Cold-hardiness strategies of some adult and immature insects overwintering in interior Alaska

C‘omp. Bdwm. Physiol. Printed in Great Britain.

Vol. 73A.

0300-9619 81’1105’)5-10so?.00~0 $24 19x Pergamon Press Lrd

No. 4. pp. 595 to 604. 1982

COLD-HARDINESS IMMATURE

STRATEGIES OF SOME ADULT INSECTS OVERWINTERING IN INTERIOR ALASKA

AND

KEITH MILLER Institute

of Arctic

Biology,

University

of Alaska,

Fairbanks. AK 99701. USA

Abstract-i. A variety of adult and immature insects. most of which occupy highly exposed habitats. successfully overwinter in interior Alaska, 2. A number of adult beetles are freezing-tolerant. All exhibit relatively high supercooling points and synthesize large quantities of polyhydric alcohols in response to cold. 3. At least four other orders are represented by adult insects that tolerate freezing. but in contrast to adult beetles, ail exhibit relatively low supercooling points associated with the build-up of glycerol. 4. At least three species of larvae that overwinter in galls undergo a remarkable increase in supcrcooiing capability (to (21. - 60’ C) associated with synthesis of large quantities of glycerol in winter. 5. None of the larvae tested could survive freezing in spite of their high glycerol levels.

I?ITRODCICTION

The severity of climatic conditions at high latitudes has presumably resulted in the evolution of insect species in which cold-hardiness mechanisms have been stirn~ll~~ted to the highest levels of developments For the sake of’ brevity and convenience “high latitudes” refers here to those regions of the earth’s land masses characterized by long, dark, very cold winters, a definition which fits closely with Irving’s (1972) definition of the term “arctic”. Of obvious imp~~rtance for both freezing-tolerant and freezing-susceptible insects are the acute or short-term, very low min. temperatures that are characteristic of high latitude land masses. Perhaps not quite as obvious, but at least as important, are the longer-term, mean low temperatures that occur over weeks or months. Prolonged sub-freezing temperatures should have a greater impact on freezing-susceptible forms that must survive by supercooling. This is so because of the “chance” or statistical nature of the ice nucleation process in living organisms (Salt, 1950). just as in physical systems (Mason, 1956). Also, interpretation of biochemical changes associated with cold-h~~rdening may be simplified due to the absence of the rapid metabolic interconversions associated with ambient temperature fluctuations near the WC region. Minimum air temperatures from interior Alaska are some of the lowest measured in North America (Irving, 1972). and temperatures are mostly in the subfreezing region from November to March (Fig. I). Location of the University of Alaska at Fairbanks (64’N Latitude) has provided an opportunity for first-hand study of low temperature tolerance in northern insects under both natural field conditions and in the laboratory. The purpose of this paper is to summarize data on cold-hardiness that have gradually been accumulated on a variety of adult and immature insects that occur in the Fairbanks vicinity. Even in cold regions, very diverse temperature habitats are available to insects. Since we presume that the greatest fow temperature stress should lead to the greatest develop-

ment of cold-hardiness mechanisms, studies to date have concentrated mostly on species that overwinter in very exposed situations where the temperature of the insect is usually close to the air temperature. Some information on two species of freczing-tolerant Alaskan beetles has previously been published (Miller. 1969: Baust & Miller. 1970, 1971: Miller & Smith, 1975; Miller, 1978a,b). The previously published material is briefly summarized here and placed in context with other unpublished data on the above two species. Unpublished data on overwintering strategies of a varietv of other adult insects are also presented and a &al section is devoted to recent studies of immature forms that seem to defy some of our preconceived ideas of how insects should spend the winter. MATERIAL

AND

METHODS

The limited availability of some species has restricted the quantity and variety of physicochemica~ data that could be obtained, We have frequently had to set priorities for data collection based on arbitrary decisions as to what information will be most useful. Given the lack of mformation on high latitude North American insects. studies to date have been kept very basic and primarily include drtermination of supercooling points. lower lethal temperatures. content of polyhydric alcohols and simple sugars. water content and body weights. and in ~rnrn~ltur~forms, trehalose content. Melting points of body fluids were determined in two immature species that demonstrate unusual superco~~iing capability and major cations were rn~~sured in one species of freezing-tolerant beetle.

These two parameters provide the basic information needed to differentiate between the two basic coid-hardiness strategies: freezing tolerance and freezing avoidance. Supercooling points were measured by aRixing a 30, 36 or 40 gauge copper-constantan thermocouple to the abdomen. cooling the specimens at a known rate (0.24.5 C:minl. and determining the temperature at which the transient rise occurs due to the release of the heat of 595

