PHYSIOLOGICAL AND CHEMICAL ECOLOGY Ecology of Mountain Pine Beetle (Coleoptera: Scolytidae) Cold Hardening in the Intermountain West

1 2 B. J. BENTZ AND D. E. MULLINS

Environ. Entomol. 28(4): 577Ð587 (1999) ABSTRACT The mountain pine beetle, Dendroctonus ponderosae Hopkins, spends the majority of its life cycle within the phloem of pine trees, experiencing exposure to temperatures below Ϫ30ЊC in many parts of their expansive range. To better understand cold tolerance capabilities of this , seasonal patterns of cold-hardiness, as measured by supercooling points in the laboratory, were compared with seasonal patterns of host tree phloem temperatures at several geographic sites for 2 beetle generations. Larvae were found to be intolerant of tissue , and supercooling points measured appear to be a reasonable estimate of the lower limit for survival. Of the compounds analyzed, was found to be the major . No differences in supercooling points were found among instars or between larvae collected from the north and south aspect of tree boles. Both phloem temperatures and supercooling points of larvae collected from within the phloem were found to be different among the geographic sites sampled. Mountain pine beetle larvae appear to respond to seasonal and yearly ßuctuations in microhabitat temperatures by adjusting levels of cold hardening.

KEY WORDS Dendroctonus ponderosae, bark beetle, supercooling point, freeze intolerant

THE MOUNTAIN PINE beetle, Dendroctonus ponderosae tectants such as polyhydric alcohols (polyols) and Hopkins, which overwinters under the bark of pine sugars (Hamilton et al. 1985, Lee and Denlinger 1991). trees in a nonmobile stage, is unable to escape low Polyol synthesis is known to be triggered by low- temperature exposure. When escape from low tem- temperature exposure, with increasing rates at lower peratures is unavoidable, many insect species respond temperatures (Baust 1982, Storey and Storey 1983). by adjusting physiological and biochemical processes Most likely, thermoperiodic cues, represented by that enhance their tolerance to freezing temperatures. some threshold length of time at or below a particular Freeze tolerant species are able to withstand the for- temperature, promote accumulation of an adequate mation of ice in the extracellular body ßuid, whereas concentration of before the time they freeze intolerant species must avoid freezing of body are needed (Storey and Storey 1991). To more fully tissues (Salt 1961). Freeze avoidance is accomplished understand the role of cold-hardening in mountain through cold-hardening. The supercooling point re- pine beetle population dynamics, therefore, it is nec- fers to the temperature at which spontaneous nucle- essary to relate supercooling capacities as determined ation of body water occurs and ice crystals begin to in the laboratory to the thermal history of the micro- form in the insect tissue (Lee 1989). For those species habitat where larvae reside (Bale 1991). that cannot survive tissue freezing, the supercooling The microhabitat of the mountain pine beetle is the point represents a lethal temperature threshold, al- phloem of living pines, wherein the majority of the life though death may also result as a consequence of cycle is spent typically overwintering as larvae. In the exposure to temperatures above the supercooling northernmost part of their range, temperatures point (Lee 1991). The cold-hardening capacity of an below Ϫ30ЊC are not uncommon. Mountain pine bee- insect may vary with the developmental stage, nutri- tle eggs and pupae are considered the least cold tol- tional status, and duration of exposure to speciÞc low erant life-stages (Reid 1963, Reid and Gates 1970, temperatures. Amman 1973), whereas the large larvae are thought to Many physiological mechanisms involved in the be the most cold tolerant (Yuill 1941, Wygant 1942, cold-hardening process have been identiÞed includ- Somme 1964). Given that effects of temperature on ing ice-nucleating , lipoproteins and anti- developmental rate are stage-speciÞc (Bentz et al. freeze proteins, evacuation of the gut to remove po- 1991), cold-hardening capabilities may also depend on tential ice nucleating agents, ice nucleating bacteria, life-stage, with one stage more adapted for overwin- and accumulation of low molecular weight cryopro- tering than another. Although temperature has pre- viously been assigned as the most important mortality factor of the mountain pine beetle (Safranyik 1978, 1 USDA Forest Service, Rocky Mountain Research Station, Logan, UT 84321. Cole 1981), little is understood about the timing of low 2 Virginia Polytechnic Institute and State University, Blacksburg, temperatures and their effect on the population dy- VA 20461. namics of this nondiapausing species. SpeciÞc objec- 578 ENVIRONMENTAL ENTOMOLOGY Vol. 28, no. 4

Table 1. Site collection information

Site Host Elevation, m Latitude Site aspect Basal area, ft2/acre 1992Ð1993 Logan, UT LPP 2,043 41Њ 56Ј NE 210 Ranch, ID LPP 2,061 44Њ 07Ј SW 140 Moose, WY LPP 2,121 42Њ 45Ј N 100 Galena, ID LPP 2,348 43Њ 54Ј W 170 1994Ð1995 Ranch, ID LPP 2,061 44Њ 07Ј SW 170 Beaver, ID LPP 2,247 43Њ 59Ј W 160 Cedar, UT PP 2,622 37Њ 37Ј E 270

