Insect Biochemistry and Molecular Biology 30 (2000) 775–783www.elsevier.com/locate/ibmb

Stress-reactivity and juvenile hormone degradation inDrosophilamelanogasterstrains having stress-related mutations

N.E. Gruntenkoa,*, T.G. Wilsonb, M. Monastirioti c, I.Y. Rauschenbacha

a Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Division, Novosibirsk 630090, Russiab Department of Biology, Colorado State University, Fort Collins, CO 80523, USA

c Institute of Molecular Biology and Biotechnology, FORTH, 711-10 Heraklion, Crete, Greece

Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000

Abstract

Juvenile hormone (JH) degradation was studied under normal and stress conditions in young and matured females ofDrosophilamelanogasterstrains having mutations in different genes involved in responses to stress It was shown that (1) the impairment inheat shock response elicits an alteration in stress-reactivity of the JH system; (2) the impairment JH reception causes a decreaseof JH-hydrolysing activity and of stress-reactivity in young females, while in mature ones stress reactivity is completely absent;(3) the absence of octopamine results in higher JH-hydrolysis level under normal conditions and altered JH stress-reactivity; (4)the higher dopamine content elicits a dramatic decrease of JH degradation under normal conditions and of JH stress-reactivity.Thus, the impairments in any component of the Drosophila stress reaction result in changes in the reponse of JH degradationsystem to stress. The role of JH in the development of the insect stress reaction is discussed. 2000 Elsevier Science Ltd. Allrights reserved.

Keywords: Drosophila melanogaster; ts403; Met; Tbh; ebony; Juvenile hormone; Stress reactivity

1. Introduction

Juvenile hormone (JH), a sesquiterpenoid involved inthe regulation of developmental transitions and repro-duction in insects (reviews: Riddiford and Ashburner,1991; Nijhout, 1994; Wyatt and Davey, 1996), is wellknown to play a main role in the development of theinsect stress reaction (reviews: Cymborowski, 1991 Rau-schenbach 1991, 1997). Two other important compo-nents of this multi-faceted response are the metabolismof biogenic amines, dopamine (DA) and octopamine(OA), and the heat shock response (HSR) (Orchard andLoughton, 1981; Davenport and Evans, 1984; Woodringet al., 1989; Hirashima et al., 1993, 1999; Rauschenbachet al. 1993, 1997; Rauschenbach, 1997; Sukhanova etal., 1997; Khlebodarova et al., 1998).

We have previously shown that the JH metabolic sys-

* Corresponding author. Tel.:+383-2-333-526; fax:+383-2-331-278.

E-mail address:kiseleva@bionet.nsc.ru (N.E. Gruntenko).

0965-1748/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.PII: S0965-1748 (00)00049-7

tem of wild type females ofDrosophila melanogasterandD. virilis responds to stress conditions (termed herestressors) with a decrease in JH-hydrolysing activity.Males do not respond to stressors in this manner(Rauschenbach et al. 1995, 1996). The metabolic sys-tems of DA and OA respond to stress, in both sexes, byan increase in the amine content and by a decrease inthe activity of their synthetic enzymes (Rauschenbach etal., 1993; Hirashima et al., 1999). We have also demon-strated that a mutation disturbing the development of thestress reaction inD. virilis also elicits the impairment ofHSR (Khlebodarova et al., 1998).

How do impairments of the different components ofthe stress reaction, such as HSR and the metabolism ofDA and OA, affect JH metabolism inD. melanogasterfemales under normal and stress conditions? Previouswork has demonstrated that biogenic amines areinvolved in the regulation of JH biosynthesis andsecretion by the corpora allata and that the expressionof some HSR genes is JH dependent (Piulachs andBelles, 1989; Thompson et al., 1990; Berger et al., 1992;Granger et al., 1996).

