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Rhizobia are soil-dwelling bacteria that can form N2-fixing symbioses with legume plant species (Fabaceae). These bacteria are globally distributed; however, few studies have examined the genomics of rhizobia that live in cold environments. Here, we isolated and characterized three rhizobial strains from legume nodules collected at a pair of distant low Arctic tundra and boreal forest sites in northern Canada. Phylogenetic and average nucleotide identity measurements suggested that the three strains are members of the genus Mesorhizobium, and that each strain represents a novel genospecies. Intriguingly, whereas most mesorhizobia contain the classical determinants of nodulation and nitrogen fixation on their chromosome, whole genome sequencing revealed that all three strains carry these genes on large symbiotic megaplasmids of ∼750 to ∼1000 kb. Phylogenetic and sequence analyses of the common nodulation genes revealed highly conserved alleles amongst these northern mesorhizobia, leading us to propose that they belong to a novel symbiovar that we termed symbiovar oxytropis. Interestingly, these nod gene alleles are uncommon in mesorhizobia isolated from similar plant hosts in other climatic regions, suggesting potential functional adaptive differences.

At high latitudes, lake whitefish (Coregonus clupeaformis) and others in the closely relatedCoregonusspecies complex (CSC), including cisco (C. autumnalisandC. sardinella), can be diadromous, seasonally transitioning between freshwater lakes and the Arctic Ocean. CSC skin- and intestine microbiomes were collected, facilitated by Inuit fishers at sites on and around King William Island, Nunavut, at the northern range limits of lake whitefish. Community composition was explored using 16S rRNA gene sequencing, with significant differences in microbiota dispersions depending on fishing site salinity for lake whitefish intestine and skin, as well as cisco skin. Overall, lake whitefish intestine communities appeared more variable than cisco and had higher Shannon diversity, suggesting that lake whitefish and their microbiomes could be more susceptible to environmental stress possibly leading to dysbiosis. Although cisco condition was similar among distinct seasonal habitats, the higher average lake whitefish condition in freshwater rivers suggests that fishing these diadromous whitefish in estuaries may be optimal from a sustainable fishery perspective. Taken together, the impact of changing habitats on fish condition and different microbial composition may inform new approaches to CSC health in fisheries and aquaculture, in addition to being relevant for northern Indigenous peoples with subsistence and economic interests in these resources.

Although intestinal microbial communities from anadromous Arctic char (Salvelinus alpinus (Linnaeus, 1758)) in Kitikmeot, Nunavut, Canada, differ depending on the timing and location of capture, determinants of gut microbiota in other wild Arctic salmonids are largely unknown. Using high-throughput 16S rRNA gene sequence data, we compared intestinal microbiota from Arctic char to those from a related and sympatric salmonid, lake whitefish (Coregonus clupeaformis (Mitchill, 1818)). Shifts in lake whitefish gut microbial community composition were observed between brackish water and freshwater, similar to impacts of salinity reported previously for Arctic char. Despite these similarities, gut community profiles for the two salmonids differed, with whitefish having higher diversities and increased proportions of taxa affiliated with potential pathogens. Geography seemed to have a greater impact on freshwater whitefish gut microbiota than on corresponding Arctic char. Additionally, microbiota diversity was significantly more affected by feeding behavior in whitefish compared with sympatric Arctic char. As sampled whitefish were at their northern range limits and grew slowly, we speculate that they, and their microbial consortia, could be more vulnerable to certain abiotic and biotic factors than Arctic char, which are well adapted to conditions found in these high latitude environments and have the most northern distribution of any freshwater fish.

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temperatures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to SCS. Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of subzero SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 °C) and at −6 °C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 °C, those stored at −6 °C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable subzero storage may not be effective for increasing storage times.

Arctic Indigenous Peoples are among the most exposed humans when it comes to foodborne mercury (Hg). In response, Hg monitoring and research have been on-going in the circumpolar Arctic since about 1991; this work has been mainly possible through the involvement of Arctic Indigenous Peoples. The present overview was initially conducted in the context of a broader assessment of Hg research organized by the Arctic Monitoring and Assessment Programme. This article provides examples of Indigenous Peoples' contributions to Hg monitoring and research in the Arctic, and discusses approaches that could be used, and improved upon, when carrying out future activities. Over 40 mercury projects conducted with/by Indigenous Peoples are identified for different circumpolar regions including the U.S., Canada, Greenland, Sweden, Finland, and Russia as well as instances where Indigenous Knowledge contributed to the understanding of Hg contamination in the Arctic. Perspectives and visions of future Hg research as well as recommendations are presented. The establishment of collaborative processes and partnership/co-production approaches with scientists and Indigenous Peoples, using good communication practices and transparency in research activities, are key to the success of research and monitoring activities in the Arctic. Sustainable funding for community-driven monitoring and research programs in Arctic countries would be beneficial and assist in developing more research/monitoring capacity and would promote a more holistic approach to understanding Hg in the Arctic. These activities should be well connected to circumpolar/international initiatives to ensure broader availability of the information and uptake in policy development.

In the region of King William Island, Nunavut, in the Canadian high Arctic, populations of salmonids including Arctic char (Salvelinus alpinus), cisco (Coregonus autumnalis and C. sardinella) as well as lake whitefish (C. clupeaformis) are diadromous, overwintering in freshwater and transitioning to saline waters following ice melt. Since these fish were sampled at the same time and from the same traditional fishing sites, comparison of their skin structures, as revealed by 16S rRNA gene sequencing, has allowed an assessment of influences on wild fish bacterial communities. Arctic char skin microbiota underwent turnover in different seasonal habitats, but these striking differences in dispersion and diversity metrics, as well as prominent taxa involving primarily Proteobacteria and Firmicutes, were less apparent in the sympatric salmonids. Not only do these results refute the hypothesis that skin communities, for the most part, reflect water microbiota, but they also indicate that differential recruitment of bacteria is influenced by the host genome and physiology. In comparison to the well-adapted Arctic char, lake whitefish at the northern edge of their range may be particularly vulnerable, and we suggest the use of skin microbiomes as a supplemental tool to monitor a sustainable Indigenous salmonid harvest during this period of change in the high Arctic.

