Effects of low temperature on diapausing Aglais urticae and inachis io (Lepidoptera: Nymphalidae): Cold hardiness and overwintering survival

J. Insect Physiol.

Vol. 35, No. 4, pp. 277-281, 1989

0022-1910/89 $3.00 + 0.00 Copyright 0 1989 Pergamon Press plc

Printed in Great Britain. All rights reserved

EFFECTS

OF LOW TEMPERATURE ON DIAPAUSING AGLAIS URTICAE AND INACHIS IO (LEPIDOPTERA: NYMPHALIDAE): COLD HARDINESS AND OVERWINTERING SURVIVAL A. S. FULLIN and J. S. BALE Department of Pure and Applied Biology, Agricultural Sciences Building, University of Leeds, Leeds LS2 9JT, England (Received 5 August 1988; reked

16 September 1988)

Abstraract-Two species of nymphalid butterflies, Agluis urticae and Inachis io were exposed to four different temperature regimes (10, 2, - Y’C, and cycling - S/lWC) during diapause to determine patterns of cold hardiness and overwintering survival. Supercooling ability increases in all groups from cessation of feeding to 100 days in diapause, but the lowest temperature regime does not produce the lowest mean crytallisation temperature. Pre and post-diapause feeding reduce supercooling as does surface water. Fresh weight loss follows a temperature-dependent pattern with greatest weight loss at 10” and least at - 5°C in both species. Mortality is highest at lo” accounting for 80% of I. io after 100 days and 75% of A. urticae after 170 days. Mortality at - 5 and - S/lo”C differs between species; in A. urticue only 30% had died after 170 days in both groups but in I. io a sharp rise in mortality after 100 days resulted in 65% mortality after 155 days in both regimes. There was no evidence of individuals freezing in the subzero regimes. Initial wet weights of survivors are significantly higher than those of non-survivors at all temperatures. Implications for assessment of cold hardiness in insects are discussed. Key Word Index: Overwintering, cold hardiness, Agfais, Znachis, supercooling, butterfly

INTRODU(XON

Insects employ a number of strategies to increase their chances of survival during winter. The accumulation of energy reserves in the form of lipids and the induction of a state or reduced metabolic activity are the most common adaptations in response to such a long period without food. Additionally, most insects increase their winter cold hardiness as an aid to survival at subzero temperatures. Insects are classified as freeze tolerant or intolerant based on the ability of the species to survive freezing of their extracellular fluids (Salt, 1961). Freeze-tolerant species are able to survive extracellular ice formation within the body tissues, whereas freezing-intolerant insects are killed by freezing and rely on supercooling to avoid this lethal event. More recently, Knight et al. (1986) found that some aphids, although able to supercool extensively, die after a brief exposure to sub-zero temperatures above their crystallisation temperature (often referred to as the supercooling point). Assessment of cold-hardiness is complicated in freezing-intolerant insects by variations in supercooling during diapause (Mansingh and Smallman, 1972). This may reflect changes in the levels of cryoprotective substances in response to the nutritional and diapause status of the population, or to variation in preceding weather conditions. In addition, supercooling may be reduced by the presence of internal ice nucleators such as food in the gut (Salt, 1953), or by surface moisture causing inoculative freezing through the cuticle (Salt, 1956). Most research on insect cold hardiness has centred on short-term exposures to low temperatures and few

studies have attempted to assess the effects of prolonged exposure throughout the winter period. In one study, Turnock et al. (1983) found that exposure of diapausing pupae of Mamestra ConJigurata to temperatures between -5 and -20°C over 140 days reduced survival in the post-diapause stages. If reduced survival is common after longer term exposure to low temperature then previous assumptions about levels of mortality in overwintering populations based on short-term exposures and supercooling data may be erroneous (Bale, 1987). The temperate nymphalid buttedies, Agluis urticue and In&is io are widely distributed on the European continent but A. urticae extends to higher latitudes and altitudes. Both species overwinter in imaginal diapause after seeking out semi-sheltered sites amongst vegetation, or in tree-holes and buildings. The survival of these species at moderate temperature (4°C) during this period has already been studied (Pullin, 1987). The aims of the research described in this paper are 2-fold. Firstly to determine the cold hardiness of adults at specific stages before, during and after the overwintering process, and to assess mortality during long-term exposure to temperatures above the crystallisation temperature; and secondly to investigate the influence of interactions between diapause, nutritional status and abiotic factors on overwintering. MATERIALS AND METHODS Rearing

