Journal of Thermal Biology 25 (2000) 363±371 www.elsevier.com/locate/jtherbio

Heat shock proteins in white¯ies, an insect that accumulates sorbitol in response to heat stress

Michael E. Salvucci*, Dawn S. Stecher, Thomas J. Henneberry

US Department of Agriculture, Agricultural Research Service, Western Cotton Research Laboratory, 4135 E. Broadway Road, Phoenix, AZ, 85040-8830, USA Received 22 July 1999; accepted 15 October 1999

Abstract

1. The heat shock response was examined in the silverleaf white¯y. Hsp70 and Hsp90 were the major polypeptides synthesized by white¯ies in response to heat stress. 2. The amounts of Hsp70 and Hsp60 protein and Hsp70 mRNA were unchanged in response to a diurnal increase in temperature, whereas sorbitol content increased eight-fold. That steady-state levels of Hsps did not increase with higher rates of synthesis suggests that Hsps turn over faster in heat-stressed white¯ies. 3. Hsp70 transcript levels were highest when nutrient deprivation accompanied heat stress. Thus, Hsps appear to be especially important for heat-stressed white¯ies when sorbitol synthesis is limited nutritionally. 7 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Heat shock proteins; Hsp; Bemisia; Sorbitol; Polyols; Heat stress; Thermotolerance

1. Introduction temperature and humidity extremes of the environ- ment. Still, the temperature of a mature leaf of a well- In the desert southwest of the US, silverleaf white- watered cotton plant can exceed 358C (Lu et al., 1997), ¯ies, Bemisia argentifolii, Bellows and Perring, increase with even higher temperatures occurring on drought- in population size during the summer (Henneberry et stressed leaves (Radin et al., 1994). al., 1998) when air temperatures often exceed 428C and Previous studies from this laboratory have shown relative humidities can be less than 10% (Brown and that white¯ies and aphids, two very small phloem-feed- Russell, 1987). By feeding on the lower surface of ing insects, accumulate polyhydric alcohols (polyols) in leaves, white¯ies are protected to some extent from the response to high temperature (Wolfe et al., 1998; Hen- drix and Salvucci, 1998; Salvucci et al., 1999). Polyol synthesis in these insects is catalyzed by an NADPH- Abbreviations: Hsp, heat shock protein; KR, ketose re- dependent ketose reductase (KR). For white¯ies, ac- ductase; DIG, digoxigenin; DTT, dithiothreitol; PMSF, phe- cumulation of sorbitol occurs under ®eld conditions in nylmethylsulfonyl ¯uoride. * Corresponding author. Tel.: +1-602-379-3524; fax: +1- response to a diurnal increase in temperature (Wolfe et 602-379-4509. al., 1998). Sorbitol and other polyols are well-known E-mail address: [email protected] (M.E. Salvucci). solvent modi®ers that protect the native structure of

0306-4565/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S0306-4565(99)00108-4 364 M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371 proteins from thermal denaturation (Back et al., 1979; Temperature was measured with a Type T needle ther- Henle et al., 1982). Recent evidence with white¯ies is mocouple. White¯ies were frozen immediately follow- consistent with the idea that sorbitol acts as a thermo- ing collection and stored at 808C for later analysis. protectant in vivo, protecting white¯y proteins from thermal denaturation (Salvucci, 2000). 2.2. Heat stress under laboratory conditions A common response to heat stress in all organisms, including insects (Denlinger and Yocum, 1998) is the White¯ies were placed on cotton plants at 25 and synthesis of Hsps (reviewed in Parsell and Lindquist, 408C or in tubes without a source of nutrition at 408C 1993; Somero, 1995). These proteins are molecular as described previously (Salvucci, 2000). White¯ies chaperones that bind to and stabilize unfolded proteins were collected from the leaves and immediately frozen (Hendrick and Hartl, 1993; Parsell and Lindquist, and stored at 808C. 1993). Hsps are distributed ubiquitously, occurring in Drosophila melanogaster Meigen were kindly pro- both thermophilic (Trent et al., 1990) and psychrophi- vided by Dr. Winifred Doane (Arizona State Univer- lic (Deegenaars and Watson, 1998) organisms. For sity). For heat shock experiments, approximately 25 most organisms, Hsps are synthesized when ambient adults were placed in an Erlenmeyer ¯ask. The tem- temperatures exceed the normal temperature optimum perature of the ¯ask was increased 18C every 5 min of the organism (Parsell and Lindquist, 1993). Studies from 25 to 378C and then held at 378C for 1 h. For an with transgenic organisms, including Drosophila (Feder unstressed control, Drosophila were placed in a ¯ask et al., 1996; Krebs and Feder, 1997b), have shown that that was maintained at 258C for 2 h. Drosophila were Hsps improve thermal ®tness (Parsell and Lindquist, frozen immediately following each treatment and 1993). Since white¯ies accumulate sorbitol in response stored at 808C. to high natural temperatures (Wolfe et al., 1998), it was of interest to determine if the heat shock response 2.3. Measurement of sorbitol content of this insect was suppressed, particularly in response to the diurnal increase in temperature that these insects The sorbitol content of white¯ies collected at various regularly encounter. Included in the present study were times of the day was determined as described pre- measurements of Hsp60, a prokaryotic chaperone that viously (Wolfe et al., 1998; Salvucci, 2000). is a major protein synthesized by the endosymbiotic bacteria of aphids (Ishikawa, 1990). 2.4. Protein synthesis in vivo