Recent climate warming in the Arctic is enhancing microbial decomposition of soil organic matter, which may result in globally significant greenhouse gas releases to the atmosphere. To better predict future impacts, bacterial and fungal community structures in both the bulk soil and the rhizosphere of Arctic birch, Betula glandulosa, were determined in control, greenhouse summer warming, and annual factorial nitrogen (N) and phosphate (P) addition treatments twelve years after their establishment. DNA sequence analyses at multiple taxonomic levels consistently indicated substantial bulk soil and rhizosphere microbial community differences among the fertilization treatments but no significant greenhouse effects. These results suggest that climate warming will likely increase the activity rates of soil microbial decomposers but without substantially altering the structure of either the bacterial or fungal communities. Differential abundance testing revealed changes in ectomycorrhizal fungal species of the genus Thelephora in both bulk soil and rhizosphere, with increases in their relative abundance in P and N + P amended plots compared with warming and controls. Because birch is the principal low Arctic ectomycorrhizal host, our results suggest that these fungi may promote this shrub’s competitiveness where tundra soil nutrient availability is enhanced by warming or other means, ultimately contributing to arctic vegetation “greening.”

The model forage crop

The N2-fixing symbiosis between rhizobia and legumes is negatively impacted by numerous stresses, including low temperatures. To identify genomic features and biochemical pathways of rhizobia that could foster improved symbiotic function under low temperatures, we isolated and characterized three Mesorhizobium strains from legume nodules collected at two distant northern Canadian sites. Whereas the classical determinants of nodulation and nitrogen fixation are located on the chromosome of most mesorhizobia, whole genome sequencing revealed that these genes are on a large symbiotic megaplasmid in all three of the newly isolated strains. A pangenome-wide association study identified 25 genes putatively associated with mesorhizobia isolated from arctic or subarctic environments, with the genomic location of many of these genes implying a relationship with legume symbiosis. Phylogenetic and sequence analyses of the common nodulation genes revealed alleles that are highly conserved amongst mesorhizobia isolated from northern climates but uncommon in mesorhizobia isolated from similar plant hosts in other climatic regions, suggesting potential functional adaptive differences and the horizontal transfer of these alleles between northern rhizobia. We speculate that nod sequence divergence was driven by climatic factors, and that the encoded proteins may be particularly stable and/or active at low temperatures.

Knowledge coevolution is the process through which information is generated by joining knowledge systems in an inclusive and iterative way to facilitate self-determination of communities and promote cultural resilience. A central and practical component of this framework is the fostering of progress towards improved co-management and community led research. Here, we illustrate a knowledge coevolution framework in the context of a major five-year genomics and food security fishery research project in Gjoa Haven, Nunavut. We highlight the process, changes in research objectives, logistical requirements, mutual benefits, and challenges associated with northern collaborative research, and what lessons we have learned from the process. Knowledge coevolution could be linked to more inclusive and effective fishery co-management in Nunavut and possibly elsewhere. Further, the research process appears to have reinforced Indigenous knowledge and Western science without merging these distinct knowledge systems. Here, we strive to provide readers with concrete examples of knowledge coevolution and encourage research groups to incorporate and improve these practices in future projects and in adaptive fishery co-management. We further call on funding agencies to place more value, and thus budgetary priority, on activities related to ongoing consultation, engagement, dissemination, and implementation of project outcomes. Qaujimaningi maruk ajingingituk pivallianirijanginnik pigiarutauplunni tukisikanirutit saqipaliasurmata nunalit nangminiq aulajungnaliquplugit iliqusinginniglu saqipalliatitsiquplugit aulajungnalirlutik nangminiq. Qitianittuq amma ilulirijauplunni piliriangujup pivallianiq turangajuq aqiumakanirnirmut aulatauninganut amma nunalingnut aulataujuq qaujisarniq. Ukua tava takutijutauniaqtut qaujimaningi maruk ajingingituk pivallianirijanginnik pilirianguniaqtunnik ilulinginnik angijut aragunni tallimani qaujisarnirmut amma niqiqatiarnirmut iqalulirinirmi qaujisarniq Uqsuqtummi, Nunavummi. Nalunaiqsipluta pilirianguniaqtunnik, asiangurninginnik qaujisarniup iluanni, aturiaqaqtunik piqutinik, angiqatigingnirmi, amma ilautittinirmik ukiuqtaqtumiunik qaujisarnirmut, iliniataujunik piliriarmit. Qaujimaningi maruk ajingingituk pivallianirijanginnik ilinganajarmat aulatauninganut kajusitiarnirmullu iqaluliriniup mianirijauninganni Nunavummi asinginnilu. Amma suli, qaujisarniq aqiktausimaplunni nunaqaqqaqsimajut tukisianinginnut amma qaplunat tukisianinginnut katitausimangiłutik tapkuak ajingingituk qaujimaniujut. Uvanni piliriaqaqpugut tukisikaniquplugit uqalimaqtut tungaviqatiaqtummik ukturaulaujunik qaujimaningi maruk ajingingituk pivallianirijanginnik amma ikajuqtuipluta timisiujunnik qaujisaqtinik atausingurlutik aqigiarlutiglu piliriangusuqtut sivunirmi amma atuliqtitaulutik iqalungnik aulatauninganut. Tuksiralaurrapta kinaujannik aturniaqtunnik timiujunnit, sivulliutitauplutik qinaujat aturiaqaqtut kajusijummut uqaqatiqarnirmut ilautittinirmut, asinginnullu tusaqtittinirmik atuliqtittinirmullu piliriangujunnik.

