Integrating the 3 Dimensions of Biodiversity: New Standards for Documenting Biodiversity of Ciliates and Strategies for Accessing and Sharing Data

Title: Ciliate biodiversity studies in Italy - DIVERSITY AND BIOINDICATIVE VALUE OF SOIL CILIATE COMMUNITIES • Soil ciliate studies in Italy……from past to present! • An overview of the ongoing soil (ciliates) bioindication projects and initiatives in Italy • Two study cases: “BioPrint project” (Marche Region) and the “Soil Mapping project” (Lombardia Region) and the amazing P2R site! •A glimpse to new species/genera from soils of Italy •Exploring Soil Ciliate Diversity in Italy: A Synthetic Evaluation •What’s next

Polyphenolics free DNA isolation and optimization of PCR-RAPD for fennel (Foeniculum vulgare Mill.) from mature and young leaves

Molecular analysis of fennel (Foeniculum vulgare Mill.) strictly relies on high yield, and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide (CTAB) protocol introducing several modifications such as inclusion of variable percentage of polyvinylpyrrolidone (PVP, 2 - 6%), different molarity of sodium chloride (NaCl, 1.5 - 4 M), activated charcoal (1 to 2%), and pre-heated buffer (65°C) with and without liquid N2 extraction for the mature leaves. In contrast, the CTAB protocol without any additional anti-oxidants using liquid N2 extraction was performed for the fresh young leaf tissue. Optimization of polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD) conditions included 10X PCR buffer compositions such as (NH4)2SO4 with 0.1% (v/v) Tween 20 over KCl buffer with and without 0.8% (v/v) Nonidet P40 and an ‘optimized’ buffer which contains KCl and (NH4)2SO4, MgCl2 (2.5 mM), Taq enzyme (1 to 1.5 U), annealing temperature of 37 and 42°C and PCR reaction volume of 10 and 25 μl. The results show that DNA isolated from fresh young leaves were superior in quality and quantity over mature (stored) leaves and was amenable to optimized PCR-RAPD conditions.Keywords: Activated charcoal, cetyl trimethyl ammonium bromide (CTAB)-DNA, Foeniculum vulgare, NaCl, polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD), PCR buffer, polyphenolics, polyvinylpyrrolidone (PVP

Polyphenolics free DNA isolation and optimization of PCR-RAPD for fennel (Foeniculum vulgare Mill.) from mature and young leaves

Molecular analysis of fennel (Foeniculum vulgare Mill.) strictly relies on high yield, and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide (CTAB) protocol introducing several modifications such as inclusion of variable percentage of polyvinylpyrrolidone (PVP, 2 - 6%), different molarity of sodium chloride (NaCl, 1.5 - 4 M), activated charcoal (1 to 2%), and pre-heated buffer (65°C) with and without liquid N2 extraction for the mature leaves. In contrast, the CTAB protocol without any additional anti-oxidants using liquid N2 extraction was performed for the fresh young leaf tissue. Optimization of polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD) conditions included 10X PCR buffer compositions such as (NH4)2SO4 with 0.1% (v/v) Tween 20 over KCl buffer with and without 0.8% (v/v) Nonidet P40 and an ‘optimized’ buffer which contains KCl and (NH4)2SO4, MgCl2 (2.5 mM), Taq enzyme (1 to 1.5 U), annealing temperature of 37 and 42°C and PCR reaction volume of 10 and 25 μl. The results show that DNA isolated from fresh young leaves were superior in quality and quantity over mature (stored) leaves and was amenable to optimized PCR-RAPD conditions

Palynological origin, Total Phenolic and Flavonoid contents and Antioxidant Properties of honey bee-collected pollen from Matelica (Marche, Italy): A seasonal analysis