Both species of butterfly were reared in the laboratory following the method of Pullin (1987) over one 277

A. S. PULLINand

278

generation after field collection from southern England. Imaginal diapause was induced in adults by exposing late-instar larvae and adults to a 12 h light-12 h dark cycle (Pullin, 1986, 1988). Overwintering

On emergence adults were kept in 20 x 20 x 10 cm containers covered with muslin and fed on 10% honey solution which was absorbed onto tissue paper layed on the muslin. A period of 10 days at 20°C was allowed for feeding followed by 5 days acclimation at 10°C. All butterflies were then weighed, sexed, and placed in individually labelled Petri dishes. Each adult was allocated to one of four temperature regimes: 10, 2, - 5°C and a cycling regime of - 5 and 10°C. The latter was achieved by transferring butte&es between -5 and 10°C incubators for 8 h a day for 5 out of every 7 days. All specimens were kept at 60-80% r.h. in constant dark. A second period of acclimation (5 days at 2°C) was given to the -5 and -5/lO’C groups to ensure that no freezing occurred due to food remaining in the gut. All individuals were weighed and checked for mortality every 14 days. After periods of 1, 3 and 6 months samples of IO individuals were removed from each group for crystallisation temperature determinations. Experiments were discontinued in some groups when survival was low. Crystallisation

temperature measurements

Individuals were taken at a number of stages during the overwintering process for assessment of supercooling ability, after suitable preparation, as detailed in Table 1. Crystallisation temperatures were measured using thermocouples linked to a thermocouple converter monitored by a microcomputer (Bale et al., 1984). Butterflies and thermocouples were restrained in a paper envelope inside a test tube suspended in a low temperature bath (Haake F3) containing ethylene glycol and cooled from 10 to - 25°C at 0.5”C min-‘. After freezing, some individuals were warmed to room temperature and examined for signs of recovery as evidence of freeze tolerance. To test whether inoculative freezing due to extracuticular water significantly reduces the crystallisation temperature, adults in mid-diapause at 2°C were sprayed with small droplets of water prior to the supercooling procedure. Pre-freeze

mortality

Diapausing adults were tested for their tolerance to temperatures above their crystallisation temperature. Effects of short exposures were investigated using Table I. Stages and preparatory stage Pupal Newly emerged Feeding stage Early diapause 1 month diapause 3 months diapause 6 months diapause Postdiapause unfed Postdiapause fed Water inoculated

J. S. BALE

adults in diapause for 1 month at 2°C. Separate samples of 30 individuals of each species were cooled to - 5, - 10 and - 15°C for 1 min using the same apparatus as above and then warmed to room temperature. Mortality was recorded after 0, 24, 48 and 72 h at 5°C. Groups of I. io adults were used after 4 months in diapause at 2°C to quantify mortality after longer and successive exposures to pre-freeze temperatures. Three groups of 30 individuals were cooled to - 15°C and held at this temperature for 1, 8 and 18 h after which they were warmed to room temperature. Another group was given four successive exposures to - 15°C of 30 min duration between which they were warmed to room temperature for another 30min. After each exposure all groups were kept at 5°C for 72 h to test for mortality. RESULTS No individuals of either species survived below their crystallisation temperature. Pre-freeze experiments on individuals in diapause for I month produced mortality in only one group; 1. io adults exposed to - 15°C for 1 min showed 23% mortality. However, the crystallisation temperature of some individuals at the same stage of diapause was above - 15°C and thus freezing cannot be excluded as a cause of this mortality. Longer and successive exposures to - 15°C on I. io adults 4 months into diapause produced no mortality. It appears that the crystallisation temperature is indicative of the lower lethal limit of A. urticae and I. io in short and medium term exposures to low temperature under the experimental conditions. The means and standard errors of the crystallisation temperature for the two species during diapause are shown in Fig. 1. The pattern of change in the mean is similar in both species. Extensive supercooling ability (- 19 to -22°C) is evident in newly emerged adults but this is reduced (to - 10°C) once feeding has taken place, and is only partially regained after 5 days of cold acclimation and starvation. Subsequently, at 10°C there is a rapid increase in supercooling of I. io which is not so evident in A. urticae. In general supercooling increases in both species between day 15 and day 40 at all temperatures and shows a further increase between day 40 and day 100. Beyond 100 days this acclimation response continues in I. io (except at 10°C where there were few survivors after 100 days) but in A. urticae supercooling remains constant. The lowest temperature regime (- 5°C) is not the optimum for acclimation. In

