Levels Of Cold Tolerance Research Articles

Identification and validation of cold responsive microRNAs of Hevea brasiliensis using high throughput sequencing

Cold stress is one of the major abiotic factors that influence the productivity and geographical distribution of many agriculturally important crops like Hevea brasiliensis. Cultivation of H. brasiliensis in India is being extended to northeastern regions, where low temperature during winter adversely affects its survival, growth, and productivity. Developing cold-tolerant genotypes is a primary requisite to maximize the productivity under such challenging environmental conditions. However, lack of methods for early evaluation of cold tolerance in the newly developed clones and the extensive time required for assessing their tolerance in the field are major constraints for clonal selection. The present study was initiated with an objective to identify and characterize cold stress responsive miRNAs from H. brasiliensis that show stronger association with cold tolerance. Next generation sequencing using Illumina HiSeq method revealed the expression of 21 and 29 conserved miRNA (from clone RRIM 600) families in cold-stressed and control samples, respectively. Forty-two novel miRNAs were identified from this study. Upon differential expression analysis, eight conserved miRNAs were found commonly expressed in both the samples. When expression analyses were performed subsequently with six selected miRNAs in two Hevea clones (viz. RRII 105 and RRIM 600), miR169 showed a strong association with cold tolerance. miRNAs such as miR482 and miR159 also exhibited association with cold tolerance. This study suggests the possibility of employing these miRNAs as markers for cold tolerance after validation in more number of genotypes with varying levels of cold tolerance.

Thermal ecology of Galaxias platei (Pisces, Galaxiidae) in South Patagonia: perspectives under a climate change scenario

The native freshwater fish Galaxias platei shows a wide latitudinal distribution in Patagonia, being found on both sides of the Andes. Currently, climate change poses one of the main threats to native fish, and its effects are appearing faster in high southern latitudes. The aim of this work was to analyse the possible effects of climate change in G. platei through its thermal responses. We hypothesized that juveniles of this species would be affected by indirect rather than by direct consequences of climate change. We determined the thermal tolerance polygon using Critical Thermal Methodology and preferred temperatures using a thermal gradient. Additionally, we evaluated routine metabolic rate using stop-flow respirometry. Results showed an intermediate to large polygon, with a non-negligible portion acquired through acclimation. Preferred temperatures and routine metabolic rates were positively related to acclimation temperature. Results suggest that G. platei thermal tolerance is dependent on its prior thermal history, have a eurythermal nature but maintain high levels of cold tolerance. Moreover, G. platei would be better suited at maintaining homeostasis at highest temperatures where more energy could be available for growth. This is the first time that thermal ecology data are registered for this species at its southernmost distribution.

Seasonal variation in basal and plastic cold tolerance: Adaptation is influenced by both long- and short-term phenotypic plasticity.

Understanding how thermal selection affects phenotypic distributions across different time scales will allow us to predict the effect of climate change on the fitness of ectotherms. We tested how seasonal temperature variation affects basal levels of cold tolerance and two types of phenotypic plasticity in Drosophila melanogaster. Developmental acclimation occurs as developmental stages of an organism are exposed to seasonal changes in temperature and its effect is irreversible, while reversible short‐term acclimation occurs daily in response to diurnal changes in temperature. We collected wild flies from a temperate population across seasons and measured two cold tolerance metrics (chill‐coma recovery and cold stress survival) and their responses to developmental and short‐term acclimation. Chill‐coma recovery responded to seasonal shifts in temperature, and phenotypic plasticity following both short‐term and developmental acclimation improved cold tolerance. This improvement indicated that both types of plasticity are adaptive, and that plasticity can compensate for genetic variation in basal cold tolerance during warmer parts of the season when flies tend to be less cold tolerant. We also observed a significantly stronger trade‐off between basal cold tolerance and short‐term acclimation during warmer months. For the longer‐term developmental acclimation, a trade‐off persisted regardless of season. A relationship between the two types of plasticity may provide additional insight into why some measures of thermal tolerance are more sensitive to seasonal variation than others.

Cold Tolerance of Pityophthorus juglandis (Coleoptera: Scolytidae) From Northern California.

