Directory UMM :Data Elmu:jurnal:P:Postharvest Biology and Technology:Vol21.Issue1.2000:

(1)

Physiological responses of insects to heat

Lisa G. Neven *

USDA-ARS Yakima Agricultural Research Laboratory,5230Konnowac Pass Road,Wapato,WA98951,USA

Received 19 April 2000; accepted 21 August 2000

Abstract

Postharvest quarantine treatments using high temperatures have been developed for various commodities. There are a wide range of insect pests that are the target of these treatments. In order to make heat treatments effective against these pests, the effects of high temperatures on insect physiology must be understood. Insects, being poikilothermic, are particularly sensitive to heat. Nearly every system studied in insects has demonstrated sensitivity to heat. Studies on the effects of heat in insect metabolism demonstrate some adaptability to thermally challenging environments. Respiration, as to be expected, is also effected by heat, and as the body temperature of the insect increases, there are concomitant increases in both metabolism and respiration up to a critical thermal limit. The effects of heat on the nervous and endocrine systems is another area where elevated temperatures wreck havoc. Changes in behavior and development have been documented as resulting from heat treatments. Among the most studied responses of insects to heat is the elicitation of heat shock proteins. The impact of these proteins on thermotolerance are still being investigated. Models of thermal damage leading to the death of the insect are discussed as well as current studies in describing the events of thermal death. Published by Elsevier Science B.V.

Keywords:Heat; Insects; Respiration; Heat shock; Metabolism

www.elsevier.com/locate/postharvbio

1. Introduction

Insects are known to live in a wide range of thermal climates, but there is very little variability in the maximum temperature (40 – 50°C) which they can survive (Heinrich, 1981). Postharvest heat treatments to disinfest fresh and stored prod-ucts have been used for more than 60 years (Jones, 1940). The successful application of these treatments relies on a delicate balance between commodity tolerance and insect intolerance.

Un-derstanding how high temperatures affect insect mortality can lead to the formulation of more effective treatments.

Many experiments have been done in which insects have been transferred from one tempera-ture to another and observations made on the effects on metabolism. Clarke (1967) classified these changes in temperature in engineering terms as ‘step function’ and ‘ramp function’. Step func-tion refers to a change from one temperature to another as rapidly as possible. An example would be water bath studies in which insects are im-mersed directly into heated water (or other aqueous medium) (Sharp and Chew, 1987; Jang,

* Tel.: +1-509-4546556; fax:+1-509-4545646.

E-mail address:neven@yarl.ars.usda.gov (L.G. Neven).

0925-5214/00/$ - see front matter Published by Elsevier Science B.V. PII: S 0 9 2 5 - 5 2 1 4 ( 0 0 ) 0 0 1 6 9 - 1

(2)

Ramp-function is when a slower rate of change in temperature occurs (Fig. 1). An example would be in-fruit heat treatments or controlled water bath treatments (Shellie et al., 1997; Neven, 1998a,b).

Ramp-function heat treatments can reveal,

through examination of the response curve, what mechanisms may be involved in thermal tolerance and indicate whether the tolerance limits of the insect are wider in response to a ramp- than to a step-function (Clarke, 1967). Where applicable, these terms are used below to describe the effects of various heat treatments on insect physiology. In this review, the effects of heat on insect metabolism, respiration, nervous and endocrine systems, and the role of heat shock proteins (hsps) in thermal tolerance are discussed.

2. Metabolism

Due to the exothermic nature of insects, metabolic rate is extremely dependent upon envi-ronmental temperature. Optimal growth and de-velopment of insects falls within a fairly broad range of temperatures. For example, for codling moth the range is between 10 and 30°C (Rock and Shaffer, 1983). Acute changes in temperature, as experienced in postharvest quarantine treatments,

nonfeeding larvae of Cochliomyia macellaria, which results in an increase in polyols and polyphosphates (Meyer, 1978).

One of the most important effects of ture change is on enzymes. Changes in tempera-ture may affect the binding of a substrate to the enzyme, causing a shift in the Michaelis constant

Km, thereby effecting immediate metabolic

com-pensation (Hochachka and Sommero, 1984). Ele-vated temperatures may also influence the rate of enzymatically catalyzed reactions by determining the proportion of molecules in a given population that possess sufficient energy (energy of activa-tion,Ea; enthalpy of activation, DH) to react and

form an activated complex (Hoffman, 1984). The temperature dependence of the barrier free energy of activation is proposed as a possible molecular basis for spontaneous temperature compensation (Hoffman, 1984). Changes in the fluidity of the phospholipid bilayer of membranes may also af-fect the activity of membrane-bound enzymes (Hochachka and Sommero, 1984). Temperature may also influence the interaction between en-zymes and modulating metabolites (Fig. 2), and affect the conformation of an enzyme, which may in turn alter the kinetic properties of the enzyme (Fig. 3). This effect is probably the most critical in situations where temperatures reach above 40°C.