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In this work, we analysed the mutationsts403, Met,Tbh and ebony (e)with respect to the response of JH-degradation system to stress. The recessive temperaturesensitive lethal mutationl(l)ts403 results in the failureof heat shock protein (HSP)83 and HSP35 to beexpressed, and a number of HSP70 proteins are only par-tially expressed (Evgen’ev and Denisenko, 1990).Met27

is a null allele of theMethoprene-tolerantgene thatshows resistance to the toxic effects of both JH and aJH analog, methoprene. The mechanism of the resistanceappears to be altered JH reception(Wilson and Fabian,1986; Shemshedini and Wilson, 1990).Met27 completelylacksMet transcript and is clearly a null allele (Wilsonand Ashok, 1998).TbhnM18 is a null mutation at theTyr-amine b-hydroxylaselocus, which results in completeabsence of the tyramineβ-hydroxylase protein andblockage of octopamine biosynthesis (Monastirioti et al.,1996).e is postulated to be the mutation ofN-b-alanyldopamine synthetasegene, based on the fact thate hastwice as much DA as normal (Hodgetts, 1972; Hodgettsand Konopka, 1973; Ramadan et al., 1993).

Here we asked whether these mutations would affectthe decrease in JH degradation occurring inD. mel-anogasterwhen stressed. In order to answer this ques-tion, we studied the JH degradation in individuals ofts403,n Met27, TbhnM18 andStestrains (carringl(l)ts403,Met27, Tbh ande mutations, respectively), under normaland stress conditions, and compared their stress-reac-tivity (calculated as percent change in JH hydrolysisunder stress compared to hydrolysis under normalconditions) with that in a number of wild type and lab-oratory strains.

We demonstrated (1) thatts403 females respond tostress by a decrease in JH degradation, as occurs in wildtype females, but that their stress-reactivity significantlydiffers from that of wild type; (2) that in youngv Met27

females, similar to wild type flies, JH hydrolysis isdecreased upon stress, but their stress-reactivity is sig-nificantly lower than in wild type; (3) that JH degra-dation is unaffected in olderv Met27 females understress; (4) thatTbhnM18 females show a significantlyhigher JH-hydrolysis level and different stress-reactivitythan does the wild type; and (5) that youngStefemalesdemonstrate significantly lower JH-hydrolysis andstress-reactivity, compared to the wild type.

2. Materials and methods

2.1. Drosophila strains

The followingD. melanogasterstrains were used: thewild type laboratory strainCanton S; wild type iso-female strain 921500 from a natural population ofGorno-Altaisk; laboratory balancer strainFirst MultipleSeven(FM7); vermilion (n) strain from which then

Met27 strain was derived; laboratory balancer strainIn(2LR)Cy/L; In(3LR)D/Sb, carrying morphologicalmutations with recessive lethal actionCurly, Lobe(chromosome 2) andDichaete, Stubble(chromosome 3;hereafter termedCyLDSb); strain ts403 carrying therecessive temperature sensitive lethal mutationl(l)ts403(Arking, 1975); strainn Met27 carrying a null allele ofthe Methoprene-tolerantgene (Wilson and Ashok,1998); strainTbhnM18 carrying a null mutation at theTyr-amine b-hydroxylaselocus (Monastirioti et al., 1996);and the laboratorySte strain carrying thee mutation.Cultures were raised on standard medium(Rauschenbach et al., 1987) at 25°C, and adults weresynchronized by eclosion. Flies were subjected to stressat 38°C for 3 h, and were subsequently frozen in liquidnitrogen and stored at220°C.

2.2. JH hydrolysis

JH hydrolysis was measured by the assay of Ham-mock and Sparks, 1977. A fly was homogenized on icein 30 µl of 0.1 M Na-phosphate buffer, pH 7.4, contain-ing 0.5 mM phenylthiourea. The homogenates were cen-trifuged for 5 min at 12,000 rpm, and samples of thesupernatant (10µl) were utilized for the reaction. A mix-ture consisting of 0.1µg unlabeled JH-III (Sigma) and12,500 dpm3H labeled JH-III (17.4 Ci/mmol at C-10,NEN Research Products, Germany) was used as sub-strate. The reaction was carried out in siliconized tubesin 100 µl of incubation mixture for 3 h, and it wasstopped by the addition of 250µl heptane and 50µl ofa solution containing 5% ammonia and 50% methanol(V/V). The tubes were shaken vigorously and centri-fuged at 12,000 rpm for 10 min. Samples (100µl) ofboth aqueous and heptane phases were placed in vialscontaining dioxane scintillation fluid and counted. Con-trol experiments have shown a linear substrate–reactionrelationship (Gruntenko et al., 1999), as well as the factthat measured activity is proportional to homogenate (i.e.enzyme) concentration (Rauschenbach, 1991; unpub-lished data).

