Generating high-quality plant and fish reference genomes from field-collected specimens by optimizing preservation.

Sample preservation often impedes efforts to generate high-quality reference genomes or pangenomes for Earth's more than 2 million plant and animal species due to nucleotide degradation. Here we compare the impacts of storage methods including solution type, temperature, and time on DNA quality and Oxford Nanopore long-read sequencing quality in 9 fish and 4 plant species. We show 95% ethanol largely protects against degradation for fish blood (22 °C, ≤6 weeks) and plant tissue (4 °C, ≤3 weeks). From this furthest storage timepoint, we assemble high-quality reference genomes of 3 fish and 2 plant species with contiguity (contig N50) and completeness (BUSCO) that achieve the Vertebrate Genome Project benchmarking standards. For epigenetic applications, we also report methylation frequency compared to liquid nitrogen control. The results presented here remove the necessity for cryogenic storage in many long read applications and provide a framework for future studies focused on sampling in remote locations, which may represent a large portion of the future sequencing of novel organisms.

The Molecular Effect of Wearing Silver-Threaded Clothing on the Human Skin.

With growing awareness that what we put in and on our bodies affects our health and wellbeing, little is still known about the impact of textiles on the human skin. Athletic wear often uses silver threading to improve hygiene, but little is known about its effect on the body's largest organ. In this study, we investigated the impact of such clothing on the skin's chemistry and microbiome. Samples were collected from different body sites of a dozen volunteers over the course of 12 weeks. The changes induced by the antibacterial clothing were specific for individuals, but more so defined by gender and body site. Unexpectedly, the microbial biomass on skin increased in the majority of the volunteers when wearing silver-threaded T-shirts. Although the most abundant taxa remained unaffected, silver caused an increase in diversity and richness of low-abundant bacteria and a decrease in chemical diversity. Both effects were mainly observed for women. The hallmark of the induced changes was an increase in the abundance of various monounsaturated fatty acids (MUFAs), especially in the upper back. Several microbe-metabolite associations were uncovered, including Cutibacterium, detected in the upper back area, which was correlated with the distribution of MUFAs, and Anaerococcus spp. found in the underarms, which were associated with a series of different bile acids. Overall, these findings point to a notable impact of the silver-threaded material on the skin microbiome and chemistry. We observed that relatively subtle changes in the microbiome result in pronounced shifts in molecular composition. IMPORTANCE The impact of silver-threaded material on human skin chemistry and microbiome is largely unknown. Although the most abundant taxa remained unaffected, silver caused an increase in diversity and richness of low-abundant bacteria and a decrease in chemical diversity. The major change was an increase in the abundance of various monounsaturated fatty acids that were also correlated with Cutibacterium. Additionally, Anaerococcus spp., found in the underarms, were associated with different bile acids in the armpit samples. Overall, the impact of the silver-threaded clothing was gender and body site specific.

Standardized multi-omics of Earth's microbiomes reveals microbial and metabolite diversity.

Despite advances in sequencing, lack of standardization makes comparisons across studies challenging and hampers insights into the structure and function of microbial communities across multiple habitats on a planetary scale. Here we present a multi-omics analysis of a diverse set of 880 microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry). We used standardized protocols and analytical methods to characterize microbial communities, focusing on relationships and co-occurrences of microbially related metabolites and microbial taxa across environments, thus allowing us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of an evolving community resource. We demonstrate the utility of this database by testing the hypothesis that every microbe and metabolite is everywhere but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment, whereas the relative abundances of microbially related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner. We additionally show the power of certain chemistry, in particular terpenoids, in distinguishing Earth's environments (for example, terrestrial plant surfaces and soils, freshwater and marine animal stool), as well as that of certain microbes including Conexibacter woesei (terrestrial soils), Haloquadratum walsbyi (marine deposits) and Pantoea dispersa (terrestrial plant detritus). This Resource provides insight into the taxa and metabolites within microbial communities from diverse habitats across Earth, informing both microbial and chemical ecology, and provides a foundation and methods for multi-omics microbiome studies of hosts and the environment.

