Comp. Biochem. Physiol., 1978. Vol. 59A, pp. 69 to 72. Pergamon Press. Printed in Great Britain

THE ROLE OF ICE NUCLEATORS IN THE FROST TOLERANCE OF OVERWINTERING QUEENS OF THE

BALD FACED HORNET

JOHN G. DUMAN AND JEAN L. PATTERSON Department of Biology, Faculty of Science, Toho University, Funabashi 274, Japan

(Received 3 March 1977)

Abstract--1. Overwintering queens of the bald faced hornet, Vespula maculata, are tolerant of ice forma- tion in their body fluids down to temperatures of approximately - 14°C.

2. Contributing to this frost tolerance is a high concentration of the cryoprotectant glycergl in the overwintering queens. An additional factor is the presence in the hemolymph of macromolecular ice nucleating agents which function to induce harmless extracellular ice formation at comparatively high temperatures (-4.6°C), and thereby prevent lethal intracellular ice formation.

3. These ice nucleating agents have molecular weights greater than 3500 and are probably proteina- ceous .

I N T R O D U C T I O N

The overwintering phases of many polar and temper- ate zone insects are well known for their abilities to withsta_nd subzero environmental temperatures (Asa- hina, 1966, 1969; Salt, 1961). Some of these insects are capable of surviving the freezing of their extracel- lular body fluids. Most of these freeze-tolerant species overwinter in the larval or pupal forms; however, several freeze-tolerant insects are now known to over- winter as adults. The freeze-tolerant species which have been investigated to date all belong to either the order Coleoptera (beetles) or the order Hymenop- tera (bees, ants and wasps)(Miller, 1969; Asahina & Ohyama, 1969; Ohyama & Asahina, 1970, 1972; Baust & Miller, 1972b; Miller & Smith, 1975; Zachar- iassen & Hammel, 1976).

The large papery nests of the bald faced hornet, Vespula maculata (Hymenoptera, Vespidae) generally contain up to 100 individuals by late summer (Evans & Eberhard, 1970). The colony consists of an egg-lay- ing queen and'female workers throughout the early summer. Males and future queens are produced late in the nesting season. The males die shortly after mat- ing in late summer. However, the inseminated future queens hibernate individually, generally in or under large partially decomposed logs. This article concerns the adaptations which enable V. maculata queens to successfully overwinter in spite of subzero environ- mental temperatures.

MATERIALS AND METHODS

Overwintering V. maculata queens were collected from their hiberaculae in and under large decomposing logs in oak forests near South Bend, IN. Future queens were col- lected from nests in late summer.

Hemolymph samples were collected by puncturing the cuticle with a 23 gauge needle in the dorsal mid-line of the abdomen. Hemolymph was then collected in glass ca- pillary tubes (10 #1).

The melting points of the hemolymph samples were determined by a modification of the technique of Ramsay & Brown (1955).

69

The supercooling points of the hemolymph were measured in the same sealed capillary tubes in which melt- ing point determinations were made. The sample was placed in a well-controlled (+0.02°C) refrigerated/dcohol bath at 0°C. The sample was monitored microscopically through a viewing port in the bath. The bath temperature was then lowered at a rate of 0.4°C/min until spontaneous nucleation was observed. The temperature at which this freezing occurred was taken as the supercooling point of the sample. Supercooling points of whole hemolymph and hemolymph diluted 1/100 with distilled water were determined. In addition, the supercooling points of heat- treated and dialyzed hemolymph were measured. Heat- treatment of the diluted hemolymph consisted of immer- sion of the sample in boiling water (100°C) for 5 min. The sample was then centrifuged and the supernatant saved for supercooling point determinations. The dialysis was done against distilled water at 4°C for 24 hr. The dialysis tubing used had a molecular weight cutoff of 3500 (Spec- trapor).

Winter hemolymph was treated with a non-specific pro- tease purified from Streptomyces griseus (Sigma Chemical Co., Type VI bacterial protease).

The organismal supercooling point, the temperature at which spontaneous nucleation occurs when the insect is supercooled below its equilibrium freezing point, was determined using a thermoelectric technique. A thermistor was'attached with paraffin to the cuticle in the anterior middorsal region of the abdomen. The thermistor was con- nected to a YSI model 42 SC telethermometer and a recorder. The insect was then placed in a freezing chamber and the temperature was lowered at a rate of 0.4°C/min. The supercooling point was easily recognized as the tem- perature at which a rapid increase in the body temperature of the insect occurred due to the release of the heat of fusion as the body fluids froze (Salt, 1966). All individuals were held without food for 24 h r prior to supercooling point determination.

