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Dinoflagellates do not have a typical TATA-binding protein (TBP), a subunit of the general transcription factor TFIID complex. Instead, they have a TBP-like factor (TLF) that has been shown to bind TTTT instead of TATA in vitro. The ability of TLF to act as a functional replacement of TBP in vivo has never been assessed, however. Here, we show that a dinoflagellate TLF can drive expression of a reporter gene controlled by a budding yeast promoter whose TATA box was mutated to TTTT. TLF is thus able to bind and activate the yeast RNA polymerase and appear to function normally in the TFIID complex.

Carotid plaque vulnerability features beyond the degree of stenosis may play a key role in the pathogenesis and recurrence of ischemic cerebrovascular events. This study sought to compare intraplaque hemorrhage (IPH) as a marker of plaque vulnerability in symptomatic patients with mild ( We included patients who experienced ischemic cerebrovascular events with no other identifiable sources and underwent carotid endarterectomy for mild (n=32), moderate (n=47), and severe (n=58) carotid artery stenosis. The degree of stenosis and imaging hallmarks were assessed by computed tomography angiography or magnetic resonance angiography. Plaque specimens were stained with hematoxylin and eosin and Movat pentachrome staining. Carotid plaques of patients with mild stenosis had a higher extent of IPH (%) on tissue analysis compared with patients with moderate (mild, 15.7% [interquartile range, 7.8%–26.7%]; moderate, 3.9% [0.0%–9.2%]; P P P Our major finding is the association of IPH with mild carotid artery stenosis based on histological analysis. The current study may suggest that IPH potentially plays a role in the mechanism of stroke in patients with nonobstructive carotid stenosis.

Aims Circulating progenitor cells (CPCs) play a role in vascular repair and plaque stability, while osteocalcin (OC) expressing CPCs have been linked to unstable plaque and adverse cardiovascular outcomes. However, their role in cardiac allograft vasculopathy (CAV) has not been elucidated. This cohort study aimed to investigate the contribution of CPCs on CAV progression and cardiovascular events after heart transplantation. Methods and results A total of 80 heart transplant patients (mean age 55 ± 14 years, 72% male) undergoing annual intravascular ultrasound (IVUS) had fresh CPCs marked by CD34, CD133, and OC counted in peripheral blood using flow cytometry, on the same day as baseline IVUS. CAV progression was assessed by IVUS as the change (Δ) in plaque volume divided by segment length (PV/SL), adjusted for the time between IVUS measurements (median 3.0, interquartile range (IQR) [2.8, 3.1] years), and was defined as ΔPV/SL that is above the median ΔPV/SL of study population. Major adverse cardiac events (MACE) was defined as any incident of revascularization, myocardial infarction, heart failure admission, re-transplantation, stroke and death. Patients with higher CD34+CD133+ CPCs had a decreased risk of CAV progression (odds ratio 0.58, 95% confidence interval [CI] [0.37, 0.92], p = 0.01) and MACE (hazard ratio [HR] 0.79, 95% CI [0.66, 0.99], p = 0.05) during a median (IQR) follow up of 8.0 years (7.2, 8.3). Contrarily, higher OC+ cell counts were associated with an increased risk of MACE (HR 1.26, 95% CI [1.03, 1.57], p = 0.02). Conclusions Lower levels of CD34+CD133+ CPCs are associated with plaque progression and adverse long-term outcomes in patients who underwent allograft heart transplantation. In contrast, higher circulating OC+ levels are associated with adverse long term outcomes. Thus, CPCs might play a role in amelioration of transplant vasculopathy, while OC expression by these cells might play a role in progression. Translational perspective The results of the current study suggest lower levels of circulating CD34+CD133+ cell levels are associated with cardiac allograft vasculopathy progression and future adverse cardiovascular events, while higher OC+ cell levels are associated with a greater risk of future cardiovascular events. Further studies confirming our findings might elucidate the role of circulating progenitor cells in the pathophysiology of CAV. Moreover, our findings might support the use of circulating progenitors as biomarkers, as well as the notion of cell therapy as potential treatment option for CAV, a disease with severe burden and limited treatment options.

Lightning talk

Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.

