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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.

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.”

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.

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.

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.

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.

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.

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.

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.

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor.

The fate of engineered zero-valent copper nanoparticles (Cu NPs) in soils collected from geographically-distinct regions of the continental United States and incubated under controlled conditions was investigated with respect to NP affinity for soil surfaces and changes in speciation, as well as their impact on bacterial communities. Soil geochemical properties had a great influence on Cu NP migration and transformation. Translocation of Cu NPs was low in soils enriched in organic matter and high in clay and sandy soils. X-ray absorption spectroscopic analysis showed that the highest rates for transformation to Cu ions and adsorption complexes was in acidic soils. Although there was some change in overall bacterial community richness at the level of order in experimental soil, the level of perturbation was evident in side-by-side comparisons of orders using a 50% microbial community change value (MCC(50)). This assessment revealed that generally, Sphingomonas, known for its importance for remediation, and Rhizobiales, symbiotic partners with certain plants appeared susceptible to Cu NPs and their transformation products. The ecological importance of organisms from these orders and its greater vulnerability to Cu NPs suggests need for future targeted studies.

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.

The antimicrobial properties of engineered silver nanoparticles (AgNPs) have led to their wide use in diverse consumer products. Ampicillin too, acts as a broad-spectrum antibiotic and thus is prescribed for the treatment of many common infections, but with the problematic emergence of ampicillin-resistant bacteria. As a consequence, there has been some interest in the combination of these two distinct chemistries prompted by the clinical challenge of resistance. Prior to trials of combination therapy, however, it is important to understand the impact on human microbiomes. Here we investigated the effect of ampicillin and AgNPs, both individually and in a combined therapy on a human intestinal ecosystem known as a defined experimental community (DEC-60). The DEC-60 consortia was co-treated with a concentration of AgNPs (50 mg/L) known to have a minimal impact, and a broad range of the antibiotic up to the clinical dose (6 mg/L). The addition of AgNPs to sub-clinical doses of ampicillin (0.06 and 0.6 mg/L) had a significantly impact (p

The potential for hydrate inhibition in a stirred autoclave at a subcooling of 2.3 K has been tested on two bacterial isolates. Chryseobacterium sp. C14 survives multiple freeze–thaw events and inhibits ice recrystallization, and Escherichia coli has neither of these properties but is a biofilm producer. Both strains showed methane hydrate inhibition, with a significant reduction in total hydrate formed. Chryseobacterium delayed hydrate nucleating time, to a similar level found for the commercial kinetic inhibitor, polyvinylpyrrolidone (PVP; 0.2 wt %). However, in the presence of E. coli, in comparison to PVP, the time to nucleation was almost tripled and the hydrate growth rate was reduced by half. Because of the variation inherent with the microbial samples, likely a result of the complexities associated with varying cell numbers and metabolic activity, numerous experiments were required. Analysis of the distinct hydrate growth profiles shown by the two bacterial strains indicated that different molecular sources were likely responsible for the observed inhibition. As a working hypothesis, it is suggested that the inhibition observed by Chryseobacterium cultures was partially due to ice recrystallization inhibition properties, while that of E. coli may be due to secreted macromolecules and the effect of biofilm.

Despite numerous applications for functionalized carbon nanotubes (fCNTs) in consumer products, such as electronics, and food packaging, as well as their development as drug delivery vehicles, the consequence of their uptake by living systems has been understudied. In particular, the impact of fCNTs on early development of different species is largely unknown. Here we investigated the effect of ingested hydroxyl-fCNTs on reproduction and development in two model organisms: Drosophila and CD-1 mice. While fCNTs had no measurable impact on Drosophila, a single oral dose of fCNTs (10 mg/kg) administered to pregnant CD-1 dams during organogenesis significantly increased the number of resorptions and resulted in fetal morphological and skeletal abnormalities. The observed difference between the responses of these two models likely reflects their physiology and/or differences in administration. This research underscores the need to examine the effects of fCNTs on reproductive health and development before the opportunities for maternal exposure by fCNTs increase further.

