Insect Cold-Hardiness and Ice Nucleating Active Microorganisms Including Their Potential Use for Biological Control RICHARD E

REVIEW Insect Cold-hardiness and Ice Nucleating Active Microorganisms Including Their Potential Use for Biological Control RICHARD E. LEE JR,* MARCIA R. LEE,? JANET M. STRONG-GUNDERSON$

Received 20 July 1992

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.

Insect cold-hardiness Supercooling point Ice nucleation Ice nucleating active bacteria and fungi Biological control

INTRODUCTION lethal temperature. However, without careful testing it must not be assumed that a given species can survive at Insects rely on a variety of ecological and physiological adaptations to survive low temperatures. Recent articles temperatures above the supercooling point: some insects have reviewed biochemical, physiological and ecological die due to cold shock, a form of chilling injury that occurs in the absence of ice formation, at temperatures aspects of insect cold-hardiness and overwintering 10°C or more above the supercooling point (Lee and biology (Baust and Rojas, 1985; Zachariassen, 1985; Denlinger, 1985; Knight et al., 1986; Bale, 1987; Lee et Tauber et al., 1986; Bale, 1987; Danks, 1987; Cannon al., 1987). Even for those insects that survive the freezing and Block, 1988; Storey and Storey, 1988; Lee, 1989; of their body water, the supercooling point results in a Somme, 1989; Block, 1990; Duman et al., 1991). In major change of physiological state. During freezing, addition, Lee and Denlinger (1991) recently edited a only water molecules join the growing ice lattice, while book on insects at low temperature. the excluded solutes become concentrated in the extra- Since most insects are unable to survive internal ice cellular fluids (Mazur, 1984). Also, cells are progressively formation, a key factor in their overwintering survival dehydrated, and metabolism is depressed as tissues is the regulation of the temperature at which they spontaneously freeze, termed the supercooling point or become anoxic (Storey and Storey, 1988). This review will summarize factors that influence the temperature of crystallization (Lee, 1991). This supercooling capacity and ice nucleation in insects. temperature is significant to both freeze-tolerant and Specifically, we will discuss the role of ice nucleating freeze-intolerant insects. Freeze-intolerant insects do not active microorganisms, primarily bacteria, in the regu- survive internal ice formation; consequently, for them lation of the supercooling capacity of insects. Lastly, we the supercooling point represents the absolute lower will discuss the potential for the use of ice nucleating microorganisms in the biological control of insect pests. *Department of Zoology, Miami University, Oxford, OH 45056 U.S.A. TDepartment of Microbiology. Miami University, Oxford, OH 45056 SUPERCOOLING AND ICE NUCLEATION U.S.A. fPresent address: Environmental Science Division, Oak Ridge Na- It is possible to cool small volumes of purified water tional Laboratory, Oak Ridge, TN 37831-6038, U.S.A. to approx. - 39°C without freezing (Angell, 1982). Even 2 REVIEW

a few microliters of tap water in a capillary tube readily form, and lose their ice nucleating activity when they are supercool (i.e. remain unfrozen at temperatures below dissolved in water (Vali, 1991). Certain insects produce the melting point) to - 15 to - 20°C before spontaneous soluble ice nucleating proteins and lipoproteins that are ice nucleation occurs. active in the range of - 6 to - 9°C (Duman et al., 1991). In aqueous systems ice nucleation requires the pres- Ice nucleating active bacteria and fungi are the most ence of nuclei upon which water molecules can aggregate efficient heterogeneous ice nucleators known (Table 1); until a sufficient size is reached to promote ice crystal these microorganisms induce freezing at temperatures as growth (Knight, 1967). If the nucleus is composed solely high as - 1°C. of water molecules, the process is referred to as homo- Since insects are essentially small volumes of water it geneous nucleation (Angell, 1982). It is generally be- is perhaps not surprising that many insects supercool lieved in insects and other biological systems that ice many degrees below the melting point of their body nucleation is initiated by a heterogeneous mechanism fluids before ice nucleation begins. Using thermocouples (Franks, 1985). In this case water molecules accumulate or other temperature sensing devices placed in contact on a seed nucleus other than water. with the insect cuticle, the limit of supercooling is The most efficient ice nucleator within the aqueous readily detected by the release of the latent heat of system determines the temperature at which ice nucle- crystallization as body water freezes. The supercooling ation begins. A commonly used criterion for identifying point of most freeze-intolerant insects varies seasonally. a relatively efficient heterogeneous ice nucleator is the Although values in summer are often between -4 and capacity to nucleate water at temperatures at or above -8°C as the insect cold-hardens in the autumn the - 10°C. A representative list of heterogeneous nucle- supercooling point may decrease to -20°C or lower ators is provided in Table 1. Inorganic compounds and (Somme, 1982; Lee, 1991). Several gall-forming insects non-proteinaceous organic compounds function as in Alaska depress their supercooling points to - 60°C in efficient nucleators only when they are in the crystalline winter (Miller, 1982).

