Correlative influence of gut appearance, water content and thermal hysteresis on whole body supercooling point of adult bark beetles, Ips acuminatus Gy11

Camp. Biochem.Physiol. Vol. 112A,No. I,pp.207-214.1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0300-9629/95 $9.50 + 0.00

Pergamon 0300-%2!495)ooo6&2

Correlative influence of gut appearance, water content and thermal hysteresis on whole body supercooling point of adult bark beetles, Ips acurninatus Gy11 Unn Gehrken University of Oslo, Department Oslo 3, Norway

of Biology, Division of Zoology, P.O. 1050 Blindern N-0316

Adults of the bark beetle Zps acmzinatus rdy upon supercooling for overwintering success. In preparation for winter, individuals stop feeding, and increase their supercooling capacity. Isolated alimentary canals froze within the temperature range of intact adults, and the agents responsible for nucleation were of a proteinaceous nature. Enhanced supercooling from -12 to - 17T could be attributed to the evacuation of residual food from the gut and a reduction in water content, while a further shift to -2OOC was closely correlated with the level of thermal hysteresis. The principle carbohydrate present during autumn, the disaccharide trehalose, increased from 10 mM at the end of August to 90 mM in November, and seemed to account for a decrease in supercooling points of approximately 1.5OC. Key words: Supercooling; Thermal hysteresis.

Freeze-sensitive;

Comp. Biochem. Physiol. 112A, 207-214,

Ice-nucleating

agents; Water content; Gut content;

1995.

Introduction Arthropods hibernating in cold regions are generally able to withstand fairly low sub-zero temperatures for long periods. Freezing is fatal in the majority of species, and they enhance their supercooling capacity to reduce the probability of freezing. In contrast, decreases in supercooling capacity are believed to protect freeze-tolerant animals by ensuring extracellular nucleation in the range of -5 to - 10°C induced by haemolymph borne ice-nucleating proteins (Zachariassen, 1985). In preparation for winter, freeze-susceptible insects commonly depress the temperature at which ice spontaneously nucleates within their Correspondence to: U. Gehrken, University of Oslo, Depart-

ment of Blindern Received 7 accepted

Biology, Division of Zoology, P.O. Box 1050 N-0316 Oslo 3, Norway. September 1993; revised 30 January 1995; 31 January 1995.

body, termed the supercooling point. Increase in supercooling capacity is closely related to cessation of feeding, particularly among microarthropods (Cannon and Block, 1988). The gut content itself may freeze and contain efficient nucleators (Salt, 1966, 1968; Krunic, 1971; Bakken, 1985; Fields and McNeil, 1988), and recently Shimada (1989) provided direct evidence that ice nucleation begins in the gut of the freeze-tolerant Trichiocampus populi prepupa. The hypothesis that the feeding stages of insect species are generally less cold-hardy than the non-feeding stages (Salt, 1961), has been confirmed by a number of investigators (Sannme, 1982). However, a number of other investigations revealed no correlation between gut content and supercooling. The mean supercooling points of two species of prostigmated mites ranged from -23 to -27°C during feeding (Somme, 1978), and similar low