Summary Intestinal microbial communities from 362 anadromous Arctic char (Salvelinus alpinus) from the high Arctic Kitikmeot region, Nunavut, Canada, were characterized using high‐throughput 16S rRNA gene sequencing. The resulting bacterial communities were compared across four seasonal habitats that correspond to different stages of annual migration. Arctic char intestinal communities differed by sampling site, salinity and stages of freshwater residence. Although microbiota from fish sampled in brackish water were broadly consistent with taxa seen in other anadromous salmonids, they were enriched with putative psychrophiles, including the nonluminous gut symbiont Photobacterium iliopiscarium that was detected in >90% of intestinal samples from these waters. Microbiota from freshwater‐associated fish were less consistent with results reported for other salmonids, and highly variable, possibly reflecting winter fasting behaviour of these char. We identified microbiota links to age for those fish sampled during the autumn upriver migration, but little impact of the intestinal content and water microbiota on the intestinal community. The strongest driver of intestinal community composition was seasonal habitat, and this finding combined with identification of psychrophiles suggested that water temperature and migratory behaviour are key to understanding the relationship between Arctic char and their symbionts.

The efficacy of needle-shaped nano-hydroxyapatite (nHA; Ca10(PO4)6(OH)2) as a phosphate (Pi) fertilizer was evaluated as well as its impact on soil and soybean (Glycine max) bacterial and fungal communities. Microbial communities were evaluated in soy fertilized with nHA using ITS (internal transcribed spacer) and 16S rRNA high-throughput gene sequencing. Separate greenhouse growth experiments using agriculturally relevant nHA concentrations and application methods were used to assess plant growth and yield compared with no Pi (−P), soluble Pi (+P), and bulk HA controls. Overall, nHA treatments did not show significantly increased growth, biomass, total plant phosphorus concentrations, or yield compared with no Pi controls. Soil and rhizosphere community structures in controls and nHA treatment groups were similar, with minor shifts in the nHA-containing pots comparable to bulk HA controls at equal concentrations. The implementation of nHA in an agriculturally realistic manner and the resulting poor soy growth advises that contrary to some reports under specialized conditions, this nano-fertilizer may not be a viable alternative to traditional Pi fertilizers. If nano-phosphate fertilizers are to achieve their conjectured agricultural potential, alternative nHAs, with differing morphologies, physicochemical properties, and interactions with the soil matrix could be investigated using the evaluative procedures described.

Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, each Brachypodium AFP gene encodes two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models suggesting that ice-binding motifs on the ~13 kDa AFPs can “spoil” nucleating arrays on the ~120 kDa bacterial ice nucleating proteins that form ice at high sub-zero temperatures, consistent with decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Strikingly, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22), with structural modelling suggesting this was due to affinity of the LRR domains to flg22. Thus, these genes play distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins’ ability to adsorb to ice as well as attenuating bacterial ice nucleation and the host immune response.

The model forage crop, Brachypodium distachyon, has a family of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were previously targeted for knockdown using a constitutive CaMV 35S promoter and the resulting transgenic Brachypodium showed reduced antifreeze activity and a greater susceptibility to freezing. However, the transgenic plants also showed developmental defects with shortened stem lengths and were almost completely sterile, raising the possibility that their reduced freeze tolerance could be attributed to developmental deficits. A cold-induced promoter from rice (prOsMYB1R35) has now been substituted for the constitutive promoter to generate temporal miRNA-mediated Brachypodium antifreeze protein knockdowns. Although transgenic lines showed no apparent pleiotropic developmental defects, they demonstrated reduced antifreeze activity as assessed by assays for ice-recrystallization inhibition, thermal hysteresis, electrolyte leakage, leaf infrared thermography, and leaf damage after infection with an ice nucleating phytopathogen. Strikingly, the number of cold-acclimated transgenic plants that survived freezing at -8 °C was reduced by half or killed entirely, depending on the line, compared to cold-acclimated wild type plants. Although these proteins have been studied for almost 60 years, this is the first unequivocal demonstration in any organism of the utility of antifreeze protein function and their contribution to freeze protection, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from accessible cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced freezing tolerance.

Bacterial ice nucleation proteins (INPs) can cause frost damage to plants by nucleating ice formation at high sub-zero temperatures. Modeling of Pseudomonas borealis INP by AlphaFold suggests that the central domain of 65 tandem sixteen-residue repeats forms a beta-solenoid with arrays of outward-pointing threonines and tyrosines, which may organize water molecules into an ice-like pattern. Here we report that mutating some of these residues in a central segment of P. borealis INP, expressed in Escherichia coli, decreases ice nucleation activity more than the section’s deletion. Insertion of a bulky domain has the same effect, indicating that the continuity of the water-organizing repeats is critical for optimal activity. The ~10 C-terminal coils differ from the other 55 coils in being more basic and lacking water-organizing motifs; deletion of this region eliminates INP activity. We show through sequence modifications how arrays of conserved motifs form the large ice-nucleating surface required for potency.

Shifts in microbiota undoubtedly support host plants faced with abiotic stress, including low temperatures. Cold-resistant perennials prepare for freeze stress during a period of cold acclimation that can be mimicked by transfer from growing conditions to a reduced photoperiod and a temperature of 4 °C for 2–6 days. After cold acclimation, the model cereal, Brachypodium distachyon, was characterized using metagenomics supplemented with amplicon sequencing (16S ribosomal RNA gene fragments and an internal transcribed spacer region). The bacterial and fungal rhizosphere remained largely unchanged from that of non-acclimated plants. However, leaf samples representing bacterial and fungal communities of the endo- and phyllospheres significantly changed. For example, a plant-beneficial bacterium, Streptomyces sp. M2, increased more than 200-fold in relative abundance in cold-acclimated leaves, and this increase correlated with a striking decrease in the abundance of Pseudomonas syringae (from 8% to zero). This change is of consequence to the host, since P. syringae is a ubiquitous ice-nucleating phytopathogen responsible for devastating frost events in crops. We posit that a responsive above-ground bacterial and fungal community interacts with Brachypodium’s low temperature and anti-pathogen signalling networks to help ensure survival in subsequent freeze events, underscoring the importance of inter-kingdom partnerships in the response to cold stress.