treatment of A&is urficae and Inochis io used in crystallisation

temperature measurements

Pm-treatment Pupal cuticle was allowed to harden for 2 days at 20°C after which pupae were acclimiated at 5°C for 7 days Emergent adults were left at 20°C for I day to allow excretion of meconial fluid After emergence adults were fed at 20°C for IO days Adults were fed at 20°C for IO days and then starved for 5 days at 10°C After feeding and acclimation adults were kept for 1 month in each of four temperature regimes -5, 2, 10 and -5/lO”C After feeding and acclimation adults were kept for 3 months as above After feeding and acclimation adults were kept for 6 months as above Diapause was broken after 6 months by placing adults in a long photoperiod at 15°C for 7 days Diapause was broken after 6 months by transferring adults to room temperature and feeding for 7 days Adults in mid-diapause were sprayed with water until covered with small droplets prior to cooling

Effects of low temperature

TIME IN DIAPAUSE (DAYS)

Fig. 1, Changes in mean (+ SE) crystalhsation temperature during diapause in Agluis urticne (a) and Inuchis io (b) adults. Temperature and developmental conditions are as follows: P pupa 20°C N newly emerged adult 2O”C, F fed adult 20°C A acclimated at 10°C for 5 days; diapausing at A -5”C, l 2”C, a 10°C 0 -5/lO’C, 2 postdiapause unfed 15°C. X postdiapause fed 20°C I water inoculated whilst diapausing at 2°C.

fact I. io shows the smallest increase in supercooling at -YC, and beyond 100 days this is also true for A. urticae. The cycling regime ( - 5/ 1O’C) produces a

0.0 Cl

40

-0

TIME IN DIA~AuSE (DAYS)

Fig. 2. Mean (It SE) survival in diapausing Ag/uis urticue (a) and Znachisio (b) adults under four temperature regimes; A -5°C

.

2°C W WC, 0 -5/lOYI.

279

TIME IN DIAPAUSE (DAYS)

Fig. 3. Mean (+ SE) fresh weight loss in diapausing Aglais urticae (a) and InacI~is io (b) adults under four temperature regimes; A -5°C

l 2°C W 10°C U -5/lO”C.

mean crystallisation temperature intermediate to the constant regimes of -5°C and 10°C in I. io but significantly lower in A. urticue. Extensive supercooling ability is retained into the postdiapause phase, but when feeding recommences the mean crystallisation temperature) rises by approx. 10°Cin both species. Acclimated pupae of both species show a similar supercooling ability to adults in earlydiapause. Water inoculation causes a significant increase in the mean crystallisation temperature of both species (t-test) and also an increase in the range. After water inoculation 72% of A. urticae and 48% of I. io froze at - 10°C or above compared with the highest individual supercooling points of - 15.3 and - 159°C respectively for the dry groups at 2°C. The survival of A. urticae adults under the four temperature regimes is shown in Fig. 2(a). Percentage survival is similar at 2, -5 and -5jlO’C with approx. 70% alive after 170 days. At 10°C 70% survive for 110 days but less than 25% are alive after 170 days. Weight loss in survivors to 170 days [Fig. 3(a)] is highest at 10°C with a 25% decrease in fresh weight after 90 days. The -5/lO”C group lost 25% fresh weight in 110 days whereas both the 2 and -5°C groups had lost only 20% fresh weight after 170 days. Survival of I. io adults [Fig. 2(b)] is also lowest at 10°C with less than 20% alive after 100 days compared with 60% survival after 180 days at 2°C. Both the -5 and -5/lO”C regimes show verly little mortality up to 80 days followed by a rapid increase to 155 days when less than 35% survived in both groups. Inspection of butterflies prior to removal from the -5°C incubator indicated that no individuals subsequently found to be dead, had been killed by

