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In preparation for winter many insects enhance the supercooling capacity of their body fluids by 25°C or more, thereby avoiding the lethal effects of tissue freezing. A primary factor limiting supercooling capacity is the presence of nucleating agents that catalyze ice formation at high subzero temperatures. Two species of ice nucleating active (INA) bacteria, Enterobacter agglomerans and Enterobacter taylorae, the latter with previously unknown ice nucleating activity, were isolated from the gut of two species of field-collected beetles, Ceratoma trifurcata and Hippodamia conuergens. Ingestion of these INA bacteria greatly diminished the capacity of our insect model, H. convergens, to supercool and caused freezing at temperatures as high as -15°C. Removal or masking of endogenous INA bacteria may be a major factor in the cold-hardening of freeze intolerant insects for winter survival. Furthermore, these bacteria may provide a novel biological insecticide to control overwintering pest insects by decreasing their natural capacity to supercool.

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This report provides further evidence that a fi'eeze-dried, concentrated form of Pseudomonas sgringae, an ice-nucleating active bacteria, reduces the cold tolerance of stored grain insect pests. Application of ice-nucleating bacteria to wheat or corn that contained insect pests decreased the insects' supercooling capacity: after treatment with 100 ppm of P. sqringae the mean supercooling points of five insect species increased from 4.7 to ll.9'C above untreated controls. Treatment with P. syringae also decreased the capacity of insects to survive a 24-h exposure to subzero temperatules. Decreases in eold tolerance were observed in eight species of stored grain pests: Indianmeal moth larvae, Plodia interpunctellu (Htibner); red flotir beetle adults, Tribolium castaneun'L (Herbst); fiat grain beetle adults, Crgptolestes pusillus (Schonherr); rusty grain beetle adults, Crgptolestes ferrugineus (Stephens) ; Cibbium psVlloides (Czenpinski); lesser grain borer adults, Rhgzopertha dominica (F.); yellow mealworm lawae, Tenebri,o molitor (L.); and granary weevil adults, Sitophilus granarius (L.). Results ofthis study provide further support for the use of ice-nucleating active bacteria as biological insecticides to kill overwintering insects by decreasing their low temperature tolerance. The approach may be particularly appropdate for the control ofa variety ofinsect pests in lestricted areas such as grain bins.