Inoculative freezing in overwintering tenebrionid beetle, Bolitophagus reticulatus Panz

J. InsectPhysiol.Vol.37, No. 9, pp.683-687,1991 Printed in Great Britain.

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INOCULATIVE FREEZING IN OVERWINTERING TENEBRIONID BEETLE, BOLITOPHAGUS RETICULATUS PANZ U. GEHRKEN,’A. STMMME,~R. LUNDHEIM~ and K. E. ZACHARIASSEN~ ‘Department of Biology, Division of Zoology, University of Oslo, P.O. Box 1050,Blindem N-0316 Oslo 3 and 2Department of Zoology, University of Trondheim, N-7055 Dragvoll Trondheim, Norway (Received 30 April 1991)

Abstract-Adults of the tenebrionid beetle Bolitophagus reticulatus are able to endure prolonged sub-zero temperatures both as freeze-tolerant and freeze-avoiding. The switch between the two strategies is linked to the moisture content of the hibemaculum. Accumulation of low molecular weight substance8 parallels depression of haemolymph melting points down to - 6.W. The supercooling point is lowered to - 30°C during winter, and enhanced supercooling below -20°C was closely correlated to the depression of haemolymph melting points. Thermal hysteresis-producing antifreeze proteins, however, were not present. Spontaneous freezing at the supercooling point proved fatal, whereas initiation of freezing by contact with external ice ensured protective extracellular freezing. At temperatures 4-5°C above mean supercooling point, survival was always poorer in the inoculated frozen specimens than in the extensively supercooled specimens. Thus, the present study does not imply that freeze tolerance is more efficient than freeze avoidance in promoting cold hardiness. Key Word Index:

survival; tenebrionid

Cold hardiness; beetle

inoculative

INTRODUCTION

The tenebrionid beetle Bolitophagus reticulatus hibernates as adults and larvae in tinderfungus Polyporus fomentarius which grows on stems of dead birches. During the winter, the tinderfungus is usually situated above the snow, thus exposing the enclosed beetles to the full range of ambient temperature fluctuations. The winter hardiness strategy of adult B. reticulatus includes a depression of the supercooling point to - 30°C and the accumulation of glycerol in multimolal concentrations (Zachariassen, 1982). Since freezing at the whole body supercooling point proved fatal, Zachariassen (1980) assumed that freeze avoidance is the main strategy for winter survival in this species. A surface-dry insect exposed to falling temperature will supercool until ice is spontaneously formed in its body fluid. In contrast, an insect in surface contact with freezing water may freeze at substantially higher temperatures as a result of the inoculation of its haemolymph by the external ice. Inoculative freezing has been studied in a few species only, and is supposed to occur in insects overwintering in moist habitats. Danks (1971) found that larvae of several chironomid species frozen by inoculation survived well following cooling to -4°C. Adult carabid beetles Pelophila borealis frozen by inoculation survived cooling to - 5°C but cooling to - 10°C proved

freezing; freeze-tolerant;

freeze-susceptible;

fatal (Ssmme, 1974). Overwintering in dry surroundings, however, adults of P. borealis supercooled to below -20°C (Ostby and Snmme, 1972). Furthermore, inoculative freezing has been found to ensure protective freezing to - 15°C in prepupae of Sciara sp. (Tanno, 1977) and to about -80°C in larvae of Chymoyza costata (Shimida and Riihimaa, 1988) overwintering in contact with moisture. Experimental studies by Gehrken and SBmme (1987), however, showed that inoculative freezing is avoided in ice-enclosed eggs of the stonefly Arcynopteryx compacta. The natural habitat of B. reticulatus is often extremely wet, and the aim of the present study was to elucidate how the species survives sub-zero temperature exposure in intimate contact with freezing water.

MATERIALS AND METHODS

P. fomentarius colonized by adult was collected from two different locations in Norway on three occasions during the winter. The first sample of 45 specimens was collected near Trondheim (63”, 25’N; lo”, 25’E) on 7 April 1989. The second and third samples of about 50 beetles were obtained from Ssrumsand (59’, 59’N; 1l”, 15’E) on 27 January and 4 February 1990. Supercooling points and haemolymph melting and freezing points, relative water content and survival

Tinderfungus

B.

