Morphology and morphogenesis of a novel stylonychid ciliate from India

The morphology and morphogenesis of a novel stylonychid ciliate isolated from a water sample collected from the Thane Creek, Mumbai, India, were investigated. The new species looked similar in morphology to the well known Tetmemena pustulata, however, on detailed investigation some non-overlapping differences in the ciliature were identified, i.e., fine differences in the number of cirri in marginal rows and the number of bristles in dorsal kineties of the Indian population in comparison with the population described by Wirnsberger et al. 1985, which justified the separation at the species level. A detailed description based on the examination of specimens in vivo and after protargol impregnation from raw culture is presented. Furthermore, the study also compares the resting cyst of the new species with related congeners. In this regard, the study extends the concept of cyst species to hypotrich ciliates, which thus far have been known only for spathidiids

Description and Molecular Phylogeny of a Novel Hypotrich Ciliate from the Soil of Marche Region, Italy; Including Notes on the MOSYSS Project

The morphology and morphogenesis during cell division of a new stylonychine hypotrich, Rigidocortex quadrinucleatus n. sp., were investigated using live observation and protargol staining. The new species was isolated from soil samples collected from an organic farm in the Marche Region, Italy, in framework of the MOSYSS project. Rigidocortex quadrinucleatus is characterized as follows: cell size about 180 9 80 lm in vivo; four ellipsoidal macronuclear nodules; 44 adoral membranelles: 18 fronto-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse, and five transverse cirri; dorsal kinety 3 with multiple fragmentation; resting cyst with hyaline ridges. Rigidocortex quadrinucleatus mainly differs from the type species R. octonucleatus in having four (vs. eight) macronuclear nodules. Rigidocortex quadrinucleatus can be easily confused with Sterkiella cavicola since both have a rather similar ventral ciliature; however, they can be separated by the slightly higher number of cirri in the left marginal row that runs along the posterior cell’s margin in R. quadrinucleatus. Morphogenesis on the ventral surface is highly similar to that of Sterkiella species, but differs significantly on the dorsal surface (multiple vs. simple fragmentation of dorsal kinety 3). Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to S. cavicola

The soil ciliated protozoa Cyrtoymena tetracirrata as bioindicators of soil health: cytotoxicity of single and bimetallic mixtures of heavy metals and antioxidant defenses

As far as we known, the releases of heavy metals (HMs) to the environments and particularly in agricultural soils, has increased globally in the last few years. Although, a number of reviews have analysed the effects of HM contaminated soils using various microbial test organisms, few studies have focused on soil ciliated protozoa as potential bioindicators of soil health. Thus, in this study we have evaluated the cytotoxic effects and the presence of antioxidant activities in the soil ciliate Cyrtoymena tetracirrata exposed for 24hrs to both single (Cd, Cu, and Zn) and bimetallic mixtures (Cd + Zn, Cd + Cu, and Cu + Zn) of HMs. Ecotoxicological tests showed, LC20 values to Cd, Cu, and Zn of 0.53, 0.22 and 23.0 mg L-1, respectively and, LC50 values of 1.16, 0.37 and 32.7 mg L-1, respectively. The order of toxicity was Cu > Cd >> Zn. Using the Concentration Addition model approach we found that the Cd + Zn bimetallic mixtures treatments were able to prevalently produce antagonistic effects in comparison to the other mixtures (Cd + Cu, and Cu + Zn). With regards to the analysis of the antioxidant activities, the total phenolic contents (TPC) were significantly higher in the ciliates exposed to Cu at the LC20 value previously identified (extracellular: P ≤ 0.01) and to Cd at the LC20 value (intracellular: P ≤ 0.001). Higher α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging activities were found at the Cd and Zn LC50 values for both the extracellular and intracellular contents (P ≤ 0.05). Hydroxyl scavenging activity (HRSA), unveiled that higher levels of antioxidants were produced following exposures to Cd LC50 (extracellular: P ≤ 0.001) and Cu LC50 values (intracellular: P ≤ 0.001). Overall the present study reveals, that C. tetracirrata has the potential to be used as bio-indicator of HM polluted soils

Ciliated Protist Communities in Soil: Contrasting Patterns in Natural Sites and Arable Lands across Italy

