Skin ice nucleators and glycerol in the freezing-tolerant frog <Emphasis Type="Italic">Litoria ewingii</Emphasis>

The brown tree frog (Litoria ewingii) is the only known Southern Hemisphere vertebrate that can survive full-body freezing. Freezing challenges living organisms in many ways, with ice formation in the body producing a suite of physical and metabolic stresses which can damage cells and tissues. The present study looked at two mechanisms that address some of these stresses: cryoprotectants and ice nucleating agents (INAs). Skin secretions from L. ewingii were sampled along with microhabitat substrate and tested for the presence of INAs, which help control ice formation in the body. L. ewingii plasma was tested for seasonal and freezing-induced changes in both glucose and glycerol, which may have a cryoprotective role in freezing-tolerant frogs. Glycerol levels increased on freezing and decreased on thawing, while glucose levels did not change on freezing but increased on thawing. This suggests that glycerol may be acting as a cryoprotectant, although levels are low compared to other frogs. A clear seasonal change was seen in INA activity, with greater activity in winter than in summer. While potent INAs came from the microhabitat substrate, this work has shown for the first time that skin secretions also contain active INAs.     

Experimental studies of ice nucleation in an Antarctic springtail (Collembola, Isotomidae)

Ice nucleation was studied in field-fresh and acclimated (4 degrees C without food for 11-20 days) samples of the springtail Cryptopygus antarcticus Willem (Collembola, Isotomidae) at Rothera Research Station, Adelaide Island on the Antarctic Peninsula. Ice nucleator activity (INA) was measured by a freezing droplet technique in addition to supercooling point (SCP) profiles and polyol, sugar, and water contents. Field and acclimated samples showed bimodal SCP distributions with a distinct high group (HG; mean SCP -8 to -10 degrees C) and low group (LG: mean SCP -23 to -25 degrees C), which were significantly different. Acclimation at 4 degrees C increased the proportion of individuals in the LG relative to that in the HG without significant effects on the mean SCP of both groups. INA of the HG was significantly greater than that of the LG, and acclimation further reduced the INA of the LG. The number of active ice nucleator agents (INAs) calculated for the HG of field samples increased by 23-100 times over the temperature range -5 to -8 degrees C compared to only 7 times for the LG over the same range. These differences were accentuated in the acclimation experiments. Glucose and galactose were the main carbohydrates in both field and acclimated springtails, with the latter compound occurring in almost twice the concentration in the LG compared with that in the HG. Acclimation reduced the concentration of both compounds (glucose by 77% and galactose by 54%), whereas water content increased significantly. Digestion of food may have continued during acclimation at 4 degrees C, which could reduce the LG INA. Lowering of temperature over time is more likely to elicit a cold hardening response than constant temperature acclimation. INA numbers calculated at the nucleation temperatures for C. antarcticus samples were higher in the LG than in the HG. However, inactivation of INAs may be a key mechanism underlying cold hardening in this species, either by sequestration within the cellular matrix or by being only seasonally active.     

Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA

We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4′-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9°C per modification when the IPN was added as a dangling end and up to 6.7°C per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8°C for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes.     

Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury

Ice nucleation temperatures of individual leaves were determined by a tube nucleation test. With this assay, a direct quantitative relationship was obtained between the temperatures at which ice nucleation occurred on individual oat (Avena sativa L.) leaves and the population sizes of ice nucleation active (INA) bacteria present on those leaves. In the absence of INA bacteria, nucleation of supercooled growth-chamber grown oat leaves did not occur until temperatures were below approximately −5°C. Both nucleation temperature and population size of INA bacteria were determined on the same individual, field-grown oat leaves. Leaves with higher ice nucleation temperatures harbored larger populations of INA bacteria than did leaves with lower nucleation temperatures. Log₁₀ mean populations of INA bacteria per leaf were 5.14 and 3.51 for leaves with nucleation temperatures of −2.5°C and −3.0°C, respectively. Nucleation frequencies (the ratio of ice nuclei to viable cells) of INA bacteria on leaves were lognormally distributed. Strains from two very different collections of Pseudomonas syringae and one of Erwinia herbicola were cultured on nutrient glycerol agar and tested for nucleation frequency at −5°C. Nucleation frequencies of these bacterial strains were also lognormally distributed within each of the three sets. The tube nucleation test was used to determine the frequency with which individual leaves in an oat canopy harbored large populations of INA bacteria throughout the growing season. This test also predicted relative frost hazard to tomato (Lycopersicon esculentum Mill) plants.     