The significance of the differences between the datasets was tested by Student’st-test. Sample size variedfrom 12 to 28 individuals for each measurement in allexperiments.

3. Results

3.1. JH degradation in 1-day old ts403 and Canton Sfemales under normal and heat stress conditions

JH-hydrolysing activity in 1-day old females of strainsCanton Sand ts403under normal and stress conditionsare shown in Fig. 1. The data indicate that under normalconditions, JH-hydrolysing activity ints403 females

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Fig. 1. Hydrolysis of [3H]JH-III in 1-day-old females ofCanton Sand ts403strains ofD. melanogasterunder normal and stress (38°C,3 h) conditions. Means±SE.

does not differ from that inCanton Sones. The data ofFig. 1 also demonstrate thatts403 females respond tostress as well asCanton Sdo: exposure to 38°C evokesin females of both strains a significant (P,0.001)decrease in JH-hydrolysing activity compared to controlfemales maintained at 25°C.

3.2. JH degradation in 1-day oldn Met27 and nfemales under normal conditions and under heat stress

JH-hydrolysis in 1-day old females of bothn and nMet27 strains under normal and stress conditions areshown in Fig. 2. They indicate that under normal con-ditions n Met27 females show a significantly (P,0.01)

Fig. 2. Hydrolysis of [3H]JH-III in 1-day-old females ofn and nMet27 strains ofD. melanogasterunder normal and stress (38°C 3 h)conditions. Means±SE.

lower JH-hydrolysing activity than don females.Exposure to 38°C causes females of bothn and Met27strains to show a significant (P,0.001) decrease in JH-hydrolysis level, compared to control females kept at25°C.

3.3. JH degradation in 1-day-old TbhnM18 and Stefemales under normal conditions and under heat stress

The levels of JH-hydrolysing activity in 1-day-oldfemales ofTbhnM18 and Ste strains under normal andstress conditions are shown in Fig. 3, together with thatof Canton S. The data reveal that under normal con-ditions, the level of JH degradation in females ofTbhnM18

strain is significantly higher than that inCanton S(P,0.001). In contrast,Stefemales are distinguished bya lower level of JH-hydrolysing activity compared toCanton S(P,0.001). The data in Fig. 3 also show thatTbhnM18 and Ste females respond to heat stress as doCanton S: exposure to 38°C elicits in females of all threestrains a decrease in the level of JH degradation com-pared to control females (P,0.001).

Fig. 3. Hydrolysis of [3H]JH-III in 1-day-old females ofCanton S,TbhnM18 and Ste strains ofD. melanogasterunder normal and stress(38°C 3 h) conditions. Means±SE.

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3.4. JH degradation under normal and stressconditions in l-day-old females of wild type (bothCanton S and strain 921500) and laboratory (FM7,nand CyLDSb) strains

It can be seen in Fig. 4 that 1-day-old females ofCan-ton S strain are characterized by a level of JH degra-dation similar to that of the iso-female strain921500andof laboratory strainsFM7, n and CyLDSb, which haveno mutations relating with any components of the stressreaction (differences betweenCanton Sand other strainsare insignificant). Heat treatment of females of all thesestrains results in a significant (P,0.001) lowering of thelevel of JH degradation (compared to control femaleskept at 25°C).

3.5. JH degradation in 6-day-old Canton S and ts403females under normal conditions and under heat stress

Since JH degradation can controlDrosophila repro-duction under normal and heat stress conditions(Rauschenbach et al., 1996), we further measured thelevel of JH-hydrolysing activity in 6-day-old females ofCanton Sand ts403 strains. As seen in Fig. 5, undernormal conditions the JH-hydrolysing activity in maturets403 females does not differ from that ofCanton S.Females of both strains show lower JH degradation(0.01) after heat stress (38°C, 3 h).

3.6. JH degradation under normal and stressconditions in 5-day-oldn Met27 and n females

Under normal conditions, the level of JH degradationin 5-day-old n Met27 females is the same as that inn

Fig. 4. Hydrolysis of [3H]JH-III in l-day-old females of921500, Canton S, FM7, n and CyLDSbstrains ofD. melanogasterunder normal andstress (38°C, 3 h) conditions. Means±SE.

