Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae

The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at -2.5 degrees C, significantly higher than their parent strains (active at approx -10 degrees C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were -3 to -4 degrees C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of -7 degrees C, and more than 95% died after 12 h at -5 degrees C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields.     

Gut colonization by an ice nucleation active bacterium, Erwinia (Pantoea) ananas reduces 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 of Erwinia (Pantoea) ananas and Pseudomonas syringae was determined. Mean SCP of the guts of silkworm larvae ingesting INA strains of E. ananas ranged from -2.5 to -2.8 degrees C, being 5 degrees C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain of E. ananas, which can grow well in the gut, was -4.7 degrees C at 3 days after treatment, being 6.5 degrees C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain of P. syringae, which cannot grow in the gut, was -9.0 degrees C at 3 days after treatment, rising by only 2.5 degrees C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain of E. ananas froze and eventually died when exposed to -6 degrees C for 18 h, while only 36% of the larvae ingesting the INA strain of P. syringae, or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains of E. ananas reduced remarkably the cold hardiness of the insects. These findings suggest that INA strains of E. ananas could be effective as a potential biological control agent of insect pests.     

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.     

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.     

Interactions between desiccation resistance,host-plant contact and the thermal biology of a leaf-dwelling sub-antarctic caterpillar,Embryonopsis halticella (Lepidoptera: Yponomeutidae)

During May 1997 thermal tolerance, supercooling point (SCP), low and high temperature survival, and desiccation resistance were examined in field-fresh Embryonopsis halticella Eaton larvae from Marion Island. SCPs were also examined in acclimated larvae, larvae starved for seven days, larvae within their leaf mines, and in larvae exposed to ice crystals. Field-fresh larvae had a critical minimum temperature (CT(Min)) and critical maximum temperature (CT(Max)) of 0 degrees C and 39.7 degrees C, respectively. Mean SCP of field-fresh caterpillars was -20.5 degrees C and this did not change with starvation. Field-fresh larvae did not survive freezing and their lower lethal temperatures (70% mortality below -21 degrees C) and survival of exposure to constant low temperatures (100% mortality after 12hrs at -19 degrees C) indicated that they are moderately chill tolerant. SCP frequency distributions were unimodal for field-fresh larvae, but became bimodal at higher acclimation temperatures. Contact with ice-crystals caused an increase in SCP (-6.5 degrees C), but contact with the host plant had less of an effect at higher subzero temperatures. It appears that the remarkable desiccation resistance of the larvae is selected for by the absence of a boundary layer surrounding their host plant, caused by constant high winds. This suggests that the low SCPs of E. halticella larvae may have evolved as a consequence of pronounced desiccation resistance.     

冰核细菌在我国北方玉米上的消长动态规律

研究证明,菠萝欧文氏菌(Erwinia ananas)为我国北方玉米上优势冰核细菌种类,占总体INA细菌95 %以上。采用定量定性和定期取样分离方法,首次研究INA细菌在玉米上的消长动态规律。结果表明:玉米不同生长发育阶段是影响INA细菌在玉米上数量分布和消长动态变化的重要因素,以抽雄至成熟期间分布INA细菌数量最多,高达10 7～10 8CFU/ g,比拔节至抽雄期高出2～3个数量级,比苗期至拔节期高出4～5个数量级;同时还指出,玉米不同播期,对INA细菌数量分布影响显著,差异很大,其中INA细菌分布数量消长变化,以正常播种(1.9×10 7CFU / g) >中期播种(7.9×10 5CFU/ g) >晚期播种(5 .0×10 4 CFU/ g) ;研究指出,处于抽雄至成熟期间的玉米上分布的INA细菌数量最多,因此期间(8月上旬至9月下旬) ,气温逐渐降低,昼夜温差大,田间结露多,加上玉米处于成熟阶段,抗INA细菌能力弱,这些因素有利于低温(5～2 0℃范围内生长)型INA细菌生长繁殖,故使INA细菌分布数量最多     

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.     