TABLE 1. Maximal threshold of ice nucleating activity of representative heterogeneous ice nucleators Threshold of ice nucleating Nucleator activity (“C) Reference Inorganic compounds Fluorophlogopite and other micas and silicas -1 to -9 Shen et al. (1977) Silver iodide crystals -4 Vonnegut (1947) Organic compounds Amino acid crystals -4 to -7 Power and Power (1962); Gavish et al. (1992) Cholesterol and other steroid crystals -5 to -7 Head (1961) Insect ice nucleating proteins/lipoproteins -6 to -9 Zachariassen and Hammel (1976); Neven er al. (1989) Microorganisms Bacteria Plant epiphytes Pseudomonas syringae -1 Maki et al. (1974); Vali et al. (1976) Pseudomonas fiuorescens -2 Lindow (1982) Pseudomonas virid$ava -3 Paulin and Luisetti (1987) Erwinia herbicola -2 Lindow et al. (1978a) Xanthomonas campestrir pv. -2 Kim et al. (1987) translucens Insect gut flora Enterobacter agglomerans” -2 Lee et al. (1991) Enterobacier tayloraeb -2 Lee et al. (1991) Erwinia herbicola’ > -10 Kaneko et al. (1991a,b) Frog gut flora Pseudomonas jluorescens -2 Lee M. R. et al. (1992a) Pseudomonas putida -2 Lee M. R. et al. (1992a) Fungi Fusarium avenaceum -2 Pouleur et al. (1991) Fusarium acuminatum -5 Pouleur et al. (1991) Lichen mycobiont Rhizoplaca chrysoleuca -2 Kieft (1988) Insect gut flora Fusarium sp.’ -5 Tsumuki et al. (1992)

“Isolated from Hippodamia convergens (Coleoptera) and Cerotoma trifurcata (Coleoptera). bIsolated from Plutella xylostella (Lepidoptera). ‘Isolated from Chilo suppressalis (Lepidoptera). REVIEW 3