Fig. 5. Effect of short term heat stress (38°C, 3 h) on JH-hydrolysingactivity in 6-day-old females ofts403andCanton Sstrains ofD. mel-anogaster. Means±SE.

females. After heat stress, maturen Met27 females showno changes in the level of JH metabolism compared withmaturen females (Fig. 6) which respond to stress witha significant decrease in JH-hydrolysing activity(P,0.001).

3.7. JH degradation in 6-day old TbhnM18 and CantonS females under normal conditions and under heatstress

Under normal conditions, the level of JH degradationin females of theTbhnM18 strain is significantly higher

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Fig. 6. Effect of short term heat stress (38°C 3 h) on JH-hydrolyzingactivity in 5-day-old females ofn andn Met27 strains ofD. melanogas-ter. Means± SE.

(P,0.001) than that ofCanton S(Fig. 7). It is clear thatmatureTbhnM18 females respond to heat stress by a sharpdecrease in JH-hydrolysing activity (P,0.001).

3.8. The stress-reactivity of the JH degradation systemin young and mature D. melanogaster females

To characterize the stress-reactivity of the JH degra-dation system, we calculated the percent decrease of JH-hydrolysing activity for each stressed female relative tothe value under normal conditions (every experimentvalue was related to the average value for the controlgroup, since it is impossible to determine the JH-hydrolysing activity of the same individual under bothcontrol and stress conditions). As seen in Fig. 8, 1-day-old females of wild type (921500and Canton S) andlaboratory (FM7, n and CyLDSb) strains have similarstress-reactivity (the differences between strains are notsignificant). On the other hand, it is also apparent fromthe data of Fig. 8, that 1-day-old females having stress-related mutations (ts403, n Met27, TbhnM18 andSte) havelower levels of stress-reactivity (P,0.05 for ts403,P,0.01 forn Met27 andP,0.001 forTbhnM18 andSte).

We further analysed the stress-reactivity in maturefemales (6-day-oldCanton S, ts403andTbhnM18 strainsand 5-day-oldFM7, n and n Met27 strains). It is clearfrom the data in Fig. 9 that the stress-reactivity of matureTbhnM18 and ts403 females is significantly higher thanthat of wild type (Canton S) and laboratory (FM7 andn) strains (P,0.001 forts403andP,0.05 forTbhnM18).The stress-reactivity ofn Met27 females is insignificant.

Fig. 7. Effect of short term heat stress (38°C, 3 h) on JH-hydrolysingactivity in 6-day-old females ofTbhnM18 and Canton Sstrains ofD.melanogaster. Means±SE.

4. Discussion

In adult female insects, JH controls reproduction byregulation of the growth of previtellogenic and/or vitel-logenic follicles, maturation of ovaries, stimulation andmaintenance of vitellogenesis, uptake of vitellogeninsfrom hemolymph to oocytes, and oviposition (Shapiro etal., 1986; Roe et al., 1987; Adams and Filipi, 1988;Bownes 1989, 1994; Khlebodarova et al., 1996; Rausch-enbach et al., 1996; Soller et al., 1999). JH must bepresent at high levels to initiate maturation of ovariesand stimulate vitellogenesis, and then at lower levels tomaintain vitellogenesis (Jowett and Postlethwait, 1981;Raikhel and Lea, 1985; Postlethwait and Parker, 1987;Bownes, 1989; Soller et al., 1999).

For completion of normal egg development and forthe onset of oviposition, the JH titer must be decreasedin some insects (Riddiford, 1970; Temin et al., 1986;

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Fig. 8. Stress-reactivity of JH-degradation system in young females of921500, Canton S, FM7, n, CyLDSb, ts403, n Met27, TbhnM18 and Stestrains ofD. melanogaster. Means±SE.

Fig. 9. Stress-reactivity of JH degradation system in matured femalesof Canton S, FM7, n, ts403, n Met27 and TbhnM18 strains ofD. mel-anogaster. Means±SE.

Shapiro et al., 1986; Khlebodarova et al., 1996; Rausch-enbach et al., 1996; Soller et al., 1999). Then Met27

characteristics revealed earlier and in the present studyare consistent with this requirement. Indeed,nMet27 flieshave reduced oogenesis (Wilson and Ashok, 1998) anddecreased fertility (Gruntenko et al., 2000) under normal

conditions. It is possible thatn Met27 females have anelevated JH level resulting from decreased JH-hydrolys-ing activity (see Figs. 2 and 6) or that the impaired JHreception in this strain may prevent JH-titre-mediatedregulation of both the JH degradation system and oogen-esis. In both cases fertility would be disturbed.