The role of ice nucleation active bacteria in supercooling of citrus tissues

The frost sensitivity of Citrus sinensis in relation to the presence of biogenic ice nuclei was studied. In commercially managed citrus groves the ice nucleation active (INA) bacterium Pseudomonas syringae reached 6 × 10⁴ colony forming units (CFU) leaf⁻¹, a population sufficiently high to catalyze ice formation. However, a transient loss of bacterial nucleation activity was noticeable at subzero field temperatures, followed by resumption as temperatures rose. This loss was apparently due to a temporary transition of INA to ice nucleation inactive (INI) bacteria. Field application of Bordeaux mixture, copper hydroxide, streptomycin, and 2-hydroxypropylmethanethiolsulfonate (HPMTS), resulted in reduction of INA bacterial populations to detectability (≤ 10² CFU leaf⁻¹) limits. However, the corresponding reduction in ice nucleation events in treated samples as compared to controls at nucleation temperature ≥−3°C was not as dramatic. It ranged from approximately 7% in samples treated with the bactericide HPMTS, to 35% in samples treated with chemicals possessing combined bactericidal - fungicidal action (coppers). Since a quantitative relationship exists between ice nucleation events on individual leaves and the INA bacterial populations harbored by these leaves, these results suggest the co-existence of a bacterial and a proteinaceous, yet non-bacterial ice nucleating source in citrus, both active at ≥−3°C.     

The ice Nucleation Activity of Pseudomonas fluorescens Migula and its Inhibition by Various Chemicals

The ice nucleation activity (INA) of three strains of Pseudomonas fluorecens, nos 553, 554 and 606, isolated by the Institute for Pathogen Diagnostics in Ascherleben, Germany, was determined. Under equal growth conditions and at given test temperatures the ice nucleation frequency spectra of the isolates differed slightly. The fraction of cells which acted as ice nuclei increased with falling temperatures. Below ?5°C the nucleation frequency rose from 10^-8 to 10^-3. Between, 0 and ?10°C only a fraction of approximately 2 to 5 × 10^-3 cells performed ice nucleation activity. Fifteen newly synthesized chemicals showed no or only a very slight intrinsic INA at ?5°C and ?7°C. The compounds were used as antinucleators against INA-exhibiting bacteria. In INA-exhibiting suspensions of isolate 553 bacterial ice nuclei were reduced after treatment with the 15 compounds. Dependent on the compounds, a nucleation frequency of ?8.32 to ?5.10 was detected at ?5°C. At ?7°C, the frequency amounted to ?7.89 to ?5.05. As the temperature was lowered to ?10°C in bacterial suspensions which were treated with 9 (of the 15) compounds, a remainder of 1.79 to 5.91 × 10^-6 cells retained ice nucleation activity. The most pronounced inhibitory effect was noted for the compounds 1989/6255, 1989/6436 and 1990/6158. In a 10-fold dilution of isolate 553 the compound 1989/6153 inhibited ice nucleation between 0 and 10°C so strongly that it was about 100 times below the control. The ‘tube-freezing’ method showed that on excised corn leaves treated with 1989/6259 and 1990/6155, the bacterial INA decreased while the super-cooling was more pronounced. ‘Frostgard’, 1986/6205, 1986/6199 and 1989/6259 inhibited most INA-exhibiting bacteria on corn seedlings. Compared to inoculated plants, a significantly higher percentage of treated plants survived at ?2 and ?3°C.     