683

reticula&s

U. GEHRKEBet al.

684

after inoculative freezing were determined immediately after the beetles were brought into the laboratory. The supercooling point of dry-surface individuals removed from the fungus and cooled at a rate of l”C/min, was measured using a 0.2mm dia copper constantan thermocouple connected to Goerz Metrawatt SE 120 recording potentiometer. Each determination of mean supercooling points was based on measurements from 13 or 14 beetles. The inoculative freezing was established in adults kept on moistened pieces of tinderfungus and attached to the 0.2 dia copper constantan thermocouple. Freezing of adults was initiated by contact with freezing water, and the water nucleated at temperatures ranging from -0.5 to - 2.l”C in the course of subsequent cooling at 0.2”C/min. The frozen adults were furthermore cooled to various sub-zero temperatures and then rewarmed to 0°C at the same rate as they had been cooled. Four individuals were used for each freezing regime. Melting and freezing points were determined using a Clifton nanolitre osmometer (temperature controlled range from -9.5 to +O.SC). Each haemolymph sample was obtained from individual beetles the supercooling point of which had previously been determined. The presence of antifreeze proteins in the haemolymph of B. reticulatus was determined by looking for a difference between the haemolymph freezing and melting points (i.e. thermal hysteresis). Each determination of mean melting point was based on measurements from 7-11 haemolymph samples. Whole body weight was measured by weighing individual larvae to the nearest mg using a Sauter RE 1614 balance. The difference between fresh and dry weight following drying at 70°C to a constant weight (24 h) was taken as the water content of B. reticulatus. The mean relative water content represented measurements from three beetles. Survival following inoculative freezing was studied in beetles transferred to moistened vials 3°C. Measures of survival were restricted to observations of co-ordinated walking made over subsequent period of 2 weeks. Survival was moreover studied in groups of eight surface-dry beetles exposed to temperatures 45°C above mean supercooling point in April 1989 as well as January and February 1990. The specimens were held in a MK Climatic Cabinet (Eurotherm) and the temperature of the cabinet

lowered by l”C/min to the specific temperature at which they were held for 24 h. Two sample t-tests were employed in the comparison of two means to infer whether differences existing between the two samples. Simple regression analyses were used to consider the relationship between whole body supercooling point. Differences P Q 0.05.

point and haemolymph melting were considered significant when

RESULTS

The mean ( f. SE) supercooling and melting points as well as the relative water content of adult B. reticufatus sampled from tinderfungus in the beginning of April 1989, at the end of January and the beginning of February 1990 are given in Table 1. Dry-surface adults removed from tinderfungus in April had supercooling points ranging from -7.3 to -22.O”C, but the extreme lower value was far from the mean (- ll.S’C). The corresponding haemolymph melting points ranged between -0.9 and -4.l”C. The extreme lower value was measured in the haemolymph of a beetle supercooling to about - 11°C. The relative water content of beetles in April ranged between 64X&66.8%, and the mean value (&SE) was equal to 65.3 + 0.60%. The beetles removed from tinderfungus at the end of January had substantially lower (P < 0.001) supercooling and melting points than those sampled in April, and the mean values ( f SE) was equal to - 29.8 f 0.34”C and - 5.2 + 0.26”C respectively. Also the relative water content was markedly lower (P < 0.01) than that of specimens sampled in April. Note that the supercooling and melting points were significantly higher (P < 0.01) in the adults collected 8 days later, at the beginning of February. In contrast, the mean relative water content of specimens sampled from tinderfungus in February was similar to that of animals sampled 8 days earlier. The supercooling points were closely correlated (P < 0.01) with the respective melting point with the exception of specimens supercooling to temperatures above -20°C (Fig. 1). The regression equation has the formulae Y = - 18.8 + 1.85X, rz = 0.92. All dry-surface specimens used for measurements of supercooling points in April 1989 as well as in January and February 1990 were killed by freezing. A difference between melting and freezing

Table 1. Mean (*SE) supercooling point (SCP), melting point (MP) and relative water content of adult B. reticulatussampled out of doors on three occasions Sampling date I Apr. 27 Jan. 4 Feb.

