specialized mitochondrion-related organelles (MROs) of various shapes were observed. The MROs may have completely lost the electron transport chain (ETC) complexes I, III, IV, and V and only retained succinate dehydrogenase subunit A (SDHA) of complex II. The tricarboxylic acid (TCA) cycle was also incomplete. It can be inferred that the hypoxic intestinal environment has led to the specialization of the mitochondria in B. polyvacuolum. Moreover, carbohydrate-active enzymes (CAZymes), including carbohydrate esterases, enzymes with a carbohydrate-binding module, glycoside hydrolases, and glycosyltransferases, were identified, which may constitute evidence that B. polyvacuolum is able to digest carbohydrates and starch. These findings can improve our knowledge of the energy metabolism and adaptive mechanisms of B. polyvacuolum.

Sequencing and analysis of the complete mitochondrial genomes of Toona sinensis and Toona ciliata reveal evolutionary features of Toona.

BACKGROUND: Toona is a critical genus in the Meliaceae, and the plants of this group are an asset for both restorative and restorative purposes, the most flexible of which are Toona sinensis and Toona ciliata. To concentrate on the advancement of mitochondrial(Mt) genome variety in T.sinensis and T.ciliata, the Mt genomes of the two species were sequenced in high throughput independently, after de novo assembly and annotation to construct a Mt genome map for comparison in genome structure. Find their repetitive sequences and analyze them in comparison with the chloroplast genome, along with Maximum-likelihood(ML) phylogenetic analysis with 16 other relatives. RESULTS: (1) T. sinensis and T.ciliata are both circular structures with lengths of 683482 bp and 68300 bp, respectively. They share a high degree of similarity in encoding genes and have AT preferences. All of them have the largest Phe concentration and are the most frequently used codons. (2) Both of their Mt genome are highly preserved in terms of structural and functional genes, while the main variability is reflected in the length of tRNA, the number of genes, and the value of RSCU. (3) T. siniensis and T. ciliata were detected to have 94 and 87 SSRs, respectively, of which mononucleotides accounted for the absolute proportion. Besides, the vast majority of their SSRs were found to be poly-A or poly-T. (4)10 and 11 migrating fragments were identified in the comparison with the chloroplast genome, respectively. (5) In the ML evolutionary tree, T.sinensis and T.ciliata clustered individually into a small branch with 100% support, reflecting two species of Toona are very similarly related to each other. CONCLUSIONS: This research provides a basis for the exploitation of T.sinensis and T.ciliata in terms of medicinal, edible, and timber resources to avoid confusion; at the same time, it can explore the evolutionary relationship between the Toona and related species, which does not only have an important practical value, but also provides a theoretical basis for future hybrid breeding of forest trees, molecular markers, and evolutionary aspects of plants, which has great scientific significance.

An Extremely Streamlined Macronuclear Genome in the Free-Living Protozoan Fabrea salina.

Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there are two distinct nuclei in ciliates with separate germline and somatic cell functions. Here, we assembled a near-complete macronuclear genome of Fabrea salina, which belongs to one of the oldest clades of ciliates. Its extremely minimized genome (18.35 Mb) is the smallest among all free-living heterotrophic eukaryotes and exhibits typical streamlined genomic features, including high gene density, tiny introns, and shrinkage of gene paralogs. Gene families involved in hypersaline stress resistance, DNA replication proteins, and mitochondrial biogenesis are expanded, and the accumulation of phosphatidic acid may play an important role in resistance to high osmotic pressure. We further investigated the morphological and transcriptomic changes in the macronucleus during sexual reproduction and highlighted the potential contribution of macronuclear residuals to this process. We believe that the minimized genome generated in this study provides novel insights into the genome streamlining theory and will be an ideal model to study the evolution of eukaryotic heterotrophs.

"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 Euplotechlamydia quinta," a novel chlamydia-like bacterium hosted by the ciliate Euplotes octocarinatus (Ciliophora, Spirotrichea).

Our knowledge of ciliate endosymbiont diversity greatly expanded over the past decades due to the development of characterization methods for uncultivable bacteria. Chlamydia-like bacteria have been described as symbionts of free-living amoebae and other phylogenetically diverse eukaryotic hosts. In the present work, a systematic survey of the bacterial diversity associated with the ciliate Euplotes octocarinatus strain Zam5b-1 was performed, using metagenomic screening as well as classical full-cycle rRNA approach, and a novel chlamydial symbiont was characterized. The metagenomic screening revealed 16S rRNA gene sequences from Polynucleobacter necessarius, three previously reported accessory symbionts, and a novel chlamydia-like bacterium. Following the full-cycle rRNA approach, we obtained the full-length 16S rRNA gene sequence of this chlamydia-like bacterium and developed probes for diagnostic fluorescence in situ hybridizations. The phylogenetic analysis of the 16S rRNA gene sequences unambiguously places the new bacterium in the family Rhabdochlamydiaceae. This is the first report of chlamydia-like bacterium being found in Euplotes. Based on the obtained data, the bacterium is proposed as a new candidate genus and species: "Candidatus Euplotechlamydia quinta."

