Seasonal and geographical variation in diapause and cold hardiness of the Asian corn borer, Ostrinia furnacalis

Asian corn borer, Ostrinia furnacalis （Guenee）, is a key corn pest in the Asian-Western Pacific countries. It overwinters as flail-grown larvae in plant stalks or in a spun-silk covering located in the plant debris in the temperate regions of China. Supercooling point （SCP） and survival rate after low sub-zero temperature treatment were assessed for field-collected populations in the laboratory using a cool bath with a 1 ℃/min cooling rate until -40℃. Mean SCPs were varied among geographical populations, with a significant decline from -22.7℃ of Haikou, the multivoltine tropical population in the south, to -28.5℃ of Gongzhuling, the univoltine temperate population in the northeast of China. In addition, there was more than 1℃ difference in SCP between Gongzhuling univoltine and bivoltine populations that were from the same geographic origin. Mean SCPs of the Guangzhou population fluctuated over the year, with significantly lower SCPs in winter than in other seasons, which correlated with a significantly higher proportion of diapausing larvae in winter than in other seasons. Over 41% of overwintering larvae from the northeast population could withstand to be supercooled for a few minutes to the low sub-zero temperature of-40℃, but only 6.7% of their southern counterparts did so. The findings from this study suggest that O. furnacalis mostly takes advantage of freeze avoidance as diapausing larvae for overwintering in the southern region, whereas it exhibits freeze tolerance in diapause in the northeastern region.

Ca^(2+)-homeostasis Differs Between Plant Species with Different Cold-tolerance at 4 ℃ Chilling

A comparative study was carried out on the EM_cytochemical localization of calcium and Ca 2+ _ATPase activity in the suspension_cultured cells between the chilling_sensitive maize ( Zea mays L. cv. Black Mexican Sweet) and chilling_insensitive Trititrigia ( Triticum sect. Trititrigia mackey) at 4 ℃ chilling. When maize and Tyititrigia cells were cultured at 26 ℃, electron microscopic observations revealed that the electron_dense calcium antimonate deposits, an indication of the calcium localization, were localized mainly in the vacuoles, and few was found in the cytosol and nuclei. The electron_dense cerium phosphate deposits, an indication of Ca 2+ _ATPase activity, were abundantly distributed on the plasma membrane (PM). When the cells from both species were cultured at 4 ℃ for 1 and 3 h, an elevation of Ca 2+ level in the cytosol and nuclei was observed, whereas the cerium phosphate deposits on the PM showed no quantitative difference from those of the 26 ℃_cultured cells, indicating that the enzymatic activities were not altered during these chilling periods. However, there was a distinct difference in the dynamics of the Ca 2+ distribution and the PM Ca 2+ _ATPase activity between maize and Trititrigia when chilled at 4 ℃ for 12, 24 and 72 h. In maize cells, a large number of Ca 2+ deposits still existed in the cytosol and nuclei, and the PM Ca 2+ _ATPase became less and less active, and even inactive at all. In Trititrigia cells, the increased cytosolic and nuclear Ca 2+ ions decreased after 12 h chilling. By chilling up to 24 and 72 h, the intracellular Ca 2+ concentration had been restored to a similar low level as those of the warm temperature_cultured cells, while the activity of the PM Ca 2+ _ATPase maintained high. The transient cytosolic and nuclear Ca 2+ increase and the activities of PM Ca 2+ _ATPase during chilling are discussed in relation to plant cold hardiness.