This study represents the first investigation of soil ciliate diversity and community structure in the Marche region, Italy, encompassing both natural sites and agro-ecosystems. The main aims were (i) to assess the ability of ciliates to discriminate between different types of land uses, i.e., arable lands and possible farming management practices [organic (ORG) vs. conventional (CON)], and forest (FOR) sites; and (ii) to investigate the relationships among ciliate communities and abiotic parameters at the studied sites. Soil samples were collected twice from 10 sites (5 forest (FOR) (natural soils) and 5 arable lands under different agricultural management systems (3 ORG (minimum tillage) and 2 CON (sod seeding)). Ciliate communities were studied using qualitative (non-flooded Petri dish) and quantitative methods (ciliate counts from permanent slides). Soil chemical-physical (texture, CEC, N, OM, C/N) parameters were also measured. Qualitative ciliate analysis allowed us to identify a total of 59 species representing 33 genera, 20 families, 13 orders, and 7 classes. ORG sites were the richest in species followed by CON and FOR. Multivariate analysis showed statistically significant differences between natural sites (FOR) and agricultural sites, and between ORG and CON management farming systems. CCA analysis revealed a positive correlation between the ciliate species and silt, clay, and pH in ORG sites, and sand, organic carbon, organic matter, total nitrogen, C/N ratio, and CEC (cation exchange capacity) in FOR sites, suggesting the significance of these parameters in shaping the ciliate communities. Altogether, these results showed the bioindicative potential of ciliate communities in discriminating between natural sites (FOR) and arable lands, and their capacity to discriminate, at least preliminarily, between different soil management systems (ORG vs. CON). Furthermore, this study highlights the high diversity of soil ciliates and their response to habitat variabilit

Ciliated protists as indicators of ecosystem health: opportunities, challenges, and case studies in Italy

This communication aims to provide an overview of the outcomes and the challenges encountered in using ciliated protists as indicator of ecosystem health in the framework of several projects conducted in Italy since 2009. Ciliates (and, in general, protistan microorganisms) in spite of the key roles they play in the microbial loop, still constitute a neglected component of the biodiversity, which is rarely included in ecological risk assessment plans. Ciliates are important bacterial and fungal feeders and thus, they significantly contribute to channel nutrients up to the food web. Furthermore, numerous ciliate species can be easily cultured with a short generation time, such as those selected for the setting up of our ecotoxicological experimentation (i.e., Coleps hirtus, Euplotes aediculatus, and Rigidohymena tetracirrata). Thus, in this context, the present communication aims to highlight the usefulness of ciliates as test organisms for the development of simple and costeffective “prognostic” assays (i.e., able to detect sub-lethal toxicant effects of various environmental matrices) based on the exploitation of antioxidant responses as well as of “diagnostic” assays (i.e., able to detect overall ecosystem impairment) based on the analysis of the community structures of ciliates in arable soils to evaluate the potential impact of different agricultural managements

Biological indicators of soil health in organic viticultures: A case study in the Verdicchio terroir of Matelica (Italy)