Mechanical and bioprosthetic valves that are currently applied for replacing diseased heart valves are not fully efficient. Heart valve tissue engineering may solve the issues faced by the prosthetic valves in heart valve replacement. The leaflets of native heart valves have a trilayered structure with layer-specific orientations; thus, it is imperative to develop functional leaflet tissue constructs with a native trilayered, oriented structure. Its key solution is to develop leaflet scaffolds with a native morphology and structure. In this study, microfibrous leaflet scaffolds with a native trilayered and oriented structure were developed in an electrospinning system. The scaffolds were implanted for 3 months in rats subcutaneously to study the scaffold efficiencies in generating functional tissue-engineered leaflet constructs. These in vivo tissue-engineered leaflet constructs had a trilayered, oriented structure similar to native leaflets. The tensile properties of constructs indicated that they were able to endure the hydrodynamic load of the native heart valve. Collagen, glycosaminoglycans, and elastin─the predominant extracellular matrix components of native leaflets─were found sufficiently in the leaflet tissue constructs. The residing cells in the leaflet tissue constructs showed vimentin and α-smooth muscle actin expression, i.e., the constructs were in a growing state. Thus, the trilayered, oriented fibrous leaflet scaffolds produced in this study could be useful to develop heart valve scaffolds for successful heart valve replacements.

Decellularized pericardial tissue is a strong candidate for a TEHV material as ECM is present to guide cellular infiltration and fixed porcine and bovine pericardial tissue have existing use in bioprosthetic heart valves. In this work, we compare the mechanical and microstructural properties of decellularized-sterilized (DS) porcine, bovine, and bison pericardial tissues with respect to use as a TEHV. HE staining was used to verify removal of cellular content post-decellularization and to evaluate collagen fiber structure. Additionally, uniaxial and biaxial tension testing were used to compare mechanical performance and, for the latter, acquire constitutive model parameters for subsequent finite element (FE) modeling. HE staining revealed complete removal of cellular content and good collagen fiber structure. Tensile testing showed comparable mechanical strength between the three DS pericardial tissues and considerably stronger mechanical properties compared to native tissues. Bovine and bison DS pericardial tissues showed the strongest mechanical performance in the FE models with bison demonstrating the overall best mechanical characteristics. The increased thickness of bovine and bison tissues coupled with the strong mechanical behavior and ECM structure indicates that these materials will be resistant to damage until sufficient cellular infiltration has occurred such that damaged tissue can be repaired.

As is true for many other aspects, genome architecture, evolution, and function in dinoflagellates are enigmatic and, in the meantime, continuous inspiration for scientific quests. Recent third-generation sequencing and Hi-C linkage analyses brought new insights into the spatial organization of symbiodiniacean genomes, revealing the topologically associated domains, discrete gene clusters and their cis and trans orientations, and relationships with transcription. Where do these new findings bring us in dinoflagellate genomics? Here, we aim to place these new results in the backdrop of the long history of research on this topic and in the context of what critical questions remain to be pursued in the future. The new data suggest, pending verification of other complete chromosome assemblies, a potential evolutionary trend in chromosome number decrease and length increase within the Symbiodiniaceae. While questions remain about the mechanics of the three-dimensional chromosome structure and cell cycle-related DNA replication, the mechanisms of gene transcription and genome size evolution, these latest findings set new starting points for further inquiries.

Dinoflagellates are a vital diverse family of unicellular algae widespread in various aquatic environments. Typically large genomes and permanently condensed chromosomes without histones make these organisms unique among eukaryotes in terms of chromatin structure and gene expression. Genomic and transcriptomic sequencing projects have provided new insight into the genetic foundation of dinoflagellate behaviors. Genes in tandem arrays, trans-splicing of mRNAs and lower levels of transcriptional regulation compared to other eukaryotes all contribute to the differences seen. Here we present a general overview of transcription in dinoflagellates based on previously described work.