Winter survivorship of insects is determined by a combination of physiological, behavioral, and microhabitat characteristics. We characterized the cold tolerance of the walnut twig beetle, Pityophthorus juglandis Blackman, a domestic alien invasive bark beetle that vectors a phytopathogenic fungus. The beetle and fungus cause thousand cankers disease in species of Juglans and Pterocarya. The disease is spreading in the United States of America (USA) and Italy. Contact thermocouple thermometry was used to measure the supercooling points of adults and larvae and lower lethal temperatures of adults from a population from northern California. Supercooling points ranged from -12.2 °C to - 25.0 °C for adults and -13.6 °C to - 23.5 °C for larvae; lower lethal temperatures of adults ranged from -14 °C to - 23 °C. We found seasonal changes in adult supercooling points in fall, winter, and spring. The supercooling point for males was 0.5 °C colder than for females over all months and 1 °C colder in the winter than in other seasons. The cold-tolerance strategy shifted in P. juglandis adults from freeze intolerance (December 2013 and January 2014) to partial freeze tolerance (February 2014). An intermediate level of cold tolerance with a plastic response to cold partially explains survival of P. juglandis outside of its native range in the southwestern USA. In addition, we characterized the relationship between minimum air temperatures and minimum phloem temperatures in two Juglans spp. in northern California and Colorado and characterized portions of the native geographic range of eastern black walnut, J. nigra L., that may be too cold currently for this insect to persist.

Natural variation in CTB4a enhances rice adaptation to cold habitats

Low temperature is a major factor limiting rice productivity and geographical distribution. Improved cold tolerance and expanded cultivation to high-altitude or high-latitude regions would help meet growing rice demand. Here we explored a QTL for cold tolerance and cloned the gene, CTB4a (cold tolerance at booting stage), encoding a conserved leucine-rich repeat receptor-like kinase. We show that different CTB4a alleles confer distinct levels of cold tolerance and selection for variation in the CTB4a promoter region has occurred on the basis of environmental temperature. The newly generated cold-tolerant haplotype Tej-Hap-KMXBG was retained by artificial selection during temperate japonica evolution in cold habitats for low-temperature acclimation. Moreover, CTB4a interacts with AtpB, a beta subunit of ATP synthase. Upregulation of CTB4a correlates with increased ATP synthase activity, ATP content, enhanced seed setting and improved yield under cold stress conditions. These findings suggest strategies to improve cold tolerance in crop plants.

Response of maize hybrids from heterotrophic to autotrophic phase of development in cold conditions

Maize ( Zea mays L.) is a crop of a tropical region with sensitivity to low temperature. This study was conducted to evaluate the performance of experimental maize hybrids in winter months of northern India in field conditions. Data were recorded for survival rate, leaf length and width, seedling height and leaf colour. The analysis of pooled data from 3- leaf stage to 6-leaf stage showed hybrids131023 x 131026 and NS76B x EL-CML-1 as best performers. Hybrids with relatively high leaf appearance rate, less cold injury symptoms showed good level of cold tolerance in terms early seedling growth under cold stress. These secondary traits could be used in selection index for improvement of tropical maize for low temperature adaptation.

Large-scale evaluation of pea (Pisum sativum L.) germplasm for cold tolerance in the field during winter in Qingdao

As a cool-season crop, pea (Pisum sativum L.) can tolerate frost at the vegetative stage but experiences yield loss when freezing stress occurs at the reproductive stage. Cold-tolerance improvement of pea varieties is important for stable yield and expansion of the winter pea planting area. Under natural low-temperature conditions during winter in Qingdao, Shandong, China, we evaluated the cold tolerance of 3672 pea germplasm accessions in the field and categorized them as displaying high resistance (214), moderate resistance (835), or susceptibility (2623). The highly and moderately resistant genotypes were validated in the following year. We found that genotypes from the winter production region showed higher cold tolerance than genotypes from the spring production region. The accessions identified as having high levels of cold tolerance are recommended as potential genetic resources in cold-tolerance breeding of pea.

Identification and physiological characterization of two sister lines of indica rice (Oryza sativa L.) with contrasting levels of cold tolerance

Exposure to low temperature during germination and vegetative growth is a limiting factor to the establishment and development of rice seedlings. Higher cold tolerance of japonica than indica subspecies is well documented. However, reports of cold tolerance in indica genotypes are rare. We screened a large number of indica rice genotypes for cold tolerance during germination and initial vegetative growth. The indica genotypes IRGA 959-1-2-2F-4-1-4-A and IRGA 959-1-2-2F-4-1-4-D-1-CA-1, derived from the same cross, were characterized, respectively, as tolerant and sensitive to low temperature. Indexes of photosynthetic performance during light absorption were heavily affected by cold in both genotypes, but recovered after cold exposure only in the tolerant genotype. Activities of the antioxidant enzymes SOD and CAT (at the vegetative stage) and CAT and APX (at the germination stage) were higher in the tolerant than in the sensitive genotype. Expression of 20 genes previously related to cold response in rice was evaluated. Expression of OsLIP9 and OsWCOR413 were higher in the tolerant genotype upon or prior to cold exposure, respectively. The two sister lines show different molecular and physiological responses to low temperature stress. Further in-depth studies with these lines may help to identify new cold tolerance mechanisms in rice.