Nowadays honey-bee collected pollen represents an apicultural product of great commercial interest due to its high nutritional value. Pollen is, in fact, generally referred to as a super-food; it contains high level of proteins (from 10% up to 40%), lipids (1-13%), carbohydrates (13-55%), minerals and vitamins (in particular of the B group) [1]. In addition, honey bee-pollen possesses antioxidant properties (mostly depending on the phenolic compounds) which are known to be very effective in fighting against free radicals; therefore, honey-bee collected pollen has high potential for both nutritional and medical applications [2]. However, the chemical composition of bee-pollen, may significantly changes according to the palynological and geographical origin, climatic conditions and month of collection [3-4]. Therefore, all these factors differently contribute to the final properties and biological activities of the bee-pollen (and thus, in turn, its therapeutic effects and nutritional properties). In this context, the present study aims to assess the palynological origin, total phenolic contents (TPC), total flavonoid content (TFC) and antioxidant properties of multifloral honey bee-collected pollen from a rural location in the area of municipality of Matelica (Marche Region, Italy). The project, realized in collaboration with the Cooperativa Apicoltori Montani of Matelica, has involved the analysis of multifloral pollen samples on a seasonal basis (from April to September 2016) by means of the following in vitro assays: a) the total phenolic contents (TPC) determined by the the Folin-Ciocalteu methods [5] using gallic acid as the standard compound; the total flavonoid contents determined by the aluminium chloride colorimetric assay developed by [6] using quercetin as reference standard and b) the 1, l-diphenyl-2-picrylhydrazyl (DPPH) Scavenging Assay [7] and Hydroxyl Radical Scavenging Assay (HRSA) [8] for the analysis of the antioxidant properties. All these assays were realized using lyophilized bee pollen ethanolic extracts (LBPE). Qualitative and quantitative palynological characterization of the 4 pollen samples, showed the presence of a total of more than 20 plant families, with Fabaceae, Rosaceae, Salicaceae, Brassicaceae and Asteraceae being the most abundant (from 20% up to 33%). Results indicated that there were significant differences (p ≤ 0.001) among pollen samples for total phenolic and flavonoid contents. TPC values ranged from 11.8 (September) to 23.8 (May) to mg GAE/gr,; TFC contents ranged from 5.98 (September) to 14.14 (May) mg QE/g. Furthermore, results of the DPPH and HRSA scavenging activities indicated that the lyophilized bee pollen ethanolic extracts have significant antioxidant properties. In particular, the highest TPC and TFC contents and antioxidant properties were recorded for the May pollen samples. The present study represent the first “seasonal fingerprint” of pollen properties in Italy. In final, our study aims to give a) a contribution to the economic valorization of this important local natural resource; and, b) to possibly define seasonal/monthly baseline values (i.e minimum and maximum values) for bee pollen properties in the area of Matelica. References [1] Almeida-Muradian L.B., Pamplona L.C., Coimbra S. and Barth O.M. Chemical composition and botanical evaluation of dried bee pollen pellets. J. Food Comp. Anal. 2005, 18, 105-111. [2] Denisow, B., Denisow-Pietrzyk, M. Biological and therapeutic properties of bee pollen: a review. J. Sci. Food Agr., 2016, 4303-4309 [3] Almaraz-Abaraca, N., Campos, M.G., Ávila-Reyes, J.A. Variability of antioxidant activity among honey-bee collected pollen of different botanical origin, J. Sci. Tech. Amer. 2004, 29, 574-578. [4] Szczesna, T., Rybak-Chielewska, H., Chmielewski, W. Sugar composition of pollen loads harvested at different periods of the beekeeping season, J. Apic. Sci. 2002, 46, 107-115. [5] Vattem AD, Shetty K (2002) Solid-state production of phenolic antioxidants from Cranberry pomace by Rhizopus oligosporus. Food Biotechnol, 2002, 16, 189–210 [6] Zhishen, J., T. Mengcheng, and Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999, 64, 555-559 [7] Yildirim A, Mavi A, Kara AA Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. J. Agri.c Food Chem. 2001, 49, 4083–4089 [8] Ravindran C, Varatharajan GR, Rajasabapathy R, Vijayakanth S, HarishKumar A, Ram M. A role for antioxidants in acclimation of marine derived pathogenic fungus (NIOCC 1) to salt stress. Microb. Pathog. 2012, 168–17

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

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

The Alpaca Melanocortin 1 Receptor: Gene Mutations, Transcripts, and Relative Levels of Expression in Ventral Skin Biopsies

The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T) were observed in coding region and three in the 3UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation

Effect of Compost Added with Compostable Plastic Packaging on Wheat Health and its Rhizosphere Microbiome

Aim: Packaging waste is steadily increasing in Europe. Thus, compostable plastics (CPs) may represent an environmentally friendly alternative to petroleum-based plastics for the development of innovative packaging solutions. However, the potential impact of CPs presence in compost on soil ecosystems and crop health, still needs to be assessed. The present investigation aims to evaluate and compare the effects produced by "standard" compost (C) obtained from urban organic waste (UOW) and UOW added with 3% compostable bioplastics (CV), on wheat health and its rhizosphere microbiome. Method: Field experiments were conducted at Agricultural Research Station, CERMIS, Italy. During 2021-2022, three treatments (C, CV, and B -no compost) with five replicates each, were tested. Soil chemical-physical properties were measured before and after composts distribution to evaluate changes in nutrient concentrations and presence of possible pollutants. At the flowering stage, ten plants and associated rhizospheric soils were collected in each plot. Morphological and productive wheat traits were measured. Rhizosphere bacterial communities were analysed by 16S rRNA metabarcoding. Results: No significant differences between treatments were detected by means of wheat traits. C and CV compost fertilization significantly changed the bacterial communities with respect to the untreated control. But no significant differences were found between the C and CV communities, which also showed similar composition. This demonstrates the equivalence of the two composts. Conclusions: Results showed that CPs addition do not negatively affect compost quality neither wheat nor microbiome health. Thus, CPs represent a sustainable alternative to conventional plastic that will be crucial in the green European transition

Influence of Organic and Conventional Management Systems on Soil Microarthropods in Protected and Non-Protected Areas

Aim: The EU Biodiversity Strategy 2030 aims to increase land-protected areas at 30% and organic farming at 25% of agricultural lands. But which measure could be more effective in preserving soil biodiversity? The aim of the study is, therefore, to assess soil health of arable lands under organic and conventional managements in Non-protected (NPAorg) and Protected (PAcon) areas of Marche region (Italy) and compare the influence of the applied farming practices on soil microarthropods in two seasons, characterized by different intensities of soil management practices: spring (lower) and autumn (higher). Method: Soil health has been assessed through the Biological Quality of Soil index based on arthropods (QBS-ar). Novel approaches (QBS-ab and FAI indices) which consider microarthropods’ abundance in the index calculation, have been also applied. Density (ind/m2), Acari/Collembola ratio, % of Oribatid mites on total mites, biodiversity indices, correlations with chemical-physical parameters, and ordination analysis (nMDS) have been evaluated. Results: In both seasons, different communities have been found according to management and, particularly, PAcon sites showed significantly higher levels of biodiversity compared to NPAorg. However, in autumn, microarthropod communities present higher stability in NPAorg sites, showing an opposite trend and fewer fluctuations of the indices compared to PAcon. Conclusions: PA, even in conventional managed soils, seem to enhance soil biodiversity, while organic farming in NPA, confers a higher resilience to soil, making microarthropod communities more stable. Results showed that agricultural intensity reduction combined with the increased integration of agroecosystems in protected areas may represent an effective, and sustainable measure to preserve soil biodiversity and its ecological services

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