LPP, logdepole pine (Pinus contorta Douglas); PP, ponderosa pine (P. ponderosa Laws). tives of this study were the following: to determine Campbell ScientiÞc, Logan, UT). A sudden tempera- supercooling points of Þeld collected mountain pine ture increase (Ն0.5ЊC), which is associated with for- beetle larvae and associated microhabitat temperature mation of an ice lattice in insect tissue, indicated the regimes, to determine the physiological mechanisms supercooling point of each individual instar. After the that allow cold tolerance in mountain pine beetle supercooling point was determined, each larvae was larval stages, and to characterize the pattern of low placed at room temperature and the head capsule temperature tolerance among geographic regions. width measured to determine instar (Logan et al. 1998). After several hours at room temperature, larvae were gently probed to evoke a response. If larvae are Materials and Methods freeze-tolerant, they can survive the freezing process Supercooling Point Determination. Mountain pine and therefore would respond to probing after warm- beetles of different instars were collected from several ing up. Otherwise, if larvae are dead after the freezing infested trees at 4 sites during the 1992Ð1993 genera- process, we assumed they were freeze-intolerant. tion, and 3 sites during the 1994Ð1995 generation (Ta- To assess if larvae were alive at temperatures above ble 1). Individuals were collected from 1 site, Ranch, the measured supercooling point, 16 larvae were both years. All larvae were collected above the snow cooled (as above) to Ϸ4ЊC above the mean super- line from either the north or south aspect of the bole. cooling point determined for 16 additional larvae Samples were collected 6 times during each genera- taken from the same tree. The larvae were slowly tion from the same trees. On each sample date, larvae returned to room temperature and allowed to sit for were removed live from under the bark, placed in petri 24 h. After this period, larval survival was determined. dishes with moistened Þlter paper, and the dishes Supercooling data were analyzed separately for packed in insulated coolers for transportation to the each generation. Larvae from each site were pooled by laboratory. We also recorded the proportion of larvae month of collection. A three-way analysis of variance that were dead at the time of collection. In 1992Ð1993, (ANOVA) with subsampling, blocked by site, was larvae were shipped via overnite express mail to Vir- performed using month, aspect on the tree, and instar ginia Polytechnic Institute and State University as the factors (SAS, PROC GLM, SAS Institute 1989). (VPI&SU), Blacksburg, VA, for supercooling point Although larvae were collected from 2 to 3 trees at determination. In 1994Ð1995, all supercooling point each site on each sample date, this separation was not analysis was performed at the Forestry Sciences Lab- maintained in the supercooling analysis and a measure oratory in Logan, UT. In 1994Ð1995, a subsample from of tree replication at each site was therefore not avail- 3 sample dates were shipped to VPI&SU for polyol able. An experimental error term was calculated as the analysis. interaction between site and the treatment effects The same technique for determining supercooling (aspect, instar, and month). This experimental error points was used both years. Using forceps, individual term included the error caused by subsampling and larvae were positioned between 2 copper constantan was used in calculating the F statistic. Interaction thermocouples (COC0Ð003, Omega Technologies, terms that included cells with empty data were ex- Stamford, CT) resting on the surface of a cold plate cluded from the analysis. The StudentÐNewmanÐ (Stir-Kool Cold plate [SK-31], Thermoelectrics Un- Keuls method was used to compare treatment means. limited, Wilmington, DE) connected to a Fisher Iso- Temperature Measurements. Beetle ßight, host col- temp Circulator (13-874-72A, Pittsburgh, PA) that had onization, and brood emergence were monitored at been cooled to Ϸ0ЊC. Eight larvae were analyzed at 1 each site. Upon successful host colonization, temper- time. Temperatures among thermocouples were sta- ature probes were placed in the phloem of 4 infested bilized by placing an insulating box over the cold plate. trees at each site, 1.4 m above the ground on the north The temperature of the cold block was reduced by and south side of each tree. Temperature probes con- Ϸ1.5ЊC per minute. Measurements of top and bottom sisted of 30-gauge chromega/constantan thermocou- larval temperature were automatically recorded every ple wire (Type E-nickel-chromium and copper-nickel second using a data micrologger (21ϫ Datalogger, wire, Omega Technologies, Stamford, CT) connected August 1999 BENTZ AND MULLINS:ECOLOGY OF MOUNTAIN PINE BEETLE COLD HARDENING 579

