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At high latitudes, lake whitefish (Coregonus clupeaformis) and others in the closely related Coregonus species complex (CSC) including cisco (C. autumnalis and C. 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 and microbiota distinctly grouped depending on fishing site salinity. Overall, lake whitefish intestine communities were more variable than those of the two cisco with higher Shannon diversity, suggesting that lake whitefish and their microbiomes could be susceptible to environmental stress possibly leading to dysbiosis. Lake whitefish showed lower condition (K) in the ocean than in freshwater rivers, whereas cisco condition was similar among distinct seasonal habitats. Taken together, the impact of changing habitats on fish condition and microbial composition may inform 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.

Concern about salt levels in freshwater habitats receiving road de-icer runoff has inspired the development of “eco-friendly” formulations that are intended to be less toxic to aquatic organisms, but few experiments have determined that these products are environmentally benign. Mesocosms containing lake water were established for 6 weeks to compare traditional road salt with two newer de-icers, one an inorganic mixture of chloride salts and the second of beet extract and brine. Amplicon sequencing and algal blocking sequences facilitated the identification of differentially impacted bacterial taxa. Ironically, although there was only a minor effect on bacterial structure at high road-salt concentrations, there was an increased relative abundance of salt-resistant genera in the mixed-salt formulation. After amendment with the beet brine de-icer, there was a turnover of taxa coincident with a 68-fold decrease in dissolved oxygen, with decreased diversity and displacement by anaerobic genera indicating a shift across a threshold to a new, apparently stable state, suggesting mesocosm recovery was unlikely. Overall, although we applaud the sentiment behind the formulation of less-damaging “eco-friendly” de-icers, they appear to have more negative environmental impacts than the traditional road salt that they were made to replace.

Ice nucleation proteins have the same tandemly arrayed water-organizing motifs seen in some antifreeze proteins, but on a larger scale. The authors show that mutation, interruption, and truncation of these arrays reduce ice nucleation activity indicating that the two protein types share a common mechanism.

As sugar play a primary role in food flavor and biochemical reaction during the industrial manufacture of dry cured meat products, we aim to evaluate the function of sugar and how the quality changed with the addition of sugar on dry cured sausage after three months of preservation, and the corresponding effect on acidity value and the total acid value. The roles of sugars’ effects on FA hydrolysis and flavor formation are required to be assessed. We selected the addition of sugar with the gradient contents from 3% to 15%, we found that the total acid, acidity value, peroxide value, FA and organic acid were significantly affected. Mono unsaturated fatty acids (MUFA) was the main content of FAs, following by saturated FA and polyunsaturated FA. C18:1n9c was the major FA, following by C16:0 and C18:0. Six kinds of organic acids in dry-cured sausages containing different concentrations of sugar were analyzed by HPLC and the total organic acid reached a maximum of 12.15% by after adding 6% sugar. Acetic acid was 8.06% and lactic acid was 2.56%. This study concludes that sugar is the main factor in flavor formation of dry cured sausage and affects the sausage quality for three months in storage.

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.

Abstract The model forage crop, Brachypodium distachyon, has a cluster of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were targeted for knockdown using a cold‐induced promoter from rice (prOsMYB1R35) to drive miRNA. The transgenic lines showed no apparent pleiotropic developmental defects but had reduced antifreeze activity as assessed by assays for ice‐recrystallization inhibition, thermal hysteresis, electrolyte leakage, and leaf infrared thermography. 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 with cold‐acclimated wild type plants. In addition, more leaf damage was apparent at subzero temperatures in knockdowns after infection with an ice nucleating pathogen, Pseudomonas syringae. Although antifreeze proteins have been studied for almost 60 years, this is the first unequivocal demonstration of their function by knockdown in any organism, and their dual contribution to freeze protection as well as pathogen susceptibility, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced pathogen and freezing tolerance.

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.”

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.

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.

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.

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.

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.

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.

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 4–32 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.

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.

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.

Climate change is already altering the landscape at high latitudes. Permafrost is thawing, the growing season is starting earlier, and, as a result, certain regions in the Arctic may be subjected to an increased incidence of freeze-thaw events. The potential release of carbon and nutrients from soil microbial cells that have been lysed by freeze-thaw transitions could have significant impacts on the overall carbon balance of arctic ecosystems, and therefore on atmospheric CO2 concentrations. However, the impact of repeated freezing and thawing with the consequent growth and recrystallization of ice on microbial communities is still not well understood. Soil samples from three distinct sites, representing Canadian geographical low arctic, mid-arctic and high arctic soils were collected from Daring Lake, Alexandra Fjord and Cambridge Bay sampling sites, respectively. Laboratory-based experiments subjected the soils to multiple freeze-thaw cycles for 14 days based on field observations (0 °C to −10 °C for 12 h and −10 °C to 0 °C for 12 h) and the impact on the communities was assessed by phospholipid fatty acid (PLFA) methyl ester analysis and 16S ribosomal RNA gene sequencing. Both data sets indicated differences in composition and relative abundance between the three sites, as expected. However, there was also a strong variation within the two high latitude sites in the effects of the freeze-thaw treatment on individual PLFA and 16S-based phylotypes. These site-based heterogeneities suggest that the impact of climate change on soil microbial communities may not be predictable a priori; minor differential susceptibilities to freeze-thaw stress could lead to a “butterfly effect” as described by chaos theory, resulting in subsequent substantive differences in microbial assemblages. This perspectives article suggests that this is an unwelcome finding since it will make future predictions for the impact of on-going climate change on soil microbial communities in arctic regions all but impossible.