Large-scale phylogenetic analysis provides insights into the diversification and evolution of sessilid peritrich ciliates (Protista: Ciliophora).

The Peritrichia is a speciose and morphologically distinctive assemblage of ciliated protists that was first observed by Antonie van Leeuwenhoek over 340 years ago. In the last two decades, the phylogenetic relationships of this group have been increasingly debated as morphological and molecular analyses have generated contrasting conclusions, mainly owing to limited sampling. In the present study, we performed expanded phylogenetic analyses of 152 sessilid peritrichs collected from 14 different provinces of China and 141 SSU rDNA peritrich sequences from GenBank. The results of the analyses revealed new divergent relationships between and within major clades that challenge the morphological classification of this group including, (1) the recovery of four major phylogenetically divergent clades in the monophyletic order Sessilida, (2) aboral structures such as the stalk and spasmoneme were evolutionary labile, (3) the stalk or/and spasmoneme was lost in each divergent clade indicating that parallel evolution occurred in sessilid peritrichs and (4) the life cycle and habit drive the diversity of aboral structures as well as diversification and evolution in peritrichs.

New contributions to the Cyrtophoria ciliates (Protista, Ciliophora): Establishment of new taxa and phylogenetic analyses using two ribosomal genes.

Periphytic ciliates play a vital role in the material cycle and energy flow of microbial food web, however, their taxonomy and biodiversity are inadequately studied given their high species richness. Two new and one little known species, viz. Derouxella lembodes gen. et sp. nov., Cyrtophoron multivacuolatum sp. nov., and Cyrtophoron apsheronica Aliev, 1991, collected from coastal waters of China, were investigated using modern methods. Derouxella gen. nov. can be recognized by having dorsoventrally flattened body, a podite, one fragmented preoral kinety, two parallel circumoral kineties, and somatic kineties progressively shortened from right to left. Morphological classification and phylogenetic analyses based on nuclear small subunit ribosomal RNA (nSSU rRNA) and mitochondrial small subunit ribosomal RNA (mtSSU rRNA) gene sequence data inferred that Derouxella gen. nov. occupies an intermediate position between Hartmannulidae and Dysteriidae. Cyrtophoron multivacuolatum sp. nov. is characterized by large body size, the numbers of somatic kineties and nematodesmal rods, and having numerous contractile vacuoles. The genus Cyrtophoron and the poorly known species C. apsheronica were redefined. Even with the addition of newly obtained nSSU rRNA and mtSSU rRNA gene sequences of Cyrtophoron, the family Chlamydodontidae was still recovered as a monophyletic group, the monophyly of Cyrtophoron was supported too.

Application of one-step inner-recycling counter-current chromatography for the preparative separation and purification of chemical constituents from the rhizome of Bergenia ciliate (haw.) Sternb.

Bergenia ciliata (haw.) Sternb, the renowned pharmaceutical plant in Jammu and Kashmir of Pakistan, is widely applied in treating different illnesses including diabetes, diarrhea, and vomiting. This work employed an efficient one-step inner-recycling counter-current chromatography for preparative separating and purifying compounds with similar partition coefficients from the rhizome of Bergenia ciliate (haw.). Five compounds, including quercetin rhamnodiglucoside (1), quercetin-3-O-rutinoside (2), bergenine (3), kaempferol (4), and palmatic acid (5), were successfully separated using the optimized biphasic solvent system that contained ter-butylmetylether/n-butanol/acetonitrile/water (2:2:1:5, v/v) with the purities over 98%. Mass spectrometry and nuclear magnetic resonance were conducted for structural identification. As a result, our proposed strategy might be applied in separating compounds with similar partition coefficients, which was advantageous with regard to the less solvent and time consumption, and the increased number of theoretical plates.

First report of Alternaria alternata causing leaf spot on Toona ciliate in China.