WHAT FACTORS REGULATE THE SUPERCOOLING In a number of freeze-tolerant insects, ice nucleating CAPACITY OF INSECTS? proteins and lipoproteins are found in the hemolymph of overwintering insects (Zachariassen and Hammel, 1976; The capacity for extensive supercooling is closely tied Duman et al., 1991). These proteinaceous nucleators to the small size of insects. That supercooling potential function as heterogeneous ice catalysts that are active in is inversely related to fluid volume is well established the range of - 6 to - 9°C (Table 1). This ice nucleating from physical studies (Angell, 1982). Insect eggs, activity of these proteins remains high even in the Collembola and mites weighing tens to hundreds of presence of high concentrations of polyhydric alcohols micrograms commonly exhibit supercooling points in and sugars (Lee et al., 1981; Duman et al., 1991). the range of -20 to -40°C (see review by Somme, Some freeze-tolerant insects have relatively high 1982). Generally larger insects have a more limited supercooling points, even though they lack efficient capacity to supercool and freeze at temperatures be- nucleating agents in the hemolymph. One such example tween - 5 and - 20°C. The inverse relationship between is the overwintering third-instar larva of the goldenrod insect mass (i.e. fluid volume) and supercooling capacity gall fly, Eurosta solidaginis. During the summer these is also evident intraspecifically among larval stages of the larvae are freeze-intolerant and supercool to tempera- mealworm Tenebrio molitor (Johnston and Lee, 1990). tures below - 12°C (Morrissey and Baust, 1976). During Consistent with the effect of body size on supercooling the autumn the larvae acquire freeze-tolerance and capacity is the observation that the larger, cold-adapted supercooling points rise to approx. -9°C. Soluble ice amphibians and reptiles, even freezing intolerant ones, nucleators must be lacking because the hemolymph generally freeze at -3°C or higher (Schmid, 1982; readily supercools to - 18°C (Bale et al., 1989). Research Costanzo et al., 1988; Storey and Storey, 1988). in our laboratory revealed large crystalloid spheres, Another factor enhancing insect supercooling is the identified as calcium phosphate, in the Malpighian accumulation of low-molecular-weight polyols and tubules that have ice nucleating activity closely matching sugars. Although glycerol is the most common cryo- the supercooling point of the intact insect (Lee R. E. protectant in overwintering insects, other commonly et al., 1992b). We have also shown that uric acid, calcium reported compounds are sorbitol, mannitol, glucose, phosphate and other crystals commonly formed in fructose and trehalose. The accumulation of glycerol, insects have significant ice nucleating activity. Since a sometimes in multimolar concentrations, frequently cor- variety of crystals are known in insects, these com- relates with increased supercooling capacity (Salt, 1959; pounds may represent a new class of ice nucleating Somme, 1964; Baust and Miller, 1972; Young and Block, agents that regulate the supercooling point. 1980). In his 1985 review, Zachariassen concluded that Aside from ice-nucleating proteins/lipoproteins and polyols depress the supercooling point approximately these newly reported ice nucleating crystals, the identifi- twice as much as they colligatively depress the melting cation of other specific classes of ice nucleators in point. freeze-susceptible insects remains elusive. A number of Not all insects use cryoprotectants to depress the investigators, however, have attempted to identify the supercooling point. Even in the absence of accumulated location at which freezing begins. Baust and Zachari- cryoprotectants, many species supercool to - 20°C assen (1983) reported ice nucleators associated with the (Zachariassen, 1985; Lee, 1991), apparently by removing cell matrix of warm-acclimated beetles, Rhagium inquisi- or inactivating heterogeneous ice nucleating agents. tor; the ice nucleating activity of these nucleators was For example, in the flesh fly, Sarcophaga crassipalpis, similar to the supercooling point, -8°C of the intact supercooling points decrease from - 11 to -23°C beetle. Nucleation was also examined in the excised legs during the larval-pupal transition in non-diapause- of insects (Salt, 1968). Recently, Tsumuki and Konno destined flies (Lee and Denlinger, 1985). This enhance- (1991) identified muscle and epidermis as the primary ment of supercooling capacity occurs when glycerol site of freezing in overwintering larvae of the rice stem titers are decreasing, not increasing (Lee et al., borer, Chile suppressalis. 1987). Albeit these reports identify ice nucleation in other Another mechanism by which insects freeze is inocula- anatomical locations, the gut is the most frequently tive freezing (Salt, 1963). In this case contact with reported site of ice nucleation. In large part, this idea external ice catalyzes ice nucleation with body fluids. In stems from the observation that feeding is associated some species wetting of the cuticle severely limits super- with an elevation of the supercooling point (Salt, 1936, cooling capacity (Danks, 1971; Fields and McNeil, 1988; 1966). Furthermore, cessation of feeding and emptying Bale et al., 1989; Layne et al., 1990). In the freeze- of the gut are often associated with increases in super- tolerant cranefly (Tip& sp.) freezing begins at an unusu- cooling capacity (Cannon and Block, 1988). Using cryo- ally high temperature of - 3°C due to inoculative freez- microscopy, Shimada (1989) observed that ice ing (Gehrken and Southon, 1992). In the drosophilid, nucleation began in the lumen of the anterior midgut, Chymomyza costata, diapausing larvae freezing by con- and once freezing began the ice lattice rapidly grew tact with external ice survive, while isolated larvae across the gut wall into the haemocoel. supercool to -20°C and die upon freezing (Enomoto, Based on the ice nucleating activity of a number of 1981; Shimada and Riihimaa, 1988). inorganic crystals (Table l), it has been suggested that 4 REVIEW food or incidentally ingested dust particles within the gut Repetitive primary structure and possibly tertiary struc- may function as ice nucleating agents (see review by ture of one ice-nucleating protein, encoded by the inaZ Cannon and Block, 1988). Others have speculated that gene sequence, is believed to be crucial to the structure ice nucleating bacteria in the gut may regulate the of the ice nucleating template (Green and Warren, 1985; supercooling point (Lindow, 1982; Somme, 1982; Baust Wolber et al., 1986). Analysis of the consensus sequence and Rojas, 1985; Cannon and Block, 1988; Strong- of ice nucleation proteins encoded by the genes iceE, Gunderson et al., 1989). Only recently has this notion inaW, and inaZ from E. herbicola, P. jluorescens and been confirmed (Strong-Gunderson et al., 1990a; P. syringue respectively, has revealed consistent simi- Kaneko et al., 199la, b; Lee et al., 1991). larities and conserved features (Warren et al., 1986; Warren and Corotto, 1989). Furthermore, evidence has been presented that the lipid, phosphatidylinositol, ICE NUCLEATING MICROORGANISMS is a component of the ice nucleation site and is syn- thesized by an enzymatic translation product, phos- Bacteria phatidylinositol synthetase, of the ice nucleating genes A search for atmospheric ice nuclei led researchers to (Kozloff et al., 1984). decaying vegetation as a primary source of efficient The threshold temperature is the highest temperature nuclei (Schnell and Vali, 1972, 1973). In turn, this study at which an individual cell functions as an ice nucleus led to the discovery of ice nucleating activity in Pseu- within a specified time interval (Warren, 1987). Within domonas syringae isolated from decaying leaves a given bacterial culture with presumably identical (Maki et ul., 1974; Vali et al., 1976). Subsequently, genotypes, threshold temperatures may vary greatly several other species of epiphytic ice nucleating bacteria from cell-to-cell. Cells with ice nucleation activity at were discovered, including Erwinia herbicola and Pseu- the highest temperatures are uncommon relative to ones domonas juorescens (see reviews of Lindow, 1982, active at lower temperatures. For example, in a popu- 1983). These Gram-negative, rod-shaped, asporogenous lation of 1 million bacterial cells, only one cell might be bacteria are the most efficient ice nucleators known active at - 2°C whereas 1000 cells are active at -6°C catalyzing ice formation at temperatures as high as and 10,000 cells have activity at - 10°C. Consequently -1°C (Table 1). the ice nucleating activity of bacterial populations is The molecular biology of ice nucleating bacteria is the usually reported as cumulative ice nucleation spectra of subject of several recent reviews (Warren, 1987; Warren threshold temperatures. These spectra describe the pro- and Wolber, 1987; Wolber and Warren, 1989). Ice portion of cells that are active as ice nuclei at a particular nucleating active bacteria synthesize large outer-mem- temperature. The most common method for reporting brane proteins (120 kDa) that apparently aggregate these data is the freezing droplet assay of Vali (1971) in within the outer wall, associate with membrane lipid and which the activity of a bacterial suspension is reported carbohydrate components, and, subsequently, function as [log(ice nuclei/cell)]. as a template for the formation of small ice crystal seeds, The majority of reported ice nucleating active bacteria called “ice nuclei” with sizes estimated to range from are ubiquitous, pathogenic epiphytes that not only nu- 150 kDa for activity at about - 12°C to 19,000 kDa for cleate water in direct contact with them, but also induce activity at -2°C (Corotto et al., 1986; Warren et al., freezing of plant tissues. The significance of ice nucleat- 1986; Govindarajan and Lindow, 1988; Wolber and ing active bacteria in promoting frost-injury in plants is Warren, 1989; Mueller et a/.,1990). Cell-free ice nuclei, clearly established. Each year these bacteria are respon- which are borne on outer membrane vesicles, may be sible for substantial amounts of frost-related crop losses shed by ice nucleating active strains of E. herbicola into throughout the world (Lindow et al., 1982a, b; Lindow, the surrounding environment and possess ice nucleation 1983). In 1985 frost-related losses were estimated at one activity at -3°C (Phelps et al., 1986). Bacterial ice billion dollars (Lindow, 1985). The recognition that nucleating activity is heat labile, sensitive to 65°C these bacteria are responsible for frost damage to crops inactivated by heavy metal ions, such as Hg+, extreme has led to a number of control strategies including pHs and sulfhydryl reagents. Activity is stable to the use of bactericides and ice nucleation inhibitors lyophilization, and variably affected by certain bacterio- (Lindow, 1983). A particularly novel approach is the tidal antibiotics, such as streptomycin, a protein-inhibit- application of genetically engineered, antagonistic, non- ing antibiotic (Kozloff et al., 1983; Maki et al., 1974; ice nucleating bacteria to selected crops in an attempt to Anderson and Ashworth, 1986). Culture conditions, competitively displace natural populations that have particularly the growth medium, and incubation tem- activity (Lindow, 1983; Warren et al., 1986). Not surpris- perature affect bacterial ice nucleating activity (Lindow ingly, the release of genetically engineered organisms et al., 1978a; Rogers et al., 1987; Pooley and Brown, into the environment has proved controversial; however, 1991). Addition of 2.5% glycerol to the growth medium, initial studies suggest that this approach is feasible incubation at 22-24°C and low temperature condition- (Halvorson et al., 1985; Lindow, 1985; Lindow et al., ing at 5°C increase ice nucleating activity (Lindow et al., 1989). Plants colonized with ice minus mutant strains 1978a, b; Rogers et al., 1987). Genes responsible for ice of P. syringae before challenge with ice plus strains nucleation have been isolated and partially sequenced. had fewer ice nucleation active bacteria, and reduced REVIEW 5