Arbuscular mycorrhizal fungi (AMF) are obligatory plant symbionts that live underground, so few studies have examined their response to light. Responses to blue light by other fungi can be mediated by White Collar-1 (WC-1) and WC-2 proteins. These wc genes, together with the frequency gene (frq), also form part of the endogenous circadian clock. The clock mechanism has never been studied in AMF, although circadian growth of their hyphae in the field has been reported. Using both genomic and transcriptomic data, we have found homologs of wc-1, wc-2, and frq and related circadian clock genes in the arbuscular mycorrhizal fungus Rhizoglomus irregulare (synonym Rhizophagus irregularis). Gene expression of wc-1, wc-2, and frq was analyzed using RT-qPCR on RNA extracted from germinating spores and from fungal material cultivated in vitro with transformed carrot roots. We found that all three core clock genes were expressed in both pre- and post-mycorrhizal stages of R. irregulare growth. Similar to the model fungus Neurospora crassa, the core circadian oscillator gene frq was induced by brief light stimulation. The presence of circadian clock and output genes in R. irregulare opens the door to the study of circadian clocks in the fungal partner of plant-AMF symbiosis. Our finding also provides new insight into the evolution of the circadian frq gene in fungi.

Fixed pericardial tissue is commonly used for commercially available xenograft valve implants, and has proven durability, but lacks the capability to remodel and grow. Decellularized porcine pericardial tissue has the promise to outperform fixed tissue and remodel, but the decellularization process has been shown to damage the collagen structure and reduce mechanical integrity of the tissue. Therefore, a comparison of uniaxial tensile properties was performed on decellularized, decellularized‐sterilized, fixed, and native porcine pericardial tissue versus native valve leaflet cusps. The results of non‐parametric analysis showed statistically significant differences (p .05). Scanning electron microscopy confirmed that valvular endothelial and interstitial cells colonized the decellularized pericardial surface when seeded and grown for 30 days in static culture. Collagen assays and transmission electron microscopy analysis showed limited reductions in collagen with processing; yet glycosaminoglycan assays showed great reductions in the processed pericardium relative to native cusps. Decellularized pericardium had comparatively low mechanical properties among the groups studied; yet the stiffness was comparatively similar to the native cusps and demonstrated a lack of cytotoxicity. Suture retention, accelerated wear, and hydrodynamic testing of prototype decellularized and decell‐sterilized valves showed positive functionality. Sterilized tissue could mimic valvular mechanical environment in vitro, therefore making it a viable potential candidate for off‐the‐shelf tissue‐engineered valvular applications.

Dinoflagellate chromatin is unique among eukaryotes, as the chromosomes are permanently condensed in a liquid crystal state instead of being packed in nucleosomes. However, how it is organized is still an unsolved mystery, in part due to the lack of a comprehensive catalog of dinoflagellate nuclear proteins. Here, we report the results of CHromatin Enrichment for Proteomics (CHEP) followed by shotgun mass spectrometry sequencing of the chromatin-associated proteins from the dinoflagellate Lingulodinum polyedra. Our analysis identified proteins involved in DNA replication and repair, transcription, and mRNA splicing, and showed a low level of contamination by proteins from other organelles. A limited number of proteins containing DNA-binding domains were found, consistent with the lack of diversity of these proteins in dinoflagellate transcriptomes. However, the number of proteins containing RNA-binding domains was unexpectedly high supporting a potential role for this type of protein in mediating gene expression and chromatin organization. We also identified a number of proteins involved in chromosome condensation and cell cycle progression as well as a single histone protein (H4). Our results provide the first detailed look at the nuclear proteins associated with the unusual chromatin structure of dinoflagellate nuclei and provide important insights into the biochemical basis of its structure and function.

Circadian clocks are nearly ubiquitous timing mechanisms that can orchestrate rhythmic behavior and gene expression in a wide range of organisms. Clock mechanisms are becoming well understood in fungal, animal, and plant model systems, yet many of these organisms are surrounded by a complex and diverse microbiota which should be taken into account when examining their biology. Of particular interest are the symbiotic relationships between organisms that have coevolved over time, forming a unit called a holobiont. Several studies have now shown linkages between the circadian rhythms of symbiotic partners. Interrelated regulation of holobiont circadian rhythms seems thus important to coordinate shifts in activity over the day for all the partners. Therefore, we suggest that the classical view of "chronobiological individuals" should include "a holobiont" rather than an organism. Unfortunately, mechanisms that may regulate interspecies temporal acclimation and the evolution of the circadian clock in holobionts are far from being understood. For the plant holobiont, our understanding is particularly limited. In this case, the holobiont encompasses two different ecosystems, one above and the other below the ground, with the two potentially receiving timing information from different synchronizing signals (Zeitgebers). The arbuscular mycorrhizal (AM) symbiosis, formed by plant roots and fungi, is one of the oldest and most widespread associations between organisms. By mediating the nutritional flux between the plant and the many microbes in the soil, AM symbiosis constitutes the backbone of the plant holobiont. Even though the importance of the AM symbiosis has been well recognized in agricultural and environmental sciences, its circadian chronobiology remains almost completely unknown. We have begun to study the circadian clock of arbuscular mycorrhizal fungi, and we compile and here discuss the available information on the subject. We propose that analyzing the interrelated temporal organization of the AM symbiosis and determining its underlying mechanisms will advance our understanding of the role and coordination of circadian clocks in holobionts in general.