Detached cotton leaves were labeled with 35S for 3 h 2. Materials and methods at low light (30 mmol photons m2 s1) by placing the petiole in water containing 0.125 mCi of [35S]Met/Cys 1 2.1. Heat stress under glasshouse conditions (Amersham Life Science, Arlington Heights, IL) . White¯ies were acclimated to either 25 or 408C by pla- White¯ies were raised through at least ®ve gener- cing approximately 200 individuals in clip cages on un- ations on cotton plants (Gossypium hirsutum L., cv. labeled cotton leaves in the light (300 mmol photons 2 1 Coker 100A glandless) in either a ``high temperature'' m s ). For acclimation and treatment at 408C, or a ``cooled'' glasshouse. The ``high temperature'' white¯ies were maintained on an unlabeled leaf for 1 h glasshouse reached a maximum air temperature of at 258C, and then the temperature was steadily 428C, similar to the temperatures that occur in the increased to 408C over an hour. White¯ies were held desert southwest of the US during the summer months at 408C on the unlabeled leaf for 1 h and were then (Brown and Russell, 1987). The ``cooled'' glasshouse transferred to a labeled leaf for 2 h at 408C. For ac- was limited to a maximum air temperature of 308C. climation and treatment at 258C, white¯ies were main- White¯ies were collected in the early morning when tained at 258C for 3 h on an unlabeled leaf and were leaf temperatures in both glasshouses were at their then transferred to a labeled leaf for 2 h at 258C. In minimum (i.e., 25±278C) and again at mid-day when vivo labeling of white¯y proteins was also conducted leaf temperatures in the ``cooled'' and ``high tempera- with white¯ies from the high temperature and cooled ture'' glasshouse were 28 and 36±398C, respectively. glasshouses (see above) by transferring white¯ies at mid-day to a labeled leaf at 40 or 258C, respectively. To analyze labeled polypeptides, labeled white¯ies 1 Mention of a trademark, proprietary product, or vendor were collected by aspiration and immediately frozen at does not constitute a guarantee or warranty of the product by 808C. Approximately 100 frozen white¯ies were the US Department of Agriculture and does not imply its extracted in 150 mM Mops-KOH, pH 7, containing approval to the exclusion of other products or vendors that 5 mM MgCl2, 2 mM EDTA, 5 mM DTT, 1 mM may also be suitable. PMSF and 20 mM leupeptin (extraction bu€er). Cold M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371 365 methanol was added to the homogenate to a ®nal con- and the Hsp60, Hsp70 and KR polypeptides were centration of 80% (v/v). After incubation overnight at detected immunologically using monospeci®c primary 208C, precipitated protein was collect from the and alkaline phosphatase-conjugated secondary anti- methanolic extracts by centrifugation for 10 min at bodies (Salvucci et al., 1998). Antibodies to KR have 13,000 g. Precipitated protein was dried in vacuo, and been described previously (Salvucci et al., 1998). suspended in bu€er containing SDS (Salvucci et al., Monoclonal antibodies to Synechococcus Hsp60 and 1998). The suspension was heated for 2 min at 908C, chicken oviduct Hsp70 were obtained from StressGen centrifuged for 1 min at 13,000 g and the supernatant Biotechnologies Corp. (Victoria, BC, Canada). was then electrophoresed in 7.5 to 15% SDS±PAGE gels (Salvucci et al., 1998). Following electrophoresis, 2.7. Northern blot analyses of RNA expression polypeptides were transferred to PVDF membrane by electroblotting overnight at 100 mA (Salvucci et al., Ten micrograms of total RNA, isolated as described