numbers of ice nuclei active at -5°C (Lindow, 1987). ICE NUCLEATING ACTIVE MICROORGANISMS AS Furthermore, plants treated with ice minus strains NORMAL FLORA IN THE INSECT GUT had less frost injury. Field studies also demonstrated Bacteria significant reductions in colonization by ice plus strains on plants previously colonized by ice minus strains Two independent research groups have recently (Lindow, 1987). isolated ice nucleating active bacteria from the gut of Early in most discussions of ice nucleating micro- insects (Strong-Gunderson et al., 1990a; Kaneko et al., organisms the question arises as to the selective advan- 1991a, b; Lee et al., 1991). These reports constitute the tage of this trait in natural populations. Although no first demonstrations of close associations between ice definitive answer is available, a number of hypotheses nucleating active bacteria and animals (Table 1). have been proposed. Since injured plants are more easily Several species of ice nucleating active bacteria have invaded by plant pathogens, including P. syringae, been isolated from the gut of the beetles, Cerotoma Lindow (1983) suggested that the ice nucleating activity trifurcata and Hippodamia convergens collected during of epiphytic bacteria may facilitate bacterial invasion the summer (Strong-Gunderson et al., 1990a, b; Lee into plant tissues and, thus, increase their access to et al., 1991). We identified strains of Enterobacter nutrients. Another contention is that the ice nucleation agglomerans and Enterobacter taylorae with ice nucleat- trait may promote condensation onto airborne bacteria ing activity. Ice nucleating activity has not previously that have been carried into the upper atmosphere been reported in E. taylorae. The droplet freezing assay and, thereby, rescue them as they return to earth in was used to characterize the ice nucleating activity of precipitation (Warren, 1987). Kieft (1988) suggested these two species. Both species exhibited maximal that lichens may benefit from their mycobiont ice threshold activities of approx. -2°C (Fig. 1). These nucleating activity by increasing their available water spectra differ significantly from that of the non-ice through condensation. It is also possible that this trait nucleating bacterium Escherichia coli. The ice nucleating enhances low temperature survival of bacteria, since the activities of the two Enterobacter species were only ice nucleation phenotype is more common in areas slightly less than that observed in the highly efficient experiencing frost than those that do not (Hirano and epiphytic bacterium, P. syringae (Fig. 1). Upper, 1990). Freeze-tolerant plants may benefit from We confirmed the ice nucleating activity of E. agglom - epiphytic ice nucleating bacteria by insuring that plant erans and E. taylorae in vivo by feeding bacterial suspen- tissues begin to freeze at high subzero temperatures sions to our insect model, the lady beetle H. convergens (Lindow, 1982). This process initiates freezing in the (Strong-Gunderson et al., 1990a, b; Lee et aE., 1991). extracellular fluids, thus avoiding lethal intracellular Overwintering adults of H. convergens are freeze- freezing. intolerant and supercool to approx. - 16°C (Lee, 1980; Bennett and Lee, 1989), but when beetles were fed Fungi bacterial suspensions of the ice nucleating active bacteria Ice nucleating activity is not restricted to bacteria. of insect origin, their supercooling points increased Epilithic lichens from three genera, Rhizoplaca, Xantho- by 12-13°C (Table 2). Indeed, some beetles froze at parmelia and Xanthoria have ice nucleating activity at temperatures as high - 1.5”C. temperatures in the range of -2.3 to - 8°C (Kieft, Both E. taylorae and E. agglomerans are members 1988). Kieft and Ruscetti (1990) associated ice nucleat- of the Enterobacteriaceae, a family of asporogenous, ing activity with protein from axenic cultures of the Gram-negative, facultative anaerobic rods. Members lichen fungus, Rhizoplaca chrysoleuca. Lichen and bac- of this group are commonly found as digestive tract terial ice nucleation sites are similar in both molecular size and in a characteristic logarithmic increase in size with a linear increase in nucleation temperature (Kieft and Ruscetti, 1992). Unlike bacteria, ice nuclei from Rhiz. chrysoleuca exhibit activity over a wide pH range from 1.5 to 12, are relatively heat stable, and their activity does not require membrane lipids (Kieft and Ruscetti, 1990). These latter observations suggest significant differences between the nature of fungal and bacterial ice nuclei. Ice nucleation activity has also been reported in the free-living fungi, Fusarium avenaceum and Fusarium acuminatum (Pouleur et al., 1991). Ice nucleation 0 - 10 20 - 30 activity occurs at temperatures as high as -2S”C in Temperature (“C) F. avenaceum. Like Rhiz. chrysoleuca, the Fusarium species retain ice nucleating activity at a wide range FIGURE 1. Comparison of the ice nucleating activity of the epiphytic bacterium, P. syringae, with E. agglomerans and E. taylorae isolated of pHs (l-l 3), and following heat treatment of up to from insects. Data from a non-ice nucleating bacterium, E. coli, are 70°C. included for comparison. Data from Lee et al. (1991). 6 REVIEW