Estimating Broad Sense Heritability and Investigating the Mechanism of Genetic Transmission of Cold Tolerance Using Mannitol as a Measure of Post-freeze Juice Degradation in Sugarcane and Energycane (Saccharum spp.).

In approximately 25% of the sugarcane-producing countries worldwide, conventional sugarcane (Saccharum spp. hybrids) is exposed to damaging freezes. A study was conducted during the 2009 and 2010 harvest seasons to compare late-season freeze tolerance among three groups: commercial Louisiana sugarcane genotypes, early generation genotypes selected for cold tolerance in the U.S. Department of Agriculture sugarcane breeding programs at Houma, LA, and Canal Point, FL, and potential energycane genotypes selected for high total biomass per acre. Mannitol concentrations in cane juice following freezing temperatures were determined to evaluate levels of cold tolerance. Genotypes selected for cold tolerance in Houma, LA, had significantly more late-season freeze tolerance than commercial sugarcane genotypes and genotypes selected in Canal Point, FL. Genotypes showing the most cold tolerance were Ho02-146 and Ho02-152, and those that were most highly susceptible were US87-1006 and US87-1003 (early-generation breeding genotypes) and L99-233 (commercial genotype). Broad-sense heritability for late-season cold tolerance in the two-year study was estimated at g(2) = 0.78. The enzymatic mannitol analysis successfully differentiated high-fiber energycane genotypes from those from other sources.

Reduced light and moderate water deficiency sustain nitrogen assimilation and sucrose degradation at low temperature in durum wheat

The rate of carbon and nitrogen assimilation is highly sensitive to stress factors, such as low temperature and drought. Little is known about the role of light in the simultaneous effect of cold and drought. The present study thus focused on the combined effect of mild water deficiency and different light intensities during the early cold hardening in durum wheat (Triticum turgidum ssp. durum L.) cultivars with different levels of cold sensitivity.The results showed that reduced illumination decreased the undesirable effects of photoinhibition in the case of net photosynthesis and nitrate reduction, which may help to sustain these processes at low temperature. Mild water deficiency also had a slight positive effect on the effective quantum efficiency of PSII and the nitrate reductase activity in the cold. Glutamine synthesis was affected by light rather than by water deprivation during cold stress. The invertase activity increased to a greater extent by water deprivation, but an increase in illumination also had a facilitating effect on this enzyme. This suggests that both moderate water deficiency and light have an influence on nitrogen metabolism and sucrose degradation during cold hardening. A possible rise in the soluble sugar content caused by the invertase may compensate for the decline in photosynthetic carbon assimilation indicated by the decrease in net photosynthesis. The changes in the osmotic potential can be also correlated to the enhanced level of invertase activity. Both of them were regulated by light at normal water supply, but not at water deprivation in the cold. However, changes in the metabolic enzyme activities and osmotic adjustment could not be directly contributed to the different levels of cold tolerance of the cultivars in the early acclimation period.

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A.

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions.

ICE1 demethylation drives the range expansion of a plant invader through cold tolerance divergence.

Cold tolerance adaption is a crucial determinant for the establishment and expansion of invasive alien plants into new cold environments; however, its evolutionary mechanism is poorly understood. Crofton weed (Ageratina adenophora), a highly invasive alien plant, is continuously spreading across subtropical areas in China, north-eastward from the first colonized south-western tropical regions, through cold tolerance evolution. Close relations between the cold tolerance levels of 34 populations, represented by 147 accessions, and the latitude, extreme lowest temperature, coldest month average temperature, and invasion period have provided direct insight into its cold tolerance divergence. A comparative study of the CBF pathway, associated with the cold tolerance enhancement of cold-susceptible CBF1-transgenic plant, among four geographically distinct crofton weed populations revealed that the CBF pathway plays a key role in the observed cold tolerance divergence. Four epialleles of the cold response regulator ICE1 ranged from 66 to 50 methylated cytosines, representing a 4.4% to 3.3% methylation rate and significantly corresponding to the lowest to highest cold tolerance levels among these different populations. The significant negative relation between the transcription levels of the primary CBF pathway members, except for CBF2, and the methylation levels among the four populations firstly demonstrates that the demethylation-upregulated transcription level of CBF pathway is responsible for this evolution. These facts, combined with the cold tolerance variation and methylation found among three native and two other introduced populations, indicate that the ICE1-demethylated upregulation of cold tolerance may be the underlying evolutionary mechanism allowing crofton weed to expand northward in China.