Shifts in microbiota undoubtedly support host plants faced with abiotic stress, including low temperatures. Cold-resistant perennials prepare for freeze stress during a period of cold acclimation that can be mimicked by transfer from growing conditions to a reduced photoperiod and a temperature of 4 °C for 2–6 days. After cold acclimation, the model cereal, Brachypodium distachyon, was characterized using metagenomics supplemented with amplicon sequencing (16S ribosomal RNA gene fragments and an internal transcribed spacer region). The bacterial and fungal rhizosphere remained largely unchanged from that of non-acclimated plants. However, leaf samples representing bacterial and fungal communities of the endo- and phyllospheres significantly changed. For example, a plant-beneficial bacterium, Streptomyces sp. M2, increased more than 200-fold in relative abundance in cold-acclimated leaves, and this increase correlated with a striking decrease in the abundance of Pseudomonas syringae (from 8% to zero). This change is of consequence to the host, since P. syringae is a ubiquitous ice-nucleating phytopathogen responsible for devastating frost events in crops. We posit that a responsive above-ground bacterial and fungal community interacts with Brachypodium’s low temperature and anti-pathogen signalling networks to help ensure survival in subsequent freeze events, underscoring the importance of inter-kingdom partnerships in the response to cold stress.

Long-term space missions will expose crew members, their cells as well as their microbiomes to prolonged periods of microgravity and ionizing radiation, environmental stressors for which almost no earth-based organisms have evolved to survive. Despite the importance of maintaining genomic integrity, the impact of these stresses on DNA polymerase-mediated replication and repair has not been fully explored. DNA polymerase fidelity and replication rates were assayed under conditions of microgravity generated by parabolic flight and compared to earth-like gravity. Upon commencement of a parabolic arc, primed synthetic single-stranded DNA was used as a template for one of two enzymes (Klenow fragment exonuclease+/−; with and without proofreading exonuclease activity, respectively) and were quenched immediately following the 20 s microgravitational period. DNA polymerase error rates were determined with an algorithm developed to identify experimental mutations. In microgravity Klenow exonuclease+ showed a median 1.1-fold per-base decrease in polymerization fidelity for base substitutions when compared to earth-like gravity (p = 0.02), but in the absence of proofreading activity, a 2.4-fold decrease was observed (p = 1.98 × 10−11). Similarly, 1.1-fold and 1.5-fold increases in deletion frequencies in the presence or absence of exonuclease activity (p = 1.51 × 10−7 and p = 8.74 × 10−13), respectively, were observed in microgravity compared to controls. The development of this flexible semi-autonomous payload system coupled with genetic and bioinformatic approaches serves as a proof-of-concept for future space health research.

As mercury emissions continue and climate-mediated permafrost thaw increases the burden of this contaminant in northern waters, Inuit from a Northwest passage community in the Canadian Arctic Archipelago pressed for an assessment of their subsistence catches. Sea-run salmonids (n = 537) comprising Arctic char (Salvelinus alpinus), lake trout (S. namaycush), lake whitefish (Coregonus clupeaformis), and cisco (C. autumnalis, C. sardinella) were analyzed for muscle mercury. Methylmercury is a neurotoxin and bioaccumulated with fish age, but other factors including selenium and other elements, diet and trophic level as assessed by stable isotopes of nitrogen (δ15N) and carbon (δ13C), as well as growth rate, condition, and geographic origin, also contributed depending on the species, even though all the fish shared a similar anadromous or sea-run life history. Although mean mercury concentrations for most of the species were ~0.09 µg·g−1 wet weight (ww), below the levels described in several jurisdictions for subsistence fisheries (0.2 µg·g−1 ww), 70% of lake trout were above this guideline (0.35 µg·g−1 ww), and 19% exceeded the 2.5-fold higher levels for commercial sale. We thus urge the development of consumption advisories for lake trout for the protection of pregnant women and young children and that additionally, periodic community-based monitoring be initiated.

In order to survive sub-zero temperatures, some plants undergo cold acclimation where low, non-freezing temperatures and/or shortened day lengths allow cold hardening and survival during subsequent freeze events. Central to this response is the plasma membrane, where low-temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first plasma membrane proteome of cold-acclimatedBrachypodium distachyon, a model species for the study of monocot crops. A time course experiment investigated cold acclimation-induced changes in the proteome following two-phase partitioning plasma membrane enrichment and label-free quantification by nano-liquid chromatography mass spectrophotometry. Two days of cold acclimation were sufficient for membrane protection as well as an initial increase in sugar levels, and coincided with a significant change in the abundance of 154 proteins. Prolonged cold acclimation resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained cold acclimation response elicited over several days. A meta-analysis revealed that the identified plasma membrane proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress and salt resistance suggesting crosstalk between stress responses, such that cold acclimation may prime plants for other abiotic and biotic stresses. The plasma membrane proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.

The identification of food fish bearing anthropogenic contaminants is one of many priorities for Indigenous peoples living in the Arctic. Mercury (Hg), arsenic (As), and persistent organic pollutants including polychlorinated biphenyls (PCBs) are of concern, and these are reported, in some cases for the first time, for fish sampled in and around King William Island, located in Nunavut, Canada. More than 500 salmonids, comprising Arctic char, lake trout, lake whitefish, and ciscoes, were assayed for contaminants. The studied species are anadromous, migrating to the ocean to feed in the summers and returning to freshwater before sea ice formation in the autumn. Assessments of muscle Hg levels in salmonids from fishing sites on King William Island showed generally higher levels than from mainland sites, with mean concentrations generally below guidelines, except for lake trout. In contrast, mainland fish showed higher means for As, including non-toxic arsenobetaine, than island fish. Lake trout were highest in As and PCB levels, with salmonid PCB congener analysis showing signatures consistent with the legacy of cold-war distant early warning stations. After DNA-profiling, only 4&ndash, 32 Arctic char single nucleotide polymorphisms were needed for successful population assignment. These results support our objective to demonstrate that genomic tools could facilitate efficient and cost-effective cluster assignment for contaminant analysis during ocean residency. We further suggest that routine pollutant testing during the current period of dramatic climate change would be helpful to safeguard the wellbeing of Inuit who depend on these fish as a staple input to their diet. Moreover, this strategy should be applicable elsewhere.

Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, Brachypodium AFP genes encode two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models which indicate that ice-binding motifs on the ~13 kDa AFPs can “spoil” nucleating arrays on the ~120 kDa bacterial ice nucleating proteins used to form ice at high sub-zero temperatures. These models are consistent with the experimentally demonstrated decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Additionally, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22). Structural models suggested that this was due to the affinity of the LRR domains to flg22. Overall, it is remarkable that the Brachypodium genes play multiple distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins’ ability to adsorb to ice as well as to attenuate bacterial ice nucleation and the host immune response.

The characterization of ice-binding proteins (IBPs) from plants can involve many techniques, a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition, an activity characteristic of plant IBPs. Two related procedures are described, both of which can be used to demonstrate and quantify ice-binding activity. First, is the traditional "splat" assay, which can easily be set up using common laboratory equipment, and second, is our modification of this method using superhydrophobic coated sapphire for analysis of multiple samples in tandem. Thermal hysteresis is described as another method for quantifying ice-binding activity, during which ice crystal morphology observations can be used to provide clues about ice-plane binding. Once ice-binding activity has been evaluated, it is necessary to verify IBP identity. We detail two methods for enriching IBPs from complex mixtures using ice-affinity purification, the "ice-finger" and "ice-shell" methods, and we highlight their advantages and limitations for the isolation of plant IBPs. Recombinant IBP expression, necessary for detailed ice-binding analysis, can present challenges. Here, a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to IBPs. Together, these methods, and a few considerations critical to success, can be used to assist researchers wishing to isolate and characterize IBPs from plants.

The action of three distinct recombinant antifreeze proteins (AFPs) as methane hydrate inhibitors was examined using a recently-developed reactor. Compared with traditional approaches, this reactor uses minimal reactant volumes and short experimentation times to assess phase equilibria, kinetics and morphology of a hydrate system in a single experiment (3-in-1). Two of the recombinant AFPs are considered highly active with respect to the inhibition of ice: ‘Maxi’, a fish AFP, and a beetle AFP (TmAFP). The third protein from a grass, is classified as a low activity AFP (LpAFP). ‘Maxi’, an AFP that has not been tested previously as a hydrate inhibitor, slowed hydrate growth rates up to an order of magnitude compared to pure water. TmAFP and LpAFP also exhibited kinetic inhibition, but were less effective than ‘Maxi’. In the presence of AFPs, hydrate films were thinner and showed a single growth mechanism compared to multiple crystal growth mechanisms observed in control experiments. The addition of TmAFP generated large irregular hydrate halos that propagated outside the original water boundary. Halo propagation was somewhat less prominent with LpAFP, and was not observed with ‘Maxi’. Although, none of the AFP’s showed thermodynamic inhibition properties, ‘Maxi’ appeared to form clusters of hydrate which remained metastable in the liquid–vapour region.

Low-temperatures pose extreme challenges to crops causing significant economical impacts. Frosts are responsible for more than 30% of weather-related insured crop losses in some temperate climate jurisdictions, but are particularly devastating for small holdings and communities reliant on a bountiful harvest. Low-temperatures are also frequently accompanied by other abiotic and biotic stresses, including pathogen attacks. Some pathogens have sub-zero temperature optima, while others leverage low-temperatures to promote freezing at high sub-zero temperatures by way of ice-nucleating proteins in order to access intracellular nutrients. To survive low-temperatures and the attendant risks, various plant species have evolved complex and intricate signaling networks, molecular mechanisms, and physiological changes, in addition to symbiotic relationships with microbiota. Enhancing low-temperature survival and pathogen-induced freezing tolerance in cold susceptible, agriculturally significant crops is an attractive area of research with immense translatable value to all aspects of society. This area of research will be particularly important in our near future as climate change increases the unpredictability of frosts, particularly in the spring and autumn. Against this backdrop, the world population continues to grow while arable land remains finite and wealth inequality exacerbates food poverty. In this review, we examine plant (i) low-temperature stress, (ii) cold acclimation responses, particularly in crops (iii) antifreeze proteins, and (iv) frost-associated pathogens. Lastly, we suggest integrated approaches to improve crop frost tolerance.

In order to survive subzero temperatures, some plants undergo cold acclimation (CA) where low, nonfreezing temperatures, and/or shortened day lengths allow cold-hardening and survival during subsequent freeze events. Central to this response is the plasma membrane (PM), where low temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first PM proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time-course experiment investigated CA-induced changes in the proteome following two-phase partitioning PM enrichment and label-free quantification by nano-liquid chromatography-mass spectrophotometry. Two days of CA were sufficient for membrane protection as well as an initial increase in sugar levels and coincided with a significant change in the abundance of 154 proteins. Prolonged CA resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained CA response elicited over several days. A meta-analysis revealed that the identified PM proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress, and salt resistance suggesting crosstalk between stress responses, such that CA may prime plants for other abiotic and biotic stresses. The PM proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.

