The embryonic zebrafish is a powerful tool for high-throughput screening of chemicals. While this model has significant potential for use in safety assessments and chemical prioritization, a lack of exposure protocol harmonized across laboratories has limited full model adoption. To assess the potential that exposure protocols alter chemical bioactivity, we screened a set of eight chemicals and one 2D nanomaterial across four different regimens: (1) the current Tanguay laboratory’s standard protocol of dechorionated embryos and static exposure in darkness; (2) exposure with chorion intact; (3) exposure under a 14 h light: 10 h dark cycle; and (4) exposure with daily chemical renewal. The latter three regimens altered the concentrations, resulting in bioactivity of the test agents compared to that observed with the Tanguay laboratory’s standard regimen, though not directionally the same for each chemical. The results of this study indicate that with the exception for the 2D nanomaterial, the screening design did not change the conclusion regarding chemical bioactivity, just the nominal concentrations producing the observed activity. Since the goal of tier one chemical screening often is to differentiate active from non-active chemicals, researchers could consider the trade-offs regarding cost, labor, and sensitivity in their study design without altering hit rates. Taken further, these results suggest that it is reasonably feasible to reach agreement on a standardized exposure regiment, which will promote data sharing without sacrificing data content. There are tens of thousands of chemicals available for commerce in the United States, but relatively few have been adequately assessed for health effects . The inaDanio rerio) is a useful tool for rapid chemical bioactivity screening, capable of producing concentration–response data for hundreds of chemicals in a matter of days [in vitro screening platforms.The embryonic zebrafish . While some chemical–condition combinations decreased the EC50, others increased the EC50 when compared to the Tanguay laboratory’s standard exposure regimen.In this study, we used a systematic approach to vary three exposure conditions across a diverse suite of test agents to determine if the bioactivity response profile would vary. With only one exception, each test agent exhibited bioactivity concordantly across the conditions; however, each condition did affect the concentration–response relationship, as determined by the concentration estimated to induce abnormal morphology in 50% of the animals (ECOW). OW.High-throughput screening should be sensitive enough to detect the bioactivity of compounds across diverse chemical classes and with a range of physiochemical properties. For the soundness of this study, it was important to select test agents that represent this variety. We selected test materials to cover diverse chemical categories and a range of known physiochemical properties, including photolability and octanol–water coefficients (log KDanio rerio) [Specific pathogen-free wild type 5D zebrafish (o rerio)  were reao rerio) . Fish we2, 1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, and 0.7 mM NaHCO3 buffered with 1 M NaOH to pH 7.2 [Embryos were collected using an internal embryo collection apparatus, sorted by similar developmental stage  and kepto pH 7.2 . EmbryosEmbryos were enzymatically dechorionated at 4 hpf . Brieflyn = 12 for each concentration). Exposure solutions were normalized to 1% by volume dimethyl sulfoxide (DMSO).Chemicals were dispensed into 96-well plates pre-loaded with embryos and EM using an HP D300 or D300e Digital Dispenser. For each chemical, embryos were exposed to initial range-finding concentrations of 0, 1, 2.54, 6.45, 16.4, 35, 74.8, and 100 µM  and incubated at 28 ± 1 °C overnight on an orbital shaker at 235 RPM . At 24 aBecause multi-walled carbon nanotubes (MWCNT) aggregate and cannot be accurately dispensed in the HP D300 or D300e Digital Dispenser, MWCNT master stock solutions were made in EM at 1000 µg/mL. Agglomeration/aggregation of MWCNTs before and after sonication was visually compared between MWCNTs suspended in EM and in ultrapure water. No difference was observed between the two media, so EM was selected as the media for MWCNT exposures to improve background survival of the zebrafish during exposure. To reduce material agglomeration, stock solutions were sonicated using a Fisher Scientific 60 Sonic Dismembrator for 6 min at 14 °C and stored briefly at 4 °C until just before exposure. Immediately before exposure, solutions were again sonicated for 6 min and simple dilutions were made by adding MWCNT solution to EM to achieve concentrations 10× above the test exposure concentrations. A total of 10 µL of the diluted stock was dispensed into each appropriate well of a 96-well plate pre-loaded with 90 µL EM and an embryo as described in n = 16 for each concentration). Definitive testing concentrations were chosen as in n = 48 for each concentration.Embryos were exposed to MWCNTs at initial range-finding concentrations of 0, 10, 23.2, 50, 75, and 100 µg/mL  was assessed and embryos were screened for mortality and 4 morphology endpoints. Plates were then returned to the dark incubator until 120 hpf when larval photomotor response (LPR) was assessed and embryos were screened for additional mortality and incidence of abnormality in 18 morphology endpoints .The embryonic photomotor response (EPR) assay was conducted at 24 hpf, taking care to not expose the test plates to visible light prior to the assay . BrieflyThe larval photomotor response (LPR) assay was conducted at 120 hpf when the 96-well plates of larvae were placed into ZebraBox behavioral analysis chambers (Viewpoint Life Sciences) and larval movement tracked with ZebraLab motion analysis software for 18 min across 3 cycles of 3 min light: 3 min dark. The distance moved by each larva was integrated over 6 s binning periods, averaged for the test concentration, and area under the curve was calculated and compared to the on-plate control group as previously described .It should be noted that while both the EPR and LPR assays assess photomotor response, EPR is strictly based on the response of photoreceptors within the hindbrain prior to eye development while LPR, on the other hand, is driven by visual stimulus. While both responses represent a behavioral effect, the mechanism leading to each effect can differ for a single compound. Additionally, the EPR assay takes place at 24 hpf, which may be before the affected system/tissue is developed enough for the chemical to have the effect(s) observed at 5 dpf; therefore, a response in either assay is considered a “hit” independently of the other.For all assessments, data were uploaded under a unique well-plate barcode into a custom LIMS, Zebrafish Acquisition and Analysis Program (ZAAP), database and analyzed using custom R scripts that were executed in the LIMS background .n = 36  or 48 (MWCNT exposures) for each concentration.Embryos were exposed to each test agent under 4 different conditions: standard exposure regimen (described above), chorion-on, light/dark cycle incubation, and daily solution renewals . EmbryosThe chorion-on condition was exactly as described for the standard exposure regimen with the omission of the chorion removal process.For the light/dark incubation treatment, embryos were incubated under a 14-h light: 10-h dark schedule for the entire exposure period with light exposure from bulbs characterized by the following specifications: 34 Watt, 2700 Lumen, Color Rendering Index 62, Color Temperature 4200 K. Light intensity was measured at 120 ± 10 lux. The remainder of the exposure proceeded exactly like the standard exposure regimen.For the daily renewal treatment, we replenished exposure solutions every 24 h see . The remExposure solutions were renewed at 24, 48, 72, and 96 hpf using a series of repeated 1:1 dilutions with EM using a 96-well Rainin Liquidator. A total of 100 µL of fresh medium was loaded into the wells in addition to the original exposure solution. Then, 100 µL of the diluted solution was removed and discarded, taking care to not disturb the animals. This was repeated 6 times for a total of 7 dilutions to reach a concentration < 1% of the original exposure solution. For MWCNT exposures, 10 dilutions were performed to help disperse agglomerated particles and 110 µL was removed during the tenth dilution for a final volume of 90 µL to accommodate 10 µL of the 10× replenishing solution. Chemical or MWCNT replenishing solutions were dispensed into the wells and plates were freshly sealed and returned to the dark 28 °C incubator. In rare instances of larvae accidentally lost in the renewal procedure, they were omitted from the final analyses.n = 36–48) for every test agent–condition combination. 50) was estimated using the drc package [50 for each test agent–condition combination was compared to the EC50 for the corresponding standard condition. Fold change was calculated between the standard condition and varied condition for each test agent and any fold change below −1.25 or above 1.25 was considered a true effect of the condition based on the observation that background effects up to 20% can be normally observed in dechorionated embryos. In this study, the highest average rate of background effects for any condition was approximately 12.2%, making the selected fold change cut-off sufficiently conservative.To assess effects on morphology, percent incidence of abnormalities for each endpoint was calculated  . EPR wasggplot2  package In teleosts, the chorion is an acellular, semipermeable membrane surrounding a developing embryo. It provides some degree of mechanical protection from environmental disturbances and facilitates gas and ion exchange. Guidelines within the OECD FET test address the potential for the chorion to serve as a chemical barrier, though it does not make recommendations for mechanical or enzymatic removal of the chorion . For bioOur second motivation for advocating the removal of the chorion is to facilitate the measurements of endpoints and to reduce the potential confounding effects of chemical-mediated hatching failure. Non-invasive microscopic imaging of embryonic structures is simply easier when the developing embryo is not encased in a round rolling configuration, as dechorionated embryos generally lay flat and are easily manipulated for consistent orientation for imaging. Additionally, since the EPR assay captures contralateral tail bends, the presence of the chorion may reduce the measurable activity, resulting in a decrease in sensitivity . It is wAn intact chorion influenced early life stage behavior responses at both 24 and 120 hpf. At 24 hpf, the presence of the chorion eliminated E-phase hypoactivity for estradiol, pyrene, and retene and induced hypoactivity for MWCNTs in the E phase (EPR). The observed lack of response with the chorion on further supports the recommendation of conducting bioactivity studies with dechorionated embryos. At 120 hpf, chlorpyrifos and estradiol induced hypoactivity in the D phase, while pyrene induced hyperactivity in the L phase and permethrin induced hyperactivity in both the D and L phases (LPR) with chorionated embryos . Figures50 could not be