Glycerol concentrations of the hemolymph were measured using Calbiochem glycerol reagents. This enzy- matic technique is based on the method of Eggstein & Kreutz (1966) as modified by Bucolo & David (1973).

The lower lethal temperatures of the insects were deter- mined by holding groups of individuals (8-14) at various temperatures for 24 hr. At the end of this time period, the insects were removed from the cold chamber and held at room temperature for 24 hr after which time the indivi- duals were checked for movement and ability to orient

70 JOHN G. DUMAN AND JEAN L. PATTERSON

Table 1. Comparison of the melting points and glycerol concentrations of the hemolymph, organismal supercooling points and lower lethal temperatures of queen V. maculata in late

summer and winter

Organismal Melting supercooling Lower lethal

point point Glycerol temperature (°C) (°C) (mg ~o) (°C)

Summer -0.72 + 0.08 (10) -3.8 + 1.1 (9) 12 + 5 (10) - 4 Winter -2.88 + 0.65(12) -4.6 + 0.8(13) 3,788 + 2038(9) -14

Values indicate the mean + S.D. Numbers in parentheses indicate the sample size.

normally. The temperature at which 50% of the individuals died was taken as the lower lethal temperature.

RESULTS

The melting points of the hemolymph of overwin- tering V. maculata queens were significantly lower than those of future queens collected in late summer (Table 1). This indicates the presence of high concen- trations of solute in the winter. As Table 1 shows, glycerol was present in high levels in the hemolymph of overwintering queens (~ = 3.788 mg ~ ) while the glycerol concentrations of the summer queens was much lower (~ = 12 nag 9/o~ The range of hemolympli glycerol concentrations in the winter queens was 1726-6700 mg ~.

The organismal supercooling points of the winter and summer queens were not significantly different (Table 1). However, the lower lethaF.temperature of the winter queens was considerably lower than that of the summer queens. In addition, the lower lethal temperature of the winter individuals was much lower than the supercooling points of this same group. This shows that the overwintering queens are freeze toler- ant. In summer, however, the lower lethal tempera- ture and the supercooling point were virtually identi- cal, thus indicating that in summer the future queens are not freeze tolerant.

The supercooling point of winter hemolymph diluted 1/100 with distilled water (-4.1°C) was nearly identical to that of undiluted winter hemolymph

Table 2. Comparison of the hemolymph supercooling points of summer (A) and winter (B) V. maculata queens with that of distilled water (C). Effects of heat-treatment (E), dialysis (F) and treatment with a proteolytic enzyme (G) on the supercooling point of a pooled winter hemo-

lymph sample (D)

Supercooling point Sample (°C)

(A) Summer (10)* (B) Winter (10)* (C) Distilled watert (D) Winter (pooled sample)i" (E) D above + heati" (F) D + dialysisi" (G) D + proteolytic enzymei"

- 4 . 0 + 0.2 -4.1 + 0.3

- 1 7 . 4 + 2.4 - 3 . 8 _ 0 . 2

- 1 6 . 7 -t- 2.8 --4.5 -t- 0.3

- 1 5 . 8 _ 3.4

* Values indicate the mean + s.d. Numbers in paren- theses indicate sample size.

i" Values indicate mean + S.D. of five individual deter- minations on the sample.

(-4.2°C) and both were considerably higher than the supercooling point of distilled water (Table 1). To facilitate the handling of the small hemolymph samples the samples were diluted 1/100 with distilled water. The supercooling points of the hemolymph of both summer and winter queens (Table 2) agree closely with the organismal supercooling points of summer and winter queens (Table 1). This ind!cates that ice nucleation originates from motes in the hemolymph. In addition, the supercooling points of the hemolymph of winter and summer queens were not significantly different (Table 2).

Heat-treatment of the diluted hemolymph for 5 min at 100°C lowered the supercooling point nearly to that of distilled water (Table 2, D,E). The supercool- ing point of dialyzed hemolymph was not significantly lower than that of non-dialyzed hemolymph (Table 2,D,F). Treatment of winter hemolymph with a non- specific bacterial protease significantly lowered the supercooling point (Table 2,G).

