Exploration and Retrieval of Virus-Related Molecular Data Using ExTaxsI: The Monkeypox Use Case.

Retrieval and visualization of biological data are essential for understanding complex systems. With the increasing volume of data generated from high-throughput sequencing technologies, effective and optimized data visualization tools have become indispensable. This is particularly relevant in the COVID-19 postpandemic period, where understanding the diversity and interactions of microbial communities (i.e., viral and bacterial) constitutes an important asset to develop and plan suitable interventions.In this chapter, we show the usage and the potentials of ExTaxsI (Exploring Taxonomy Information) tool to retrieve viral biodiversity data stored in National Center for Biotechnology Information (NCBI) databases and create the related visualization. In addition, by integrating different functions and modules, the tool generates relevant types of visualization plots to facilitate the exploration of microbial biodiversity communities useful to deep dive into ecological and taxonomic relationships among different species and identify potential significant targets.Using the Monkeypox virus as a case study, this work points out significant perspectives on biological data visualization, which can be used to gain insights into the ecology, evolution, and pathogenesis of viruses. Accordingly, we show the potentiality of ExTaxsI to organize and describe the available/downloaded data in an easy, simple, and interpretable way allowing the user to interact dynamically with the visualization plots through specific filters, zoom, and explore functions.

Aquaculture ecosystem microbiome at the water-fish interface: the case-study of rainbow trout fed with Tenebrio molitor novel diets.

BACKGROUND: Sustainable aquaculture relies on multiple factors, including water quality, fish diets, and farmed fish. Replacing fishmeal (FM) with alternative protein sources is key for improving sustainability in aquaculture and promoting fish health. Indeed, great research efforts have been made to evaluate novel feed formulations, focusing especially on the effects on the fish gut microbiome. Few studies have explored host-environment interactions. In the present study, we evaluated the influence of novel insect-based (Tenebrio molitor) fish diets on the microbiome at the water-fish interface in an engineered rainbow trout (Oncorhynchus mykiss) farming ecosystem. Using 16S rRNA gene metabarcoding, we comprehensively analyzed the microbiomes of water, tank biofilm, fish intestinal mucus, fish cutis, and feed samples. RESULTS: Core microbiome analysis revealed the presence of a highly reduced core shared by all sample sources, constituted by Aeromonas spp., in both the control and novel feed test groups. Network analysis showed that samples were clustered based on the sample source, with no significant differences related to the feed formulation tested. Thus, the different diets did not seem to affect the environment (water and tank biofilm) and fish (cutis and intestinal mucus) microbiomes. To disentangle the contribution of feed at a finer scale, we performed a differential abundance analysis and observed differential enrichment/impoverishment in specific taxa, comparing the samples belonging to the control diet group and the insect-based diet group. CONCLUSIONS: Omic exploration of the water-fish interface exposes patterns that are otherwise undetected. These data demonstrate a link between the environment and fish and show that subtle but significant differences are caused by feed composition. Thus, the research presented here is a step towards positively influencing the aquaculture environment and its microbiome.

Red mark syndrome: Is the aquaculture water microbiome a keystone for understanding the disease aetiology?

Aquaculture significantly contributes to the growing demand for food worldwide. However, diseases associated with intensive aquaculture conditions, especially the skin related syndromes, may have significant implications on fish health and industry. In farmed rainbow trout, red mark syndrome (RMS), which consists of multiple skin lesions, currently lacks recognized aetiological agents, and increased efforts are needed to elucidate the onset of these conditions. Most of the past studies were focused on analyzing skin lesions, but no study focused on water, a medium constantly interacting with fish. Indeed, water tanks are environmental niches colonized by microbial communities, which may be implicated in the onset of the disease. Here, we present the results of water and sediment microbiome analyses performed in an RMS-affected aquaculture facility, bringing new knowledge about the environmental microbiomes harbored under these conditions. On the whole, no significant differences in the bacterial community structure were reported in RMS-affected tanks compared to the RMS-free ones. However, we highlighted significant differences in microbiome composition when analyzing different samples source (i.e., water and sediments). Looking at the finer scale, we measured significant changes in the relative abundances of specific taxa in RMS-affected tanks, especially when analyzing water samples. Our results provide worthwhile insight into a mostly uncharacterized ecological scenario, aiding future studies on the aquaculture built environment for disease prevention and monitoring.