596

KEITH MILLER

6

m 5

-4o.m

-6OI

JUL

AUG

I

1

SEP

OCT

I

I

I

NOV

DEC

I

J*N

I

FEB

1971

Fig. 1. Monthly

I

APR

,

MAY

JUN

tw~prrrrturcs

Whole body weights were measured for w’ater content. analysis of polyhydric alcohols. and SCP determinations. Specimens over 3 mg were weighed to the nearest 0.1 mg and specimens under 3 mg were weighed to the nearest 0.01 mg. Water content of whole insects was measured by drying to constant weight in an oven at 8&100 C (adults) or at 60-70 C (immatures). Problems have been encountered in gall larvae with loss of polyhydric alcohols during oven-drying and freeze-drying was used for some water content determinations of immature specimens. Hemolymph m.p. was determined by withdrawing ~1 samples in a capillary tube. heat-sealing the top. inverting and sealing the open end with oil. centrifuging to remove cells and particulates. quick-freezing with pressurized freon to - 50 ‘C. then determining melting temperature in a temperature controlled methanol bath. The portion of the tube containing the frozen sample was kept immersed in the bath and observed with a magnifying glass while the bath was warmed at approx. 0.5 C;min. The phase change was readily observed and gave m.p. accuracies of kO.2 C. Some gall insect hemolymph exhibited remarkable supercooling capability and such samples had to be quick-frozen by dipping the (sealed) capillary tube with its sample into a Neslab Cryocool bath at -75 C. ut~u/~.srs-po/~oIs,

trrhulosr

md mqjor

,

1972

In order to interpret meaningfully lethality data it is essential to measure habitat temperatures. Since most of the Alaskan insects so far studied overwinter in exposed situations above the snowline, max.-min. thermometers placed in collecting areas usually provided close estimates of habitat temperatures. Thermocouples (30 or 36 gauge) placed beneath bark or in galls have been used to provide supplemental information on effect of solar insulation or other microclimatic variations.

Biochrr~~icul

1

air temperatures at Fairbanks, Alaska showing typical seasonal changes period (Climatological Data, US Department of Commerce).

fusion at onset of freezing. Controlled cooling rates for supercooling point (SCP) or lethal temperature determinations were obtained with a synchronous motor-controlled freezer or cooling bath (Lauda UK-30). or a Neslab system using a Cryocool 100 unit with an Extrol insulated bath and ETP-3 temperature programmer. For lethality tests most specimens were held for I hr at the min. temperature. Rewarming rates varied from 0.5 to 5 C,‘min. Specimens used in lethal temperature tests were held for a minimum of three days after testing to determine if injury had occurred (some immatures were held for weeks or months). Any injury was considered to be potentially lethal and post-test specimens were considered to have survived only if they demonstrated normal behavior such as coordinated walking, feeding. etc. Hahitut

MAR

over a one-gear

under 0.05 mg. Application of greater quantities puts results in an increasingly non-linear portion of the curve for sample weight vs spot size. The occurrence of sorbitol and threitol in Upis crmmhoides required the use of gas-liquid chromatography of methylated derivatives for identification of these polyols (Miller & Smith. 1975). Trehalose was determined quantitatively using a slight modification of the anthrone chromatographic technique described by Wyatt & Kalf (1957). Major cations (sodium. potassium. calcium and magnesium) were determined in hemolymph samples of Lipis crrurnhoides using atomic absorption spectrophotometry (Perkin -Elmer. 303). Hemolymph samples were first treated with concentrated nitric and perchloric acid to digest proteins. Due to the small sample sizes. carefully cleaned and rinsed polyethylene sample tubes had to be used for all dilutions for sodium. Lanthanum was added to improve calcium and magnesium analysis. RESULTS AND

DISCUSSION

Before 1968. freezing tolerance in insects had been reported to occur only in immature forms (Salt, 1961: Somme. 1964. 1965: Asahina. 1966. 1969). Initial studies of cold-hardy Alaskan insects thus concentrated on a search for freezing-tolerant species that overwintered as adults. The first adult species found to be freezing-tolerant was a small carabid beetle, Pterostichrrs hrericorrtis (Coleoptera :Carabidae) that overwinters in decaying stumps and downed timber (Miller. 1969: Baust & Miller, 1970). Although its habitat is normally at least partly protected by snou cover during the coldest part of winter. some populations of P. hrericomis are exposed to nearly the full impact of winter cold. and because of their relatively high winter supercooling point (Fig. 2). must tolerate

curium

Polyhydric alcohols in hemolymph or whole body. alcohol extracts were quantitatively determined using the basic paper chromatographic technique described by Perkins & Aronoff (1959). We have found this technique to be reliable for quantitative use if samples and standards are kept

-51

1

AuG

SEP

OCT

NO”

DEC

JAN

FEB

MAR

APR

MAY

Fig. 2. Seasonal variation in SCP in the freezing tolerant carabid beetle. Ptwostichus hrrricorrlis. Values are mean with SE in bars.

Cold-hardy Table

I. Some physical

and chemical

Weight (mg)

Species

Alaska

properties

of adult

SCP (Q

591

insects overwintering

insects from interior

Lower lethal temp.

Alaska*

Possible cryoprotectant (“, fresh body wtl

( C)

Coleoptera: Carabidae Ptvrostichus

hrericornis

1.5

Tenebrionidae UpIs wrurnhoities Chrysomelidae

I52

sp.

-II

-70

Glycerol,

l4”,,

-6

-60

Sorbitol.

X”,,; threitol,

or colder

Glycerol,

?I’,,

-8

-45

I9

-2X

SCP’!

Glycerol,

I2”,,

Ostomci ,jkrruyirlcu

41

-12

-55

Glycerol.

X”,,

Cucujidae Cuclcjlts c~luriprs

37

Phrurorc~

9.6

3”,,

Scolytidae

Dcwdroctrmlrs rufipf3ii.s Ostomatidae

or colder

_ 55 or colder

~

- 34 or colder _ 34 or colder

Sorbitol. Glycerol.