Analysis of Differential Expression Patterns of mRNA and Protein During Cold-acclimation and De-acclimation in Arabidopsis

Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through the process of cold acclimation (CA). In contrast to CA, the acquired freezing tolerance is rapidly reduced during cold de-acclimation (DA) and plants resume growth after sensing warm temperatures. In order to better understand plant growth and development, and to aid in the breeding of cold-tolerant plants, it is important to decipher the functional mechanisms of the DA process. In this study, we performed comparative transcriptomic and proteomic analyses during CA and DA. As revealed by shotgun proteomics, we identified 3987 peptides originating from 1569 unique proteins and the corresponding mRNAs were analyzed. Among the 1569 genes, 658 genes were specifically induced at the transcriptional level during the process of cold acclimation. In order to investigate the relationship between mRNA and the corresponding protein expression pattern, a Pearson correlation was analyzed. Interestingly, 199 genes showed a positive correlation of mRNA and protein expression pattern, indicating that both their transcription and translation occurred during CA. However, 226 genes showed a negative correlation of mRNA and protein expression pattern, indicating that their mRNAs were transcribed during CA and were stored for the subsequent DA step. Under this scenario, those proteins were specifically increased during DA without additional transcription of mRNA. In order to confirm the negative correlation of mRNA and protein expression patterns, qRT-PCR and western blot analyses were performed. Mitochondrial malate dehydrogenase 1 (mMDH1) exhibited a negative correlation of mRNA and protein levels, which was characterized by CA-specific mRNA induction and protein accumulation specifically during DA. These data indicate that the expression of specific mRNAs and subsequent accumulation of corresponding proteins are not always in accordance under low temperature stress conditions in plants.

Nuclear and chloroplast diversity and phenotypic distribution of rice (Oryza sativa L.) germplasm from the democratic people's republic of Korea (DPRK; North Korea).

BackgroundRice accounts for 43% of staple food production in the Democratic People’s Republic of Korea (DPRK). The most widely planted rice varieties were developed from a limited number of ancestral lines that were repeatedly used as parents in breeding programs. However, detailed pedigrees are not publicly available and little is known about the genetic, phenotypic, and geographical variation of DPRK varieties.ResultsWe evaluated 80 O. sativa accessions from the DPRK, consisting of 67 improved varieties and 13 landraces. Based on nuclear SSR analysis, we divide the varieties into two genetic groups: Group 1 corresponds to the temperate japonica subpopulation and represents 78.75% of the accessions, while Group 2 shares recent ancestry with indica varieties. Interestingly, members of Group 1 are less diverse than Group 2 at the nuclear level, but are more diverse at the chloroplast level. All Group 2 varieties share a single Japonica maternal-haplotype, while Group 1 varieties trace maternal ancestry to both Japonica and Indica. Phenotypically, members of Group 1 have shorter grains than Group 2, and varieties from breeding programs have thicker and wider grains than landraces. Improved varieties in Group 1 also show similar and/or better levels of cold tolerance for most traits, except for spikelet number per panicle. Finally, geographic analysis demonstrates that the majority of genetic variation is located within regions that have the most intensive rice cultivation, including the Western territories near the capital city Pyungyang. This is consistent with the conscious and highly centralized role of human selection in determining local dispersion patterns of rice in the DPRK.ConclusionsDiversity studies of DPRK rice germplasm revealed two genetic groups. The most widely planted group has a narrow genetic base and would benefit from the introduction of new genetic variation from cold tolerant landraces, wild accessions, and/or cultivated gene pools to enhance yield potential and performance.