1998). Membranes were rinsed in H2O and the labeled above, was separated under denaturing conditions on polypeptides were visualized by direct autoradiog- formaldehyde-containing agarose gels (Sambrook et raphy. The molecular weight of individual polypeptides al., 1989). Following electrophoresis, the RNA was was determined using an image acquisition densi- transferred to a nylon membrane (Boehringer Man- tometer calibrated with known molecular weight stan- nheim, Indianapolis, IN). Membranes were baked at dards (Salvucci et al., 1992). 808C for 1 h and then probed with a DIG-labeled oli- For two-dimensional electrophoresis, approximately 200 white¯ies were extracted as described above and total protein was precipitated with 80% methanol. The dried protein pellets were suspended in a solution con- taining 8 M urea, 1% SDS, 0.02% nonidet-40 and 0.01% 2-mercaptoethanol and separated by isoelectric focusing and SDS±PAGE as described previously (Duncan and Hershey, 1984). Gels were stained with Coomassie Blue followed by silver stain.

2.5. In vitro translation of white¯y mRNA

Total RNA was isolated from white¯ies by using the Triazol method as described by the manufacturer (Gibco BRL-LifeTechnologies, MD). Poly A+ RNA was isolated using magnetic oligo dT beads (Dyna- beads Oligo (dT)25, Dynal Inc., Oslo, Norway) and 40 mg was translated in the presence of [35S]Met/Cys using a wheat germ system (Amersham Life Sciences). Labeled polypeptides were separated by SDS±PAGE as described above.

2.6. Western blot analyses of protein expression

For Western blots, white¯ies were homogenized at 48C in extraction bu€er and the protein concentration of the solution was determined (Bradford, 1976). Ali- Fig. 1. In vivo labeling of white¯y Hsps with [35S]Met/Cys. quots of the homogenates containing 80 mg of protein White¯ies were acclimated on an unlabeled cotton leaf for 1 h were supplemented with acetone to 80% (v/v). After at either 25 (lane 1) or 408C (lane 2) and were then trans- 1hat208C, the extracts were centrifuged for 10 min ferred to a labeled leaf at the same temperature. White¯ies at 13,000 g. The supernatants were discarded and the were collected from the cooled (lane 3) and the high tempera- ture (lane 4) glasshouses and were transferred to a labeled leaf protein pellets were dried and then suspended in bu€er at 25 and 408C, respectively. Following labeling, equal num- containing SDS, dithiothreitol and sucrose (Salvucci et bers of white¯ies were extracted and their proteins separated al., 1998). After heating to 1008C for 2 min, the by SDS±PAGE. Labeled polypeptides were visualized by extracts were centrifuged for 1 min and the super- autoradiography. The arrows indicate the positions of Hsps. natants were electrophoresed in 12% SDS±PAGE gels The lines to the left of the ®gure indicate the positions of (Salvucci et al., 1998). Polypeptides were electrophor- standard proteins of 97 and 66 kDa (marked) and 116, 45, 31, etically transferred to Immobilon-P PVDF membrane 21 and 14 kDa (unmarked). 366 M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371 gonucleotide according to the manufacturer's instruc- 3. Results tions (Boehringer Mannheim, Indianapolis, IN). A DIG-labeled riboprobe to Hsp70 was synthesized 3.1. Synthesis of Hsps in white¯ies using the Genius 4 Kit (Boehringer Mannheim, India- napolis, IN). The template for riboprobe synthesis was The major di€erence in protein labeling between a plasmid containing an insert that encodes for Droso- adult white¯ies labeled with [35S]Met/Cys at 40 and phila Hsp70 EST GM05226 (Research Genetics Inc., 258C was increased synthesis at 408C of a 93 and a 73 Huntsville, AL). Transcripts for Hsp70 were detected kD polypeptide (Fig. 1). The molecular weights of on blots using a chemiluminescence detection system these polypeptides corresponded to Hsp90 and Hsp70, after hybridizing with the DIG-labeled oligonucleotides two of the major Hsps (reviewed in Parsell and Lind- as described by the manufacturer (Boehringer Man- quist, 1993). Increased synthesis of these two polypep- nheim, Indianapolis, IN). The relative amounts of each tides at 408C occurred when white¯ies were transcript were determined by whole-band analysis conditioned in the laboratory on cotton leaves for 1 h using an image acquisition densitometry (Salvucci et at 408C prior to labeling as well as when white¯ies al., 1992). were transferred directly from the high temperature glasshouse to a labeled leaf without conditioning (Fig. 1). White¯ies transferred from the high tempera- ture glasshouse contained less total label than those conditioned on a leaf in the laboratory, indicating that the transfer from glasshouse to laboratory interrupted feeding. RNA isolated from white¯ies exposed to 40 and 258C produced similar pro®les of labeled polypeptides when translated in vitro (Fig. 2). At both tempera- tures, the 93 and 73 kD Hsps appeared to be among the translation products. However, these polypeptides were signi®cantly enriched among the translation pro- ducts of mRNA from the 408C white¯ies.