5” “: glycerol. 9”,,: sorhitol

-40

or colder?

Glycerol.

21” 0

Lepidoptera: Butterflies: Nymphalidae Nytnphnlis Pdyymia

utttioptu

‘60 100

sp.

Moths: Oecophoridae /Vftrrr~rhi/&

c~i~~jfioiwllrr

4.9

-20 -25

-25,,-36t

3”,,

Diptera: Mycetophilidae jllycc,tophild

sp

4.3

-33

-40

or colder?

Glycerol.

14””

5.0

-l5:-36t

-40

or colder

Glycerol,

l6”,,

-32

-40

or colder’?

Neuroptera: Hemerobiidae Hrrwrohius

sirmdans

Homoptera: Cicadellidae T~~phl0c~ho sp.

0.77

* All values are for soecimens collected during winter months. mid-November

t Dual SCP.

to mid-March.

’

freezing. A large percentage of the population is tolerant to about -7O’~C (Miller, 1969), although some have tolerated temperatures to - 87°C without apparent damage. Supercooling points in P. hrekvtwis decline from a summer mean near -6 C to a winter mean of about - I I C (Fig. 2). The decrease in SCP parallels an increase in glycerol cont. (Baust & Miller. 1970, 1972), but is of smaller extent than the decrease in SCP seen in most immatures (Salt, 1959; Somme. 1964. 1965: Asahina. 1969) that exhibit similar increases in glycerol content. The small size of P. hreckwrr~is (7 mg) requires specimen pooling for most biochemical determinations. This fact led to a search for larger freezing-tolerant species. A large ( 150 mg) tenebrionid beetle, Upis cernmhoides (Coleoptera:Tenebrionidae) is found under loose bark of dead standing spruce, birch and aspen in the boreal forest of interior Alaska and northern Canada. Its habit of overwintering above the snowline means that Upis can be exposed to winter temperatures as low as - 55’C. Supercooling points in winter Upis average -6.2’C (Table 1). indicating that this species must also be freezing-tolerant. Laboratory tests confirm that Upis can tolerate freezing to about

-60 C, which is just below the lowest temperature recorded for interior Alaska (Miller. 1978b). Upis cerctrdwidrs seems to be quite unique in several aspects of its cold-hardiness characteristics. It is extremely sensitive to cooling rate. and requires rates below 0.3 C,/min for full development of freezing tolerance; an increase in rate to just 0.35~C, min will greatly increase mortality at - 50°C (Miller. lY7Xb). Another unusual feature of LIpis is its synthesis of the two pol~ols. sorbitol and threitol. when exposed to cold. Wmter cont. of sorbital in freshly collected specimens average about 0.5 M (X”,, of wet body weight) while threitol cont. average 0.25 M (3”” of wet body weight) (Miller. 1978a). The small, freezing-tolerant carabid. Pterostichus hrericomis, is not able to resynthesize glycerol once the beetles have been warm-acclimated in the spring (Baust & Miller, 1972). but Upis can synthesize sorbito1 and threitol during cold acclimation in spring or at any other season. Figure 3 shows the increase in sorbitol and threitol levels in previously warm-acclimated Upis that had lost all of their polyols and then were progressively exposed to lower temperatures during March. Threitol synthesis is seen to lag behind sorbitol. Figure 3 also demonstrates that the build-up

KtlTH

59x

Fig. 3. Concentration of hemolymph polyols in the tenebrionid beetle C’pis wrrrdwidrs during progressive acclimation to cold during March. Before cold acclimation. beetles were exposed to 22’ C for one week and had lost all polyols.

of threitol continues after the beetles are frozen, even at -14 C. A change in polyol (glycerol) levels has also been reported in frozen Ptero.stichus hrrcicorr~is (Baust & Miller. 1970) and in the arctic beetle, Prtko ~rnicric~rnns (Coleoptera:Pythidae) (Ring & Tesar. 1980). Analysis of major cations in the hemolymph of Upis has shown that sizeable changes in concentration occur from summer to winter (Table 2). It is not possible yet to state the importance of such changes in ion concentration for cold-hardiness in Upis. but such major shifts may reflect major changes in many chemical and physical properties. Of possible interest is the effect of calcium on protoplasmic viscosity (Clark. 1958). A two-fold decrease in calcium from summer to winter could cause a decrease in viscosity of cytoplasm during winter. which could be advantageous for continued diffusion of materials and cellular reactions at low temperature. Calcium is also probably the most important ion affecting membrane permeability (Clark. 1958). and a winter decrease in calcium would tend to increase membrane permeability and allow more rapid movement of water out of the cells during freezing. Another small beetle. Plrr~~tora sp. (Coleoptera: Chrysomelidae) has been found overwintering in rather large numbers beneath loose aspen bark. It has been found mainly on hillsides where. due to temperature inversions that commonly occur during clear, cold Table 2. Summer hemolymph