Interest is growing in understanding not only the impact of individual nanoparticles (NPs) on ecosystems but also the effect of NP mixtures. In the present study, the impact of a combination of three different NPs, silver, copper, and silica (all at 0.022%, w/w), on an arctic microbial community was investigated. After adding the NPs, soil microcosms were incubated for 176 d, and subsequent estimates of microbe diversity were obtained using culture-dependent and culture-independent assessments. The treated soil appeared to show a reduction in the ability to use carbohydrate and amino acid substrates and demonstrated an altered pattern of major fatty acid peaks. Polymerase chain reaction–denaturing gradient gel electrophoresis showed consistent differences in the pattern of predominant rRNA gene sequences. Although this is an initial investigation of soil contaminated with mixed NPs, these results demonstrate that even at the relatively modest concentrations used such pollutants have the potential to disrupt microbial communities. Environ. Toxicol. Chem. 2012;31:131–135. © 2011 SETAC

Arctic vegetation communities vary greatly over short distances due to landscape heterogeneities in topography and hydrological conditions, but corresponding patterns and controls for soil microbial communities are not well understood. We characterized and compared the most abundant phylotypes within replicate soil microbial communities (n = 4) underlying the four principal vegetation types in Canadian low Arctic tundra (dry heath, birch hummock, tall birch, and wet sedge) using denaturing gradient gel electrophoresis (DGGE) of small subunit rRNA genes. We identifi ed 10 major bacterial phylotypes. Although most were present in all soil samples, their relative abundances diff ered signifi cantly and consistently according to vegetation type. By contrast, the fungal communities of all vegetation types were dominated by two common phylotypes. Th e communities of major archaea (11 identifi ed) diff ered substantially among some of the vegetation types and even among replicate patches of the same vegetation type, indicating large spatial heterogeneities that could not be attributed to the infl uence of vegetation type. Bacterial and fungal communities in all vegetation types were dominated by Acidobacteria and Zygomycota, respectively. Archaeal communities were dominated by Euryarchaeota in tall birch and wet sedge although both Euryarchaeota and Th aumarchaeota were abundant in the birch hummock and dry heath soils. We conclude that vegetation type exerts a strong infl uence on soil bacterial community structure, and a relatively small and varying infl uence on archaeal and fungal communities in low Arctic tundra. Finally, variation in bacterial community structure among the vegetation types was correlated with soil soluble N and N mineralization potential, suggesting a close association between the relative abundances of dominant soil bacteria and N availability across low Arctic tundra.

The folic acid analog methotrexate (MTX), a competitive inhibitor of dihydrofolate reductase (DHFR), is used to treat a variety of cancers and autoimmune disorders. However, MTX also causes a wide range of toxic effects in healthy cells and is an established teratogen. Efforts to "rescue" the defects caused by MTX by administering a folate analog or by transgenic expression of a DHFR with an altered affinity for MTX have been attempted in a variety of mammals but limited protection was conferred. As a result, our understanding of the effect of MTX at the molecular genetic level remains incomplete and, in addition, continued mammalian sacrifice is not ideal. Due to the similarity of teratogenic effects produced by MTX in Drosophila melanogaster these insects were transformed with DHFR alleles to determine if rescue could be achieved. The resulting "MTX-resistant" flies were subsequently used to investigate changes in gene expression in response to MTX using semiquantitative reverse transcription PCR. The majority (12/14) of key transcripts that were affected in MTX-exposed females including transcripts involved in cell cycle, defense response, and transport were "rescued" in the "MTX-resistant" transgenic flies. These studies illustrate the utility of this invertebrate model for the investigation of molecular effects of MTX-induced teratogenicity, MTX-resistant DHFRs for gene therapy techniques, and teratogenic protection.