Soil health represents the foundation for the production of healthy food. Soil conditions influence the soil capacity for agricultural production and the provision of key ecosystem services. Healthy soils are key contributors to biodiversity and are a prerequisite for sustainable development. According to van Bruggen & Semenov [1] a healthy soil can be defined as “a stable soil, with resilience to stress, high biological diversity, and high levels of internal cycling of nutrients”. Soil biodiversity constitutes one of the main components of agroecosystems, being involved in the delivery of several essential ecosystem services such as, among others, nutrient cycling, soil formation, pest and pollution control. Thus, soil biodiversity indicators can be used by governments and farmers to monitor soil health and ecosystem functioning under various land uses and management practices. At present, organic wine represents an emerging market that is showing a high potential for growth. There is, in fact, a growing worldwide interest and attention for environmental friendly products and sustainable agricultural practices which may ensures both the increase of profitability and the improvement of life and food safety. In this scenario, the aim of our study was to assess the long-term effects of organically managed vineyards on soil health by means of two bioindicators: ciliated protozoa and microarthropods. Ciliated protozoa are eukaryotic microorganisms which constitutes an essential component of the soil microbial loop. By feeding on bacterial biomass ciliates play an essential role in the liberation of nutrients in the plant rhizosphere [2]. Soil microarthropods play crucial roles in regulating ecosystem processes and functions by influencing organic matter decomposition (pre-decomposers) and facilitating the microbial activity, soil porosity and water infiltration [3]. Both ciliates and microarthropods are very sensitive to changes in their habitat and fluctuations in their communities can affect the food web and the energy transfer within the soil ecosystem. Thus, the monitoring of the structure of microarthropod and ciliate communities represents a valuable tool to assess soil health and functioning. The study was realized in the terroir of Verdicchio di Matelica (Marche, Italy), on 3 vineyard plots belonging to the commercial wine farm Collestefano, that were organically managed since 1992 (V92, 19 years), 1998 (V98, 13 years) and 2009 (V09, 2 years) respectively. In each vineyard, soil samples (0-10 cm depth) were taken every month from March to October 2011. For microarthropods, the measured biological parameters were: the Soil Biological Quality (QBS-ar) index [4], abundances of biological and euedaphic forms (BF & EF) and diversity indices. Soil samples were collected in both disturbed (tillage) and not-disturbed (no-tillage) inter-rows. In this regards, the wine farm adopted an alternate management of the tractor-rows on either side of the vine row with tillage and non-tillage annual cover crop species (clover, common vetch and oats). For ciliates: abundances and diversity indices were measured and soil samples were randomly collected in the whole sampling area for a total of 3 sampling (May, June and July 2011). For ciliates, the results of the multivariate analysis (Cluster Analysis and Non-metric Multidimensional scaling) seem to indicate that the most stable habitat for ciliates is represented by the older V92 followed by the V98 and the V09 vineyards. For microarthropods, the comparison between tilled and no-tilled inter-rows in the vineyards V92, V98 & V09 by mean of QBS-ar values and abundances of FB and EF, shows that exsclusively in the oldest V92 vineyard no significant differences were detected between the tilled and no-tilled inter-rows with respect to QBS, EF and BF values by the non parametric Kruskal-Wallis (K-W) test. On the contrary, significant differences between the two differently managed inter-rows were detected for the V98 & V09 vineyards. Thus, and in agreement with ciliate indications, in the V92 the microartrhopod communities remain more stable than in the other vineyards irrespective of the type of disturbance applied (tillage). Overall, our analysis shows that the biological communities experience less fluctuations (>stable) in the “older” vineyards compared with the “younger” V09 vineyard (V92>V98>V09). As reported also by other authors [4-5], this effect may be due to the greater resilience of the soils of the vineyards V92 and V98 possible achieved during the long term organic management. In final, this study helps in evaluating the long term effects of common organic vineyard floor management practices as well as, in the definition of possible biotic baseline values to evaluate soil health in vineyards. Furthermore, and in agreement with the guidelines of the European Commission and the Food and Agriculture Organization (FAO) of the United Nations, our study contribute to raising awareness among stakeholders and policy makers on the importance of soil biodiversity in preserving soil health (and food safety) and in assisting them to select, promote and stimulate adequate sustainable farming practices by which to reduce the environmental impact of agriculture and move the world toward a more sustainable food future. References [1] A.H.C. van Bruggen, A.M. Semenov. In search of biological indicators for soil health and disease suppression. App. Soil Ecol. 2000, 15, 13-24 [2] W. Foissner, W. Soil protozoa as bioindicators in agroecosystems, with enphasis on farming practices, biocides and biodiversity. Agric, Ecos. and Env. 1997, 62:,93-103. [3] C. Menta. Soil Fauna Diversity, Function, Soil Degradation, Biological Indices, Soil Restoration in "Biodiversity Conservation and Utilization in a Diverse World" edited by Gbolagade Akeem Lameed, InTech Publishing, 2012, 59-94 [4] V. Parisi, C. Menta. C. Gardi, C. Jacomini, E. Mozzanica.. Microarthropod Communities as a Tool to Assess Soil Quality and Biodiversity: a new Approach in Italy. Agric. Ecos and Env. 2005, 105, 323-333. [4] L.F. Elliot, JM Lynch. Biodiversity and soil resilience in “Soil Resilience and Sustainable Land Use”. D.J. Greenland and I. Szabolcs Eds CAB International, Wallingford UK, 1994, 353-364 [5] F. Stagnari, G. Perpetuini, R. Tofalo, G. Campanelli, F. Leteo, U.D. Vella, M.Schirone, G. Suzzi, M. Pisante. Long-term impact of farm management and crops on soil microorganisms assessed by combined DGGE and PLFA analyses. Frontiers in Microbiology, 2014, 1-1