207

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U. Gehrken

iments. In addition, measurements of mid- and hind-gut food residue, relative water content and the concentration of low molecular weight substances were obtained. Similar measurements were also obtained from pupae and newly-emerged adults during warm acclimation in October. Survival of 10 individuals was tested after 14 days at - 5 or - 10°C whenever beetles were sampled from the two holding regimes in August, September and October. Experiments were also performed to see if the gut itself contained efficient nucleators. A sample of beetles was collected immediately after the transfer to the University of Oslo and by carefully removing the plate surrounding the anus the attached gut followed. The small wound was then sealed with paraffin. The whole body supercooling point of individuals was measured both before and after their mid- and hind-gut had been so removed. The supercooling points of excised alimentary tracts were obtained after determining the whole body supercooling point and the level of gut filling. The alimentary tracts were dissected out under paraffin oil and cut anterior to proventriculus. Measurements of supercooling point were obtained before and after heating to 100°C for 3 min. Individual alimentary tracts were heated in a water bath contained in separate glass tubes under paraffin oil. The alimentary tracts dissected out under paraffin oil were grouped into one of seven categories according to the level of gut filling. Alimentary tracts filled all the way posterior to the proventriculus were classed as category 6. Those with food residues all the way to the malpighian tubules (i.e. residuals present only in the hind-gut) were classed as category 3. An empty gut was classed category 0. The level of gut filling of lo-12 animals was recorded whenever beetles were obtained from the two Materials and Methods regimes and following every step during the Logs of Scats pine colonized by I. acuminatus warm acclimation experiments. Supercooling points were measured with a were collected near Kongsberg (59”40’) SE Norway in August. Cold tolerance was monitored at 0.2 mm diameter thermocouple connected to a various intervals in beetles sampled both from Goerz Metrawatt SE 120 recording potentiometer, at a cooling rate of l”C/min. Samples of logs maintained out of doors at the University haemolymph were measured, contained in of Oslo and in a cold-room at 3°C. In addition, 1.3 mm diameter glass capillaries sandwiched the effects of feeding and starving were studied in individuals collected from the cold-room on between two droplets of paraffin oil. The technique for measuring haemolymph melting and 16 September and with specimens obtained from the outdoor regime on 17 October. They freezing points, the presence and quantity of low were held at 21°C in continuous darkness, either molecular weight substances and the relative water content of I. acuminatus have been dewithin logs (access to food) or housed individuscribed earlier (Gehrken, 1984). The concenally in moist glass vials without access to food. The effect of feeding on the cold-tolerance was tration of low molecular weight substances was given in mM/g beetle water content. Each detertested in the first 4 days following transfer. mination of whole body and haemolymph Whole body supercooling points and haemolymph supercooling points, as well as melting supercooling points represents the mean of 20 and 10 measurements, respectively. Mean and freezing points, were measured in all exper-

supercooling points were demonstrated in the collembolan Crypturgus antarcticus when given a diet of purified green algae (Ssmme and Block, 1982). The particularly pronounced rise in the supercooling point of the non-feeding, ladybird beetle Coleomegilla maculeata in spring suggested that a compliment of nucleators remained within individuals during overwintering (Baust and Morrissey, 1975). Zachariassen (1982) concluded that nucleating agents were synthesized during warm acclimation in the tenebrionid beetle Bolitophagus reticulatus and that intracellular compartments were the most probable site for these nucleators. The cerambycid beetle Rhagium inquistor had fecal pellets in the gut which contained nucleating agents that had no effect on supercooling while overwintering (Baust and Zachariassen, 1983). Although depression of supercooling points is critical for freeze-intolerant insects to survive the winter, a number of studies have demonstrated that additional mechanisms of cold hardening may be required to survive exposure to temperatures near the supercooling point (Lee and Denlinger, 1985; Bale, 1987; Lee et al., 1987). Previous studies have suggested seasonal changes in survival at temperature near the supercooling point in adult Zps acuminatus (Gehrken, 1984, 1989). The present study was therefore conducted to survey the cold tolerance of adult Zps acuminatus during autumn. In the winter, this freeze-susceptible species has the capacity to accumulate thermal hysteresis producing antifreezes and high concentrations of ethylene glycol that promote supercooling from -20 to -35°C (Gehrken, 1984). Experiments were undertaken to study the various factors affecting supercooling in fed and starved individuals.

209

Influences on supercooling point of adult bark beetles

haemolymph melting and freezing points, the concentration of low molecular weight substances and relative water content represent measurements from five beetles. The significance of differences between two means were assessed by two-sample t-tests and a paired-sample t-test. Simple regression analyses were employed to consider the relationship between whole body supercooling points and gut filling, as well as between whole body supercooling points and relative water content. BMDPl R-multiple regression analysis (Dixon and Brown, 1981) was applied to measurements of whole body supercooling points, levels of gut filling and melting and freezing point difference (thermal hysteresis). Differences were considered significant at P < 0.05.

