Pergamon J. rherm. Biol. Vol. 22, No. I. pp. l-9. 1997

9, 1997 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain

PII: 80306-4565(%)000289 0306-4565197 $17.00 + 0.00

NECROTIC FRUIT: A NOVEL MODEL SYSTEM FOR THERMAL ECOLOGISTS

MARTIN E. FEDER*

Department of Organismal Biology & Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, IL, 60637, U.S.A.

(Receioed 22 July 1996; accepted in recked ,fbrm 28 September 1996)

Abstract-l. Necrotic fruits, which a diversity of small organisms (e.g. larval Drosophila melanogaster) can inhabit, routinely attained temperatures greater than 35°C and as high as 52°C when in direct sunlight for 1-2 h. 2. Fruit color, integument, water loss and size each influenced the kinetics and magnitude of solar heating of fruit. 3. Within-fruit spatial variation in temperature was < 5°C at a given time, and often much less. 4. These findings suggest that organisms inhabiting isolated fruit experience stressful if not lethal temperatures. ((2’ 1997 Published by Elsevier Science Ltd. All rights reserved

Key Word Indes: Color; Drosophila melanogaster; evaporative cooling; fruit; heat-shock protein; heat exchange; microclimate; necrotic fruit; solar heating; stress; temperature

INTRODUCTION

In contrast to the frequency with which ecophysiolo- gists study adaptation to high temperature (Hoffmann and Parsons, 1991), reports of animals encountering intolerably high temperatures in nature are surprisingly rare. Behavior often enables animals to avoid thermal stress or circumvent it when encountered; when behavioral avoidance will not suffice, physiological capacities and tolerances have often evolved to withstand the range of temperatures that a population or species encounters in nature. Accordingly, animal ecophysiologists are usually limited to studying the successful outcome of natural selection for avoidance or tolerance of thermal stress, rather than the process that yields this outcome. In response, some evolutionary physiologists have resorted to studies of natural selection in the laboratory. An alternative research strategy is to identify natural or semi-natural circumstances in which animals undergo thermal stress. The present study reports on a natural model of high temperature stress that may be ripe for exploitation by thermal biologists.

Necrotic (i.e. fallen or rotting) fruit harbors a rich and diverse arthropod fauna, which consumes the

*E-Mail: m-feder@uchicago.edu Tel: 312 702-8096. Fax: 312 702-0037.

I

host fruit and/or inhabits it. While in the fruit, small organisms will experience whatever temperatures the fruit imposes on them. In particular, if a necrotic fruit is exposed to direct sunlight, solar heating can impose an enormous heat load on any indwelling arthropods. Prior work has primarily examined this possibility only indirectly. A large agricultural literature examines “sunscald” in fruits and fleshy vegetables that are still attached to the parent plant (Barber and Sharpe, 1971), and reports temperatures that are similar to if not greater than those in the present study. McKenzie and McKechnie (1979) measured temperatures of larval Drosophila in a pile of grape residues, and reported a mode of 25-30°C and a range of 10-45”C. By releasing and recapturing Drosophila of a strain in which eye color of adults is related to body temperature during pupation, Jones et al. (1987) inferred that pupae experienced temperatures of IS-31°C. Sampsell (1977) observed small numbers of necrotic apples, with results similar to those of the present study. The present work examines the temperatures of necrotic fruit outside of the laboratory. It first characterizes variation in temperatures of necrotic fruit; then seeks to define the contributions of fruit exposure, size, color and the integument to this variation; and finally documents that fruit in nature experiences the same thermal variation as experimental fruit.

2 M. E. Feder

Similar circumstances may ensue for insects in galls or in fruit still attached to the parent plant (Layne, 1991, 1993; Patiiio et al., 1994), in terrestrial vertebrate ectotherms in diurnal retreats under it-isolated cover objects (Huey et al., 1989; Spotila et al., 1989) and presumably in aquatic organisms trapped in small insolated pools or in the intertidal zone (Hofmann and Somero, 1995). As will be seen, however, a particular advantage of necrotic fruit as a study system is its ease of manipulation, both in the laboratory and in the field, and its exploitability in natural and semi-natural experiments.