Active layer detachments (ALDs) are permafrost disturbances associated with climate change and increased seasonal warming. Such perturbations result from thawing of the upper permafrost and downslope movement of the overlying thawed material, including the active layer. ALDs have the potential to impact soil microbial community composition and function in arctic soil ecosystems. Here we report an initial investigation of an ALD located at Cape Bounty on Melville Island in the Canadian High Arctic. We examined soil nutrient content as well as microbial community structure using denaturing gradient gel electrophoresis and sequencing. Soil from the disturbed site showed changes in microbial communities with strikingly different fungal community composition compared to soils from an adjacent undisturbed site. These community changes were correlated with enhanced levels of dissolved organic carbon, microbial carbon, total dissolved nitrogen, and microbial nitrogen. The Nitrososphaerales—an order of am...

Titanium dioxide (TiO2) nanoparticles (NPs) are used as an additive (E171 or INS171) in foods such as gum, candy and puddings. To address concerns about the potential hazardous effects of ingested NPs, the toxicity of these food-grade NPs was investigated with a defined model intestinal bacterial community. Each titania preparation (food-grade TiO2 formulations, E171-1 and E171-6a) was tested at concentrations equivalent to those found in the human intestine after sampling 1-2 pieces of gum or candy (100 250 ppm). At the low concentrations used, neither the TiO2 food additives nor control TiO2 NPs had an impact on gas production and only a minor effect on fatty acids profiles (C16:00, C18:00, 15:1 w5c, 18:1 w9c and 18:1 w9c, p < 0.05). DNA profiles and phylogenetic distributions confirmed limited effects on the bacterial community, with a modest decrease in the relative abundance of the dominant Bacteroides ovatus in favor of Clostridium cocleatum (-13% and +14% respectively, p < 0.05). Such minor shifts in the treated consortia suggest that food grade and nano-TiO2 particles do not have a major effect on human gut microbiota when tested in vitro at relevant low concentrations. However, the cumulative effects of chronic TiO2 NP ingestion remain to be tested.

Plants exposed to sub-zero temperatures face unique challenges that threaten their survival. The growth of ice crystals in the extracellular space can cause cellular dehydration, plasma membrane rupture and eventual cell death. Additionally, some pathogenic bacteria cause tissue damage by initiating ice crystal growth at high sub-zero temperatures through the use of ice-nucleating proteins (INPs), presumably to access nutrients from lysed cells. An annual species of brome grass, Brachypodium distachyon (Bd), produces an ice-binding protein (IBP) that shapes ice with a modest depression of the freezing point (~0.1 °C at 1 mg/mL), but high ice-recrystallization inhibition (IRI) activity, allowing ice crystals to remain small at near melting temperatures. This IBP, known as BdIRI, is unlike other characterized IBPs with a single ice-binding face, as mutational analysis indicates that BdIRI adsorbs to ice on two faces. BdIRI also dramatically attenuates the nucleation of ice by bacterial INPs (up to −2.26 °C). This ‘anti-nucleating’ activity is significantly higher than previously documented for any IBP.

Fruit flies, Drosophila melanogaster, have been traditionally valued as a simple model system due to their easy and inexpensive culture, their relatively compact genome, and the variety of available genetic tools. However, due to similarities of their neurological and developmental pathways with those of vertebrates, Drosophila also offers advantages for developmental toxicity assays. The ability to distinguish the effects of a toxicant on adult females, males, and the developing offspring adds to the usefulness of this model. Here we describe key techniques to screen chemicals and other potential emerging toxicants such as nanoparticles on adult Drosophila female and male reproductive success. In addition, assessments of relative toxicity can be revealed by viability assays at each developmental stage from the embryo to the pharate, or preemergent, adult.

Fruit flies, Drosophila melanogaster, have been traditionally valued as a simple model system due to their easy and inexpensive culture, their relatively compact genome, and the variety of available genetic tools. However, due to similarities of their neurological and developmental pathways with those of vertebrates, Drosophila also offers advantages for developmental toxicity assays. The ability to distinguish the effects of a toxicant on adult females, males, and the developing offspring adds to the usefulness of this model. Here we describe key techniques to screen chemicals and other potential emerging toxicants such as nanoparticles on adult Drosophila female and male reproductive success. In addition, assessments of relative toxicity can be revealed by viability assays at each developmental stage from the embryo to the pharate, or preemergent, adult.

Repeatable experiments with accurate data collection and reproducible analyses are fundamental to the scientific method but may be difficult to achieve in practice. Several flexible, open-source tools developed for the R and Python coding environments aid the reproducibility of data wrangling and analysis in scientific research. In contrast, analogous tools are generally lacking for earlier stages, such as systematic labelling and processing of field samples with hierarchical structure (e.g. time points of individuals from multiple lines or populations) or curating heterogenous data collected by different researchers over several years. Such tools are critical for modern research given trends toward globally distributed collaborators using higher-throughput technologies. As a step toward improving repeatability of methods for the collection of biological samples, and curation of biological data, we introduce the R package baRcodeR and the PyTrackDat pipeline in Python. The baRcodeR package provides tools for generating biologically informative, hierarchical labels with digitally encoded 2D barcodes that can be printed and scanned using low-cost commercial hardware. The PyTrackDat pipeline integrates with baRcodeR output to build a web interface for sample management and tracking along with data collection and curation. We briefly describe the application of principles from baRcodeR and PyTrackDat in three large research projects, which demonstrate their value to (i) help document sampling methods, (ii) facilitate collaboration and (iii) reduce opportunities for human errors and omissions that could otherwise propagate through downstream data analysis to compromise biological inference.

