Effects of feeding sulfate trace minerals above recommendations on nutrient digestibility, rumen fermentation, lactational performance, and trace mineral excretion in dairy cows.

Most trace minerals (TM) are fed above dairy cow requirements in commercial herds but their fate and effects on dairy cows have not been well documented. In this study, we evaluated the effects of feeding short-term sulfate TM above recommendations on apparent total-tract digestibility of nutrients, rumen fermentation characteristics, serum concentrations, milk yield and composition as well as milk, fecal, and urinary TM excretion in mid-lactation dairy cows. Eight multiparous Holstein cows [average body weight: 684 (SD: 29) kg at 82 (SD: 10) days in milk] in a quadruple 2 × 2 crossover design were fed a basal diet, differing in sulfate TM supplement concentrations, to provide either 0.11, 17, and 63 (control; CON) or 0.95, 114, and 123 (high trace minerals; HTM) mg of dietary Co, Mn, and Zn/kg of dry matter, respectively. Each experimental period had a 21-d adaptation to the diet, followed by a 10-d sample collection period. Feed ingredients and total feces and urine were collected during 4 consecutive d and rumen fluid was collected 0, 1, 2, 4, and 6 h relative to feeding. Milk yield was recorded daily and milk samples were collected on 4 consecutive milkings. Ingestion of Co, Mn, and Zn was higher for HTM compared with CON group by 216, 233, and 93%, respectively. Dry matter intake averaged 25.0 (SE = 0.6) kg/d, and apparent total-tract digestibility of major nutrients was similar between treatments. There was no measurable effect of HTM on ruminal pH, major volatile fatty acids, and protozoa counts. Isovalerate molar proportion was 9.4% greater for HTM compared with CON group. Neither milk yield (43.5 kg/d; SE = 0.8) nor milk fat and protein concentrations differed between treatments. Milk urea nitrogen concentration was significantly higher for HTM (11.7 mg/dL) compared with CON group (9.7 mg/dL; SE = 0.7). Fecal excretion of Co, Mn, and Zn increased by 223, 198, and 75%, respectively, for HTM compared with CON group. Urinary excretions of TM were marginal compared with feces, and only urinary Co and Mn were significantly higher for HTM than CON cows as similarly obtained for serum Co and Mn concentrations. Milk TM yields were not modified by treatments. In summary, short-term dietary sulfate TM supply over the recommendation did not improve cow performance but significantly increased fecal TM excretion, which could have impacts on TM accumulation in soils where manure is applied and could potentially result in leaching into nearby watersheds. Further studies are needed to evaluate the impact of high fecal TM excretion on the environment using the One Health approach. Moreover, the impacts of TM oversupply on milk production and cow health should be evaluated by long-term experiments.

Deep Cyanobacteria Layers: An Overlooked Aspect of Managing Risks of Cyanobacteria.

The risk of human exposure to cyanotoxins is partially influenced by the location of toxin-producing cyanobacteria in waterbodies. Cyanotoxin production can occur throughout the water column, with deep water production representing a potential public health concern, specifically for drinking water supplies. Deep cyanobacteria layers are often unreported, and it remains to be seen if lower incident rates reflect an uncommon phenomenon or a monitoring bias. Here, we examine Sunfish Lake, Ontario, Canada as a case study lake with a known deep cyanobacteria layer. Cyanotoxin and other bioactive metabolite screening revealed that the deep cyanobacteria layer was toxigenic [0.03 µg L-1 microcystins (max) and 2.5 µg L-1 anabaenopeptins (max)]. The deep layer was predominantly composed of Planktothrix isothrix (exhibiting a lower cyanotoxin cell quota), with Planktothrix rubescens (exhibiting a higher cyanotoxin cell quota) found at background levels. The co-occurrence of multiple toxigenic Planktothrix species underscores the importance of routine surveillance for prompt identification leading to early intervention. For instance, microcystin concentrations in Sunfish Lake are currently below national drinking water thresholds, but shifting environmental conditions (e.g., in response to climate change or nutrient modification) could fashion an environment favoring P. rubescens, creating a scenario of greater cyanotoxin production. Future work should monitor the entire water column to help build predictive capacities for identifying waterbodies at elevated risk of developing deep cyanobacteria layers to safeguard drinking water supplies.