Playing "hide and seek" with the Mediterranean monk seal: a citizen science dataset reveals its distribution from molecular traces (eDNA).

Animal conservation relies on assessing the distribution and habitat use of species, but for endangered/elusive animals this can prove difficult. The Monk Seal, Monachus monachus, is one of the world's most endangered species of pinniped, and the only one endemic to the Mediterranean Sea. During recent decades, direct observations have been few and scattered, making it difficult to determine its distribution away from the Aegean Sea (core distribution area of the post-decline relict population). This study relies on environmental DNA (eDNA) analysis to detect the presence of the Monk Seal in 135 samples collected in 120 locations of the central/western Mediterranean Sea, spanning about 1500 km longitudinally and 1000 km latitudinally. A recently described species-specific qPCR assay was used on marine-water samples, mostly collected during 2021 by a Citizen Science (CS) project. Positive detections occurred throughout the longitudinal range, including the westernmost surveyed area (Balearic archipelago). The distribution of the positive detections indicated six "hotspots", mostly overlapping with historical Monk Seal sites, suggesting that habitat-specific characteristics play a fundamental role. We applied single-season occupancy models to correct for detection probability and to assess the importance of site-specific characteristics. The distance from small islets and protected (or access-restricted) areas was correlated negatively with the detection probability. This novel molecular approach, applied here for the first time in an extensive CS study, proved its potential as a tool for monitoring the distribution of this endangered/elusive species.

The Microbiome of the Built Environment: The Nexus for Urban Regeneration for the Cities of Tomorrow.

Built environments are, for most of us, our natural habitat. In the last 50 years, the built-up area has more than doubled, with a massive biodiversity loss. The undeniable benefits of a city providing all the basic needs to a growing population showed longer-term and less obvious costs to human health: autoimmune and non-communicable diseases, as well as antimicrobial resistance, have reached unprecedented and alarming levels. Humans coevolved with microbes, and this long-lasting alliance is affected by the loss of connection with natural environments, misuse of antibiotics, and highly sanitized environments. Our aim is to direct the focus onto the microbial communities harbored by the built environments we live in. They represent the nexus for urban regeneration, which starts from a healthy environment. Planning a city means considering, in a two-fold way, the ecosystem health and the multidimensional aspects of wellbeing, including social, cultural, and aesthetic values. The significance of this perspective is inspiring guidelines and strategies for the urban regeneration of the cities of tomorrow, exploiting the invaluable role of microbial biodiversity and the ecosystem services that it could provide to create the robust scientific knowledge that is necessary for a bioinformed design of buildings and cities for healthy and sustainable living.

It's a Long Way to the Tap: Microbiome and DNA-Based Omics at the Core of Drinking Water Quality.

Microbial communities interact with us and affect our health in ways that are only beginning to be understood. Microorganisms have been detected in every ecosystem on Earth, as well as in any built environment that has been investigated. Drinking water sources, drinking water treatment plants and distribution systems provide peculiar microbial ecological niches, dismantling the belief of the "biological simplicity" of drinking water. Nevertheless, drinking water microbiomes are understudied compared to other microbiomes. Recent DNA sequencing and meta-omics advancements allow a deeper understanding of drinking water microbiota. Thus, moving beyond the limits of day-to-day testing for specific pathogenic microbes, new approaches aim at predicting microbiome changes driven by disturbances at the macro-scale and overtime. This will foster an effective and proactive management of water sources, improving the drinking water supply system and the monitoring activities to lower public health risk. Here, we want to give a new angle on drinking water microbiome research. Starting from a selection of 231 scientific publications on this topic, we emphasize the value of biodiversity in drinking water ecosystems and how it can be related with industrialization. We then discuss how microbiome research can support sustainable drinking water management, encouraging collaborations across sectors and involving the society through responsible research and innovation.

SKIOME Project: a curated collection of skin microbiome datasets enriched with study-related metadata.