The toxicity of heavy metals in algal monocultures is well studied and is mediated by reactive oxygen and nitrogen species (ROS/RNS). However, little is known about the toxicity of heavy metals and the mechanisms involved in mixed cultures. Here we examine the oxidative stress and toxic effects of Cu

Protein levels were assessed in the dinoflagellate Lingulodinium polyedra over the course of a diurnal cycle using a label-free LC-MS/MS approach. Roughly 1700 proteins were quantitated in a triplicate dataset over a daily period, and 13 were found to show significant rhythmic changes. Included among the proteins found to be most abundant at night were the two bioluminescence proteins, luciferase and luciferin binding protein, as well as a proliferating cell nuclear protein involved in the nightly DNA replication. Aconitase and a pyrophosphate fructose-6-phosphate-1-phosphotransferase were also found to be more abundant at night, suggestive of an increased ability to generate ATP by glucose catabolism when photosynthesis does not occur. Among the proteins more abundant during the day were found a 2-epi-5-epi-valiolone synthase, potentially involved in synthesis of mycosporin-like amino acids that can act as a “microbial sunscreen”, and an enzyme synthesizing vitamin B6 which is known to protect against oxidative stress. A lactate oxidoreductase was also found to be more abundant during the day, perhaps to counteract the pH changes due to carbon fixation by facilitating conversion of pyruvate to lactate. This unbiased proteomic approach reveals novel insights into the daily metabolic changes of this dinoflagellate. Furthermore, the observation that only a limited number of proteins vary support a model where metabolic flux through pathways can be controlled by variations in a select few, possibly rate limiting, steps. Data are available via ProteomeXchange with identifier PXD006994.

The control of transcription is poorly understood in dinoflagellates, a group of protists whose permanently condensed chromosomes are formed without histones. Furthermore, while transcriptomes contain a number of proteins annotated as transcription factors, the majority of these are cold shock domain proteins which are also known to bind RNA, meaning the number of true transcription factors is unknown. Here we have assessed the transcriptional response to light in the photosynthetic species Symbiodinium kawagutii. We find that three genes previously reported to respond to light using qPCR do not show differential expression using northern blots or RNA-Seq. Interestingly, global transcript profiling by RNA-Seq at LD 0 (dawn) and LD 12 (dusk) found only seven light-regulated genes (FDR = 0.1). qPCR using three randomly selected genes out of the seven was only able to validate differential expression of two. We conclude that there is likely to be less light regulation of gene expression in dinoflagellates than previously thought and suggest that transcriptional responses to other stimuli should also be more thoroughly evaluated in this class of organisms.

There is increasing interest in the possibility of sexual recombination in dinoflagellates, especially those symbiotic with coral, since recombination may be able to augment genetic diversity and reduce levels of coral bleaching. Several previous studies have addressed this in Symbiodinium by querying sequence databanks with a list of 51 genes termed a meiosis detection toolkit. Here, we have constructed an expanded list of 307 genes involved in meiosis in budding yeast. We find the genes involved in the major regulatory steps in yeast meiosis are also found in dinoflagellates, as are many of the genes involved in recombination. In contrast, few genes involved in forming the synaptonemal complex or forming spores are conserved. We further note that the meiosis-related genes absent in dinoflagellates are also as a general rule absent from other protists in the closely related apicomplexa and the ciliates. We conclude the symbiotic dinoflagellates are as able to undergo meiosis as are other protists.

