Mechanisms underlying insect freeze tolerance

Freeze tolerance – the ability to survive internal ice formation – has evolved repeatedly in insects, facilitating survival in environments with low temperatures and/or high risk of freezing. Surviving internal ice formation poses several challenges because freezing can cause cellular dehydration and mechanical damage, and restricts the opportunity to metabolise and respond to environmental challenges. While freeze‐tolerant insects accumulate many potentially protective molecules, there is no apparent ‘magic bullet’ – a molecule or class of molecules that appears to be necessary or sufficient to support this cold‐tolerance strategy. In addition, the mechanisms underlying freeze tolerance have been minimally explored. Herein, we frame freeze tolerance as the ability to survive a process: freeze‐tolerant insects must withstand the challenges associated with cooling (low temperatures), freezing (internal ice formation), and thawing. To do so, we hypothesise that freeze‐tolerant insects control the quality and quantity of ice, prevent or repair damage to cells and macromolecules, manage biochemical processes while frozen/thawing, and restore physiological processes post‐thaw. Many of the molecules that can facilitate freeze tolerance are also accumulated by other cold‐ and desiccation‐tolerant insects. We suggest that, when freezing offered a physiological advantage, freeze tolerance evolved in insects that were already adapted to low temperatures or desiccation, or in insects that could withstand small amounts of internal ice formation. Although freeze tolerance is a complex cold‐tolerance strategy that has evolved multiple times, we suggest that a process‐focused approach (in combination with appropriate techniques and model organisms) will facilitate hypothesis‐driven research to understand better how insects survive internal ice formation.     

Environmental regulation and physiological basis of freezing tolerance in woody plants

Cold acclimation of plants is a complex process involving a number of biochemical and physiological changes. The ability to cold acclimate is under genetic control. The development of freezing tolerance in woody plants is generally triggered by non-freezing low temperatures but can also be induced by mild drought or exogenous abscisic acid, as well as by short photoperiod. In nature, the extreme freezing tolerance of woody plants is achieved during sequential stages of cold acclimation the first of which is initiated by short photoperiods and non-freezing low temperatures, and the second by freezing temperatures. Although recent breakthroughs have increased our knowledge on the physiological molecular basis of freezing tolerance in herbaceous species, which acclimate primarily in response to non-freezing low temperatures, very little is known about cold acclimation of woody plants. This article attempts to review our current understanding of the physiological aspects that underline cold acclimation in woody plants.     

昆虫耐寒性研究

昆虫是变温动物,气候变化是造成种群季节消长的基本原因之一。尤其在不良的低温环境中,昆虫耐寒力的高低是其种群存在与发展的种要前提,昆虫对低温的适应能力及其机理也因而成为昆虫生态学和生物进化研究中的一个深受重视的问题,本文论述了与耐寒性直接相关的过冷却点昆虫的抗寒对策,明确了昆虫耐寒性的一些基本概念,一方面从环境影响昆虫的角度对耐寒性的一般规律,如季节性变化,地理变异快速冷驯化的作用等做了简要的概念括,另一方面阐述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等做了简要的概括,另一方面简单述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等。强调昆虫与环境相互作用过程中的生态生理适应,并指出昆虫耐寒性应当与生活史中别的因素联系起来,这样才能对耐寒性有一个更加全面的理解。     

Low-Temperature Stress: Implications for Chickpea (Cicer arietinum L.) Improvement

Chickpea is the third major cool season grain legume crop in the world after dry bean and field pea. Chilling and freezing range temperatures in many of its production regions adversely affect chickpea production. This review provides a comprehensive account of the current information regarding the tolerance of chickpea to freezing and chilling range temperatures. The effect of freezing and chilling at the major phenological stages of chickpea growth are discussed, and its ability for acclimation and winter hardiness is reviewed. Response mechanisms to chilling and freezing are considered at the molecular, cellular, whole plant, and canopy levels. The genetics of tolerance to freezing in chickpea are outlined. Sources of resistance to both freezing and chilling from within the cultivated and wild Cicer genepools are compared and novel breeding technologies for the improvement of tolerance in chickpea are suggested. We also suggest future research be directed toward understanding the mechanisms involved in cold tolerance of chickpea at the physiological, biochemical, and molecular level. Further screening of both the cultivated and wild Cicer species is required in order to identify superior sources of tolerance, especially to chilling at the reproductive stages.     