Large amounts of data from microbiome-related studies have been (and are currently being) deposited on international public databases. These datasets represent a valuable resource for the microbiome research community and could serve future researchers interested in integrating multiple datasets into powerful meta-analyses. However, this huge amount of data lacks harmonization and it is far from being completely exploited in its full potential to build a foundation that places microbiome research at the nexus of many subdisciplines within and beyond biology. Thus, it urges the need for data accessibility and reusability, according to findable, accessible, interoperable and reusable (FAIR) principles, as supported by National Microbiome Data Collaborative and FAIR Microbiome. To tackle the challenge of accelerating discovery and advances in skin microbiome research, we collected, integrated and organized existing microbiome data resources from human skin 16S rRNA amplicon-sequencing experiments. We generated a comprehensive collection of datasets, enriched in metadata, and organized this information into data frames ready to be integrated into microbiome research projects and advanced post-processing analyses, such as data science applications (e.g. machine learning). Furthermore, we have created a data retrieval and curation framework built on three different stages to maximize the retrieval of datasets and metadata associated with them. Lastly, we highlighted some caveats regarding metadata retrieval and suggested ways to improve future metadata submissions. Overall, our work resulted in a curated skin microbiome datasets collection accompanied by a state-of-the-art analysis of the last 10 years of the skin microbiome field. Database URL:  https://github.com/giuliaago/SKIOMEMetadataRetrieval.

ExTaxsI: an exploration tool of biodiversity molecular data.

BACKGROUND: The increasing availability of multi-omics data is leading to regularly revised estimates of existing biodiversity data. In particular, the molecular data enable novel species to be characterized and the information linked to those already observed to be increased with new genomics data. For this reason, the management and visualization of existing molecular data, and their related metadata, through the implementation of easy-to-use IT tools have become a key point to design future research. The more users are able to access biodiversity-related information, the greater the ability of the scientific community to expand its knowledge in this area. RESULTS: In this article we focus on the development of ExTaxsI (Exploring Taxonomy Information), an IT tool that can retrieve biodiversity data stored in NCBI databases and provide a simple and explorable visualization. We use 3 case studies to show how an efficient organization of the available data can lead to obtaining new information that is fundamental as a starting point for new research. Using this approach highlights the limits in the distribution of data availability, a key factor to consider in the experimental design phase of broad-spectrum studies such as metagenomics. CONCLUSIONS: ExTaxsI can easily retrieve molecular data and its metadata with an explorable visualization, with the aim of helping researchers to improve experimental designs and highlight the main gaps in the coverage of available data.

DNA-Based Herbal Teas' Authentication: An ITS2 and psbA-trnH Multi-Marker DNA Metabarcoding Approach.

Medicinal plants have been widely used in traditional medicine due to their therapeutic properties. Although they are mostly used as herbal infusion and tincture, employment as ingredients of food supplements is increasing. However, fraud and adulteration are widespread issues. In our study, we aimed at evaluating DNA metabarcoding as a tool to identify product composition. In order to accomplish this, we analyzed fifteen commercial products with DNA metabarcoding, using two barcode regions: psbA-trnH and ITS2. Results showed that on average, 70% (44-100) of the declared ingredients have been identified. The ITS2 marker appears to identify more species (n = 60) than psbA-trnH (n = 35), with an ingredients' identification rate of 52% versus 45%, respectively. Some species are identified only by one marker rather than the other. Additionally, in order to evaluate the quantitative ability of high-throughput sequencing (HTS) to compare the plant component to the corresponding assigned sequences, in the laboratory, we created six mock mixtures of plants starting both from biomass and gDNA. Our analysis also supports the application of DNA metabarcoding for a relative quantitative analysis. These results move towards the application of HTS analysis for studying the composition of herbal teas for medicinal plants' traceability and quality control.

Plant biodiversity assessment through pollen DNA metabarcoding in Natura 2000 habitats (Italian Alps).

Monitoring biodiversity is of increasing importance in natural ecosystems. Metabarcoding can be used as a powerful molecular tool to complement traditional biodiversity monitoring, as total environmental DNA can be analyzed from complex samples containing DNA of different origin. The aim of this research was to demonstrate the potential of pollen DNA metabarcoding using the chloroplast trnL partial gene sequencing to characterize plant biodiversity. Collecting airborne biological particles with gravimetric Tauber traps in four Natura 2000 habitats within the Natural Park of Paneveggio Pale di San Martino (Italian Alps), at three-time intervals in 1 year, metabarcoding identified 68 taxa belonging to 32 local plant families. Metabarcoding could identify with finer taxonomic resolution almost all non-rare families found by conventional light microscopy concurrently applied. However, compared to microscopy quantitative results, Poaceae, Betulaceae, and Oleaceae were found to contribute to a lesser extent to the plant biodiversity and Pinaceae were more represented. Temporal changes detected by metabarcoding matched the features of each pollen season, as defined by aerobiological studies running in parallel, and spatial heterogeneity was revealed between sites. Our results showcase that pollen metabarcoding is a promising approach in detecting plant species composition which could provide support to continuous monitoring required in Natura 2000 habitats for biodiversity conservation.