Results Mean ( f SE) whole body supercooling point of I. acuminatus sampled out of doors on 25 August was - 11.9 +_0.39”C and all individuals had food throughout the gut, posterior to the proventriculus (category 6). After removal of their alimentary tracts, the supercooling point dropped significantly (P < 0.001) to - 16.5 &O.l7”C. There were marked reductions (P < 0.01) in mid- and hind-gut content after 1 and 4 weeks

at 3°C and evacuation of residual food was complete by 8 October (Fig. 1). In contrast, the level of food residuals remained high throughout September in I. acuminatus maintained outdoors (Fig. 2). The significant reduction (P c 0.02) in gut filling from early October implied cessation of feeding, and evacuation of residual food was complete by 10 November. Thus, beetles maintained out of doors cleared their guts 1 month later than those held at 3°C. Relative water content of I. acuminatus was gradually reduced from about 60% to about 50% (Figs 1 and 2). The drop, which corresponded to a loss of 33.3% of the body water, accompanied gut evacuation. Accordingly, dehydration was completed a month later in beetles held outdoors than in specimens maintained at 3°C. The regression equation for the effect of water content on supercooling has the formula Y = - 34.8 + 0.35X, r* = 0.85 (P < 0.00001). Carbohydrate-polyol screening by gas chromatography indicated that the major carbohydrate present in beetles during autumn was the disaccharide trehalose, which increased from 10 mM at the end of August to about 90 mM by November. Specimens at 3°C steadily accumulated trehalose during the autumn (Fig. l), while those held outdoors had relatively 6

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constant levels throughout September that did not rise significantly (P < 0.01) until October (Fig. 2). In both conditions the increase in trehalose coincided with the reduction in gut content. While beetles invariably died during 14 days at - 10°C in August, September and October, survival was always 100% at -5°C. Enhanced supercooling from about - 12 to - 17°C coincided with a reduction in gut contents. A marked depression of whole body supercooling points occurred in early September after 1 week at 3°C (Fig. 1). Comparing consecutive data from individuals maintained outdoors, substantial decreases (P < 0.01) in whole body supercooling points were demonstrated in early October and again in mid-October (Fig. 2). The mean supercooling points of haemolymph remained almost unaltered until November. Throughout September and October, haemolymph supercooling points corresponded to the respective whole body supercooling point of beetles stored at 3°C (Fig. 1). In contrast, the August and September haemolymph means were consistently lower (P c 0.01) than the respective whole body means of animals maintained out of doors (Fig. 2). The haemolymph melting points were gradually lowered from -0.9 in August to - 1.5”C by the end of October. The loss of 33.3% of the

water content accounted for a melting point depression of 0.5”C, whereas the increase in trehalose from 10 to 90 mM accounted for a depression of melting point of about 0.15”C. The melting point depression of trehalose was deduced from the known effect of mM concentration of sucrose, with the same molecular weight as trehalose (Wolf et al., 1972). Noticeable differences between haemolymph melting and freezing points (thermal hysteresis) were demonstrated on 1 November in beetles stored at 3°C (Fig. 1) and on 5 November in beetles maintained out of doors (Fig. 2). Production of ethylene glycol occurred a few days later on 5 November at 3°C and on 10 November at outdoor conditions. In both regimes, a substantial decline in whole body supercooling points coincided with the production of thermal hys(P < 0.01) and ethylene glycol teresis (P < 0.001) (Figs 1 and 2). Alterations in haemolymph supercooling points (P < 0.002) and melting points (P < 0.01) occurred only after the onset of ethylene glycol accumulation. Evidence that feeding occurred upon transfer to 2 1“C comes from the fresh grass in the natal galleries and an increase in contents of the mid-hindgut. Beetles transferred from 3°C on 16 September had marked increase in food residuals (P < 0.01) after 1 day at 21°C while a subsequent reduction in levels occurred after 3