Northern populations of Arctic char (Salvelinus alpinus) can be anadromous, migrating annually from the ocean to freshwater lakes and rivers in order to escape sub-zero temperatures. Such seasonal behavior demands that these fish and their associated microbiomes adapt to changes in salinity, temperature, and other environmental challenges. We characterized the microbial community composition of anadromous S. alpinus, netted by Inuit fishermen at freshwater and seawater fishing sites in the high Arctic, both under ice and in open water. Bacterial profiles were generated by DNA extraction and high-throughput sequencing of PCR-amplified 16S ribosomal RNA genes. Results showed that microbial communities on the skin and intestine of Arctic char were statistically different when sampled from freshwater or saline water sites. This association was tested using hierarchical Ward's linkage clustering, showing eight distinct clusters in each of the skin and intestinal microbiomes, with the clusters reflecting sampling location between fresh and saline environments, confirming a salinity-linked turnover. This analysis also provided evidence for a core composition of skin and intestinal bacteria, with the phyla Proteobacteria, Firmicutes, and Cyanobacteria presenting as major phyla within the skin-associated microbiomes. The intestine-associated microbiome was characterized by unidentified genera from families Fusobacteriaceae, Comamonadaceae, Pseudomonadaceae, and Vibrionaceae. The salinity-linked turnover was further tested through ordinations that showed samples grouping based on environment for both skin- and intestine-associated microbiomes. This finding implies that core microbiomes between fresh and saline conditions could be used to assist in regulating optimal fish health in aquaculture practices. Furthermore, identified taxa from known psychrophiles and with nitrogen cycling properties suggest that there is additional potential for biotechnological applications for fish farm and waste management practices.

Sub-zero temperatures put plants at risk of damage associated with the formation of ice crystals in the apoplast. Some freeze-tolerant plants mitigate this risk by expressing ice-binding proteins (IBPs), that adsorb to ice crystals and modify their growth. IBPs are found across several biological kingdoms, with their ice-binding activity and function uniquely suited to the lifestyle they have evolved to protect, be it in fishes, insects or plants. While IBPs from freeze-avoidant species significantly depress the freezing point, plant IBPs typically have a reduced ability to lower the freezing temperature. Nevertheless, they have a superior ability to inhibit the recrystallization of formed ice. This latter activity prevents ice crystals from growing larger at temperatures close to melting. Attempts to engineer frost-hardy plants by the controlled transfer of IBPs from freeze-avoiding fish and insects have been largely unsuccessful. In contrast, the expression of recombinant IBP sequences from freeze-tolerant plants significantly reduced electrolyte leakage and enhanced freezing survival in freeze-sensitive plants. These promising results have spurred additional investigations into plant IBP localization and post-translational modifications, as well as a re-evaluation of IBPs as part of the anti-stress and anti-pathogen axis of freeze-tolerant plants. Here we present an overview of plant freezing stress and adaptation mechanisms and discuss the potential utility of IBPs for the generation of freeze-tolerant crops.

Certain organisms survive low temperatures using a range of physiological changes including the production of antifreeze proteins (AFPs), which have evolved to adsorb to ice crystals. Several of these proteins have been purified and shown to also inhibit the crystallization of clathrate hydrates. They have been found to be effective against structure II (sII) hydrates formed from the liquid tetrahydrofuran, sI and sII gas hydrates formed from single gases, as well as sII natural gas hydrates using a mixture of three gases, as assessed using a variety of instrumentation including stirred reactors, differential scanning calorimetry, nuclear magnetic resonance, Raman spectroscopy, and X-ray powder diffraction. For the most part, AFPs are equal to or more effective than the commercial kinetic hydrate inhibitor (KHI) polyvinylpyrolidone, even under field conditions where saline and liquid hydrocarbons are present. Enclathrated gas analysis has revealed that the adsorption of AFPs to the hydrate surface is distinct from tested commercial KHIs and results in properties that should make these proteins more valuable in some field applications. Efforts to overcome the difficulties of recombinant protein production are ongoing, but in silico models of AFP adsorption to hydrates may offer the opportunity to design commercial KHIs for hydrocarbon recovery and transport with all the attributes of these AFP ”green inhibitors”, including their benefits for human and environmental safety.

The Arctic is experiencing rapid vegetation changes, such as shrub and tree line expansion, due to climate warming, as well as increased wetland variability due to hydrological changes associated with permafrost thawing. These changes are of global concern because changes in vegetation may increase tundra soil biogeochemical processes that would significantly enhance atmospheric CO 2 concentrations. Predicting the latter will at least partly depend on knowing the structure, functional activities, and distributions of soil microbes among the vegetation types across Arctic landscapes. Here we investigated the bacterial and microeukaryotic community structures in soils from the four principal low Arctic tundra vegetation types: wet sedge, birch hummock, tall birch, and dry heath. Sequencing of rRNA gene fragments indicated that the wet sedge and tall birch communities differed significantly from each other and from those associated with the other two dominant vegetation types. Distinct microbial communities were associated with soil pH, ammonium concentration, carbon/nitrogen (C/N) ratio, and moisture content. In soils with similar moisture contents and pHs (excluding wet sedge), bacterial, fungal, and total eukaryotic communities were correlated with the ammonium concentration, dissolved organic nitrogen (DON) content, and C/N ratio. Operational taxonomic unit (OTU) richness, Faith's phylogenetic diversity, and the Shannon species-level index ( H ′) were generally lower in the tall birch soil than in soil from the other vegetation types, with pH being strongly correlated with bacterial richness and Faith's phylogenetic diversity. Together, these results suggest that Arctic soil feedback responses to climate change will be vegetation specific not just because of distinctive substrates and environmental characteristics but also, potentially, because of inherent differences in microbial community structure.