Tolerance of freezing in caterpillars of the New Zealand Magpie moth (Nyctemera annulata)

In most insects known to tolerate freezing, the adaptation has been completely canalized and permanently incorporated into the genotype, either as a perennial or seasonal phenotypic switch. The exceptions to this (i.e. insects for which the adaptation is, in some manner, incomplete) represent examples of considerable evolutionary interest. To date, the few examples known of incomplete adaptation are readily identified by survival metrics. Caterpillars of the New Zealand Magpie moth (Nyctemera annulata Boisduval) represent a previously undescribed stage in the adaptive continuum of freeze tolerant insects from freeze avoidance to tolerance: a form of freeze tolerance that is intermediate between partial and complete freeze tolerance, the relative ‘incompleteness’ of which, is only apparent using indices of extended fitness (successful metamorphosis). This intermediate form is characterized by: the capacity to mechanistically tolerate equilibrium freezing (>75% survival); a narrow survival envelope below equilibrium freezing temperatures (3–4 °C); and a limited ability to complete metamorphosis after freezing (approximately 27% emergence). The low temperature capabilities of these caterpillars provide support for the hypothesis that the capacity to mechanistically tolerate internal extracellular ice formation by freeze tolerant holometabolous insects is acquired prior to the metabolic adaptations necessary to enable continuation of the life cycle.     

植物抗寒及其基因表达研究进展

植物经过逐渐降低的温度从而提高抗寒能力 ,这个过程被人们称为低温驯化。植物低温驯化过程是一个复杂的生理、生化和能量代谢变化过程 ,这些变化主要包括膜系统的稳定性、可溶性蛋白的积累和小分子渗透物质 ,比如脯氨酸、糖等 ,这些变化中的一些是植物抗寒必需的 ,而另外一些变化不是必需的。主要对冷害和低温生理生化变化、低温诱导表达基因的功能和作用、低温驯化的调节机制及其信号转导方面进行了综述。通过差别筛选 c DNA文库的方法已经鉴定了许多低温诱导表达、进而提高植物抗寒能力的基因 ,其中有脱水素、COR基因和 CBF1转录因子等。低温信号的感受、转导和调节表达是低温驯化的关键环节 ,低温信号的转导过程与干旱胁迫之间具有一定的交叉 ,这为利用 ABA等来提高植物抗寒能力成为可能 ,相信不久的将来人们可以通过提高植物抗寒能力从而增加经济产量成为现实。     

枣园害虫、捕食性和中性昆虫群落结构及动态研究

对山西省太谷地区枣园的害虫、捕食性和中性昆虫群落结构及动态进行研究,结果表明,不同年份枣园的害虫、捕食性和中性昆虫种类与数量均有明显差异(P<0.05),且树上明显大于地面.不同年份捕食性和中性昆虫与害虫的物种数和个体数比例也不同,其物种数和个体数随季节的暖和冷而增加和减少.相同亚群落不同季节的垂直分层结构相似程度不同,不同亚群落在同一季节的垂直分层相似性也不同.总体上垂直分层明显.枣园害虫、捕食性和中性昆虫的多样性指数随季节变化而波动.捕食性和中性昆虫与害虫数量起伏跟随紧密,总体呈极显著相关(r=0.9833,P<0.05).层次间差异明显,以中层相关最显著(r=0.9887,P<0.01)     

Expression of freezing tolerance in the interspecific F1 and somatic hybrids of potatoes

 The expression of freezing tolerance was examined in interspecific F₁ and somatic hybrids of potatoes using 20 species and 34 different combinations between hardy and sensitive species. In the field, the frost tolerance of hybrids resembled either that of the hardy parent, the sensitive parent, or the parental mean, depending on the species combination and the genomic ratio (ratio of the number of sets of chromosomes contributed from each parent). Similar phenomena were observed when the non-acclimated freezing tolerance (NA) and the acclimation capacity (ACC) (two independent genetic components of freezing tolerance) were evaluated separately under controlled environments. In general, the expression level of freezing tolerance was higher in hybrids with more genomes contributed from the hardy parent than from the sensitive parent. In addition, the effectiveness or combining ability of genes conferring freezing tolerance from the hardy species also showed some influence on the expression of freezing tolerance. All three parameters, namely NA, ACC and acclimated freezing tolerance (AA) (NA plus ACC), were significantly correlated to the frost tolerance exhibited in the field. This indicates that the controlled freezing test used in this study could provide a good estimate of field performance. The implications of these results in breeding for freezing tolerance in potatoes are discussed. Received: 21 July 1998 / Accepted: 29 September 1998     