Influences

on supercooling

and 4 days of warm acclimation (Fig. 3). However, the evidence for feeding was weak in individuals brought in from the field in October. Food throughout the digestive tract was only seen in three out of 11 beetles after 1 days at 21°C (Fig. 4). In beetles held at 21°C without access to food, food evacuation was completed within 4 days in September (Fig. 3) as well as in October (Fig. 4). Mean relative water content of fed and starved I. acuminatus remained almost unaltered during 4 days at 21°C (Figs 3 and 4). Trehalose levels decreased, whereas haemolymph melting points remained unaltered. The supercooling capacity of beetles decreased following 1 days with access to food, but increased almost to the initial level during the next 3 days. The rise in supercooling points was only concomitant (P < 0.001) in September (Fig. 3) and not October (Fig. 4). In contrast, starvation and subsequent evacuation of food residuals during the course of warm acclimation had no marked effect on supercooling in September (Fig. 3) or in October (Fig. 4). Also mean haemolymph supercooling points remained unaltered, and the measures from feeding animals were consistent with those from starved specimens. A marked difference (P < 0.01) between whole body and haemolymph supercooling points was only observed after 1, 2 and 3 days of feeding in

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Fig. 3. Mean (k SE) whole body (0) and haemolymph (0) supercooling points, haemolymph melting point (A), relative water content (+), concentration of trehalose (0) as well as the median level of gut filling posterior to proventriculus (0) of I. acumina6us taken from 3°C in the middle of September and held at 21°C for 4 days with (A) or without access to food (B).

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Fig. 4. Mean (k SE) whole body (0) and haemolymph (0) supercooling points, haemolymph melting point (a), relative water content (+), concentration of trehalose (0) as well as the median level of gut filling posterior to proventriculus (0) of I. acuminarus taken from the out door supply in the middle of October and held at 21°C for 4 days with (A) or without access to food (B).

September and after 1 and 2 days of feeding in October. The influence of gut filling levels and thermal hysteresis on supercooling is given by the following equation Y,,, = - 15.8 +0.51X,,, r2 =0.90 (P < 0.00001). The 0.99z,, , BMDPlR-multiple regression analysis was only applied to data from individuals lacking ethylene glycol in their body fluids. Incorporating only data from specimens with thermal hysteresis, the multiple regression line has the Y,,, = - 17.2 +0.1X,,, - 0.962,,, formula r2 = 0.94, (P (Cat) < 0.01, P (TH) < 0.0001). Whether or not food residuals had lost a nucleating effect when evacuated posterior to the Malpighian tubules was determined on data from beetles with no thermal hysteresis. The influence of food residuals all the way posterior to proventriculus (category 4-6) on supercooling is best described by the formula Ysc, = -20.1 + 1.08Xcat(4_6jrr* = 0.56, (P < O.OOOOl),while gut content posterior to the Malpighian tubules (category O-3) is best described by Y,,, = (P < 0.002). - 17 + 0.31Xcatc&,), r2 = 0.26 Analysis of variance of regression coefficients in high and low category groups showed that slopes and/or intercepts differ between groups (P -L 0.0008). Apparently, qualitative differences existed between groups, and the nucleating effect of gut content was substantially greater

212

U. Gehrken Table 1. Mean (+ SE) whole body and haemolymph supercooling points (SCP), haemolymph melting point (MP), relative water content and trehalose concentration of pupae and newly hatched adult IDSacuminatus transferred from outdoor logs on 17 October and held at 21°C. Both newly hatched adults with (I) and without (II) gut filling were tested Haemolymph

Stage Pupae Adult 1 Adult II

Whole body SCP (“C) - 19.7+0.13 - 19.6 f 0.15 - 5.8 + 0.30

SCP (“C)

MP (“C)

Water content (%)

Trehalose (mM)