3. Respiration

The overall metabolic rate of an insect is often measured as oxygen consumption or carbon diox-ide evolution. To assess the effects of temperature on insect metabolism a relationship between body weight and respiration over a range of tempera-tures can be calculated by the van’t Hoff equation.

Q10=

10/(T₂−T₁)

whereR2is the rate at any temperatureT2(in °C)

and R1 is the rate at any lower temperature T1.

The formula indicates that the Q10 is temperature

dependent. An example of the difference in

respi-Fig. 1. Comparison of two ramp-function heat treatments of apples using two different rates of heating. The graph shows core (C) and surface (S) temperatures of size 100 ‘Delicious’ apples subjected to heating rates of 100 and 12°C/h (Neven, unpublished).

(3)

Fig. 2. Influence of experimental temperature (ET) on the apparent Km of phosphoenolpyruvate (PEP) of pyruvate kinase (PK) from muscle and fat body of three cricket species:Gryllus campestris()Gryllus bimaculatus(), andAcheta domesticus

(). AT acclimation temperatures (After Hoffman and Marstatt, 1977). Reprinted from Journal of Thermal Biology, Vol. 2, Hoffman and Marstatt, pp. 203 – 207, 1977, with permission from Elsevier Science.

Fig. 3. Arrhenius plots of PK activity from muscle and fat body of Acheta domesticus after periods at various acclimation temperatures (After Hoffman and Marstatt, 1977). Reprinted from Journal of Thermal Biology, Vol. 2, Hoffman and Marstatt, pp. 203 – 207, 1977, with permission from Elsevier Science.

point, respiration decreases (Fig. 5). It is unclear whether this is an active suppression of respiration, supposedly to conserve stored energy and to buy time in case the temperature lowers and normal metabolism can resume, or if it is an effect of compromised metabolism due to the effects of heat on the respiratory system. Once the insect reaches peak respiration, it is able to recover (Table 2) (Neven, 1998b). However, death occurs soon after respiration rates begin to drop, even if the insect is returned to normal thermal conditions, indicating that systemic cell death is occurring.

An adequate supply of oxygen is essential for insects to survive heat stress. Research with light brown apple moth,Epiphyas posti6ittana(Whitting et al., 1991), red flour beetle, Tribolium casta -ration rates of insects at constant temperatures and

insects under acute heat treatments is given in Neven (1998b). Fifth instar codling moths under constant temperatures over the normal growing range (10 – 30°C) have a Q10 of 1.49 (Table 1).

Codling moths subjected to acute heat treatments had a maximal carbon dioxide evolution rate of 4.88ml/mg/min at 45°C. This correlated to aQ10of

2.4 when calculated from the respiration rate at 30°C (Fig. 4).

Respiration increases in response to increasing temperature up to a critical upper limit. After this

Table 1

Respiration rates of fifth instar codling moth at constant temperatures. (From Neven, 1998b)

Temperature Respiration rate

mlCO2/mg/min

10°C 0.581

0.926 15°C

20°C 1.098

25°C 1.146

1.296 30°C

(4)

Fig. 4. Regression and actual data of respiration rates of 5th-instar codling moth at constant temperatures. Line indi-cates the regression (y=0.3485+0.033054x; R2₌_0.91) (From Neven, 1998b).

tion, a lack of ATP would render these proteins relatively inactive. Other effects of an anoxic envi-ronment may be observed in the inability to me-tabolize lipids and carbohydrates to maintain elevated metabolism, inability to synthesize proteins, and inability to alter fatty acids in the

phospholipid bilayer to maintain membrane

integrity.

4. Nervous system

Changes in temperature affect the central ner-vous system by differential effects on various nerves. Studies on Periplaneta americana group these nerve chord responses into four classes: (1) those that increase firing frequency with an in-crease in temperature; (2) those that show tran-sient changes in firing pattern; (3) those that are responsive over a limited range of temperatures; and (4) those unaffected by temperature changes (Kerkut and Taylor, 1957).