280

A. S. PLILLIN and J. S. BALE

Table 2. Comoarison

of initial weinhts of survivors

and non-survwxs

to the end of the exoerimental

ueriod

Wet weight (mg) after feeding and acclimation /nachir io Aglai.s urficae ..~__

Temperature (’ C) Survived: Mean N SE Died: Mean N SE I-test

-5 230 I2 10.0 190 30 4.4 P < 0.01

2 211 23 7.6 191 18 x.3 P < 0.01

IO 240 8 17.1 197 45 4.5 P < 0.05

freezing. Fresh weight loss is highest at 10°C [Fig. 3(b)] with a 25% reduction after 55 days. The order of groups with respect to weight loss is the same as in A. urticae with 25% fresh weight loss at 95 days at -5jlO’C and 165 days at 2°C. Only a 16% fresh weight loss was recorded at -5’C up to the end of the experiment after 155 days. Initial fresh weights of those surviving the experimental period are significantly greater in all groups than of those not surviving. The mean initial fresh weight of survivors is highest at 10 and lowest at 2’.C in both species (Table 2). DISCUSSION

Both species are evidently freezing intolerant under the experimental conditions. No pre-freeze mortality was recorded after short term, medium term of successive exposures to subzero temperatures above the crystallisation temperature suggesting that the latter is a valid measure of their cold-hardiness. Diapausing adults can supercool sufficiently to avoid freezing in all but the most severe of continental European winters, particularly in semi-sheltered sites. A mean crystallisation temperature of - 21 .O”C was recorded for A. urticae from Leningrad (LozinaLozinskii, 1974) suggesting that no increase in supercooling occurs at the northerly extent of its range. The supercooling ability of the mid-pupal stage, that does not overwinter, would probably be sufficient to ensure survival of a proportion of the population. However, if pupae are overwintered at 5°C adults fail to emerge and they eventually die (personal observation). The level of supercooling in the pupae may either represent a partial metabolic preparation for diapause as an adult, or the inherent ability of the non-diapausing stages to supercool. In comparison with pupae, newly emerged adults show increased supercooling ability without acclimation suggesting an adaptive response, involving either the masking or voiding of ice nucleators, or the accumulation of cryoprotectants. The voiding of meconial fluid during the first 24 h after eclosion seems likely to have eliminated some possible sites of nucleation. Feeding appears to adversely affect the supercooling ability of newly emerged adults. Liquid diets containing sugars do not reduce supercooling in some aphids @‘Doherty and Bale, 1985), but in some Collembola and mites inhibition of double-distilled water alone greatly reduces supercooling suggesting that ice nucleators in the gut may be unmasked (Cannon er al., 1985; Cannon, 1986). Acclimation in

-5:10 224 I4 x.4 191 ?R 5.1 P
-5 170 44 3.5 144 I9 4.2 P < 0.001

2 168 47 3.5 147 17 5.4 P
10 179 I4 4.4 I57 50 3.6 P < 0.001

-5/lO 171 44 3.0 142 20 4.6 P < 0.001

the nymphalid butterflies appears to be a complex process, possibly involving voiding of liquid from the gut, which is evidently not complete after 5 days at 10°C. Metabolic adjustments which increase cold hardiness may be slow, especially at low temperatures when the metabolic rate is reduced. In contrast the cycling regime combining the inducing effect of low temperature exposure and high temperature periods during which metabolism is increased may explain the more rapid decrease in mean crystallisation temperature in the - 5jlO”C regime in both species. The difference in response of the 10°C groups between species is unexplained but may reflect the high mortality in I. io at this temperature. A significant proportion of the overwintering population may be vulnerable to temperatures as high as - 10°C during the first few weeks of diapause and this may partially explain why both species enter diapause well in advance of the normal onset of cold weather in autumn. Presumably the same phenomenon associated with liquid food intake is responsible for the increase in mean crystallisation temperature in postdiapause feeding groups. The level of cold hardiness achieved by both species during diapause is severely reduced if water accumulates on the surface of the insect. From the crystallisation temperature distribution of water inoculated groups it is apparent that freezing is more of a chance process occurring at temperatures as high as -5.l’C whilst some individuals are not frozen at - 15°C. The dense hairs covering the cuticle, particularly on the thorax, may help to isolate water droplets, but its effectiveness is clearly limited in most cases. The semi-sheltered sites sought by overwintering adults may therefore be most valuable for their protection from moisture rather than from cold. If dry sites are found neither species is likely to suffer high levels of mortality from freezing. Mild overwintering temperatures may be deleterious to both species over long periods. Weight loss is rapid and energy reserve depletion is a likely cause of mortality (Pullin, 1987). This suggests that to some insect species abnormally mild winters may be a comparable threat to winters which are colder than average. Although the crystallisation temperature may be an adequate measure of cold-hardiness for short to medium term low temperature exposure this does not appear to be the case for the entire overwintering period. The crystallisation temperature has also been found to be a poor indicator of low temperature tolerance in other insect species. Lee and Denlinger (1985) found that the ability of Surcophaga crassipal-