calculated for the MWCNT treatment under any condition, when compared to the standard exposure regime, the presence of the chorion during exposure completely eliminated incidences of abnormal morphology as displayed in While an ECThis study illustrates that chorion status can significantly affect a chemical or nanomaterial’s bioactivity, and that, for traditional small molecules, the effect is more complex than simple size exclusion. The exact mechanism is unknown and likely different for each substance, as indicated by increased bioactivity for some chemicals and decreased bioactivity for others with intact chorions.p-dioxin (TCDD), benzene [Some evidence suggests the chorion pores increase in size throughout development, potentially altering chemical uptake at critical developmental periods . Others  benzene , trichlo benzene , and die benzene , potentiSome laboratories have expressed concern about the need to conduct chemical exposures under a light:dark (LD) cycle  to maintIn this study, chemical exposures under a 14 h light: 10 h dark cycle potentiated the incidence of morphological effects associated with abamectin and chlorpyrifos and inhibited the incidence of morphological effects associated with permethrin. The profiles of specific morphological effects for each chemical were not greatly altered under a LD cycle as compared to the standard (dark) exposure condition, though the concentration eliciting a response was altered for some endpoints under this treatment. See 50 for incidence of morphological effects of chlorpyrifos was dramatically lowered from 64.4 µM under dark conditions to 34.1 µM under an LD cycle (Chemical photodegradation of labile compounds could be accelerated when using light:dark cycling during screening, which could confound data interpretation. If a chemical rapidly degrades to less toxic intermediates due to co-exposure with light, its bioactivity may be underestimated. Alternatively, if the photo intermediates are more bioactive than the parent compound, its bioactivity may be overestimated. In this study, the incidence of morphological effects was altered for abamectin, chlorpyrifos, and permethrin, all of which are known to be photolabile. Specifically, the ECLD cycle . A previExposure techniques such as static, daily renewal, and flow-through exposures each present pros and cons in early life stage chemical bioactivity screening. Static exposures are less labor-intensive, require less test chemicals, and avoid repeated disturbance of the developing organism, a potential but unquantified stressor on development. However, static exposures face limitations from poor chemical solubility, lability, and rapid metabolism, which may each dramatically curtail the intended exposure period. Without a priori knowledge of these chemical limitations, generally the case in high-throughput library screens, static exposures achieve higher throughput and lower cost at the potential expense of fewer bioactivity hits. Daily renewal exposures may reduce throughput, certainly increase costs, but also maximize the likelihood of hit detection.In this study, daily renewal of the test solution potentiated the incidence of morphological effects associated with abamectin, chlorpyrifos, permethrin, pyrene, and retene. There were no chemicals for which the daily renewal condition reduced the associated bioactivity relative to the standard condition . For eacOW value above 4.4 (The potential for metabolism and bioaccumulation should be considered when deciding on an exposure regimen. The bioactivity of a chemical that accumulates in the body will likely be potentiated by a renewal regimen while a chemical that is readily metabolized early in the exposure period may no longer be available in the solution to induce effects on later-developing tissues, therefore lowering perceived bioactivity in static exposures. Of the nine test agents, five exhibited greater incidence of morphological effects with chemical renewal. Each of these chemicals has a log Kbove 4.4 , which mOW may be useful to consider when choosing an exposure regimen.Most chemical screening studies report nominal water concentrations and do not analyze concentrations of parent chemicals or their metabolites in the exposure medium or in the organism’s body. The OECD FET test makes recommendations that exposure regimen may be adapted for volatile, easily degradable, or adsorptive substances and that exposure concentrations should be verified at the beginning and end of the test for static exposures and at the beginning and end of each exposure interval when using a renewal regimen . While t50 value, in some cases quite significantly. Therefore, if the goal were to identify active molecules, with the exception of MWCNTs, any of the evaluated protocols in this study would suffice assuming important endpoints are reproducibly measurable and that users of the data understand the EC50 values will vary across laboratories. Perhaps predictably, daily chemical renewals altered the EC50 to the greatest degree since the total amount of chemical presented to the fish is 400% higher in a 5-day study compared to static non-renewal design. Therefore, for some chemicals, daily renewal would be expected to lead to higher bioaccumulation. However, by utilizing this exposure strategy, researchers may increase the sensitivity of high-throughput screening, by detecting bioactive compounds at lower nominal water concentrations. In some cases, this advantage is offset by the need to use higher quantities of precious or limited chemicals. The chorion represented a perfect barrier to MWCNTs. With chorions removed, MWCNTS are highly toxic, but when left intact, the fish appeared indistinguishable from controls indicating that for nanomaterials, to avoid false negatives, we recommend chorions be removed. Taken further, these results suggest that it is reasonably feasible to reach agreement on a standardized exposure regimen, which will promote data sharing without sacrificing data content. Future refinement of tools capable of predicting the chemical behavior in high-throughput screening zebrafish plates may allow the field to developed assays tailored to chemical characteristics, thus maximizing the reproducibility and translation of zebrafish screening data.This study systematically evaluated the impact of changing exposure conditions on chemical bioactivity by varying the common variables of chorion status, lighting, and chemical renewal. Importantly, we screened the same endpoints in a single laboratory and using the same fish strain, permitting direct data comparisons between experiment designs. Since all three variable conditions altered the concentration-response of the test agents, though not in the same way for each, the results indicate that study design indeed influenced chemical concentration responses. However, in every case except for MWCNTs, each exposure regimen concordantly identified the bioactive compounds, though depending on the chemical, the exposure protocol altered the EC


The dancing sessions of approximately 75 min were performed twice a week and each session was based on five Scottish dances performed in 3 sets. Heart rate (HR) measurements were obtained during all dances to evaluate whether the intervention achieves the criteria of moderate to vigorous aerobic exercise intensity. Body composition, waist circumference, and HR during Chester Step test were measured before and after dancing intervention. HR achieved during individual dances ranged from 64 ± 5% to 80 ± 5% of HRmax and the mean HR of the five dances corresponded to 72 ± 7% of HRmax. Post-intervention mean HR was lower throughout Level 2  and Level 3  of the Chester test compared with baseline values. The intervention had no impact on body weight or body fat but reduced waist circumference . Thus, traditional Scottish dancing should be advocated to sedentary postmenopausal women, emphasising its potential in meeting current physical activity recommendations in relation of weekly duration and exercise intensity and improving cardiorespiratory fitness.This study investigated the efficacy of participation in culture-specific dancing to meet current physical activity recommendations and increase cardio-respiratory fitness in postmenopausal women. Sedentary postmenopausal women (n = 24), aged 63 ± 8 years and with BMI of 28 ± 3 kg/m Despite the estimation that physical inactivity is responsible for 1 in 10 deaths globally . In the The evidence based on data from countries representing 96% of the world’s population suggest that, in almost every country, women are less physically active than men and that a substantial gender gap has changed little over time ,7,8,9. IDespite evidence that participation in exercise improves markers of cardiovascular health in postmenopausal women ,15,16, nThis study aimed to investigate the efficacy of participation in traditional Scottish dancing to meet current PA recommendations in relation to weekly duration and intensity of exercise sessions in postmenopausal women. It additionally, it investigated whether participation in traditional Scottish dancing can modify body fatness and central obesity measured by waist circumference and increase cardio-respiratory fitness, a robust predictor of cardiovascular risk and other adverse health outcomes. Our decision to investigate efficacy of Scottish traditional dancing is related to the evidence that PA levels are concern in Scotland, where only 12% of women aged over 65 achieve the current guidelines .2) or obesity (BMI > 30 kg/m2), and briefed individually by an investigator who provided them with a participant information sheet outlining the requirements of the study. Participants were required to read and consent to the risk assessments associated with the study, and to complete a PA readiness questionnaire (PAR-Q) and a health-screening questionnaire prior to commencing the study. Women were invited to participate if answers to all PAR-Q question were negative. Participants were required to be non-smokers, with stable body weight for two months prior to the study enrolment, free of nutritional supplementation or following any specific diet. Women were not eligible if they had suffered from myocardial infarction, heart failure, or active angina in the previous 6 months or had blood pressure greater than 160/90mmHg. Participants with chronic illness, eating disorders and history of gastrointestinal operations were also excluded. The Ethics Committee of the College of Medical Veterinary and Life Sciences of the University of Glasgow approved the study (Project No 2012051). Recruitment and data collection started in August 2012 and study was completed in March 2013. Thirty-nine women were initially recruited . Although all women met the inclusion criteria, 7 did not participate due to unsuitability of class times. Therefore, 32 participants started the intervention.The participants in this study were healthy postmenopausal women, aged 50–80 years. Participants were previously sedentary, i.e., performed little or no organized PA and were spending most waking hours sitting. Participation in PA was estimated from recall diaries reflecting organized and non-organized activities  over two months prior to participation in the dancing