Table 2. Results of ANOVA used to determine differences in Each sample was weighed and placed in microcen- daily maximum, minimum, and average phloem temperatures trifuge tubes with 20 ␮l of 70% ethanol: 30% distilled among sites and between tree bole aspects water. A sample consisted of 3Ð4 small larvae in the Between N and S Among November collection, and a large single larva in the Source tree bole aspect sites January and March collections. The samples were ho- F (df ϭ 1, 3,037) PF(df ϭ 3, 3,037) P mogenized with a probe tip, and the probe rinsed with 150 ␮l of the 70% ethanol: 30% distilled water solution. 1992Ð1993 The samples were then centrifuged for 3 min at Max 31.84 0.001 5.09 0.002 ϫ ␮ Min. 0.46 0.498 32.56 0.001 5,600 g. A 150- l aliquot of the extracting solvent Avg 2.14 0.144 13.85 0.001 was then transferred to a 0.5-ml reaction vial and 1994Ð1995 evaporated to dryness at room temperature using a Max 51.42 0.001 27.05 0.001 nitrogen evaporator. Under a stream of nitrogen, 0.1 Min. 0.83 0.363 119.91 0.001 ml of the derivatizing agent (MTBSTFA ϩ TBDMCS, Avg 4.35 0.037 52.88 0.001 99:1 [N-(tert-Butyldimethylsilyl)-N-methyl-trißuora- cetamide, with 1% TBDMSCI]; Aldrich, Milwaukee, WI) and 24 ml of acetonitrile were added to the via a 20-gauge chromega/constantan wire to a mi- reaction vial, vortexed, and placed on a heat block that crologger (see above). Phloem temperatures were had been heated to a temperature of 100ЊC. After 2 h measured every 15 min and averaged and recorded for the samples were removed and allowed to cool. Each each hour, beginning just after successful colonization sample was again evaporated to dryness at room tem- and ending during beetle emergence and ßight the perature using a nitrogen evaporator. Hexane was following year (Ϸ365 d). Ambient air temperatures added to attain a volume of 0.15 ml. The samples were were measured 1.4 m above the ground, using a then diluted as necessary for GC-MS analysis. Stan- shielded temperature probe placed on the north side dards for glycerol, sorbitol, and dulcitol were derivat- of a tree. Hourly temperatures of all 4 trees at a site ized using the same technique. Results are reported in were Þrst averaged by north and south bole aspect. ␮g/g of live wet tissue weight. Daily maximum, minimum, and average temperatures were then computed for a site, and by tree aspect Results within a site. Polyol synthesis is often dependent on temperature Daily maximum phloem temperatures on north and acclimation that is a complex combination of the du- south aspects of tree boles were signiÞcantly different ration and intensity of temperatures experienced in at all sites both years (Table 2). Daily average tem- the insectÕs microhabitat. To relate recorded phloem peratures were signiÞcantly different between the temperatures and polyol synthesis, cooling and heat- north and south aspects of tree boles in 1994Ð1995, ing units were calculated using trapezoid integration although not in 1992Ð1993. Daily minimum phloem above a lower threshold. Because polyol synthesis is temperatures between tree bole aspects were not sig- typically triggered by temperatures around 0Ð5ЊC niÞcantly different at any site either year. There were (Baust and Miller 1970, Storey and Storey 1991), these signiÞcant differences in daily maximum, minimum, values were used as lower thresholds in calculating and average phloem temperatures (north and south temperature units. Heat (Ͼ0or5ЊC) and cold (Ͻ0or aspect averaged) among all sites, both years (Table 2) 5ЊC) units were calculated for each day then summed (Figs. 1 and 2). Cumulated heating and cooling units over the life of the generation. Accumulation for all were also signiÞcantly different among sites and sam- sites and both generations was begun on Julian date ple months both years (Table 3). Winter phloem tem- 224, an average start date for oviposition at all sites. peratures at the Ranch site were colder in 1992Ð1993 ANOVA was performed to test for signiÞcant differ- than in 1994Ð1995. A total of 1,060 cold units was ences in accumulated cooling and heating units across accumulated at the Ranch site in 1992Ð1993 (Fig. 3B) sites and months, separated by generation year. compared with 847 in 1994Ð1995 (Fig. 4B), and the Polyol Analysis. Several techniques were employed average winter supercooling point in 1992Ð1993 to measure hemolymph polyols of mountain pine bee- (-34.0ЊC) was signiÞcantly lower than in 1994Ð1995 tle larvae. Initial high-performance thin-layer chro- (-29.9ЊC) (t ϭϪ3.466, df ϭ 52, P Ͻ 0.000). matgraphy analysis identiÞed relative positions for As a result of developmental progression with time sorbitol, dulcitol, and glycerol. Gas chromatography and sampling error, not all instars were represented in (GC) combined with mass spectrometery (MS) were all samples (Tables 4 and 5). For both generations, then employed to more accurately measure which supercooling points among sample months and sites compounds were present. Mountain pine beetle lar- were signiÞcantly different (Table 6). The signiÞcant vae were collected from infested lodgepole pine trees interaction between site and month in 1992Ð93 implies at the following 3 times during the life cycle: (1) 23 that the observed differences varied among the sites November 1994, (2) 11 January 1995, and (3) 8 March depending on sample month (Fig. 3C and 4C). In 1995. In the Þeld, larvae were place in petri dishes with 1992Ð93, mean comparisons showed that average su- slightly moistened Þlter paper, packed in coolers with percooling points among all sites were different (df ϭ ice, and shipped to VPI&SU via overnite express mail. 428, P Ͻ 0.05). In 1994Ð95, only the average super- Upon arrival, larvae were immediately frozen. cooling point at the Cedar site was signiÞcantly dif- 580 ENVIRONMENTAL ENTOMOLOGY Vol. 28, no. 4