Toona ciliate is an excellent timber and ornamental tree cultivated in China (Li et al. 2018). In May 2018, a leaf spot disease was observed on the foliage of T. ciliate in Nanchang city, Jiangxi province. Disease incidence averaged approximately 40%. Initial symptoms were small, brown spots with yellow halos, then the spots gradually enlarged and coalesced to form large lesions. To identify the pathogen, thirty pieces (5 × 5 mm) from the lesion margins were surface sterilized in 70% ethanol (30 s), then in 3% NaOCl (1 min), and finally rinsed three times with sterile water. The pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Fourteen strains with similar morphological characters were isolated, and three representative isolates (MT-2, MT-5, MT-8) were used for morphological and molecular characterization. The colonies on PDA were gray to brown after 7 days. Ovoid or elliptical conidia were brown to light-brown in color with a short beak, 1-5 diaphragms, and 0-3 mediastinum. The diameter of these conidia were thick (18.2-47.4×7.9-15.1 µm, n= 100). The morphological characteristics of three isolates matched those of Alternaria sp. with straight or curved primary conidiophores with obclavate, long ellipsoid conidia (Woudenberg et al. 2013). The internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), small subunit (SSU), large subunit (LSU), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) (Woudenberg et al. 2013) and Alternaria major allergen gene (Alt a 1) (Woudenberg et al. 2014) were amplified by using the following primer pairs ITS1/ITS4, GPD-1/GPD-2, NS1/NS4, LR0R/LR05, RPB2-5F2/fRPB2-7cR, EF1-728F/EF1-986R and Alt-f/Alt-r, respectively. The sequences were deposited in GenBank (ITS: ON459540, ON459541, ON459542; GAPDH: ON427936, ON427937, ON427938; SSU: ON422107, ON422108, ON422109; LSU: ON422110, ON422111, ON422112; RPB2: ON427939, ON427940, ON427941; TEF1: ON427933, ON427934, ON427935; Alt a 1: ON427942, ON427943, ON427944). A maximum likelihood and Bayesian posterior probability-based analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, GAPDH, SSU, LSU, RPB2, TEF1, Alt a 1) placed three isolates in the clade of Alternaria alternata (Fr.) Keissl. The three isolates were identified as A. alternata based on morphological and molecular characteristics. For pathogenicity tests, 10 T. ciliate plants (two leaves each, n=20) grown outdoors were pin-pricked with a sterile needle and inoculated with a drop of spore suspension (106 conidia per mL) in July. Another 20 healthy leaves were inoculated with sterile water as the control. All the inoculated leaves were wrapped with plastic bags to keep them moist for 2 days. The pathogenicity tests were repeated twice. The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic for 10 days after inoculation. The same fungus was re-isolated from the lesions, confirming Koch's postulates. The pathogen was previously reported to cause leaf spots on Aquilegia flabellata (Garibaldi et al. 2022), Chrysanthemum morifolium (Luo et al. 2022), Liriodendron chinense × tulipifera (Jin et al. 2021) and so on. To our knowledge, this is the first report of A. alternata associated with leaf spot disease on T. ciliate in China. This disease may potentially decrease the value of ornamental T. ciliate plants under favorable conditions and proper management strategies should be applied.

Genome-Wide Identification of G Protein-Coupled Receptors in Ciliated Eukaryotes.

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and play important roles in many physiological processes. As a representative group of protozoa, ciliates represent the highest stage of eukaryotic cell differentiation and evolution in terms of their reproductive mode, two-state karyotype, and extremely diverse cytogenesis patterns. GPCRs have been poorly reported in ciliates. In this study, we identified 492 GPCRs in 24 ciliates. Using the existing classification system for animals, GPCRs in ciliates can be assigned to four families, including families A, B, E, and F. Most (377 members) belong to family A. The number of GPCRs is extremely different in different ciliates; the Heterotrichea ciliates usually have more GPCRs than other ciliates. Parasitic or symbiotic ciliates usually have only a few GPCRs. Gene/genome duplication events seem to play important roles in the expansion of the GPCR superfamily in ciliates. GPCRs in ciliates displayed seven typical domain organizations. GPCRs in an ortholog group are common and conserved in all ciliates. The gene expression analysis of the members in this conserved ortholog group in the model ciliate, Tetrahymena thermophila, suggested that these GPCRs play important roles in the life cycle of ciliates. In summary, this study provides the first comprehensive genome-wide identification of GPCRs in ciliates, improving our understanding of the evolution and function of GPCR in ciliates.

Crystal structure of the pheromone Er-13 from the ciliate Euplotes raikovi, with implications for a protein-protein association model in pheromone/receptor interactions.

In the ciliate Euplotes raikovi, water-borne protein pheromones promote the vegetative cell growth and mating by competitively binding as autocrine and heterologous signals to putative cell receptors represented by membrane-bound pheromone isoforms. A previously determined crystal structure of pheromone Er-1 supported a pheromone/receptor binding model in which strong protein-protein interactions result from the cooperative utilization of two distinct types of contact interfaces that arrange molecules into linear chains, and these into two-dimensional layers. We have now determined the crystal structure of a new pheromone, Er-13, isolated from cultures that are strongly mating reactive withculturessource of pheromone Er-1.The comparison between the Er-1 and Er-13 crystal structuresreinforces the fundamental of the cooperative model of pheromone/receptor binding, in that the molecules arrange into linear chains taking a rigorously alternate opposite orientation reflecting the presumed mutual orientation of pheromone and receptor molecules on the cell surface. In addition, the comparison provides two new lines of evidence for a univocal rationalization of observations on the differentbehaviourbetween the autocrine and heterologous pheromone/receptor complexes. (i) In the Er-13 crystal, chains do not form layers which thus appear to be an over-structureunique tothe Er-1 crystal, not essential for the pheromone signalling mechanisms. (ii) In both crystal structures, the intra-chain interfaces are equally derived from burying amino-acid side-chains mostly residing on helix-3 of the three-helical pheromonefold. This helix is thus identified as the key structural motif underlying the pheromone activity, in line with its tight intra- and interspecificstructuralconservation.