Effecten van een integrale wijkaanpak genaamd ‘Even Buurten’ op de (gezondheidsgerelateerde) kwaliteit van leven en welzijn van ouderen

Integrated neighborhood approaches (INAs) are increasingly advocated to support community-dwelling older people; their effectiveness however remains unknown. We evaluated INA effects on older people’s (health-related) quality of life (HRQoL) and well-being in Rotterdam. We used a matched quasi-experimental design comparing INA with “usual” care and support. Community-dwelling people (aged ≥70) and control subjects (n?=?186 each) were followed over a one-year period (measurements at baseline, 6 and 12 months). Primary outcomes were HRQoL (EQ-5D-3L, SF-20) and well-being (SPF-IL). The effect of INA was analysed with generalized linear mixed modeling of repeated measurements, using both an “intention to treat” and “as treated” approach. The results indicated that pre-intervention participants were significantly older, more often single, less educated, had lower incomes and more likely to have ≥1 disease than control subjects; they had lower well-being, physical functioning, role functioning, and mental health. No substantial difference in well-being or HRQoL was observed between the intervention and control group after 1 year. The lack of effects of INA highlights the complexity of integrated care and support initiatives.     

Influence of water activity on the ice-nucleating activity of Pseudomonas syringae

Pseudomonas syringae is known as a biological ice-nucleating agent. The bacterium has the unusual property of increasing the temperature at which water freezes by a few degrees. However, the ice-nucleating activity (INA) always remains lower for in vitro cultivated cells, than for cells grown in planta. We examined the effects of the hydrophobic environment and of water availability, on the in vitro growth and INA of P. syringae. The hydrophobic environment was modified by addition of fatty acids, vegetable oils or silicone oil to the culture medium. Addition of olive oil (1%), or traces of silicone oil in the culture medium had a positive effect upon the expression of INA. Variations in water activity from 0.990 to 0.988 by addition of sugar beet fibres or sodium chloride in the culture medium were followed by an increase in INA. This study suggested that control of the medium’s water activity must be considered as an important parameter for optimization of INA in P. syringae. Received 16 June 1998/ Accepted in revised form 02 September 1999     

多粘类芽胞杆菌KM2501-1杀南方根结线虫活性产物研究

【目的】南方根结线虫(Meloidogyne incognita)是一种危害严重的土传性植物病原线虫,给农业生产造成了巨大的经济损失,前期研究发现多粘类芽胞杆菌(Panebacillus polymyxa) KM2501-1具有很好的温室防治南方根结线虫效果,且可产生多种挥发性杀线虫活性物质,但对其非挥发性产物是否有杀线虫活性没有研究。本研究拟进一步分离鉴定其产生的杀线虫活性代谢产物,发掘新的杀线虫药物。【方法】对菌株KM2501-1进行液体发酵并离心收集发酵上清液,通过硅胶柱层析、高效液相色谱分离等方法得到高纯度的杀线虫活性物质,并通过液相色谱质谱联用分析、核磁共振等技术鉴定杀线虫活性物质的结构。【结果】生物活性检测显示,多粘类芽胞杆菌KM2501-1发酵上清液具有较强的南方根结线虫触杀活性,并能有效抑制南方根结线虫卵孵化,体外杀线虫效率高达87.66%,抑制卵孵化效率达92.26%。结构鉴定结果显示多粘类芽胞杆菌产生的杀线虫活性物质为环二肽类物质cyclo (Pro-Phe),800 mg/L的cyclo(Pro-Phe)杀线虫效率达84.75%。进一步的显微观测结果表明,与对照组相比,活性物质cyclo(Pro-Phe)处理后的根结线虫肠道组织紊乱、结构发生破坏。【结论】多粘类芽胞杆菌KM2501-1产生的cyclo (Pro-Phe)是一个具有杀线虫新功能的活性物质,其可能通过破坏线虫肠道杀死线虫。     

Effect of an Ice-Nucleating Activity Agent on Subzero Survival of Nematode Juveniles