and winter concentrations of cations in Upis crru&vi&.s

weather. it is not usually subjected to temperatures that are as severe as in the bottom lands. Field and laboratory observations indicate almost IOO”,, survival at -45~C and the rapid recovery of the beetles at these temperatures indicates that they can probably survive much lower temperatures. Glycerol build-up is associated with freezing tolerance m this species. but maximum levels of glycerol are the lowest observed in any freezing-tolerant species I have tested (3”” of fresh body weight). Two other species of beetles have been collected beneath loose tree bark in winter and are undoubtedly freezing-tolerant. These are O.storntr fbwyirlctr (Coleoptera:Ostomatidae) and the widespread C~$ra clnript~s (Coleoptera: Cucujidae) (Table I ). It is unfortunate that so far only two specimens of the latter species have been collected in interior Alaska since c‘. chiprs has been studied with respect to cold-hardiness in the more temperate climate of Indiana by Duman (1979). He found about 3”” glycerol in adult beetles in March. however freezing tolerance was only present to - I@ C. In interior Alaska the beetles must tolerate freezing to -55 C. The only overwintering adult beetle so far studied in Alaska that demonstrates little tolerance to freezing is the spruce bark beetle Deridroctornrs rujiperlis (Coleoptera:Scolytidae). Pertinent to the lack of freezing tolerance is the fact that the SCP is much lower than that of the other beetles. Drnrlrc~c.tor~~t,s rufiprlis is common in newly fallen timber and appears to require a relatively humid habitat (Miller & Werner, unpublished observations). Its habitat is thus protected during winter by ground contact and snow cover. Some typical mid-winter temperatures of Dendroctomrs in white spruce are given in Table 3. The adult beetles must rely on supercooling and freezing avoidance in order to survive the winter. As will be discussed later, in contrast to the adults, some spruce beetle larvae can tolerate at least mild freezing. The fact that all of the freezing-tolerant Alaskan beetles have high SCP (above - I2 C) lends further support to the theory first clearly stated by Zachariassen & Hammel (1976) that nucleating agents with their attendant high SCP. are essential to the development of freezing tolerance in the Coleoptera. In contrast, the other adult insects from interior Alaska that are freezing-tolerant all have relatively low SCP.

major

Concentration in hemolymph (mg.ml I* Winter Summer

Cation

MILLER

Lepidopteru--hutterPi~s At least three species of butterflies, all from the Nymphalidae. are known to overwinter in interior Alaska as adults (Philip. personal COmmUnifamily

Sodium

0.61 + 0.26 (0.026 M)

0.36 + 0.16 (0.016 M)

Potassium

0.91 + 0.23 (0.023 M)

I.55 * 0.70 (0.040 M)

Calcium

0.51 * 0.09 (0.013 M)

0.25 * 0.04 (0.006 M)

Dec.

1.34 * 0.21 (0.055 M)

0.96 + 0.14 (0.040 M)

Jan.

Table 3. Exterior body temperatures of Drndrocfr~~us rufipenis beneath white spruce bark in contact with the ground-~ interior Alaska upland site. 1979-80

Month

Magnesium

*All theses.

values

are mean

f SD. Molality

given

in paren-

Mean M&X. Min. Mean MU. Min.

Air temp. (’ C) -?I -9 -3s -23 +3 -41

Specimen

temp.

Snow depth

( C)

(cm)

- 17 - 16 - 19 -13 -9 - IU

30 23 I5 33 37 33

Cold-hardy Table 4. Survival

of adult artificial

butterflies kept overwinter hib~rn~lculum

in an

Alaska

insects

599

most commonfy in upland areas. it is possible that some indivjdu~~is could overwinter without freezing. Again. long-term SCP would tend to he higher. so freezing tolerance appears to be necessary for survival.