TABLE 2. Effect of treatment with ice nucleating active microorganisms on the supercooling point of insects Supercooling point (“C) (x k SEM) Insect (stage) INA Microorganism Untreated Treated Reference Coleoptera Cryptolestes ferrugineus (adult) Pseudomonas syringae” -17.0+ 1.0 -8.1 f 0.5 Fields (1991) Cryptolestes pusillus (adult) P. syringae” -14.0+ 1.0 -12.0* 1.5 Fields (1992) Diabrotica undecimpunctata P. syringae b -7.5 k 0.8 - 3.2 k 0.2 Strong-Gunderson et al. (unpublished data) howardi (adult) Gibbium psylloides (adult) P. syringae’ - 10.7 + 0.9 -6.0 + 0.5 Lee R. E. et a/. (1992b) Hippodamia convergens (adult) P. syringae b - 16.0 + 0.5 -2.8 + 0.2 Strong-Gunderson et al. (1990b) Erwinia herbicolad - 16.0 & 0.5 -4.4 + 0.6 Strong-Gunderson et al. (1990b) Enterobactor agglomeransd - 16.0 + 0.5 -3.1 f 0.1 Lee et al. (1991) Enterobactor tayloraed - 16.0 f 0.5 -4.3 f 0.4 Lee et al. (1991) Fusarium acuminatume - 14.9 + 0.5 -11.0+0.7 Lee M. R. et al. (1992b) Oryzaephilus surinamensis (adult) P. syringaea -13.7+ 1.9 -11.0* 1.3 Fields (1992) Plodia interpunctella (larva) P. syringae ’ - 10.3 f 0.4 - 5.4 + 0.5 Lee R. E. et al. (1992b) Rhyzopertha dominica (adult) P. syringaeC -15.2kO.6 -3.3 & 0.1 Lee R. E. et al. (1992b) Sitophilus granarius (adult) P. syringaeC - 14.3 f 0.8 - 7.8 f 0.5 Fields (1992) S. granarius (adult) P. syringae’ -15.7+ 1.0 -8.0 + 0.6 Lee R. E. et al. (1992b) Tenebrio molitor (larva) P. syringaeC - 16.0 ) 0.7 - 5.4 + 0.7 Strong-Gunderson et al. (unpublished data) T. molitor (adult) P. syringaer -15.1 kO.6 -2.7 + 0.3 Strong-Gunderson et al. (unpublished data) Tribolium castaneton (adult) P. syringae’ - 13.9 + 0.8 -4.7 + 0.4 Lee R. E. et al. (1992b) P. syringaea - 12.3 * 1.0 -5.8 k 0.3 Fields (1992) Diptera Sarcophaga crassipalpis (larva) P. syringaet - 13.8 _+0.9 -3.6 + 0.1 Strong-Gunderson et al. (unpublished data) Hemiptera Lygus sp. (adult) P. syringae b -20.0 f 0.5 -8.7 & 1.0 Strong-Gunderson and Lee (unpublished data) Lepidoptera Chilo suppressalis (larva) Fusarium sp.8 -20.1 f 0.9 -5.7 & 0.6 Tsumuki et al. (1992) Galleria mellonella (larva) P. syringae’ - 10.4 f 0.1 -4.0 f 0.3 Strong-Gunderson et al. (unpublished data)