We believe that our data obtained onTbhnM18 femalesin this study also agree with the idea that a high JH titreimpedes ovipositon. Earlier it was shown thatTbhnM18

females are sterile: although they mate, they retain fullydeveloped eggs (Monastirioti et al., 1996). It was sug-gested that this phenotype is connected with the fact thatin the absence of OA the function of oviductal muscleis impaired (Monastirioti et al., 1996) based on the find-ing that OA modulates activity of the oviductal musclein two orthopteran species (Kalogianni and Theophilidis,1993). The experiments of Thompson et al. (1990) dem-onstrated that OA inhibits JH biosynthesis in the adultfemale cockroach,Diploptera punctata. If a similar situ-ation exists inD. melanogaster, the absence of OA inTbhnM18 females would result in the increased JH syn-thesis. Such an absence of down regulation of JH pro-duction by OA should result in higher JH production,which would elicit an increase in JH-hydrolysing activityfor maintenance of the JH titre. Indeed,TbhnM18 femaleswere shown to have levels of JH degradation almosttwice those inCanton S(see Figs. 3 and 7), althoughthe increased JH-hydrolysing activity is not enough tolower the JH titer to alevel permitting oviposition. On

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the other hand, it is also possible that altered JH metab-olism in these flies is related to a hither to unidentifiedphenotype/process.

Evidence supporting the regulation of JH metabolismby biogenic amines inDrosophilacomes from our dataon Stestrain. The flies carryinge mutation are knownto have twice as much DA as wild type (Hodgetts, 1972;Hodgetts and Konopka, 1973; Ramadan et al., 1993).Because DA can influence JH secretion (Piulachs andBelles, 1989; Granger et al., 1996) we might expect theJH titre to be affected, with consequent changes in JH-hydrolysing activity in females ofStestrain. Indeed,Stefemales have a level of JH degradation almost a half thatof Canton S(see Fig. 3).

We cannot exclude the possibility that the differencesbetweenSte, TbhnM18 andCanton Sfemales in the levelof JH-hydrolysis are the result of strain polymorphism.However, four strains without any stress-relatedmutations (921500, FM7, n and CyLDSb) were exam-ined and were found to have JH-hydrolysing activitysimilar to that of Canton S, under normal conditions.Hence, the high JH-hydrolysis level inTbhnM18 femalesand low level inSte apparently result from the corre-sponding mutations.

The reason to investigate JH metabolism in thets403strain, with its impairment of HSR, was the existing evi-dence on the regulation of the expression of heat shockproteins (hsp) genes by the combined action of the hor-mones JH and 20-OH-ecdysone. Inhibition of the ecdys-teroid peak at pupariation by a temperature shift of theconditionally ecdysteroid-deficientD. melanogasterstrain ecd-1 results in a block of hsp26 RNA and adecline in hsp83 RNA level; subsequent addition ofexogenous 20-OH-ecdysone restores expression of bothgenes (Thomas and Lengyel, 1986). JH was reported toinhibit in a dose-dependent manner the ecdysteroneinduction of the small hsp genes ofDrosophila,expressed in cultured cells (Berger et al., 1992). Our datarevealed that the block in HSR expression does not affectJH degradation under normal conditions (see Figs. 1and 5).

How do the mutations in the different components ofthe D. melanogasterstress-reaction effect the stress-reactivity of the JH degradation system? We have pre-viously demonstrated that exposure ofDrosophilafemales to stress results in a sharp decrease of the JH-hydrolysing activity and as a consequence, the onset ofoviposition by young females is delayed 24 h, whilemature females cease oviposition for two days(Rauschenbach et al. 1995, 1996). We have also shownthat in certainD. virilis andD. melanogasterstrains thatdo not respond to stress, the level of JH-hydrolysis infemales was significantly lower compared to that of wildtype. This level does not alter upon heat stress(Rauschenbach et al. 1995, 1996; Gruntenko et al.,1999).