Evolution of freezing susceptibility and freezing tolerance in terrestrial arthropods

Arthropods have evolved various adaptations to survive adverse seasons and it has long been discussed why some arthropods are freezing-susceptible and some are freezing-tolerant. However, which mode of frost resistance came first during the course of evolution? A commonly held opinion is that no choice of strategy has been offered in evolution, because each species of arthropod may have its own evolutionary and natural history, leading to cold-hardiness. Freezing tolerance is more frequent in holometabolous insect orders and partially used by certain vertebrates, like some terrestrially hibernating amphibians and reptiles. Supported by phylogenetic, ontogenetic and ecological arguments, we suggest here that freezing tolerance is more recent than freezing susceptibility in the course of arthropods evolution. In addition, we observe that three basic modes of freezing resistance in insect species exist in the field: (i) permanent or year-round freezing-susceptible species, (ii) alternative or seasonal freezing-susceptible/freezing-tolerant species, (iii) permanent or year-round freezing tolerant species.     

EPT昆虫群落分布与环境因子的相关性

为了研究EPT昆虫与水环境因子的相关性,在2006年6-7月和9-10月分季在丹江口水库两条主水源河流上分设5个采样点,对EPT昆虫和水质采样检测。结果共检测到EPT昆虫780头,为12科16种(或种团),水质理化指标7项。简单相关分析与复相关分析都表明,氮浓度、磷浓度、生化需氧量与EPT昆虫密度呈极显著正相关（P<0.01）,与EPT昆虫的种数呈极显著负相关（P<0.01）。典型相关分析表明,EPT昆虫组与环境因子组有极显著的正相关（P<0.0001）,EPT昆虫组主要目为蜉蝣目和襀翅目,环境因子组主要因子为氮浓度、磷浓度、生化需氧量。由此得出：即使在低污染、低营养程度的水环境下,EPT昆虫与环境因子也表现出群体的显著相关性,环境因子对EPT昆虫分布有重要影响。     

Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.     

Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana

Freezing injury is a major factor limiting the geographical distribution of plant species and the growth and yield of crop plants. Plants from temperate climates are able to increase their freezing tolerance during exposure to low but non‐freezing temperatures in a process termed cold acclimation. Damage to cellular membranes is the major cause of freezing injury in plants, and membrane lipid composition is strongly modified during cold acclimation. Forward and reverse genetic approaches have been used to probe the role of specific lipid‐modifying enzymes in the freezing tolerance of plants. In the present paper we describe an alternative ecological genomics approach that relies on the natural genetic variation within a species. Arabidopsis thaliana has a wide geographical range throughout the Northern Hemisphere with significant natural variation in freezing tolerance that was used for a comparative analysis of the lipidomes of 15 Arabidopsis accessions using ultra‐performance liquid chromatography coupled to Fourier‐transform mass spectrometry, allowing the detection of 180 lipid species. After 14 days of cold acclimation at 4°C the plants from most accessions had accumulated massive amounts of storage lipids, with most of the changes in long‐chain unsaturated triacylglycerides, while the total amount of membrane lipids was only slightly changed. Nevertheless, major changes in the relative amounts of different membrane lipids were also evident. The relative abundance of several lipid species was highly correlated with the freezing tolerance of the accessions, allowing the identification of possible marker lipids for plant freezing tolerance.     

Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield

Solanum tuberosum is a frost-sensitive species incapable of cold acclimation. A brief exposure to frost can significantly reduce its yields, while hard frosts can completely destroy entire crops. Thus, gains in freezing tolerance of even a few degrees would be of considerable benefit relative to frost damage. The S . tuberosum cv. Umatilla was transformed with three Arabidopsis CBF genes ( AtCBF1-3 ) driven by either a constitutive CaMV35S or a stress-inducible Arabidopsis rd29A promoter. AtCBF1 and AtCBF3 over-expression via the 35S promoter increased freezing tolerance about 2 °C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter reached the same level of freezing tolerance as the 35S versions within a few hours of exposure to low but non-freezing temperatures. Constitutive expression of AtCBF genes was associated with negative phenotypes, including smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production. While imparting the same degree of freezing tolerance, control of AtCBF expression via the stress-inducible promoter ameliorated these negative phenotypic effects and restored tuber production to levels similar to wild-type plants. These results suggest that use of a stress-inducible promoter to direct CBF transgene expression can yield significant gains in freezing tolerance without negatively impacting agronomically important traits in potato.     

桉树枝瘿姬小蜂的耐寒性测定

桉树枝瘿姬小蜂Leptocybe invasa Fisher&LaSalle是一种新入侵的检疫性有害生物,为了明确其对极端低温的耐受性,以了解其适生范围,测定了桉树枝瘿姬小蜂幼虫、蛹、成虫及不同地区、不同寄主条件下雌雄成虫以及广东、广西、海南3省6地越冬幼虫12—3月的过冷却点和冰点。结果表明,不同虫态的过冷却点和冰点由低到高顺序为:蛹<幼虫<成虫。蛹的过冷却点和冰点分别为(-24.93±0.10)℃、(-22.81±0.14)℃,成虫的过冷却点和冰点为(-20.93±0.24)℃和(-17.33±0.27)℃。随着纬度的升高,桉树枝瘿姬小蜂的过冷却点和冰点都呈现降低的趋势。海南地区不同寄主桉树枝瘿姬小蜂过冷却点从低到高的顺序排列为:湛-201<小叶桉<广林9号。在12—3月,桉树枝瘿姬小蜂的越冬幼虫过冷却点和冰点随着环境温度的升高而升高,以广东广州地区1月份的越冬幼虫过冷却点和冰点为最低,其数值分别为(-25.44±0.17)℃和(-24.04±0.21)℃,个体过冷却点的最低值为-26.9℃。由实验结果可知,桉树枝瘿姬小蜂蛹和幼虫的耐寒力最强,以幼虫和蛹越冬。地区、寄主、温度对其耐寒力均有显著的影响,而且其有向现疫区以北的区域扩散的潜能。     

Differentiation of freezing tolerance and vernalization responses in Cruciferae exposed to a low temperature

High levels of freezing tolerance were induced in leaves of different Cruciferae species including Brassica napus, Arabidopsis thaliana, Barbarea vulgaris, Thlaspi arvenses and Descurainia sophia by low-temperature acclimation. Concomitantly, the amount of total RNA doubled in these three species. Analyses of methylation patterns and dosage of rRNA genes were carried out to determine whether or not alterations occur in this DNA during development of freezing tolerance. Hybridizations of Southern transfers with an rDNA probe revealed two additional EcoRI sites in purified DNA isolated from freezing-tolerant leaves of winter B. napus cv Jet Neuf and D. sophia (both of which require low temperatures for vernalization), but not in isolates of A. thaliana or spring B. napus cv Topas. An increase of rDNA cistrons was also observed in both B. napus cv Jet Neuf and D. sophia but not in A. thaliana or B. napus cv Topas upon cold acclimation. These results suggest that low temperature induced amplification of rDNA and the differential methylation of EcoRI sites may possibly be related to the vernalization process but may not be related to the development of freezing tolerance. However, the higher activity of RNA polymerase (2.5 times more) observed upon cold acclimation may explain the concomitant increase in total RNA and may be related to the development of freezing tolerance in the Cruciferae.     

Evidence for a freezing tolerance-growth rate trade-off in the live oaks (Quercus series Virentes) across the tropical-temperate divide

? It has long been hypothesized that species are limited to the north by minimum temperature and to the south by competition, resulting in a trade-off between freezing tolerance and growth rate. We investigated the extent to which the climatic origins of populations from four live oak species (Quercus series Virentes) were associated with freezing tolerance and growth rate, and whether species fitted a model of locally adapted populations, each with narrow climatic tolerances, or of broadly adapted populations with wide climatic tolerances. ? Acorns from populations of four species across a tropical-temperate gradient were grown under common tropical and temperate conditions. Growth rate, seed mass, and leaf and stem freezing traits were compared with source minimum temperatures. ? Maximum growth rates under tropical conditions were negatively correlated with freezing tolerance under temperate conditions. The minimum source temperature predicted the freezing tolerance of populations under temperate conditions. The tropical species Q. oleoides was differentiated from the three temperate species, and variation among species was greater than among populations. ? The trade-off between freezing tolerance and growth rate supports the range limit hypothesis. Limited variation within species indicates that the distributions of species may be driven more strongly by broad climatic factors than by highly local conditions.     

