Diversity and Bioindicative value of soil and sediment ciliate communities from Italy: An investigation on agricultural and natural soils, and Frassasi cave sediments (Italy)

Phylum Ciliophora comprises a vast variety of unicellular eukaryotic microorganisms, which possess cilia in one or other stages of their life cycle, belonging to the kingdom Protista. The group emerged as a distinct evolutionary line from the main eukaryotic tree more than one billion years ago [...]. The phylum includes over 8000 described species, ranging in size from 10 to 2000 μm. Most of them are free living, few groups also live as commensals or parasites in or on other animals, e.g. the commensal ciliates present in rumen of vertebrates, some species colonize the surface of crustaceans and water beetles, some live in complete association with other organisms, like Balantidium, which are known to infect humans and fish such as Ichtyophthirius [...]. The cilia serve for locomotion and food acquisition and occur throughout the cell cycle in all members of the phylum, except of the Suctoria, in which they are absent in the adult stage. They are variable in number and arrangement, distributed over the body surface in a species specific manner and derived from an infraciliature consisting of the kinetosomes (basal bodies) with three characteristic fibrillar structures in the cytoplasm the kinetodesmal fibril, the post ciliary microtubular ribbon, and the transverse microtubular ribbon. One of the main distinguishing features of ciliates is the nuclear dimorphism: a polyploidy DNA-rich macronucleus (except in the group Karyorelictea, where it is diploid and does not divide) and a diploid generative micronucleus. The life cycles of ciliates are complex and clearly separate sexual processes from asexual reproduction. Ciliates reproduce asexually by transverse binary fission separating the anterior division product (the proter) from the posterior division product (the opisthe). The sexual process, called conjugation, involves the temporary union of two cells of complementary mating types to exchange gametic nuclei; however, this phenomenon is unknown (unobserved) in various groups. Up to date, about 4,500 valid free-living ciliate morphospecies have been described, but the true number may be as many as 30,000 [...] it is estimated that 8090% of the ciliate biological species are still undiscovered. The small size of the ciliates and their high abundances were used as argument for the ''ubiquity model'' by Finlay et al. (1996), i.e., everything is everywhere, but the environment selects. However, this assumption underestimates the biodiversity of ciliates because it refutes biogeography in microscopic organisms. In contrast, Foissner et al. (2007) proposed the ''moderate endemicity model'', which takes into account presence of cyst species, cryptic species, and about 30% endemic species. Ciliates are essential component of microbial communities in both aquatic and terrestrial environments, where they are integral constituents of trophic chains and nutrient [...]. In particular, ciliate communities exert critical roles in the soil ecosystem; several studies have shown that the grazing activity of ciliates can significantly influence the soil bacterial community, thus exerting secondary effects on plant growth [...]. Soil provides a number of critical ecosystem services and the soil biodiversity supports a multiplicity of ecosystem functions and processes which deliver benefits to people. Soil host more than one fourth of all living species in the earth and the main key functions of soil biodiversity are related of the three major bio-geochemical cycles on earth: nutrient, carbon and water cycling. To this regard, ciliates play key roles in the transfer of matter and energy within the ''microbial soil loop'' by feeding on bacterial biomass. Therefore, the community structure and dynamics of soil protozoa can provide powerful means for assessing and monitoring changes in natural and human-influenced environments [...]. In this context, the present work thesis has a twofold purpose: first, to assess the bioindicative value of soil ciliate communities in natural and man-influenced soil systems (e.g. agroecosystems) (chapter1), and second, to explore the diversity of ciliates in natural and agricultural sites (chapter2) as well as in the sediments of the Frasassi cave complex (chapter3). To this regard the first chapter of the thesis work presents the results in analysing the soil ciliate communities of sites exposed to various degrees of soil disturbances, i.e. agricultural sites (organic and conventional) and natural sites with the main aims to evaluate their potential as indicators of soil quality. These activities have been realized in the framework of two projects: a) The BioPrint Pilot project (2010-2011) ''Ciliate communities as a tool to assess soil quality in agroecosystems (Organic and conventional farms) and natural sites'' Marche region, Central Italy, (chapter1: part1), and b) The ''Soil Mapping project'' (2012-2014), Lombardia Region, North Italy (chapter1: part2). First Chapter (Part 1) - The BioPrint Pilot Project Marche Region. Currently, few studies have been addressed to the analysis of soil ciliate communities and in particular, in agricultural soils (organic and conventional). In the BioPrint