The nervous system is important in the regula-tion of the insect’s response to high temperature by: (1) involvement of the perception of environ-mental temperature; (2) integration of this infor-mation with other sources (i.e. motor neurons, sensillia); (3) adjusting the patterns of insect be-havior which will influence activity of the en-docrine glands and patterns of muscular activity of the body (Clarke, 1967). The perception of environmental temperatures by the nervous sys-tem is accomplished primarily through the sensil-lae, but may also occur through the antennae, arolia and pulivilli of the legs, and some chemosensors are sensitive to thermal changes (Clarke, 1967).

5. Endocrine system

The role of the endocrine system in response to thermal change is not clear. Obviously, changes in nervous function will ultimately affect the activity of the endocrine system. Many of the changes in insect development and reproduction may result

neum (Sondersom et al., 1992), Mediterranean fruit fly, Ceratitis capaitata (Moss and Jang, 1991), the flesh fly Sarcophaga crassipalpis

(Yocum and Denlinger, 1994), and codling moth,

Cydia pomonella (Neven and Mitcham, 1996) has demonstrated that anoxic conditions make insects more sensitive to high temperature (Fig. 6). Oxy-gen deprivation appears to interfere with the physiological mechanisms involved in insect ther-mal tolerance. An interesting effect of anoxia on insects during a heat stress may be exemplified by the actions of heat shock proteins (hsps).HSP70

requires ATP in order to fold and release nascent

Fig. 5. Respiratory response of 5th-instar codling moth to a simulated heat treatment profile. (From Neven, 1998b). PR-12 (- -) is respiration expressed as ml CO2/mg/min (y-axis), temperature profile ( ) (2ndy-axis).

(5)

Table 2

Respiration rates and peak respiration times of fifth instar codling moths during simulated fruit heat treatments (From Neven, 1998b)b

Peak rate Peak timea

Treatment type Recovery times Time to 42°C (min) LT95(min)

Min9se mlCO2/min/mg

3/4 Peak Full peak 3.2590.23 51.794.6 32.792.9 97

MFA 44°C 126.395.0 292.0

3.6690.17 29.592.6 32.292.9

124.093.0 68

MFA 46°C 190.4

106.492.5

MFA 46°C 4.0590.25 22.291.9 25.292.2 66 145.4

4.3190.23 28.7910.8 29.692.6

MFA 46°C 121.293.7 73 170.7

4.1190.15 31.392.7 40.593.6

109.392.2 56

MFA 48°C 120.7

116.592.8

MFA 48°C 4.3890.18 30.792.7 42.793.8 75 162.7

VFA 44°C 105.792.6 4.2890.17 24.392.1 38.293.4 58 153.8 4.8890.17 33.292.9 33.893.0

89.493.2 42

VFA 46°C 88.1

90.193.5

VFA 48°C 4.4390.13 39.093.4 39.593.5 38 74.9

VFA 48°C 85.594.8 4.3590.20 39.793.5 33.592.9 32 66.9 a_{Time required to reach maximum respiration.}

b_{MFA: Moist Forced Air treatment, non-condensing environment. VFA: Vapor Forced Air treatment, condensing environment.}

from changes in the endocrine system. For exam-ple, when codling moth were reared at elevated temperatures, a decline in fertility was observed as the duration of exposure to the elevated tempera-ture was increased (Proverbs and Newton, 1962; White, 1981). Another example comes from re-search with codling moth and the effects of prestorage heat treatments on larval mortality (Neven and Rehfield, 1995). A ramping function heat treatment at 4°C per hour to a final tempera-ture of 38°C and held there for 4 days did not kill fifth instar codling moth. However, a supernumer-ary molt to a 6th instar was observed. With the first example, the elevated temperature could have affected the endocrine system and prevented mat-uration of germ cells and perhaps inhibited the deposition of vitellin in the eggs. In the case of the supernumerary molt, it is most likely that the levels of juvenile hormone were too high to permit the formation of pupae and continuation of devel-opment. There are numerous examples in the literature on abnormalities, defects, and delays in development in response to thermal stress (see Denlinger and Yocum, 1998). Many of these may be attributed to the effects on the endocrine sys-tem, whereas others may be an effect of high

temperatures on DNA. Additional effects of heat on the endocrine systems of insects include: (1) decrease in PTTH levels due to a decrease in the size of the procerebral A1 neurosecretory cells (Jankovi-Hladni et al., 1983); (2) changes in JH titer (Rauschenbach, 1991); (3) effects on JH es-terase (Rauschenbach, 1991); (4) changes in ecdysone titer (Rauschenbach, 1991); and (5) changes in AKH and other hormones (Ivanovi, 1991).

