Insights into freshwater ciliate diversity through high throughput DNA metabarcoding.

The freshwater bodies of India are highly biodiverse but still understudied, especially concerning ciliates. Ciliates constitute a significant portion of eukaryotic diversity and play crucial roles in microbial loops, nutrient recycling, and ecosystem maintenance. The present study aimed to elucidate ciliate diversity in three freshwater sites in the Delhi region of India: Okhla Bird Sanctuary (OBS), Sanjay Lake (SL), and Raj Ghat pond (RJ). This study represents the first investigation into the taxonomic diversity and richness of freshwater ciliates in India using a high-throughput DNA metabarcoding approach. For the analysis, total environmental DNA was extracted from the three freshwater samples, followed by sequencing of the 18S V4 barcode region and subsequent phylogenetic analyses. Operational taxonomic units (OTU) analyses revealed maximum species diversity in OBS (106), followed by SL (104) and RJ (99) sites. Ciliates from the classes Oligohymenophorea, Prostomatea, and Spirotrichea were dominant in the three sites. The study discusses the ability of the metabarcoding approach to uncover unknown and rare species. The study highlights the need for refined reference databases and cautious interpretation of the high-throughput sequencing-generated data while emphasizing the complementary nature of molecular and morphological approaches in studying ciliate diversity.

DNA barcoding, an effective tool for species identification: a review.

DNA barcoding is a powerful taxonomic tool to identify and discover species. DNA barcoding utilizes one or more standardized short DNA regions for taxon identification. With the emergence of new sequencing techniques, such as Next-generation sequencing (NGS), ONT MinION nanopore sequencing, and Pac Bio sequencing, DNA barcoding has become more accurate, fast, and reliable. Rapid species identification by DNA barcodes has been used in a variety of fields, including forensic science, control of the food supply chain, and disease understanding. The Consortium for Barcode of Life (CBOL) presents various working groups to identify the universal barcode gene, such as COI in metazoans; rbcL, matK, and ITS in plants; ITS in fungi; 16S rRNA gene in bacteria and archaea, and creating a reference DNA barcode library. In this article, an attempt has been made to analyze the various proposed DNA barcode for different organisms, strengths & limitations, recent advancements in DNA barcoding, and methods to speed up the DNA barcode reference library construction. This study concludes that constructing a reference library with high species coverage would be a major step toward identifying species by DNA barcodes. This can be achieved in a short period of time by using advanced sequencing and data analysis methods.

Molecular characterization and transcriptional modulation of stress-responsive genes under heavy metal stress in freshwater ciliate, Euplotes aediculatus.

Heavy metal pollutants in the environment are increasing exponentially due to various anthropogenic factors including mining, industrial and agricultural wastes. Living organisms exposed to heavy metals above a certain threshold level induces deleterious effects in these organisms. To live in such severe environments, microbes have developed a range of tolerance mechanisms which include upregulation of stress-responsive genes and/or antioxidant enzymes to detoxify the metal stress. Single cell eukaryotic microorganisms, i.e., ciliates, are highly sensitive to environmental pollutants mainly due to the absence of cell wall, which make them suitable candidates for conducting ecotoxicological studies. Therefore, the present investigation describes the effects of heavy metals (cadmium and copper) on freshwater ciliate, Euplotes aediculatus. The activities of antioxidant enzymes, i.e., catalase and glutathione peroxidase in E. aediculatus were determined under heavy metal exposure. Besides, the expression of stress-responsive genes, namely, heat-shock protein 70 (hsp70) and catalase (cat), has also been determined in this freshwater ciliate species under metal stress. The present study showed that the enzyme activity and the expression of these genes increased with an increase in the heavy metal concentration and with the duration of metal exposure. Also, these stress-responsive genes were sequenced and characterized to comprehend their role in cell rescue.

Characterization of Euplotes lynni nov. spec., E. indica nov. spec. and description of E. aediculatus and E. woodruffi (Ciliophora, Euplotidae) using an integrative approach.

Four species belonging to the genus Euplotes have been investigated, namely: E. lynni nov. spec., E. indica nov. spec., E. aediculatus, and E. woodruffi. All populations are from India and were investigated using morphological and molecular markers. The phylogenetic relationships were inferred from small subunit ribosomal rRNA gene (SSU rRNA), internal transcribed spacer (ITS) region, and mitochondrial cytochrome c oxidase subunit I (COI) gene. Predicted secondary structure models for two new species using the hypervariable region of the SSU rRNA gene and ITS2 region support the distinctness of both species. Morphological characters were subjected to principal component analysis (PCA) and genetic variations were studied in-depth to analyze the relatedness of the two new species with their congeners. An integrative approach combining morphological features, molecular analysis, and ecological characteristics was carried out to understand the phylogenetic position of the reported species within the different clades of the genus Euplotes.

Indicators for assessment of soil quality: a mini-review.