Host biology, ecology and the environment influence microbial biomass and diversity in 101 marine fish species.

Fish are the most diverse and widely distributed vertebrates, yet little is known about the microbial ecology of fishes nor the biological and environmental factors that influence fish microbiota. To identify factors that explain microbial diversity patterns in a geographical subset of marine fish, we analyzed the microbiota (gill tissue, skin mucus, midgut digesta and hindgut digesta) from 101 species of Southern California marine fishes, spanning 22 orders, 55 families and 83 genera, representing ~25% of local marine fish diversity. We compare alpha, beta and gamma diversity while establishing a method to estimate microbial biomass associated with these host surfaces. We show that body site is the strongest driver of microbial diversity while microbial biomass and diversity is lowest in the gill of larger, pelagic fishes. Patterns of phylosymbiosis are observed across the gill, skin and hindgut. In a quantitative synthesis of vertebrate hindguts (569 species), we also show that mammals have the highest gamma diversity when controlling for host species number while fishes have the highest percent of unique microbial taxa. The composite dataset will be useful to vertebrate microbiota researchers and fish biologists interested in microbial ecology, with applications in aquaculture and fisheries management.

A comparison of six DNA extraction protocols for 16S, ITS and shotgun metagenomic sequencing of microbial communities.

Microbial communities contain a broad phylogenetic diversity of organisms; however, the majority of methods center on describing bacteria and archaea. Fungi are important symbionts in many ecosystems and are potentially important members of the human microbiome, beyond those that can cause disease. To expand our analysis of microbial communities to include data from the fungal internal transcribed spacer (ITS) region, five candidate DNA extraction kits were compared against our standardized protocol for describing bacteria and archaea using 16S rRNA gene amplicon- and shotgun metagenomics sequencing. The results are presented considering a diverse panel of host-associated and environmental sample types and comparing the cost, processing time, well-to-well contamination, DNA yield, limit of detection and microbial community composition among protocols. Across all criteria, the MagMAX Microbiome kit was found to perform best. The PowerSoil Pro kit performed comparably but with increased cost per sample and overall processing time. The Zymo MagBead, NucleoMag Food and Norgen Stool kits were included.

Fine scale transitions of the microbiota and metabolome along the gastrointestinal tract of herbivorous fishes.

BACKGROUND: Gut microorganisms aid in the digestion of food by providing exogenous metabolic pathways to break down organic compounds. An integration of longitudinal microbial and chemical data is necessary to illuminate how gut microorganisms supplement the energetic and nutritional requirements of animals. Although mammalian gut systems are well-studied in this capacity, the role of microbes in the breakdown and utilization of recalcitrant marine macroalgae in herbivorous fish is relatively understudied and an emerging priority for bioproduct extraction. Here we use a comprehensive survey of the marine herbivorous fish gut microbial ecosystem via parallel 16S rRNA gene amplicon profiling (microbiota) and untargeted tandem mass spectrometry (metabolomes) to demonstrate consistent transitions among 8 gut subsections across five fish of the genus of Kyphosus. RESULTS: Integration of microbial phylogenetic and chemical diversity data reveals that microbial communities and metabolomes covaried and differentiated continuously from stomach to hindgut, with the midgut containing multiple distinct and previously uncharacterized microenvironments and a distinct hindgut community dominated by obligate anaerobes. This differentiation was driven primarily by anaerobic gut endosymbionts of the classes Bacteroidia and Clostridia changing in concert with bile acids, small peptides, and phospholipids: bile acid deconjugation associated with early midgut microbiota, small peptide production associated with midgut microbiota, and phospholipid production associated with hindgut microbiota. CONCLUSIONS: The combination of microbial and untargeted metabolomic data at high spatial resolution provides a new view of the diverse fish gut microenvironment and serves as a foundation to understand functional partitioning of microbial activities that contribute to the digestion of complex macroalgae in herbivorous marine fish.