Cold hardiness adaptations of codling moth, cydia pomonella

The cold hardiness adaptations of natural and laboratory reared populations of the codling moth, Cydia pomonella, were examined. Hemolymph, gut, and whole body supercooling points (SCPs), 24-h LT50s, polyhydroxy alcohol concentrations, hemolymph freezing points, and hemolymph melting points were determined. Nondiapausing codling moth larvae do not have appreciable levels of ice nucleators in the hemolymph or gut. Whole body supercooling points were higher than hemolymph supercooling points. For nondiapausing larvae, LT50s were significantly higher than both the whole body and the hemolymph supercooling points, indicating the presence of chill sensitivity. As the larvae left the food source and spun a cocoon, both hemolymph and whole body SCPs decreased. Diapause destined larvae had significantly lower hemolymph SCPs than nondiapausing larvae, but whole body SCPs were not significantly different from nondiapausing larvae of the same age. The LT50s of diapause destined and diapausing larvae were significantly lower than that of nondiapausing larvae. Codling moths are freezing intolerant, with LT50s close to the average whole body supercooling point in diapause destined and diapausing larvae. The overwintering, diapausing larvae effectively supercool to avoid lethal freezing by removal of ice nucleators from the gut and body without appreciable increase of antifreeze agents such as polyols or antifreeze proteins.     

Freezing activities of flavonoids in solutions containing different ice nucleators

In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 μL droplets at 0.2 °C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT(50)) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT(50) were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT(50) between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p<0.05) in the range of 1.4-4.2 °C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 °C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 °C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 °C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 °C (no compounds with significant difference at p<0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 °C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water.     

Cold hardiness of Apteropanorpa tasmanica Carpenter (Mecoptera: Apteropanorpidae)

There are very few investigations of cold hardiness in native Australian insects, and no such studies on insects from Tasmania. The Apteropanorpidae is a family of wingless Mecoptera endemic to Tasmania, comprising four described species that can be active in winter. In this study, we used infrared video thermography to investigate the physiological and behavioural responses of Apteropanorpa tasmanica to fast (0.3 degrees Cmin(-1)) and slow (0.03 degrees Cmin(-1)) rates of temperature reduction down to -10 degrees C. No adults survived cooling to -10 degrees C at either cooling rate. Mean supercooling points (SCPs) from fast cooling were -7.0 and -4.6 degrees C in 2002 and 2003, respectively. Ice nucleation always began in the abdomen, however, the position of nucleation within the abdomen varied between individuals. There was no relationship between SCP and body length, and no significant difference in SCPs between males and females. Stress-induced fast walking began when insects reached approximately -1.5 degrees C. Cooling rate did not affect the SCP or the temperature at which the behavioural stress response began. Adults survived for only short periods of time in the supercooled state; however they survived in the laboratory for up to 60 days at 4 degrees C, indicating their longevity at more favourable temperatures. Members of the Apteropanorpidae are adapted to the relatively warm, maritime climate currently influencing Tasmania.     

Effects of summer frost exposures on the cold tolerance strategy of a sub-Antarctic beetle

The sub-Antarctic beetle Hydromedion sparsutum (Coleoptera, Perimylopidae) is common locally on the island of South Georgia where sub-zero temperatures can be experienced in any month of the year. Larvae were known to be weakly freeze tolerant in summer with a mean supercooling point (SCP) around -4 degrees C and a lower lethal temperature of -10 degrees C (15min exposure). This study investigated the effects of successive freezing exposures on the SCP and subsequent survival of summer acclimatised larvae. The mean SCP of field fresh larvae was -4.2+/-0.2 degrees C with a range from -1.0 to -6.1 degrees C. When larvae were cooled to -6.5 degrees C on 10 occasions at intervals of 30min and one and four days, survival was 44, 70 and 68%, respectively. The 'end of experiment' SCP of larvae surviving 10 exposures at -6.5 degrees C showed distinct changes and patterns from the original field population depending on the interval between exposure. In the 30min interval group, most larvae froze between -6 and -8 degrees C, a depression of up to 6 degrees C from the original sample; all larvae were dead when cooling was continued below the SCP to -12 degrees C. In the one and four day interval groups, most larvae froze above -6 degrees C, showing no change as a result of the 10 exposures at -6.5 degrees C. As with the 30min interval group, some larvae froze below -6 degrees C, but with a wider range, and again, all were dead when cooled to -12 degrees C. However, in the one and four day interval groups, some larvae remained unfrozen when cooled to -12 degrees C, a depression of their individual SCP of at least 6 degrees C, and were alive 24h after cooling. In a further experiment, larvae were cooled to their individual SCP temperature at daily intervals on 10 occasions to ensure that every larva froze every day. Most larvae which showed a depression of their SCP of 2-4 degrees C from their day one value became moribund or died after six or seven freezing events. Survival was highest in larvae with SCPs of -2 to -3 degrees C on day one and which froze at this level on all 10 occasions. The results indicate that in larvae in which the SCP is lowered following sub-zero exposure, the depression of the SCP is greatest in individuals that do not actually freeze. Further, the data suggest that after successive frost exposures in early winter the larval population may become segregated into two sub-populations with different overwintering strategies. One group consists of larvae that freeze consistently in the temperature range from -1 to -3 degrees C and can survive multiple freeze-thaw cycles. A second group with lower initial SCPs (around -6 degrees C), or which fall to this level or lower (down to -12 degrees C) after freezing on one or more occasions, are less likely to freeze through extended supercooling, but more likely to die if freezing occurs.     