Alterations of Intracellular Ca^(2+) Concentration and Ultrastructure in Spruce Apical Bud Cells during Seasonal Transition

Potassium antimonite was used to localize Ca2+ in the apical bud cells of spruce from July 1999 to May 2000. During the period of active growth (July 14), Calcium precipitates, an indication of Ca2+ localization, were mainly distributed in vacuoles, intercellular spaces and cell walls. Few Ca2+ deposits localized in the cytosol and nucleus, showing a low level of the cytosolic and nuclear Ca2+ concentration in the warm summer. In August, some Ca2+ deposits appeared in the cytosol and nuclei, indicating that Ca2+ influx occurred in the cytosol and nucleus as the day length became shorter. From September to November, high levels of the cytosolic and nuclear Ca2+ remained. During the mid-winter (December and January), the distribution of Ca2+ deposits and the ultrastructures in the cells were altered dramatically. Plasmolysis occurred in many cells due to the protoplasmic dehydration. In addition plasmalemma invagination and nuclear chromatin aggregation also occurred. A large number of Ca2+ deposits appeared in the space between the plasmalemma and the cell wall. And also some Ca2+ deposits were distributed in the plastids. However, few Ca2+ deposits were observed in the cytosol and nuclei. By spring of the next year (May), when plants were de-acclimated and resumed active growth, Ca2+ subcellular localization essentially restored to that observed in July of the last year, i.e., the cells contained low cytosolic and nuclear Ca2+ concentrations; Ca2+ deposits were mainly distributed in the vacuoles, cell walls and intercellular spaces. The relationships between the seasonal changes of intracellular Ca2+ concentration and the development of dormancy/cold acclimation, as well as plasmolysis associated with dormancy and cold hardiness were discussed.

Major physiological adjustments in freezing-tolerant grey tiger longicorn beetle(Xylotrechus rusticus) during overwintering period

1Xylotrechus rusticus （Linnaeus） is one of the most destruc-tive woodborers in northeast China;it damages poplar and is listed as a domestic forestry quarantine pest. Overwintering larvae were collected during October 2012 and March 2013 in Harbin, China, to quantify indi-cators related to the insect’s overwintering strategy and the major cryo-protectants. The supercooling points （SCPs）, which ranged from-14.7＆#176;C to -2.9＆#176;C, were higher than the lethal temperatures of LT50 （-33.64＆#176;C） and LT99 （-40.17＆#176;C） after 24 h exposure. , also the minimum mean daily temperature （-24.5＆#176;C） and mean monthly temperature （-18.0＆#176;C） at the sampling site in January during 2008-2012. Thus, X. rusticus is a typical freezing-tolerant insect. Glycerol serves as a major cryoprotectant for overwintering larvae , because it was the only polyol accumulated during the winter and it also had a significant negative correlation with the SCP （p=0.033, R=0.907）. The glycogen and lipid are major sources of ener-gy and their levels decreased substantially in the middle of overwintering, when glycogen had a significant correlation with the SCP （p= 0.006, R=0.971） whereas the lipid contents did not. Moreover, inter-conversions between glycerol and glycogen, as well as mannose and glycogen, were suggested by their negative correlations. The water content did not change obviously during the winter and was not correlated with the SCP. The free amino acids in the hemolymph and the total protein contents of the bodies of larvae changed significantly during winter, although both had no correlations with the SCP.

Assessing frost hardiness of Pinus bungeana shoots and needles by electrical impedance spectroscopy with and without freezing tests

Aims Nursery and forest operations require that frost hardiness results be produced faster than can be provided by controlled freezing tests.There is a great challenge to develop a rapid method for predicting frost hardiness that might not necessitate controlled freezing tests.The aim of this study was to examine the assessment of the frost hardiness of shoots and needles of Pinus bungeana by electrical impedance spectroscopy(EIS)with and without controlled exposure to freezing.Methods The frost hardiness of current-year shoots and needles of P.bungeana in an 8-year-old provenance field trial was measured at Shisanlin Nursery in Beijing,China,from September 2006 to January 2007 by means of EIS and conventional electrolyte leakage(EL).In the same plants,but without controlled freezing test,were monitored the EIS parameters in current-year shoots and needles.Important Findings The results showed that(i)after controlled freezing tests,the frost hardiness estimated by EIS parameters(extracellular resistance,re,and membrane time constant,sm)was significantly correlated with the frost hardiness assessed by EL method(r=0.95)and(ii)for the samples not exposed to controlled freezing treatment,the relaxation time s1 for shoots and b for needles had greater correlations with the frost hardiness estimated by EL after controlled freezing tests relative to the other parameters(r=
0.90 for shoots and r=0.84 for needles,respectively).The parameters re of shoots and needles and sm of needles might be applied for measuring frost hardiness of samples after exposed to controlled freezing tests.The frost hardiness results can be obtained within 48 h.The parameters s1 of shoots and b of needles could be used for estimating the frost hardiness of samples without using a controlled freezing test.The frost hardiness results can be obtained within 24 h.

