细菌冰核提高印度谷螟过冷却点的研究

印度谷螟（Ｐｌｏｄｉａｉｎｔｅｒｐｕｎｃｔｅｌｌａ）是一种不耐结冰的昆虫,在冬季它通过降低过冷却点以避免结冰。现已查明,冰核活性细菌能显著提高植物的过冷却点,导致许多作物在较高的温度下发生霜冻害。本文也评明细菌冰核能显著提高印度谷螟幼虫的过冷却点。对照的平均过冷却点是－１７．６℃;分别用０．１ｇ和１ｇ细菌冰核与１ｋｇ面粉混合后进行处理,平均过冷却点分别比对照提高了１２．８℃和１３．６℃。研究结果支持这样的观点：细菌冰核有可能成为一种在冬季使用的、杀灭不耐结冰害虫的生物制剂。     

冰核真菌削弱赤拟谷盗抗寒力的初步研究

赤拟谷盗Tribolium castaneum是不耐结冰的害虫,在冬季它通过降低过冷却点以避免结冰造成的致命伤害。冰核活性细菌能显著提高昆虫的过冷却点,使之在较高的零下温度发生结冰。试验证明冰核活性真菌也能显著提高赤拟谷盗的过冷却点。对照组平均过冷却点为-14.9℃。用10 g/L的冰核真菌制剂喷洒虫体,风干后测定,平均过冷却点提高到-4.8℃。用0.1 g/L处理后至少在7天内过冷却点保持较高。这些结果表明冰核真菌可能成为一种在冬季使用的、防治不耐结冰害虫的促冻杀虫剂。     

不同冷藏条件对食蚜瘿蚊过冷却点和冰点的影响

【目的】食蚜瘿蚊Aphidoletes aphidimyza(Rondani)是一种捕食性天敌,其幼虫对蚜虫具有较好的控制潜能。研究不同冷藏条件对食蚜瘿蚊耐寒能力的影响,为食蚜瘿蚊的低温冷藏技术提供了理论依据。【方法】利用热电偶原理,测定食蚜瘿蚊在不同冷藏条件下的过冷却点和结冰点。【结果】25℃条件下,食蚜瘿蚊各虫态的过冷却点及结冰点存在显著差异（P<0.05）,其中2龄幼虫过冷却点（﹣26.18℃）和结冰点（﹣24.98℃）最低,雄成虫过冷却点（﹣22.95℃）和结冰点（﹣21.86℃）最高。在不同变温条件下冷藏食蚜瘿蚊蛹,其过冷却点在冷藏10、20和30 d时均上升且高于对照,冷藏40 d时低于对照;同一冷藏时长下不同冷藏方式间过冷却点的差异均不显著（P>0.05）。食蚜瘿蚊蛹在5℃条件下冷藏50d,每天定时7℃中断4h的结冰点（﹣23.09℃）最低,冷藏30d的结冰点（﹣21.15℃）最高;食蚜瘿蚊蛹在5℃条件下冷藏40 d,每天定时9℃中断4 h的结冰点（﹣22.66℃）最低,冷藏20 d时的结冰点（﹣20.95℃）最高;同一冷藏时长下3种冷藏方式间结冰点差异均不显著（P>0.05）。【结论】食蚜瘿蚊2龄幼虫的耐寒能力最强,雌成虫的耐寒能力要高于雄成虫;变温贮存和恒定低温均适合食蚜瘿蚊的低温贮存。     

冰核真菌研究进展

水在０℃以下仍未结冰的现象称为水的过冷却作用,小体积纯净水的过冷却温度可接近－４０℃。引起水从液态向固态转变的物质称冰核,不同种类冰核催化活性差异很大,非生物冰核可在－１０℃左右诱发小体积水结冰,而自然界中活性最强的冰核来自生物体,已报道某些细菌（如Pseudomonas syringae和Erwinia hericola的一些菌株）可产生在－１℃～－２℃下催化水结冰的冰核。研究者将一些可以产生在－５℃以上具有冰核活性冰核的生物称为冰核生物。自１９７４年Ｍａｋｉ［１］首次从赤杨树叶中分离到在－２℃～－５℃下诱发植物结冰发生霜冻的…     