Soil quality is the competence of soil to perform necessary functions that are able to maintain animal and plant productivity of the soil. Soil consists of various physical, chemical, and biological parameters, and all these parameters are involved in the critical functioning of soil. There is a need for continuous assessment of soil quality as soil is a complex and dynamic constituent of Earth's biosphere that is continuously changing by natural and anthropogenic disturbances. Any perturbations in the soil cause disturbances in the physical (soil texture, bulk density, etc.), chemical (pH, salinity, organic carbon, etc.), and biological (microbes and enzymes) parameters. These physical, chemical, and biological parameters can serve as indicators for soil quality assessment. However, soil quality assessment cannot be possible by evaluating only one parameter out of physical, chemical, or biological. So, there is an emergent need to establish a minimum dataset (MDS) which shall include physical, chemical, and biological parameters to assess the quality of the given soil. This review attempts to describe various physical, chemical, and biological parameters, combinations of which can be used in the establishment of MDS.

Soil ciliates of the Indian Delhi Region: Their community characteristics with emphasis on their ecological implications as sensitive bio-indicators for soil quality.

The present investigation aims to study the diversity of ciliates from different habitats in and around Delhi, India, and the correlation of this diversity with soil quality {agricultural lands (site 1 and 2), dump yards (site 3 and 4), sewage treatment plant (site 5), residential land (site 6), landfill (site 7) and barren land (site 8)}. Various physicochemical parameters of the different soil samples were studied and analysed for soil texture, interstitial water, pH, conductivity, total organic carbon, total organic matter, total nitrogen and phosphorous content, using standard protocols. Seventeen ciliate taxa belonging to four classes, seven orders, ten families, and 17 genera were recorded, with the maximum number of species (eleven) belonging to the class Spirotrichea. Ciliate diversity was highest at sites 5 and 6 and lowest at sites 1 and 2. Spathidium sp. was the dominant species in the conditioned land (site 8), while the ciliate Colpoda sp. was present in all the sites examined, showing the highest population density in the sewage treatment plant site (site 5). Statistical analysis showed that ciliate diversity was positively correlated to physicochemical parameters such as interstitial water, total organic matter and organic carbon, total nitrogen and total phosphorous content. Analyses of spirotrichs/colpodids (S/C) ratio and diversity indices implied that the habitat conditions of sites 1, 2, 3 and 8 are relatively unfavourable for soil ciliates to flourish; while sites 4, 5, 6 and 7 provided more favourable conditions. The ubiquity of ciliate distribution suggests their important role in the soil food webs and nutrient cycling, and their community structure and specific characteristics appear to be of major importance for soil formation. A full understanding of soil ciliate diversity and physicochemical parameters helps to inform best practice for improving soil quality as well as conservation practices for sustainable development and management of farms and cultivated lands. In conclusion, ciliate diversity serves as an important and sensitive bio-indicator for soil quality.

Expression and molecular characterization of stress-responsive genes (hsp70 and Mn-sod) and evaluation of antioxidant enzymes (CAT and GPx) in heavy metal exposed freshwater ciliate, Tetmemena sp.

Response of heavy metals namely cadmium (Cd) and copper (Cu) on the expression of stress responsive genes in the fresh water ciliate, Tetmemena sp. (single cell eukaryote) was studied. Stress responsive genes include heat shock protein genes and genes involved in antioxidant defence system. Quantitative real time PCR (qRT-PCR) was employed to evaluate the effects of Cd and Cu on the expression of cytosolic hsp70 and Mn-sod genes. Increase in the expression of these genes was observed after exposure with the heavy metals. The macronuclear cytosolic hsp70 and Mn-sod (SOD2) genes were also sequenced and characterized using various bioinformatics tools. In antioxidant defence system, the superoxide dismutase (SOD) family is a first line antioxidant enzyme group involved in catalysing reactive oxygen species (ROS) to hydrogen peroxide and molecular oxygen. Influence of Cd and Cu on the activity of SOD has already been reported by our group. Therefore, the enzymatic activities of antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPx) were studied in the presence of Cd and Cu and there was significant increase in activity of these enzymes in concentration dependent manner. This study suggests that cytosolic hsp70, Mn-sod and the antioxidant enzymes such as CAT and GPx can be used as effective molecular biomarkers for heavy metal toxicity and Tetmemena sp. can be used as potential model for understanding the molecular response to heavy metal contamination in aquatic ecosystems.

Techniques and tools for species identification in ciliates: a review.

Ciliates are highly divergent unicellular eukaryotic organisms with nuclear dualism and a highly specialized ciliary pattern. They inhabit all biotopes and play crucial roles in regulating microbial food webs as they prey on bacteria, protists and even on microscopic animals. Nevertheless, subtle morphological differences and tiny sizes hinder proper species identification for many ciliates. In the present review, an attempt has been made to elaborate the various approaches used by modern day ciliate taxonomists for species identification. The different approaches involved in taxonomic characterization of ciliates such as classical (using live-cell observations, staining techniques, etc.), molecular (involving various marker genes) and statistical (delimitation of cryptic species) methods have been reviewed. Ecological and behavioural aspects in species identification have also been discussed. In present-day taxonomy, it is important to use a 'total evidence' approach in identifying ciliates, relying on both classical and molecular information whenever possible. This integrative approach will help in the mergence of classical methods with modern-day tools for comprehensive species description in future.