Some organisms that experience subzero temperatures, such as insects, fish, bacteria, and plants, synthesize antifreeze proteins (AFPs) that adsorb to surfaces of nascent ice crystals and inhibit their growth. Although some AFPs are globular and nonrepetitive, the majority are repetitive in both sequence and structure. In addition, they are frequently encoded by tandemly arrayed, multigene families. AFP isoforms from the mealworm beetle, Tenebrio molitor, are extremely potent and inhibit ice growth at temperatures below -5 degrees C. They contain a 12-amino acid repeat with the sequence TCTxSxxCxxAx, each of which makes up one coil of the beta-helix structure. TxT motifs are arrayed to form the ice-binding surface in all three known insect AFPs: the homologous AFPs from the two beetles, T. molitor and Dendroides canadensis, and the nonhomologous AFP from the spruce budworm, Choristoneura fumiferana. In this study, we have obtained the cDNA and genomic sequences of additional T. molitor isoforms. They show variation in the number of repeats (from 6 to 10) which can largely be explained by recombination at various TCT motifs. In addition, phylogenetic comparison of the AFPs from the two beetles suggests that gene loss and amplification may have occurred after the divergence of these species. In contrast to a previous study suggesting that T. molitor genes have undergone positive Darwinian selection (selection for heterogeneity), we propose that the higher than expected ratio of nonsynonymous-to-synonymous substitutions might result from selection for higher AT content in the third codon position.

As an initial screen for microorganisms that produce ice-active macromolecules, ice-affinity was used to select microorganisms from soil consortia originating from three temperate regions. Once selected and subsequently purified to single colonies, these microbes were putatively identified by 16S ribosomal RNA sequencing and assayed for various ice-active properties. Ice-affinity selection appeared to select for bacteria with ice-associating activities: inhibition of ice recrystallization; ice nucleation; ice shaping. Although none of these activities were observed in Paenibacillus amyloliticus C8, others such as Chryseobacterium sp. GL8, demonstrated both ice recrystallization inhibition and ice-shaping activities. Pseudomonas borealis DL7 was classified as a type I ice nucleator, Flavobacterium sp. GL7, was identified as a type III ice nucleator and Acinetobacter radioresistens DL5 demonstrated ice recrystallization inhibition. In all, 19 different culturable bacteria were selected from the thousands of microbes in late-summer collected soil samples. Many of the selected microbes have been previously reported in glacial ice cores or polar sea ice, and of five isolates that were further characterized, four showed ice-associating activities. These results indicate the significant potential of ice-affinity selection even with temperate climate soils, suggesting that sampling in more extreme and remote areas is not required for the isolation of ice-active bacteria.

Not surprisingly, in the spruce budworm, Choristoneura fumiferana, antifreeze protein (AFP) gene expression is most abundant in the second instar, overwintering stage. However, low level RNA and protein expression was also found in the sixth instar larvae, a summer stage. In situ hybridization further confirmed the presence of AFP mRNA in sixth instar midgut tissues. Sequencing of cDNAs corresponding to ‘‘summer-expressed’’ transcripts revealed an isoform that was not apparent in a cDNA library made to second instar larvae. Although similar to AFP cDNAs obtained from overwintering larvae, this AFP-like isoform (CfAFP6) has two Cys substitutions. Since AFPs from this species fold into a b-helix that is stabilized by disulfide bonds, it was of interest to determine if this summerexpressed isoform had AFP activity. No thermal hysteresis activity was found when CfAFP6 was cloned and expressed in E. coli, even after in vitro denaturation and refolding. As well, there was no activity detected when the sequence of a known, active isoform was changed to mimic the Cys substitutions in CfAFP6. Since CfAFP6 does not appear to contribute to freeze resistance, its apparent absence in the overwintering second instar should not in itself be considered curious. r 2006 Elsevier Ltd. All rights reserved.

Cold hardening treatment ‐ a brief exposure to low temperatures ‐ can protect certain insects against subsequent exposure to temperatures sufficiently low to cause damage or lethality. Microarray analysis to examine the changes in transcript abundance associated with cold hardening treatment (0 ° C for 2 h followed by 30 min recovery at 25 ° C) was undertaken in Drosophila melanogaster in order to gain insight into this phenomenon. Transcripts associated with 36 genes were identified, a subset of which appeared to be also differentially expressed after heat shock treatment. Quantitative RT-PCR was used to independently determine transcript abundance of a subset of these sequences. Taken together, these assays suggest that stress proteins, including Hsp23, Hsp26, Hsp83 and Frost as well as membrane-associated proteins may contribute to the cold hardening response.