Variation in Survival and Gut Microbiome Composition of Hatchery-Grown Native Oysters at Various Locations within the Puget Sound.

The Olympia oyster (Ostrea lurida) of the Puget Sound suffered a dramatic population crash, but restoration efforts hope to revive this native species. One overlooked variable in the process of assessing ecosystem health is association of bacteria with marine organisms and the environments they occupy. Oyster microbiomes are known to differ significantly between species, tissue type, and the habitat in which they are found. The goals of this study were to determine the impact of field site and habitat on the oyster microbiome and to identify core oyster-associated bacteria in the Puget Sound. Olympia oysters from one parental family were deployed at four sites in the Puget Sound both inside and outside of eelgrass (Zostera marina) beds. Using 16S rRNA gene amplicon sequencing of the oyster gut, shell, and surrounding seawater and sediment, we demonstrate that gut-associated bacteria are distinct from the surrounding environment and vary by field site. Furthermore, regional differences in the gut microbiota are associated with the survival rates of oysters at each site after 2 months of field exposure. However, habitat type had no influence on microbiome diversity. Further work is needed to identify the specific bacterial dynamics that are associated with oyster physiology and survival rates. IMPORTANCE This is the first exploration of the microbial colonizers of the Olympia oyster, a native oyster species to the West Coast, which is a focus of restoration efforts. The patterns of differential microbial colonization by location reveal microscale characteristics of potential restoration sites which are not typically considered. These microbial dynamics can provide a more holistic perspective on the factors that may influence oyster performance.

Clean room microbiome complexity impacts planetary protection bioburden.

BACKGROUND: The Spacecraft Assembly Facility (SAF) at the NASA's Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom. RESULTS: Cleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter. CONCLUSION: This study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays. Video abstract.

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

Non-targeted tandem mass spectrometry enables the visualization of organic matter chemotype shifts in coastal seawater.

Urbanization along coastlines alters marine ecosystems including contributing molecules of anthropogenic origin to the coastal dissolved organic matter (DOM) pool. A broad assessment of the nature and extent of anthropogenic impacts on coastal ecosystems is urgently needed to inform regulatory guidelines and ecosystem management. Recently, non-targeted tandem mass spectrometry approaches are gaining momentum for the analysis of global organic matter composition (chemotypes) including a wide array of natural and anthropogenic compounds. In line with these efforts, we developed a non-targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) workflow that utilizes advanced data analysis approaches such as feature-based molecular networking and repository-scale spectrum searches. This workflow allows the scalable comparison and mapping of seawater chemotypes from large-scale spatial surveys as well as molecular family level annotation of unknown compounds. As a case study, we visualized organic matter chemotype shifts in coastal environments in northern San Diego, USA, after notable rain fall in winter 2017/2018 and highlight potential anthropogenic impacts. The observed seawater chemotype, consisting of 4384 LC-MS/MS features, shifted significantly after a major rain event. Molecular drivers of this shift could be attributed to multiple anthropogenic compounds, including pesticides (Imazapyr and Isoxaben), cleaning products (Benzyl-tetradecyl-dimethylammonium) and chemical additives (Hexa (methoxymethyl)melamine) and potential degradation products. By expanding the search of identified xenobiotics to other public tandem mass spectrometry datasets, we further contextualized their possible origin and show their importance in other ecosystems. The mass spectrometry and data analysis pipelines applied here offer a scalable framework for future molecular mapping and monitoring of marine ecosystems, which will contribute to a deliberate assessment of how chemical pollution impacts our oceans.

Feasibility of using alternative swabs and storage solutions for paired SARS-CoV-2 detection and microbiome analysis in the hospital environment.