DISCUSSION

Although the overwintering queens of V. maculata are frost tolerant, a lower lethal temperature of - 14°C classifies this species as only moderately frost resistant. Other species, such as the Alaskan Carabid beetle, Pterostichus brevicornis, survive temperatures down to -40°C or lower (Baust & Miller, 1970). Therefore, the choice of insulated hibernaculae in large decomposing logs must be an important factor in the overwintering capabilities of the Vespula queens. Air temperatures below -14°C are quite common during winter in the South Bend, IN, area and therefore the insulation provided by decomposing logs and any snow cover which might be present (Mail, 1932; Wellington, 1950; Baust, 1976) would seem to be critical.

Baust (1973) has pointed out that many factors are involved in the frost tolerance of insects. The in- creased concentrations of glycerol, and other polyols, in winter in many frost-resistant species have been well documented (Salt, 1957; Somme, 1964, 1965, 1969; Baust & Miller, 1970, 1972a, b; Frankos & Platt, 1976; Miller & Smith, 1975), and there is little doubt that glycerol functions as a cryoprotectant in many of these species, including V. maculata queens. However, not all frost-tolerant insects have high gly- cerol concentrations, and not all insects with high gly- cerol concentrations are frost resistant (Asahina, 1969). In addition, injection of high concentrations of glycerol into frost susceptible diapausing insects

The role of ice nucleators

fails to induce frost tolerance in these individuals (l 'akehara & Asahina, 1960; Tanno, 1963; Asahina, 1969). It is obvious then that high glycerol levels alone do not result in frost tolerance.

Formation of intracellular ice seems to invariably result in death, even in freeze-hardy individuals (Asa- hina et al., 1954; Asahina, 1956; Meryman, 1956). In frost-tolerant insects, ice formation is restricted to t h e extracellular fluid except when the insect is artificially subjected to very fast cooling rates (Asahina, 1969). The cell membrane acts as a barrier to ice propaga- tion and therefore prevents nucleation of the intracel- lular fluid by the extracellular ice (Chambers & Hale, 1932; Asahina, 1961). In adult insects an important contributing factor to frost tolerance appears to be the ability to induce extracellular ice formation at relatively high temperatures (above -10°C). Over- wintering Vespula queens likewise had high super- cooling points which were generally less than 2°C below the equilibrium freezing points of the hemo- lymph (Table 1). This high supercooling point appears to result from the presence in the hemolymph of ice nucleating agents similar to those recently described by Zachariassen & Hammel (1976). The function of these ice nucleating agents, as hypothesized by Zachariassen & Hammel (1976), appears to be to in- duce ice formation in the hemolymph at high tem- peratures and thereby inhibit lethal intracellular ice formation.

The V. maculata ice nucleating agent has a molecu- lar weight greater than 3500 and exhibits a loss of activity when subjected to either heat or treatment with a bacterial protease. Although not conclusive, this preliminary evidence indicates that the nucleating agent is probably proteinaceous.

Supercooling is a probabilistic event which is not well understood (Salt, 1966). Therefore, repetitive determinations on the same sample may often show considerable variation. Comparison of the supercool- ing points and standard deviations of samples which contain the active protein nucleator with those which do not (Table 2) indicates that not only does the nuc- leator raise the supercooling point of water, but that it also decreases the variability of the temperature at which spontaneous nucleation occurs. Salt (1966) and Zachariassen & Hammel (1976) came to similar conclusions.

The supercooling points of both the whole organism and of the hemolymph were approximately equal both in winter and summer Vespula queens, and therefore the nucleating agents seem to be present in both seasons. This is in contrast to the situation with the Tenebrionid beetles which produce the hemolymph nucleating agents only in winter. How- ever, this is not too surprising when we recall that the future Vespula queens are not produced until late summer and that within a few weeks after their emer- gence, freezing temperatures are likely to occur.

In conclusion, the overwintering queens of the bald faced hornet, Vespula maculata, survive the winter by (1) seeking hibernaculae'which will provide some in- sulation from the extremes of air temperatures and (2) becoming tolerant to ice formation in their extra- cellular fluids down to temperatures of approximately - 1 4 ° C . This frost tolerance involves (a) the produc- tion of high levds of the cryoprotectant glycerol and

in the bald faced hornet 71

(b) the presence in the hemolymph of macromolecular ice nucleating agents which insure that ice will form in the extracellular fluid at reasonably high tempera- tures (-4.6°C), thereby preventing lethal intracellular ice formation.

Acknowledoements--Special thanks to Dr. Quentin Ross for his help in collecting Vespula nests.

This work was supported by N.I.H. Biomedical Sciences Support Grant FR/RR-07033-10.

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