“Treated with 1OOOppm dry, powdered P. syringae. bMisted with lo9 bacteria/ml water. ‘Treated with IOOppm dry, powdered P. syringae. dIngestion of 2 x lo9 bacteria/ml water. “Misted with 3 mg/ml water. ‘Misted with lo* bacteria/ml water. EIngestion. microflora in insects (Cruden and Markovetz, 1987; species, some pupae that fed as larvae on germinating Mead et al., 1988). Our most frequent bacterial isolate, radish seeds had relatively higher supercooling points in E. agglomerans, was phenotypically classified in the the range of - 5 to - 12°C (Kaneko et al., 1989). When broad, heterogeneous “E. agglomerans-Erwinia her- these pupae were held for 5 days at 5°C the proportion bicola complex”. Although historically Erw. herbicola of individuals having high supercooling points increased. was considered a synonym of E. agglomerans, it is now This result suggests an enhancement of ice nucleating known that some bacteria phenotypically placed within activity due to low temperature conditioning similar to this complex are not closely related genotypically that previously observed in Erw. herbicola (Rogers et al., (Brenner et al., 1984). Since phenotypic biochemical 1987). In contrast, larvae reared on aseptic radish identification schematics are not yet established to seeds produced pupae that did not have elevated super- enable one to identify to species a new isolate initially cooling points. Based upon this study Kaneko et al. placed within this broad complex, this problem has been (1991a) recently isolated three bacterial isolates with ice addressed by the creation of 13 DNA-DNA hybridiz- nucleating activity from pupae. These isolates were later ation groups, based on genotypic relatedness within this identified as Erw. herbicola (Kaneko et al., 1991b). complex (Brenner et al., 1982, 1984). DNA genotypic relatedness studies are currently in progress to determine Fungi the taxonomic identity of our ice nucleating active An ice nucleating active fungus has also been shown isolates. Studies of this type are critical for a full to influence the supercooling capacity of an insect. In understanding of the relationship between ice nucleating rice stem borer larvae, Chilo suppressalis, the supercool- active bacteria isolated from insects and their relation- ing points of the gut (-8.4”C) and the frass (-6S”C) ship to epiphytic ice nucleating active bacteria. correspond closely to the supercooling point of the An ice nucleating active bacterium was isolated from whole body, -8.3”C (Tsumuki and Konno, 1991). Other pupae of the diamondback moth, Plutella xylostella body compartments supercool to - 12°C or lower. Fur- (Kaneko et al., 1991a, b). In a preliminary study of this ther investigation isolated an ice nucleating active fungus REVIEW 1