Fig. 1. Maximum and minimum phloem temperatures (T, ЊC) at 4 sites (AÐD) in 1992Ð1993 with the mean (Ñ) and range (‚, ƒ) of associated larval supercooling points (SCP) (ЊC). ferent from the Ranch and Beaver sites (df ϭ 517, P Ͻ the average supercooling point measured for indi- 0.05). No signiÞcant differences in supercooling points viduals 8 d later. No mortality was observed at this were observed among instars nor among individuals time. Assigning mortality factors in the Þeld was collected from the north and south bole aspect in difÞcult, however, because of the rapid desiccation either generation (Table 6). Although the proportion and decline of larval carcasses. Consequently, in st nd of 1 and 2 instars in winter samples of lodgepole both generations we observed very little mortality rd th pine hosts were lower than proportions of 3 and 4 during Þeld sampling. During the spring especially, instars at the same time, all instars were observed in many individual supercooling points were warmer samples throughout the winter and into May and June than minimum phloem temperatures experienced at in both hosts. that time (Figs. 1 and 2). Supercooling points in the There was no response from any individual allowed fall were always lower than Ϫ10ЊC despite the ab- to warm to room temperature after supercooling point sence of any temperatures below a threshold of 0 or determination. Observed survival was 100% for the 16 5ЊC (Figs. 3B and 4B). In the middle of February larvae that were cooled to temperatures just above the (around Julian day 45), small increases in temper- supercooling point. Њ A similar seasonal trend was observed in mean ature units above 0 C were associated with a raising supercooling points in both generations, for all in- of the supercooling point. Although cooling units stars, at all sites except Logan. Values were approx- were still being accumulated, the rate of accumu- imately Ϫ10 to Ϫ20ЊC in early fall, dropped to Ϫ25 lation during this time was decreasing, whereas heat to Ϫ35ЊC in January, and increased again in the units were constant or only slightly increasing. spring and early summer (Figs. 1 and 2). At all sites The major polyol found in larval mountain pine except Beaver, the associated daily minimum beetles was glycerol, with negligible concentrations of phloem temperatures, a representation of the mi- sorbitol or dulcitol. There were no signiÞcant differ- crohabitat of larvae, were above the average super- ences found in glycerol levels of larvae collected from cooling point for larvae on each sample date (Figs. the north and south aspects of tree boles. Glycerol 1 and 2). Around Julian day 1 at the Beaver site, levels (mean Ϯ SE) in the 3 samples collected in minimum phloem temperatures were slightly below November (6.8 Ϯ 3.0 ␮g/g), January (8.6 Ϯ 4.0 ␮g/g), August 1999 BENTZ AND MULLINS:ECOLOGY OF MOUNTAIN PINE BEETLE COLD HARDENING 581

Fig. 2. Maximum and minimum phloem temperatures (T, ЊC) at 3 sites (AÐC) in 1994Ð1995 with the mean (Ñ) and range (‚, ƒ) of associated larval supercooling points (SCP) (ЊC). 582 ENVIRONMENTAL ENTOMOLOGY Vol. 28, no. 4