Fig. 6. Mortality of 5th-instar codling moth in infested cherries following heat treatments with and without controlled atmo-spheres. Values represent mean corrected mortality9SE (From Neven and Mitcham, 1996).

(6)

The elicitation of specific groups of proteins in response to an acute heat treatment in insects has received considerable attention in recent years (Yocum and Denlinger, 1992; Benedict et al., 1993; Gallie, 1993; Parsell et al., 1994; Marin et al., 1994; Vuister et al., 1994; Coleman et al., 1995; Feder et al., 1997). Most of the research has centered on Drosophila (Solomon et al., 1991; Vuister et al., 1994; Feder et al., 1997), but more and more information on heat shock proteins (hsps) in other insects is becoming available. HSPs are characterized in relation to their func-tion and molecular weight (Pardue, 1988). Most of the literature on insect heat shock response centers on the intermediate group of stress proteins in the 60 to 80 kDa range.

Sarcophaga crassipalpis, conferred thermal toler-ance to a subsequent normally lethal heat treat-ment of 90 min at 45°C. This thermal tolerance decayed over time, but lasted 72 h, beyond the time over which the originally induced HSP70s were degraded (24 h). This was an excellent exam-ple of how pre-conditioning of an insect can confer thermal tolerance to a subsequent higher thermal treatment, but this effect is not necessar-ily being related to hsps; other factors may partic-ipate in thermal tolerance.

Fig. 7. Graphical depiction of the effects of cumulative ther-mal damage on whole multicellular organisms.

7. Models of thermal damage

What actually causes an organism to ultimately die in response to a heat treatment? Denlinger and Yocum (1998) discuss two current theories, one by Roti Roti (1982) in which it is suggested that the effects of heat on macromolecules are the culprit, and the other by Bowler (1987) which points to damage of the cell membrane as the critical event. However, these views negate the effects of heat on the whole organism. Denlinger and Yocum (1998) point out the hierarchy of thermal resistance: macromolecules\cells\ tis-sues\whole organism (Fig. 7). The progression explains a phenomenon we often see in assessing heat treatments on insects, that of delayed mortal-ity. The insect may seem ‘alive’ following a heat stress, but may fail to complete development un-der normal growing conditions.

There has been relatively little research done on the effects of heat stress on insect cells. Inferences on the cellular effects can be drawn from other systems. As temperature increases the pH and ion concentrations are altered, and there are also dramatic effects on macromolecules such as proteins, DNA, RNA, lipids, and carbohydrates, and on cellular structures such as cell and nuclear membranes, mitochondria and ribosomes. In gen-eral, as temperature increases, pH decreases. The ratio of free hydrogen ions is increased as temper-ature increases at a ratio of 0.015 – 0.2 pH units per °C. This change in pH affects the function of

(7)

(Hochachka and Sommero, 1984). Heat can also alter the structure of proteins and nucleic acids by destabilizing weak interactions such as van der Waals, ionic and hydrogen bonds. The Tm of many nucleic acids is around 50 – 52°C. Ribo-somal RNA, being shorter in length, is more susceptible to changes in temperature than longer polymers. DNA is particularly vulnerable to heat damage. Lesions in DNA can occur at temperatures above 42°C in Chinese hamster cells (Warter and Brizgys, 1987). These lesions are easily repaired once the cells are returned to normal temperatures. However, if temperatures remain elevated, DNA repair enzymes might not be able or available to repair the damage.

Membranes of the cell, mitochondria, micro-somes and nuclei are also vulnerable to thermal damage due to the effects on the phospholipid bilayer and other lipid components. Alteration of the liquid-crystaline fluidity of the membrane can alter the ionic balance of the cell, electric

potential, and function of membrane-bound

proteins.

The cuticle is also sensitive to temperature changes. The wax of the cuticle is important in protecting the insect from its external environ-ment and maintaining water balance. High tem-peratures can alter the wax complex to become

more fluid and may lead to desiccation (Hep-burn, 1985). The effects of high temperature on insect mortality in low humidity environments may be compounded with desiccation stress (Fig. 8) (Beament, 1959). However, a high tempera-ture treatment in a highly saturated environment may lead to drowning, primarily due to the loss of cuticular protection of the spiracles leading to the tracheoles.