3.2. Sorbitol levels in white¯ies experiencing diurnal changes in temperature

White¯ies maintained on cotton plants in a glass-

Fig. 2. In vitro translation of mRNA from unstressed and heat-stressed white¯ies. PolyA+ RNA was isolated from white¯ies that were acclimated on a cotton leaf for 1 h at Fig. 3. Diurnal changes in leaf temperature of cotton plants either 40 (lane 1) or 258C (lane 2) and 40 mg of each was in glasshouses set at two di€erent temperatures. Leaf tempera- translated in a wheat germ system. Labeled polypeptides were tures was measured during the course of a typical summer separated by SDS±PAGE and visualized by autoradiography. day in the high temperature (*) and cooled (Ð Ð) glass- The arrows indicate the positions of Hsps. The numbers and houses. The arrows and numbers in parentheses indicate the lines to the right of the ®gure indicate the positions and mol- various times that white¯ies were collected from the leaves ecular weights in kilodaltons of standard proteins. (see Table 1). M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371 367

Table 1 ¯y polypeptides (Fig. 4). According to the supplier, the Leaf temperature and sorbitol content of white¯ies in the high Hsp70 antibodies recognize both the constitutive and temperature and cooled glasshouses at the times of sampling induced forms of Hsp70 in several mammalian species,

a b as well as in frogs, chickens, yeast, Drosophila and Time of day Leaf temperature Sorbitol content tunicates. These antibodies recognized two polypep- (a.m.) (8C) (nmol white¯y1) tides of about 72 and 70 kD on blots of white¯y poly- 6 (1)c 26 0.1820.09 peptides. Commercially available antibodies to Hsp90 11:40 (2) 37 1.5620.77 did not recognize an Hsp90 cognate among white¯y 11:40 (3) 28 0.3420.11 polypeptides, consequently they were not used in the study (data not shown). The lack of recognition indi- a The numbers in parentheses refer to the numbered arrows cated that either commercial Hsp90 antibodies did not in Fig. 3. cross-react with white¯y Hsp90 or white¯ies do not b Mean2SEM of three separate samples, each consisting of contain an Hsp90 cognate. 50±75 white¯ies. c White¯ies from the high temperature glasshouse had Leaf temperatures and sorbitol content at this sampling similar levels of Hsp70 and Hsp60 at 5:30 and 11:40 time were identical in the high temperature and cooled glass- houses. a.m. (Fig. 4), and there were no major di€erences in the level of the sorbitol-synthesizing protein, KR. Simi- larly, Hsp60, Hsp70 and KR protein levels were equiv- house experienced diurnal changes in leaf temperature alent in white¯ies collected from the high temperature from a minimum of 268C at 5:30 a.m. to a maximum and cooled glasshouses at mid-day (Fig. 5). White¯ies near mid-day of 37±398C in the high temperature exposed to 25 and 408C for 1 h on a cotton plant in glasshouse or 288C in the cooled glasshouse (Fig. 3). the laboratory also had similar levels of Hsp70 (data For white¯ies in the high temperature glasshouse, sor- not shown). Comparison of the polypeptide pro®les of bitol levels increased more than eight-fold from 5:30 to 25 and 408C white¯ies by one- and two-dimensional 11:40 a.m. (Table 1). In contrast, sorbitol levels electrophoresis showed that there was no di€erence in increased less than two-fold over this same time period the amounts or distribution of the polypeptides detect- for white¯ies in the cooled glasshouse. able with Coomassie Blue or silver staining (data not shown). 3.3. Levels of Hsp and KR protein in white¯ies experiencing diurnal changes in temperature 3.4. Levels of Hsp70 mRNA in white¯ies experiencing diurnal changes in temperature Commercially available antibodies to Hsp70 and Hsp60 recognized polypeptides of the appropriate The levels of Hsp70 transcripts were similar in the apparent molecular weights on Western blots of white- morning and at mid-day in white¯ies from the high temperature glasshouse (Fig. 6). In contrast, white¯ies feeding on cotton leaves in the laboratory had about ten-fold higher levels of Hsp70 transcripts when exposed to 40 compared with 258C. White¯ies that were exposed to 408C in the absence of a source of nutrition had even higher levels of Hsp70. Transcript levels for Hsp70 were similar after feeding in the lab- oratory at 25, 33 and 368C and only increased when the temperature was increased to 408C (data not shown). Hsp70 mRNA was almost undetectable in Drosophila adults exposed to 258C, but increased to a very high level after exposure for 1 h to 378C.