programme. Prospective participants were recruited via online and leaflet advertisement, targeting women with a BMI in the category for overweight , designed to achieve current PA recommendations in relation to weekly duration of exercise . The danTANITA TBF−310, Cranela, UK) was used to measure weight, BMI (weight (kg)/ height (m)2), fat mass and fat-free mass. For all measurements, participants required to be fasted, wearing minimal clothing and no shoes.Height was measured via a Stadiometer model . Waist circumference was obtained at the end of normal expiration at the narrowest point of the abdomen using a standard waist measuring tape. The anthropometric measurements were taken twice on both occasions (before and after intervention), according to International Society for the Advancement of Kinanthropometry (ISAK) standards . Bioelecmax), with HRmax being calculated as 220 −age. If 80% of maximal HR was reached during a level, yet participants appeared to be in no great physical strain, the level was completed before terminating the test. During pre- and post- training intervention, Chester step tests were conducted in the morning, at approximately the same room temperature. Prior to the first Chester test, participants were advised to drink some water. This amount of water was recorded and replicated during the second test.Cardio-respiratory fitness was assessed via a modified version of the Chester step test . The tesThe dancing programme was designed and facilitated by a Scottish dance expert. The 4-week dancing intervention required participants to attend 2 sessions per week, each lasting approximately 90 min including breaks. All sessions consisted of three sets and each set was comprised of 5 traditional Scottish dances lasting 4–5 min, with a 1–2 min break permitted between dances. A break of 2–4 min was allowed between sets. Thus, active dancing during each of the sessions lasted for approximately 75 min. Each set was designed to vary exercise intensity in the following pattern: low , moderate (Gay Gordons), vigorous (Canadian Barn Dance), moderate (Gay Gordons), and vigorous (Strip the Willow). As the intervention was specifically tailored for the current participants, techniques which were considered too strenuous, such as spins, were replaced with easier stepping movements. HR and RPE were measured at the very end of five dances of the second and third set of the sessions conducted in week 3 and week 4, when participants were familiar with the routines. Towards of the end of each dance, randomly selected participants were reminded and helped to take HR and RPE measurements and asked to report these measurements to the researcher. The obtained data were used to calculate group mean and SD values. Dancing sessions were performed in the late afternoon, with dancing session starting at approximately 18:00. Each exercise session was supervised by the researcher.t-tests. Statistical significance was considered when p < 0.05. Categorical data were analysed using Fishers Exact test. Statistical analysis was conducted by Minitab . Adherence to the dancing programme was calculated from the participation logs by using the following formula: (the number of sessions completed/ the number of sessions prescribed) × 100%.The data collected were tested for normality via the Anderson–Darling test. Since all variables were normally distributed, the differences between pre- and post-intervention values were determined via paired The dancing programme was completed by 28 women and the study achieved an adherence rate of 89 ± 13%. Two participants failed to attend the post-intervention assessment session and 2 did not supply full data. Thus, the results are presented on 24 women (75% of participants who enrolled the dancing programme). Baseline characteristics of the 24 women are presented in max) for each dance and the corresponding exercise intensity according to criteria established by the American College of Sport Medicine for [max), followed by intermediate intensity dances; Strip the Willow (75 ± 5% HRmax), Virginia Reel (73 ± 5% HRmax) and the Gay Gordons (70 ± 3% HRmax), and St. Bernards Waltz was the least intensive dance (64 ± 5% HRmax). The average HR throughout the dancing sessions was 113 ± 11 bpm, corresponding to 72 ± 7% HRmax.The intensity of each of the five dances used in the intervention was estimated from HR recordings conducted on a subset of participants (n = 14). A total of 815 HR and 818 RPE measurements were recorded throughout the intervention. cine for . The Canp = 0.006).Pre- and post-intervention measures of body weight, body composition, BMI, and waist circumference are displayed in p = 0.005) and Level 3 . Mean HR of the first three levels of step test was significantly lower post-intervention . There was no statistical difference in RPE throughout the first three levels of the Chester Step test.The current study demonstrated that, for sedentary postmenopausal women, participation in traditional Scottish dancing provides a novel strategy for meeting current PA recommendations, in relation to the weekly duration and intensity of aerobic exercise ,4,5,6. Wmax which represents moderate intensity exercise according to the ACSM [The 4-week Scottish dancing programme required participants to attend two weakly 90-min sessions, with the active dancing component consisting of 75 min. The dances chosen for the programme were specifically tailored to vary the intensity of the exercise sessions, ranging from light- to-moderate to vigorous intensity. According to the ACSM , the HR the ACSM . Thus, othe ACSM . We notethe ACSM , the higAs low cardiorespiratory fitness is an independent predictor of cardiovascular risk, and cardiovascular and total mortality ,26,27, oIt was unsurprising that body weight and body fatness measurements were unchanged by the dancing intervention. Alternative dancing and exercise studies in older women have also failed to find a significant reduction in body fat and BMI after aerobic training, ,32. We nThis study has limitations. Admittedly, cardiorespiratory fitness is most accurately measured via a maximal oxygen uptake test, which measures the rate of oxygen consumption during either exercising on a treadmill or a bike at an intensity that increases every few minutes until exhaustion and is designed to achieve a maximal effort . HoweverThe current study highlights that suggests that the pragmatic minimum target of 150 min a week of well-structured, culture-specific (Scottish) dancing programme can not only sufficiently meet PA guidelines, but additionally improve cardiorespiratory fitness and achieved significant reduction in waist circumference, whilst enjoying a low drop-out rate and high rate of adherence in postmenopausal women. Data of this study, however, cannot be used to suggest that participation in a Scottish dancing programme favourably impacts markers of cardiovascular risk and other aspects of health in older women. Thus, this preliminary study indicates that further randomised controlled trials investigating the longer effects of traditional dancing on cardiorespiratory fitness and cardiovascular risk factors in more variable group of postmenopausal are warranted.


Microcoleus asticus sp. nov. Our studies of geosmin gene presence in Bacteria, revealed a scattered distribution among Cyanobacteria, Actinobacteria, Delta and Gammaproteobacteria, covering different niches. Careful analysis of the bacterial geosmin gene and gene tree suggests an ancient bacterial origin of the gene, that was probably successively lost in different time frames. The high sequence similarities in the cyanobacterial geosmin gene amidst freshwater and soil strains, reinforce the idea of an evolutionary history of geosmin, that is intimately connected to niche adaptation.Cyanobacteria are ubiquitous organisms with a relevant contribution to primary production in all range of habitats. Cyanobacteria are well known for their part in worldwide occurrence of aquatic blooms while producing a myriad of natural compounds, some with toxic potential, but others of high economical impact, as geosmin. We performed an environmental survey of cyanobacterial soil colonies to identify interesting metabolic pathways and adaptation strategies used by these microorganisms and isolated, sequenced and assembled the genome of a cyanobacterium that displayed a distinctive earthy/musty smell, typical of geosmin, confirmed by GC-MS analysis of the culture’s volatile extract. Morphological studies pointed to a new Oscillatoriales soil ecotype confirmed by phylogenetic analysis, which we named  It represents a prokaryotic group with huge diversity; in morphology presenting from unicellular to filamentous forms with specialized cells; chemo-diverse since they produce a panoply of bioactive compounds: antifungal4, anticancer5, antibacterial3, antiviral5, algicidal6, and antioxidant7, as well as toxic compounds noxious to the health of humans and wildlife9. Two of the most common volatile organic compounds produced by Cyanobacteria are 2- Methylisoborneol  and Geosmin (PubChem CID:29746)12. These compounds are frequently described in fresh and brackish waters Cyanobacteria, as they cause taste and odor problems in drinking water, fish and shellfish14. Although non-toxic to humans, these compounds change the perception of safety by the public, causing major economic losses in both sectors14.Cyanobacteria can be found in marine, brackish and freshwaters exhibiting both planktonic and benthic lifestyles, covering almost all ranges of temperatures and salinities, in symbiosis with a variety of organisms, in soil, caves, deserts, and buildings, and are among the oldest life forms that evolved throughout timeNorcardia cummidelens; N. fluminea; Streptomyces Luridiscabiei; S. albidoflavus - since they are typical soil inhabitants16. Other known producers of geosmin are fungi - Penicillium discolour, P. crustosum, P. expansum, Botrytis cinerea, and Chaetomium sp. - that are also present in soil and associated with fruit and vegetable spoilage17. Geosmin is also produced by higher plants, such as red beet18 and cactus flowers19 and is thought to be implicated in defense mechanisms, pollination, and competition22.Geosmin is greatly associated with terrestrial environments. It is assumed that the organisms responsible for the gross production of geosmin in soil are Bacteria belonging to actinomycetes - 23 and recently the unicellular Synechococcales Coelosphaerium sp.13, debunking the idea that only filamentous Cyanobacteria could produce geosmin25. Most of the knowledge on cyanobacterial producers is from freshwaters, probably due to the blooms related to odor and musty taste issues, while terrestrial, brackish and marine producers are rarely described or absent25. In freshwaters, the most frequent reported producers are the Nostocales (Dolichospermum and Aphanizomenon) and the Oscillatoriales (Phormidium and Tychonema)25 and in the soil, Nostocales of the genus Nostoc in symbiotic associations with cycads, lichens, mosses, and liverworts26.The species of Cyanobacteria producing geosmin are diverse, belonging to several distinct taxons, namely, multicellular strains of Oscillatoriales, Nostocales and Synechococcales29 or may be synonymous