Exploring Changes in the Microbiota of Aedes albopictus: Comparison Among Breeding Site Water, Larvae, and Adults.

The mosquito body hosts highly diverse microbes, which influence different physiological traits of both larvae and adults. The composition of adult mosquito microbiota is tightly linked to that of larvae, which are aquatic and feed on organic detritus, algae and prokaryotic microorganisms present in their breeding sites. Unraveling the ecological features of larval habitats that shape the structure of bacterial communities and their interactions with the mosquito host is still a poorly investigated topic in the Asian tiger mosquito Aedes albopictus, a highly invasive species that is vector of numerous arboviruses, including Dengue, Chikungunya, and Zika viruses. In this study, we investigated the composition of the bacterial community present in the water from a natural larval breeding site in which we separately reared wild-collected larvae and hatched eggs of the Foshan reference laboratory strain. Using sequence analysis of bacterial 16S rRNA gene amplicons, we comparatively analyzed the microbiota of the larvae and that of adult mosquitoes, deriving information about the relative impact of the breeding site water on shaping mosquito microbiota. We observed a higher bacterial diversity in breeding site water than in larvae or adults, irrespective of the origin of the sample. Moreover, larvae displayed a significantly different and most diversified microbial community than newly emerged adults, which appeared to be dominated by Proteobacteria. The microbiota of breeding site water significantly increased its diversity over time, suggesting the presence of a dynamic interaction among bacterial communities, breeding sites and mosquito hosts. The analysis of Wolbachia prevalence in adults from Foshan and five additional strains with different geographic origins confirmed the described pattern of dual wAlbA and wAlbB strain infection. However, differences in Wolbachia prevalence were detected, with one strain from La Reunion Island showing up to 18% uninfected individuals. These findings contribute in further understanding the dynamic interactions between the ecology of larval habitats and the structure of host microbiota, as well as providing additional information relative to the patterns of Wolbachia infection.

Fermented food products in the era of globalization: tradition meets biotechnology innovations.

Omics tools offer the opportunity to characterize and trace traditional and industrial fermented foods. Bioinformatics, through machine learning, and other advanced statistical approaches, are able to disentangle fermentation processes and to predict the evolution and metabolic outcomes of a food microbial ecosystem. By assembling microbial artificial consortia, the biotechnological advances will also be able to enhance the nutritional value and organoleptics characteristics of fermented food, preserving, at the same time, the potential of autochthonous microbial consortia and metabolic pathways, which are difficult to reproduce. Preserving the traditional methods contributes to protecting the hidden value of local biodiversity, and exploits its potential in industrial processes with the final aim of guaranteeing food security and safety, even in developing countries.

Tasting the differences: Microbiota analysis of different insect-based novel food.

Traceability, quality and safety of edible insects are important both for the producers and the consumers. Today, alongside the burst of edible insects in western countries, we are facing a gap of knowledge of insect microbiota associated with the microbial ecosystems of insect-based products. In this context, High-Throughput DNA Sequencing (HTS) techniques can give insight into the carryover of insect microbiota into final food products. In this study, we investigated the microbiota composition of insect-based commercial food products, applying HTS techniques coupled with bioinformatic analysis. The work aimed to analyse the microbiota variability of different categories of some insect-based commercial food products made of A. domesticus (house cricket), T. molitor (mealworm beetle), and A. diaperinus (lesser mealworm or litter beetle), including commercial raw materials and processed food items, purchased via e-commerce from different companies. Our data revealed that samples cluster per insect species based on microbiota profile and preliminary results suggested that a small number of prevalent bacteria formed a "core microbiota" characterizing the products depending on the insect. This microbial signature can be recognized despite the different food processing levels, rearing conditions and selling companies. Furthermore, differences between raw and processed food made of the same insect or similar product produced by different companies was found. These results support the application of HTS analysis for studying the composition of insect-based commercial food products in a wider perspective, for food traceability and food quality control.