Cold tolerance and cold hardening strategy of the Japanese pine sawyer Monochamus alternatus （Coleoptera： Cerambycidae）

The Japanese pine sawyer, Monochamus alternatus, is an important pine forest pest and vector transmitting the pine wilt nematode that causes pine wilt disease. Low temperatures in autumn, winter and spring often differentially affect mortality of M. alternatus larvae. In this paper, we mainly compared the differences of mortality and cold hardening of larvae from different seasons, based on supercooling point （SCP） and cumulative probability of individuals freezing （CPIF）. The cold hardening of the larvae from autumn, winter and spring seasons were largely different. Correlations between mortality and CPIF of autumn and spring larvae were highest on day 1/4, and gradually decreased with prolonged exposure duration. This beetle＇s death mainly resulted from freezing in short exposure duration. However, the correlation between mortality and CPIF of winter larvae increased gradually with the prolonged exposure duration. Death did not mainly result from freezing in long exposure duration. Autumn larvae are more susceptible and adaptable than winter and spring larvae. Winter larvae have a slight freeze-tolerance trend. Our research showed that M. alternatus came into complex cold-hardening strategies under natural selection. Freeze avoidance is the primary strategy; with prolonged exposure duration to above SCP or 〈 0℃, chill tolerance is more important; this is followed by freeze tolerance during harsh winters.

Cold acclimation increases Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae) survival during exposure to freezing temperatures

The Asian citrus psyllid,Diaphorina citri,is the vector of the pathogenic bacteria that causes Huanglongbing,the most devasting disease affecting citrus worldwide.As cultivation of citrus tends to expand northward,the tolerance of psyllids to freezing temperatures needs to be investigated.While mortality of D.citri to cold temperature has been previously studied,much less is known regarding the acclimation potential of psyllids to cold temperatures.We first evaluated cold resistance of D.citri depending on color morph and sex.Subsequently,we compared mortality of D.citri at−4°C for 10 hours between unacclimated psyllids,psyllids that were gradually acclimated to cold over 1 or 2 weeks,and psyllids intermittently acclimated also for 1 or 2 weeks.We did not find major differences in cold resistance between color morphs and sex.We found that after 1 week of gradual acclimation,the survival of psyllids at−4°C increased to 86%,compared to 20%in the control group.Survival did not increase after the second week of gradual acclimation.Although intermittent acclimation improved survival compared to the control group,it was less effective than gradual acclimation with a survival at 30%and 70%after 1 and 2 weeks of acclimation,respectively,although this difference with gradual acclimation was not significant at week 2.These data show that gradual cold acclimation allows D.citri to survive brief periods below freezing.It may serve as a mechanism responsible for increasing northern establishment of this pest.

Chill injury in the eggs of the migratory locust, Locusta migratoria （Orthoptera： Acrididae）： the time-temperature relationship with high-temperature interruption

Mortality of the overwintering egg of the migratory locust, Locustamigratoria L., was attributed to chill injury because of its occurrence well above the egg' ssupercooling point. In this study, two parameters, upper limit of chill injury zone (ULCIZ) and sumof the injurious temperature (SIT), were used to examine the locust egg's cold hardiness. The valueof ULCIZ for the locust egg is 1.06 ± 0.54℃, and the SIT is -329.7 (hour ? degree). Thesuperoxide dismutase (SOD) and catalase (CAT) activities changed dramatically after cold stress,indicating that oxygen and hydroxide free radicals are probably efficiently detoxified at lowtemperatures. It was suggested that the nature of chill injury in locust egg might be a complex ofmetabolic disorder and a non-proportional decrease in enzymatic reaction and transports, because theLDH activity at low temperature increased significantly and the ATPase activity decreased withprolonged duration of exposure to low temperatures. The results from high temperature interruptionrevealed that the high temperature intervals significantly increased the survival of locust eggs.