Spruce budworm (Choristoneura) species survive sub-zero winter temperatures by producing antifreeze proteins (AFPs) encoded by a multigene family of short and long isoforms. We report in this study the first analysis of antifreeze proteins from related Choristoneura sister species. The additional thirty amino acid insert found in the longer AFP isoforms maintains the proteins beta-helix and original fifteen amino acid (Thr-X-Thr) repeat motif. Analysis of the beta-helix region shows more divergent residues surround the conserved Thr residues. Maintaining the beta-helix structure and conserved Thr residues appear to be paramount for AFP function and surviving sub-zero winter temperatures. Two other species within the same lepidopteran clade, Ditrysia, do not appear to contain any AFP-like sequences.

The effect of Type I fish antifreeze protein (AFP) from the winter flounder, Pleuronectes americanus (Walbaum), (WfAFP) on the formation of tetrahydrofuran (THF) clathrate hydrate was studied by observing changes in THF crystal morphology and determining the induction time for nucleation. AFP retarded THF clathrate-hydrate growth at the tested temperatures and modified the THF clathrate-hydrate crystal morphology from octahedral to plate-like. AFP appears to be even more effective than the kinetic inhibitor, polyvinylpyrrolidone (PVP). Recombinant AFP from an insect, a spruce budworm, Choristoneura fumiferana (Clem.), moth, (Cf) was also tested for inhibition activity by observation of the THF-hydrate-crystal-growth habit. Like WfAFP, CfAFP appeared to show adsorption on multiple THF-hydrate-crystal faces. A protein with no antifreeze activity, cytochrome C, was used as a control and it neither changed the morphology of the THF clathrate-hydrate crystals, nor retarded the formation of the hydrate. Preliminary experiments on the inhibition activity of WfAFP on a natural gas hydrate assessed induction time and the amount of propane gas consumed. Similar to the observations for THF, the data indicated that WfAFP inhibited propane-hydrate growth. Taken together, these results support our hypothesis that AFPs can inhibit clathrate-hydrate growth and as well, offer promise for the understanding of the inhibition mechanism. PACS No.: 87.90ty

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular β-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Δ-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its β-helical structure and hence the size of its ice-binding face.

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.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a β-roll domain with eight loops of 14–15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein’s inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.

The first insect cDNA and genomic sequences encoding pyrroline 5-carboxylate reductase (EC 1.5.1.2) have been isolated from Drosophila melanogaster. The cDNA sequence was identified by interspecies complementation of an E. coli proline auxotroph and encodes a protein 280 amino acids in length with 25–41% identity to pyrroline 5-carboxylate reductases isolated from other organisms. The corresponding gene is single copy and is located at cytological position 91E-F, and in one of the P1 clones in that region. With a single 61-bp intron, and an impressively small 135- to 200-bp region that presumably acts as a bidirectional promoter, the gene itself shows remarkable economy. The calculated molecular weight of 29,700 predicts that the native enzyme is likely an octomer. Sequencing of the promoter region and expression studies, as well as the known function of the enzyme in redox regulation and the high levels of free proline in insects, suggest that this housekeeping gene encodes an enzyme with a crucial role in intermediary metabolism.

The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys-rich and Thr-rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5. 5 degrees C (DeltaT = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11-13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10-fold and 18-fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis > 3 degrees C. Exposure of small larvae (11-13 mg) to 4 weeks of cold (4 degrees C) caused an approximately 20-fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 degrees C. Exposure of large larvae (100-120 mg) to cold caused 12-fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 degrees C. Thus, thermal hysteresis increased to similar levels (> 4 degrees C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased > 20-fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.

The hyperactive antifreeze protein from the beetle, Tenebrio molitor, is an 8.5-kDa, threonine-rich protein containing 16 Cys residues, all of which are involved in disulfide bonds. When produced by Escherichia coli, the protein accumulated in the supernatant in an inactive, unfolded state. Its correct folding required days or weeks of oxidation at 22 or 4 degrees C, respectively, and its purification included the removal of imperfectly folded forms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional (1)H and two-dimensional (1)H total correlation spectroscopy spectra were particularly helpful in establishing the characteristics of the fully folded antifreeze in comparison to less well-folded forms. The recombinant antifreeze had no free -SH groups and was rapidly and completely inactivated by 10 mM DTT. It had a thermal hysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of approximately 1.0 degrees C at 10-20 mg/ml. The circular dichroism spectra of the beetle antifreeze had a superficial resemblance to those of alpha-helical proteins, but deconvolution of the spectra indicated the absence of alpha-helix and the presence of beta-structure and coil. NMR analysis and secondary structure predictions agree with the CD data and are consistent with a beta-helix model proposed for the antifreeze on the basis of its 12-amino-acid repeating structure and presumptive disulfide bond arrangement.