As the present data show, each of the mutations stud-ied elicits some alteration in the stress reactivity of theJH degradation system. Mature females of wild type andlaboratory strains without any disturbances in theirstress-reaction demonstrate significantly lower stress-reactivity than younger ones (see Figs. 8 and 9). In con-trast, matureTbhnM18 females demonstrate higher stress-reactivity than younger ones. Moreover, this responsediffers from that in wild type: in mature females it ishigher (see Fig. 9) and in young ones, lower (see Fig.8). The stress-reactivity ints403 females does notchange with age in contrast to wild-type (see Figs. 8and 9). YoungStefemales demonstrate the lowest stress-reactivity (see Fig. 8). Youngn Met27 females also havedecreased stress-reactivity compared toMet27 flies of thesame age (see Fig. 8). Maturen Met27 females show themost essential differencies from wild type: their stress-reactivtity is insignificant (see Fig. 9).

In summary this work suggests that JH may play thekey role in the development of the insect stress-reaction.

Acknowledgements

This study was supported by grants from the RussianFundamental Research Foundation and the SiberianBranch of the Russian Academy of Sciences for YoungProminent Scientists. Dr. Gruntenko was the recipient ofa travel award from the Organizing Committee of theSeventh International Conference on the Juvenile Hor-mones.

References

Adams, T.S., Filipi, P.A., 1988. Interaction between juvenile hormone,20-hydroxyecdysone, thecorpus cardiacum–allatumcomplex, andthe ovaries in regulating vitellogenin levels in the housefly,Muscadomestica. Journal of Insect Physiology 34, 11–19.

Arking, R., 1975. Temperature sensitive cell-lethal mutants ofDroso-phila: Isolation and characterisation. Genetics 80, 519–523.

Berger, E.M., Goudie, K., Klieger, L., Berger, M., DeCato, R., 1992.The juvenile hormone analogue, methoprene, inhibits ecdisteroneinduction of small heat shock protein gene expression. Develop-mental Biology 151, 410–418.

Bownes, M., 1989. The roles of juvenile hormone, ecdysone and theovary in the control ofDrosophila vitellogenesis. Journal of InsectPhysiology 35, 409–413.

Bownes, M., 1994. The regulation of yolk-protein genes, a family ofsex differentiation genes inDrosophila melanogaster. BioEssays16, 745–752.

Cymborowski, B., 1991. Effects of cold stress on endocrice system inGalleria melonella. In: Ivanovic, J., Jankovic-Hladni, M. (Eds.),Hormones and Metabolism in Insect Stress. CRC Press, BocaRaton, FL, pp. 99–114.

Davenport, A.K., Evans, P.D., 1984. Stress-induced changes in octopa-mine levels of insect haemolymph. Insect Biochemistry 14, 135–143.

Evgen’ev, M.B., Denisenko, O.M., 1990. The influence of ts mutationon heat-shock-inducible genes inDrosophila melanogaster. III.

782 N.E. Gruntenko et al. / Insect Biochemistry and Molecular Biology 30 (2000) 775–783

Expression of HSP70 cognate proteins. Russian Journal of Genetics26, 266–271.

Granger, N.A., Sturgis, S.L., Ebersohl, R., Geng, C., Sparks, T.C.,1996. Dopaminergic control of corpora allata activity in the larvaltobacco hornworm,Manduca sexta. Archives of Insect Biochemis-try and Physiology 32, 449–466.

Gruntenko, N.E., Khlebodarova, T.M., Sukhanova, M.JH., Vasenkova,I.A., Kaidanov, L.Z., Rauschenbach, I.YU., 1999. Prolonged nega-tive selection ofDrosophila melanogasterfor a character of adapt-ive significance disturbs stress reactivity. Insect Biochemistry andMolecular Biology 29, 445–452.

Gruntenko, N.E., Khlebodarova, T.M., Vasenkova, I.A., Sukhanova,M.J., Wilson, T.G., Rauschenbach, I.Y., 2000. Stress-reactivity ofa Drosophila melanogasterstrain with impaired juvenile hormoneaction. Insect Physiology 46, 451–456.

Hammock, B.D., Sparks, T.C., 1977. A rapid assay for insect juvenilehormone esterase activity. Analytical Biochemistry 82, 573–579.

Hirashima, A., Nagano, T., Takeya, R., Eto, M., 1993. Effect of larvaldensity on whole-body biogenic amine levels ofTribolium free-mani Hinton. Comparative Biochemical Physiology 106C, 457–461.

Hirashima, A., Sukhanova, M.JH., Kuwano, E., Rauschenbach, I.YU.,1999. Alteration of biogenic amines inDrosophila virilis understress. Drosophila Information Service 82, 30–31.