(Fusarium sp.) from the gut (Tsumuki et al., 1992). clear. The freezing-intolerant lady beetle, H. conuergens, Sterile larvae fed a non-ice nucleating fungus, Penicillium overwinters as an adult in reproductive diapause (Lee, sp., supercool extensively to below -20°C while larvae 1980; Bennett and Lee, 1989). Although these beetles are fed a mycelial suspension of Fusarium have supercooling unlikely to experience subzero temperatures in summer, points near -5°C. Furthermore, application of the actively feeding adults have relatively high supercooling fungus to the larval surface produced a similar elevation points of -7°C possibly due to the presence of in the supercooling point. E. agglomerans and E. taylorae found in the gut during the summer (Lee et al., 1991). To enhance supercooling, Other animals having ice nucleating active bacteria either these bacteria must be evacuated from the gut Ice nucleating active microorganisms may also play prior to overwintering or the expression of this pheno- a role in the freezing tolerance of intertidal marine type in the gut flora must be diminished or eliminated. invertebrates. In a preliminary report, Loomis (1990) Since the members of the genus Enterobacter are com- described the presence of an ice nucleator in the palial monly found in the insect gut, the ice nucleating trait fluid, that was not present in hemolymph, of the bivalve may be incidental to the primary role of these micro- mollusc, Geukensia demissa. The presence of an ice organisms. Many insects evacuate the gut in preparation nucleating bacterium was suggested because filtration for overwintering, thus the ice nucleating bacteria may significantly reduced the ice nucleating activity of the be removed allowing the insect to supercool in winter. palial fluid. Additional circumstantial evidence was the isolation of ice nucleating active bacteria from sea USE OF ICE NUCLEATING MICROORGANISMS water surrounding the bivalve. Ice nucleating activity FOR BIOLOGICAL CONTROL is known from marine microorganisms; Fall and Schnell (1985) identified a marine pseudomonad that The rationale for the use of ice nucleating micro- was phenotypically similar to P. fluorescens. organisms for biological control is based on their poten- Recent studies in our laboratory have demonstrated tial for reducing the cold-hardiness of insect pests. In the association of ice nucleating active bacteria and practice these microorganisms would increase winter freeze-tolerant vertebrates (Lee M. R. et al., 1992a). P. mortality by decreasing the effectiveness of natural fluorescens, E. agglomerans and P. putida were isolated mechanisms of supercooling. With this application from the gut of the freeze-tolerant wood frog, Rana in mind, a number of investigators have examined the syluatica, a species that routinely encounters and sur- use of ice nucleating microorganisms to manipulate vives freezing in nature (Table 1). Ice nucleating activity the supercooling point and thereby decrease the cold was previously undescribed in P. putida. tolerance of insects. Since ice nucleating active microorganisms are normal flora in a variety of insects and are associated with other Bacteria freeze-tolerant animals, the question arises as to the Ingestion of ice nucleating active bacteria causes a ecological and evolutionary significance of this relation- rapid and significant increase in the supercooling points ship. In freeze-tolerant insects, they may function to of insects (Strong-Gunderson et al., 1990a, b). Adults of promote ice nucleation at warm sub-zero temperatures H. convergens that ingested water or a non-ice nucleating (Lee, 1991). In this case they would function like ice active bacterium, Escherichia coli, have supercooling nucleating proteins/lipoproteins that limit supercooling point values of - 15°C or lower. In contrast, beetles that of the insect’s body fluids, insuring that ice formation are fed bacterial suspensions of P. syringae and Erw. begins in the extracellular compartment (Zachariassen herbicola (1 O9bacteria/ml) have supercooling points that and Hammel, 1976; Duman and Horwath, 1983). Insect are 12-14°C higher. Even beetles that are allowed to ice nucleating proteins/lipoproteins are relatively less drink for as few as 10 s show an immediate elevation in efficient nucleators compared to ice nucleating active the supercooling point. The magnitude of the increase is bacteria (Table 1). Ice nucleating active microorganisms related to the concentration of the bacterial solution would function even better in this role, since even though ingested; a P. syringae concentration of 106cells/ml only a portion of the cells are active at the highest produced a mean supercooling point of - 10°C while a subzero temperatures (about -2”C), only one cell in concentration of lO’cells/ml resulted in a value of the population need be phenotypically active at this - 3.9”C. Following ingestion of bacteria the supercool- temperature to nucleate an ice crystal that will then ing points remain elevated for at least 7 days. spread throughout the insect’s body. Ice nucleating In retrospect it is perhaps not surprising that the microorganisms that regulate the supercooling point of ingestion of a potent ice nucleator should cause a freeze-tolerant insects, as in the case of the rice stem dramatic decrease in the supercooling capacity of an borer (Tsumuki and Konno, 1991; Tsumuki et al., 1992) insect. However, it is surprising that the application of represent a mutualistic relationship in which the micro- these bacteria to the surface of the insect should have organism enhances the freeze tolerance of the insect and an equally dramatic effect on the supercooling point. by doing so preserves its host and food source. When suspensions of P. syringae were misted onto The significance of the presence of ice nucleating beetles whose mouths had been sealed, the supercooling active microorganisms in freeze-intolerant insects is un- points increased similar to that observed following 8 REVIEW ingestion (Strong-Gunderson et al., 1989, 1992). Even (Fields, 1991). However, the use of low temperatures for beetles that were misted with P. syringae and allowed to insect control is generally slower acting than chemical dry had elevated supercooling points for at least 3 days application and is often costly in terms of electrical power following the treatment. That surface application of a for ventilation and refrigeration (Fields, 199 1). potent nucleator rapidly and significantly increases Fields (1991) proposed the use of ice nucleating active supercooling points has obvious significance for the bacteria to control stored grain insects. He used a potential use of these bacteria as biological insecticides. freeze-dried, killed preparation of P. syringae to reduce A variety of insects have been tested for the effect of the cold-hardiness of the freeze-intolerant rusty grain ice nucleating active bacteria on their supercooling points beetle, Cryptolestes ferrugineus (Fields, 199 1). Supercool- (Table 2). All four species of ice nucleating active bacteria ing point increases were directly related to the concen- that were applied as living suspensions have the potential tration of bacteria applied. At the highest concentration to significantly elevate the supercooling point. Dusting tested (1000 ppm), supercooling points were - 8.1”C, an insects with a freeze-dried, killed preparation of P. increase of 11°C above that of untreated controls. syringue also causes a significant increase in the super- Survival at subzero temperatures closely correlated with cooling point (Fields, 1991; Lee R. E. et al., 1992b). This the supercooling point data; exposure for 24 h at - 10°C effect is widespread as 15 species from four orders of caused only 8% mortality in untreated control groups, insects show an increase in the supercooling point follow- while 81% of the beetles treated with 1000 ppm of P. ing the application of ice nucleating bacteria to their syringae died. In a simulation of field conditions in surface. The magnitude of the increase in the super- granaries, Fields (1992) treated C. ferrugineus with vari- cooling point differs among insects. Treatment with P. ous concentrations of P. syringue; application of as little syringue elevated the supercooling point by only 2°C in as 10 ppm produced significantly reduced insect survival. the flat grain beetle, Cryptolestes pusillus (Fields, 1992) Additional studies in our laboratory further examined whereas values increased by 11.9”C in the lesser grain the effect of P. syringae on the cold tolerance of a variety borer, Rhyzopertha dominica (Lee R. E. et al., 1992b). of stored grain pests (Lee R. E. et al., 1992b). The application of 100 ppm of ice nucleating active bacteria Fungi to wheat or corn containing insect pests markedly Recent studies in our laboratory demonstrated that decreased the insect’s supercooling capacity. Following surface application of the filamentous ice nucleating treatment with 100 ppm of P. syringue, the mean super- active fungus Fusarium acuminatum suspensions elevate cooling points of five species increased by as much as the supercooling point of H. convergens (Lee M. R. et al., 11.9”C above untreated controls (Table 2). Treatment 1992b). Using an aqueous suspension of F. acuminatum with P. syringae also decreased the insect’s capacity to (0.01 g/3 ml sterile distilled water) 50% of the 10 ~1 drops survive a 24-h exposure to -5°C in every stored grain freeze at - 6.1 “C or warmer temperatures. When beetles pest tested: Indian meal moth larvae [Plodiu inter- were misted with this suspension, their supercooling punctella (Hubner)], red flour beetle adults [Tribolium points increased slightly from - 14.9”C (misted with castaneum (Herbst)], flat grain beetle adults [C. pusillus water only) to - 11.O”C. In an attempt to further increase (Schonherr)], rusty grain beetle adults [C. ferrugineus the supercooling point, we added surfactants to the (Stephens)], Gibbium psylloides (Czenpinski), lesser grain fungal suspension. A 1% solution of Tween 80 caused an borer adults [R. dominica (F.)], yellow mealworm larvae elevation of the supercooling point to - 13.3”C. In [Tenebrio molitor (L.)] and granary weevil adults contrast, when the fungal suspension contained a 1% solution of Tween 80 the supercooling point increased to - 5.8”C. Application of Tween 20, Triton X-100, and gum arabic with the fungal suspension had minimal effect in elevating the supercooling point. These preliminary results suggest that surfactants used in combination with ice nucleating active microorganisms may be useful in the development of protocols for the control of some insect pests (Lee M. R. et al., 1992b).