Effects of low temperature pis pupae

to survive sub-zero temperatures above the crystallisation temperature was dependent on the time they had been in diapause. Both a constant - 5°C and a cycling regime of - 5 and 10°C cause substantial mortality in I. io adults over a short period of time around 100 days suggesting a period in diapause development which is less tolerant to low temperature exposure. This is not apparent in A. urticae and a comparison of the changes in physiological status of the two species is the subject of a later paper. Mortality appears to be related to initial weight of individuals suggesting a nutritional or physiological basis for their susceptibility to low temperature observed in longer term exposures. A. urticae survives in more northerly zones than i. io where long periods of subzero temperatures are common during the winter months, and its greater tolerance of the -5°C regime may be a pointer to the reason for this distribution. Similarly it is possible that such temperatures exclude I. io from these areas. At the other extreme, southern and oceanic areas with mild winters may also be unsuitable.

Acknowledgement-This GR,ID 75441 to JSB.

work was funded

by SERC

Grant

REFERENCES

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Cannon R. J. C., Block W. and Collett G. D. (1985) Loss of supercooling ability in Cryptopygus antarcticus (Collembola: Isotomidae) associated with water uptake. Cryoletts 6, 73-80. Knight J. D., Bale J. S., Franks F., Mathias S. and Baust J. G. (1986) Insect cold hardiness: supercooling points and pre-freeze mortality. Cryoletts 7, 194203. Lee R. E. and Denlinger D. L. (1985) Cold tolerance in diapausing and non-diapausing stages of the flesh fly, Sarcophaga crassipalpis. Physiol. Ent. 10, 309-315. Lozina-Lozinskii L. K. (1974) Studies in Cryobiology. Wiley, New York. Mansingh A. and Smallman B. N. (1972) Variation on polyhydric alcohol in relation to diapause and coldhardiness in the larvae of Isia isabella. J. Insect Physiol. 18, 1565-1571. G’Doherty R. and Bale J. S. (1985) Factors attectmg the cold hardiness of the peach potato aphid M_vzus persirae. A. appi. Biol. 106, 219-228. Pullin A. S. (1986) Effect of photoperiod and temperature on the life-cycle of different populations of the peacock butterfly Inachis io. Ent. exp. appl. 41, 237-242. Pullin A. S. (1987) Adult feeding time, lipid accumulation, and overwintering in Aglais urticae and Inachis io (Lepidoptera: Nymphalidae). J. Zool., Land. 211, 631641. Pullin A. S. (1988) Environmental cues and variable voltinism patterns in Aglais urticae (L.) (Lepidoptera: Nymphalidae). Ent. Gaz. 39, 101-112. Salt R. W. (1953) The influence of food on cold hardiness of insects. Can. Ent. 85, 261-269. Salt R. W. (1956) Influence of moisture content and temperature on the cold hardiness of hibernating insects. Can. J. Zool. 34, 283-294. Salt R. W. (1961) Principles of insect cold-hardiness. A. Rev. Em. 6, 55-74. Turnock W. J., Lamb R. J. and Bodnaryk R. P. (1983) Effects of cold stress during pupal diapause on the survival and development of Mamestra conjigurata (Lepidoptera: Noctuidae). Oecologia 56, 185-192.