MATERIALS AND METHODS

General

Over-ripe fruit was obtained from grocers. This was primarily apples (M&s domestica), apricots, bananas (Muss spp.), peaches (pubescent skin) and nectarines (glabrous skin) (Prunus persica), plums (Prunus spp.), and tomatoes (Lycopersicon esculen- turn). This fruit attracted insects. For example, in both the laboratory and the field, Drosophila adults visited this fruit and oviposited on it, and larvae thrived within it when given an opportunity. Fruit and environmental temperatures were measured with copperconstantin thermocouples and either a Cole-Parmer Model 8112-20, Wescor TH-65, or Physitemp BAT- 12 meter. Thermocouple junctions were either made from 2430 gauge wire or a Cole-Parmer needle microphobe (H-08505-95, time constant 1 s). Care was taken not to alter a fruit’s temperature by contact with the investigator’s hands, by shading it, or by otherwise altering its thermal environment. Dates of each experiment are reported; the prevailing weather on each date is a matter of public record (National Oceanic and Atmospheric Administration, 1994a, b). All field sites were in the Central Time Zone of North America, and times are given in Central Daylight Time (CDT).

Experiment I

Fruit was dispersed across an old field-second growth forest ecotone at Sundown Meadows, Countryside, Cook County, IN, USA. Fruit was thrown behind the investigator. Bananas were peeled before dispersal. This process positioned fruit in diverse microhabitats; some were exposed and unshaded throughout the day, some were surrounded by sparse grass, some were shaded by low shrubs during part of the day and some were in deep shade. Fruit was dispersed on 14 June 1994 and left overnight. Beginning at 11:OO CDT on the following day, temperatures were measured repeatedly for 45 different fruits. Typically, fruit temperature was

measured at 1 cm depth for the upper surface exposed to air and the lower surface contacting the substrate, as well as several locations deeper within the fruit. Air temperature in sun and deep shade were also measured. At the conclusion of temperature measurements, the size of each fruit was estimated with a ruler.

Experiment 2 [ejbct of fruit color in peaches]

On 30 June 1994, nine peaches of comparable size (5.0-5.3 cm diameter) were assigned to two groups according to their color: five red peaches and four yellow peaches, Typically, yellow coloration precedes red coloration during ripening, although no differ- ences were evident in the firmness of the flesh. Thermocouple wires were passed beneath the skin of each peach, with the junctions I cm beneath the skin at opposite sides of the peach or next to the pit. Wire was fixed to the peach where it emerged from the skin with hot melt glue. These peaches were left in the laboratory overnight. At approximately 10: 15 CDT on the following day, temperatures were determined and the peaches were then placed on grass lawn on the campus of The University of Chicago. Peaches were oriented so that the skin above one subsurface thermocouple faced directly towards the sun and that the skin above the other rested on the grass substrate. Temperatures of each peach were taken at regular intervals, as was the shaded air temperature, the temperature of adjacent sunlit soil and the tempera- ture of soil in deep shade. Peaches were moved occasionally to sunlit substrate as the sun and shadows moved.

Experiment 3 [ejfect of fruit color in apples]

Beginning on 7 August 1994, Experiment 2 was repeated with 5 red (Rome Beauty variety) and 5 yellow (Golden Delicious variety) apples. Apple mass was 201.0 + 12.8 g (X k SD), and did not differ between colors (Mann-Whitney u-test, p > 0. I).

Experiment 4 [eflects of mass and experimental manipulation qf fruit integument]

Beginning 18 August 1994, Experiment 3 was repeated in part with apples in a 2 x 2 design. Red apples (Rome Beauty variety) of equivalent color and ripeness were sorted into 2 groups each of 4 large apples [257.7 + 10.0 g (2 + SD)] and 4 small apples [41.5 + 7.5 g (X + SD)]. Among-group variation in mass was significant for neither large nor small apples (p > 0.7 in each case; Kruskal-Wallis test). A large mass group and a small mass group were randomly assigned to each of two treatments, control and partially peeled, in which approximately 40% of the

Necrotic fruit temperatures 3

skin was removed in a contiguous patch. All groups were thereafter treated as were the peaches in Experiment 2; i.e. they were instrumented with thermocouples and placed on sunlit soil the following day while their temperatures were recorded.