The remaining adult insects representing the above three orders all share the same habitat as the adult cation). Some cold-hardiness data have been obtained moth and several species of beetles: the) overwinter for two species, ~~i~tp~z~~li.~ mtictptr and P[)~~~J~I~~~~ sp. beneath loose aspen or spruce bark (prim~trify aspen) Unfortunately, no specimens have yet been found in on dead standing trees in upland areas. All of the their natural winter habitat. so inform~ltioI1 must be adult insects found utilizing this habitat seem to have based on specimens obtained just prior to the butterno preference as to the side of the tree on which they flies entering their hibernacula (fate September or hibernate, This tinding is in contrast to some studies in which insects were seen to hibernate on the sunny early October) or just after they emerge in April, or side (Dennys. 1917) or non-sunny side (Dasch, 1971; from specimens kept in artificial overwinter hihernaoufa. The fatter approach has yielded the best eviAfford, 1969) (all cited in Danks. 1978). The neuropteran, Hr~~~wrohitrs .sirmrhs (Neuropdence for freezing tolerance in X. rrrrrioptr and PO/JY~Otera:Hemerobiidae), shares many features with the t7irr spp. freezing-toi~r~~nt moth, ~~~~~~f~~zj~~~~~. ~~~~Ff~~~~~~i~{s has A total of 6 P[)~~~~o}~i~~ and I7 j~~~j~~?~~~~~i,s Mere also been unpredictabfe in laboratory lethal temperaplaced in a plain wooden box which in turn rested on ture tests, though a few individuals have survived the ground in a four-sided. screened enclosure. Maxi-50 ‘C without apparent damage. It is much fess mum and min. temperatures in the box were checked common than .~~~~~f~r~zj~~~~~. but individu~~l adults that with a max.-min. thermometer at 1~2 month intervals from October to May. The minimum temperature are collected from beneath bark in April are invariably active and able to fly if the air temperature is reached in the box wits -.3X5 C. On 12 May all above 0 ‘C, so even in a mild winter they must tolerate specimens were checked for viability. The results are given in Table 4. As indicated in Table 1. both .‘VJW- -4O’“C. ~~~iz~rob~~~salso exhibits a dual SCP: an initial. small point at - 15 k YC and the major SCP at pi7di.s and PoI~~/c~nitr produce moderate amounts of _ 36 & 5.6’C. Glycerol concentration glycerol and sorbitol when exposed to cold. Most imaverages 16”,, portantly. mean SCP for !~~,~~z~j7~~~i.~ and ~~~~~~/~~~~i~~ of fresh body weight in winter. During some years relatively large numbers of are -20 and - 25’ C, both well above the min. temgnats (Myc~ophila sp.) (Diptern: Mycetophifidae) can perature to which the butterflies were exposed in the artificial hibernaculum. Also. the mean SCP given are be found in upland areas beneath loose aspen bark. Specimens collected during October quickly revive for very short-term cooling. The long-term cooling and become active %$>henreu>armed at 10 C, but durexperienced by overwintering adults would undoubting mid-winter (fate November to February) gnats edly cause them to freeze at higher temperatures. as shown by Salt (1950). it is interesting to note here that brought into the laboratory and rewarmed at 20 C usually fail to revive. Gnats collected later in March specimens of ~~~~fp~ju~j.s~~~7~i~~~7~~ have been obtained from southern California where temperatures have or April are active at outdoor temperatures near 0°C never fallen to 0 ‘C. but the ability of the adult butterand can ffy when brought into the laboratory at 20-C. flies to produce polyofs during cold acclimation is still The gnats, just ~1s the moth, ~~~rt~rj~~~d~~,and facepresent (Miller. in preparation). Gng, ~~~?t~r~~i~~s, thus appear to enter a state of winter dormancy which cannot usually be broken by simple rewarming. The most obvious means by which A small moth. ,~urt~r~zild~i ~i~~~~~~z~~l~ (Lepidopthe dormant state could be broken in the natural situtera:Oecophoridae), can be found in large numbers ation is by cyclic ~varming and cooling, by photoperbeneath loose aspen and spruce hark during autumn. iodic effects, or by some combination of thermo- and winter and early spring. This species exhibited freezphotoperiodic stimuli. ing toferance in onfy occasional indivjdu~~ls during To test the possibility that temper~lture cycling was mid-winter fower lethality tests done in the iaborathe important stimulus, specimens of all three species tory. yet for years large numbers of the adult moths were collected in February and subjected to an aftercould be found actively moving or able to fly when nating series of temperatures between + S and - 18-C exposed in their natural sub-bark habitat in early for four days. then held at 5’C for an ~~ddition~~l two spring (late March or April). Recent tests have shown weeks. The results were as follows, that freezing survival in the laborator? is much improved if the moths are kept in a humId atmosphere NO. No. during rewarming. The adults unquestionably survive Species recovered tested outdoor winter temperatures of -40 C. Gnat (i\f~~topl~iltr sp.) 18 33 The adult moths almost invariably exhibit a dual Lacewing (Htvierc~hirts simrrhs) 18 3 SCP. A small portion of the body. probably an Moth i&f ~~~~~~~zj~~~ ~i/i~~~~~~~~~~~~~15 4 appendage. freezes first (mean SCP -25 i 7‘C) and the major portion of the insect freezes at -36 t 5.7”C. Supercooling points in this species From these results it is apparent that cyclic temperahave been as fow as -49 C, and because it is found ture changes by themselves are able to break the

I
600 Table

5. Some

physical

and

chemical

properties

of overwintering

irnrn~t;ir~

insects from interior

Alaska* Fresh hod> wt lmgl

SCP

Lower lethal temp.

( c‘)

( Cl

-56

SCPf

-58

SCP

12Y

-56

SCP

2.13

-3x

SCP

16.X

-31

SCP

-3

Below -25

Major polyol or sugar (I’,,fresh body wt)

Giyccrol. 73”,, Trehnlose. I._i”,, Glycerol. 24”,, Trehalose. I”,,

Glycerol.

* All data are from freshly collected. t Supercooling point. ‘; Data from Werner

winter-acclimatized

specimens:

16”,,

Trehulosc, IX’,,

Glycerol. 1”,,

all larvae except for R~I~~~~~~‘~P~~~~~I.

( 1978): pupar only.

winter dormancy in the three species tested. It is probable that other combinations of temperatures would be even more effective in promoting survival and much work remains to be done to clarify the mechanisms involved in both the init~~~ti[~n and termin~ltion of the dormant or non-responsive state seen in some overwintering adult northern insects. Circumstantial evidence also exists for freezing tolerance in one other order, the No~optcrtr. A lcafhopper. probably of the genus 7)p/1oc~rhrr (family Cicadellidae), has just been found overwintering beneath loose aspen bark. Only a few individuals have been tested for SCP and the mean is relatively low: -31.6 k UC. No data arc yet available on potyols or sugars and the small size of the species will require that sizeable numbers of specimens be found in the future to permit additional chemical and physical analyses. It is certain that freezing-tolerant adult insects belonging to other orders will be found in Alaska and throughout the arctic and subarctic regions. The Hymenoptera are undoubtedly represented. In interior Alaska, isolated, Llnidenti~ed wasps have been found alive beneath loose bark in late winter but have not yet been found in numbers sufficient for coldhardiness testing. Mild freezing tolerance has been reported in four species of Hymenoptera (three wasps. one ant) from northern Japan (Ohyama & Asahina, 1972) and Duman & Patterson (1978) have found that overwintering queens of the bald-faced hornet. V’G&u ~~xKI&~Q.could tolerate freezing to - 14 C. In E’q~uicc. nucleators. possibly proteins. appear to be important to deveiopment of freezing tolerance (Duman & Patterson. 1978).