Life can persist under severe osmotic stress and low water activity in hypersaline environments. On Mars, evidence for the past presence of saline bodies of water is prevalent and resulted in the widespread deposition of sulfate and chloride salts. Here we investigate Spotted Lake (British Columbia, Canada), a hypersaline lake with extreme (>3 M) levels of sulfate salts as an exemplar of the conditions thought to be associated with ancient Mars. We provide the first characterization of microbial structure in Spotted Lake sediments through metagenomic sequencing, and report a bacteria-dominated community with abundant Proteobacteria, Firmicutes, and Bacteroidetes, as well as diverse extremophiles. Microbial abundance and functional comparisons reveal similarities to Ace Lake, a meromictic Antarctic lake with anoxic and sulfidic bottom waters. Our analysis suggests that hypersaline-associated species occupy niches characterized foremost by differential abundance of Archaea, uncharacterized Bacteria, and Cyanobacteria. Potential biosignatures in this environment are discussed, specifically the likelihood of a strong sulfur isotopic fractionation record within the sediments due to the presence of sulfate reducing bacteria. With its high sulfate levels and seasonal freeze-thaw cycles, Spotted Lake is an analog for ancient paleolakes on Mars in which sulfate salt deposits may have offered periodically habitable environments, and could have concentrated and preserved organic materials or their biomarkers over geologic time.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

An ice nucleating protein (INP) coding region with 66% sequence identity to the INP of Pseudomonas syringae was previously cloned from P. borealis, a plant beneficial soil bacterium. Ice nucleating activity (INA) in the P. borealis DL7 strain was highest after transfer of cultures to temperatures just above freezing. The corresponding INP coding sequence (inaPb or ina) was used to construct recombinant plasmids, with recombinant expression visualized using a green fluorescent protein marker (gfp encoding GFP). Although the P. borealis strain was originally isolated by ice-affinity, bacterial cultures with membrane-associated INP-GFP did not adsorb to pre-formed ice. Employment of a shuttle vector allowed expression of ina-gfp in both Escherichia coli and Pseudomonas cells. At 27 °C, diffuse fluorescence appeared throughout the cells and was associated with low INA. However, after transfer of cultures to 4 °C, the protein localized to the poles coincident with high INA. Transformants with truncated INP sequences ligated to either gfp, or an antifreeze protein-gfp fusion showed that the repetitive ice-nucleation domain was not necessary for localization. Such localization is consistent with the flanking residues of the INP associating with a temperature-dependent secretion apparatus. A polar location would facilitate INP-INP interactions resulting in the formation of larger aggregates, serving to increase INA. Expression of INPs by P. borealis could function as an efficient atmospheric dispersal mechanism for these soil bacteria, which are less likely to use these proteins for nutrient procurement, as has been suggested for P. syringae.

There is concern that waste waters containing manufactured metal nanoparticles (NPs) originating from consumer goods, will find their way into streams and larger water bodies. Aquatic invertebrates could be vulnerable to such pollution, and here we have used fruit flies, Drosophila melanogaster, as a model invertebrate, to test for the effect of NPs on fitness. Both copper NP and microparticle (MP)-containing medium slowed development, reduced adult longevity and decreased sperm competition. In contrast, ingestion of silver resulted in a significant reduction in developmental success only if the metal particles were nanosized. Ag NP-treatments resulted in reduced developmental success as assessed by larval and pupal survival as well as larval climbing ability, but there was no impact of silver on adult longevity and little effect on reproductive success. However, Cu NPs generally appeared to be no more toxic to this invertebrate model than the bulk counterpart. The impact of silver ingestion in larvae was further investigated by 454 pyrosequencing of the 16S rRNA genes of the midgut flora. There was a striking reduction in the diversity of the gut microbiota of Ag NP-treated larvae with a rise in the predominance of Lactobacillus brevis and a decrease in Acetobacter compared to control or Ag MP-treatment groups. Importantly, these experiments show that perturbation of the microbial assemblage within a metazoan model may contribute to Ag NP-mediated toxicity. These observations have implications for impact assessments of nanoparticles as emerging contaminants.

Economic concerns associated with the recovery of non-conventional hydrocarbon reserves include unexpected ice as well as ice-like gas hydrate formation. Antifreeze proteins (AFPs) inhibit ice growth, and experiments with fish, plant, and insect AFPs have shown promise of effective gas hydrate inhibition in lab-scale experiments. If produced on an industrial scale, AFPs could provide a more environmentally friendly alternative to kinetic inhibitors, but a large-scale production of these AFPs is not currently feasible. We believe that these difficulties could be surmounted by the production of microbial AFPs, but to date, only a few such proteins have been identified and purified, and none of these are associated with hydrocarbon reserves. Here, we have used ice-affinity and freeze-thaw stress to select microbes derived from oil and gas formation water, or produced water, as a source of anaerobic microbial communities. Ice-affinity successfully incorporated anaerobic bacteria under aerobic conditions, and the mixed culture had ice-associating properties. Under these conditions, ice-affinity selection does not result in cultivatable isolates, but similar, cultivable microbes were obtained following freeze-thaw selection under anaerobic conditions. Since these mixed cultures inhibited the growth of ice crystals, they also have the potential to inhibit hydrate growth. Overall, freeze-thaw selection provides a promising first step towards the isolation of microbes capable of the inhibition of ice and gas hydrate growth, for possible application for energy exploration and recovery at high-latitudes and in-deep, cold waters.

The performance of polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap) as kinetic gas hydrate inhibitors in saline solutions and with heptane was evaluated using high pressure microdifferential scanning calorimetry, as well as with a new apparatus, consisting of two high pressure stainless steel crystallizers. Although PVP and PVCap were found to prolong natural gas hydrate induction time in saline solutions, nucleation was followed by catastrophic hydrate crystal growth. PVP was found to be more effective in this case, since this hydrate growth was modestly slower. The addition of n-heptane to the natural gas in the system created a 4th phase. This resulted in increased induction time and a slowing of hydrate growth relative to the gas mixture. Unexpectedly, in the presence of n-heptane, addition of kinetic hydrate inhibitors (KHIs) decreased induction time, but catastrophic growth did not occur. Here PVCap was more effective than PVP in both prolonging the induction time and decreasing the rate of hydrate crystal growth. Once formed, however, hydrate decomposition took longer and proceeded in two steps in the presence of n-heptane. This observation has profound applications on the use these KHIs under ocean field conditions. In the case of hydrate blockages, our observations that hydrate dissociation started later with the KHIs and complete dissociation took longer could have far reaching economic implications for industry. © 2013 Elsevier Ltd. All rights reserved.