Pilot project, the first one to be performed in Italy, using ciliates as bioindicators of soil quality, the ciliate communities in different soil habitats (agroecosystems and natural sites) located in a hill area (300-550 m asl) of the province of Macerata and Ancona (Marche region), were investigated. The specific aims of the project were: a) to evaluate the capacity of ciliates to discriminate between different types of land uses (natural and agricultural sites), different farming management practices (conventional and organic) and to assess relationships among ciliate community and abiotic parameters Soil samples were collected twice from 10 sites (5 agricultural and 5 natural sites). Diversity (H'), evenness (J'), abundances, colpodid/polyhymenophoran (C/P) ratio of soil ciliate communities were studied by means of qualitative (non-flooded Petri dish) and quantitative methods. Soil chemical-physical (texture, CSC NPK, OM, C/N, soil moisture and temperature) were also measured at each site. In total of 58 species belonging to 7 classes, 13 orders, and 33 genera were identified. Dominant ciliates species belonging to genera Colpoda, Gonostomum, Oxytricha, Halteria and Dileptus were identified. Interestingly, poorly known /new records of more than 12 species of ciliates from the Region Marche were identified, testifying the substantial lack of knowledge of soil ciliates diversity in Italy. The species richness ranged from 14 to 31 and it was higher in agricultural soils as compared with the natural habitats. These findings highlight the high bio-indicative potential of ciliate species number, supporting the intermediate disturbance hypothesis. Univariate and multivariate analysis showed statistically significant differences between natural sites and agricultural sites, as well as between the organic and conventional management farming systems, as well as correlations between the distributions of species with environmental parameters indicating the importance of these parameters in shaping the ciliate communities in the different site types. (Manuscript under preparation). First Chapter (Part 2) - The ''Soil Mapping project'' Lombardia Region. The main objective of the ''Soil Mapping'' project was to evaluate the potential of soil ciliate communities to discriminate between different levels of soil contamination/disturbances in four industrial sites (Incinerator; SIN, Site of National Interest; Plant of regeneration of exhausted oils; Cement factory) of Lombardia Region. Since that this interdisciplinary project funded by Lombardia region is still ongoing, therefore, only the preliminary qualitative and quantitative data related to the analysis of community structure of soil ciliates at the four investigated sites will be presented in the second part of the thesis. Furthermore, the surprising data regarding one of the sampled sites known as the P2R site is presented. Among the four sampled areas, the Parona sites surprised more for the presence of an elevated number of new species/genera. The species richest site was the paddy field known as P2R in which two new genera and five new species were identified. In particular, the 14% of the P2R ciliate species appear to be new and restricted to this sampling site (Daizy Bharti, Santosh Kumar, and Antonietta La Terza, unpublished results). Second chapter of the present study reports the novel genera/species and the poorly known species, which has been identified in the course of the study on the community structure of ciliates from Marche and Lombardia Regions. The chapter includes those species for which the detailed morphometric, morphogenetic, and molecular data has been collected (i.e., published and manuscript under preparation). Briefly, these include: (1) A new genus Paraparentocirrus sibillinensis n. gen., n. sp., which was found in soil samples of a beech forest stand within the National Park of Sibillini Mountains, Marche region, Italy. The new species is mainly characterized by a semi-rigid body, an undulating membrane in the Oxytricha pattern, six fronto-ventral rows, absence of transverse cirri, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kinety 4. Further, details on the morphogenesis and molecular phylogeny based on SSU rDNA were studied. (Published in Journal of Eukaryotic microbiology, 2014) (2) A new species Pseudouroleptus plestiensis n. sp., isolated from soil samples collected from the uplands of Colfiorito, Umbria region, Italy. The morphology of interphase, morphogenesis, and molecular phylogeny inferred from SSU rDNA sequences of this ciliate were studied. The novel species is mainly characterized by the following: absence of cortical granules, left fronto-ventral row consisting of about 2740 cirri, right frontoventral row of about three to seven cirri forming a short row to the right of the rear portion of the left frontoventral row; and four dorsal kineties with caudal cirri at the end of kineties 1 and 2. The morphogenesis of the novel species is similar to that of Pseudouroleptus caudatus caudatus. Phylogenetic analyses based on SSU rRNA gene sequences consistently placed the novel species within the family Oxytrichidae Ehrenberg, 1838, clustering with P. caudatus and the genus Strongylidium. (Published in International Journal of Systematics and Evolutionary microbiology, 2014). (3) Species of