Hodgetts, R.B., 1972. Biochemical characterization of mutants affect-ing the metabolism ofβ-alanin in Drosophila. Journal of InsectPhysiology 18, 937–947.

Hodgetts, R.B., Konopka, R.J., 1973. Tyrosine and catecholaminemetabolism in wild-typeDrosophila melanogasterand a mutant,ebony. Journal of Insect Physiology 19, 1211–1220.

Jowett, T., Postlethwait, J., 1981. Hormonal regulation of synthesis ofyolk proteins and a larvae serum protein (LSP) inDrosophila. Nat-ure 292, 633–635.

Kalogianni, E., Theophilidis, G., 1993. Centrally generatedrhythmicactivity and modulatory function of the oviductal dorsal unpairedmedian (DUM) neurones in two orthopteran species (CalliptamusSP. andDecticus albifrons). Journal of Experimental Biology 174,123–138.

Khlebodarova, T.M., Ankilova, I.A., Gruntenko, N.E., Sukhanova,M.ZH., Rauschenbach, I.YU., 1998. The impairments in Droso-phila stress reaction correlates with the alterations in heat shockresponse. Dokl. Akademia Nauk (Russia) 361, 184–l86.

Khlebodarova, T.M., Gruntenko, N.E., Grenback, L.G., Sukhanova,M.Z., Mazurov, M.M., Tomas, B.A., Hammock, B.D., Rauschen-bach, I.Y., 1996. A comparative analysis of juvenile hormone meta-bolyzing enzymes in two species ofDrosophila during develop-ment. Insect Biochemistry and Molecular Biology 26, 829–835.

Monastirioti, M., Linn, C.E. Jr., White, K., 1996. Characterization ofDrosophila Tyramineb-hydroxylasegene and isolation of mutantflies lacking octopamine. Journal of Neuroscience 16, 3900–3911.

Nijhout, F., 1994. Insect Hormones. Princeton University Press, Prin-ceton, NJ.

Orchard, I., Loughton, B.G., 1981. Octopamine and short-term hyperli-paemia in the locust. Genetic and Comparative Endocrinology 45,175–180.

Piulachs, M.D., Belles, H., 1989. Stimulatory activity of cycteamineon juvenile hormone release in adult females of the cockroach,Blattela germanica. Comparative Biochemistry and Physiology94A, 795–798.

Postlethwait, J.H., Parker, J., 1987. Regulation of vitellogenesis inDrosophila. In: O’Connor, J.D., Alan, R. (Eds.), Molecular Biologyof Invertebrate Development. Liss Inc, New York, pp. 29–42.

Raikhel, A.S., Lea, A.O., 1985. Hormone-mediated formation of theendocytic complex in mosquito oocytes. Genetics and ComparativeEndocrinology 57, 422–423.

Ramadan, H., Alawi, A.A., Alawi, M.A., 1993. Catecholamines in

Drosophila melanogaster(wild type and ebony mutant) decuticala-rized retinas and brains. Cell Biology International 17, 765–771.

Rauschenbach, I.Y., 1991. Changes in ecdysteroid and juvenile hor-mone under heat stress. In: Ivanovic, J., Jankovic-Hladni, M. (Eds.)Hormones and Metabolism in Insect Stress. CRC Press, BocaRaton, FL, pp. 115–148.

Rauschenbach, I.YU., 1997. Stress response in insects: mechanism,genetic control, and role in adaptation. Russian Journal of Genetics33, 942–949.

Rauschenbach, I.Y., Lukashina, N.S., Maksimovsky, L.F., Korochkin,L.I., 1987. Stress-like reaction ofDrosophila to adverse environ-mental factors. Journal of Comparative Physiology 157, 519–531.

Rauschenbach, I.Y., Serova, L.I., Timochina, I.S., Chentsova, N.A.,Schumnaja, L.V., 1993. Analysis of differences in dopamine con-tent between two lines ofDrosophila virilis in response to heatstress. Journal of Insect Physiology 39, 761–767.

Rauschenbach, I.Y., Khlebodarova, T.M., Chentsova, N.A., Grun-tenko, N.E., Grenback, L.G., Yantsen, E.I., Filipenko, M.L., 1995.etabolism of the juvenile hormone inDrosophilaadults under nor-mal conditions and heat stress. Journal of Insect Physiology 41,179–189.