So, what is the real cause of insect mortality following a heat treatment? There is no indica-tion in the literature that can point to any one cause. However, recent research may begin to elucidate a cause. Thermal death kinetic studies with codling moth shows an interesting trend (Tang et al., 2000). For larvae of different in-stars and eggs, the TDT curves have different intercepts but the same slope with an activation energy of 500 kJ/mole. Preliminary work with other immature quarantine pests also show acti-vation energies of 500 kJ/mole (Tang, pers. comm.). Whether this can be related to a single event (i.e. breakdown of the mitochondria, dis-ruption of cellular membranes, denaturation of proteins and/or nucleic acids), or a summation of separate events remains to be seen. Further testing on individual cells, macromolecules, and other isolated systems will be needed to discern this phenomenon. Other work with scanning calorimetry may also help to describe energy balance of insects subjected to anoxic environ-ments and thermal stress (Zhou et al., 2000). There is an increase in the metabolic heat rate (measured in W) of codling moth and omnivo-rous leaf roller pupae as the temperature is raised from 10 to 30°C. However, at tempera-tures above 40°C, there is a drop in the heat rate (Mitcham, unpublished data), indicating ei-ther a protective mechanism of energy conserva-tion or the inability to produce an adequate supply of ATP to support elevated metabolism.

It is apparent that whatever system is investi-gated as being affected by heat, differences will be found. The key is to determine the critical or most sensitive point in the system, which can be manipulated to make heat treatments most effec-tive in controlling insects.

Fig. 8. Cuticular water permeability as related to changes in temperature. (--) denotes pupae ofTenebrio molitor, (--) larvae ofRhodnius prolixus, (--) pupae of the butterflyPieris rapae(After Beament, 1959). Reproduced with the kind per-mission of Company of Biologists Ltd. (J. Exp. Biol. 36 (1959) 391 – 442).

(8)

Beament, J.W.L., 1959. The waterproofing mechanism in arthropods. The effect of temperature on cuticle permeabil-ity in terresterial insects and ticks. J. Exp. Biol. 36, 391 – 442.

Benedict, M.Q., Cockburn, A.F., Seawright, J.A., 1993. The Hsp70 heat-shock gene family of the mosquito Anopheles albimanus. Insect Mol. Biol. 2, 93 – 102.

Bowler, K., 1987. Cellular heat injury. Are membranes in-volved? In: Bowler, K., Fuller, B.J (Eds.), Temperature and Animal Cells. Soc. Experimental Biol Symposium 41. Cambridge, England, pp. 157 – 185.

Chiang, H., Terlecky, S.R., Plant, C.P., Dice, J.F., 1994. A role for a 70-kilodaton heat shock protein in lysosomal degradation of intracellular proteins. Science 246, 382 – 385.

Clarke, K.U., 1967. Insects and temperature. In: Rose, A.H. (Ed.), Thermobiology. Academic Press, London, pp. 293 – 352.

Coleman, J.S., Heckathorn, S.A., Hallberg, R.L., 1995. Heat shock proteins and thermotolerance: Linking molecular and ecological perspectives. Tree 10, 305 – 306.

Denlinger, D.L., Yocum, G.D., 1998. Physiology of heat sensitivity. In: Hallman, G.J., Denlinger, D.L. (Eds.), Tem-perature Sensitivity in Insects and Application in Inte-grated Pest Management. Westview Press, Boulder, CO, pp. 7 – 54.

Feder, M.E., Blair, N., Figueras, H., 1997. Natural thermal stress and heat-shock protein expression in Drosophila

larvae and pupae. Functional Ecology 11, 90 – 100. Gallie, D.R., 1993. Regulation of translation during

heat-shock (Abstract). J. Plant Phys. 102, 34.

Heinrich, B., 1981. Ecological and evolutionary perspectives. In: Heinrich, B. (Ed.), Insect Thermoregulation. Wiley, New York, pp. 236 – 302.

Hepburn, H.R., 1985. Structure of the Integument. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiol-ogy, Biochemistry and PharmacolPhysiol-ogy, vol. 3, pp. 1 – 58. Hoffman, K.H., 1984. Metabolic and enzyme adaptation to

temperature. In: Hoffman, K.H. (Ed.), Environmental Physiology and Biochemistry of Insects. Springer Verlag, Berlin, pp. 1 – 32.

Hoffman, K.H., Marstatt, H., 1977. The influence of tempera-ture on catalytic efficiency of pyruvate kinase of crickets (Orthoptera: Gryllidae). J. Therm. Biol. 2, 203 – 207. Hochachka, P.W., Sommero, G.N., 1984. Temperature

Adap-tation. In: Hochachka, P.W., Sommero, G.N. (Eds.), Bio-chemical Adaptation. Princeton University Press, Princeton, NJ, pp. 355 – 449.