Microcystin concentrations and congener composition in relation to environmental variables across 440 north-temperate and boreal lakes.

Understanding the environmental conditions and taxa that promote the occurrence of cyanobacterial toxins is imperative for effective management of lake ecosystems. Herein, we modeled total microcystin presence and concentrations with a broad suite of environmental predictors and cyanobacteria community data collected across 440 Canadian lakes using standardized methods. We also conducted a focused analysis targeting 14 microcystin congeners across 190 lakes, to examine how abiotic and biotic factors influence their relative proportions. Microcystins were detected in 30 % of lakes, with the highest total concentrations occurring in the most eutrophic lakes located in ecozones of central Canada. The two most commonly detected congeners were MC-LR (61 % of lakes) and MC-LA (37 % of lakes), while 11 others were detected more sporadically across waterbodies. Congener diversity peaked in central Canada where cyanobacteria biomass was highest. Using a zero-altered hurdle model, the probability of detecting microcystin was best explained by increasing Microcystis biomass, Daphnia and cyclopoid biomass, soluble reactive phosphorus, pH and wind. Microcystin concentrations increased with the biomass of Microcystis and other less dominant cyanobacteria taxa, as well as total phosphorus, cyclopoid copepod biomass, dissolved inorganic carbon and water temperature. Collectively, these models accounted for 34 % and 70 % of the variability, respectively. Based on a multiple factor analysis of microcystin congeners, cyanobacteria community data, environmental and zooplankton data, we found that the relative abundance of most congeners varied according to trophic state and were related to a combination of cyanobacteria genera biomasses and environmental variables.

Spatio-temporal connectivity of the aquatic microbiome associated with cyanobacterial blooms along a Great Lake riverine-lacustrine continuum.

Lake Erie is subject to recurring events of cyanobacterial harmful algal blooms (cHABs), but measures of nutrients and total phytoplankton biomass seem to be poor predictors of cHABs when taken individually. A more integrated approach at the watershed scale may improve our understanding of the conditions that lead to bloom formation, such as assessing the physico-chemical and biological factors that influence the lake microbial community, as well as identifying the linkages between Lake Erie and the surrounding watershed. Within the scope of the Government of Canada's Genomics Research and Development Initiative (GRDI) Ecobiomics project, we used high-throughput sequencing of the 16S rRNA gene to characterize the spatio-temporal variability of the aquatic microbiome in the Thames River-Lake St. Clair-Detroit River-Lake Erie aquatic corridor. We found that the aquatic microbiome was structured along the flow path and influenced mainly by higher nutrient concentrations in the Thames River, and higher temperature and pH downstream in Lake St. Clair and Lake Erie. The same dominant bacterial phyla were detected along the water continuum, changing only in relative abundance. At finer taxonomical level, however, there was a clear shift in the cyanobacterial community, with Planktothrix dominating in the Thames River and Microcystis and Synechococcus in Lake St. Clair and Lake Erie. Mantel correlations highlighted the importance of geographic distance in shaping the microbial community structure. The fact that a high proportion of microbial sequences found in the Western Basin of Lake Erie were also identified in the Thames River, indicated a high degree of connectivity and dispersal within the system, where mass effect induced by passive transport play an important role in microbial community assembly. Nevertheless, some cyanobacterial amplicon sequence variants (ASVs) related to Microcystis, representing less than 0.1% of relative abundance in the upstream Thames River, became dominant in Lake St. Clair and Erie, suggesting selection of those ASVs based on the lake conditions. Their extremely low relative abundances in the Thames suggest additional sources are likely to contribute to the rapid development of summer and fall blooms in the Western Basin of Lake Erie. Collectively, these results, which can be applied to other watersheds, improve our understanding of the factors influencing aquatic microbial community assembly and provide new perspectives on how to better understand the occurrence of cHABs in Lake Erie and elsewhere.

Metatranscriptomic Sequencing of Winter and Spring Planktonic Communities from Lake Erie, a Laurentian Great Lake.

Previous reports suggest planktonic and under-ice winter microbial communities in Lake Erie are dominated by diatoms. Here, we report the assembled metatranscriptomes of 79 Lake Erie surface water microbial communities spanning both the winter (28 samples) and spring (51 samples) months over spatial, temporal, and climatic gradients in 2019 through 2020.