Genome-scale cold stress response regulatory networks in ten Arabidopsis thalianaecotypes

BackgroundLow temperature leads to major crop losses every year. Although several studies have been conducted focusing on diversity of cold tolerance level in multiple phenotypically divergent Arabidopsis thaliana (A. thaliana) ecotypes, genome-scale molecular understanding is still lacking.ResultsIn this study, we report genome-scale transcript response diversity of 10 A. thaliana ecotypes originating from different geographical locations to non-freezing cold stress (10°C). To analyze the transcriptional response diversity, we initially compared transcriptome changes in all 10 ecotypes using Arabidopsis NimbleGen ATH6 microarrays. In total 6061 transcripts were significantly cold regulated (p < 0.01) in 10 ecotypes, including 498 transcription factors and 315 transposable elements. The majority of the transcripts (75%) showed ecotype specific expression pattern. By using sequence data available from Arabidopsis thaliana 1001 genome project, we further investigated sequence polymorphisms in the core cold stress regulon genes. Significant numbers of non-synonymous amino acid changes were observed in the coding region of the CBF regulon genes. Considering the limited knowledge about regulatory interactions between transcription factors and their target genes in the model plant A. thaliana, we have adopted a powerful systems genetics approach- Network Component Analysis (NCA) to construct an in-silico transcriptional regulatory network model during response to cold stress. The resulting regulatory network contained 1,275 nodes and 7,720 connections, with 178 transcription factors and 1,331 target genes.ConclusionsA. thaliana ecotypes exhibit considerable variation in transcriptome level responses to non-freezing cold stress treatment. Ecotype specific transcripts and related gene ontology (GO) categories were identified to delineate natural variation of cold stress regulated differential gene expression in the model plant A. thaliana. The predicted regulatory network model was able to identify new ecotype specific transcription factors and their regulatory interactions, which might be crucial for their local geographic adaptation to cold temperature. Additionally, since the approach presented here is general, it could be adapted to study networks regulating biological process in any biological systems.

Divergent selection in a maize population for germination at low temperature in controlled environment: study of the direct response, of the trait inheritance and of correlated responses in the field

Improving cold tolerance in maize (Zea mays L.) is an important breeding objective, allowing early sowings which result in many agronomic advantages. Using as source the F(2) population of B73×IABO78 single cross, we previously conducted four cycles of divergent recurrent selection for high (H) and low (L) cold tolerance level, evaluated as the difference (DG) between germination at 9.5°C and at 25°C in the germinator. Then, we pursued the divergent selection in inbreeding from S(1) to S(4). This research was conducted to study (1) the direct response to selection (by testing ten S(4) L and ten S(4) H lines), (2) the trait inheritance (in a complete diallel scheme involving four L and four H lines), (3) the associated responses for cold tolerance in the field (at early and delayed sowings) and (4) the responses for other traits, by testing the ten L and the ten H lines at usual sowing. Selection was effective, leading to appreciable and symmetric responses for DG. Variation among crosses was mainly due to additive effects and the ability to predict hybrid DG based on parental lines DG was appreciable. Associated responses for cold tolerance traits in the field were noticeable, though the relationship between DG and these traits was not outstanding. High tolerance was also associated with early flowering, short plants, less leaves, low kernel moisture, red and thin cob, and flint kernels. These divergently selected lines can represent valuable materials for undertaking basic studies and breeding works concerning cold tolerance.

Cold resistance depends on acclimation and behavioral caste in a temperate ant

Adjusting to low temperatures is important for animals living in cold environments. We studied the chill-coma recovery time in temperate ant workers (Temnothorax nylanderi) from colonies collected in autumn and spring in Germany. We experimentally acclimated these ant colonies to cold temperatures followed by warm temperatures. As expected, cold-acclimated workers recovered faster from freezing temperatures, but subsequent heat acclimation did not change the short recovery times observed after cold acclimation. Hence, either heat acclimation improves cold tolerance, possibly as a general response to stress, or at least it does not negate enhanced cold tolerance following cold acclimation. Colonies collected in spring showed similar cold tolerance levels to cold-acclimated colonies in the laboratory. Next, we compared the chill-coma recovery time of different worker castes and found that exterior workers recovered faster than interior workers. This difference may be related to their more frequent exposure to cold, higher activity level, or distinct physiology. Interior workers were also heavier and showed a higher gaster-to-head ratio and thorax ratio compared to exterior workers. An obvious difference between exterior and interior workers is activity level, but we found no link between activity and cold tolerance. This suggests that physiology rather than behavioral differences could cause the increased cold tolerance of exterior workers. Our study reveals the importance of acclimation for cold tolerance under natural and standardized conditions and demonstrates differences in cold tolerance and body dimensions in monomorphic behavioral castes of an ant.