of inedibility and presence of noxious microorganisms for fruit flies and humans29. In fact, humans smell and taste geosmin at very low concentrations and this compound has always received attention from various sectors: public health, academia, industrial and commercial14. Geosmin is recognized for the pleasant earthy smell in the air, especially when it rains and can give a freshness note to a perfume. However, when associated with water, food, and beverages the pleasant odor becomes an unpleasant earthy or musty taste14.Animals are not indifferent to geosmin, presenting behaviors of both attraction and repulsion towards it. The geosmin odor may represent the presence of freshwater, as it happens with camels and eelsgeoA) that encodes for a bi-functional domain enzyme, with both the N and the C-terminal parts containing two metal-binding motifs typical of sesquiterpene synthases. These two motifs require Mg2+ as the catalyst for the formation of complex terpenoid molecules like geosmin30, and are responsible for the cooperative binding of the three catalyst metal ions, as well as the positioning of the substrate molecule for the cyclization reactions cascade that follows32. The protein domains share significant similarities and contain typical metal-binding motifs of class I terpenoid cyclases. The N-terminal part is responsible for the ionization and cyclization of farnesyl diphosphate (FPP) into germacradienol and inorganic pyrophosphate molecules, and the C-terminal part mediates the reactions of protonation, cyclization, and fragmentation of the precursor germacradienol molecules into geosmin and acetone34.The molecular basis of geosmin production is geosmin synthase gene  (758 aa in length), followed by two cyclic nucleotide-binding genes (cnb) (471 and 469 aa in length each) of the Crp/Fnr-type global transcription regulators , polyketide synthase (PKS), hybrid NRPS/PKS gene clusters and ribosomally synthesized and post-translationally modified peptides (RiPPs), we were able to identify the complete gene cluster for the synthesis of terpenoid geosmin, composed of the geosmin synthase gene (DarB was detected as well as genes with shared similarity), trichamide, and the mixed PKS/NRPSs nostophycin, nostopeptolide, and jamaicamide. Furthermore, we also identified the existence of two unknown terpene biosynthesis gene clusters showing the potential for alternative pathways for the synthesis of terpenoid compounds and other secondary metabolites that are still unknown.Other genes that participate in the synthesis of known cyanobacterial secondary metabolites were also detected, albeit, in none of them, the complete cluster was found. The incompletes gene clusters identified would code for: benzenodiol resorcinol, cyanopeptolin . Figure To better illustrate geosmin presence/production in Bacteria, it was fundamental to tackle different questions: How frequent is the gene spread in terms of phyla?; What species possess the genetic machinery to produce geosmin and even niche occupancy?; Can we trace the evolutionary history of the gene in Bacteria? To do so, we compiled a set of geosmin gene sequences, collected by BLAST searching public databases for complete and partial putative geosmin gene sequences. Our first outcome, was that sequences were restricted to only three bacterial phyla: Cyanobacteria, Actinobacteria, and Proteobacteria represented by two classes, Delta and Gammaproteobacteria. Besides being restricted, the geosmin synthase was not evenly distributed in those groups Fig. , with juMyxococcus fulvus. Moreover, of the 3 Gammaproteobacteria strains, 2 are related to bacterial infections in edible mushrooms. Actinobacteria are mostly terrestrial strains, but 2 out of the 13 actinobacterial strains are associated with insects, while 1 was identified in a human lung infection. Figure The environmental sources of our set of bacterial strains are also depicted in Fig. Taking into account the number of geosmin producers used in our phylogenetic study, we found that, of the 36 Cyanobacteria analyzed, 21 are known geosmin producers, whilst 2 out of the 14 Deltaproteobacteria strains have been tested positive regarding the synthesis of this metabolite. Furthermore, among the set of 13 Actinobacteria strains, there are 3 known producers of geosmin, while there is no information regarding the production of geosmin by the 3 Gammaproteobacteria strains in our dataset. In fact, to our knowledge, this is the first report of geosmin synthase gene in Gammaproteobacteria.35. To better understand the evolutionary history of this gene in Bacteria, we compared the geosmin gene tree presented in Fig. 35. Some differences in tree topology were visible between these two phylogenetic trees regarding the positioning of Cyanobacteria, Actinobacteria, Delta and Gammaproteobacteria. The geosmin synthase gene tree shows a close relationship between Cyanobacteria and Deltaproteobacteria, while in the species tree this closer relationship is not clearly visible. Nevertheless, Actinobacteria appears to be the most ancient phylum to harbor the geosmin gene as well as the most ancient of the four taxa divisions in the species tree. We decided to analyze these two phylogenies using a tree reconciliation algorithm to obtain support for a possible scenario for the evolutionary history of the geosmin gene in Bacteria  is replaced in Cyano II by isoleucine and the 4th residue, a phenylalanine in Cyano I, is replaced by a leucine in 2 strains of Cyano II. Still, none of the strains in Cyano II were tested for geosmin production, so more tests should be made in order to clarify the impact of these amino acid modifications.Overall, the gene tree topology matched the major phyla divisions of a 16S rRNA based species tree for Bacteria2+ binding motifs, but it is clear that in terms of the broader analyzed protein sequence Leptolyngbya sp. A2 is distinct from other cyanobacterial sequences, sharing with them 77 to 82% similarity, while strains in Cyano I share amongst them 83% to 100% similarity. Strains in Cyano II share high similarity values, 91 to 100%, and lower values with Leptolyngbya sp. A2 (78 to 79%). Leptolyngbya sp. A2, a MIB and geosmin producer from freshwater23 has its highest similarity values with the Nostocales strains in this study except for Fischerella muscicola and Calothrix sp. NIES-2100. The Cyano I group also reveals the existence of two clusters formed both by gene sequence and niche, where the freshwater strains in the clade Aphanizomenon/Anabaena/Dolichospermum share 94% to 100% sequence similarity and soil-related strains that include all Calothrix and Nostoc strains in our set, Oscillatoria sp. PCC 6506, F. muscicola and Cylindrospermum stagnale share amongst them 90% to 100% similarity. Microcoleus asticus sp. nov. IPMA8 lies in an intermediate group formed by strains from soil and aquatic origin: Oscillatoria sp. 327–2, Oscillatoria sp. PCC 9240, Planktothrix sp. 328 Fischerella sp. PCC 9431 and Phormidium sp. P2r, where the range of similarity values starts at lower values, from 87% to 100%.Cyano I group harbors cyanobacterial strains that share the same residue sequence of the MgArchangium strains and C. fuscus (74 to 81%). In fact, the Deltaproteobacteria phylum shows a tight proximity among its gene sequences with similarity values ranging from 76 to 100%. Regarding Actinobacteria, the Streptomyces sp. clade forms a closely related group with clear differences in the protein sequence to the other actinobacterial strains, despite their similarities of the geosmin synthase metal-binding motifs. The Pseudomonas strains , which we identified by in silico search, have low similarity values with all sequences from the other bacterial phyla, with the lowest similarity values (61%) with Deltaproteobacteria’s strains M. xanthus DK 1622 and C. coralloides DSM 2259 and Actinobacteria’s S. roseum and S. cattleya. The best similarity values with the Pseudomonas strains were identified for Deltaproteobacteria C. fuscus (67 to 70%) and Cyanobacteria Fischerella sp. PCC 9431 (69 to 70%). Taken as reference the Cyano I group, Actino is the group with more modifications which in turn occur with low frequency (L by V in the first site and L by I and Q by E in the second binding site), while Cyano II group has two modifications both in the second binding site: a complete replacement of L with I and F with L. Delta and Gamma groups have both one amino acid replacement: E by Q in the second binding site and Y/H in the first binding site, respectively.Analysis of the geosmin gene tree highlights the probable close evolutionary history of geosmin synthase gene of Cyanobacteria with the Deltaproteobacteria gene, with higher similarity values shared with the two We quantified the selection pressures on geosmin gene using GARD algorithm to identify recombination breakpoints, which could increase the false points of positive selection pressure. This pre-analysis identified one recombination point in the amino-acid alignment. We then tested three different algorithms; SLAC, FUBAR and MEME and in all of them the global value of dN/dS is significantly higher than 1. We identified most codon sites as positive selection sites, indicative of strong diversifying selection pressure of the geosmin gene in Bacteria. The SLAC analysis points to pervasive (refereeing to the whole phylogeny) positive selection in 28 out of 269 sites and negative selection in solely 1 site  while the FUBAR test also points to positive (35 sites) over purifying (21 sites) pervasive selection . The MEME algorithm, used for the identification of adaptive evolution in individual sites, shows positive selection in 55 sites , confirming our hypothesis that several sites in the geosmin gene were subjected to positive or diversifying selection pressure.The novel specimen is a terrestrial, free-living filamentous Cyanobacteria without heterocysts and akinetes. It forms dense mats in culture conditions in both liquid and solid media. In the liquid, the mat is both submerged attached to the bottom of the flasks and walls and also at the surface of the liquid forming aggregates with pockets of air Fig. . In soliFilament separation is by means of necridia and two types of these cells are present in this species Fig. . One typIn cell ultrastructure it is visible the constrictions at cross-cell walls Fig.  and reguOscillatoria nigro-viridis PCC 7112. In an effort to refine and validate this primary identification, we performed a close-up phylogenetic analysis, using the 16S rRNA gene, widely represented in genomic databases, which confirmed a close phylogenetic relationship of the isolate with other strains from the family Microcoleaceae  clade, as presented in Fig. Microcoleus vaginatus FGP-2 and Oscillatoria nigro-viridis PCC 7112. The similarity matrix constructed from the set of 64 genes of these three genomes shows differences of 3% with M. vaginatus FGP-2 and 7% with O. nigro-viridis PCC 7112 (supplementary Table in silico DNA–DNA hybridization (DDH) between our isolate and its closest relative Microcoleus vaginatus FGP-2. For the ANI38 analysis results showed that average nucleotide identity is91.71% (Table 39 (GLM-based) was 46.40% with a distance of 0.0805 with a probability that DDH > 70% (same species) of 10.65% and probability that DDH > 79% (same subspecies) of 2.28% and a difference in % G + C of 0.44.To perceive the phylogenetic context of this soil isolate, it is required to find its kindred strains. Our primary 16S rRNA based phylogenetic analysis Fig.  using a eae Fig. . Finallyeae Fig.  present 40. Microcoleus asticus C. Churro, A.P. Semedo-Aguiar, R.B. Leite sp. nov.The description was made under the provisions of the International Code of Nomenclature for algae, fungi and plants Here designatedEtymology: of/located in a city, city, urbanType Locality: Lisbon city center, urban area, Av. Infante Santo, coordinates: 38°42′33.7″N 9°10′00.3″W, Portugal.Nostoc sp. in a street flowerbed from an urbanized area.Habitat: Cyanobacterial mat in soil dominated by http://phycobank.org/102090.Description: Filaments form dark olive-green mats, only entangled when the densely mat is formed, otherwise, solitary, motile and enwrapped in a single sheath. The sheath is prominent, firm, colourless and hyaline. Filaments are cylindrical, heteropolar (straight and/or attenuated towards the ends) and constricted at the cross-walls. Apical cells can be broadly rounded to conical with or without calyptra. Cell content is granulated with the cell inclusions visible in light microscopy. Lipid droplets are located near the cross-walls, polyphosphate granules and polyhedral bodies are in the center of the cell and cyanophycin granules are mainly near the cross-walls, but can also be scattered in the cytoplasm. Gas vacuoles are absent. Cells are wider than long and rarely isodiametric; 2.90–7.62 (5.87) µm wide and 1.82–6.05 (3.95) µm long. Cell division is in one plane and several divisions occur simultaneously. Filament dispersal is by means of motile hormogonia or long filament breakage by the aid of necridia. Necridia can be formed by the degradation of only one cell or by a group of adjacent cells. The thylakoids have a fasciculate arrangement. The fascicules run in parallel and form curves to fully spherical formations. The genome data is available at DDBJ/EMBL/GenBank under the accession number: PRJNA531034. Phycobank registration Diagnosis: The main distinctive morphological characters are the type of fasciculated thylakoid arrangement and the type of necridia cells.https://coicatalogue.uc.pt/index.php.Holotype: Held in the Herbarium of University of Coimbra (COI), Department of Life Sciences, University of Coimbra Coimbra, Portugal in a metabolically inactive state in the form of preserved material with the strain identifier COI00097100 and the information available online at: Microcoleus asticus are held in in the Collection of Microalgae of University of Coimbra (ACOI) at the Department of Life Sciences, University of Coimbra, Coimbra, Portugal and at the Culture Collection of Cyanobacteria of the Portuguese Institute for Sea and Atmosphere, I. P. , Av. Alfredo Magalhães Ramalho, 6, 1495–165 Algés under the strain identifiers ACOI 3416 and IPMA8 respectively. Preserved isotypes are held in the Herbarium of University of Coimbra (COI), Department of Life Sciences, University of Coimbra Coimbra, Portugal with the strain identifiers COI00097101; COI00097102; COI00097103 and the information available online at: https://coicatalogue.uc.pt/index.php.Isotypes: Cultures of Microcoleus vaginatus/Microcoleus autumnalis42 clade was confirmed and named Microcoleus asticus. The strong support of our phylogenetic and the results of average nucleotide identity, and DNA-DNA hybridization, allowed us to confirm that it is a new species with close proximity to Microcoleus vaginatus FGP-2, a free-living isolate collected from a desert-soil crust43 and a member of the Microcoleaceae IV family 45. The ANI values were below the 95–96% similarity threshold47 and DDH estimate also below 70% similarity threshold for species boundaries48 indicating that our isolate is a different species from M. vaginatus. The main distinctive morphological characters are the type of fasciculated thylakoid arrangement and the type of necridia cells. The curved and spherical formations of the thylakoid membranes that resemble the centers for thylakoid connectivity described by Nevo et al.49 are not described for Microcoleus. Spherical thylakoid formations are present in several distinct Cyanobacteria50, but none of the reported combinations of spatial, directional, and morphological rearrangement of the thylakoids are similar to the ones present in Microcoleus asticus sp. nov50. Necridia cells are also different in Microcoleus asticus sp. nov., beside the separation disc formed by one cell that is typically described44, this species also presents fragmentation by a group of degrading cells. Surprisingly the resulting filaments have tapered ends. Tapered end cells is usually attributed to filament maturation rather than newly separated filaments in Oscillatoriales51. All the other morphological characters are congruent with the genus Microcoleus sensu stricto with the type species Microcoleus vaginatus (Vaucher) Gomont ex Gomont (1892). Nevertheless, morphology is quite similar within some groups of the order Oscillatoriales (ex. the genus Phormidium) so, genetic information is necessary for the distinction42. Microcoleus is worldwide distributed and mainly associated with soil, aerophytic and epiphytic habitats and is one of the main constituents of biological soil crusts54. In soil environments Cyanobacteria play an important ecological role providing a nitrogen source in symbiotic relationships and producing phytohormones required for plant growth and development56, in soil particle aggregation, erosion reduction, increasing water penetration and retention and nutrient recycling60. Although the knowledge in the diversity of soil Cyanobacteria is increasing, much is still unknown regarding species composition and bioactive compound production.We have isolated and sequenced the genome of a soil Cyanobacteria that is able to produce geosmin. Based on a phylogenetic study and morphological analysis a new representative of the in silico mining of the genomic data identified a complete geosmin synthesis gene cluster, and the analytical analysis for volatile compounds allowed us to conclude that Microcoleus asticus sp. nov. IPMA8 is actively producing geosmin.The Our primary search for the geosmin synthase gene in bacterial public genomic databases allowed the identification of this gene in Gammaproteobacteria - which to our knowledge, is reported here for the first time - as well as in Deltaproteobacteria, Actinobacteria, and Cyanobacteria. Our efforts to map the presence of the geosmin gene in Bacteria revealed a restricted distribution, to these three phyla, crossing distinct genera from different niches, suggesting a possible rich evolutionary history of the gene. In our analysis the congruence between the bacterial species tree and the gene tree confirms a probable ancient origin of geosmin gene in Bacteria, that might have resulted from a mixture of evolutionary processes difficult to disentangle but that we identified as dominated by gene loss.44. The gene cluster in Cyanobacteria is a mostly conserved one; to our knowledge the only cyanobacterial strain for which a different cluster organization was detected is Phormidium sp. (Pr_1 and Pr_2)61. The apparent disparity of the Cyano II group when compared with the straightforward divisions in the species tree could be explained with minor specific alterations in taxon and niche, since geosmin genes studied here appear to come from a common ancient Cyanobacteria and thus are orthologous genes. The geosmin gene cluster has two global transcription regulator genes, known to modulate cellular signals associated with responses to environmental stress62. The high conservation of the arrangement of the cluster, in Cyanobacteria, could thus indicate its high importance in controlling environmental adaptation, which is supported by the results described in other studies, indicating that the synthesis of geosmin and other volatile organic compounds, could be related with defense/offense mechanisms towards other microorganisms64. In Nostoc punctiforme PCC 73102, formation of geosmin results from conversion of the sesquiterpene precursor farnesyl diphosphate. However, upon expression of the enzyme in E. coli, no geosmin is produced, but instead synthesis of alternative products of geosmin synthases, such as germacradienol, germacrene D, and germacrene A, were observed, suggesting a dependency for environmental factors or other enzymes and metabolites for the active production of geosmin65.It is noteworthy that even in Cyanobacteria the distribution of geosmin gene is not heterogeneous, from the eight orders described for this Phylum, the Oscillatoriales, Nostocales and Synechococcales harbor the majority of known geosmin producers66. So, the inferences made from these patterns of evolution cannot be made separately, since these are probably the result of distinct evolutionary events acting on these phyla either simultaneously or in different time frames.Cyanobacteria and Deltaproteobacteria have very conserved and similar gene architecture, with a three gene operon assembly, whilst in Gammaproteobacteria and Actinobacteria this construction is not widely conserved. When circumspect geosmin synthase sequences, we find that Actinobacteria representatives are the most distant phylogenetic relative to Cyanobacteria while Deltaproteobacteria’s protein sequences are the closest see Fig. . Otherwi25. However, as stated by Jutner and Watson25 salinity by itself is not impeditive of geosmin production since Cyanobacteria from brackish and high salinity environments are able to produce it. Additionally, there is only one marine organism in our data set carrying the full geosmin gene cluster which is the Deltaproteobacteria Myxococcus fulvus HW-167. Myxococcus are mostly associated with terrestrial environments and marine specimens have only recently been discovered68. From an evolutionary perspective the salinity barrier in habitat expansion can be difficult to overcome66. Nevertheless, according to Sánchez-Baracaldo 201569 the origin of marine planktonic cyanobacteria lies in freshwaters and benthic marine ancestors. The brackish water Nodularia spumigena CCY9414, for example, evolved from freshwater relatives by the end of the Pre-Cambrian period69. This strain carries the geosmin gene (geoA accession Number: WP_063873980.1). So, there is the possibility that marine cyanobacterial geosmin producers are already present in marine environments or perhaps the geosmin gene had no specific advantage in the sea and was lost throughout time. Interestingly, in Cyanobacteria it is noticeable the high sequence similarity of the geosmin gene among strains from freshwater environments as well as higher values for strains from soil symbiotic related environments, reinforcing that the evolutionary history of this gene could be related to niche adaptation.Even though half of the strains in our cyanobacterial dataset are associated to soil related environments (18 out of 36), a considerable part (15), has an aquatic origin and only one is found in brackish waters. It is also evident that free living terrestrial Cyanobacteria that carry the