Toxins and Other Bioactive Metabolites in Deep Chlorophyll Layers Containing the Cyanobacteria Planktothrix cf. isothrix in Two Georgian Bay Embayments, Lake Huron.

The understanding of deep chlorophyll layers (DCLs) in the Great Lakes-largely reported as a mix of picoplankton and mixotrophic nanoflagellates-is predominantly based on studies of deep (>30 m), offshore locations. Here, we document and characterize nearshore DCLs from two meso-oligotrophic embayments, Twelve Mile Bay (TMB) and South Bay (SB), along eastern Georgian Bay, Lake Huron (Ontario, Canada) in 2014, 2015, and 2018. Both embayments showed the annual formation of DCLs, present as dense, thin, metalimnetic plates dominated by the large, potentially toxic, and bloom-forming cyanobacteria Planktothrix cf. isothrix. The contribution of P. cf. isothrix to the deep-living total biomass (TB) increased as thermal stratification progressed over the ice-free season, reaching 40% in TMB (0.6 mg/L at 9.5 m) and 65% in South Bay (3.5 mg/L at 7.5 m) in 2015. The euphotic zone in each embayment extended down past the mixed layer, into the nutrient-enriched hypoxic hypolimnia, consistent with other studies of similar systems with DCLs. The co-occurrence of the metal-oxidizing bacteria Leptothrix spp. and bactivorous flagellates within the metalimnetic DCLs suggests that the microbial loop plays an important role in recycling nutrients within these layers, particularly phosphate (PO4) and iron (Fe). Samples taken through the water column in both embayments showed measurable concentrations of the cyanobacterial toxins microcystins (max. 0.4 µg/L) and the other bioactive metabolites anabaenopeptins (max. ~7 µg/L) and cyanopeptolins (max. 1 ng/L), along with the corresponding genes (max. in 2018). These oligopeptides are known to act as metabolic inhibitors (e.g., in chemical defence against grazers, parasites) and allow a competitive advantage. In TMB, the 2018 peaks in these oligopeptides and genes coincided with the P. cf. isothrix DCLs, suggesting this species as the main source. Our data indicate that intersecting physicochemical gradients of light and nutrient-enriched hypoxic hypolimnia are key factors in supporting DCLs in TMB and SB. Microbial activity and allelopathy may also influence DCL community structure and function, and require further investigation, particularly related to the dominance of potentially toxigenic species such as P. cf. isothrix.

The Lake Erie HABs Grab: A binational collaboration to characterize the western basin cyanobacterial harmful algal blooms at an unprecedented high-resolution spatial scale.

Monitoring of cyanobacterial bloom biomass in large lakes at high resolution is made possible by remote sensing. However, monitoring cyanobacterial toxins is only feasible with grab samples, which, with only sporadic sampling, results in uncertainties in the spatial distribution of toxins. To address this issue, we conducted two intensive "HABs Grabs" of microcystin (MC)-producing Microcystis blooms in the western basin of Lake Erie. These were one-day sampling events during August of 2018 and 2019 in which 100 and 172 grab samples were collected, respectively, within a six-hour window covering up to 2,270 km2 and analyzed using consistent methods to estimate the total mass of MC. The samples were analyzed for 57 parameters, including toxins, nutrients, chlorophyll, and genomics. There were an estimated 11,513 kg and 30,691 kg of MCs in the western basin during the 2018 and 2019 HABs Grabs, respectively. The bloom boundary poses substantial issues for spatial assessments because MC concentration varied by nearly two orders of magnitude over very short distances. The MC to chlorophyll ratio (MC:chl) varied by a factor up to 5.3 throughout the basin, which creates challenges for using MC:chl to predict MC concentrations. Many of the biomass metrics strongly correlated (r > 0.70) with each other except chlorophyll fluorescence and phycocyanin concentration. While MC and chlorophyll correlated well with total phosphorus and nitrogen concentrations, MC:chl correlated with dissolved inorganic nitrogen. More frequent MC data collection can overcome these issues, and models need to account for the MC:chl spatial heterogeneity when forecasting MCs.