E.uperiment 5 [day-to-day variation in fruit tempera- tures]

Between 30 June and 15 July 1994, a variety of fruits were outfitted with thermocouples and placed in the sun on the subsequent day as in Experiment 2. Temperatures were recorded throughout the day.

E.yprriment 6 [microsputial variation in fruit tempera- tures]

Two red peaches and two red plums of approximately equal size (5 cm diameter) were chosen. A Z-cm diameter patch of skin was carefully cut away from the side of 1 peach and 1 plum, with care taken to disturb the underlying flesh as little as possible. Fruits were then placed within 1 m of one another on a homogenous patch of sunlit grass lawn on the campus of The University of Chicago, with the center of the peeled patch (or its equivalent position on the unpeeled fruit) oriented towards the sun. The center of the patch was used to define a grid of 13 points spaced at intervals of 1 cm circumference in both directions horizontally and vertically. Each point was then sampled as rapidly as possible with the Cole Parmer needle microprobe inserted I mm, 1 cm, and 2 cm into the fruit.

Temperatures were measured in Garwood Or- chards, La Porte, IN, U.S.A., 41’ 36.4’N, 86” 43.2’ W. In the orchard, fruit trees are ca. 3 m apart in north-south rows. which are 6-7 m apart. The understory is grass and annual weeds, IO-30 cm in height Temperatures were measured of fruit that was on the ground. either underneath trees or between the rows. Fruit location before measurement was determined by natural abscission, the activity of orchard workers, or by animal activity, but not by the investigators. Fruit was chosen for measurement to reflect the full range of potential thermal variation, and included fruit that had been in deep shade for some time. fruit newly exposed to sun or shade, and fruit that had been in direct sunlight for some time.

RESULTS

Experiment 1 characterized variation in fruit temperatures on a given day at a given site. Temperatures of fruits that were in sites exposed to sunlight usually exceeded 35’C and were almost

always warmer than shaded air temperature (Fig. I). Fruits on the floor of the second-growth forest, by contrast, were typically cooler than air temperature and did not exceed 30°C (see B and D in Fig. IA, and D and F in Fig. 1B). In some cases, changes in shading due to the sun’s apparent movement affected these patterns. For example, fruit B in Fig. 1B was shaded until late in the observation period. and fruit A in Fig. IC became shaded in the midpoint of the

I

*&_........*...._..............--.--------- -m B B B

9

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B. Peach

s 0 D D

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C. Nectarine (A-D) and Plum (E)

D. Banana

BO li0 li0 240 Time (minutes after 11:00 local time)

Fig. I. Temperatures within necrotic fruit (Experiment I). Fruit was randomly distributed on 14 June 1994. and temperatures were measured on the following day. Brackets indicate th: minimum and maximum temperature within each fruit at a given time; records for a given fruit are connected at the midpoint of these brackets and are designated by a common letter. The broken line and solid circles indicate shaded air temperature. Time of day is given

in minutes after I I:00 CDT.

4 M. E. Feder

Local time (minutes after midnight)

Fig. 2. Effects of fruit color on temperatures of peaches. Solid lines indicate records for 5 red peaches; broken lines indicate records for 4 yellow peaches. Time of day is given

in minutes after midnight CDT.

observation period. Fruits also differed by species. The highest temperature observed (52°C) and the bulk of the records above 45°C were in tomatoes (Fig. lA), whereas bananas seldom exceed 40°C (Fig. 1D).

The foregoing observations began in late morning, by which time fruit had already equilibrated with its surroundings. Experiments 2-5 (Figs. 224) provided information on the rate of equilibration by examining fruit acutely transferred from the laboratory to a

sunlit substrate outside of the laboratory. In each experiment, natural air temperature increased during the day and fluctuated with wind and the passage of clouds. Temperatures of insolated fruit increased similarly but usually exceeded air temperature, sometimes by as much as 14°C. Temperatures of insolated fruit typically increased rapidly for the first l-2 h after transfer from the laboratory (see also Feder ef al., 1997a), and thereafter varied little unless the weather changed significantly. In Experiment 5, however, fruit never achieved temperatures resem- bling a steady-state (Fig. 3B).