BACKGROUND: Determining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a synthetic swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs (including plastic and wood shafts and various head materials including cotton, synthetic, and foam) and one clinical-grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients including COVID-19+ patients. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent. RESULTS: Compared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2-4× higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved synthetic (SYN) swab. The limit of detection (LoD) of SARS-CoV-2 from floor samples collected using the consumer-grade plastic (CGp) or research-grade plastic The Microsetta Initiative (TMI) swabs was similar or better than the SYN swab, further suggesting that swab type does not impact RNA recovery as measured by the abundance of SARS-CoV-2. The LoD for TMI was between 0 and 362.5 viral particles, while SYN and CGp were both between 725 and 1450 particles. Lastly microbiome analyses (16S rRNA gene sequencing) of paired samples (nasal and floor from same patient room) collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type, but instead driven by the patient and sample type. CONCLUSIONS: Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer-grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity and lack of antibiotics in these samples makes it possible to perform concomitant microbiome analyses. Video abstract.

Impacts of the Marine Hatchery Built Environment, Water and Feed on Mucosal Microbiome Colonization Across Ontogeny in Yellowtail Kingfish, Seriola lalandi.

The fish gut microbiome is impacted by a number of biological and environmental factors including fish feed formulations. Unlike mammals, vertical microbiome transmission is largely absent in fish and thus little is known about how the gut microbiome is initially colonized during hatchery rearing nor the stability throughout growout stages. Here we investigate how various microbial-rich surfaces from the built environment "BE" and feed influence the development of the mucosal microbiome (gill, skin, and digesta) of an economically important marine fish, yellowtail kingfish, Seriola lalandi, over time. For the first experiment, we sampled gill and skin microbiomes from 36 fish reared in three tank conditions, and demonstrate that the gill is more influenced by the surrounding environment than the skin. In a second experiment, fish mucous (gill, skin, and digesta), the BE (tank side, water, inlet pipe, airstones, and air diffusers) and feed were sampled from indoor reared fish at three ages (43, 137, and 430 dph; n = 12 per age). At 430 dph, 20 additional fish were sampled from an outdoor ocean net pen. A total of 304 samples were processed for 16S rRNA gene sequencing. Gill and skin alpha diversity increased while gut diversity decreased with age. Diversity was much lower in fish from the ocean net pen compared to indoor fish. The gill and skin are most influenced by the BE early in development, with aeration equipment having more impact in later ages, while the gut "allochthonous" microbiome becomes increasingly differentiated from the environment over time. Feed had a relatively low impact on driving microbial communities. Our findings suggest that S. lalandi mucosal microbiomes are differentially influenced by the BE with a high turnover and rapid succession occurring in the gill and skin while the gut microbiome is more stable. We demonstrate how individual components of a hatchery system, especially aeration equipment, may contribute directly to microbiome development in a marine fish. In addition, results demonstrate how early life (larval) exposure to biofouling in the rearing environment may influence fish microbiome development which is important for animal health and aquaculture production.

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.

One goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols. Accession numbers: Raw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub ( github.com/justinshaffer/Extraction_test_MagMAX ). Methods summary: To allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

The Southern Bluefin Tuna Mucosal Microbiome Is Influenced by Husbandry Method, Net Pen Location, and Anti-parasite Treatment.