Table 3. Results of ANOVA used to determine differences in aspects of tree boles were not signiÞcantly different. cooling and heating units among sites and sample months Minimum phloem temperatures within the lodgepole pine from which larvae were collected also were not Among sites Among months Source ϭ ϭ signiÞcantly different between the aspects. Southern F (df 3, 457) PF(df 6, 457) P exposures of tree boles had signiÞcantly warmer tem- 1992Ð1993 peratures, but this had no consistent effect on super- Cooling units 254.40 0.001 12,609.15 0.001 cooling capacity of the larvae. Minimum phloem tem- Heating units 394.61 0.001 6,988.80 0.001 peratures, which typically occur around sunrise, 1994Ð1995 appear to have more of an effect on larval cold-hard- Cooling units 3,124.54 0.001 1,517.73 0.001 ening than daily maximum temperatures. Supercool- Heating units 2,319.71 0.001 1,229.38 0.001 ing points of larvae may indeed be inßuenced by changing temperatures within a 24-h period, although this effect was not observed in our monthly samples. and March (10.6 Ϯ 6.4 ␮g/g) were not signiÞcantly Previous laboratory research with other has different. shown that short-term exposure to high temperatures may decrease the capacity to supercool (Lee et al. 1987). If this physiological phenomena occurs in the Discussion mountain pine beetle, with periodic warming Larvae that were cooled to temperatures just above trends could result in lower survival rates than winters the measured supercooling point survived, suggesting with consistent cold temperatures, especially so on that mortality above the measured supercooling point southern bole aspects. is minimal, and that the supercooling point is a rea- Daily maximum, minimum, and average phloem sonable measure of cold tolerance for this insect. To temperatures were signiÞcantly different among the truly understand mortality above the supercooling sites both years (Table 2). Accumulated temperature point however, long-term effects will need to be as- units, both above and belowa0and5ЊC threshold, sessed. Additional tests wherein larvae are kept at were also signiÞcantly different among all sites (Table temperatures just above the supercooling point for 3). These differences imply that larvae from at least extended periods will need to be performed to con- some geographic sites were exposed to different mi- clusively determine if larvae can indeed survive tem- crohabitat temperature regimes. Observed differ- peratures above the supercooling point. The lack of ences in temperature regimes among the sites may be response from any individual allowed to warm to room contributing to the differences observed in larval su- temperature after supercooling point determination percooling points among the sites, and could also im- suggests that the mountain pine beetle is freeze in- ply differential mortality. The most striking differ- tolerant and cannot survive tissue freezing. Several ences were observed between the Cedar and Beaver other scolytid bark beetles have been found to be and Ranch sites in 1994Ð1995. In the middle of winter, freeze intolerant (Ring 1977, Gehrken 1984, Miller larval supercooling points at Cedar were as much as and Werner 1987). 6ЊC higher than supercooling points at either Beaver All mountain pine beetle instars were found to over- or Ranch (Fig. 4C). The Cedar site, which was the winter, although higher proportions of 3rd and 4th instars only site composed of all ponderosa pine hosts, was were observed during winter months. Although Amman located at the highest elevation (2,622 m), lowest (1973) and Langor (1989) observed higher mortality in latitude (37ЊC), and had the fewest accumulated cool- smaller larvae (e.g., 1st and 2nd instars) during the winter ing units. Cold units accumulated at the Cedar site months, and mountain pine beetle instars have been were only a 3rd of the cold units accumulated in found to exhibit stage-speciÞc development rates (Bentz lodgepole pine at the other sites monitored that year et al. 1991), we found no signiÞcant differences among (Fig. 4B). The Cedar site also had the highest mean instars in their capacity to cold-harden. This is in contrast supercooling points at all sample times except in April to a study with the weevil Hypera punctata (F.) where a (Fig. 2A). Because of a high metabolic cost (Danks positive correlation between supercooling points and 1978), maintaining elevated levels of cold-hardiness larval body weight was found, suggesting that larger (low supercooling points) is not advantageous at times instars have less capacity to cold-harden (Watanabe and when it is not required. Differences observed between Tanaka 1997). Although we did not measure size of the Cedar and Ranch and Beaver sites indicate that larvae within an instar, adult Douglas Þr beetles, Den- mountain pine beetle larvae may have the capacity to droctonus pseudotsugae Hopkins, and mountain pine bee- metabolize only amounts of cryoprotectants needed, tles emerging from bolts kept at cool, as compared with given a particular thermal history. The thresholds re- warm, temperatures were larger (Atkins 1967, Amman quired to trigger this , as well as other and Cole 1983). The Douglas Þr beetles reared at the cold-hardening mechanisms, is unclear. Although su- colder temperatures also had proportionately greater percooling points of eggs were not analyzed, we ob- content. served viable eggs in ponderosa pine at the Cedar site Although others have observed differences in cold- on all sample dates, whereas viable eggs were found no hardening capacity between aspects (Gehrken and later than October in any samples from lodgepole Zachariassen 1978), supercooling points of mountain pine. Observed differences in cold-hardening capacity pine beetle larvae collected from the north and south between the Cedar and both Ranch and Beaver sites August 1999 BENTZ AND MULLINS:ECOLOGY OF MOUNTAIN PINE BEETLE COLD HARDENING 583

Fig. 3. (A) Accumulated heat units (Ͼ0ЊC) and (B) accumulated cold units (Ͻ0ЊC) at 4 sites in 1992Ð1993 and (C) associated mean supercooling points (SCP) (ЊC) and standard errors. Sites: E, Ranch; Ⅺ, Galena; ‚, Logan; छ, Moose. may also be inßuenced, at least in part, by selective Observed seasonality in the cold-hardening re- pressures on larvae because of tree host nutrition and sponse suggests that mountain pine beetle larvae re- other host-speciÞc factors which differ between pon- spond to ßuctuating temperatures in the microhabit derosa and lodgepole pines. (Figs. 1 and 2), as described for several scolytid bark 584 ENVIRONMENTAL ENTOMOLOGY Vol. 28, no. 4