cluster, including our specimen, are more common than previously reported. This implies that Cyanobacteria are likely important contributors for the geosmin production in soil. Cyanobacteria from marine environments with the genetic capability to synthesize geosmin were not identified in our survey, which is congruent to what has been described in other studiesSome questions can be posed at this time: Could these similarities in the metal-binding motifs and the broader protein sequence imply that the geosmin gene was acquired by Cyanobacteria from Actinobacteria? But what about the Cyano II group, was it a later acquisition to the cyanobacterial phylum?70. Also in Cyanobacteria, others have proposed a similar evolutionary history for the cyanotoxin microcystin, synthesized by several genera, proposing that negative selection was the main driver for the sporadic distribution of microcystin synthesis in this phylum: the toxin was present in ancient cyanobacteria and was successively lost throughout the species tree, refuting the lateral gene transfer hypothesis for this gene cluster71. These two examples are specific of Cyanobacteria and of distinct genes, which have their on environmental queues, but show that distinct evolutionary processes could be pressuring and shaping bacterial genomes and particularly the geosmin profile in Bacteria. We identified only three Pseudomonas strains, with the geosmin gene, which we do not know if are effective geosmin producers. Interestingly, several studies on geosmin biodegradation have identified strains of Pseudomonas, Sphingopyxis, Chryseobacterium, Sinorhizobium, Stenotrophomonas, Novosphingobium  with potential to be used in bioremediation of geosmin and 2-MIB in aquaculture and water treatment facilities, often in bacterial consortiums in specific niches75. Several bacteria evolved to rely on less abundant and less explored carbon sources as their primary carbon source like monoterpenes and other volatile organic compounds76, which could ultimately result in losing the ability to synthesize terpenoids like geosmin, in specific environments. How this gene was kept and/or lost by distinct members of Bacteria, especially in the Phyla we studied here, is a result of many evolutionary processes that acted simultaneously or in different time frames or in different environments and different microorganisms. Our data support that this gene, was present in an ancient bacterium, probably an ancestor of Actinobacteria, but due to a rich evolutionary history was successively lost by several groups of Bacteria, resulting in this patchy presence in the taxa discussed.A congruence test of the geosmin gene tree with a well-supported Bacteria species tree revealed no evidence for any specific pattern of evolution, instead, it appears that selection is acting towards the elimination of genes that were probably representing excessive energy costs. Nevertheless, our scrutiny of the natural selection pressures on the geosmin gene, shows clear evidence of strong positive selection when tested for both whole bacterial phylogeny and codon sites. These results support our hypothesis of an early presence of the geosmin gene in the bacterial kingdom, which was successively lost in the most derived branches of the species tree. The fixation of beneficial mutations, from positive natural selection, ultimately leads to differences among species and adaptation to different niches. Many bacterial strains have kept the geosmin gene and the ability to synthesize the metabolite, as it is clear by the present-day producers in Actinobacteria, Deltaproteobacteria and Cyanobacteria. Recent studies describe that Cyanobacteria kept the geosmin gene through purifying selection29. Nevertheless, the exact function of this compound and the adaptive advantage in “retaining” this gene cluster in Bacteria is still unknown. It is clear the near absence of marine geosmin-producing organisms, which pushes us to pursue the cunning halotolerant geosmin gene carrier Cyanobacteria. In fact, our study demonstrates that bacterial, as well as cyanobacterial, soil representatives could still be identified, diversifying the origins of geosmin producers that could have biotechnological uses. Over the last years, the increasing number of reports of geosmin occurrence in freshwaters states them as significant contributors for the production of this organic volatile compound in this environment. Likely, the increasing reports on terrestrial Cyanobacteria producing geosmin, indicates that they are also key players of its production in soil. Our study demonstrates that geosmin in soil can have multiple cyanobacterial origins resulting from intrinsic factors and environmental drivers during evolution.Current knowledge on geosmin distribution, points to a bacterial originated soil compound that is strongly precepted by animals and also endogenously produced by higher plants, where terpenoids have been linked to defense mechanismsNostoc sp. from an urbanized area in Lisbon, Portugal. A sample of the Nostoc sp. mat was collected and washed with Z8 culture medium. The washings were observed under the inverted microscope Leica® DMi8 and cyanobacterial filaments were isolated by micromanipulation with a glass capillary. Only one filament was picked up at each time, washed in new culture medium several times and placed in a culture flask to propagate. Successfully established cultures were maintained at 20 ± 1 °C with a light intensity of 10 μmol·photons·m−2·s−1 and a 12:12 h light: dark cycle.The cyanobacterial specimen used in this study was obtained from soil mats of the Cyanobacteria 44. The ultrastructure aspects analyzed were: arrangement and vacuolization of the thylakoids, type of cell division and cellular inclusions44. Captures were taken with a Leica® DFC7000T digital camera using a Leica® DMi8 inverted fluorescent microscope at 1000× magnification. The cell measurements were performed using LAS V4.12.0 Leica® software 2017, at least 500 measurements were done in 100 different filaments. The description and identification of the Cyanobacteria specimen was done based on the following manuals and bibliography77.The specimen was studied using both optical and electron microscopy. The morphological characters evaluated were: filament color and shape, cell dimensions (length and width), constrictions at the cross-cell walls, shape of apical cells, presence/absence of sheath, false branching, calyptra and necridiaCell viability was accessed in live cells using the ReadyProbes™ Cell Viability Imaging Kit (Blue/Red) from Invitrogen™ composed of NucBlue® Live reagent and propidium iodide following the manufacturer’s instructions. The cells were visualized under the Leica® DMI8 inverted fluorescent microscope and photographs were taken with a Leica® DFC7000T camera.et al.78 with minor modifications. Briefly, cell material was prefixed in 2.5% glutaraldehyde in Z8 culture medium for 30 min at room temperature and then transferred to 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4 °C. Cell material was washed three times with 0.1 M cacodylate buffer and immobilized in 2% low melting point agarose. Post-fixation was in 1% osmium tetroxide, 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 3 h on the ice and protected from light. Cells were then washed three times in 0.1 M cacodylate buffer for 10 min. To increase cell contrast, cells were incubated with 0.5% uranyl acetate for 1 h in the dark at room temperature and rinsed with distilled water for 15 min. Cells were dehydrated in a graded ethanol series  at 4 °C, with a final step of propylene oxide for 10 min. After dehydration, cells were embedded in Spurr’s low-viscosity epoxy resin overnight, placed in flat embedding silicone rubber molds and polymerized at 60 °C for 24 h. Sections were post-stained with 2% uranyl acetate (5 min in the dark) and lead citrate (5 min). Sections were examined with a Hitachi® H-7650 transmission electron microscope.The cell ultrastructure was analyzed with transmission electron microscopy following the method describe in Churro 79.The DNA was extracted from the cultures using the DNeasy Plant Mini Kit, Quiagen®. The total DNA concentration was quantified using the Qubit™ Fluorometric Quantitation, Thermo Fisher Scientific®. The DNA libraries were created using Nextera DNA Library Preparation Kit, the purified DNA was sequenced on an Illumina NextSeq. 500  and the sequencing was executed by Instituto Gulbenkian de Ciência at the Genomics Facility in Oeiras, Portugal. Genome assembly procedures were done as we have previously described for other genomic studies80. We used QUAST81 to calculate the quality statistics of the genome assemblies. Annotation of the genome was done with PROKKA v1.1182 and RAST v2.083 using default parameters to identify putative genes (coding and non-coding sequences). Identification of orthologs proteins clusters was accomplished with eggNOG v4.584 and the identification of CRISPR repeats, typical in Cyanobacteria, was performed with CRISPR Recognition Tool CRT v1.185, considering a minimum of 3 repeat units. The prediction of transmembrane topology and signal peptide sites was done using Phobius86. To evaluate the presence and possible origin of prophage sequences, identification and annotation of these sequences were performed using PHASTER87. The quantitative assessment and annotation completeness of the assembled genome was performed with BUSCO v388, by comparison with BUSCO’s orthologues database set for Cyanobacteria, odb9. We used the online tool antiSMASH v4.089 to identify the presence of non-ribosomal peptide synthase (NRPS), polyketide synthase (PKS), hybrid NRPS/PKS gene clusters and other domains of secondary metabolites produced by Cyanobacteria.The genome was assembled using SPAdes v3.6.10Microcoleus asticus sp. nov. IPMA8 in the cyanobacterial phylum a phylogenetic tree of the 16S rRNA gene sequences of the 118 strains from a public dataset of cyanobacterial 16S rRNA genes collected in CyanoType v.1 database90. This is a curated database of 16S rRNA gene sequences from 317 relevant cyanobacterial strains, which aided to our initial identification of kin strains of Microcoleus asticus sp. nov. IPMA8, for which we used the compressed set of 118 representative cyanobacterial strains. 