Marcia R. Lee

Insect Cold-hardiness and Ice Nucleating Active Microorganisms Including Their Potential Use for Biological Control

Abstract Since most insects are unable to survive internal ice formation a key factor in their overwintering survival is the regulation of the temperature at which they spontaneously freeze. To enhance their supercooling capacity overwintering insects eliminate endogenous ice nucleators, accumulate low-molecular-weight polyols and sugars, and synthesize hemolymph antifreeze proteins. A number of freeze-tolerant species contain proteins/lipoproteins or insoluble crystals that are ice nucleating active at relatively high subzero temperatures. Only recently have ice nucleating active bacteria and fungi been identified as normal flora in the gut of insects. These microorganisms are the most efficient class of heterogeneous ice nucleators that have been found in insects. Ice nucleating active microorganisms can regulate the supercooling capacity of insects when ingested or applied topically. These unique microorganisms may offer a novel means for the biological control of insect pests during the winter.

LONG-TERM REDUCTION OF COLD HARDINESS FOLLOWING INGESTION OF ICE-NUCLEATING BACTERIA IN THE COLORADO POTATO BEETLE, LEPTINOTARSA DECEMLINEATA

We investigated the effect of ingestion of ice-nucleating bacteria on the supercooling capacity and cold hardiness of the Colorado potato beetle (Leptinotarsa decemlineata Say), a freeze-intolerant species that overwinters as adults in shallow, terrestrial burrows. Ingestion of ice-nucleating bacteria (Enterobacter agglomerans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae), fed on slices of potato tuber, caused an abrupt decrease in supercooling capacity. No change occurred in the supercooling capacity of beetles fed Escherichia coli, as this species lacks ice-nucleating activity. Ingestion rates showed that tubers treated with different species were equally palatable. During diapause induction beetles evacuated food from their guts, but nevertheless retained sufficient ice-nucleating bacteria to diminish supercooling. Beetles fed P. fluorescens and P. putida exhibited reduced supercooling even after an 8-wk exposure to simulated winter conditions. Furthermore, P. fluorescens was isolated 10-wk post-ingestion from diapausing beetles. Our data suggest that ingested bacteria may be retained by insects during entry into diapause and that the cold hardiness of candidate crop pests, such as L. decemlineata, may be reduced by feeding them ice-nucleating bacteria prior to winter diapause.

Identification of Ice-Nucleating Active Pseudomonas fluorescens Strains for Biological Control of Overwintering Colorado Potato Beetles (Coleoptera: Chrysomelidae)

Abstract 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 106 or 103 bacterial cells per beetle had significantly elevated supercooling points, from –4.5 to –5.7°C and from –5.2 to –6.6°C, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was –9.2°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.

Sensitivity of partially purified ice nucleation activity of Fusarium acuminatum SRSF 616.

Factors that affect bacterial ice nucleation, including growth medium, growth phase, nutrient deprivation, and cold-temperature exposure, were investigated in the ice nucleation active (INA) fungus Fusarium acuminatum SRSF 616. Ice nucleation activity remained relatively constant throughout the growth cycle, and the cell-free culture supernatant consistently displayed higher ice nucleation activity than the hyphal pellet. Although nutrient starvation and low-temperature exposure enhance bacterial ice nucleation activity, reducing the concentration of C, N, or P in synthetischer nährstoffarmer broth (SNB) did not increase fungal ice nucleation activity, nor did exposure to 4°C or 15°C. From the SNB supernatant, selected INA chromatography fractions were obtained that demonstrated increased sensitivity to proteinase K and heat compared with culture supernatant. We propose that partial purification of the fungal ice nuclei resulted in removal of low-molecular-weight stabilizing factors.

Effect of biological ice nucleators on insect supercooling capacity varies with anatomic site of application

Abstract 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. syringae 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 treatments 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.

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.

Using ice-nucleating bacteria to reduce winter survival of Colorado potato beetles: Development of a novel strategy for biological control

Publisher Summary Most insects are not able to survive internal ice formation. Thus, a key factor in their winter survival is the regulation of the temperature at which they freeze. This temperature is termed the supercooling point (SCP) or the temperature of crystallization. A major factor in the overwintering survival of insect pests is their ability to seasonally enhance their cold tolerance by increasing their capacity to supercool and, thus, avoid the lethal effects of internal ice formation. Any agent that limits the supercooling capacity of a freeze-intolerant insect will increase the likelihood of injury or death following exposure to subzero temperatures. In the 1970s, a unique class of biological nucleators, icenucleating active (INA) bacteria, was discovered. These bacteria are remarkable for their ability to catalyze ice nucleation at temperatures as high as -1 to -2°C. Ice-nucleating activity is conferred by the presence of ina genes, which code for ice nucleating proteins localized on the bacteriums outer membrane. This chapter reviews a paper that uses Colorado potato beetle, Leptinotarsa decemlineata, (Coleoptera: Chrysomelidae) as a model system to address basic questions regarding the seasonal regulation of cold tolerance and insect–microbial interactions. These studies provide a foundation for novel approaches in biological control by manipulating insect cold-hardiness and overwintering survival using INA microorganisms.