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.

One of the principal environmental adaptations of certain fishes inhabiting polar and northern coastal waters is the synthesis of antifreeze proteins (AFPs). AFPs bind to and prevent the growth of nascent ice crystals, thus depressing the serum freezing point. The transgenic expression of AFP holds great promise for conferring freeze resistance to commercially important plant and animal species. Since fish at the greatest risk of freezing have multiple AFP gene copies in order to synthesize higher levels of this protein, we have evaluated this evolutionary strategy as a way to maximize AFP expression in a model transgenic host, the fruit fly Drosophila melanogaster. A construct in which AFP genes of the Atlantic wolffish are fused to the Drosophila yolk protein 1,2 promoter/enhancer region was transferred to flies through P-element mediated transformation. Several independent transgenic fly lines were used in genetic crosses to obtain multi-insert lines. Haemolymph freezing point depression (thermal hysteresis) was greater in homozygotes relative to heterozygotes for a given insert. Similarly, multi-insert lines consistently displayed greater haemolymph AFP activity than the single insert lines from which they were derived. The thermal hysteresis value obtained with a fly line harboring 8 AFP gene copies, 0.43 °C, represents the highest such value to date recorded in a transgenic host, and is even higher than the levels found in some AFP-producing fish.

Using Drosophila as a host, we have examined the effect that the presence of an intron has on the accumulation of a processed transgene mRNA. To provide a model system that was free from major position effects, two fish antifreeze protein (AFP) transgenes were arranged in divergent transcriptional orientation from a central Drosophila yolk protein promoter/enhancer region and introduced into the flies by P-element transformation and/or mobilization. In this way the organization of both the structural genes and the promoter elements mimicked their natural arrangements. When one member of the fish AFP transgene pair had its single 180 bp intron deleted, there was a 2- to 11-fold (average 5-fold) decrease in its mRNA level compared to that generated from the control gene containing the fish AFP intervening sequence. When the deleted intron was replaced by a 70 bp intervening sequence originating from the yolk protein 1 gene, mRNA accumulation was restored to its original level. Even for the streamlined genome of Drosophila, where the intron number and size are generally reduced compared to mammals, the presence of an intervening sequence appears to facilitate mRNA accumulation.

Levels of a 28 kDa hemolymph protein, desiccation stress protein (dsp28), in the mealworm beetle, Tenebrio molitor, increase in response to desiccation and cold stress and are also developmentally regulated under nonstress conditions. Dsp28 mRNA is produced in the fat body, and its abundance changes dramatically throughout development. Transcript abundance increases throughout larval development, drops at pupation, and increases again in adults, the highest levels being found in females. The juvenile hormone (JH) analogue, methoprene, increased dsp28 transcript accumulation in pupae, suggesting that changing JH titres have a role in developmental expression and also perhaps contribute to dsp28 regulation during conditions of environmental stress. Genomic DNA, containing the entire dsp28 coding region plus 1.3 kb of upstream sequence, was isolated and potential regulatory sequences, including putative JH response elements, were identified. © 1996 Wiley-Liss, Inc.

Three malathion carboxylesterase (MCE) phenotypes have been described previously in Lucilia cuprina adults: low, intermediate, and high. The MCE specific activities of adults with the intermediate and high phenotypes are 21- and 33-fold higher than those with the low phenotype, but the high phenotype is almost 1000-fold more resistant to malathion than either of the other phenotypes. Here we show that MCE activity also peaks in Day 4 larvae from three lines representative of these phenotypes. The MCE activity of the low-line larvae is only 2.7- and 4-fold lower than those of the intermediate and high-line larvae, respectively. The relatively high MCE activity of the low-line larvae is largely explained by their crop activity. Assays in the presence of esterase inhibitors reveal three distinct types of MCE activity across the three lines and two developmental stages. Activity in the low-line adults is not completely inhibited by paraoxon and is classified as a subclass II carboxylesterase. The MCE activities in the larvae of the low and intermediate lines and adults from the intermediate line are inhibited by low concentrations of paraoxon, classifying them as subclass I carboxylesterases. The MCE activities in the high-line larvae and adults are also classified as subclass I carboxylesterases, but they are more sensitive to inhibition by triphenyl phosphate than those in the other two lines. These data suggest that MCE in the malathion-resistant high line may be structurally different from the MCEs in the malathion-susceptible intermediate and low lines.