16S rRNA gene sequences were separately aligned with MAFFT91 and trimmed using Gblocks v0.91b93. ML phylogeny values were calculated with IQTree94, using the best-fit model of substitution proposed by this algorithm, for 1.000 bootstraps under GTR + I + G model. Subsequently, to refine the phylogenetic relations of our strain and validate this gene tree, we calculated a phylogenetic tree of 12 Oscillatoriales strains using a set of 12 fully sequenced genomes, which was built from the concatenation of 64 gene sequences common to all 12 genomes  using the online tool http://ggdc.dsmz.de/faq.php#qggdc2439.Genome similarity analysis were done by average nucleotide identity (ANI) using the online tool Cylindrospermum stagnale PCC 7417, with a cut-off value of 50% identity on similarity. We collected complete and partial sequences of geosmin synthase sequences from available databases  from 66 representative bacterial strains from four distinct phyla. Several putative gene sequences from the three bacterial phyla were identified, which including 36 cyanobacterial gene sequences, three Gammaproteobacteria, 14 Deltaproteobacteria and 13 Actinobacteria strains covering all identified genus and strains of known geosmin producers and non-producers. Gene sequences were aligned with MAFFT and trimmed with Gblocks resulting in an alignment of 273 amino acid positions, which included the N-terminal part of gene. This alignment was used to calculate the phylogenetic tree for geosmin synthase gene and a sesquiterpene cyclase (CAM04173.1) was used as outgroup98. To infer ML phylogenies, we used IQTree to calculate and support the trees for 1.000 bootstraps under LG + I + G4 model, the model proposed by this algorithm. To corroborate this tree, Bayesian phylogenies were calculated with MrBayes v3.2 for 100.000 generations using the same model and a discarded burn-in rate of 25% of the initial generations.To interrogate the phylogenetic relationships of the geosmin synthase genes in Bacteria kingdom we inferred a gene tree with geosmin synthase genes available on Genbank (accessed on Oct. 2018). Candidate geosmin synthase genes were identified by BLAST comparison with a reference geosmin synthase gene from 100 and the graphical representation was done with the online tool Evolview102.To evaluate sequence similarities between the geosmin synthase genes, in our set of 66 Bacteria strains, we compared the calculated geosmin gene tree described in the previous section, with a heatmap representing pairwise sequence similarities values between all the gene sequences. This percent identity matrix was calculated, with the amino acid trimmed alignment, using CLUSTAL OMEGA online tool103. The detection possible recombination was performed with Genetic Algorithm for Recombination Detection (GARD)104, a method that identifies recombination, and can be used as a pre-analysis to the inference selection tests performed afterwards.To detect adaptive evolution in the bacterial geosmin gene, the rates of synonymous and non-synonymous substitutions were estimated using algorithms in the Datamonkey.org web-server105.We used the protein alignment prepared for the geosmin gene distribution analysis in the 3 bacterial phyla which we back translated to DNA, as suggested by the software’s documentation, using EMBOSS Backtranseq V6.6.0 online tool103 algorithms SLAC (Single-Likelihood Ancestor Counting) and FUBAR (Fast Unconstrained Bayesian AppRoximation). Finally, MEME (Mixed Effects Model of Evolution) performed a test for the identification of individual sites also subject to positive or purifying selection. The global ratio of non-synonymous nucleotide substitutions (dN) to synonymous nucleotide substitutions (dS) was employed as an indicator of positive or negative selection pressure. Diversifying or positive selection dominates when the value of dN/dS is >1, dN/dS >1 implies purifying or negative selection while dN/dS = 1 is suggestive of neutral selection pressure.To detect the possibility of adaptive (positive) or purifying (negative) selection selection in the geosmin gene in Bacteria, we used Datamonkey web-server−1 and maintained for 8 min, then was increased to 230 °C at 25 °C min−1 and maintained for 1 min.; carrier gas (He) 2.00 mL.min−1, interface and ion source temperature in MS were at 250 °C. Mass spectra were acquired in Electron Ionization (EI) mode at 70 eV. in a m/z range between 29–299 with a scan speed of 555 scans s−1. Geosmin in samples was detected in Single Ion Monitoring (SIM) mode using characteristic ions m/z 111, 112, 125 and 126. The geosmin solution  was used as standards for GC-MS analysis. Dilution series from 100 µg ml−1 to 5 ng ml−1 were prepared for the geosmin standard to test the response and sensitivity of the GC-MS method. Geosmin was identified using the mass spectra libraries, NIST 21, 27, 107, 147 and Wiley 229.The Cyanobacteria culture was tested for geosmin production using SPME GC-MS at iBET - Instituto de Biologia Experimental e Tecnológica by the Food and Health Division Laboratory . Solid phase microextraction (SPME) was used for the extraction of volatile compounds from Cyanobacteria cultures. Briefly, 2.5 mL of sample  were measured to a 10 mL headspace vial (La-Pha-Pack®) and were capped with a white PTFE silicone septum . The SPME operating conditions were: extraction temperature 40 °C for 40 min, rotating speed 250 rpm, agitator during 10 s, desorption time 5 min at 260 °C. Analysis were carried out in a GCMS-QP2010 Plus (Shimadzu®) equipped with an AOC-5000 autosampler (Shimadzu®). A divinylbenzene/Carboxen/polydimethylsiloxane (DVB/Car/PDMS) fiber  was used for headspace SPME sampling. For the analysis a capillary column ZB-5MSi  capillary column 30 m, 0.25 mm (IS), 0.25 µm (film thickness) was used. The working conditions were: injector temperature: 260 °C, injection mode: splitless, detector temperature: 250 °C. High-purity helium (≥99.999%) was used as the carrier gas, column oven temperature was kept at 50 °C for 3 min, increased to 180 °C at a rate of 8 °C minSupplementary Information.


Salivary microbiome composition can change following exposure to environmental toxicants, e.g., heavy metals. We hypothesized that levels of salivary nutrients and metals would correlate with salivary microbiome composition and be associated with dental decay. Here we assess the salivary concentrations of 5 essential minerals , 4 metals with some evidence of normal physiological function , and 12 with known toxicity , and their associations with salivary microbiome composition and dental decay in 61 children and adults. 16 metals were detected in 54% of participants; 8 were found in all. Marked differences in salivary bacterial taxa were associated with levels of antimony, arsenic, and mercury, after adjusting for multiple testing. Further, antimony levels were associated with the presence of decayed teeth. Thus, salivary metal levels, even at low concentrations, may impact oral health. However, toxicity is a concern for bacteria, as some metals—such as cadmium and copper—are bactericidal at certain concentrations3. Further, even essential metals can be toxic, depending on the dose . Some studies have assessed salivary metals, but the number of publications and the scope of metals assessed are limited. Lead8 and mercury9 have been of particular interest, but there are some reports on salivary iron, magnesium, and zinc10.When certain toxic metals, e.g., lead or cadmium, are measured in blood or urine the resulting levels can be compared to guidelines for concentrations of concernTwo major gaps in the literature motivated us to quantify salivary concentrations of 21 metals among 61 healthy individuals, to describe associations between salivary essential mineral and metal levels with composition of the salivary microbiome, and describe the associations between metal levels and dental decay. First, lack of information on how metals affect the oral bacterial community, and second, the lack of a comprehensive study of multiple metals in saliva and their impact on the oral microbiome, and third, limited studies of the associations of metal levels and the presence of dental decay.The 61 participants included children (21% aged 7–17) and adults (79% aged 18–45) from 37 families . The average age was 26.8 years (SD: 9.39 years); 60% were female; 43% were from West Virginia and the remainder from Pennsylvania in the United States. About one-quarter (24.6%) of participants had an annual income less than $10,000, and 38.6% earned between $10,000 and $35,000 per year. A history of dental decay was common: 98% of adults and 69% of children had one or more decayed, missing or filled teeth, and 75% of adults and 31% of children had an untreated dental lesion.The minimum limit of detection (LOD) for most salivary minerals and metals was between 0.1 and 0.5 parts per billion (ppb), with zinc having the highest LOD at 30 ppb. Table We observed differences in salivary levels for some metals by age and income but none by sex. Using the Wilcoxon rank-sum test, there was a significantly higher median level of cesium among adults (0.55 ppb) than among children (0.34 ppb) (p = 0.02), but no other metals. Levels of copper were higher among those with incomes in the $10,000–35,000 range compared to those with < $10,000 or those > $35,000  , there were no significant differences in alpha diversity of the oral microbiome and tertile of metal exposure ]11. After Benjamini–Hochberg correction, the relative abundance of one or more bacterial taxa varied by exposure level for three metals: antimony, arsenic, and mercury .The relative abundance of 66% of the taxa differed by age using the ALDEX2 method  analysis allowed us to detect these metals at very low concentrations, and detection was observed in most individuals. We found novel associations between high levels of three metals  and the relative abundance of selected bacteria in the saliva, and between antimony and presence of an untreated dental lesion. Antimony is commonly found in flame- retardant materials, paint, enamels, glass and pottery. It is also an alloy used in the production of batteries and bullets. Although dietary exposure to antimony is low, it may be found in drinking water 10. Our mean salivary lead concentrations were also somewhat higher than that reported in a low-income population of 969 Michigan African Americans where the mean salivary lead levels were 2.4 ± 0.13 ppb, respectively7 compared to our overall mean of 14.94 ± 38.5 ppb. These differences might reflect difference in collection—the Polish and Michigan studies used unstimulated saliva collected following a rinse with sterile water and we used stimulated saliva, or differences in exposure by study population. Our study contributes to an emerging field of salivary metals exposure assessment by providing consistent measurement ranges to previously documented metals and benchmarking ranges for newly measured metals.The salivary metal concentrations we observed for lead, copper, manganese, zinc, cadmium, and mercury are generally consistent with those reported in previous studies. A study among 29 healthy Polish adults who did not smoke, use dietary supplements or have known metal exposure, used plasma induction for salivary metal detection of some of the same metals13. This is potentially relevant as the oral and gut microbiomes are predictive of each other14. However, our sample size was too small to evaluate this association.Our observed associations between microbiome taxa and arsenic and mercury probably are due to their known toxicity at low doses and in multiple systems. Further, mercury is used in dental amalgams, so we might expect those with multiple fillings to be more likely to have an untreated dental lesion. In a murine model, zinc deficiency and arsenic exposure individually resulted in changes in the diversity of the gut microbiome and together acted synergisticallyLactobacillus increased in relative abundance in the presence of antimony. The Lactobacilli are acid producers, and have long been associated with dental decay15. Additionally, Granulicatella—whose relative abundance has been negatively associated with dental caries16—decreased in abundance