Juveniles of five species of nematodes, Caenorhabditis elegans, Panagrellus redivivus, Pratylenchus agilis, Pristionchus pacificus, and Distolabrellus veechi, were added to solutions with (treatment) and without (control) a commercial ice-nucleating activity (INA) agent. Ten-microliter droplets of the solutions containing the juveniles were placed on glass microscope slides and transferred to a temperaturecontrolled freeze plate where the temperature was reduced to -6 to -8 °C. At this temperature, the droplets containing the INA agent froze while those without the agent remained liquid. After 2 minutes, the temperature of the plate was raised to 24 °C, and the slides were examined with a light microscope to determine the viability of the juveniles. The results showed that usually most juveniles (43% to 88%, depending on species) in solutions that did not contain the INA agent (controls) were active, indicating that the juveniles were capable of supercooling and were thereby protected from the subzero temperatures. Alternatively, less than 10% of the juveniles that had frozen for 2 minutes in solutions containing the INA agent remained viable, indicating that inoculative freezing of the solution was lethal to the supercooled juveniles. Our results suggest that, in geographical areas where winter temperatures may not be sufficiently low or sustained to freeze soil, the addition of an INA agent may help induce ice nucleation and thereby reduce the populations of nematode species that are unable to survive when the soil solution is frozen.     

Bacterial ice nucleation: a factor in frost injury to plants

Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia herbicola (Löhnis) Dye are sufficient to incite frost injury to sensitive plants at −5°C. The ice nucleation activity of the bacteria occurs at the same temperatures at which frost injury to sensitive plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about −2°C, whereas the leaves themselves, i.e. without INA bacteria, contain nuclei active only at much lower temperatures. The temperature at which injury to plants occurs is predictable on the basis of the ice nucleation activity of leaf discs, which in turn depends on the number and ice nucleation activity of their resident bacteria. Bacterial isolates which are able to incite injury to corn at −5°C are always active as ice nuclei at −5°C. INA bacteria incited frost injury to all of the species of sensitive plants tested.     

Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203

Summary The ice nucleation gene inaZ, from Pseudomonas syringae S203, was manipulated to produce a series of defined rearrangements in its coding sequence without changing the reading frame. The effects of these mutations on the ice nucleation phenotype were determined in a heterologous host, Escherichia coli K12. Deletions which disrupted the periodicity of 16 codons, in a repetitive region of inaZ, caused the frequencies of ice nuclei in the bacterial population to be significantly depressed; the nuclei with thresholds at warmer temperatures were most affected. In contrast, when the periodicity was left intact, deletions and duplications in the same region had only slight effects on nucleation activity. Deletions removing part or all of one of the nonrepetitive regions (that encoding the amino-terminal domain of the InaZ protein) did not abolish nucleation activity, but caused it to be limited to cooler threshold temperatures. In contrast, the non-repetitive carboxy-terminal domain of the InaZ protein was shown to be essential for ice nucleation at all temperatures. The differential requirements (for periodicity, and for the amino-terminus) in forming nuclei with different thresholds may be significant for understanding what determines the threshold temperature of an ice nucleus.     

Characterization and Quantification of Intrinsic Ice Nucleators in Winter Rye (Secale cereale) Leaves

Extracellular ice formation in frost-tolerant organisms is often initiated at specific sites by ice nucleators. In this study, we examined ice nucleation activity (INA) in the frost-tolerant plant winter rye (Secale cereale). Plants were grown at 20[deg]C, at 5[deg]C with a long day, and at 5[deg]C with a short day (5[deg]C-SD). The threshold temperature for INA was -5 to -12[deg]C in winter rye leaves from all three growth treatments. Epiphytic ice nucleation-active bacteria could not account for INA observed in the leaves. Therefore, the INA must have been produced endogenously. Intrinsic rye ice nucleators were quantified and characterized using single mesophyll cell suspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in mesophyll cell suspensions exhibited a threshold ice nucleation temperature of -7[deg]C and occurred infrequently at the rate of one nucleator per 105 cells. Rye cells were treated with chemicals and enzymes to characterize the ice nucleators, which proved to be complexes of proteins, carbohydrates, and phospholipids, in which both disulfide bonds and free sulfhydryl groups were important for activity. Carbohydrates and phospholipids were important components of ice nucleators derived from 20[deg]C leaves, whereas the protein component was more important in 5[deg]C-SD leaves. This difference in composition or structure of the ice nucleators, combined with a tendency for more frequent INA, suggests that more ice nucleators are produced in 5[deg]C-SD leaves. These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.     

