The majority of overwintering insects avoid lethal freezing by lowering the temperature at which ... more The majority of overwintering insects avoid lethal freezing by lowering the temperature at which ice spontaneously nucleates within their body fluids. We examined the effect of ice-nucleating-active bacteria on the cold-hardiness of the lady beetle, Hippodamia convergens , a freeze-intolerant species that overwinters by supercooling to ca. −16°C. Topical application of the ice-nucleating-active bacteria Pseudomonas syringae increased the supercooling point to temperatures as high as −3°C. This decrease in cold tolerance was maintained for at least 3 days after treatment. Various treatment doses (10 8 , 10 6 , and 10 4 bacteria per ml) and modes of action (bacterial ingestion and topical application) were also compared. At the highest concentration of topically applied P. syringae , 50% of the beetles froze between −2 and −4°C. After topical application at the lowest concentration, 50% of the individuals froze by −11°C. In contrast, beetles fed bacteria at this concentration did not ...
Most overwintering insects do not survive internal freezing and must avoid low temperatures or en... more Most overwintering insects do not survive internal freezing and must avoid low temperatures or enhance the capacity of their body fluids to supercool to survive low temperature exposure. Recent reports have demonstrated that the application of ice-nucleating active microorganisms markedly diminishes supercooling. Topical application of as little as 20 ppm of a suspension of a freeze-dried preparation of the ice-nucleating active bacterium Pseudomonas syringae van Hall was sufficient to elevate the mean supercooling point of thc overwintering adults of the Colorado potato beetle, Leptinotarsa decemlineata (Say), from-8.7 to-4.7"C. Previous reports have demonstrated that topical application of these biological icc nucleators to insects whose mouths have been sealed still reduces supercooling capacity; however, the anatomic,J route by which these agents make contact with body water is unclear. Application of the P. syringae suspension to the ventral abdomen did not significantly increase the supercooling point (-5.5°C) compared with beetles treated with the non-ice-nucleating active (eontrol) bacterium Escherichia coli (Migula). However, application of the icc-nucleating agent to the thoracic spiracle, ventral cervix, or abdominal spiracle elevated supercooling point values above those of beetles treated on the ventral abdomen. These data are instructive in the development of methods for the use of ice-nucleating active microorganisms for the biological control of overwintering pests.
Topical application of ice nucleating active (INA) bacteria or fungi decreases the cold tolerance... more Topical application of ice nucleating active (INA) bacteria or fungi decreases the cold tolerance of freeze-intolerant insects by raising their supercooling points (SCPs). However, the route by which INA agents come in contact with insect body water is unknown. To determine their effect on the SCP, we topically applied a suspension of INA Pseudomonas syringae to four anatomic sites of the freeze-intolerant lady beetle, Hippodamia convergens. Aqueous suspensions of either cultured or lyophilized, ultraviolet irradiated (UVI) P. syriugae produced significantly higher mean SCPs than control treatments when applied to the thoracic spiracle of the insect,-7.7 and-5.6'C, respectively, compared with the control treatment's mean SCP of-14.9'C. Application of an aqueous suspension of UVI P. syringae to three other anatomic sites on the beetle produced less dramatic and more varied increases in mean SCP. Application of the INA fungus Fusarium avenaceum to the thoracic spiracle significantly elevated the mean SCP to approx.-10°C. Application of powdered UVI P. syringae to the thoracic spiracle resulted in a SCP increase from-14.9 to-4.6'C, the most dramatic increase in this study. These results indicate that the efficacy of INA microorganisms in elevating the SCP varies with the microorganism and its site of application. Supercooling capacity Pseudomonas syringae Fusarium avenaceum Ice nucleating active Hippodamia convergens Microbial. 54, 16788168 1. Knight C. A. and Duman J. G. (1986) Inhibition of recrystalization of ice by insect thermal hysteresis proteins: a possible cryoprotective Acknowledgements-We thank Jon Costanzo and the anonymous role. Cryobiology 23, 256262. reviewers for critically reviewing an earlier draft of this manuscript. We
Laboratory studies were conducted to identify ice-nucleating active bacterial strains able to ele... more 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 ßuids, 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 signiÞcantly elevated supercooling points, from Ð 4.5 to Ð5.7ЊC and from Ð5.2 to Ð 6.6ЊC, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was Ð9.2ЊC. When sampled at 2 and 12 wk after ingestion, only beetles fed P. fluorescens F26-4C and 88 Ð335 still had signiÞcantly 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 sufÞcient for establishment of the bacterium in the gut. Persistence of these bacteria in Colorado potato beetles long after ingestion was also conÞrmed 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 signiÞcantly reduce their winter survival, thereby reducing local populations and, consequently, crop damage.
The supercooling point (SCP) of an insect model, the lady beetle Hippodamia convergens Guérin-Men... more 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.