4. Discussion

In the present study, white¯ies raised in the high Fig. 4. E€ect of diurnal temperature changes on the levels of temperature glasshouse experienced air and leaf tem- Hsp70, Hsp60 and KR protein in white¯ies. White¯ies were peratures that were similar to those that are encoun- collected from the high temperature glasshouse at the indi- tered naturally during the summer growing season in cated times and the proteins were analyzed on Western blots. the desert southwest of the US (Brown and Russell, Equal amounts of protein were loaded in each lane. 1987; Lu et al., 1997). Under these conditions, the sor- 368 M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371

Fig. 5. E€ect of temperature on the levels of Hsp70, Hsp60 and KR protein in white¯ies at noon. White¯ies were collected at noon from cotton plants in the cooled (leaf temperature=288C) and the high temperature (leaf temperature=398C) glasshouse and the proteins were analyzed on Western blots. Equal amounts of protein were loaded in each lane.

bitol content of the white¯ies increased eight-fold in re- sponse to a diurnal increase in leaf temperature from 26 to 388C. In contrast, the sorbitol content of the white¯ies increased only two-fold when leaf tempera- tures increased from 268C in the morning to only 288C at mid-day. Thus, temperature appears to be the major factor responsible for diurnal changes in sorbitol con- tent of white¯ies (Wolfe et al., 1998; Salvucci et al., 1999). In vivo labeling of white¯y proteins at 25 and 408C showed that the rate of synthesis of Hsp70 and Hsp90 increased at the higher temperature. However, there was no increase in the steady-state amount of Hsp70 in white¯ies exposed to elevated temperatures. Wood et al. (1998) reported a similar discrepancy in heat- shocked lampreys, suggesting that either the masking of inducible Hsp70 by constitutive Hsp70 or a lack of antibody recognition could explain why increased syn- thesis of Hsp70 was not accompanied by higher amounts of protein on Western blots. These expla- Fig. 6. E€ect of temperature on the levels of Hsp70 transcripts in white¯ies and Drosophila. Ten micrograms of total RNA nations do not appear to be valid for white¯ies since from white¯ies (A) and Drosophila (B) were analyzed on Hsp70 antibodies recognized multiple forms of Hsp70 Northern blots using a probe prepared to Drosophila Hsp70. on Western blots of white¯y polypeptides. Also, separ- (A) White¯ies were collected from cotton plants in the high ation of white¯y proteins by one- or two-dimensional temperature glasshouse in the morning (lane 1) and at mid- gel electrophoresis gave no indication of an increase in day (lane 2) when leaf temperatures were 25 and 368C, re- the amounts of Hsps in response to heat stress. Thus, spectively. In a separate experiment, white¯ies were exposed the turnover rate of Hsp70 in white¯ies must be faster to 25 (lane 3) or 408C (lane 4) for 1 h in the laboratory while at higher temperatures since heat stress increased the feeding on a cotton plant. White¯ies were also incubated for 1 h at 408C in the absence of a source nutrition (lane 5). (B) rate of synthesis (Fig. 1), but did not a€ect the steady- Adult Drosophila were incubated for 1 h at 25 (lane 1) and state level of protein. This conclusion does not pre- 378C (lane 2). Panel B1 and B2 are two exposures of the same clude the likely possibility (Joplin and Denlinger, 1990; blot. Krebs and Feder, 1997a) that the amount of Hsp70 in M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371 369 white¯ies increases in response to heat shock, but that to protect the virus from degradation during its pas- the increase occurs in certain tissues, without a notice- sage from the hemocoel. Thus, Hsp60 appears to be able e€ect on the total Hsp70 pool. required for aspects of the symbiosis unrelated to heat In contrast to the results with white¯ies, the amount stress, perhaps explaining its presence in unstressed of Hsp70 protein in both the larval and the adult white¯ies and aphid. forms of Drosophila increases nearly a 1000-fold in re- In a previous study, an analogy was drawn between sponse to heat shock (Velazquez et al., 1983). Interest- polyol synthesis in white¯ies and aphids and trehalose ingly, Hsp70 is barely detectable in unstressed synthesis in yeast (Salvucci, 2000). The relationship Drosophila (Velazquez et al., 1983; Krebs and Feder, between Hsps and trehalose in yeast has been