APPLICATION OF ICE NUCLEATING BACTERIA TO STORED PRODUCT INSECT PESTS Exposure to temperature extremes is a commonly used T. molitor S. granarius P. interpunctella C. pusillus means of controlling stored-product insects (Mullen and FIGURE 2. Survival of stored grain insect pests exposed to 100 and Arbogast, 1979; Hunter and Taylor, 1980; Fields, 1991, IOOOppm of dry, powdered P. syringae in wheat for 24 h at 23°C 1992). Since this approach relies on a physical means of before 24 h exposure to - 5°C. Insects (n = 28-30) used were larvae of control it offers obvious advantages over the use of T. molitor and P. interpunctella and adults of S. granarius and C. pusillus. All differences between untreated control and groups treated pesticides, such as malathion and phosphine, with respect with P. syringae were significant at P < 0.05. Asterisk indicates no to residues in foods and safety concerns for applicators survivors. Data from Lee R. E. et al. (1992b). REVIEW 9

[Sitophilus granarius (L.)] (Fig. 2). Insects, such as C. ferrugineus were treated with various concentrations C. pusillus, that showed significant mortality in the of P. syringae for 1 or 7 days at 30°C prior to subzero control group at -5°C experienced even greater mor- exposure (Fields, 1991). Ice nucleation activity at three tality with the application of P. syringae. In some cases, concentrations of P. syringae decreased by 33-40% increasing the treatment dose from 100 to 1000 ppm between day 1 and 7. It is interesting to note that, despite increased mortality by 25-50%. Although this study this decrease in activity, mortality was highly correlated used non-cold-acclimated insects, no evidence to date with the index of ice nucleating activity during this suggests that cold acclimation decreases the effectiveness interval (Fig. 3). Possibly increased bacterial application of ice nucleating active bacteria in decreasing the super- could be used to reach a specific level of mortality. It cooling capacity of insects (Strong-Gunderson et al., would be desirable for ice nucleating activity to be 1990a, b; Fields, 1991, 1992; Lee R. E. et al., 1992b). maintained in granaries over extended periods because The use of ice nucleating active microorganisms as this would allow treatment of the grain at the time it was biological insecticides requires that insects be exposed to being loaded into the granaries in the autumn. Several temperatures below their supercooling points. Probably months are often required in the northern U.S. and they would be most effective and economical against Canada before grain cools to subzero temperatures. freeze-intolerant pests that overwinter in aggregated Alternatively this problem could be addressed by devel- or restricted microhabitats that naturally experience oping bacterial or fungal strains or formulations that subzero temperatures during the winter. However, it retain ice nucleating activity at high temperatures. is possible that refrigeration could be combined with It may also be possible to augment the effectiveness of the application of the biological nucleator. Since these ice nucleating active microorganisms in elevating the microorganisms are field isolates, their use circumvents supercooling point. Inoculative freezing involves water the problems associated with the release of genetically on or possibly in the insect cuticle. In the wheat stem engineered organisms or toxic synthetic compounds into sawfly, Cephus cinctus, the addition of a detergent to the environment. Furthermore, since this control the water applied to the cuticle increased inoculative method would be applied after the growing season when freezing as compared to soaking the sawfly with water microhabitat temperatures of the insect fall below 0°C alone (Salt, 1963). Using a similar approach with plants, it would be particularly attractive in its compatibility Bunster et al. (1989) reported that the surfactant Tween with other control measures of integrated pest manage- 80, added to a bacterial suspension, increased the wet- ment programs. tability of the leaf cuticle and facilitated homogeneous A current limitation of the use of ice nucleating active distribution of the bacteria across the surface. Since leaf bacteria for biological control is their susceptibility to a and insect cuticles are both hydrophobic, the utilization loss of activity upon warming. Bacterial cultures con- of surfactants in the bacterial suspensions may increase ditioned at low temperatures have greater ice nucleating cuticle wettability, thereby increasing the efficacy of activity than ones held at higher temperatures (Rogers inoculative freezing. As discussed previously, the et al., 1987). Similarly, the activity of concentrated, addition of a surfactant to F. acuminatum suspensions freeze-dried and killed preparations of P. syringae, significantly increased the supercooling point as com- such as the ones used by Fields (1991) and Lee R. E. pared to the use of the fungal suspension alone (Lee et al. (1992b), are also high-temperature sensitive. This M. R. et al., 1992a). In addition to these synthetic temperature sensitivity was evident when adults of surfactants, natural biosurfactants produced by bacteria

100

80 0 - i E * Y = -4.75 +9.83x R"2 = 0.930 h 60- .s

E! 40- z 2O-

0 I- , 0 2 4 6 8 Index of Ice Nucleating Activity FIGURE 3. Effect of ice nucleating activity of P. syringue on the mortality of the rusty grain beetle, C. ferrugineus exposed to low temperature. Index of ice nucleating activity is the log of the number of ice nucleating sites per gram of P. syringae. Wheat containing C. ferrugineus was treated with various concentrations of P. syringue and held for 1 or 7 days at 30°C prior to exposure to - 10°C for 24 h. Data are from Table 2 of Fields (1991). 10 REVIEW

might be combined in culture with ice nucleating active insect pest with these bacteria or fungi? If so, could or microorganisms to promote inoculative freezing. We how rapidly would insects develop resistance to this type have observed that, in general, strains of Pseudomonas of control? The answers to these questions will enhance fluorescens (a less efficient nucleator than P. syringae) our understanding of natural interactions between in- have a much weaker surface tension than P. syringae sects and ice nucleating microorganisms as well as aid in strains. Even though a particular ice nucleating active the evaluation of their potential for use in biological strain is a weaker nucleator, it may be more effective as control. a transcuticular inoculant because of its inherent ability REFERENCES to lower water’s surface tension. By promoting the spread of ice nucleating microorganism over the insect, Anderson J. A. and AshworthE. N. 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