Toxic benthic freshwater cyanobacterial proliferations: Challenges and solutions for enhancing knowledge and improving monitoring and mitigation.

1. This review summarises knowledge on the ecology, toxin production, and impacts of toxic freshwater benthic cyanobacterial proliferations. It documents monitoring, management, and sampling strategies, and explores mitigation options. 2. Toxic proliferations of freshwater benthic cyanobacteria (taxa that grow attached to substrates) occur in streams, rivers, lakes, and thermal and meltwater ponds, and have been reported in 19 countries. Anatoxin- and microcystin-containing mats are most commonly reported (eight and 10 countries, respectively). 3. Studies exploring factors that promote toxic benthic cyanobacterial proliferations are limited to a few species and habitats. There is a hierarchy of importance in environmental and biological factors that regulate proliferations with variables such as flow (rivers), fine sediment deposition, nutrients, associated microbes, and grazing identified as key drivers. Regulating factors differ among colonisation, expansion, and dispersal phases. 4. New -omics-based approaches are providing novel insights into the physiological attributes of benthic cyanobacteria and the role of associated microorganisms in facilitating their proliferation. 5. Proliferations are commonly comprised of both toxic and non-toxic strains, and the relative proportion of these is the key factor contributing to the overall toxin content of each mat. 6. While these events are becoming more commonly reported globally, we currently lack standardised approaches to detect, monitor, and manage this emerging health issue. To solve these critical gaps, global collaborations are needed to facilitate the rapid transfer of knowledge and promote the development of standardised techniques that can be applied to diverse habitats and species, and ultimately lead to improved management.

Picoplankton accumulate and recycle polyphosphate to support high primary productivity in coastal Lake Ontario.

Phytoplankton can accumulate polyphosphate (polyP) to alleviate limitation of essential nutrient phosphorus (P). Yet polyP metabolisms in aquatic systems and their roles in P biogeochemical cycle remain elusive. Previously reported polyP enrichment in low-phosphorus oligotrophic marine waters contradicts the common view of polyP as a luxury P-storage molecule. Here, we show that in a P-rich eutrophic bay of Lake Ontario, planktonic polyP is controlled by multiple mechanisms and responds strongly to seasonal variations. Plankton accumulate polyP as P storage under high-P conditions via luxury uptake and use it under acute P stress. Low phosphorus also triggers enrichment of polyP that can be preferentially recycled to attenuate P lost. We discover that picoplankton, despite their low production rates, are responsible for the dynamic polyP metabolisms. Picoplankton store and liberate polyP to support the high primary productivity of blooming algae. PolyP mechanisms enable efficient P recycling on ecosystem and even larger scales.

Meteorological and Nutrient Conditions Influence Microcystin Congeners in Freshwaters.

Cyanobacterial blooms increasingly impair inland waters, with the potential for a concurrent increase in cyanotoxins that have been linked to animal and human mortalities. Microcystins (MCs) are among the most commonly detected cyanotoxins, but little is known about the distribution of different MC congeners despite large differences in their biomagnification, persistence, and toxicity. Using raw-water intake data from sites around the Great Lakes basin, we applied multivariate canonical analyses and regression tree analyses to identify how different congeners (MC-LA, -LR, -RR, and -YR) varied with changes in meteorological and nutrient conditions over time (10 years) and space (longitude range: 77°2'60 to 94°29'23 W). We found that MC-LR was associated with strong winds, warm temperatures, and nutrient-rich conditions, whereas the equally toxic yet less commonly studied MC-LA tended to dominate under intermediate winds, wetter, and nutrient-poor conditions. A global synthesis of lake data in the peer-reviewed literature showed that the composition of MC congeners differs among regions, with MC-LA more commonly reported in North America than Europe. Global patterns of MC congeners tended to vary with lake nutrient conditions and lake morphometry. Ultimately, knowledge of the environmental factors leading to the formation of different MC congeners in freshwaters is necessary to assess the duration and degree of toxin exposure under future global change.

Reduction of industrial iron pollution promotes phosphorus internal loading in eutrophic Hamilton Harbour, Lake Ontario, Canada.