ASSESSMENT OF BIOLOGICAL SOIL QUALITY IN LONG-TERM ORGANICALLY MANAGED VINEYARDS IN THE VERDICCHIO TERROIR OF MATELICA (MARCHE, ITALY)

Soil biodiversity constitutes one of the main component of agroecosystems, being involved in the delivery of several essential ecosystem services such as, among others, nutrient cycling, soil formation, pest and pollution control. Thus, soil biodiversity indicators can be used by governments and farmers to monitor soil quality and ecosystem functioning under various land uses and management practices. At present, organic wine represents an emerging market that is showing potential for growth. There is, in fact, a growing worldwide interest and attention for environmental friendly products and sustainable agricultural practices. Organic farming employs a set of farming practices that contribute in preserving soil quality. In this scenario, the aim of our study was to assess the long-term effects of organically managed vineyards on soil quality by means of two bioindicators: ciliated protozoa and microarthropods. Ciliated protozoa are eukaryotic microorganisms which constitutes an essential component of the soil microbial loop. By feeding on bacterial biomass ciliates play an essential role in the liberation of nutrients in the plant rhizosphere. Soil microarthropods contribute to the organic matter degradation and dispersal, affect the soil porosity, aeration, water infiltration, modifying and improving soil fertility. Both ciliates and microarthropods are very sensitive to changes in their habitat and fluctuations in their communities can affect the food web and the energy transfer within the soil ecosystem. Thus, the monitoring of the structure of microarthropod and ciliate communities represent a valuable tool to assess soil quality and functioning. The study was realized in the terroir of “Verdicchio di Matelica” (Marche, Italy), on three vineyards that were organically managed since 1992, 1998 and 2009 respectively. In each vineyard, soil samples (0-10 cm depth) were taken every month from March to October 2011. In addition, soil chemical-physical (texture, soil moisture, pH, NPK, OM, C/N, Cu), were measured in each site. For microarthropods, the measured biological parameters were: the Soil Biological Quality (QBS-ar) index, abundances of biological (BF) and euedaphic forms (EF) and diversity indices. Soil samples were collected in both disturbed (tillage) and not-disturbed (no-tillage) inter-rows. For ciliates: abundances and diversity indices were measured and soil samples were randomly collected in the whole sampling area. The results of the multivariate data analysis (Cluster Analysis, CA; non-metric Multi-Dimensional Scaling, nMDS) and diversity indices (H’,J,d) indicate that the most stable habitat for ciliates and microarthropods is represented by the “older” (V92) followed by the V98 and the “younger” V09 vineyards. Collectively, the data seem to indicate that the long-term organic management of the soil contributes to global soil quality in vineyards at least in this particular pedoclimatic area and for the investigated bioindicators. Furthermore, this study helps in the definition of possible biotic baseline values to evaluate soil quality and health in vineyards

Soil biodiversity and sustainable vineyards: hints from the analysis of microarthropods and ciliated protozoa communities