the genus Sterkiella; includes brief description of two populations of the poorly known species S. tricirrata from Italy and India, their comparison with a population of S. histriomuscorum having four transverse cirri from Italy, and description of a new species S. tetracirrata from India. (Manuscript on S. tetracirrata under revision in European Journal of Protistology; Manuscript on populations of S. histriomuscorum and S. tricirrata under preparation). (4) A gonostomatid ciliate Neogonostomum paronensis n. gen., n. sp., isolated from soil samples of a paddy field (P2R site), Lombardia region, Italy. Neogonostomum paronensis n. gen. is mainly characterized by a tailed body, AZM and undulating membrane in Gonostomum pattern, frontoventral cirri arranged in pairs, presence of transverse and pretransverse cirri, and the absence of caudal cirri. Morphologically, the new species is very similar to Gonostomum namibiense in having a tailed body and frontoventral cirral pairs. Phylogenetic analyses based on the SSU rDNA sequences supports the congenerecity of the new species and G. namibiense, and the establishment of this new genus (Submitted in Journal of Eukaryotic microbiology, 2014). (5) Brief, redescription of Stylonychia harbinensis based on morphology, morphogenesis, and SSU rDNA gene sequences were studied. Important characters which differentiate S. harbinensis and the closely related species Stylonychia ammermanni includes mainly the size of micronucleus. Data on the unique resting cyst of S. harbinensis is also presented. This poorly known species has been isolated from the P9R site (Parona sampling area, Soil Mapping project) (Manuscript under preparation). (6) Brief, description of Apokeronopsis italiensis n. gen. n. sp. isolated from the P10R site, Parona sampling area; the new genus is mainly characterized by a large body size, frontal ciliature in oxytrichid pattern i.e., three frontal cirri and one buccal cirrus, and undulating membrane in indistinct Steinia pattern. Dorsal surface with multiple fragmentation and dorsomarginal rows. (Manuscript under preparation). Overall the results presented in Chapter 1 and Chapter 2 allowed to verify the bioindicative potential of ciliate communities in discriminating between natural sites and agroecosystems, as well as their capacity to further discriminate between different management systems (organic vs. conventional). Furthermore, this study also allowed identifying about 25 new species/genera using classical and molecular analysis. Seven poorly known species were also identified. In particular a total of 13 new species/genera were identified in Lombardia region (Soil Mapping Project) and 12 new species/genera were identified in Marche region (Bioprint, Mosys and other projects). More than 50% of the identified species were not present in the checklist of ciliate species of Italian fauna. These results stress the ignorance of soil ciliate diversity and highlight the importance of an internationally concerted research effort for decoding ciliates diversity and their functional roles in soil under different land use systems. Chapter three of the present report discuss the community structure of ciliates from the habitat with extreme climatic conditions a karst system known as the Frasassi cave complex (Genga, AN, Italy). The sulfide-rich Frasassi cave complex host a still largely uncharacterized micro-biota whom study might offer an intriguing view on the solutions adopted by different species to survive and interact with each other's in such a harsh environment. The aim of the study was to study the community structure and observe behavioural and morphological differences of ciliates from the Frasassi caves. Four sampling locations within Frasassi caves were selected namely Pozzo dei Cristalli, Lago Verde, Ramo Sulfureo, and Grotta Sulfurea. Periodic sampling were realised from 2009 to 2011 in the form of water-sediments. In total, 33 ciliate species were identified as well as one flagellate species, one amoeboid species and one heliozoan. The ciliates species belonged to 9 classes, 15 orders and 23 genera and were studied in detail. Distinct ciliate communities were obtained during different sampling occasion. This could be due to various environmental challenges and favourable habitats, which leads to ciliate dispersal within these microhabitats. It was observed that some species e.g. Urocentrum turbo, Coleps hirtus hirtus, Oxytricha sp., Euplotes sp. and Aspidisca turrita showed adaptation for the cave environment (Photo-sensitivity, sulphur tolerance, feeding behaviour, morphological difference). Molecular comparisons based on SSU rDNA for some species were done to verify differences between the strains from cave and outside; it was concluded that genes other than SSU rDNA are required to better resolve the differences between the same species from different habitats. Further, contribution (the study of morphology and molecular analysis of the Coleps hirtus cave strain) was made for a publication in Journal of Eukaryotic microbiology, 2014 [...]. Overall the results presented in Chapter 3 provide a platform for various in-depth studies of ciliates to understand potential role in ecosystem functioning, nature of chemical compounds secreted, dispersal pattern, and adaptations within cave. (Manuscript under preparation).