Relative water content (%)

SCP (“C) -11.5* 1.34(14) -29.8 f 0.34 (13) -24.5 f 0.53 (13)

-1.8*0.27(11) - 5.2 f 0.26 (7) -2.8 f 0.42 (7)

65.3 f 0.60 (3) 50.2 f 0.65 (3) 50.8 f 1.19 (3)

The number of individuals tested is given within parentheses.

Inoculative freezing of B. reticulatus points (thermal hysteresis) was never observed in the haemolymph collected from overwintering 8. reticulatus.

Table 2 shows the mean ( + SE) supercooling point of B. reticulatus in surface contact with freezing water. The mean supercooling points (+_SE) of adults inoculated by freezing water in April 1989, January and February 1990 were equal -2.4 k 0.16, -5.5 -t_0.06 and -3.6 + O.l2”C, respectively. The inoculation temperature was significantly changed (P < 0.01) during the investigation period and corresponded to temperatures slightly below that of the mean melting point in April, January and February (Table 1). Therefore, inoculative freezing occurs almost without supercooling in adult B. reticulatus. The relationship between mean inoculation temperatures and mean haemplymph melting points from April, January and February had the formulae Y = -0.93 +0.89X, r2 = 0.99, P < 0.001. The freeze tolerance of inoculated beetles changed during the investigation period (Table 2). Inoculated frozen beetles always survived to - 5 and -6°C in April. In contrast, survival was not seen among the four inoculated specimens cooled to -75°C. They “survived” freezing but were fatally injured, and died during the subsequent period of 2 weeks at 3°C. At the end of January, 100% of the inoculated frozen adults survived to -23°C. Only two out of four survived cooling to -25°C; the remaining had been lethally injured. The freeze tolerance of beetles was poorer 8 days later, at the beginning of February.

685

Table 2. Mean (f SE) inoculation temperature of groups of four wet-surface adult B. reticulafus and the per cent survival following freezing to various sub-zero temperatures Sampling date 7 Apr.

27 Jan.

4 Feb.

Inoculation temperature (“C)

Exposure temperature (“C)

-2.4 + 0.35 -3.0 4 1.00 -2.2 f 0.13 -2.3 f 0.35 -5.3 &-0.14 -5.4 + 0.13 -5.5 f 0.20 -5.5 f 0.30 -5.6kO.13 - 5.5 + 0.09 -5.7 + 0.18 - 3.7 f 0.21 - 3.5 f 0.23 -3.7 +0.16

-2.4 f 0.35 -5.0 -6.0 -7.5 -5.3 + 0.14 -7.0 -9.0 -12.0 - 20.0 -23.0 -25.0 - 12.0 - 16.0 - 20.0

Survival W) 100 100 LOO 1: 100 100 100 t: 1: 100 25

They survived freezing to - 16°C but cooling to -20°C proved fatal. All beetles “survived” freezing but three out of four had lost the ability to move and orient normally. The survival data obtained from dry-surface specimens following exposure for 24 h at 45°C above their mean supercooling point are given in Table 3. Evidently, survival was always higher in the extensively supercooled insects than in the inoculated specimens cooled to temperatures 45°C above their mean supercooling points. In April, only four out of eight dry-surface beetles survived extensive supercooling for 24 h at - 7.5”C. All the dry-surface insects survived exposure for 24 h at - 25°C in January, and six out of eight survived extensive supercooling at -20°C in February. Since no movements were observed in the beetles which died, it was assumed that they died from freezing. DISCUSSION

;;

-15

0

-

0

Adults of B. reticulatus survive inoculated freezing, and should therefore be classified as a freezing tolerant species. Nonetheless, the lower lethal temperature of adults obtained by inoculated freezing was always a few “C higher than that attained by the extensive supercooled insects avoiding freezing. Cold hardy specimens with a haemolymph mean ( f SE) melting point of -5.2 + 0.26”C (Table 1) were inoculated at - 5.5 &-0.06”C by the ice formed in the wet tinderfungus, and 100% survived subsequent cooling to -23

e 4-

.E :: IF -20 5 :: 2 % z

-25 -

Table 3. Per cent survival of groups of eight dry-surface adult B. reticulatus exposed for 24 h to temperatures 4-5°C above their mean supercooling point (SCP)

I

I

I -7

-6

I -5

Melting

I -4

I -3

point

I

I -2

-1

I 0

PC)

Fig. 1. Individual supercooling points of B. reticuhtus as a function of the corresponding haemolymph melting points.