Aquaculture is the fastest growing primary industry worldwide. Marine finfish culture in open ocean net pens, or pontoons, is one of the largest growth areas and is currently the only way to rear high value fish such as bluefin tuna. Ranching involves catching wild juveniles, stocking in floating net pens and fattening for 4 to 8 months. Tuna experience several parasite-induced disease challenges in culture that can be mitigated by application of praziquantel (PZQ) as a therapeutic. In this study, we characterized the microbiome of ranched southern Bluefin Tuna, Thunnus maccoyii, across four anatomic sites (gill, skin, digesta, and anterior kidney) and evaluated environmental and pathological factors that influence microbiome composition, including the impact of PZQ treatment on microbiome stability. Southern bluefin tuna gill, skin, and digesta microbiome communities are unique and potentially influenced by husbandry practices, location of pontoon growout pens, and treatment with the antiparasitic PZQ. There was no significant relationship between the fish mucosal microbiome and incidence or abundance of adult blood fluke in the heart or fluke egg density in the gill. An enhanced understanding of microbiome diversity and function in high-value farmed fish species such as bluefin tuna is needed to optimize fish health and improve aquaculture yield. Comparison of the bluefin tuna microbiome to other fish species, including Seriola lalandi (yellowtail kingfish), a common farmed species from Australia, and Scomber japonicus (Pacific mackerel), a wild caught Scombrid relative of tuna, showed the two Scombrids had more similar microbial communities compared to other families. The finding that mucosal microbial communities are more similar in phylogenetically related fish species exposes an opportunity to develop mackerel as a model for tuna microbiome and parasite research.

Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation.

Microbial contamination during long-term confinements of space exploration presents potential risks for both crew members and spacecraft life support systems. A novel swab kit was used to sample various surfaces from a submerged, closed, analog habitat to characterize the microbial populations. Samples were collected from various locations across the habitat which were constructed from various surface materials (linoleum, dry wall, particle board, glass, and metal), and microbial populations were examined by culture, quantitative PCR (qPCR), microbiome 16S rRNA gene sequencing, and shotgun metagenomics. Propidium monoazide (PMA)-treated samples identified the viable/intact microbial population of the habitat. The cultivable microbial population ranged from below the detection limit to 106 CFU/sample, and their identity was characterized using Sanger sequencing. Both 16S rRNA amplicon and shotgun sequencing were used to characterize the microbial dynamics, community profiles, and functional attributes (metabolism, virulence, and antimicrobial resistance). The 16S rRNA amplicon sequencing revealed abundance of viable (after PMA treatment) Actinobacteria (Brevibacterium, Nesternkonia, Mycobacterium, Pseudonocardia, and Corynebacterium), Firmicutes (Virgibacillus, Staphylococcus, and Oceanobacillus), and Proteobacteria (especially Acinetobacter) on linoleum, dry wall, and particle board (LDP) surfaces, while members of Firmicutes (Leuconostocaceae) and Proteobacteria (Enterobacteriaceae) were high on the glass/metal surfaces. Nonmetric multidimensional scaling determined from both 16S rRNA and metagenomic analyses revealed differential microbial species on LDP surfaces and glass/metal surfaces. The shotgun metagenomic sequencing of samples after PMA treatment showed bacterial predominance of viable Brevibacterium (53.6%), Brachybacterium (7.8%), Pseudonocardia (9.9%), Mycobacterium (3.7%), and Staphylococcus (2.1%), while fungal analyses revealed Aspergillus and Penicillium dominance.IMPORTANCE This study provides the first assessment of monitoring cultivable and viable microorganisms on surfaces within a submerged, closed, analog habitat. The results of the analyses presented herein suggest that the surface material plays a role in microbial community structure, as the microbial populations differed between LDP and metal/glass surfaces. The metal/glass surfaces had less-complex community, lower bioburden, and more closely resembled the controls. These results indicated that material choice is crucial when building closed habitats, even if they are simply analogs. Finally, while a few species were associated with previously cultivated isolates from the International Space Station and MIR spacecraft, the majority of the microbial ecology of the submerged analog habitat differs greatly from that of previously studied analog habitats.

Feasibility of using alternative swabs and storage solutions for paired SARS-CoV-2 detection and microbiome analysis in the hospital environment.