Diagenetic sediment phosphorus (P) recycling is a widespread phenomenon, which causes degradation of water quality and promotes harmful algal blooms in lakes worldwide. Strong P coupling with iron (Fe) in some lakes is thought to inhibit diagenetic P efflux, despite elevated P concentrations in the sediment. In these sediments, the high Fe content leads to P scavenging on ferric Fe near the sediment surface, which increases the overall P retention. Reduced external Fe inputs in such lakes due to industrial pollution control may lead to unintended consequences for sediment P retention. Here, we study sediment geochemistry and sediment-water interactions in the historically polluted Hamilton Harbour (Lake Ontario, Canada) which has undergone 30 years of restoration efforts. We investigate processes controlling diagenetic P recycling, which has previously been considered minor due to historically high Fe loading. Our results demonstrate that present sediment P release is substantial, despite sediment Fe content reaching 6.5% (dry weight). We conclude that the recent improvement of wastewater treatment and industrial waste management practices has reduced Fe pollution, causing a decrease in diagenetically reactive Fe phases, resulting in the reduction of the ratio of redox-sensitive P and Fe, and the suppression of P scavenging on Fe oxyhydroxides.

A Bayesian risk assessment framework for microcystin violations of drinking water and recreational standards in the Bay of Quinte, Lake Ontario, Canada.

Freshwater ecosystems can experience harmful algal blooms, which negatively impact recreational uses, aesthetics, taste, and odor in drinking water. Cyanobacterial toxins can have dire repercussions on aquatic wildlife and human health, and the most ubiquitous worldwide are the hepatotoxic compounds known as microcystins. The factors that influence the occurrence and magnitude of cyanobacteria blooms and toxin production vary in space and time and remain poorly understood. It is within this context that we present a suite of statistical models, parameterized with Bayesian inference techniques, to link the retrospective analysis of important environmental factors with the probability of exceedance of threshold microcystin levels. Our modelling framework is applied to the Bay of Quinte, Lake Ontario, Canada; a system with a long history of eutrophication problems. Collectively, 16.1% of the samples of the system collected during the study period (2003-2016) exceeded the drinking water guideline of 1.5 µgL-1, while approximately 3% of recorded values exceeded the recommended recreational threshold of 20 µgL-1. Using a segmented regression model with a stochastic breakpoint of microcystin concentrations estimated at 0.54 µg L-1, we demonstrate that the environmental conditions associated with increased probability of exceedance of the drinking water standard are chlorophyll a concentration ≥7 µg L-1, water temperature ≥20 °C, ammonium concentration ≤40 µgL-1, total phosphorus concentration ≥25 µg L-1, and wind speed ≤37 km h-1. Considering the multitude of factors that can influence the ambient levels of toxins, our study argues that the adoption of probabilistic water quality criteria offers a pragmatic approach to accommodate the associated uncertainty by permitting a realistic frequency of violations. In this context, we present a framework to evaluate the confidence of compliance with probabilistic standards that stipulate less than 10% violations of microcystin threshold ambient levels.

Algal bloom response and risk management: On-site response tools.

Harmful algal blooms caused by cyanobacteria can present a risk to the safety of drinking- and recreational waters and beachfronts through the production of toxins, particularly microcystin, which are highly resilient to degradation. These blooms are difficult to predict, vary in appearance and toxicity, and can show significant spatial heterogeneity: wind- and current-borne scums can produce an order of magnitude range in toxin levels along shorelines. The growing demand for reliable, cost-effective and rapid methods to detect toxins in bloom material and reduce the risk of public exposure cannot be met by most analytical lab turnaround times. Commercial microcystin test kits are now available, but few have been rigorously field-tested or incorporated into monitoring programmes. Working with a local health agency, we evaluated two kits with different operative ranges of detection, applied to samples covering a wide range of water quality, sample matrices, and bloom composition. We compared their performance against lab analyses using Enzyme-Linked Immunosorbent and Protein Phosphatase Inhibition assays. Both kits could resolve samples with high (<10 µg/L microcystin equivalents (MCequiv)) and low/no toxins, but failed to reliably detect toxin levels between 1 and 5 µg/L, at which threshold there were few false negatives (8%) but âˆ¼ one third of the samples (32%) yielded false positives. We conclude that these kits are potentially useful for screening and informed risk management decisions e.g. on beach closures, but should be followed up with more rigorous tests where needed. We describe how, based on these results, the kits have been successfully incorporated into the routine municipal beach monitoring and advisory programme by the Hamilton Public Health Services (Ontario).