External color markedly affected temperatures of insolated fruit (Figs. 2 and 3A). With a single exception (that of a red peach depicted in Fig. 2) red fruit heated more rapidly when sunlit and attained warmer temperatures. The magnitude of this difference is striking; temperatures of most red peaches exceeded those of yellow peaches by as much as 10°C.

The mass of insolated unpeeled apples likewise affected their temperature (Experiment 4; Fig. 3B). In this experiment, the standard error of the mean temperature for a single group at a given time point averaged 0.4”C (standard deviation = 0.2”C) and was never greater than l.O”C. For apples with intact integuments, the average temperatures of the large and small groups diverged by as much as 2.8”C while heating and by more than 3.5”C while cooling at the end of the observation period. Partial removal of the peel, which is likely to have altered both absorption of solar radiation and evaporative

20 600 660 720 780 840 540 600 660 720 780 Local time (minutes after midnight) Local time (minutes after midnight)

Fig. 3. Effects of color, size and experimental manipulations of temperatures of apples. A, Effects of fruit color on temperatures. Solid lines indicate records for 5 red apples; broken lines indicate records for 5 yellow apples. Shaded air temperature is also indicated. B, Environmental temperatures and mean temperatures for the control and partially peeled apples of different sizes. Time of day is given in minutes

after midnight CDT.

Necrotic fruit temperatures

570 630 690 750 810 870 930

A Apricot 54.53 6/30/945 g B Peach 132.3 6130194 g C Peach 283.5 g 6/30/O/94 D Peach 121.87 g l/EM E Peach 266.1 g 7lY94 F Apricot 74.67 715194 g G Nectarine 112.45 g 715194 H Peach 187.0 g 7/S/94 I Mango 276.3 71594 g J Apricot 71.89 g 716194 K Nectarine 110.32 7/6/94 g L Peach 173.7 716194 g M Mango 273.1 716194 g N Apricot 69.30 g 717194 0 Nectarine 108.49 g 7/7/94 p Peach 162.8 g 717194 Q Mango 270.3 g 7/794 R Apricot 65.04 g 718194 s Nectarine 106.77 g 718194

990 T Peach 149.1 g 7/8/94

Local time (minutes after midnight) u Mango 267.fg 718194 v Peach 179.8 g 7/H/94

Fig. 4. Daily variation in fruit temperatures. Each letter refers to a temperature record for a given fruit during one day. The figure represents data for 22 different fruits, each measured on only one of 7 days of measurement from 30 June to 11 July 1994, and is intended to illustrate the enormous variation among

fruits and days. Time of day is given in minutes after midnight CDT.

cooling, dramatically decreased temperatures of apples. Fruit mass affected the temperatures of peeled apples less than the temperatures of unpeeled apples.

Experiment 5 was intended to document variation both among days and among different fruits. Figure 4. taken as a whole, demonstrates that this variation results in a great diversity of fruit temperatures. At noon, for example, fruits were as cool as 28°C or as warm as 48°C. Moreover, patterns of thermal variation themselves vary enormously. Some fruits remain cool throughout the day (e.g. V), some remain warm (e.g. G), some initially achieve high tempera- tures and then fall (e.g. K), some heat gradually, decline and then rapidly peak (e.g. D), and so on.

A waxy or shiny outer surface, as in tomatoes (Fig. 1) and in peaches sprayed with a waterproof coating (Feder et al., 1997a) increased temperatures of insolated fruit, and treatments that enhanced water loss (Fig. 3; Feder ef al., 1997a) decreased tempera- tures of insolated fruit. Experiment 6 was intended to examine whether such effects manifest themselves on a microspatial scale, and whether larvae in fruit might have an opportunity to mitigate thermal stress by seeking out cool microhabitats. As in previous experiments, plums (with a waxy, smooth outer surface) attained warmer temperatures than peaches (with a pubescent, comparatively rough surface) at comparable positions and depths (compare Al with Bl, A2 with B2 and A3 with B3 in Fig. 5). Also, removal of a 2 cm diameter circle of skin resulted in a decrease in temperature at comparable positions and depths in both peaches (compare Al with A2 and

Bl with B2 in Fig. 5). Removal of skin did not. however, create an adjacent zone of cooler tempera- tures in the exposed flesh. Variation within a fruit among the various points sampled (13 localities x 3 depths) was typically 3-5°C (Fig. 5).