Soil biodiversity constitutes one of the main components of agroecosystems, being involved in the delivery of several essential ecosystem services such as, among others, nutrient cycling, soil formation, pest and pollution control. Thus, soil biodiversity indicators can be used by governments and farmers to monitor soil quality and ecosystem functioning under various land uses and management practices. In this scenario, the aim of our study was to assess the long-term effects of organically managed vineyards on soil quality by means of two bioindicators: protozoan ciliates and microarthropods. The study was realized in the terroir of Verdicchio di Matelica (Marche, Italy), on three vineyards that were organically managed since 1992, 1998 and 2009 respectively. In each vineyard, soil samples (0-10 cm depth) were taken every month from March to October 2011. In addition, soil chemical-physical (texture, soil moisture, pH, NPK, OM, C/N, Cu), were measured in each site. For microarthropods, the measured biological parameters were: the Soil Biological Quality (QBS-ar) index, abundances of Biological (BF) and Euedaphic forms (EF) and diversity indices. Soil samples were collected in both disturbed (tillage) and not-disturbed (no-tillage) inter-rows. For ciliates: abundances and diversity indices were measured and soil samples were randomly collected in the whole sampling area. The results of the multivariate data analysis (Cluster Analysis, CA; non-metric Multi-Dimensional Scaling, nMDS) and diversity indices (H’,J,d) indicate that the most stable habitat for ciliates and microarthropods is represented by the “older” (V92) followed by the V98 and the “younger” V09 vineyards. Collectively, the data seem to indicate that the long-term organic management of the soil contributes to global soil quality in vineyards at least in this particular pedoclimatic area and for the investigated bioindicators. Furthermore, this study helps in the definition of possible biotic baseline values to evaluate soil quality/health in vineyards

THE BIOPRINT PILOT PROJECT: CILIATED PROTOZOA COMMUNITIES AS A TOOL TO ASSESS SOIL QUALITY IN AGROECOSYSTEMS AND NATURAL SITES OF MARCHE REGION (ITALY)

In the recent years the potential of ciliated protozoa, as indicators of soil quality has been highlighted by several studies. Thus, in the framework of the BioPrint Pilot Project and for the first time in Italy, we have investigated the biodiversity and the community structure of soil ciliates from agroecosystems and natural sites of Marche Region. The aims of our study were: i) to evaluate the capacity of ciliates to discriminate between different types of land uses; and ii) farming management practices; iii) to assess relationships among ciliate community and abiotic parameters. Soil samples were collected twice from 10 sites (5 natural sites: FORest; and 5 arable field: 3 ORGanic and 2 CONventional). Ciliate communities were studied by means of qualitative (non-flooded Petri dish) and quantitative methods. Soil chemical-physical (texture, CEC NPK, OM, C/N, soil moisture and temperature) parameters were measured. Qualitative ciliate analysis allowed us to identify a total of 59 species representing 29 genera and 12 orders (plus 10 new species for the science). ORG sites were the richest in species followed by CON and FOR. The mean values for H’ (2.6), d (3.4) and J (0.8) were significantly higher in ORG than in CON (H’=2.1; d=2.7; J=0.7) and FOR (H’=1.7; d=1.95; J=0.6) supporting the intermediate disturbance hypothesis. Multivariate analysis showed statistically significant differences between natural sites (FORest) and agricultural sites, as well as between the ORGanic and CONventional management farming systems. CCA analysis showed correlations between the distribution of species with environmental parameters indicating the importance of these parameters in shaping the ciliate communities in the different type sites. Altogether, these results showed the bioindicative potential of ciliate communities in discriminating between natural sites (FORests) and agroecosystems, as well as their capacity to discriminate, at least preliminary, between different management systems (ORG vs CON)

Shedding light on the polyphyletic behavior of the genus Sterkiella: The importance of ontogenetic and molecular phylogenetic approaches

Present study, investigates a poorly known species of the genus Sterkiella, i.e., S. tricirrata, based on two populations isolated from soil samples collected from the Colfiorito Regional Park, Umbria Region, Italy and from the Silent Valley National Park, India. Both populations showed a highly similar morphology, however different ontogenetic pattern in between. The study confirms the validity of the species S. tricirrata which was considered to be a species within the Sterkiella histriomuscorum complex. The main ontogenetic difference between S. tricirrata and other species of the genus Sterkiella is the different mode of formation of anlagen V and VI of the proter in the former. In the phylogenetic analyses, Sterkiella tricirrata clusters with Sterkiella sinica within the stylonychine oxytrichids, in a clade away from the type species (Sterkiella cavicola) of the genus Sterkiella. The study highlights the importance of ontogenetic as well as molecular data in shedding light on the polyphyletic behavior of the genus Sterkiella. A detailed description of S. tricirrata based on morphology, ontogenesis and molecular phylogenetic methods is presented. Further, the improved diagnosis has been provided for the genus Sterkiella and the poorly known species S. tricirrata