Background Determining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a synthetic swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs (including plastic and wood shafts and various head materials including cotton, synthetic, and foam) and one clinical grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients with 16 COVID-19+. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent. Results Compared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2-4x higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved synthetic swab. The limit of detection (LoD) of SARs-CoV-2 from floor samples collected using the CGp or TMI swabs was similar or better than the CDC standard, further suggesting that swab type does not impact RNA recovery as measured by SARs-CoV-2. The LoD for TMI was between 0-362.5 viral particles while SYN and CGp were both between 725-1450 particles. Lastly microbiome analyses (16S rRNA) of paired samples (e.g., environment to host) collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type but instead driven by the patient and sample type (floor or nasal). Conclusions Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity in these samples makes it possible to perform concomitant microbiome analysis. Keywords: COVID-19, SARS-CoV-2, RT-qPCR, swab, global health.

Temporal, Environmental, and Biological Drivers of the Mucosal Microbiome in a Wild Marine Fish, Scomber japonicus.

Changing ocean conditions driven by anthropogenic activities may have a negative impact on fisheries by increasing stress and disease. To understand how environment and host biology drives mucosal microbiomes in a marine fish, we surveyed five body sites (gill, skin, digesta, gastrointestinal tract [GI], and pyloric ceca) from 229 Pacific chub mackerel, Scomber japonicus, collected across 38 time points spanning 1 year from the Scripps Institution of Oceanography Pier (La Jolla, CA). Mucosal sites had unique microbial communities significantly different from the surrounding seawater and sediment communities with over 10 times more total diversity than seawater. The external surfaces of skin and gill were more similar to seawater, while digesta was more similar to sediment. Alpha and beta diversity of the skin and gill was explained by environmental and biological factors, specifically, sea surface temperature, chlorophyll a, and fish age, consistent with an exposure gradient relationship. We verified that seasonal microbial changes were not confounded by regional migration of chub mackerel subpopulations by nanopore sequencing a 14,769-bp region of the 16,568-bp mitochondria across all temporal fish specimens. A cosmopolitan pathogen, Photobacterium damselae, was prevalent across multiple body sites all year but highest in the skin, GI, and digesta between June and September, when the ocean is warmest. The longitudinal fish microbiome study evaluates the extent to which the environment and host biology drives mucosal microbial ecology and establishes a baseline for long-term surveys linking environment stressors to mucosal health of wild marine fish.IMPORTANCE Pacific chub mackerel, Scomber japonicus, are one of the largest and most economically important fisheries in the world. The fish is harvested for both human consumption and fish meal. Changing ocean conditions driven by anthropogenic stressors like climate change may negatively impact fisheries. One mechanism for this is through disease. As waters warm and chemistry changes, the microbial communities associated with fish may change. In this study, we performed a holistic analysis of all mucosal sites on the fish over a 1-year time series to explore seasonal variation and to understand the environmental drivers of the microbiome. Understanding seasonality in the fish microbiome is also applicable to aquaculture production for producers to better understand and predict when disease outbreaks may occur based on changing environmental conditions in the ocean.

Visualizing 'omic feature rankings and log-ratios using Qurro.

Many tools for dealing with compositional ' 'omics' data produce feature-wise values that can be ranked in order to describe features' associations with some sort of variation. These values include differentials (which describe features' associations with specified covariates) and feature loadings (which describe features' associations with variation along a given axis in a biplot). Although prior work has discussed the use of these 'rankings' as a starting point for exploring the log-ratios of particularly high- or low-ranked features, such exploratory analyses have previously been done using custom code to visualize feature rankings and the log-ratios of interest. This approach is laborious, prone to errors and raises questions about reproducibility. To address these problems we introduce Qurro, a tool that interactively visualizes a plot of feature rankings (a 'rank plot') alongside a plot of selected features' log-ratios within samples (a 'sample plot'). Qurro's interface includes various controls that allow users to select features from along the rank plot to compute a log-ratio; this action updates both the rank plot (through highlighting selected features) and the sample plot (through displaying the current log-ratios of samples). Here, we demonstrate how this unique interface helps users explore feature rankings and log-ratios simply and effectively.

Microbial Ecology of Atlantic Salmon (Salmo salar) Hatcheries: Impacts of the Built Environment on Fish Mucosal Microbiota.