Fig. 4. (A) Accumulated heat units (Ͼ0ЊC) and (B) accumulated cold units (Ͻ0ЊC) at 3 sites in 1994Ð1995 and (C) associated mean supercooling points (SCP) (ЊC) and standard errors. Sites: E, Ranch; Ⅺ, Beaver; ‚, Cedar. beetles (Hansen et al. 1980, Gehrken 1984, Miller and site that were Ͻ100 m apart were signiÞcantly colder Werner 1987, Luik and Voolma 1990). A year-to-year in 1992Ð1993 than 1994Ð1995. Larvae responded with ßuctuation in cold-hardiness at the same site in re- a mean supercooling point which was Ϫ5ЊC colder in sponse to temperature change was also observed. 1992Ð1993. A considerable amount of variation among Phloem temperatures of infested trees at the Ranch supercooling points of individual larvae on any day, as August 1999 BENTZ AND MULLINS:ECOLOGY OF MOUNTAIN PINE BEETLE COLD HARDENING 585

Table 4. 1992–1993 mean supercooling points for each instar, by site and sample date

Site Instar Sept. Nov. Jan. March May June Ranch 1st Ϫ31.1 Ϫ6.8 N/A Ϫ9.6 N/A N/A (1, 0.0) (1, 0.0) (1, 0.0) 2nd Ϫ14.5 Ϫ12.9 Ϫ35.0 Ϫ15.2 Ϫ3.3 N/A (6, 3.5) (8, 3.0) (4, 1.5) (9, 1.3) (2, 0.0) 3rd Ϫ9.7 Ϫ12.0 Ϫ33.9 Ϫ13.2 Ϫ6.6 Ϫ12.0 (9, 0.9) (4, 2.9) (11, 0.9) (13, 1.1) (7, 0.8) (11, 0.8) 4th N/A Ϫ12.6 Ϫ35.0 Ϫ7.8 Ϫ5.1 Ϫ10.6 (3, 0.9) (3, 0.4) (3, 0.5) (15, 0.5) (10, 1.2) Galena 1st Ϫ14.5 N/A Ϫ21.2 N/A Ϫ7.2 N/A (2, 5.0) (1, 0.0) (1, 0.0) 2nd Ϫ15.2 Ϫ12.7 Ϫ30.8 Ϫ14.0 Ϫ8.2 N/A (7, 2.9) (7, 1.6) (15, 1.0) (9, 1.6) (3, 1.7) 3rd Ϫ17.8 Ϫ16.1 Ϫ31.0 Ϫ16.3 Ϫ7.3 Ϫ9.1 (3, 0.7) (15, 1.8) (12, 1.1) (16, 1.0) (5, 1.2) (9, 1.7) 4th N/A Ϫ11.7 Ϫ30.2 Ϫ14.9 Ϫ5.3 Ϫ8.9 (8, 1.8) (6, 1.6) (4, 3.5) (3, 0.6) (6, 1.4) Logan 1st Ϫ27.4 N/A Ϫ13.9 N/A N/A N/A (2, 3.6) (1, 0.0) 2nd Ϫ27.4 Ϫ24.4 N/A Ϫ21.8 Ϫ10.0 Ϫ8.9 (7, 2.0) (4, 2.9) (7, 0.8) (1, 0.0) (1, 0.0) 3rd Ϫ25.9 Ϫ27.8 Ϫ32.0 Ϫ19.5 Ϫ8.9 Ϫ6.4 (6, 2.2) (16, 1.2) (1, 0.0) (16, 1.3) (10, 0.6) (2, 1.3) 4th Ϫ28.1 N/A N/A Ϫ17.7 Ϫ6.5 Ϫ7.0 (2, 2.4) (6, 1.4) (3, 0.6) (7, 0.6) Moose 1st N/A Ϫ16.2 Ϫ29.9 Ϫ14.3 N/A N/A (1, 0.0) (4, 3.7) (4, 2.9) 2nd Ϫ16.2 Ϫ15.6 Ϫ27.5 Ϫ18.0 Ϫ2.0 N/A (11, 1.4) (10, 2.4) (2, 1.2) (11, 1.2) (1, 0.0) 3rd Ϫ17.1 Ϫ15.9 Ϫ30.1 Ϫ15.6 Ϫ9.1 Ϫ6.8 (6, 1.8) (22, 1.1) (6, 3.4) (10, 1.3) (1, 0.0) (3, 0.2) 4th Ϫ17.1 Ϫ20.5 Ϫ26.5 Ϫ17.1 Ϫ5.2 Ϫ5.4 (3, 3.2) (8, 2.4) (3, 3.5) (8, 0.8) (4, 1.1) (4, 0.5)

Total sample size ϭ 467. N/A indicates no larvae were sampled at that time. Supercooling points are in ЊC. Numbers in parentheses beneath means indicate values for (sample size, Ϯ standard error).

has been found with other insects (Block 1982, Wa- sample of larvae cooled to temperatures just above the tanabe and Tanaka 1997), was also observed. measured supercooling point, suggesting a selection We observed no cold-induced mortality in the small for lower supercooling points than the minimum tem-