Sampling date 7 Apr. 27 Jan. 4 Feb.

Above mean SCP (“C) 4.0 4.8 4.5

Exposure temperature (“C) -1.5 -25.0 - 20.0

Survival (%) 50 100 75

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U. Gxmuor~ et al.

and 50% revived cooling to -25°C (Table 2). The dry-surface individuals which froze spontaneously at -29.8 * 0.34”C (Table 1) were killed by freezing probably due to intracellular ice formation. The present results suggest that initiation of freezing by contact with surrounding ice ensures protective extracellular ice formation. A correlation between supercooling and melting points was only found in B. reticulatus supercooling to at least -20°C (Fig. 1). The regression equation fitted the data presented by Zachariassen (1980) in which the supercooling points of 20 different freezeavoiding insect species were regressed against the corresponding osmolal concentration of haemolymph. Increased freeze-tolerance also paralleled enhanced supercooling below -20°C (Table 2). Among the beetles displaying supercooling points ranging from -7.3 and - 14.X and melting points ranging from -0.9 to -4.l”C (Fig. l), however, there seemed to be no correlation between supercooling and melting points (r2 = 0.06) and inoculated freezing to temperatures below - 6°C proved fatal. Zachariassen (1982) ascribed the rise in supercooling points above -20°C to the synthesis of some nucleating organic compound within B. reticulatus during the transition from the cold-acclimated to the warm-acclimated state. This study revealed that adults of B. reticulatus can endure prolonged sub-zero temperatures both as freeze-tolerant and freeze-intolerant and the switch between overwintering strategies is linked to the moisture content of the hibemaculum. The fact that the tinderfungus can be soaking wet strongly suggests that the overwintering success of B. reticulatus is linked to its ability to switch between strategies. Protective freezing is ensured by inoculation of external ice in those beetles overwintering in contact with moisture. In dry surroundings, however, avoidance of freezing is crucial. Thus, the individual cannot combine feeding and cold exposure, and potent nucleating substances in the cells and intestine must be removed prior to overwintering. The present study moreover suggests that dehydration takes place during cold hardening in overwintering B. reticulatus, and the lower relative water content of specimens in January and February agrees with findings from overwintering freeze-avoiding Zps acuminatus (Gehrken, 1984). Dehydration is enhanced by evacuation of the gut content, and the water content of freeze-avoiding species is usually reduced in overwintering stages (Somme, 1982). In freeze-tolerant species, however, dehydration is usually not part of cold hardening (Somme and Conradi-Larsen, 1979; Ring, 1982; Lee and Lewis, 1985; Rojas et al., 1986). Consequently, preparations for overwintering which provide enhanced supercooling must take place in B. reticulatus in spite of the fact that the specimen can withstand extracellular freezing. The distinction between freezing tolerant and freezing intolerant species is the paradigm of cold adap-