Studies have recently been initiated to describe some of the important physical and chemical characteristics of immature insects that overwinter in severe northern climates. Emphasis has been on several species of willow gall-forming larvae that overwinter above the snowline and are thus exposed to the most severe ambient conditions. Another species of rose gall-former that typically overwinters beneath the show has received some study and the larvae of the spruce bark beetle. which overwinters beneath the bark of fallen timber in contact with the ground or in live standing timber. often beneath the snowline, has been tested for cold-h~~rdiness. A list of species under study that overwinter in interior Alaska. and some of their physical and chemical characteristics. is given in Table 5. Data are also included from a study hy Werner ( 1978) on the gcometrid defoliater R/wrrr~nptern hcnsttrt~~(Lepidoptera :Geometridnc). which overwinters in leaf litter.

Because of their fully exposed winter habitat the three species of willow gall-formers so frir identified were expected to be freezing-tolerant. However. as Table 5 shows, none are able to tolerate freezing and depend on a remarkable capability for supercooling in order to avoid lethal ice formation. The supercooling and glycerol-producing capabilities of the three species are essentially identical. Mean SCP vary from -56 to -.58~C and a number of indi~~iduals of each species have had SCP of -61 to -63-C. At the time these SCP were first reported (Miller & Werner.

601

Cold-hardy Alaska insects Table 6. Supercooling points. hemolymph melting points and glycerol concentration* of willow cone gall larvae (~~7abd~p~~~u strohibides) in winter and summer

Season Summer Winter

Extent of supercooling

SCP f-C)

M.p.

( C)

( C)

Glycerol (molal)

-26.5 i 4.1 -56.1 k 4.7

- 1.2 & 0.2 - 19.3 + 4.1

25.3 36.8

o+o 4.8 i 1.9

* All values are mean rt SD. 1980). Ring (1980) also noted that he had observed SCP into the - 60°C region in laboratory-acclimated larvae of the same willow cone gall-former, R/&dopltugu strohiloides (Diptera:Cecidomyiidae). obtained from northern Canada. The unusual supercooling capability of all three species of willow gall larvae is apparently due to the production of very large quantities of glycerol (Table 5) and to an absence of effective ice nucleators. The water content of ~~z~lbd(~p/zLl~l~l and ~~~~~~fjo~~~ during winter is 54”,, (Miller & Werner, 1980) so the actual cont. of glycerol in aqueous solution in these species is about 4.8 molal. As shown in Table 6 the decrease in m.p. from summer to winter amounts to 18.1’ C. Based on the classical colligative relationship of a change in the m.p. of - 1.86”C per molal increase in concentration of a non-ionizing substance, the winter m.p. should be only about - 10°C. including the contributions from glycerol. trehalose, salts and other normal hemolymph constituents. Since I have full confidence in the accuracy of the m.p. determinations. either our water content measurements are too high or some. as yet unknown, factor or factors are contributing to the low m.p. The use of oven-drying to constant weight may well have caused significant loss of glycerol. which would result in water content measuretnents that were too high. The superco~~ling capacity (difference between mean SCP and mean freezing point) was considerably greater in winter than in summer (Table 6). and as has been noted in several other insects and mites (Salt, 1959; Somme. 1967: Block & Young. l979), the increase in glycerol depressed the SCP more than the m.p. Winter m.p. and SCP in the willow apical gall larvae !%ltr~t~tioltr were. respectively. - 14.5 * 4.1 C and -57.8 i 5.0 C. so the sLlpercooling capacity in this species was about 43-C. This capacity for supercooling is greater than was thought possible for insects (Salt. 1961: Ring. 1980) and equals or exceeds the limit of supercooling for pure water (Salt, 1961: Meryman, 1966). In fact pure water has been supercooled to -4O~C only in tiny droplets that are cooled very rapidly. and one would think that the heterogeneous chemical nature of insect body fluids and the slow cooling rates involved in supercooling tests used here would have resulted in less supercooling than ~3s actually observed. The apparent lack of nucleating substances in the gall insects may in part be related to the fact that they are completely separated from the usual atmospheric nucleators by their gall habitat. Feeding on the high protein plant tissue produced on the inside of the gall does not appear to introduce efficient nucleators into the insect. Evidence that atmospheric nucleators can

increase SCP in willow stem gall insects comes from the fact that Euuru adults had SCP about 8°C higher than larvae: summer larvae had a mean SCP of -25.5 k 4.8’C while adults had a mean SCP of -16 f 2 C. The extremely low winter SCP of the various willow gall larvae provides a natural system in which to test for possible effects of artificially introduced nucleators on freezing survival. As noted earlier. the winter-acclimatized larvae, though they contain extremely large quantities of glycerol. are not freezingtolerant. Injection of hemolymph taken from the freezing-tolerant beetle Upis ccru~~hoides (L’pis hemolymph equal to _7”,1 of the larval body weight introduced by microinjection) raises the mean SCP from -5X f 3 ‘C to -33 * 1 C (Miller & Werner, 1980 and manuscript submItted). In spite of the 2S’C increase in SCP of the winter-hardened larvae. after injection of Upis hemolymph none survived freezing to -60-C’. so nucleation at a temperature well above the normal SCP does not promote freezing tolerance. Freezing tolerance does occur in larvae of Brucorl crphi (Salt. 1959) in spite of the fact that the larvae supercool to about -45 C. so there is no good explanation at present as to why the Alaskim gall insects are freezing-susceptible.