Mild desiccation rapidly increases freeze tolerance of the goldenrod gall fly, Eurosta solidaginis: evidence for drought-induced RCH

Overwintering insects may experience extreme cold and desiccation stress. Both freezing and desiccation require cells to tolerate osmotic challenge as solutes become concentrated in the hemolymph. Not surprisingly, physiological responses to low temperature and desiccation share common features and may confer cross-tolerance against these stresses. Freeze-tolerant larvae of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae), experience extremely dry and cold conditions in winter. To determine whether mild desiccation can improve freeze tolerance at organismal and cellular levels, we assessed survival, hemolymph osmolality and glycerol levels of control and desiccated larvae. Larvae that lost only 6-10% of their body mass, in as little as 6 h, had markedly higher levels of freeze tolerance. Mild, rapid desiccation increased freezing tolerance at -15°C in September-collected larvae (33.3±6.7 to 73.3±12%) and at -20°C in October-collected larvae (16.7±6.7 to 46.7±3.3%). Similarly, 6 h of desiccation improved in vivo survival by 17-43% in fat body, Malpighian tubule, salivary gland and tracheal cells at -20°C. Desiccation also enhanced intrinsic levels of cold tolerance in midgut cells frozen ex vivo (38.7±4.6 to 89.2±5.5%). Whereas hemolymph osmolality increased significantly with desiccation treatment from 544±16 to 720±26 mOsm, glycerol levels did not differ between control and desiccated groups. The rapidity with which a mild desiccation stress increased freeze tolerance closely resembles the rapid cold-hardening response, which occurs during brief sub-lethal chilling, and suggests that drought stress can induce rapid cold-hardening.

The Activity of the Plant Mitochondrial Inner Membrane Anion Channel (PIMAC) of Maize Populations Divergently Selected for Cold Tolerance Level is Differentially Dependent on the Growth Temperature of Seedlings

The activity of the plant inner membrane mitochondrial anion channel (PIMAC) is involved in metabolite shuttles and mitochondrial volume changes and could have a role in plant temperature tolerance. Our objectives were to investigate (i) the occurrence and (ii) the temperature dependence of anion fluxes through PIMAC in mitochondria isolated from seedlings of three maize populations differing in terms of cold tolerance; and (iii) the relationships between the PIMAC activity kinetics and the level of cold tolerance. Populations were the source population (C0) and two populations divergently selected for high (C4H) and low (C4L) cold tolerance. Such divergently selected populations are expected to share most of their genes, with the main exception of those genes controlling cold tolerance. Arrhenius plots of PIMAC chloride fluxes showed a linear temperature dependence when seedlings were grown at 25 or 14°C, whereas a non-linear temperature dependence was found when seedlings were grown at 5°C, with or without acclimation at 14°C. The activation energy and other thermodynamic parameters of PIMAC activity varied depending on temperature treatments during seedling growth. When seedlings were grown at 14 and 5°C with acclimation, PIMAC activity of the C4H population increased, while that of C4L declined, as compared with the activities of seedlings grown at 25°C. These symmetric responses indicate that PIMAC activity changes are associated with genetically determined differences in the cold tolerance level of the investigated populations. We conclude that anion fluxes by PIMAC depend upon changes on growth temperature and are differentially related to the tolerance level of the tested populations.

Thermal biology and establishment potential in temperate climates of the aphid parasitoid, Lysiphlebus testaceipes

Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae, Aphidiinae) is a parasitic wasp which plays an important role in the biological control of a number of aphid species. Through assessment of its thermal biology and low temperature tolerance, this study ascertains the establishment potential of L. testaceipes in cool temperate climates typical of northern Europe. The developmental threshold of L. testaceipes was 5.8°C. Rearing of parasitoids at shorter day lengths and lower temperatures indicated no ability to enter a diapause state. The supercooling points (SCP) of non-acclimated and acclimated parasitoid life stages were between −24.6°C and −17.7°C, with LTemp50 temperatures approaching these values, indicating a high level of cold tolerance in short exposures. At 5°C the LTime50 of acclimated larvae within parasitized aphids was 42.8 days. Acclimated pupae continued to develop with 54% adult emergence from mummies within 60 days. Acclimated parasitoid larvae and pupae, within living and mummified aphids, continued to develop during 70 days of winter field exposure and emerging adult parasitoids were reproductively viable under field conditions. These data indicate that where suitable host species are available throughout the year, L. testaceipes would be able to establish in northern Europe.