The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Centra... more The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gö taland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T = 5°C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T c = À6.4 ± 0.8°C), significantly greater, however, than the supercooling capacity of the population from France (T c = À4.6 ± 1.4°C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T = 20°C), both populations exhibited a similar high T c (À2.0 ± 1.0°C). The lower T c of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg À1) and active (165-215 mOsm kg À1) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than À9°C. Only the strain Kluyvera sp. initiated nucleation at À9°C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of
The majority of overwintering insects avoid lethal freezing by lowering the temperature at which ... more The majority of overwintering insects avoid lethal freezing by lowering the temperature at which ice spontaneously nucleates within their body fluids. We examined the effect of ice-nucleating-active bacteria on the cold-hardiness of the lady beetle, Hippodamia convergens , a freeze-intolerant species that overwinters by supercooling to ca. −16°C. Topical application of the ice-nucleating-active bacteria Pseudomonas syringae increased the supercooling point to temperatures as high as −3°C. This decrease in cold tolerance was maintained for at least 3 days after treatment. Various treatment doses (10 8 , 10 6 , and 10 4 bacteria per ml) and modes of action (bacterial ingestion and topical application) were also compared. At the highest concentration of topically applied P. syringae , 50% of the beetles froze between −2 and −4°C. After topical application at the lowest concentration, 50% of the individuals froze by −11°C. In contrast, beetles fed bacteria at this concentration did not ...
Most overwintering insects do not survive internal freezing and must avoid low temperatures or en... more Most overwintering insects do not survive internal freezing and must avoid low temperatures or enhance the capacity of their body fluids to supercool to survive low temperature exposure. Recent reports have demonstrated that the application of ice-nucleating active microorganisms markedly diminishes supercooling. Topical application of as little as 20 ppm of a suspension of a freeze-dried preparation of the ice-nucleating active bacterium Pseudomonas syringae van Hall was sufficient to elevate the mean supercooling point of thc overwintering adults of the Colorado potato beetle, Leptinotarsa decemlineata (Say), from-8.7 to-4.7"C. Previous reports have demonstrated that topical application of these biological icc nucleators to insects whose mouths have been sealed still reduces supercooling capacity; however, the anatomic,J route by which these agents make contact with body water is unclear. Application of the P. syringae suspension to the ventral abdomen did not significantly increase the supercooling point (-5.5°C) compared with beetles treated with the non-ice-nucleating active (eontrol) bacterium Escherichia coli (Migula). However, application of the icc-nucleating agent to the thoracic spiracle, ventral cervix, or abdominal spiracle elevated supercooling point values above those of beetles treated on the ventral abdomen. These data are instructive in the development of methods for the use of ice-nucleating active microorganisms for the biological control of overwintering pests.
Topical application of ice nucleating active (INA) bacteria or fungi decreases the cold tolerance... more Topical application of ice nucleating active (INA) bacteria or fungi decreases the cold tolerance of freeze-intolerant insects by raising their supercooling points (SCPs). However, the route by which INA agents come in contact with insect body water is unknown. To determine their effect on the SCP, we topically applied a suspension of INA Pseudomonas syringae to four anatomic sites of the freeze-intolerant lady beetle, Hippodamia convergens. Aqueous suspensions of either cultured or lyophilized, ultraviolet irradiated (UVI) P. syriugae produced significantly higher mean SCPs than control treatments when applied to the thoracic spiracle of the insect,-7.7 and-5.6'C, respectively, compared with the control treatment's mean SCP of-14.9'C. Application of an aqueous suspension of UVI P. syringae to three other anatomic sites on the beetle produced less dramatic and more varied increases in mean SCP. Application of the INA fungus Fusarium avenaceum to the thoracic spiracle significantly elevated the mean SCP to approx.-10°C. Application of powdered UVI P. syringae to the thoracic spiracle resulted in a SCP increase from-14.9 to-4.6'C, the most dramatic increase in this study. These results indicate that the efficacy of INA microorganisms in elevating the SCP varies with the microorganism and its site of application. Supercooling capacity Pseudomonas syringae Fusarium avenaceum Ice nucleating active Hippodamia convergens Microbial. 54, 16788168 1. Knight C. A. and Duman J. G. (1986) Inhibition of recrystalization of ice by insect thermal hysteresis proteins: a possible cryoprotective Acknowledgements-We thank Jon Costanzo and the anonymous role. Cryobiology 23, 256262. reviewers for critically reviewing an earlier draft of this manuscript. We
Laboratory studies were conducted to identify ice-nucleating active bacterial strains able to ele... more 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 ßuids, 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 signiÞcantly elevated supercooling points, from Ð 4.5 to Ð5.7ЊC and from Ð5.2 to Ð 6.6ЊC, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was Ð9.2ЊC. When sampled at 2 and 12 wk after ingestion, only beetles fed P. fluorescens F26-4C and 88 Ð335 still had signiÞcantly 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 sufÞcient for establishment of the bacterium in the gut. Persistence of these bacteria in Colorado potato beetles long after ingestion was also conÞrmed 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 signiÞcantly reduce their winter survival, thereby reducing local populations and, consequently, crop damage.
The supercooling point (SCP) of an insect model, the lady beetle Hippodamia convergens Guérin-Men... more 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.
The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Centra... more The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gö taland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T = 5°C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T c = À6.4 ± 0.8°C), significantly greater, however, than the supercooling capacity of the population from France (T c = À4.6 ± 1.4°C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T = 20°C), both populations exhibited a similar high T c (À2.0 ± 1.0°C). The lower T c of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg À1) and active (165-215 mOsm kg À1) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than À9°C. Only the strain Kluyvera sp. initiated nucleation at À9°C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of
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