The foregoing results are for fruit that was positioned on the substrate, either randomly or intentionally, by the investigators. To ascertain whether the results reflect an unintentional bias by the investigators or are representative of necrotic fruit in a typical orchard, temperatures were taken of fruits whose position before measurement was not deter- mined by the investigators. The diversity of temperatures in such fruit (Fig. 6) is entirely consistent with the patterns of thermal variation in intentionally positioned fruit.

DISCUSSION

Whether a given thermal regime is stressful to an organism depends upon that organism’s thermal tolerance, which can vary greatly among species. The fruit fly Drosophila melanogastrr exemplifies the problems that a relatively intolerant species may face in necrotic fruit. Populations of D. melanogaster cannot persist with continuous exposure to tempera- tures above 30°C (Parsons, 1978) and temperatures between 30 and 35°C are inimical to diverse aspects of growth and reproduction (David et a/., 1983; Ashburner, 1989). Temperatures as low as 38’C are acutely lethal to larvae, with acute tolerance inversely related to the duration and magnitude of heat stress

M. E. Feder

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ature in peaches (A) and plums (B). A point chosen on the side . of each fruit defined the origin of a vertical and horizontal axis, each 6cm long along the outer

circumference of the fruit. For each fruit, temperatures were sampled at 1 cm intervals along each axis (represented in the figure by crosshatched circles) at depths of I mm (left column), I cm (center column), and 2 cm (right column). Numbers are the corresponding temperatures (in “C) sampled at each position and depth. Rows Al and Bl refer to a peach and plum with intact skin. Remaining rows are for a peach and a plum in which a 2 cm diameter circular patch of skin was removed; the location of the patch is indicated by a shaded circle. Rows Al, A2, Bl and B2 are for measurements between 14:24 and 14:39 CDT; remaining rows are for measurements between 15:14 and 15:24 CDT. Row Al is repeated from

Feder et al. (1997a).

Necrotic fruit temperatures I

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Fig. 6. Temperature variation in necrotic fruit whose position was not determined by the investigators. Tempera- tures were taken at various positions within fruit at the times of day indicated on 5 separate days in 1994 (apples only) and on 27 .luly 1995 (peaches only). Time of day is given in

minutes after midnight CDT.

(Feder ct (II.. 1997a; Krebs and Feder, 1997). Whole larvae express heat-shock proteins. a response to unfolding of native proteins (Feder et ~1.. 1995), after exposure to 34 C and warmer temperatures (Feder rt ui., 1996, 1997a; Krebs and Feder, 1997). Thus, the temperatures reported for necrotic fruit in the present study can pose serious problems for any indwelling Drosophila if not kill them outright. Although Drosophih is not an especially thermotolerant organism. the highest temperatures reported here for necrotic fruit can kill or harm many eukaryotes. Accordingly. necrotic fruit ought to be a valuable model for investigations of organismal responses to ongoing thermal stress in nature.

The present study and Feder rt a/. (1997a) elucidate some of the factors that determine both the magnitude and kinetics of thermal variation in necrotic fruit. All of these factors have had their influence characterized from first principles and have been thoroughly investigated in biological systems (Smart and Sinclair, 1976: Gates, 1980). Their implications for the thermal biology of organisms dwelling within necrotic fruit have not, however, been recognized and thus deserve emphasis: the most significant factor affecting fruit temperature is insolation, which may elevate fruit temperatures to > 20°C above air temperature (e.g. Fig. I). Fruit in deep shade, by contrast. may remain at relatively cool temperatures even on warm days. Fruit color and mass can modify the temperature rise due to insolation. The influence of color is presumably due to the increased absorption of the corresponding wavelengths of solar radiation. The influence of mass, specifically the higher temperatures attained by larger fruit, presumably reflects their larger boundary layer and lessened convective heat loss (Patiiio et al., 1994).