charac- 1997b; Feder et al., 1996), whereas unstressed white- terized using mutants in both Hsp and trehalose syn- ¯ies contain amounts of Hsp70 that are comparable to thesis (Ribeiro et al., 1997; Gross and Watson, 1998; the amount present after heat stress. Similarly, Gehring Elliott et al., 1996; De Virgilio et al., 1994). The con- and Wehner (1995) showed that signi®cant levels of sensus view appears to be that both trehalose and Hsp70 and Hsp72 occur in temperate and desert ant Hsps contribute to thermotolerance, the former ame- species prior to heat stress. These investigators pro- liorates long term heat stress while the latter are posed that the accumulation of Hsps prior to exposure required for short term repair during heat shock to high temperature preadapts the ants to heat stress. (Gross and Watson, 1998). During the summer The occurrence of high constitutive levels of Hsp70 in months, white¯ies often experience regular diurnal unstressed white¯ies appears to be an inherent charac- increases in air and leaf temperatures, sometimes teristic rather than one that is induced by daily ex- exceeding 45 and 358C, respectively (Brown and Rus- posure to high temperature since white¯ies that were sell, 1987; Lu et al., 1997). Under ®eld conditions, raised in a cooler glasshouse also had high levels of white¯ies synthesize sorbitol that probably functions as Hsp70. a thermoprotectant to protect the insect during the In Drosophila, transcript levels for Hsp70 increase diurnal exposure to high temperature (Wolfe et al., nearly 1000-fold in response to heat stress (Velazquez 1998). et al., 1983; Goto et al., 1998). In the present study, A faster rate of synthesis and turnover of Hsp70 transcript levels for Hsp70 increased in heat-stressed and Hsp90 and relatively high constitutive levels of compared with unstressed white¯ies. However, the these proteins may also provide some protection increase was only about 10-fold and occurred in the during the hottest times of the day. Previously, we laboratory when leaf temperatures reached 408C, but showed that white¯ies do not accumulate sorbitol in not in the glasshouse when the leaf temperature the absence of a source of carbohydrates or when their increased from 26 to 368C. Interestingly, the levels of nutritional source lacked sucient carbohydrate (Sal- Hsp70 transcripts were relatively high in unstressed vucci, 2000). Here we show that transcript levels for white¯ies, in contrast to unstressed Drosophila which Hsp70 were highest when nutrient deprivation ac- contain levels that are barely detectable. The character- companied heat stress. Thus, even though white¯ies ac- istics of Hsp70 expression in white¯ies was similar to cumulate sorbitol as a thermoprotectant, there may be the pattern in Ceratitis capitata larvae where transcript times when sorbitol levels are inadequate and Hsps levels for Hsp70 increased only two-fold upon heat provide the main protection against heat damage. shock (Thanaphum and Haymer, 1998). An Hsp60 homologue called symbionin is the major protein synthesized by the endosymbiotic bacteria in Acknowledgements the pea aphid, Acyrthosiphon pisum (Ishikawa, 1990). Studies with this aphid have shown that synthesis of We would like to acknowledge the expert technical Hsp60 or possibly a phosphorylated form of this pro- assistance of C. Chamberlain. We would also like to tein increases with heat stress (Morioko and Ishikawa, thank Dr. D.L. Hendrix and T. Steele for providing 1992). However, other investigators have shown that HPLC analyses of white¯y carbohydrates. The Buchnera, the endosymbiont in the aphid Schizaphis research was supported in part by grant No. 97-431 graminum contain high steady state levels of Hsp60 from Cotton Inc. that are una€ected by a 108C increase in temperature (Baumann et al., 1996). In the present study, Hsp60 was detected in white¯ies, but both its steady-state References amount and its rate of synthesis were una€ected by temperature. Recent investigations with white¯ies Back, J.F., Oakenfull, D., Smith, M.B., 1979. Increased ther- (Morin et al., 1999) and earlier investigations with mal stability of proteins in the presence of sugars and aphids (van den Heuvel et al., 1994) have shown that polyols. Biochemistry 18, 5191±5196. Hsp60 binds to virus particles and this binding appears Baumann, P., Baumann, L., Clark, M.A., 1996. Levels of 370 M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371