An efficient, high throughput and cost-effective direct aqueous injection approach for the analysis of neonicotinoid pesticides and a common metabolite in environmental water has been described here. The method determines eight neonicotinoid pesticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid, thiamethoxam) and 6-chloronicotinic acid (a common metabolite of the first generation neonicotinoids, acetamiprid, imidacloprid, nitenpyram and thiacloprid) without any sample enrichment/cleanup steps. The method detection limits are 2-8 ng/L for the neonicotinoids and 93 ng/L for 6-chloronicotinic acid. The performance of the QTRAP(®)5500 mass spectrometer was compared against a 4000QTRAP(®), and a QTRAP(®)6500, to provide insights for future method transfer among different generations of instrumentations. Critical mass spectrometric parameters such as collision energy were quite consistent among the three instruments evaluated. However, increased chemical background levels for some target compounds on the more sensitive instruments were observed. The application of differential ion mobility spectrometry combined with tandem mass spectrometry was demonstrated to have great potential in reducing chemical background and/or isobaric interferences inherited in sample matrices. This ISO 17025 accredited method was employed to quantitate neonicotinoids in Ontario stream water samples. Good correlation for analytical results of this direct aqueous injection approach and a previously published solid phase extraction approach warrant high confidence in data quality.

Dinoflagellates are unicellular eukaryotes capable of forming spectacular harmful algal blooms (HABs). Eutrophication of coastal waters by fertilizer runoff, nitrate in particular, has contributed to recent increases in the frequency, magnitude and geographic extent of HABs. Although physiological nitrate uptake and assimilation in dinoflagellates have often been measured in the field and in the laboratory, no molecular components involved in nitrate transport have yet been reported. This study reports the first identification and characterization of dinoflagellate nitrate transporters, found in the transcriptome of the bloom-forming Lingulodinium polyedrum. Of the 23 putative transporters found by BLAST searches, only members of the nitrate transporter 2 (NRT2) family contained all key amino acids known to be essential for nitrate transport. The dinoflagellate NRT2 sequences have 12 predicted transmembrane domains, as do the NRT2 sequences of bacteria, plants and fungi. The NRT2 sequences in Lingulodinium appear to have two different evolutionary origins, as determined by phylogenetic analyses. The most expressed transcript of all putative nitrate transporters was determined by RNA-Seq to be LpNRT2.1. An antibody raised against this transporter showed that the same amount of protein was found at different times over the light dark cycle and with different sources of N. Finally, global nitrate uptake was assessed using a 15N tracer, which showed that the process was not under circadian-control as previously suggested, but simply light-regulated.

The toxicity of heavy metals in algal monocultures is well studied and is mediated by reactive oxygen and nitrogen species (ROS/RNS). However, little is known about the toxicity of heavy metals and the mechanisms involved in mixed cultures. Here we examine the oxidative stress and toxic effects of Cu2+ on the green alga Dunaliella salina (DS) and the cyanobacteria Synecochoccus elongatus (SE) in both mono- and mixed cultures. We find that both species benefit in mixed cultures and acquire higher resistance to Cu2+ toxicity, with a particularly marked effect on SE. DS has a larger surface area than SE, so increases in the number of DS cells compared to SE diminishes the proportion of SE surface area exposed to Cu2+, and contributes to increasing cyanobacterial resistance in mixed cultures. However, these mixed cultures also display as an unexpected property an increased resistance of DS in mixed cultures. SE and DS cells showed significant differences on the kinetics of H2O2 production and antioxidant capacities. The integrated (overall) redox response of mixed cultures, in terms of total amount of H2O2 produced, was proportional to the total surface area of algal species exposed to Cu2+, independent of algal composition in mixed systems. However, mixed cultures display emergent properties, as the time course of H2O2 accumulation is not a simple function of the composition of the mixed cultures. Emergent properties are also observed in the speed of membrane lipid oxidation by the two species, as measured using mixed cultures in which only one of the two species is labeled using the membrane oxidation indicator C11-BODIPY581/591. We suggest that, in addition to H2O2¸ other redox signals (e.g. NO) and allelochemicals (auxins, cytokinins, etc.) may be used to construct a complex inter-species communication network. This could allow mixed algal systems, whatever their composition, to integrate their cellular responses and perform as a coherent unit against toxic Cu2+ ions.