亚洲柑橘木虱和柚喀木虱过冷却点和体液结冰点测定

亚洲柑橘木虱Diaphorina citri Kuwayama和柚喀木虱Cacopsylla citrisuga YangLi均为柑桔上的重要害虫,前者为黄龙病的主要媒介昆虫,后者也已证实为黄龙病菌的携带者。本文对这两种木虱各龄若虫和成虫的过冷却点和体液结冰点进行了测定,结果表明,柑橘木虱过冷却点和冰点均以1龄若虫最低,平均值分别为-26.32℃和-26.22℃;成虫的过冷却点和冰点最高,分别为-19.60℃和-18.71℃;其它虫态过冷却点和冰点由低到高依次为2龄若虫、4龄若虫、5龄若虫、3龄若虫。柚喀木虱过冷却点和冰点也均以1龄若虫最低,平均值分别为-25.30℃和-25.08℃;5龄若虫的过冷却点和冰点最高,平均分别为-19.50℃和-17.69℃;其它虫态过冷却点和冰点由低到高依次为2龄若虫、3龄若虫、4龄若虫、成虫。两种木虱的比较结果表明,各发育阶段的表现不一致,柚喀木虱的3龄若虫和成虫过冷却点显著低于柑桔木虱,而4龄、5龄若虫则显著高于柑桔木虱;3龄若虫体液结冰点也显著低于柑桔木虱,但1龄、4龄却显著高于柑桔木虱。柑桔木虱雌雄成虫的过冷却点和体液结冰点差异不显著。研究结果表明两种木虱的过冷却点、体液结冰点都较低,因而可能具有较强的耐寒能力。     

西藏飞蝗各发育阶段的耐寒性

西藏飞蝗Locusta migratoria tibetensis Chen是青藏高原的重要农牧业害虫。对该虫各发育阶段的过冷却点和结冰点的测定表明,西藏飞蝗卵的过冷却点和结冰点为最低,分别为-22.02℃、-16.36℃,4龄蝻的过冷却点和结冰点最高,分别是-6.46℃和-5.05℃,西藏飞蝗在甘孜以卵越冬。     

烟夜蛾滞育蛹和非滞育蛹的耐寒性

对烟夜蛾Helicoverpa assultaGuen?e不同日龄滞育蛹和非滞育蛹的过冷却点和结冰点测定结果表明,在预蛹至5日龄蛹期,过冷却点和结冰点均逐渐下降。在预蛹期和3日龄蛹期,滞育蛹的过冷却点与非滞育蛹差异不显著;5日龄蛹时,滞育蛹和非滞育蛹的过冷却点分别下降至(-15.7±1.8)℃和(-13.5±1.2)℃,结冰点分别降至(-12.7±2.7)℃和(-9.5±1.3)℃,两类蛹间的差异均达极显著水平。自然越冬后,滞育蛹的存活率显著高于非滞育蛹,在土壤不同深处越冬的蛹的存活率存在差异,距土表15cm深的蛹存活率最高。     

内蒙古河套灌区玉米与向日葵霜冻的关键温度

以玉米和向日葵幼苗为试验材料,利用MSX-2F人工模拟霜箱系统模拟自然霜冻的降温过程,通过测定幼苗叶片的温度,观测植株冻伤、死亡情况,观测河套灌区两种主要作物玉米和向日葵的过冷却点、结冰点温度,确定以上两种作物受冻的临界温度。研究结果表明:(1)玉米幼苗全部冻死的最低温度为≤-5℃;向日葵幼苗全部冻死的最低温度为≤-6℃;(2)玉米幼苗的过冷却点主要分布在-3.5—-5℃之间,而向日葵幼苗的过冷却点主要分布在-4.0—-5.5℃之间;(3)玉米叶龄、高度与组织过冷能力呈现极显著正相关,与过冷却点温度呈现极显著的负相关,与结冰点呈现显著的负相关。以上结果表明:向日葵的抗寒能力比玉米强;玉米在一定叶龄范围内,苗龄越大,高度越高,其过冷却点越低,结冰点越低,抗寒性越强。     

不同寄主植物对二点委夜蛾幼虫抗寒性的影响

【目的】探讨取食不同寄主植物对二点委夜蛾Athetis lepigone(Mschler)老熟幼虫抗寒性的影响。【方法】室内分别用棉花、花生、大豆、甘薯及玉米的幼嫩叶片饲养二点委夜蛾4龄幼虫6 d至老熟,测定二点委夜蛾老熟幼虫过冷却点、结冰点、鲜重、含水量、脂肪、糖原和山梨醇含量。【结果】二点委夜蛾老熟幼虫取食不同寄主植物叶片后结冰点、鲜重、脂肪含量、糖原含量有显著差异。取食大豆叶片后结冰点最高(-2.80℃),鲜重最低(0.056 g),脂肪含量最低(12.47%);取食棉花叶片后结冰点最低(-5.45℃),脂肪含量最高(32.12%),糖原含量最高(54.07 mg/g);取食玉米叶片后鲜重最高(0.118 g)。取食不同寄主植物叶片的二点委夜蛾老熟幼虫过冷却点、含水量、山梨醇含量没有显著差异。【结论】寄主植物对二点委夜蛾老熟幼虫过冷却点没有显著影响,而影响到其体重及脂肪和糖原含量。     