- 16.9 + 0.34 - 16.8 + 0.27 - 16.7 + 0.34

- 1.4* 0.02 - 0.8 + 0.02 - 0.8 + 0.02

71.2 + 1.39 71.4 & 1.23 73.4 f 1.24

13.5 + 0.84 8.5 +0.15 11.3kO.47

before its residuals had been evacuated pos- mid- and hind-gut, dehydration and the level of thermal hysteresis. Antifreeze protein proterior to the Malpighian tubules. duction coincided with a shift in supercooling Measurements on whole body and haemolymph supercooling points, haemolymph melt- from - 17 to -20°C while the completion of gut evacuation and dehydration coincided with ing and freezing points, relative water content and trehalose concentration in pupae and newly enhanced supercooling from approximately - 12 to - 17°C. Knowing that supercooling is emerged I. acuminatus were only carried out during warm acclimation in October (Table 1). increased by 2.3”C for each degree of melting point depression in I. acuminatus (Gehrken, Adults assumed to have emerged from pupae 1984), the effect of dehydration and trehalose during the course of warm acclimation had soft, yellow cuticles different from the hard, yellow- synthesis on supercooling was found to be merely 1.5”C. The supercooling point of the brown cuticles of older adults. Newly emerged haemolymph obtained from I. acuminatus was adults which had gut filling all the way posterior to proventriculus supercooled to -58°C. The similar to that of nucleator-free haemolymph of freeze-susceptible species in summer (Zacharisubstantially lower (P < 0.001) supercooling point at - 19.6”C of newly emerged adults with assen, 1980) and winter (Baust and Zacharimid- and hind-guts empty of food particles was assen, 1983). The small samples froze at - 16 to - 17°C from August throughout October, while similar to that of pupae. Haemolymph supercooling points and relative water contents of the excised mid- and hind-gut froze within the pupae and newly emerged adults (whether they temperature range of whole body insects. Thus, a functional nucleating site other than gut is had fed or not) did not differ significantly unlikely. The fact that the nucleating agents are (Table 1). The mean haemolymph melting point of feeding and non-feeding adults was similar temperature sensitive strongly suggests that and differed from pupal values (P < 0.01) but nucleation is not caused by the surface of only adults with empty guts had markedly lower ingested bark itself but rather by ice nucleators (P < 0.01) concentrations of trehalose. The of a proteinaceous nature. The elevation of supercooling point to - 6°C mean relative water content of pupae and newly in beetles newly emerged from pupae (Table 1) higher emerged adults was significantly (P < 0.01) than that of “older adults” (Figs 1 could not be ascribed to the quantity of food residuals per se. In fact, the somewhat ‘older and 2). The mean (*SE) supercooling point of 10 adults’ with the same level of residuals superexcised alimentary tracts in category O-3 was cooled to - 12°C (Fig. 2). I. acuminatus spend - 16.9 f 0.37”C and the corresponding value autumn and early winter in diapause, characterfor intact beetles - 16.7 + 0.27”C. Similarly, 10 ized by continued feeding, reduced respiration rates and cessation of ovarian development at mid- and hind-guts (classed category 6-4) supercooled to - 14.7 + 0.64 in agreement with an early pre-vitellogenic stage (Gehrken, 1985). In fact, feeding is associated with elevation of the intact animals at - 13.3 f 0.63”C. However, heating of the alimentary tracts to 100°C the supercooling point to -6°C during postchanged supercooling markedly (P < 0.01). The diapause development as well (Gehrken, 1984). mean supercooling point ( f SE) of those classed Therefore, the shift in nucleating efficiency category O-3 was lowered to - 19.8 + 0.2O”C probably reflects reduced feeding rates that are after heating, and that of category 4-6 was common features among diapausing species (Tauber et al., 1986). lowered to - 19.9 f 0.18°C. Qualitative differences in nucleating activity existed after the onset of diapause as well. Discu!%ion Enhanced supercooling from about 12 to 17°C The supercooling point of I. acumirzatus was merely ascribed to evacuation of food rewas closely related to the level of food in the siduals from the mid-gut in animals held at 3°C