Successful rearing of fish in hatcheries is critical for conservation, recreational fishing, commercial fishing through wild stock enhancements, and aquaculture production. Flowthrough (FT) hatcheries require more water than recirculating aquaculture systems (RAS), which enable up to 99% of their water to be recycled, thus significantly reducing environmental impacts. Here, we evaluated the biological and physical microbiome interactions of three Atlantic salmon hatcheries (RAS n = 2, FT n = 1). Gill, skin, and digesta from six juvenile fish along with tank biofilms and water were sampled from tanks in each of the hatcheries (60 fish across 10 tanks) to assess the built environment and mucosal microbiota using 16S rRNA gene sequencing. The water and tank biofilm had more microbial richness than fish mucus, while skin and digesta from RAS fish had 2 times the richness of FT fish. Body sites each had unique microbiomes (P < 0.001) and were influenced by hatchery system type (P < 0.001), with RAS being more similar. A strong association between the tank and fish microbiome was observed. Water and tank biofilm richness was positively correlated with skin and digesta richness. Strikingly, the gill, skin, and digesta communities were more similar to that in the origin tank biofilm than those in all other experimental tanks, suggesting that the tank biofilm has a direct influence on fish-associated microbial communities. Lastly, microbial diversity and mucous cell density were positively associated with fish growth and length. The results from this study provide evidence for a link between the tank microbiome and the fish microbiome, with the skin microbiome as an important intermediate.IMPORTANCE Atlantic salmon, Salmo salar, is the most farmed marine fish worldwide, with an annual production of 2,248 million metric tons in 2016. Salmon hatcheries are increasingly changing from flowthrough toward recirculating aquaculture system (RAS) design to accommodate more control over production along with improved environmental sustainability due to lower impacts on water consumption. To date, microbiome studies of hatcheries have focused either on the fish mucosal microbiota or on the built environment microbiota but have not combined the two to understand their interactions. Our study evaluates how the water and tank biofilm microbiota influences the fish microbiota across three mucosal environments (gill, skin, and digesta). Results from this study highlight how the built environment is a unique source of microbes to colonize fish mucus and, furthermore, how this can influence fish health. Further studies can use this knowledge to engineer built environments to modulate fish microbiota for beneficial phenotypes.

Atlantic Salmon (Salmo salar L., 1758) Gut Microbiota Profile Correlates with Flesh Pigmentation: Cause or Effect?

In Tasmania (Australia), during the marine phase, it has been observed that flesh pigmentation significantly drops in summer, possibly due to high water temperatures (> 20 °C). Although this deleterious effect of summer temperatures has been ascertained, there is a lack of knowledge of the actual mechanisms behind the impaired uptake and/or loss of pigments in Atlantic salmon in a challenging environment. Since the microbial community in the fish intestine significantly changes in relation to the variations of water temperature, this study was conducted to assess how the gut microbiota profile also correlates with the flesh color during temperature fluctuation. We sampled 68 fish at three time points covering the end of summer to winter at a marine farm in Tasmania, Australia. Flesh color was examined in two ways: the average color throughout and the evenness of the color between different areas of the fillet. Using 16S rRNA sequencing of the v3-v4 region, we determined that water temperature corresponded to changes in the gut microbiome both with alpha diversity (Kruskal-Wallis tests P = 0.05) and beta diversity indices (PERMANOVA P = 0.001). Also, there was a significant correlation between the microbiota and the color of the fillet (PERMANOVA P = 0.016). There was a high abundance of Pseudoalteromonadaceae, Enterobacteriaceae, Microbacteriaceae, and Vibrionaceae in the pale individuals. Conversely, carotenoid-synthesizing bacteria families (Bacillaceae, Mycoplasmataceae, Pseudomonas, Phyllobacteriaceae, and Comamonadaceae) were found in higher abundance in individuals with darker flesh color.