A small wasp. Dip1olqi.s hicwior (H ymenoptera : Cynipidae), is responsible for the formation of spherical. multi-compartmented galls that are covered with numerous spicules and that occur on the veins of leaves of the wild rose. Rostr ~~cicd~uts, Clusters of the dried galls may remain on some bushes throughout a portion or all of the winter, but most of the galls with their larvae drop to the ground or are low enough on the bushes that they are covered by snow. Table 5 shows that the mean winter SCP of the larvae is - 38’ C. so larvae that overwinter on the ground or on snow-covered bushes are able to avoid freezing. Larvae in galls that remain attached until mid-winter and are above the snowline apparently do not survive, since none were found to tolerate freezing in the laboratory if cooled below the SCP.

This serious pest of white spruce (P&t! &tcrr) and black spruce (P&V rntrricmr) in interior Alaska and northern Canada overwinters beneath the bark of live standing or recently downed trees. It may overwinter above or below the snowline. Laboratory tests of winter larvae from interior Alaska show a mean SCP of -3 1 C (Table 5). This temperature is well above

the lows that would be encountered beneath the bark of spruce projecting above the snowline. So far we have only tested larvae from downed trees, but none of these could tolerate freezing under laboratory conditions (Table 5). Laboratory tests of the closely related species that attacks Piceu eryelmwr~ii at lower latitudes show that subcortical temperatures of - 34.4 ‘C are fatal to larvae (Massey & Wygant. 1954), but Frye t’f (il. (1974) report that a severe cold spell with five days of min. temperatures of -35 to -40 C killed only XX”,, of the larvae at chest height (above the snowline) in the White Mountains of Arizona. Daily highs during the cold spell were about 15-20 C and since sub-bark temperatures were not measured it cannot be stated for certain whether or not all of the larvae were subjected to freezing temperatures. As show-n earlier in Table 3. body temperatures of &VI~~~~~~r~~~~~~.~ larvae at ground level beneath the snow did not fall below - 19~‘C when air temperatures reached -4i’C. Supercooling points begin to decline during September from summer levels near - 12’ C and reach the winter mean of -31 C by early November (Fig. 3). Large quantities of glycerol (Table 5) undoubtedI> account for the decline in SCP in autumn. Moderate amounts of trehaiose are also present. A more precise anafvsis of the importance of freezing tolerance vs freezing avoidance in overwinter survival of the overall population of ~;,~i~~r~)~~(j~~~~,~ rc!fipt~iis awaits studies of supercooling and lower lethal temperatures in individuals that remain above the snowline.

This defoliator of birch, Beth pqyrifiw. overwinters in leaf litter (Werner, 1977) und is included since it has been studied in interior Alaska (Werner. 197X) and because it shows the best evidence for freezing tolerance of immature Alaskan insects thus far studied. Only the diapausing pupae show possible freezing tolerance and considerable (40”,,) mortalit) has been found to occur after :I 15-day exposure to _ 35 ‘C (Table 5 and Werner, 1978). Since the SCP is pupw it stems near -2.5 C in winter diapausing probable that few individuals would survive prolonged temperatures much below the SCP. Measurement of temperatures in the leaf litter has clearly demonstrated ;I direct relation between survival and snos depth and ;I winter decline in SCP parallels a drop in leaf litter temperature (Werner. IY7X). A rather modest ($I,, of fresh body weight) increase in glycerol content results in a 20 C decrease in the mean SCP. again showing the disproportionately large effect that glycerol can have on supercooling. Most of the imm~lturc insects so far studied in Alaska have bvinter trchalosc levels of approx. l-2”,, of the fresh body weight (Table 5). Such cont. are somewhat higher than those found in many warmacclimated higher insects, except for the Lepidoptera (Wyatt & Kalf, 1957: Bedford. 1977; Kramer et trl.. 197X), but are comparable to Icvcls reported for some other cold-acclimated insects (Morrissq & Baust. 1976; Ring & Tesar. 1980). No major seasonal changes in trehalose levels have been observed in the larvac tested, which contrasts with the situation in cold-acclimated adults and larvae of the arctic beetlc

Qtilo ff/~tt,~j~,~~~~~is which shows a major decline in trehalose during cold exposure (Ring & Tesar, 1980). Pq‘rho UIIIU%UUS is freezing-tolerant whereas the Alaskan immatures are not. so the retention of 100 mM trehalose in the latter would assist supercooiing by at lcsst ;I small amount. The most striking evidence for the role of trehaiose in cold-hardiness has been presented by Tanno (1970) who found ;I close correlation between freezing tolerance and trehalose in the poplar sawfly, ~r~~~lio~u~?~~~~.s p~~~~~j. in which trehalose cones. may reach S’,, of the fresh body weight. Except for its apparent importance in 7: pop~li, the wide range of trehalose levels seen in various insects, and especially the high cones. in warm-acclimated Lepidoptera, (Wyatt & Kaif, 1957; Kramer et ~1.. 1978) leads to the conclusion that trehalose serves primarily as an important storage c~~rbohydrate rather than as a cryoprotect~~nt.