Influences

on supercooling

or out of doors. The lack of correlation between supercooling and level of gut filling in starved beetles which emptied their hind-gut within 4 days at 21°C might even imply that ice nucleating activity was lost at the time when food particles reached the hindgut. Even so, the enhanced supercooling after heating of excised alimentary tracts characterized category O-3 revealed that the opposite was true. A number of authors have speculated that bacteria in insect gut may regulate the supercooling point (Snrmme, 1982; Baust and Rojas, 1985; Cannon and Block, 1988). Recently, Strong-Gunderson et al. (1990) revealed that supercooling was closely correlated with increasing concentrations of ingested ice-nucleating active bacteria in the lady beetle Hippodamia convergens. Ice nucleating active bacteria are found epiphytically on plants (Vali et al., 1976; Schnell, 1976), and in the gut of insects (Lee et al., 1991). The nucleating efficiency of the gut is negligible in unfed Z. acuminatus newly emerged from pupae (Table 1). Their ability to supercool to -20°C is comparable with that of overwintering cold-hardy adults depleted of ethylene glycol during warm acclimation (Gehrken, 1984). Therefore, the shift in supercooling to - 6°C upon feeding in newly emerged adults can be largely ascribed to nucleating proteins ingested or rather synthesized during digestion. The nucleating effect of the gut filling seemed completely lost following heating. The stabilization of supercooling points at -20°C agreed with earlier measurements from overwintering beetles after a complete loss of ethylene glycol (Gehrken, 1984). Ice nucleators, therefore, are still present in the gut after a complete evacuation of food residual and completion of dehydration (Fig. 1). Thus, the unaltered supercooling in starved beetles cannot be ascribed to the retention of body water during gut evacuation at 21°C (Figs 3 and 4). The close correlation between thermal hysteresis and supercooling between - 17 and - 20°C however, might arise from coincident timing. If antifreeze proteins are to promote supercooling, then they must inhibit the ice nucleators responsible for nucleation. This may well be true in I. acuminatus which invariably reduced supercooling by about 2°C after depletion of thermal hysteresis (Gehrken, 1989). Hence, there may even be ice nucleators present in the so-called nucleator-free haemolymph. Evidence that thermal hysteresis producing antifreezes can inhibit ice nucleators and, thus, promote supercooling was achieved upon addition of fish glycoprotein antifreeze to a solution containing bacterial ice nucleators (Parody-Morreale et af., 1988). Similarly, inactivation of one of three ice-nucleating

point

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bark

beetles

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proteins in Tipula trivittata occurred upon addition of antifreeze proteins from Dendroides canadensis (Duman et al., 1991). Nucleating proteins can even enable antifreeze proteins to increase their hysteresis activity (Duman et al., 1991) and a direct interaction between the various proteins is not unlikely. Emptying of the gut and stabilization of whole body supercooling points at - 17°C during autumn seemed inadequate to allow survival for 2 weeks at - 10°C in September and October. In contrast, 70-85% survived 1 week at 6-7°C above the supercooling point in December, January and March (Gehrken, 1989). In the flesh fly Sarcophaga crassipalpis pupae (Lee and Denlinger, 1985) and the butterfly Znachis io (Pullin and Bale, 1989) alteration in survival at subzero temperatures above the supercooling point was dependent on the time they had been in diapause. The capacity of highly supercooled I. acuminatus to avoid freezing, however, cannot be ascribed to diapause development per se. Exposure at 6-7°C above supercooling point was even fatal in December, January and March after depletion of antifreeze proteins (Gehrken, 1989). Antifreeze proteins are known to act to stabilize the metastable supercooled state over long periods (Zachariassen and Husby, 1982; Duman et al., 1991), and the overall reduction in survival rates of I. acuminatus is probably ascribed to the absence of antifreeze proteins. Acknowledgements-This work was supported Iinancially by the Norwegian Research Council of Science. The author would also like to thank Dr Egil Jellum, Institute of Clinical Biochemistry, Rikshospitalet, Oslo who provided technical facilities necessary for this work.

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