with higher levels of antimony. A search for microbiome studies of antimony found only 14 studies 30.This is the first report of an association between antimony with microbiome composition and an untreated dental lesion. The two findings together suggest that the association might be important, as several species of acid-producing genus 31, we might have gotten stronger signals if we had measured dental plaque microbiome which are directly involved in cariogenesis. While these limitations should be taken into account when generalizing to other populations, they do not invalidate our major findings that low levels of many metals are detectable in the saliva, and that these levels are associated with the relative abundance of bacteria present in the saliva that are in turn associated with diseases of the oral cavity.Findings from this study should be considered in light of limitations. A relatively small sample size limited our ability to simultaneously adjust for confounding effects of age, income and history of dental decay, that may be associated with metal exposure, oral microbiome composition and/or presence of an untreated dental lesion. Additionally, our methodology did not allow us to distinguish intracellular metals from those free-floating in saliva. Further, although the salivary microbiome overlaps with that of dental plaque33 serum for copper and zinc34, and urine for chronic exposure to arsenic, cadmium, chromium, or inorganic mercury38. For other metals, there is less of a consensus as to whether blood, serum, urine, hair, or nails serve as better biomarkers, and whether the exposure is chronic or acute also impacts interpretation of biomarker concentrations. Future studies should explore the broader application of easily-obtainable saliva as a biomarker by profiling salivary metal concentrations in larger populations with known sources of exposure and explore the associations with oral health.Saliva has the potential to serve as a minimally invasive biomarker of metal exposures, especially in studies focused on oral health outcomes. For example, in studies focused on the oral microbiome or dental health as the primary outcome of interest, quantifying metals in the saliva is advantageous as these are the concentrations directly in contact with the target site of toxicity. Furthermore, saliva collection may be more feasible than urine or blood collection for epidemiological studies recruiting in remote locations  or recruiting sensitive populations . In current exposure science, some biological specimens are considered better biomarkers of metal exposure than others, such as whole blood for leadEscherichia coli, can reproduce as frequently as every 20 min under optimal conditions39, and rapidly respond to environmental exposures of all kinds—including to metals. This property might be leveraged in the future to develop diagnostics or prognostics using saliva. Further, microbiota alter the body’s ability to absorb and metabolize environmental exposures40. Motivation for continued research on the salivary microbiome is supported by the results we observed, as well as a previous study describing an association between chronic arsenic exposure and an increase in dental caries amongst children41. Dental caries remains a major public health problem: in the US 12.4 million children and 37.6 million adults have untreated caries, with substantial disparities by race and income42. The ability to measure metals and microbes in a single sample is a great strength, as it eliminates the need to extrapolate using data from other collection sites.Bacteria present in the human microbiome, such as 43. Approval was obtained from the Institutional Review Boards at the University of Pittsburgh and West Virginia University, and all relevant guidelines and regulations were adhered to during the duration of the study. Informed consent was obtained from all adult participants upon enrollment. The consent of a legal parent or guardian was obtained for all individuals with less than 18 years of age. Below we describe details for saliva exposure and microbial biosampling that occurred between 2006 and 2009, in a previous study led by co-author Dr. M.L. Marazita44.The Center for Oral Health Research in Appalachia (COHRA) is an oral health initiative in the Appalachian region of Pennsylvania and West Virginia, focused on studying oral health disparities. The COHRA1 dataset includes families with at least one biological child < 18 years of age were recruited from four counties deemed representative of rural Appalachia. In person interviews were used to obtain demographic characteristics, and qualified dentists or hygienists conducted a dental examination, physical evaluation, and microbiological and salivary sampling. The full collection protocol for this cross-sectional study was published previously44. Samples were stored in a cryogenic tube without preservative. Samples were frozen at − 80 °C until testing. DNA was isolated and sequenced in 2012. This pilot study used a subset of the saliva samples from a previous COHRA I publication44 that characterized the salivary microbiome among 173 individuals. These participants came from 49 different families, in which at least two children and at least one adult provided a saliva sample43. For this analysis we used all samples from individuals > 6 years of age, where there was sufficient stimulated saliva remaining for testing. This gave us a final sample size of 61 individuals.At the dental assessment by calibrated dental professionals, stimulated saliva was collected by having participants chew on a paraffin pellet for one minute. Participants were instructed not to eat, drink, or brush teeth at least two hours before screening and assessments were conducted about two hours into the interview. For this analysis, our outcome was the presence of one or more unfilled carious lesions44. Briefly, DNA extraction of saliva samples was completed using DNeasy Blood and Tissue kits and the automated QIAcube system . To lyse microbial cell walls, prior to DNA extraction, 80 μL of an enzyme cocktail comprised of Promega cell lysis solution , lysozyme, mutanolysin, RNase A, and lysostaphin  in 22.5:4.5:1.25:1.25:1 parts, respectively, was added to 100 μL of sample before incubation at 37 °C for 30 min. Extraction was completed using manufacturer’s instructions. DNA concentration was assessed using a Nanodrop 2000C spectrophotometer  post extraction.Microbial detection was described previously45. 3 ng of barcoded DNA from each sample was pooled into a single 1.5 mL Eppendorf tube. 140 ng of an in-house barcoded mock community consisting of 57 species with an overrepresentation of genera common and uncommon to a healthy oral microbiome were also included in the pool. Species included in the mock community were Lactobacillus spp. Peptoniphilus spp., and Propionibacterium spp. The University of Michigan DNA Sequencing Core performed sequencing of the pooled DNA using the Illumina HiSeq Platform.Thermal amplification of the V6 region of the 16 s rRNA gene was performed using eight base pair error-correcting barcodes with conserved primers using a previously published protocol46. Reads were then demultiplexed and filtered using Quantitative Insights Into Microbial Ecology (QIIME) 1.9.047. The combined final read count after filtering and demultiplexing was 178,682,752, with a range of 2,745–1,726,317 reads. The read count per sample range for our subset was 139,279–1,006,268 reads.Paired end sequences were joined using fast length adjustment of short reads (FLASH)48. We clustered our sequences into oligotypes using an iterative unsupervised Minimum Entropy Decomposition algorithm using Shannon entropy values49. Taxonomy for the oligotypes was assigned using the CORE database, specifically curated for the identification of bacteria found in the oral cavity50.The QIIME data was prepared for oligotyping using the publicly available python script q2oligo.pyIn 2018, NSF International  assessed the stored stimulated saliva samples from 61 COHRA participants to quantify concentrations of 21 metals using inductively coupled plasma mass spectrometry on a Thermo Fisher  ICAPRQ spectrometer with an CETAC ASX-520 auto sampler. The protocol was adapted from a validated urinary analysis method 56. No significant outliers were detected using this method.Statistical analyses were performed using R statistical software (version 3.4.0)57. The diversity analysis was also conducted using a rarified dataset with 100 trials for comparison. Individuals in the first and second tertiles were combined to form a “low exposure” group. We used simple descriptive statistics  to describe the distributions overall and by age. Because the distributions were highly skewed, we tested for differences in metal distribution by age and gender using the Wilcoxon Rank Sum test. Differences by personal income were tested using Kruskal–Wallis tests. For adults with missing income information and children we used the highest reported annual income from a family member in the same household when available.As there are no published standards for salivary metal concentrations considered clinically relevant, we categorized participants by tertiles of each metal. Alpha diversity for each tertile was assessed using the Shannon index and Chao1 with the Phyloseq package in R11. For each species, ALDEx2 inferred absolute abundance given the observed abundance matrix using 1,000 Monte-Carlo simulations from a Dirichlet distribution to account for the fact that the observed abundance matrix is a single realization or sample from the oral bacterial communities of participants. The effect size for each species is the difference between groups relative to an estimate of within-group dispersion58. Statistical significance of the observed effect size between each metal and the relative abundance of each taxa was summarized as a p-value corrected for the false discovery rate using the Benjamini–Hochberg procedure. For simplicity, we summarize p-values associated with ALDEx2 tests and visualize statistically differences in species using association plots of species relative abundance. The significant associations were summarized in a table. ALDEx2 does not allow adjustment for covariates. Therefore, select taxa identified in the ALDEx2 analysis and additional taxa previously reported as associated with dental decay59 were further analyzed using linear mixed models. Separate models were fit using the lme4 R package60 to predict the relative abundance of the center-log ratio transformed relative abundance of each taxa by metal level, and included a random effect for family clustering.For all metals where > 30% of participants had a value above the LOD, we next compared species abundance by tertile of metal exposure status using ALDEx2, an analysis of variance-like tool for compositional data52. Due to the low percentage of detection with beryllium, thallium, uranium, and platinum, we compared the top 33% to the lower 66% for these metals. All models were adjusted for age as a continuous variable.We explored the relationship between salivary metal exposure  and presence of an untreated dental lesion using odds ratios from logistic regression models in SAS 9.4Supplementary file 1