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.

AbstractIn 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

Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first 'IBP-knockdowns'. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03-0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to -10°C, showing a 13-22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to -8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.

The impact of silver nanoparticles (NPs) and microparticles (MPs) on bacterial and fungal assemblages was studied in soils collected from a low arctic site. Two different concentrations (0.066% and 6.6%) of Ag NPs and Ag MPs were tested in microcosms that were exposed to temperatures mimicking a winter to summer transition. Toxicity was monitored by differential respiration, phospholipid fatty acid analysis, polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequencing. Notwithstanding the effect of Ag MPs, nanosilver had an obvious, additional impact on the microbial community, underscoring the importance of particle size in toxicity. This impact was evidenced by levels of differential respiration in 0.066% Ag NP-treated soil that were only half that of control soils, a decrease in signature bacterial fatty acids, and changes in both richness and evenness in bacterial and fungal DNA sequence assemblages. Prominent after Ag NP-treatment were Hypocreales fungi, which increased to 70%, from only 1% of fungal sequences under control conditions. Genera within this Order known for their antioxidant properties (Cordyceps/Isaria) dominated the fungal assemblage after NP addition. In contrast, sequences attributed to the nitrogen-fixing Rhizobiales bacteria appeared vulnerable to Ag NP-mediated toxicity. This combination of physiological, biochemical and molecular studies clearly demonstrate that Ag NPs can severely disrupt the natural seasonal progression of tundra assemblages.

It is not known if the annual production of tonnes of industrial nanoparticles (NPs) has the potential to impact terrestrial microbial communities, which are so necessary for ecosystem functioning. Here, we have examined the consequences of adding zero valent copper and zinc oxide NPs to soil in pots that were then maintained under field conditions. The fate of these NPs, as well as changes in the microbial communities, was monitored over 162 days. Both NP types traveled through the soil matrix, albeit at differential rates, with Cu NPs retained in the soil matrix at a higher rate compared to ZnO NPs. Leaching of Cu and Zn ions from the parent NPs was also observed as a function of time. Analysis of microbial communities using culture-dependent and independent methods clearly indicated that Cu and ZnO NPs altered the microbial community structure. In particular, two orders of organisms found in rhizosphere, Flavobacteriales and Sphingomonadales, appeared to be particularly susceptible to the presence of NPs. Together, the migration of NPs through soil matrix and the ability of these potential pollutants to influence the composition of microbial community in this field study, cannot help but raise some environmental concerns.

The formation of hydrate plugs in oil and gas pipelines is a serious industrial problem and recently there has been an increased interest in the use of alternative hydrate inhibitors as substitutes for thermodynamic inhibitors like methanol. We show here that antifreeze proteins (AFPs) possess the ability to modify structure II (sII) tetrahydrofuran (THF) hydrate crystal morphologies by adhering to the hydrate surface and inhibiting growth in a similar fashion to the kinetic inhibitor poly-N-vinylpyrrolidone (PVP). The effects of AFPs on the formation and growth rate of high-pressure sII gas mix hydrate demonstrated that AFPs are superior hydrate inhibitors compared to PVP. These results indicate that AFPs may be suitable for the study of new inhibitor systems and represent an important step towards the development of biologically-based hydrate inhibitors.

Our understanding of how engineered nanoparticles (NPs) migrate through soil and affect microbial communities is scarce. In the current study we examined how metal NPs, including those from the iron triad (iron, cobalt and nickel), moved through pots of soil maintained under winter field conditions for 50 days, when mesophilic bacteria may not be dividing. Based on total metal analysis, cobalt and nickel were localized in the top layer of soil, even after exposure to high precipitation and freeze–thaw cycles. In contrast, a bimodal distribution of silver was observed. Due to high endogenous levels of iron, the migration pattern of these NPs could not be determined. Pyrosequence analysis of the bacterial communities revealed that there was no significant engineered NP-mediated decline in microbial richness. However, analysis of individual genera showed that Sphingomonas and Lysobacter were represented by fewer sequences in horizons containing elevated metal levels whereas there was an increase in the numbers of Flavobacterium and Niastella . Collectively, the results indicate that along with the differential migration behavior of NPs in the soil matrix, their impact on soil bacterial diversity appears to be dependent on environmental parameters.