柑桔大实蝇老熟幼虫和蛹过冷却点和结冰点的测定

为明确柑桔大实蝇Bactrocera minax (Enderlein)老熟幼虫和蛹的抗寒能力,对柑桔大实蝇老熟幼虫、滞育蛹和滞育解除蛹的过冷却点和结冰点进行了测定。结果显示,同一虫态不同个体间其过冷却点和结冰点具有不同程度的变异,但其变异范围均符合正态分布。柑桔大实蝇不同发育状态间的过冷却点和结冰点均具有显著差异,其中,已解除滞育进入发育状态的蛹的过冷却点和结冰点最低,分别为-11.15℃和-9.76℃;其次为滞育蛹,分别为-8.93℃和-7.63℃;而老熟幼虫最高,分别为-7.09℃和-3.43℃。此外,老熟幼虫的结冰点和过冷却点之间的差值最大为3.24℃,显著高于滞育蛹（1.30℃）和滞育解除蛹（1.39℃）。表明,倒春寒对已进入发育状态的柑桔大实蝇蛹不会造成不利影响。     

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.     

Maintenance of the supercooled state in the gut fluid of overwintering pyrochroid beetle larvae, Dendroides canadensis : role of ice nucleators and antifreeze proteins

  The effect of gut fluid ice nucleators and antifreeze proteins on maintenance of supercooling was explored in fire-colored beetle larvae, Dendroides canadensis, via seasonal monitoring of supercooling points, antifreeze protein activity and ice nucleator activity of gut fluid and/or larvae. During cold hardening in the field, freeze-avoiding larvae evacuated their guts and depressed larval supercooling points. Analysis of gut fluid indicated supercooling points and ice nucleator activity decreased, whereas antifreeze protein activity increased as winter approached. Suspensions of bacteria isolated from guts of feeding larvae collected in spring/summer had higher supercooling points than those from midwinter-collected non-feeding larvae, suggesting bacterial ice nucleators are removed from midwinter gut fluid. The ice nucleation active bacterium Pseudomonas fluorescens was isolated from gut fluid of feeding larvae but was absent in winter. When mixed with purified D.␣canadensis hemolymph antifreeze proteins (structurally similar and/or identical to those in gut fluid), the cumulative ice nucleus spectra of P. fluorescens suspensions were shifted to lower temperatures indicating an inhibitory effect on the bacteria's ice-nucleating phenotype. By extending larval supercooling capacity, both gut clearing and masking of bacterial ice nucleators by antifreeze proteins may contribute to overwintering survival in supercooled insects. Accepted: 8 August 1996     

Overwintering adaptations of the stag beetle,Ceruchus piceus: removal of ice nucleators in the winter to promote supercooling

Summary Overwintering larvae and adults of the stag beetle,Ceruchus piceus, are freeze sensitive (i.e. cannot survive internal freezing). The most commonly described cold adaptation of freeze susceptible insects involves the production of antifreezes to promote supercooling, butCeruchus piceus larvae produced only low levels of antifreezes in the winter. However, by removing ice nucleators from the gut and hemolymph in the winter the larvae were able to depress their supercooling points from approximately –7°C in the summer to near –25°C in mid-winter. The ice nucleators present in the non-winter hemolymph were identified as lipoproteins. One of these lipoproteins with ice nucleator activity was purified using flotation ultracentrifugation and anion exchange (DEAE-Sephadex) chromatography.Removal of ice nucleators to promote supercooling in winter may be energetically preferable to costly production and maintenance of high, of-ten molar, concentrations of antifreeze. Obviously the ice nucleator must normally perform a function which the insect can spare over the winter. Hemolymph lipoproteins, which generally function in lipid transport, may fit this criterion during the winter period of reduced metabolic activity.Abbreviations LP I very low density lipoprotein - LP II low density lipoprotein - PAGE polyacrylamide gel electrophoresis - SCP supercooling point     

A method for quantitative determination of ice nucleating agents in insect hemolymph

The relationship between the concentration of insect hemolymph ice nucleators in samples of 0.9% NaCl solution and the supercooling points of the samples was determined by using a dilution technique. The supercooling points were only moderately reduced following dilution by a factor of up to 10³, whereas dilution beyond this point caused a marked drop in the supercooling points. The dilution factor corresponding to a 50% reduction in the nucleating activity of native hemolymph is taken as a measure of the concentration of ice nucleators in native hemolymph.This method was used to determine the concentration of ice nucleators in the hemolymph of Eurosta solidaginis larvae from Minnesota and Texas, acclimated to different temperatures. Significant levels of nucleators were found only in larvae from Minnesota, and +5 °C was found to be the optimal temperature for nucleator formation. This comparatively high temperature optimum is interpreted as a physiological adaptation, ensuring sufficient nucleator levels in the hemolymph by the time of the first exposure to freezing temperatures in the winter.     