Sl~blbIARY

.AUD COYCI.l’SIONS

A variety of adult and immature insects successfully overwinter in interior Alaska, many in habitats that are exposed to the full impact of winter cold. Among those species that survive in the most exposed situations. the majority of adults are able to tolerate frcering while the imm~~ture larvae are freezing susccptible and survive through their ability to supercool, some to remarkably low temperatures. Freezing tolerance has been found in adult representatives from five orders of Alaskan insects, but other than the fact that ail produce quantities of polyhydric alcohols in response to cold. there are no physical or chemical properties that have invariably been found associated with their ability to tolerate i&ring. If ue consider oni4 freezillg-tol~r~~nt beetles. the c~ip~ibiI~ty of nucleating at relatively high tempcraturc (Zachariasscn & Hammel, 1976: Zachariassen, 1980) also seems to be important in Alaskan forms. but freezing-tolerant Alaskan species from other orders can tolerate freezing in spite of supercooling points as low as -36 C (Table I). Although all of the frze7inp-tolerant adults so far described from Alaska produced moderate to large quantities of polyhydric alcohols. usually giycrroi. until wc can shou clearly just how polyols act to produce freezing tolerance the evidence for the importance of polyois in confurring freezing protection will remain largely circumstantial. Chemical analysis of freeze-tolerant species has. of course. been far from exhaustive and ue should not let our preoccupation with polyhydric alcohols hamper the search for other substances of cryoprotectivc importance at this reiativciq: primitive stage in our understanding of coidhardmess. Asabina ( 19hh) has p~~stlll~lted that some pr~~t~~pi~~srnicchange may hc irnport~~I~t in the genesis of freeLing tolerance and in light of our present knowlcdgc. some tissue changes at the membrane level or molecular Icvcl arc likely to bc of critical importance to the dcvelopmcnt of frccring tolerance. The linding that the adult tenebrionid beetle. L’pis ~,~,~trllthoitlf,.s.shows rather dramatic summer to stinter changes in the levels of at1 four major cations (Table 2) gives an inktint: that many changes may occur at the molecular level during the devcl~~prne~lt of freering tolerance.

603

Cold-hardy Alaska insects The finding that several species of immature gallforming insects from interior Alaska are not able to tolerate freezing, and survive the winter in highly exposed habitats by their ability to supercool to about -60°C (Table 5), is surprising for several reasons. The amount of supercooling observed is irpprox. the same as the max. limit for supercooling in pure water under rigorous experimental conditions (Meryman, 1966), and one would not expect a biological system to be as free of nucleating material as highly purified water. The matter is complicated by the fact that in the above gall larvae and also in some other insects and mites (Somme. 1967; Block & Young, 1979) glycerol lowers the SCP about 2’C or more for every 1°C lowering of the m.p. We should not be too alarmed that several studies of physical systems (see Meryman. 1966) have indicated that solutes depress the temperature of heterogeneous nucleation to about the same extent as they depress the m.p. Biological systems are complex and there may be synergistic action between glycerol and other substances present. More studies of simple physical systems are also needed since Block & Young (1979) found that glycerol depressed the SCP of distilled water by 2.2’~‘C per degree of (calculated) m.p. depression. From the standpoint of energetics it is more et% cient to freeze at as high a temperature as possible. since freezing causes a sharp drop in metabolic rate (Schol~~llder et (II.. 1953; Salt. 1958). Also. freezingtolerant species do not generally seem to require the prodigious quantities of polyols that the gall larvae and some other freezing-avoidant species need. Zachariassen (1980) has recently noted that a given increase in polyol cont. will depress the lethal temperature to ;I greater extent in freezing-tolerant beetles than in freezing-sensitive forms and should thus favor the development of freezing tolerance, especially in species that overwinter in very cold environments. but the Alaskan willow gall larvae do not conform to the adult beetle pattern. It is particularly interesting that at least three species of larvae from two different orders should have developed nearly identical supercooling capabilities in association with very high glycerol levels. This is a striking example of parallel evolutionary adaptation. Another interesting aspect of the overwinter survival of both freezing-tolerant and freezing-susceptible northern insects is that their low temperature limits are only a little below the record minimum temperatures for a given region. This relationship holds also for overwintering insects in more temperate climates and it would be interesting to see if certain species with wide latitudinal distributions show a progressive decline in lower lethal temperature with increasing latitude. There is a need for additional studies that combine detailed information on microhabitat temperatures with controlled laboratory testing so that results of physico-chemical analyses can be related to conditions in the natural habitat. Precise low temperature lethality data are lacking for many species, perhaps due to the requirement for careful handling and testing of large numbers of specimens. The cyclic nature of many insect populations requires long-term studies of insects from a variety of habitats. Even in the restricted habitats so far surveyed in interior

Alaska, previously unseen species appear with each new season. From many stand~ints, in Alaska or elsewhere. our current knowledge of invertebrate cold-hardiness is undoubtedly very biased and is certainly very incomplete.

REFERENCES

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