A PCR method for detection of plant meals from the guts of insects

The feeding behaviour of insects is a difficult ecological interaction to study. To date, entomologists have used biochemical and molecular techniques to identify the meals of predatory insects. We present here a molecular approach to identifying the DNA of plant species in the insect gut using the ribulose bisphosphate carboxylase gene large subunit (rbcL). A reference collection of 23 plant species from the southern Jordan Valley, Israel, was genetically characterized and employed. Insects belonging to eight different families were collected in the field along with the plants upon which they were found. After collection and prior to analysis, these insects were isolated on the plants they were found upon in the laboratory. This was to ensure that the insects had only one plant meal in their gut, as multiple plant meals would require additional techniques like cloning. A blind study was performed, genetically confirming plant DNA to species level from the processed gut contents of the insects. All reference plant species could be differentiated using a 157 bp long fragment of the rbcL gene. Plant DNA was identifiable, and the meal of the respective insect was accurately determined in each case. Analyses using experimentally fed crickets, Gryllodes hebraeus, determined that plant DNA was still detectable by PCR up to 12 h post-ingestion. This research proposes the application of molecular techniques for the identification of herbivorous insect feeding behaviour to increase understanding of plant–insect interactions.     

Host‐mediated shift in the cold tolerance of an invasive insect

While many insects cannot survive the formation of ice within their bodies, a few species can. On the evolutionary continuum from freeze‐intolerant (i.e., freeze‐avoidant) to freeze‐tolerant insects, intermediates likely exist that can withstand some ice formation, but not enough to be considered fully freeze tolerant. Theory suggests that freeze tolerance should be favored over freeze avoidance among individuals that have low relative fitness before exposure to cold. For phytophagous insects, numerous studies have shown that host (or nutrition) can affect fitness and cold‐tolerance strategy, respectively, but no research has investigated whether changes in fitness caused by different hosts of polyphagous species could lead to systematic changes in cold‐tolerance strategy. We tested this relationship with the invasive, polyphagous moth, Epiphyas postvittana (Walker). Host affected components of fitness, such as larval survivorship rates, pupal mass, and immature developmental times. Host species also caused a dramatic change in survival of late‐instar larvae after the onset of freezing—from less than 8% to nearly 80%. The degree of survival after the onset of freezing was inversely correlated with components of fitness in the absence of cold exposure. Our research is the first empirical evidence of an evolutionary mechanism that may drive changes in cold‐tolerance strategies. Additionally, characterizing the effects of host plants on insect cold tolerance will enhance forecasts of invasive species dynamics, especially under climate change.     

Clinal variation in freezing tolerance among natural accessions of Arabidopsis thaliana

Low temperature represents a form of abiotic stress that varies predictably with latitude and altitude and to which organisms have evolved multiple physiological responses. Plants provide an especially useful experimental system for investigating the ecological and evolutionary dynamics of tolerance to low temperature because of their sessile lifestyle and inability to escape ambient atmospheric conditions. Here, intraspecific variation in freezing tolerance was investigated in Arabidopsis thaliana by conducting freezing tolerance assays on 71 accessions collected from across the native range of the species. Assays were performed at multiple minimum temperatures and on both cold-acclimated and non-cold-acclimated individuals. Considerable variation in freezing tolerance was observed among accessions both with and without a prior cold-acclimation treatment, suggesting that differences among accessions in cold-acclimation capacity as well as differences in intrinsic physiology contribute to variation in this phenotype. A highly significant positive relationship was observed between freezing tolerance and latitude of origin of accessions, consistent with a major role for natural selection in shaping variation in this phenotype. Clinal variation in freezing tolerance in A. thaliana coupled with considerable knowledge of the underlying genetics and physiology of this phenotype should allow evolutionary genetic analysis at multiple levels.