In winter flounder, the levels of type I antifreeze protein (AFP) and its mRNA vary seasonally by as much as 1000-fold. Elevated levels in the fall are prompted by the loss of long day-lengths, while higher spring temperatures correlate with AFP clearance. We have investigated the role of temperature on AFP accumulation using transgenic Drosophila melanogaster by expressing multiple AFP genes under control of the heat-inducible hsp70 promoter. AFP and AFP mRNA persisted far longer in flies reared at 10 degrees C compared to 22 degrees C. This difference appears to be mediated by cold-specific mRNA stability since no such temperature effect was observed with either an endogenous heat-inducible mRNA or a constitutively expressed mRNA.

Resistance to malathion in a strain of the Australian sheep blowfly is due to a 10-fold increase in malathion carboxylesterase (MCE) activity relative to a more susceptible strain. MCE was purified to apparent homogeneity from these two strains and was shown to be a monomer of 60,500, with a pI of 5.5 in both strains. Purified MCE from both populations had identical Km and Vmax values for the hydrolysis of malathion as well as for three other esterase substrates. Similarly, the kinetics of inhibition by several inhibitors were the same for the MCE from each strain. These data therefore suggest that resistance to malathion is due to a quantitative rather than a qualitative change in the MCE of the two strains. Estimation of the total MCE content in each strain showed that the resistant blowflies had nine times more MCE than the more susceptible insects. Although blowfly MCE showed greater specificity for naphthyl esters over malathion, it nevertheless hydrolyzes malathion faster than any other esterase yet isolated from an insect. This is in sharp contrast to previously studied insect strains in which organophosphate resistance has been attributed to large increases in nonspecific esterases that show very slow or no hydrolysis of the insecticides.

In the last two decades, nanoparticles (NPs) have found applications in a wide variety of consumer goods. Titanium dioxide (TiO(2)) and silver (Ag) NPs are both found in cosmetics and foods, but their increasing use is of concern due to their ability to be taken up by biological systems. While there are some reports of TiO(2) and Ag NPs affecting complex organisms, their effects on reproduction and development have been largely understudied. Here, the effects of orally administered TiO(2) or Ag NPs on reproduction and development in two different model organisms were investigated. TiO(2) NPs reduced the developmental success of CD-1 mice after a single oral dose of 100 or 1000 mg/kg to dams, resulting in a statistically significant increase in fetal deformities and mortality. Similarly, TiO(2) NP addition to food led to a significant progeny loss in the fruit fly, Drosophila, as shown by a decline in female fecundity. Ag NP administration resulted in an increase in the mortality of fetal mice. Similarly in Drosophila, Ag NP feeding led to a significant decrease in developmental success, but unlike TiO(2) NP treatment, there was no decline in fecundity. The distinct response associated with each type of NP likely reflects differences in NP administration as well as the biology of the particular model. Taken together, however, this study warns that these common NPs could be detrimental to the reproductive and developmental health of both invertebrates and vertebrates.

Antifreeze proteins (AFP) are an evolutionarily diverse class of stress response products best known in certain metazoans that adopt a freeze-avoidance survival strategy. The perennial ryegrass, Lolium perenne (Lp), cannot avoid winter temperatures below the crystallization point and is thought to use its LpAFP in a freeze-tolerant strategy. In order to examine properties of LpAFP in relation to L. perenne's life history, cDNA cloning, recombinant protein characterization, ice-binding activities, gene copy number, and expression responses to low temperature were examined. Transcripts, encoded by only a few gene copies, appeared to increase in abundance after diploid plants were transferred to 4°C for 1-2 days, and in parallel with the ice recrystallization inhibition activities. Circular dichroism spectra of recombinant LpAFP showed three clear folding transition temperatures including one between 10 and 15°C, suggesting to us that folding modifications of the secreted AFP could allow the targeted degradation of the protein in planta when temperatures increase. Although LpAFP showed low thermal hysteresis activity and partitioning into ice, it was similar to AFPs from freeze-avoiding organisms in other respects. Therefore, the type of low temperature resistance strategy adopted by a particular species may not depend on the type of AFP. The independence of AFP sequence and life-history has practical implications for the development of genetically-modified crops with enhanced freeze tolerance.