Table 5. 1994–1995 mean supercooling points for each instar, by site and sample date

Site Instar Sept. Oct. Nov. Jan. March April May June Ranch 1st N/A Ϫ21.8 N/A Ϫ22.9 N/A Ϫ22.5 Ϫ24.1 Ϫ19.7 (4, 0.6) (1, 0.0) (2, 3.1) (1, 0.0) (1, 0.0) 2nd N/A Ϫ22.9 N/A Ϫ28.4 N/A Ϫ23.3 Ϫ19.3 Ϫ14.5 (3, 0.8) (9, 1.7) (6, 0.5) (14, 1.3) (4, 1.9) 3rd Ϫ13.1 Ϫ20.4 N/A Ϫ28.8 N/A Ϫ22.7 Ϫ17.4 Ϫ14.3 (2, 4.6) (5, 1.3) (14, 1.4) (22, 0.8) (19, 1.3) (11, 1.7) 4th Ϫ16.8 Ϫ23.5 N/A Ϫ32.2 N/A Ϫ22.6 Ϫ20.8 Ϫ12.7 (14, 1.2) (20, 0.4) (17, 0.8) (22, 1.0) (21, 0.9) (22, 0.9) Beaver 1st N/A Ϫ21.7 N/A Ϫ26.1 N/A Ϫ24.2 N/A N/A (3, 0.7) (1, 0.0) (1, 0.0) 2nd N/A Ϫ23.0 N/A Ϫ25.5 N/A Ϫ20.0 Ϫ20.0 Ϫ20.7 (7, 0.5) (2, 12.4) (9, 0.6) (5, 1.3) (2, 1.5) 3rd Ϫ20.5 Ϫ22.4 N/A Ϫ30.1 N/A Ϫ21.6 Ϫ18.2 Ϫ14.2 (10, 0.4) (7, 0.2) (21, 1.2) (29, 0.6) (25, 1.1) (10, 1.8) 4th N/A Ϫ25.2 N/A Ϫ29.1 N/A Ϫ22.3 Ϫ18.6 Ϫ15.5 (21, 0.7) (15, 1.7) (15, 1.0) (26, 0.9) (27, 0.8) Cedar 1st N/A Ϫ22.4 Ϫ20.8 Ϫ23.2 Ϫ21.4 Ϫ19.7 Ϫ20.4 N/A (3, 0.9) (11, 0.4) (4, 1.60) (38, 0.3) (14, 1.2) (11, 0.6) 2nd N/A Ϫ20.2 Ϫ20.8 Ϫ23.6 Ϫ20.6 Ϫ18.5 Ϫ20.1 N/A (4, 0.6) (14, 0.5) (21, 0.6) (23, 0.5) (30, 0.8) (44, 0.3) 3rd N/A Ϫ21.1 Ϫ21.6 Ϫ22.9 Ϫ21.5 Ϫ19.6 Ϫ19.9 N/A (2, 0.2) (12, 0.4) (2, 0.4) (6, 0.6) (8, 0.4) (6, 0.5) 4th N/A N/A Ϫ21.5 Ϫ26.9 Ϫ21.7 Ϫ19.6 Ϫ21.4 N/A (9, 0.4) (5, 1.9) (4, 0.4) (7, 0.5) (1, 0.0)

Total sample size ϭ 750. N/N indicates no larvae were sampled at that time. Supercooling points are in ЊC. Numbers in parentheses beneath means indicate values for (sample size, Ϯ standard error). 586 ENVIRONMENTAL ENTOMOLOGY Vol. 28, no. 4