Determination of phthalates in fish fillets by liquid chromatography tandem mass spectrometry (LC-MS/MS): A comparison of direct immersion solid phase microextraction (SPME) versus ultrasonic assisted solvent extraction (UASE).

Due to the increasing presence of plastic and plastic associated contaminants in the aquatic environments, the monitoring of this contamination in fish products and the understanding of possible human health implications is considered urgent. However, data are still relatively scarce, mostly due to the methodological challenges in the chemical analysis: these contaminants are ubiquitous and procedural contamination from the laboratory is frequent. In this work, we compared solid-phase microextraction (SPME) to ultrasonic assisted solvent extraction (UASE) as sample preparation methods for the liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) determination of phthalates in fish fillets. UASE was carried out with an acetone-hexane (1:1) solution and according to a reference procedure aimed to obtain the exhaustive extraction of the target analytes. SPME was carried out by applying C18 fibers in direct immersion mode and by using water/methanol 20:80 mixture to desorb the aliquot required for the analysis. Overall, SPME displayed an improved control of the background contamination and enabled lower LOQs. Precision, calculated as relative standard deviation (RSD) on replicates of a reference sample, was below 24% for both the method. Analysis of real samples purchased from Italian supermarkets showed that SPME might be an efficient tool for estimating the risk associated with fish consumption.

A systematic review of feasibility studies promoting the use of mobile technologies in clinical research.

Mobile technologies, such as smart phone applications, wearables, ingestibles, and implantables, are increasingly used in clinical research to capture study endpoints. On behalf of the Clinical Trials Transformation Initiative, we aimed to conduct a systematic scoping review and compile a database summarizing pilot studies addressing mobile technology sensor performance, algorithm development, software performance, and/or operational feasibility, in order to provide a resource for guiding decisions about which technology is most suitable for a particular trial. Our systematic search identified 275 publications meeting inclusion criteria. From these papers, we extracted data including the medical condition, concept of interest captured by the mobile technology, outcomes captured by the digital measurement, and details regarding the sensors, algorithms, and study sample. Sixty-seven percent of the technologies identified were wearable sensors, with the remainder including tablets, smartphones, implanted sensors, and cameras. We noted substantial variability in terms of reporting completeness and terminology used. The data have been compiled into an online database maintained by the Clinical Trials Transformation Initiative that can be filtered and searched electronically, enabling a user to find information most relevant to their work. Our long-term goal is to maintain and update the online database, in order to promote standardization of methods and reporting, encourage collaboration, and avoid redundant studies, thereby contributing to the design and implementation of efficient, high-quality trials.

Food Tracking Perspective: DNA Metabarcoding to Identify Plant Composition in Complex and Processed Food Products.

One of the main goals of the quality control evaluation is to identify contaminants in raw material, or contamination after a food is processed and before it is placed on the market. During the treatment processes, contamination, both accidental and economically motivated, can generate incongruence between declared and real composition. In our study, we evaluated if DNA metabarcoding is a suitable tool for unveiling the composition of processed food, when it contains small trace amounts. We tested this method on different types of commercial plant products by using tnrL marker and we applied amplicon-based high-throughput sequencing techniques to identify plant components in different food products. Our results showed that DNA metabarcoding can be an effective approach for food traceability in different type of processed food. Indeed, the vast majority of our samples, we identified the species composition as the labels reported. Although some critical issues still exist, mostly deriving from the starting composition (i.e., variable complexity in taxa composition) of the sample itself and the different processing level (i.e., high or low DNA degradation), our data confirmed the potential of the DNA metabarcoding approach also in quantitative analyses for food composition quality control.

Changes in the Drinking Water Microbiome: Effects of Water Treatments Along the Flow of Two Drinking Water Treatment Plants in a Urbanized Area, Milan (Italy).