Current heart valve prostheses have limitations that include durability, inability to grow in pediatric patients, and lifelong anticoagulation. Transcatheter aortic valve replacements are minimally invasive procedures, and therefore have emerged as an alternative to traditional valve prostheses. In this experiment, the regenerative capacity of potential tissue engineered transcatheter valve scaffolds (1) acellular porcine pericardium and (2) mesenchymal stem cell-seeded acellular porcine pericardium were compared to native porcine aortic valve cusps in a rat subcutaneous model for up to 8 weeks. Immunohistochemistry, extracellular matrix evaluation, and tissue biomechanics were evaluated on the explanted tissue. Acellular valve scaffolds expressed CD163, CD31, alpha smooth muscle actin, and vimentin at each time point indicating host cell recellularization; however, MSC-seeded tissue showed greater recellularization. Inflammatory cells were observed with CD3 biomarker in native porcine pericardial tissue throughout the study. No inflammation was observed in either acellular or MSC-seeded scaffolds. There was no mechanical advantage observed in MSC-seeded tissue; however after the first week post-explant, there was a decrease in mechanical properties in all groups (p

PURPOSE: Tissue engineered heart valves (TEHV) are being investigated to address the limitations of currently available valve prostheses. In order to advance a wide variety of TEHV approaches, the goal of this study was to develop a cardiac valve bioreactor system capable of conditioning living valves with a range of hydrodynamic conditions as well as capable of assessing hydrodynamic performance to ISO 5840 standards. Methods: A bioreactor system was designed based on the Windkessel approach. Novel features including a purpose-built valve chamber and pressure feedback control were incorporated to maintain asepsis while achieving a range of hydrodynamic conditions. The system was validated by testing hydrodynamic conditions with a bioprosthesis and by operating with cell culture medium for 4 weeks and living cells for 2 weeks. RESULTS: The bioreactor system was able to produce a range of pressure and flow conditions from static to resting adult left ventricular outflow tract to pathological including hypertension. The system operated aseptically for 4 weeks and cell viability was maintained for 2 weeks. The system was also able to record the pressure and flow data needed to calculate effective orifice area and regurgitant fraction. CONCLUSIONS: We have developed a single bioreactor system that allows for step-wise conditioning protocols to be developed for each unique TEHV design as well as allows for hydrodynamic performance assessment.

Symbionts are adapted to work with corals Many corals have formed mutualistic associations with dinoflagellate symbionts, which are thought to provide nutrients and other benefits. To examine the underlying genetics of this association, S. Lin et al. sequenced the genome of the endosymbiont dinoflagellate Symbiodinium kawagutii. The genome includes gene number expansions and encodes microRNAs that show complementarity to genes within the coral genome. Such microRNAs may be involved in regulating coral genes. Furthermore, coral and S. kawagutii appear to share homologs of genes encoding specific nutrient transporters. The findings shed light on how symbiosis is established and maintained between dinoflagellates and corals. Science , this issue p. 691

Rationale Neonicotinoids (NNIs) are the fastest expanding group of pesticides in the world over the last two decades; however, they may be a significant contributing factor to bee mortality. The widespread use of NNIs makes it critical to monitor their residuals in the environment. Published methods for NNI analysis are mainly focused on agricultural and food products, and many of them only measured a portion of the commercially available NNIs. Methods Utilizing a biphenyl stationary-phase column, a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to determine eight NNIs, including acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitempyram, thiacloprid and thiamethoxam in environmental water. Two multiple reaction monitoring (MRM) transitions were monitored for each compound to ascertain true positive identification. Isotope-labelled NNIs, d3-acetamiprid, d3-clothianidin, d4-imidacloprid and d3-thiamethoxam, were used to compensate for extraction efficiency, matrix effects and instrument variability while monitoring real-time method performance. Target compounds in aqueous samples were analyzed by direct aqueous injection (DAI) or after solid-phase extraction (SPE). Results The method detection limits (MDLs) of NNIs in drinking water, surface water and groundwater were in the ranges of 50 to 190 and 2 to 7 ng/L for DAI and SPE procedures, respectively, and target compound recoveries ranged from 78 to 110%. The stability of target compounds in water samples and SPE extracts was also investigated for the first time to ensure accurate results. No obvious degradation was observed for target compounds within four weeks in either water samples or SPE extracts. Conclusions The method developed for neonicotinoid pesticide analysis is very sensitive and efficient. It provides good flexibility to meet various environmental monitoring needs and is employed for an extensive study to determine the distribution of NNIs in Ontario's water. Copyright © 2015 John Wiley & Sons, Ltd.