• 18S rDNA
• ciliates
• endosymbiont
• mitochondrial genome
• morphology
• systematics
• taxonomy
• WWTP protists

Ciliated protists are a diverse group of unicellular microscopic eukaryotes with complex structures. They are vital organisms in aquatic ecosystems, serving as key indicators of environmental health and playing essential roles in nutrient cycling and energy transfer. Ciliates stand out as a core component of wastewater treatment plants, where they significantly enhance operational efficiency and effluent quality. Since the activated sludge process was created, protists have been recognized as useful indicators. In recent years, new technologies have been developed for the optimization of wastewater treatment, and eukaryotic organisms involved in the process have been extensively investigated. However, the field of sewage protozoology has not developed rapidly synchronously, this development was constrained by the requirement of species identification, despite the successful application of modern integrative taxonomy approaches. To embark on an exploration and provide insights into the biodiversity and taxonomy of ciliates in this unique anthropogenic setting, we selected an Italian industrial wastewater treatment plant (WWTP) in San Romano, Pisa, Italy, operated by Cuoiodepur S.P.A. This study employs integrative taxonomy and Next Generation Taxonomy (NGTax) to comprehensively investigate ciliate communities. The NGTax criteria incorporate electron microscopy, endosymbiont analysis, bioinformatics, and genomic analyses, which can serve as a valuable standard for unicellular eukaryotes descriptions and redescriptions.
Over the period from May 2021 to March 2022, we isolated 37 populations belonging to 13 ciliate species from this WWTP, suggesting a potential for greater species diversity in this man-made environment. In our investigation, we conducted the morphological and 18S rDNA characterizations these populations. The objectives of our endeavours were threefold. Firstly, conduct taxonomic investigations on specific species within the identified populations to deepen our understanding of lesser-known ciliates and potentially unveil new taxa. By combining morphological and molecular approaches, we provide detailed characterization of species well-known in the literature, contributing to the creation of a high-quality DNA bank for genomic and NGT studies. Secondly, compile two definitive checklists of ciliate species present in this specific artificial habitat, which contains two main wastewater treatment lines: one for industrial wastewater and one for domestic wastewater. Thirdly, establish a substantial groundwork for future comprehensive ecological research on the ciliate community within the Cuoiodepur WWTP which employs standard quantitative and statistical methodologies.
In addition, ciliates also represent pivotal components within marine microbial food webs, exerting essential roles in the flux of materials and energy within aquatic ecosystems. We investigated five species collected from the marine aquarium in Livorno (salinity, 38‰) and three sites along the Ligurian Sea coast since June 2022. Given that ciliates have long been valued in the fields of cell biology, genetics, development and evolution, and ecology for their research usefulness. A substantial number of unknown taxa are also awaiting discovery, and some historical ambiguities must be resolved as soon as possible. Therefore, these five newly documented populations, including one new species, provide solid data of ciliate diversity, contribute to the enrichment of ciliate genetic resources. The primary results and conclusions are summarized as follows:
(1) A total of 13 ciliate species belonging to nine families Oxytrichidae, Pseudokeronopsidae, Aspidiscidae, Euplotidae, Parameciidae, Trimyemidae, Colepidae, Spirostomidae and Stentoridae were found in Cuoiodepur WWTP. Among these, one novel species in the genus Trimyema was recognized and described. Trimyema n. sp. shares the generic characters and can be separated from congeners by its unique aerobic habitat, body shape fusiform ellipsoid, number of ciliary girdles, and oral ciliature. In addition, the endosymbiont of Trimyema n. sp. was detected.
(2) The other 12 species collected from WWTP were further described with some emended diagnosis. In this context, for example, Pseudokeronopsis erythrina showed a remarkable morphological variation. The previously undocumented phenomena such as cannibalism and gigantism, which had never been found in urostylids, were disclosed in the WWTP population of P. erythrina. These new data offer an update of the previous knowledge of the species on its morphology, morphogenesis, intraspecific and intrapopulation variation of the ciliary structure, and phylogenetic and phylogenomic position, giving a hint to species determination and providing an improved taxon diagnosis.
(3) Five species of the genera Holosticha, Trachelostyla, Uronychia, Paranophrys and Mesanophorys collected from marine environments were investigated, including the novel putative pathogenic Paranophrys n. sp., isolated from a diseased jellyfish of an artificial aquarium. Paranophrys n. sp. can be characterized by small cell size, adoral zone of membranelles tripartite, and twelve or thirteen bipolar somatic kineties.
(4) 18S rDNA sequencing, comparison and phylogenetic analyses of 17 species were obtained. Among them, three species were newly sequenced internationally (Trimyema n. sp., Paranophrys n. sp. and Mesanophrys chesapeakensis). Phylogenetic analyses on Trimyema n. sp. showed it exhibited close affinities with T. minutum, and two distinct clades in the family Trimyemidae. Preliminary results on two scuticociliates suggested that Paranophrys n. sp. and M. chesapeakensis undoubtedly belong to Philasterida. Paranophrys n. sp. clusters with P. marina with full statistical support and then sister groups with the Cohnilembus lineage. All five sequences of the genus Mesanophrys group together with full statistical support as a monophyletic clade.
(5) For the first time, the complete mitochondrial genome was obtained for Pseudokeronopsis erythrina, which provided further insights to solve the phylogenetic problems of the lineages which these ciliates belong to.
This study not only advances our understanding of ciliate biodiversity and taxonomy in wastewater and marine environments through the integration of morphological and genomic data but also establishes a robust framework for future research, offering new perspectives on the application of ciliates as bioindicators and the potential for discovering species through Next Generation Taxonomy.