Ivanovi, J., 1991. Metabolic response to stressors. In: Ivanovi, J., Jankovi -Hladni, M. (Eds.), Hormones and Metabolism in Insect Stress. CRC Press, Boca Raton, FL, pp. 27 – 67. Jang, E.B., 1991. Thermal death kinetics and heat tolerance in early and late third instars of the oriental fruit fly (Diptera: Tephritidae). J. Econ. Entomol. 84, 1298 – 1303.

different factors. Comp. Biochem. Physiol. A. 74, 131. Jones, W.W., 1940. Vapor heat treatment for fruits and

veg-etables grown in Hawaii. Hawaii Agricultural Experimen-tal Station Circular No. 16, pp. 8.

Kerkut, G.A., Taylor, B.J.R., 1957. A temperature receptor in the tarsus of the cockroach,Periplaneta americana. J. Exp. Biol. 34, 486 – 493.

Marin, R., Blaker, T.W., Tanguay, R.M., 1994. Hsp 78: A prominent heat shock protein of the lepidopteranChoris

-toneura fumiferana that is immunologically unrelated to members of the major Hsp families. Archives Insect Biochem. Physiol. 25, 39 – 53.

Meyer, S.G.E., 1978. Effects of heat, cold, anaerobiosis and inhibitors on metabolite concentrations in larvae ofCal

-litroga macellaria. Insect Biochem. 8, 471 – 477.

Moss, J.I., Jang, E.B., 1991. Effects of age and metabolic stress on heat tolerance of Mediterranean fruit fly (Diptera: Tephritidae) eggs. J. Econ. Entomol. 84, 537 – 541. Neven, L.G., 1998a. Effects of heating rate on the mortality of

fifth instar codling moth. J. Econ. Entomol. 91, 297 – 301. Neven, L.G., 1998b. Respiratory response of fifth instar codling moth to rapidly changing temperatures. J. Econ. Entomol. 91, 302 – 308.

Neven, L., Mitcham, E., 1996. CATTS: Controlled atmo-sphere/temperature treatment system. A novel approach to the development of quarantine treatments. Amer. Ent. 42, 56 – 59.

Neven, L.G., Rehfield, L.M., 1995. Comparison of prestorage heat treatmetns on fifth-instar codling moth (Lepidoptera: Tortricidae) mortality. J. Econ. Entomol. 88, 1371 – 1375. Parsell, D.A., Lindquist, S., 1994. In: Morimoto, R.I.,

Tissieres, A., Georgopoulos, C., (Eds.), The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, pp. 457 – 494.

Parsell, D.A., Kowal, A.S., Singer, M.A., Lindquist, S., 1994. Protein disaggregation mediated by heat-shock protein Hsp104. Nature 372, 475 – 478.

Pardue, M.L., 1988. The heat shock response in biology and human disease: a meeting review. Genes Develop. 2, 783 – 785.

Proverbs, M.D., Newton, J.R., 1962. Effect of heat on the fertility of the codling moth, Carocapsa pomonella (L.). Canadian Entomol. 94(3), 225 – 233.

Rauschenbach, I.Y., 1991. Changes in juvenile hormone and ecdysteroid content during insect development under heat stress. In: Ivanovi, J., Jankovi-Hladni, M. (Eds.), Hor-mones and Metabolism in Insect Stress. CRC Press, Boca Raton, FL, pp. 116 – 148.

Rock, G.C., Shaffer, P.L., 1983. Developmental rates of codling moth (Lepidoptera: Olethreutidae) reared on apple at four constant temperatures. Environ. Entomol. 12, 831 – 834.

Roti Roti, J.L., 1982. Heat-induced cell death and radiosen-stization: Molecular mechanisms. In: Dethlefsen, L.A.,

(9)

Dewey, W.C. (Eds.), Proceedings of the Third Interna-tional Symposium: Cancer Therapy by Hyperthermia, Drugs and Radiation, National Cancer Institute Mono-graphs, 61, 310

Sharp, J.L, Chew, V., 1987. Time/mortality relationships for

Anastrepha suspensa(Diptera: Tephritidae) eggs and larvae submerged in hot water. J. Econ. Entomol. 80, 646 – 649. Shellie, K.C., Mangan, R.L., Ingle, S.J, 1997. Tolerance of

grapefruit and Mexican fruit fly larvae to heated controlled atmosphere. Postharvest Biol. Technol. 10, 179 – 186. Solomon, J.M., Rossi, J.M., Golic, K., McGarry, T.,

Lindquist, S., 1991. Changes in hsp70 after thermotoler-ance and heat-shock regulation in Drosophila. The New Biologist 3, 1106 – 1120.