Cold hardiness of Habrobracon hebetor (Say) (Hymenoptera: Braconidae), a parasitoid of pyralid moths

The ectoparasitoid Habrobracon hebetor (Say) attacks stored-product infesting pyralid moths that are able to overwinter under extremely cold conditions. The extent to which H. hebetor can withstand these conditions is not known, but has important implications for the ability of H. hebetor to provide long-term suppression of these pests in temperate climates. We investigated basic cold hardiness aspects of a mutant eye-color strain of H. hebetor. Feeding larvae and adults of H. hebetor had supercooling points (SCPs) at temperatures higher than those of eggs and pupae. Mean SCPs of females and males were equivalent, as were those of naked and silk-encased pupae. Feeding on honey prior to being subjected to low temperatures significantly increased the SCP of adult females by approximately 8 degrees C. Mortality of pupae and adults increased significantly whenever the temperature dropped below the mean SCP, indicating that H. hebetor does not tolerate freezing. For pupae and adults exposed to -12 and -5 degrees C, the hourly mortality rate increased with time of exposure. Pupae and adults exposed to -12 degrees C for different time intervals showed high mortality after only 1d of exposure. At -5 degrees C, none survived 12d of exposure. A better understanding of how well this parasitoid tolerates low temperatures will be useful in evaluating its potential as a biological control agent of stored-product moths in temperate regions.     

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.     

Influence of soil moisture on supercooling capacity and associated physiological parameters of overwintering larvae of rice stem borer

Influence of soil moisture on the supercooling capacity and associated physiological parameters of overwintering larvae of the rice stem borer Chilo suppressalis was examined by exposing larvae to soil moistures of 25, 50, 75 and 100% of saturated soil water content (SSWC) at ambient temperature for 30 d from December 2007 to January 2008 in Beijing, China. At the end of the exposure, supercooling points (SCPs) varied significantly among the treatments, the lowest being in the larvae exposed to soil moisture of 25% SSWC. Fresh weight was significantly higher in the larvae exposed to soil moisture of 100% SSWC than in those kept at 25 and 50% SSWC. Dry weight and body water content (% fresh weight) were not different among the treatments. Glucose and trehalose contents were markedly lower, and glycerol content was significantly higher in the larvae confined to soil moisture of 25% SSWC than in those exposed to the other soil moisture treatments. It is suggested that variation in body water content (% fresh weight) contributes to the differences in SCPs of the overwintering C. suppressalis larvae in all treatments, but the influence of soil moisture treatments on supercooling capacity are caused through changes in glycerol content.     

The relationship between gut contents and supercooling capacity in hatchling painted turtles (Chrysemys picta)

Painted turtles (Chrysemys picta) typically spend their first winter of life in a shallow, subterranean hibernaculum (the natal nest) where they seemingly withstand exposure to ice and cold by resisting freezing and becoming supercooled. However, turtles ingest soil and fragments of eggshell as they are hatching from their eggs, and the ingestate usually contains efficient nucleating agents that cause water to freeze at high subzero temperatures. Consequently, neonatal painted turtles have only a modest ability to undergo supercooling in the period immediately after hatching. We studied the limit for supercooling (SCP) in hatchlings that were acclimating to different thermal regimes and then related SCPs of the turtles to the amount of particulate matter in their gastrointestinal (GI) tract. Turtles that were transferred directly from 26 degrees C (the incubation temperature) to 2 degrees C did not purge soil from their gut, and SCPs for these animals remained near -4 degrees C for the 60 days of the study. Animals that were held at 26 degrees C for the duration of the experiment usually cleared soil from their GI tract within 24 days, but SCPs for these turtles were only slightly lower after 60 days than they were at the outset of the experiment. Hatchlings that were acclimating slowly to 2 degrees C cleared soil from their gut within 24 days and realized a modest reduction in their SCP. However, the limit of supercooling in the slowly acclimating animals continued to decline even after all particulate material had been removed from their GI tract, thereby indicating that factors intrinsic to the nucleating agents themselves also may have been involved in the acclimation of hatchlings to low temperature. The lowest SCPs for turtles that were acclimating slowly to 2 degrees C were similar to SCPs recorded in an earlier study of animals taken from natural nests in late autumn, so the current findings affirm the importance of seasonally declining temperatures in preparing animals in the field to withstand conditions that they will encounter during winter.     