Evaporative cooling can be particularly effective in damping changes in temperature that would other- wise ensue, as natural and experimental (Patifio ef al., 1994; Feder et al., 1997a) blockade of evaporation increased temperatures and both natural and experimental increases in evaporation decreased temperatures. Patiiio er ul. (1994) have shown evaporative cooling to be particularly important to wasps within figs; without it, figs overheat and kill the wasps within. These factors in combination must present a temporal and spatial mosaic of tempera- tures to ovipositing female insects and the larvae the) produce. Importantly. on cool days or at times when fruit has been in shade or darkness for long periods, the thermal cues that might alert ovipositing females to likely thermal stress will be absent.

The present study examines naturally variable fruit exposed, for the most part, to natural diel variation in heat flux and in weather. Accordingly. the findings represent exactly only those specific fruits and dates on which the study was conducted. konetheless. several points emerge: (I) although fruit was not observed on days forecast to be overcast or atypically cool, the weather was typical of summer weather in a continental temperate climate. The data rcvcal many instances in which chance events. such a~ passage of clouds or gusts of wind. clear11 influenced fruit temperatures. Such events are ;I normal component of natural thermal regimes. Data gathered in this manner are as instructive about natural thermal variation as insights gleaned from biophysical models under standard and repeatable conditions (Smart and Sinclair, 1976; Gates, 1980). (2) Although the data are variable, they are both internally and externally consistent. For diverse fruits and days, temperatures routinely achieved levels that are stressful for Drosophila larvae. This outcome ensued in fruits that were randomly (Fig. I) or deliberately (Figs. 2-5; see also Feder t’r ul. 19973) placed by the investigators and in fruits i/7 .\itu in an orchard (Fig. 6). Moreover, parallel variation in so many different fruits on so many different days suggests that the pattern of thermal variation is generally representative of small (i.e. < 250 g) fruit rather than of any specific variety. These findings also resemble those of related work on insolated fruit (Barber and Sharpe, 1971; Sampsell, 1977). (3) The experimental methodology is potentially exploitable as a means to investigate natural thermal variation and its consequences for organisms dwelling in fruit. Near-lethal or sub-lethal temperatures with natural magnitudes and kinetics can readily be imposed on fruit experimental@ infested with a study organism of choice, and the study organism readily extracted for analysis thereafter. Whereas most work on such

8 M. E. Feder

phenomena requires travel to exotic locales or then-unpublished work (Patino er al., 1994) and entree to

extreme climates (e.g. Wehner ef al., 1992), insects the literature on fruit temperatures. Research was supported

and other fruit-inhabiting organisms can be exper- by a Sabbatical Supplement Award in Molecular Studies of

imentally exposed to natural stress in one’s own back Evolution from the Alfred P. Sloan Foundation, National Science Foundation grants IBN94-082 I6 and BIR94- 19545

yard in fruit discarded by a grocer, and the host and the Louis Block Fund of the University of Chicago. fruit’s environment manipulated at will.

Although these data establish that necrotic fruit

can become hot, they do not show that insects or other animals actually occupy such fruit. Several behavioral and physiological mechanisms of in- dwelling organisms might conceivably avert thermal stress of the necrotic fruit host. Mobile organisms might seek out cool regions in otherwise warm fruit. Drosophila larvae, for example, are clearly capable of behavioral thermoregulation in the laboratory (McKenzie and McKechnie, 1979). In the present study, however, necrotic fruits often lacked sufficient thermal diversity to allow significant behavioral thermoregulation; in other cases temperatures within a necrotic fruit, while diverse, were all stressful. Another potentially beneficial behavior would be for ovipositing females to avoid warm fruits. Adult female Drosophila, for example, have the capacity for such avoidance (Fogleman, 1979; Schnebel and Grossfield, 1986). Because the bulk of egg-laying is crepuscular or nocturnal in this species (David et ul., 1983), however, the thermal cues enabling ovipositing females to deploy this capacity effectively may be absent. Drosophila do not avoid ovipositing on fruit that has been heated in the past but is cool at the time of oviposition (Feder et al., 1997b). Conceivably, large numbers of larvae in a fruit could disrupt the fruit’s integument and thereby enhance evaporative cooling; this possibility has not been investigated. The most direct evidence that Drosophilu larvae are unable to avoid thermal stress imposed by fruit, however, is body temperature measurements for larvae in situ in the field, which are consistent with the fruit temperatures in the present study (Feder, 1996; Feder et al., 1997a). Such temperatures are warm enough to cause death and to induce expression of heat-shock proteins in the field (Feder. 1996; Feder et al., 1997a). Although species other than Drosophila have yet to be characterized similarly, presumably the fauna of necrotic fruit is generally at risk of grave thermal stress and has evolved diverse responses, which await future study.