Synergism of imidacloprid and entomopathogenic nematodes against white grubs: the mechanism

Entomopathogenic nematodes and the chloronicotinyl insecticide, imidacloprid, interact synergistically on the mortality of third-instar white grubs (Coleoptera: Scarabaeidae). The degree of interaction, however, varies with nematode species, being synergistic for Steinernema glaseri (Steiner) and Heterorhabditis bacteriophora Poinar, but only additive for Steinernema kushidai Mamiya. The mechanism of the interaction between imidacloprid and these three entomopathogenic nematodes was studied in the laboratory. In vials with soil and grass, mortality, speed of kill, and nematode establishment were negatively affected by imidacloprid with S. kushidai but positively affected with S. glaseri and H. bacteriophora. In all other experiments, imidacloprid had a similar effect for all three nematode species on various factors important for the successful nematode infection in white grubs. Nematode attraction to grubs was not affected by imidacloprid treatment of the grubs. Establishment of intra-hemocoelically injected nematodes was always higher in imidacloprid-treated grubs but the differences were small and in most cases not significant. The major factor responsible for synergistic interactions between imidacloprid and entomopathogenic nematodes appears to be the general disruption of normal nerve function due to imidacloprid resulting in drastically reduced activity of the grubs. This sluggishness facilitates host attachment of infective juvenile nematodes. Grooming and evasive behavior in response to nematode attack was also reduced in imidacloprid-treated grubs. The degree to which different white grub species responded to entomopathogenic nematode attack varied considerably. Untreated Popillia japonica Newman (Coleoptera: Scarabaeidae) grubs were the most responsive to nematode attack among the species tested. Untreated Cyclocephala borealis Arrow (Coleoptera: Scarabaeidae) grubs showed a weaker grooming and no evasion response, and untreated C. hirta LeConte (Coleoptera: Scarabaeidae) grubs showed no significant response. Chewing/biting behavior was significantly increased in the presence of nematodes in untreated P. japonica and C. borealis but not in C. hirta and imidacloprid-treated P. japonica and C. borealis. Our observations, however, did not provide an explanation for the lack of synergism between imidacloprid and S. kushidai.     

The occurrence of ice nucleation activity in pathogenic bacterial species

We tested the nucleation activity (INA) of 122 strains of plant pathogenic bacteria (12 varieties and 15 subspecies) stored in collection of the University of Göttingen (GSPB). The strains are isolates from diverse host plants and different geographic regions. One-hundred and seven isolates belong to the Pseudomonads, nine to the genus Erwinia and six to the Xanthomonas. The INA was analysed by ?3°, ?5°, ?7° and ?9°C. The observed value of INA cells ranged from non-detectable to a maximum concentration of ice nuclei in a range from ?7.85 at ?5°C to ?2.63 at ?3°C in 1.82 × 10³ cfu to 3.3 × 10³ cfu per ml. The data indicated that 71 (58.2%) of the 122 strains had INA cells, and 51 (41.8%) were inactive. The highest amount of strains with INA cells we found in Pseudomonads (69). In comparison only one strain was active at Erwinia and at Xanthomonas, 46 strains were isolated from the genus Phaseolus vulgaris and 6 from the genus Beta vulgaris. The other isolates with ice active cells belonged to the 13 other plant species. The 51 inactive pathovars were isolated from 21 different culture plants. The pathogens under test were isolated in 16 different countries, mainly in Germany and USA.     

Reduction of insect cold-hardiness using ice-nucleating active fungi and surfactants

The supercooling point (SCP) of an insect model, the lady beetle Hippodamia convergens Guérin-Menéville (Coleoptera, Coccinellidae) was markedly elevated by treatment with aqueous suspensions of the filamentous, ice nucleation active (INA) fungi Fusarium avenaceum and slightly elevated by Fusarium acuminatum. Addition of the surfactant Tween 80 to the fungal suspensions further reduced the supercooling capacity of adult beetles. When used alone the surfactant Triton X-100 produced a greater SCP elevation than Tween 20 or Tween 80. The emulsifier gum arabic was ineffective in elevating beetle SCPs when applied alone and when added to INA fungal preparations it decreased their efficacy. Aqueous suspensions of both viable sporulating and viable pleomorphic (a permanent, degenerative, nonsporulating cultural state) forms of both fungal species were more effective in elevating the SCP than killed preparations except for the pleomorphic F. acuminatum suspension in which the killed form was slightly more active. Application of INA fungi applied in combination with surfactants may be useful in the development of methods for the biological control of overwintering freeze-susceptible insect pests by decreasing their capacity to avoid lethal freezing by supercooling.     