Table 6. Results of NNOVN used to determine factor effects on for bark beetles, which spend the majority of time supercooling points in 1992–1993 and 1994–1995 under the bark of host trees, temperature is the most reliable environmental cue for cold-hardening. Glyc- 1992Ð1993 1994Ð1995 Source erol was found to be the predominant cryoprotectant F df PFdf P in larval mountain pine beetle from central Idaho, Site 2.80 3, 107 0.05 3.99 2, 89 0.05 whereas levels of sorbitol and dulcitol were negligible. Month 35.51 5, 107 0.001 50.87 7, 89 0.001 Results on seasonality of this compound in larval Aspect 0.18 1, 107 0.488 1.84 1, 89 0.098 Instar 0.11 3, 107 0.839 0.94 3, 89 0.172 mountain pine beetle and its direct relation to tem- Site ϫ Month 3.36 15, 107 0.01 Ñ perature are inconclusive given our results. However, glycerol content in adult and larval spruce beetles, Ñ interaction not analyzed because of missing cells. Dendroctonus rufipennis Kirby, from Alaska (Miller and Werner 1987) and 4th-instar mountain pine beetle (Sømme 1964) from California appear to be regulated perature occurring under the bark. However, addi- by temperature, as does the ethylene glycol concen- tional research is needed in this area to ascertain if tration in Ips acuminatus Gyllenhal larvae (Gehrken larvae can withstand long periods at these tempera- 1984). Other mechanisms most likely also play a role, tures. Although the average supercooling point on a including other cryoprotectants and thermal hysterisis particular day was typically below the minimum provided by high molecular weight particles such as phloem temperature, many individual supercooling proteins and glycoproteins. points were often warmer than the minimum phloem We have investigated only 1 of the processes that temperature, especially in the spring, suggesting that may be responsible for cold-hardening and associated mortality could be occurring (Figs. 1 and 2). Addi- mortality in mountain pine beetle larval stages: freez- tionally, supercooling points in the spring were often ing and the production of glycerol which plays a role higher than those in the fall at the same site. This could in this process. Thermal history experienced by larvae be due in part to the fact that maximum phloem tem- beneath the bark of pine trees, as well as the daily peratures in the spring were generally above lower change in thermal cycles are both important in the developmental thresholds for 3rd and 4th instars (see cold-hardening process for this insect. Larval instars Bentz et al. 1991), perhaps triggering a feeding re- were found to be freeze intolerant, with seasonal sponse and concomitant decreased cold-hardening in changes in supercooling capacity associated with the spring. Spring, therefore, may be the most suscep- phloem temperatures. All instars were found to over- tible time for cold-induced mortality in this insect. winter in both lodgepole and ponderosa pine. Re- It is well documented that individuals experience gional populations of mountain pine beetles can differ greater cold-hardening capabilities when exposed to signiÞcantly in cold-hardiness, apparently because of cold temperatures (Baust and Miller 1970). However, local weather patterns. An important question that the temperature at which acclimation for cold-hard- remains to be answered is if populations of mountain ening begins appears to be species-speciÞc and me- pine beetle at southern latitudes have adapted to the diated by climate. Acclimation for some insects is local climate, or if they are capable of surviving tem- more effective at 3Ð5ЊC (Baust and Miller 1970), perature regimes experienced by populations at more whereas in others polyol production was enhanced at northern latitudes and colder sites. The amount of subzero temperatures (Ϫ5toϪ10ЊC) (Young and cold-hardening that occurs in mountain pine beetle Block 1980). An increase in the rate of temperature populations appears to be dictated by temperature acclimation below 0ЊC was correlated with a decrease regimes at each particular site. It is unclear, however, in supercooling points, although we are unable to if all populations in the intermountain region have the ascertain from our Þeld study the appropriate thresh- same capacity to cold-harden, and only do so if tem- old trigger for accumulation of cryoprotectants. In- peratures dictate it. This sensitivity of mountain pine sects also respond to changes in the duration and beetles to local microclimate emphasizes their impor- temperature of daily cycles (Beck 1991). Observed tance as biological indicators of regional climate maximum cold-hardiness occurred after the peak of change. Observed variation among individual super- daily cold units yet before the maximum accumulation cooling points within an instar, ßexibility in overwin- of cold units (Figs. 3 and 4). Cold-hardening then tering life stage, and the ability of larvae to respond decreased with a decrease in the rate of accumulation seasonally to changing temperature conditions within of cold units (below 0ЊC), whereas heat units re- the microhabitat represent important patterns of phe- mained relatively constant during this period. The rate notypic plasticity which contribute to the success of of change in daily thermal units most likely play an this important outbreak species. important role in mountain pine beetle cold-harden- ing, and a better understanding of time lags and the decay effects of temperature history on cold-hardiness Acknowledgments is needed. Keith Tignor and Karen Johnson were instrumental in Considering the diversity and complexity of the supercooling point determination anaylsis, and Sandra Gab- cold-hardening process, it is unlikely that any single bert was responsible for GC/MS analysis of hemolymph environmental cue is responsible for the complex bio- samples. Jim Vandygriff and Bridget Clayton helped with the chemical strategies that occur (Baust 1982). However, arduous winter sampling. We thank Rick Lee, Matt Ayres, August 1999 BENTZ AND MULLINS:ECOLOGY OF MOUNTAIN PINE BEETLE COLD HARDENING 587 and several anonymous reviewers for valuable comments on Logan, J. A., B. J. Bentz, and J. C. Vandygriff, and D. L. the manuscript. Turner. 1998. General program for determining instar distributions from headcapsule widths: example analysis References Cited of mountain pine beetle data. Environ. Entomol. 27: 555Ð 563. Atkins, M. D. 1967. The effect of rearing temperature on the Luik, A., and K. Voolma. 1990. peculiarities size and fat content of the Douglas-Þr beetle. Can. En- and cold-hardiness of the great spruce bark beetle, Den- tomol. 99: 181Ð187. droctonus micans (Kug.). Proc. Estonian Acad. Sci. Biol. Amman, G. D. 1973. Population changes of the mountain 39: 214Ð218. pine beetle in relation to elevation. Environ. Entomol. 2: Miller, L. 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