Influence of temperature acclimation and gut content on the supercooling ability of the land snail Cornu aspersum

The invasive land snail Cornu aspersum possesses a low ability to supercool (c. -5 degrees C in winter) and survives only minimal ice formation in its body fluids, what may limit its expansion to colder environments. In the present study, we investigated the influence of acclimation and starvation on its supercooling ability. During eight weeks, individuals were maintained at 20 degrees C, fed or starved, or placed at 5 degrees C, directly or with progressive acclimation to cold and shorter photoperiod. Temperature of crystallisation of whole individual (Tc(I)) and hemolymph (Tc(H)), mass data and gut content were recorded every two weeks. Hemolymphatic glucose and glycerol were measured at the end of experiment and occurrence of intestinal ice-nucleating agents (INA) was researched. Acclimation had no effect on Tc(I) but stimulated purging of the gut. Starvation induced a slight decrease of Tc(I) whereas a high quantity of alimentary particles in the digestive tract limited the supercooling ability. Glucose and glycerol were not synthesized in cold conditions. Mean Tc(H) was low (c. -17 degrees C), some INA being present in hemolymph of fed animals. Intestinal content of starved individuals exhibited a mean Tc of c. -6 degrees C, decreasing to c. -12 degrees after heating, suggesting the presence of organic INA.     

A sexually dimorphic response in supercooling temperature, enhanced by starvation, in the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae)

The effect of starvation on supercooling temperature (SCP) distribution was investigated in adults and larvae of Alphitobius diaperinus (Coleoptera: Tenebrionidae).

The mean values for SCPs of adults fed at 20°C were −14.5±2.4°C (31 males) and −10.3±1.3°C (29 females). The distribution of the SCPs of these control adults was unimodal. No significant differences were observed in either mean wet weight or mean dry weight between males and females.

The mean values for SCPs of adults starved for 1 month at 20°C were found to be bimodal due to sexual dimorphism. The mean SCPs for males was lower (−17±2.6°C; 28) than that for females (−11.2±1.8°C; 26). No significant differences were observed in either mean dry weight or wet weight between males and females.

The SCPs of both fed and starved larvae, kept for 1 month at 20°C were −12.3±2.4°C (fed) and −18.0±2.6°C (starved).     

Expression of AmpC beta-lactamase in Enterobacter cloacae isolated from retail ground beef, cattle farm and processing facilities

AIMS: To better understand antibiotic resistance of Enterobacter cloacae isolates originated from food animals, the phenotypic and genotypic resistance of Ent. cloacae isolates from retail ground beef, cattle farm, processing facilities and clinical settings were investigated. METHODS AND RESULTS: The ampC, ampD and ampR genes in the isolates were sequenced and analysed. beta-Lactamase activities and beta-lactamase profiles of the isolates were analysed by the enzymatic hydrolysis of nitrocefin and isoelectric focussing, respectively. The ampC gene of the Ent. cloacae isolate was cloned and transformed into Escherichia coli strains. The genomic DNA profiles of Ent. cloacae isolates were analysed by using pulse field gel electrophoresis (PFGE). Mutation at one residue (Val-54-->Ile) in the AmpR amino acid sequence was consistently found in Ent. cloacae isolates that were resistant to a broadspectrum of beta-lactam agents. The enzyme activity in the isolates was induced by cefoxitin. The pI (isoelectric point) of the enzymes produced by the test strains ranged from 8.4 to 8.9. Cloning of ampC gene of the Ent. cloacae isolate conferred the resistance to ampicillin, cephalothin and amoxicillin in recipient E. coli strains. One recipient of E. coli O157:H7 strain additionally acquired resistance to ceftiofur. The genomic analysis of Ent. cloacae isolates by PFGE showed that the isolates from various sources were genetically unrelated. CONCLUSIONS: The spread of diverse clones of AmpC-producing Ent. cloacae occurred in the ecosystem and retail products. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggested that AmpC-producing Ent. cloacae could be a contributor in spreading beta-lactamase genes in farm environments and food processing environments.     

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.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.