While safe and of high quality, drinking water can host an astounding biodiversity of microorganisms, dismantling the belief of its "biological simplicity." During the very few years, we are witnessing an exponential growth in scientific publications, exploring the ecology hidden in drinking water treatment plants (DWTPs) and drinking water distribution system (DWDS). We focused on what happens to the microbial communities from source water (groundwater) throughout the main steps of the potabilization process of a DWTP, located in an urbanized area in Northern Italy. Samples were processed by a stringent water filtration to retain even the smallest environmental bacteria and then analyzed with High-Throughput DNA Sequencing (HTS) techniques. We showed that carbon filters harbored a microbial community seeding and shaping water microbiota downstream, introducing a significant variation on incoming (groundwater) microbial community. Chlorination did not instantly affect the altered microbiota. We were also able to correctly predict (through machine learning analysis) samples belonging to groundwater (overall accuracy was 0.71), but the assignation was not reliable with carbon filter samples, which were incorrectly predicted as chlorination samples. The presence and abundance of specific microorganisms allowed us to hypothesize their role as indicators. In particular, Candidatus Adlerbacteria (Parcubacteria), together with microorganisms belonging to Alphaproteobacteria and Gammaproteobacteria, characterized treated water, but not raw water. An exception, confirming our hypothesis, is given by the samples downstream the filters renewal, which had a composition resembling groundwater. Volatility analysis illustrated how carbon filters represented an ecosystem that is stable over time, probably bearing the environmental conditions that promote the survival and growth of this peculiar microbial community.

Grape microbiome as a reliable and persistent signature of field origin and environmental conditions in Cannonau wine production.

Grape berries harbor a wide range of microbes originating from the vineyard environment, many of which are recognized for their role in the must fermentation process shaping wine quality. To better clarify the contribution of the microbiome of grape fruits during wine fermentation, we used high-throughput sequencing to identify bacterial and fungi communities associated with berries and musts of Cannonau. This is the most important cultivar-wine of Sardinia (Italy) where most vineyards are cultivated without phytochemical treatments. Results suggested that microbiomes of berries collected at four different localities share a core composition characterized by Enterobacteriales, Pseudomonadales, Bacillales, and Rhodospirillales. However, any area seems to enrich berries microbiome with peculiar microbial traits. For example, berries belonging to the biodynamic vineyards of Mamoiada were rich in Bacillales typical of manure (i.e. Lysinibacillus, Bacillus, and Sporosarcina), whereas in the Santadi locality, berries showed soil bacteria such as Pasteurellales and Bacteroidales as well as Rhodospirillales and Lactobacillales which are commonly involved in wine fermentation. In the case of fungi, the most abundant taxa were Dothioraceae, Pleosporaceae, and Saccharomycodaceae, and although the proportion of these families varied among localities, they occurred ubiquitously in all vineyards. During vinification processes performed at the same wine cellar under controlled conditions and without using any yeast starter, more than 50% of bacteria groups of berries reached musts, and each locality had its own private bacteria signature, even if Saccharomyces cerevisiae represented the most abundant fungal species. This work suggests that natural berries microbiome could be influenced by pedoclimatic and anthropologic conditions (e.g., farming management), and the fruits' microorganisms persist during the fermentation process. For these reasons, a reliable wine genotyping should include the entire holobiont (plant and all its symbionts), and bioprospecting activities on grape microbiota could lead to improved viticulture yields and wine quality.

Exploring the under-investigated "microbial dark matter" of drinking water treatment plants.

Scientists recently reported the unexpected detection of unknown or poorly studied bacterial diversity in groundwater. The ability to uncover this neglected biodiversity mainly derives from technical improvements, and the term "microbial dark matter" was used to group taxa poorly investigated and not necessarily monophyletic. We focused on such under-investigated microbial dark matter of drinking water treatment plant from groundwater, across carbon filters, to post-chlorination. We tackled this topic using an integrated approach where the efficacy of stringent water filtration (10000 MWCO) in recovering even the smallest environmental microorganisms was coupled with high-throughput DNA sequencing to depict an informative spectrum of the neglected microbial diversity. Our results revealed that the composition of bacterial communities varies across the plant system: Parcubacteria (OD1) superphylum is found mainly in treated water, while groundwater has the highest heterogeneity, encompassing non-OD1 candidate phyla (Microgenomates, Saccharibacteria, Dependentiae, OP3, OP1, BRC1, WS3). Carbon filters probably act as substrate for microorganism growth and contribute to seeding water downstream, since chlorination does not modify the incoming bacterial community. New questions arise about the role of microbial dark matter in drinking water. Indeed, our results suggest that these bacteria might play a central role in the microbial dynamics of drinking water.