Characteristics and Importance of Heterogeneous ice Nuclei Associated With Citrus Fruits

The freezing behaviour and relative importance of heterogeneousice nuclei in affecting supercooling of Citrus sinensis fruitswere studied. The size of an ice nucleation active (INA) Pseudomonassyringae pool inhabiting fruits was positively correlated withthe nucleation temperature (NT) of the plant tissue, with amean of 369 log colony forming units (CFU) fruit^–1 atNT–25C. The INA bacterial pool was responsible for 23%of the nucleation events occurring at this temperature, and29% were attributed to an additional nucleating source. Thelatter was sensitive to bacterial ice nucleation inhibitors,it occupied a different microniche from that of P. syringaenuclei, yet was neither a fungus nor any of the bacterial strainsever reported as active. Treatment with an ice nucleation inactivebacterium antagonistic to INA bacteria, a lentil lectin, a protease,and guanidine reduced mean nucleation temperatures (MNT) offruits to –258, –266, –421, and–452C,respectively, compared to a MNT–167C for the controls.Thus, the citrus-associated nucleator apparently contained activeproteinaceous components but was void of carbohydrate-like groupsreportedly encountered at or near the bacterial ice nucleatingsite. Despite the different origins of citrus nuclei, bacterialnucleation inhibitors reduced nucleation in the field by 50%at NT––25C, an effect probably exerted throughthe proteinaceous site possessed by both nuclei. Key words: Citrus, ice nucleation, INA bacteria, supercooling     

聚乙烯醇-海藻酸盐共固定化冰核活性细菌——黄单胞菌TS206的研究

冰核活性细菌固定化在食品冷冻浓缩中的应用具有重要意义,冰核活性和抗渗漏能力是衡量其性能的两个重要技术指标。研究采用PVA和海藻酸盐作为固定化载体,通过两者优良性能的互补而建立对冰核活性细菌Xanthomonas ampelina TS206的共固定化技术。结果表明,细胞投入量对冰核活性有较大影响,基础固定化条件对固定化技术指标的综合评分影响程度大小的顺序依次为：海藻酸钠浓度＞硼酸浓度＞PVA浓度＞CaCl2浓度,各因素的较优水平是：海藻酸钠浓度1%,硼酸浓度5%,PVA浓度8%,CaCl2浓度1.1%;研究还发现冰核活性与固定化凝胶珠的添加量正相关,与固化时间相关性较小,渗漏量受固定化凝胶珠的添加量和固化时间影响不显著。     

Gut Colonization by an Ice Nucleation Active Bacterium,Erwinia(Pantoea)ananasReduces the Cold Hardiness of Mulberry Pyralid Larvae

To evaluate the suitability of using ice nucleation active (INA) bacteria for the biological control of insect pests, the supercooling point (SCP) of larvae of mulberry pyralid,Glyphodes duplicalis,and silkworm,Bombyx mori,ingesting INA strains ofErwinia(Pantoea)ananasandPseudomonas syringaewas determined. Mean SCP of the guts of silkworm larvae ingesting INA strains ofE. ananasranged from −2.5 to −2.8°C, being 5°C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain ofE. ananas,which can grow well in the gut, was −4.7°C at 3 days after treatment, being 6.5°C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain ofP. syringae,which cannot grow in the gut, was −9.0°C at 3 days after treatment, rising by only 2.5°C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain ofE. ananasfroze and eventually died when exposed to −6°C for 18 h, while only 36% of the larvae ingesting the INA strain ofP. syringae,or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains ofE. ananasreduced remarkably the cold hardiness of the insects. These findings suggest that INA strains ofE. ananascould be effective as a potential biological control agent of insect pests.