Acknowledgemenrs-Julie Alipaz, Nathaniel Blair, Jerry Coyne, Jonathan Feder, Hunter Figueras, Raymond Huey, Robert Krebs, Warren Porter, and Bonnie and Bruce Sampsell contributed advice, useful discussion, technical assistance and/or equipment. The proprietors of Garwood Orchard, LaPorte, IN, kindly allowed fieldwork on their property, and Allen Herre provided access to his

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Barber H. N. and Sharpe P. J. H. (1971) Genetics and physiology of sunscald of fruit. Agr. Meteoral, 8, 175-191.

David J. R., Allemand R., Van Herrewege J. and Cohet Y. (1983) Ecophysiology: abiotic factors. In The Genetics and Biology qf Drosophila 3d (Edited by M. Ashburner, H. L. Carson & J. N. Thompson), pp. 105-170. Academic Press, Inc., London.

Feder M. E. (1996) Ecological and evolutionary physiology of stress proteins and the stress response: the Drosaphih melanogaster model. in Animals and Temperature: Phenotypic and Evolutionary Adaptation (Edited by 1. A. Johnston & A. F. Bennett), pp. 79-102. Cambridge University Press, Cambridge.

Feder M. E.. Blair N. and Figueras H. (1997a) Natural thermal stress and heat-shock protein expression in Drosophila larvae and pupae. Func. Ecol., 11, in press.

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Feder M. E., Cartaiio N. V., Miles L., Krebs R. A. and Lindquist S. L. (1996) Effect of engineering Hsp70 copy number on Hsp70 expression and tolerance of ecologi- cally relevant heat shock in larvae and pupae of Drosophih melanogaster. J. Exp. Biol. 199, 1837-1844.

Feder M. E., Parse11 D. A. and Lindquist S. L. (1995) The stress response and stress proteins. In Cell Biokagy of Trauma (Edited by J. J. Lemasters & C. Oliver), pp. 177-191. CRC Press, Boca Raton, FL.

Fogleman J. (1979) Oviposition site preference for temperature in D. melanogaster. Behav. Gen. 9, 407412.

Gates D. M. (1980) Biophysical Ecology. Springer-Verlag, New York.

Hoffmann A. A. and Parsons P. A. (1991) Evnlutionary Genetics and Environmental Stress. Oxford University Press, Oxford.

Hofmann G. E. and Somero G. N. (1995) Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus rrossulus. .I. Erp. Biol. 198, 1509-1518.

Huey R. B., Peterson C. R.. Arnold S. J. and Porter W. P. (1989) Hot rocks and not-so-hot rocks: retreat-site selection by garter snakes and its thermal consequences. Ecology IO, 93 l-944.

Jones J. 8, Coyne J. A. and Partridge L. (1987) Estimation of the thermal niche of Drosophila melanogoster using a temperature-sensitive mutation. Am. Nat. 130, 83-90.

Krebs R. A. and Feder M. E. (1997) Natural variation in the expression of the heat-shock protein Hsp70 in a population of Drosophila melanogaster, and its corre- lation with tolerance of ecologically relevant thermal stress. Evolution. 51, 1733179.

Necrotic fruit temperatures 9

Layne J. R. (1991) Microclimate variability and the eurythermic natural of goldenrod gall fly (Eurosra solidaginis) larvae (Diptera: Tephritidae). Can. J. Zoo/. 69, 614617.

Layne J. R. (1993) Winter microclimate of goldenrod spherical galls and its effect on the gall inhabitant Eurosta .solidaginis (Diptera: Tephritidae). J. Therm. Eiol. 18, 125- 130.

McKenzie J. A. and McKechnie S. W. (1979) A comparative study of resource utilization in natural populations of Drosophila melanogaster and D. simulans. Oecologia 40, 299-309.

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