Sondersom, E.L., Brandal, D.G., Mackey, B., 1992. High temperature combined with carbon dioxide enriched or reduced oxygen atmospheres for control ofTribolium cas

-taneum (Herbst) (Coleoptera: Tenebrionidae). J. Stored Product Research 28, 235 – 238.

Tang, J., Ikediala, J.N., Wang, S., Hansen, J.D., Cavalieri, R., 2000. High-temperature-short-time quarantine methods, Postharvest Biol. Technol. In press.

Vuister, G.W., Kim, S., Orosz, A., Marquardt, J., Wu, C., Bax, A., 1994. Solution structure of the DNA-binding

domain ofDrosophilaheat shock transcription factor. Nat. Struct. Biol. 1, 605 – 614.

Warter, R.L., Brizgys, L.M., 1987. Apurinic site induction in the DNA of cells heated at hyperthermic temperature. J. Cell Physiol. 133, 144 – 150.

White, L.D., 1981. Survival and reproduction of Codling Moths exposed to 33° during larval and pupal develop-ment. Ent. Exp. Appl. 29, 98 – 102.

Whitting, D.C., Foster, S.P., Maindonald, J.H., 1991. Effects of oxygen, carbon dioxide, and temperature on the mortal-ity responses ofEpiphyas posti6ittana(Lepidoptera:

Tortri-cidae). J. Econ. Entomol. 84, 1544 – 1549.

Yocum, G.D., Denlinger, D.L., 1992. Prolonged thermotoler-ance in the Flesh fly, Sarcophaga crassipalpis, does not require continuous expression or persistence of the 72 kDa heat-shock protein. J. Insect Physiol. 38, 603 – 609. Yocum, G.D., Denlinger, D.L., 1994. Anoxia blocks

thermo-tolerance and the induction of rapid cold hardening in the flesh fly Sarcophaga crassipalpis. Physiol. Entomol. 19, 152 – 158.

Zhou, S., Criddle, R.S., Mitcham, E.J., 2000a. Metabolic response ofPlatynota stultanapupae to controlled atmo-spheres and its relation to insect mortality response, J. Insect Physiol. (in press).

(1)

Fig. 4. Regression and actual data of respiration rates of 5th-instar codling moth at constant temperatures. Line indi-cates the regression (y=0.3485+0.033054x; R2₌_0.91) (From Neven, 1998b).

and denatured proteins. Since oxygen is required to produce ATP through oxidative phosphoryla-tion, a lack of ATP would render these proteins relatively inactive. Other effects of an anoxic envi-ronment may be observed in the inability to me-tabolize lipids and carbohydrates to maintain elevated metabolism, inability to synthesize proteins, and inability to alter fatty acids in the

phospholipid bilayer to maintain membrane

integrity.

4. Nervous system

5. Endocrine system

fruit fly, Ceratitis capaitata (Moss and Jang, 1991), the flesh fly Sarcophaga crassipalpis (Yocum and Denlinger, 1994), and codling moth, Cydia pomonella (Neven and Mitcham, 1996) has demonstrated that anoxic conditions make insects more sensitive to high temperature (Fig. 6). Oxy-gen deprivation appears to interfere with the physiological mechanisms involved in insect ther-mal tolerance. An interesting effect of anoxia on insects during a heat stress may be exemplified by the actions of heat shock proteins (hsps).HSP70

requires ATP in order to fold and release nascent

(2)

Table 2

Respiration rates and peak respiration times of fifth instar codling moths during simulated fruit heat treatments (From Neven, 1998b)b

Peak rate Peak timea

Treatment type Recovery times Time to 42°C (min) LT95(min)