The expression of fusion gene constructs containing Drosophila regulatory sequences and the structural portions of fish antifreeze protein genes have been examined by transfer into Drosophila melanogaster using P elements. A fusion gene, containing the enhancer, promoter, and cap site of the yolk polypeptide 1 gene, joined in the 5'-untranslated region to the structural portion of the winter flounder type I antifreeze gene, was transcribed in mature female transformants to give an mRNA of the predicted size, but no antifreeze protein was detected by Western blotting. When the same antifreeze protein gene was fused to a Drosophila hsp 70 gene regulatory region and placed downstream of the yolk polypeptide gene enhancer, appropriate expression of mRNA was directed by both gene regulatory elements. However, a translation product from this mRNA was only observed under heat shock conditions and was present at low levels. It is suggested that type I antifreeze mRNA, with its high content of alanine codons and their grouping into clusters of up to seven in a row, is poorly translated when in competition with other host mRNAs. In agreement with this hypothesis, a fusion gene construct between the yolk protein gene regulatory region and two type III antifreeze protein genes produced sub-mmolar concentrations of antifreeze protein in mature females from each of several transgenic lines analysed. The type III antifreeze protein does not have an imbalanced amino acid composition or sequence irregularities, and may be an appropriate choice for conferring freeze protection to frost-susceptible hosts by gene transfer.

We have accomplished gene transfer into embryos of Locusta migratoria, the African migratory locust. Freshly oviposited eggs were injected with circular or linear plasmids containing the Drosophila hsp70 promoter and the choramphenicol acetyltransferase (CAT) reporter gene (hsp-cat), or with circular plasmid containing the Drosophila copia promoter fused to CAT (copia-cat). Southern blot analysis showed that the hsp-cat plasmid persisted extrachromosomally for at least 8 days after injection. There was no evidence for plasmid replication. Transient expression from the introduced promoters was determined by monitoring CAT enzyme activity. After injection of hsp-cat, activity was detected at varying levels in 6–8% of day 3 and day 9 embryos. Embryos injected with copia-cat, assayed on day 3, had a greater frequency but no higher level of expression. The described gene transfer system is promising for analysis of other promoters, including those of Locusta.

Drosophila research has been and continues to be an essential tool for many aspects of biological scientific research and has provided insight into numerous genetic, biochemical, and behavioral processes. As well, due to the remarkable conservation of gene function between Drosophila and humans, and the easy ability to manipulate these genes in a whole organism, Drosophila research has proven critical for studying human disease and the physiological response to chemical reagents. Methotrexate, a widely prescribed pharmaceutical which inhibits dihydrofolate reductase and therefore folate metabolism, is known to cause teratogenic effects in human fetuses. Recently, there has been resurgence in the use of methotrexate for inflammatory diseases and ectopic or unwanted pregnancies thus, increasing the need to fully understand the cytotoxicity of this pharmaceutical. Concerns have been raised over the ethics of studying teratogenic drugs like methotrexate in mammalian systems and thus, we have proposed a Drosophila model. We have shown that exposure of female Drosophila to methotrexate results in progeny with developmental abnormalities. We have also shown that methotrexate exposure changes the abundance of many fundamental cellular transcripts. Expression of a dihydrofolate reductase with a reduced affinity for methotrexate can not only prevent much of the abnormal transcript profile but the teratogenesis seen after drug treatment. In the future, such studies may generate useful tools for mammalian antifolate “rescue” therapies.

The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 μM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 μM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 μM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.Key words: dihydrofolate reductase, Drosophila melanogaster, methotrexate.

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.