Buchnera aphidicola chaperonin GroEL during growth of Henneberry, T.J., Jech, L.F., Hendrix, D.L., 1998. Seasonal the aphid Schizaphis graminum. Curr. Microbiol. 32, 279± distribution of Bemisia argentifolii (Homoptera: 285. Aleyrodidae) honeydew sugars on Pima and upland cotton Bradford, M.M., 1976. A rapid and sensitive method for the lint and lint stickiness at harvest. Southwest Entomol. 23, quantitation of microgram quantities of proteins utilizing 105±121. the principle of protein-dye binding. Anal. Biochem. 72, van den Heuvel, J.F.J.M., Verbeek, M., van der Wilk, F., 248±259. 1994. Endosymbiotic bacteria associated with circulative Brown, P., Russell, B. 1987. Weather Data and On-Line transmission of potato leaf roll virus by Myzus persicae.J. Documentation. The Arizona Meteorological Network Gen. Virol. 75, 2559±2565. (AZMET) Computer Bulletin Board. The University of Ishikawa, H., 1990. Molecular interactions of intracellular Arizona Cooperative Extension Service, Tucson, AZ symbionts and aphids. In: Campbell, R.K., Eikenbary, (http://ag.arizona.edu/azmet/). R.D. (Eds.), Aphid-Plant Interactions. Elsevier, Deegenaars, M.L., Watson, K., 1998. Heat shock response in Amsterdam, pp. 351±361. psychrophilic and psychrotrophic yeast from Antarctica. Joplin, K.H., Denlinger, D.L., 1990. Developmental and tis- Extremophiles 2, 41±49. sue speci®c control of the heat shock induced 70 kDa re- Denlinger, D.L., Yocum, G.D., 1998. Physiology of heat sen- lated proteins in the ¯esh ¯y, Sarcophaga crassipalpis.J. sitivity. In: Hallman, G.J., Denlinger, D.L. (Eds.), Insect Physiol. 36, 239±249. Temperature Sensitivity in Insects and Application in Krebs, R.A., Feder, M.E., 1997a. Natural variation in the ex- Integrated Pest Management. Westview Press, Oxford, pp. pression of the heat-shock protein Hsp70 in a population 7±53. of Drosophila melanogaster and its correlation with toler- De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T., ance of ecologically relevant thermal stress. Evolution 51, Wiemken, A., 1994. The role of trehalose synthesis for ac- 173±179. quisition of thermotolerance in yeast I. Genetic evidence Krebs, R.A., Feder, M.E., 1997b. Tissue-speci®c variation in that trehalose is a thermoprotectant. Eur. J. Biochem. 219, Hsp70 expression and thermal damage in Drosophila mela- 179±186. nogaster larvae. J. Exp. Biol. 200, 2007±2015. Duncan, R., Hershey, J.W.B., 1984. Evaluation of isoelectric Lu, Z., Chen, J., Percy, R.G., Zeiger, E., 1997. Photosynthetic focusing running conditions during two-dimensional iso- rate, stomatal conductance and leaf area in two cotton electric focusing/sodium dodecylsulfate-polyacrylamide gel species (Gossypium barbadense and Gossypium hirsutum ) electrophoresis: variations in gel patterns with changing and their relation with heat resistance and yield. Aust. J. conditions and optimized isoelectric focusing conditions. Plant Physiol. 24, 693±700. Anal. Biochem. 138, 144±155. Morin, S., Ghanim, M., Zeidan, M., Czosnek, H., Verbeek, Elliott, B., Haltiwanger, R.S., Futcher, B., 1996. Synergy M., van den Heuvel, J.F.J.M., 1999. A GroEL homologue between trehalose and Hsp104 for thermotolerance in from endosymbiotic bacteria of the white¯y Bemisia tabaci Saccharomyces cerevisiae. Genetics 144, 923±933. is implicated in the circulative transmission of tomato yel- Feder, M.E., CartanÄ o, N.V., Milos, L., Krebs, R.A., low leaf curl virus. Virology 256, 75±84. Lindquist, S.L., 1996. E€ect of engineering Hsp70 copy Morioko, M., Ishikawa, H., 1992. Mutualism based on stress: number on Hsp70 expression and tolerance of ecologically selective synthesis and phosphorylation of a stress protein relevant heat shock in larvae and pupae of Drosophila mel- by an intracellular symbiont. J. Biochem. 111, 431±435. anogaster. J. Exp. Biol. 199, 1837±1844. Parsell, D.A., Lindquist, S., 1993. The function of heat-shock Gehring, W.J., Wehner, R., 1995. Heat shock protein syn- proteins in stress tolerance: degradation and reactivation thesis and thermotolerance in Cataglyphis, an ant from the of damaged proteins. Annu. Rev. Genet. 27, 437±496. Sahara desert. Proc. Natl Acad. Sci. USA 92, 2994±2998. Radin, J.W., Lu, Z., Percy, R.G., Zeiger, E., 1994. Genetic Goto, S.G., Yoshida, K.M., Kimura, M.T., 1998. variability for stomatal conductance in Pima cotton and Accumulation of Hsp70 mRNA under environmental its relation to improvements of heat adaptation. Proc. stresses in diapausing and nondiapausing adults of Natl Acad. Sci. USA 91, 7217±7221. Drosophila triauraria. J. Insect Physiol. 44, 1009±1015. Ribeiro, M.J., Reinders, A., Boller, T., Wiemken, A., De Gross, C., Watson, K., 1998. De novo protein synthesis is Virgilio, C., 1997. Trehalose synthesis is important for the essential for the thermotolerance acquisition in a acquisition of thermotolerance in Schizosaccharomyces Saccharomyces cerevisiae trehalose synthase mutant. pombe. Molec. Microbiol. 25, 571±581. Biochem. Molec. Biol. Int. 45, 663±671. Salvucci, M.E., 2000 Sorbitol accumulation in white¯ies: evi- Hendrick, J.P., Hartl, F.-U., 1993. Molecular chaperone func- dence for a role in protecting proteins during heat stress, tions of heat-shock proteins. Annu. Rev. Biochem. 62, J. Therm Biol. 25, 353±361. 349±384. Salvucci, M.E., Chavan, A.J., Haley, B.E., 1992. Hendrix, D.L., Salvucci, M.E., 1998. Polyol metabolism in Identi®cation of peptides from the adenine binding homopterans at high temperature: accumulation of manni- domains of ATP and AMP in adenylate kinase: isolation tol in aphids (Aphididae: Homoptera) and sorbitol in of photoanity-labeled peptides by metal chelate chroma- white¯ies (Aleyrodidae: Homoptera). Comp. Biochem. tography. Biochemistry 31, 4479±4487. Physiol. 120, 487±494. Salvucci, M.E., Wolfe, G.H., Hendrix, D.L., 1998. Henle, K.J., Nagle, W.A., Moss Jr, A.J., Herman, T.S., 1982. Puri®cation and properties of an unusual NADPH-depen- Polyhydroxy compounds and thermotolerance: a proposed dent ketose reductase from the silverleaf white¯y. Insect concatenation. Radiat. Res. 92, 445±451. Biochem. Molec. Biol. 28, 357±363. M.E. Salvucci et al. / Journal of Thermal Biology 25 (2000) 363±371 371