Min9se mlCO2/min/mg

3/4 Peak Full peak 3.2590.23 51.794.6 32.792.9 97

MFA 44°C 126.395.0 292.0

3.6690.17 29.592.6 32.292.9

124.093.0 68

MFA 46°C 190.4

106.492.5

MFA 46°C 4.0590.25 22.291.9 25.292.2 66 145.4

4.3190.23 28.7910.8 29.692.6

MFA 46°C 121.293.7 73 170.7

4.1190.15 31.392.7 40.593.6

109.392.2 56

MFA 48°C 120.7

116.592.8

MFA 48°C 4.3890.18 30.792.7 42.793.8 75 162.7

VFA 44°C 105.792.6 4.2890.17 24.392.1 38.293.4 58 153.8

4.8890.17 33.292.9 33.893.0

89.493.2 42

VFA 46°C 88.1

90.193.5

VFA 48°C 4.4390.13 39.093.4 39.593.5 38 74.9

VFA 48°C 85.594.8 4.3590.20 39.793.5 33.592.9 32 66.9

a_{Time required to reach maximum respiration.}

b_{MFA: Moist Forced Air treatment, non-condensing environment. VFA: Vapor Forced Air treatment, condensing environment.}

(3)

6. Heat shock proteins

The HSP70 family is the best characterized of the heat-inducible proteins in insects.HSP70s are thought to function as molecular chaperones, in which either nascent or denatured proteins are held in situ by the HSP70 protein, which may ‘chaperone’ the protein to the lysosome for degra-dation (Chiang et al., 1994) or help the protein re-fold after return to favorable temperatures (Parsell and Lindquist, 1994). The role ofHSP70s in thermal tolerance in insects is well documented, although, it should be noted that the effects of hsp expression are short lived, and long-term exposure to inducing temperatures exceeds the capacity of these proteins to function and confer thermotolerance. However, in the application of high temperature treatments for disinfestation, in which acute thermal stresses are applied, hsps may play a very important role. Research by Yocum and Denlinger (1992) showed that a mild heat treatment of 40°C for 2 h to the flesh fly, Sarcophaga crassipalpis, conferred thermal toler-ance to a subsequent normally lethal heat treat-ment of 90 min at 45°C. This thermal tolerance decayed over time, but lasted 72 h, beyond the time over which the originally induced HSP70s were degraded (24 h). This was an excellent exam-ple of how pre-conditioning of an insect can confer thermal tolerance to a subsequent higher thermal treatment, but this effect is not necessar-ily being related to hsps; other factors may partic-ipate in thermal tolerance.

Fig. 7. Graphical depiction of the effects of cumulative ther-mal damage on whole multicellular organisms.

7. Models of thermal damage

(4)

potential, and function of membrane-bound

proteins.

(5)

References

Beament, J.W.L., 1959. The waterproofing mechanism in arthropods. The effect of temperature on cuticle permeabil-ity in terresterial insects and ticks. J. Exp. Biol. 36, 391 – 442.

Benedict, M.Q., Cockburn, A.F., Seawright, J.A., 1993. The Hsp70 heat-shock gene family of the mosquito Anopheles albimanus. Insect Mol. Biol. 2, 93 – 102.

Chiang, H., Terlecky, S.R., Plant, C.P., Dice, J.F., 1994. A role for a 70-kilodaton heat shock protein in lysosomal degradation of intracellular proteins. Science 246, 382 – 385.

Clarke, K.U., 1967. Insects and temperature. In: Rose, A.H. (Ed.), Thermobiology. Academic Press, London, pp. 293 – 352.

Coleman, J.S., Heckathorn, S.A., Hallberg, R.L., 1995. Heat shock proteins and thermotolerance: Linking molecular and ecological perspectives. Tree 10, 305 – 306.

Feder, M.E., Blair, N., Figueras, H., 1997. Natural thermal stress and heat-shock protein expression in Drosophila

larvae and pupae. Functional Ecology 11, 90 – 100. Gallie, D.R., 1993. Regulation of translation during

heat-shock (Abstract). J. Plant Phys. 102, 34.

Heinrich, B., 1981. Ecological and evolutionary perspectives. In: Heinrich, B. (Ed.), Insect Thermoregulation. Wiley, New York, pp. 236 – 302.

temperature. In: Hoffman, K.H. (Ed.), Environmental Physiology and Biochemistry of Insects. Springer Verlag, Berlin, pp. 1 – 32.

Adap-tation. In: Hochachka, P.W., Sommero, G.N. (Eds.), Bio-chemical Adaptation. Princeton University Press, Princeton, NJ, pp. 355 – 449.

Jankovi-Hladni, M., Ivanovi, J., Nenadovi, V., Stani, V., 1983. The selective response of the prothocerebral neurosecre-atory cells of the Cerambyx cerdolarvae to the effect of different factors. Comp. Biochem. Physiol. A. 74, 131. Jones, W.W., 1940. Vapor heat treatment for fruits and