Rauschenbach, I.YU., Gruntenko, N.E., Khlebodarova, T.M.,Mazurov, M.M., Grenback, L.G., Sukhanova, M.JH., Shumnaja,L.V., Zakharov, I.K., Hammock, B.D., 1996. The role of the degra-dation system of the juvenile hormone in the reproduction ofDro-sophilaunder stress. Journal of Insect Physiology 42, 735–742.

Rauschenbach, I.Y., Sukhanova, M.Z., Shumnaya, L.V., Gruntenko,N.E., Grenback, L.G., Khlebodarova, T.M., Chentsova, N.A., 1997.Role of dopa decarboxylase and N-acetyltransferase in regulationof dopamine content inDrosophila virilis under normal and heatstress conditions. Insect Biochemistry and Molecular Biology 27,829–835.

Riddiford, L.M., 1970. Effects of juvenile hormone on the program-ming of postembryonic development in eggs of the silkworm,Hyla-phora cecropia. Developmental Biology 22, 249–263.

Riddiford, L.M., Ashburner, M., 1991. Role of juvenile hormone inlarval development and metamorphosis inDrosophila melanogas-ter. Genetics and Comparative Endocrinology 82, 172–183.

Roe, R.M., Crawford, C.L., Clifford, C.W., Woodring, J.P., Sparks,T.C., Hammock, B.D., 1987. Role of juvenile hormone metabolismduring embryogenesis of the house cricket,Acheta domesticus.Insect Biochemistry 17, 1023–1026.

Shapiro, A.B., Wheelock, G.D., Hagedorn, H.H., Baker, F.C., Tsai,L.W., Schooly, D.A., 1986. Juvenile hormone and juvenile hor-mone esterase in adult females of the mosquitoAedes aegypti. Jour-nal of Insect Physiology 32, 867–885.

Shemshedini, L., Wilson, T.G., 1990. Resistance to juvenile hormoneand in insect growth regulator inDrosophila is associated with analtered cytosolic juvenile hormone binding protein. Proceedings ofthe National Academy of Science USA 87, 2072–2076.

Soller, M., Bownes, M., Kubli, E., 1999. Control of oocyte maturationin sexually matureDrosophila females. Developmental Biology208, 337–351.

Sukhanova, M.JH., Shumnaya, L.V., Grenback, L.G., Gruntenko, N.E.,Khlebodarova, T.M., Rauschenbach, I.Y., 1997. Tyrosine decar-boxylase and dopa decarboxylase inDrosophila virilis under heatstress. Biochemical Genetics 35, 91–103.

Temin, G., Zander, M., Roussel, J.-P., 1986. Physico-chemical (GC–MS) measurements of juvenile hormone III titres duringembryogenesis ofLocusta migratoria. International Journal ofInvertebrate Reproductive Development 9, 105–112.

Thomas, S.R., Lengyel, J.A., 1986. Ecdysteroid-regulated heat-shockgene expression duringDrosophila melanogasterdevelopment.Developmental Biology 115, 434–438.

Thompson, C.S., Yagi, K.J., Chen, Z.F., Tobe, S.S., 1990. The effectsof octopamine on juvenile hormone biosynthesis, electrophysiol-ogy, and cAMP content of the corpora allata of the cockroach

783N.E. Gruntenko et al. / Insect Biochemistry and Molecular Biology 30 (2000) 775–783

Diploptera punctata. Journal of Comparative Physiology B 160,241–249.

Wilson, T.G., Ashok, M., 1998. Insecticide resistance resulting froman absence of target-sitegene product. Proceedings of the NationalAcademy of Science USA 95, 14040–14044.

Wilson, T.G., Fabian, G., 1986. ADrosophila melanogestermutantresistant to a chemical analog of juvenile hormone. DevelopmentalBiology 118, 190–201.

Woodring, J.P., McBride, L.A., Fields, P., 1989. The role of octopam-ine in handling and exercise-induced hyperglycaemia and hyperli-paemia inAcheta domesticus. Journal of Insect Physiology 41,613–617.

Wyatt, G.R., Davey, K.G., 1996. Cellular and molecular actions ofjuvenile hormone. II. roles of juvenile hormone in adult insects.Advances in Insect Physiology 26, 1–155.