Effects of environmental humidity on the survival and development of pine caterpillars, Dendrolimus tabulaeformis （Lepidoptera： Lasiocampidae）

The pine caterpillar, Dendrolimus tabulaeformis is one of the most important pests on Pinus tabulaeformis and other pine species in North China. In the present study, effects of relative humidity （RH） on the development and survival of pine caterpillars and soil moisture （SM） on their diapausing larvae were investigated. Low RH （20%） deferred the development of eggs and larvae, reduced egg hatching and larval surviving compared to 40%, 60% and 80% RH. Both low （20%） and high （100%） RH reduced egg hatching, but only 20% RH deferred the development of larvae, prolonged developmental duration and reduced the body mass and body length of larvae. The SM influenced the survival of diapausing larvae significantly. The dry treatment significantly reduced the supercooling points （SCPs）, whereas increased the mortality and reduced body mass from 56.9 to 36.5 mg and body water content from 78% to 63% after 2 weeks＇ exposure. Therefore, higher RH is more favorable for the development of early instars and survival of diapausing larvae of the pine caterpillars.

False codling moth Thaumatotibia leucotreta （Lepidoptera, Tortricidae） larvae are chill-susceptible

This study reports on the low temperature tolerance and cold hardiness of larvae of false codling moth, Thaumatotibia leucotreta. We found that larvae have mean critical thermal minima （lower limits of activity） of 6.7℃ which was influenced by feeding status. The effects of low temperature exposure and duration of exposure on larval survival were assessed and showed that the temperature at which 50% of the population survives is -11.5 ± 0.3℃ after 2 h exposure. The supercooling point （SCP, i.e., freezing temperature） was investigated using a range of cooling rates and under different conditions （feeding and hydration status） and using inoculative freezing treatments （in contact with water or orange juice）. The SCP decreased significantly from -15.6℃ to -17.4℃ after larvae were fasted for 24 h. Twenty-four hour treatments at either high or low relative humidity （95.9% or 2.4%） also significantly decreased SCP to -17.2℃ and -18.2℃ respectively. Inoculative freezing （by water contact） raised SCP from -15.6℃ to -6.8℃ which could have important implications for post-harvest sterilization. Cooling rates did not affect SCP which suggests that there is limited phenotypic plasticity of SCP during the larval life-stage, at least over the short time-scales investigated here. In conclusion, larvae of T. leucotreta are chill-susceptible and die upon freezing. These results are important in understanding this pest＇s response to temperature variation, understanding pest risk status and improving post-harvest sterilization efficacy

Divergent patterns between phenotypic and genetic variation in Scots pine

In boreal forests,autumn frost tolerance in seedlings is a critical fitness component because it determines survival rates during regeneration.To understand the forces that drive local adaptation in this trait,we conducted freezing tests in a common garden setting for 54 Pinus sylvestris(Scots pine)populations(>5000 seedlings)collected across Scandinavia into western Russia,and genotyped 24 of these populations(>900 seedlings)at>10000 SNPs.Variation in cold hardiness among populations,as measured by QST,was above 80%and followed a distinct cline along latitude and longitude,demonstrating significant adaptation to climate at origin.In contrast,the genetic differentiation was very weak(mean FST 0.37%).Despite even allele frequency distribution in the vast majority of SNPs among all populations,a few rare alleles appeared at very high or at fixation in marginal populations restricted to northwestern Fennoscandia.Genotype–environment associations showed that climate variables explained 2.9%of the genetic differentiation,while genotype–phenotype associations revealed a high marker-estimated heritability of frost hardiness of 0.56,but identified no major loci.Very extensive gene flow,strong local adaptation,and signals of complex demographic history across markers are interesting topics of forthcoming studies on this species to better clarify signatures of selection and demography.