tation in arthropods. This founding hypothesis has been challenged by Baust and Rojas (1985). A switch from tolerance to avoidance of freezing has been recorded in larvae of two beetles species, Cucu&r clwipes and Dendroides canaa’ensis (Duman, 1984; Horwath and Duman, 1984). Moreover, survival of freezing was altered by changing the rates of cooling and warming in Pterostichus brevicornis (Miller, 1969) and Vpk ceramboides (Miller, 1978) and by changing the acclimation time at low temperatures in Chrymomyza costata (Shimada and Riihimaa, 1988). Nonetheless, the mechanisms and controls of freeze tolerance are poorly understood. A loss of haemolymph nucleating proteins was the only change noted in D. canaaknsis larvae accompanying the switch from tolerance to avoidance of freezing. Indeed, the tolerance to freezing was not restored by artificial nucleation in D. canaaknsis (Horwath and Duman, 1984), and the presence of functional ice nucleating substances cannot be the only requirement. Thus, the mechanisms operating to provide freeze tolerance in B. reticulatus are still a matter of speculation. The absence of thermal hysteresis-producing proteins in over-wintering B. reticulatus is in part contradictory. For example the freeze-tolerant beetles D. canaaknsis and C. clwipes, which can spend considerable periods of the winter in a freeze-intolerant stage, produce antifreeze proteins (Duman, 1979). Antifreeze proteins may act to stabilize the metastable supercooled state by absorption to the surface of embryonic ice crystals, thereby inhibiting further growth (Zachariassen and Husby, 1982), and production of such proteins appears to be more characteristic of hibernating freeze-avoiding insects than does production of polyols (Zachariassen, 1985). Indeed, antifreeze proteins have only been demonstrated on a small number of freeze-tolerant insect species (Duman et al., 1990). The absence of antifreeze proteins make possible protective inoculative freezing in insects which lack ice-nucleating agents in their haemolymph. With such antifreezes, seeding of external ice through the body wall should have been prevented until environmental temperatures had dropped below that of the hysteresis freezing point. Apparently, the disadvantage of staying extensively supercooled over long periods without the protection of antifreeze proteins is overridden by the advantage in humid environments of freezing occurring almost without supercooling at temperatures just below the haemolymph melting point. Therefore, the ability to avoid supercooling prior to inoculation may be crucial to the freeze tolerance in B. reticulatus by preventing fatal intracellular freezing. Spontaneous freezing depends on temperature and time of exposure, and occurs more readily in the freeze-avoiding bark beetle Zps acuminatus without antifreeze proteins than in those displaying such antifreezes (Gehrken, 1989). Comparison between data on survival following extensive supercooling in

Inoculative freezing of B. reticulatus

B. reticulatus (Table 3) and Ips acuminatus contradicts this result. In fact, the survival of B. reticulatus in January and February (Table 3) was similar to that of the bark beetle displaying low molecular weight solute and antifreeze proteins when subjected for 24 h to 4-5°C above their supercooling point means. The ability of B. reticulatus to avoid freezing is probably linked to the protective value of low molecular weight substances. At equivalent depression of haemolymph melting points, the supercooling capacity was poorer in B. reticufutus (Fig. 1) than in the bark beetles with antifreeze proteins (Gehrken, 1989). Thus, the higher solute concentration needed to attain equivalent supercooling in overwintering B. reticulatus may be sufficient to protect against spontaneous freezing for at least 24 h (the length of the test period). The present results do not imply an advantage of freeze tolerance (Table 2) over freeze avoidance (Table 3). Similarly, overwintering prepupae of Sciaria sp. which supercooled to - 18”C, survived freezing to - 15°C following inoculation of external ice (Tanno, 1977) and adult P. borealis which supercooled to about - 22”C, survived inoculative freezing to -5°C (Ostby and SDmme, 1972; Smnme, 1974). However, the present findings conflict with those of Zachariassen (1985) who noted that freeze-tolerant insects are more cold hardy than freeze-avoiding insects with equivalent polyol concentration above 1500 mosmol/l in their body fluid. Indeed, the somewhat poorer survival in inoculated frozen beetles can be linked to low water content and solution effects leading to intolerable osmotic stress across the cell membrane. In addition, the rates of cooling after freezing are generally crucial to successful freeze preservation (Mazur, 1984). The constant cooling rate of 0.2”C/min used in these experiments might have been too fast to offer maximal freeze tolerance. Indeed, the inoculated individuals were not given a chance to freeze out before the temperature was lowered. Given an opportunity to freeze out at the temperature at which inoculation occurred, the temperature gradient between extra- and intracellular compartments would have been less steep and survival might have been considerably improved in B. reticulatus. Acknowledgemenr-The

present work was supported by the Norwegian Research Council for Science.

687

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