Analysis of intracellular and extracellular microcystin variants in sediments and pore waters by accelerated solvent extraction and high performance liquid chromatography-tandem mass spectrometry.

The fate and persistence of microcystin cyanotoxins in aquatic ecosystems remains poorly understood in part due to the lack of analytical methods for microcystins in sediments. Existing methods have been limited to the extraction of a few extracellular microcystins of similar chemistry. We developed a single analytical method, consisting of accelerated solvent extraction, hydrophilic-lipophilic balance solid phase extraction, and reversed phase high performance liquid chromatography-tandem mass spectrometry, suitable for the extraction and quantitation of both intracellular and extracellular cyanotoxins in sediments as well as pore waters. Recoveries of nine microcystins, representing the chemical diversity of microcystins, and nodularin (a marine analogue) ranged between 75 and 98% with one, microcystin-RR (MC-RR), at 50%. Chromatographic separation of these analytes was achieved within 7.5 min and the method detection limits were between 1.1 and 2.5 ng g(-1) dry weight (dw). The robustness of the method was demonstrated on sediment cores collected from seven Canadian lakes of diverse geography and trophic states. Individual microcystin variants reached a maximum concentration of 829 ng g(-1) dw on sediment particles and 132 ng mL(-1) in pore waters and could be detected in sediments as deep as 41 cm (>100 years in age). MC-LR, -RR, and -LA were more often detected while MC-YR, -LY, -LF, and -LW were less common. The analytical method enabled us to estimate sediment-pore water distribution coefficients (K(d)), MC-RR had the highest affinity for sediment particles (log K(d)=1.3) while MC-LA had the lowest affinity (log K(d)=-0.4), partitioning mainly into pore waters. Our findings confirm that sediments serve as a reservoir for microcystins but suggest that some variants may diffuse into overlying water thereby constituting a new route of exposure following the dissipation of toxic blooms. The method is well suited to determine the fate and persistence of different microcystins in aquatic systems.

Translational control of apolipoprotein B mRNA via insulin and the protein kinase C signaling cascades: evidence for modulation of RNA-protein interactions at the 5'UTR.

The link between hepatic insulin signaling and apolipoprotein B (apoB) production has important implications in understanding the etiology of metabolic dyslipidemia commonly observed in insulin-resistant states. Recent studies have revealed important translational mechanisms of apoB mRNA involving the 5' untranslated region (5'UTR) and insulin-mediated translational suppression via an insulin-sensitive RNA binding protein. Here, we have investigated the role of the protein kinase C (PKCs) signaling cascade in the regulation of apoB mRNA translation, using a series of chimeric apoB UTR-luciferase constructs, in vitro translation of UTR-luciferase cRNAs, and metabolic labeling of intact HepG2 cells. The PKC activator, phorbol 12-myristate 13-acetate (PMA), increased luciferase expression of constructs containing the apoB 5' UTR whereas treatment with Bis-I, a general PKC inhibitor or Go6976, a more specific PKC alpha/beta inhibitor, decreased expression, under both basal and insulin-treated conditions. These effects were confirmed to be translational in nature based on in vitro translation studies of T7 apoB UTR-luciferase constructs transcribed and translated in vitro in the presence of HepG2 cytosol treated with insulin or signaling modulators. Mobility shift experiments using cytosol treated with either PKC inhibitor (Bis-I) or activator (PMA) showed parallel changes between translation of apoB 5'UTR-luciferase constructs and the binding of a protein(s) complex migrating around 110 kDa to the apoB 5' UTR. ApoB mRNA levels were unaltered under these conditions based on real-time PCR analysis. Bis-I and Go6976 were both able to significantly decrease newly synthesized apoB100 protein in the presence or absence of insulin. Overall, the data suggests that PKC activation may induce increased mRNA translation and synthesis of apoB100 protein through a mechanism involving the interaction of trans-acting factors with the apoB 5'UTR. We postulate potential links between PKC activation as seen in insulin-resistant/diabetic states, enhanced translation of apoB mRNA, and hepatic VLDL-apoB overproduction.