Cryoprotective mechanisms in subtidally cultivated and intertidal blue mussels (mytilus edulis) from the Magdalen Islands, Québec

Centrifuged extracts from subtidally cultivated and intertidal blue mussels have higher supercooling points (up to -5.5°C) in winter than in summer (up to -12.5°C). The concentration of nucleators (as estimated by the dilution factor) is greater in winter than in summer in both groups. The nucleator concentration in the extracts of winter mussels is one to two orders of magnitude higher than that in the haemolymph of Norwegian mussels. Although these extracts show spilule-like growth of ice crystals, they caused no thermal hystersis. The seasonal variation of these cryoprotective mechanisms is similar for intertidal and cultivated mussels. However, in the spring, cultivated mussels have a lower supercooling point and a lower concentration of nucleators than their intertidal counterparts. This suggests that cultivated mussels decrease their cryoprotective capacity earlier than intertidal mussels.     

Maintenance of the supercooled state in overwintering pyrochroid beetle larvae, Dendroides canadensis : role of hemolymph ice nucleators and antifreeze proteins

Freeze-avoiding fire-colored beetle larvae, Dendroides canadensis, were monitored seasonally to explore the role of endogenous hemolymph ice nucleators and antifreeze proteins on the maintenance of supercooling. In preparation for overwintering, D. canadensis depressed hemolymph ice nucleator activity and increased thermal hysteresis activity [mean value circa 0. 5 °C (summer) versus circa 5 °C (midwinter)] resulting in decreased larval and hemolymph supercooling points [−7 °C (summer) versus −20 °C (midwinter)]. Results of gel filtration chromatography, flotation ultracentifugation and quantitative investigation of ice nucleator activity using hemolymph from summer and winter collected larvae strongly suggest that highly active protein and lipoprotein ice nucleators are removed in preparation for overwintering. Additions of either purified antifreeze proteins or midwinter hemolymph with high antifreeze protein activity to a mixture of protein or lipoprotein ice nucleators isolated from D. canadensis hemolymph inhibited the activity of these nucleators. This suggests that in addition to seasonal removal, inhibition of hemolymph ice nucleators by antifreeze proteins contributes to seasonal increases in hemolymph supercooling capacity. Accepted: 8 August 1996     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Ice crystallization by Pseudomonas syringae

Several bacterial species can serve as biological ice nuclei. The best characterized of these is Pseudomonas syringae, a widely distributed bacterial epiphyte of plants. These biological ice nuclei find various applications in different fields, but an optimized production method was required in order to obtain the highly active cells which may be exploited as ice nucleators. The results presented here show that P. syringae cells reduce supercooling of liquid or solid media and enhance ice crystal formation at sub-zero temperatures, thus leading to a remarkable control of the crystallization phenomenon and a potential for energy savings. Our discussion focuses on recent and future applications of these ice nucleators in freezing operations, spray-ice technology and biotechnological processes. Received: 21 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

Identification of ice-nucleating active Pseudomonas fluorescens strains for biological control of overwintering Colorado potato beetles (Coleoptera: Chrysomelidae)

Laboratory studies were conducted to identify ice-nucleating active bacterial strains able to elevate the supercooling point, the temperature at which freezing is initiated in body fluids, of Colorado potato beetles, Leptinotarsa decemlineata (Say), and to persist in their gut. Adult beetles fed ice-nucleating active strains of Pseudomonas fluorescens, P. putida, or P. syringae at 10(6) or 10(3) bacterial cells per beetle had significantly elevated supercooling points, from -4.5 to -5.7 degrees C and from -5.2 to -6.6 degrees C, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was -9.2 degrees C. When sampled at 2 and 12 wk after ingestion, only beetles fed P. fluorescens F26-4C and 88-335 still had significantly elevated supercooling points, indicating that these strains of bacteria were retained. Furthermore, beetle supercooling points were comparable to those observed immediately after ingestion, suggesting that beetle gut conditions were favorable not only for colonization but also for expression of ice-nucleating activity by these two strains. The results obtained from exposure to a single, low dose of either bacterial strain also show that a minimum amount of inoculum is sufficient for establishment of the bacterium in the gut. Persistence of these bacteria in Colorado potato beetles long after ingestion was also confirmed using a polymerase chain reaction technique that detected ice-nucleating active bacteria by virtue of their ina genes. Application of these ice-nucleating active bacteria to elevate the supercooling point of this freeze-intolerant insect pest could significantly reduce their winter survival, thereby reducing local populations and, consequently, crop damage.