Salvucci, M.E., Hendrix, D.H., Wolfe, G.R., 1999. E€ect of archaebacterium Sulfolobus sp. Strain B12. J. Bacteriol. high temperature on the metabolic processes a€ecting sor- 172, 1478±1484. bitol synthesis in the silverleaf white¯y, Bemisia argentifo- Velazquez, J.M., Sonoda, S., Bugaisky, G., Lindquist, S., lii. J. Insect Physiol. 45, 21±27. 1983. Is the major Drosophila heat shock protein present Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular in cells that have not been heat shocked? J. Cell Biol. 96, Cloning, A laboratory manual, 2nd ed. Cold Spring 286±290. Harbor Laboratory, New York. Wolfe, G.R., Hendrix, D.L., Salvucci, M.E., 1998. A thermo- Somero, G.N., 1995. Proteins and temperature. Annu. Rev. Physiol. 57, 43±68. protective role for sorbitol in the silverleaf white¯y. J. Thanaphum, S., Haymer, D.S., 1998. A member of the hsp70 Insect Physiol. 44, 597±603. gene family from the Mediterranean fruit ¯y, Ceratitis Wood, L.A., Brown, I.R., Youson, J.H., 1998. capitata. Insect Molec. Biol. 7, 63±72. Characterization of the heat shock response in the gills of Trent, J.D., Osipiuk, J., Pinkau, T., 1990. Acquired thermoto- sea lampreys and a brook lamprey at di€erent intervals of lerance and heat shock in the extremely thermophilic their life cycles. Comp. Biochem. Physiol. A120, 509±518.