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www.elsevier.com/locate/ibmb

Stress-reactivity and juvenile hormone degradation in

Drosophila

melanogaster

strains having stress-related mutations

N.E. Gruntenko

a,*

_{, T.G. Wilson}

_{, M. Monastirioti}

_{, I.Y. Rauschenbach}

a_{Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Division, Novosibirsk 630090, Russia} b_{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 ofDrosophila melanogaster strains having mutations in different genes involved in responses to stress It was shown that (1) the impairment in heat shock response elicits an alteration in stress-reactivity of the JH system; (2) the impairment JH reception causes a decrease of 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 degradation system to stress. The role of JH in the development of the insect stress reaction is discussed. 2000 Elsevier Science Ltd. All rights reserved.

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

1. Introduction

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

tem of wild type females of Drosophila melanogaster andD. virilisresponds to stress conditions (termed here stressors) 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, by an increase in the amine content and by a decrease in the activity of their synthetic enzymes (Rauschenbach et al., 1993; Hirashima et al., 1999). We have also demon-strated that a mutation disturbing the development of the stress reaction inD. virilisalso elicits the impairment of HSR (Khlebodarova et al., 1998).

How do impairments of the different components of the stress reaction, such as HSR and the metabolism of DA and OA, affect JH metabolism in D. melanogaster females under normal and stress conditions? Previous work has demonstrated that biogenic amines are involved in the regulation of JH biosynthesis and secretion by the corpora allata and that the expression of some HSR genes is JH dependent (Piulachs and Belles, 1989; Thompson et al., 1990; Berger et al., 1992; Granger et al., 1996).

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

is a null allele of the Methoprene-tolerant gene that shows resistance to the toxic effects of both JH and a JH analog, methoprene. The mechanism of the resistance appears to be altered JH reception(Wilson and Fabian, 1986; Shemshedini and Wilson, 1990).Met27_completely

lacks Met transcript and is clearly a null allele (Wilson and Ashok, 1998).TbhnM18_{is a null mutation at the} Tyr-amine b-hydroxylase locus, which results in complete absence of the tyramine β-hydroxylase protein and blockage of octopamine biosynthesis (Monastirioti et al., 1996). eis postulated to be the mutation of N-b-alanyl dopamine synthetase gene, based on the fact that e has twice as much DA as normal (Hodgetts, 1972; Hodgetts and Konopka, 1973; Ramadan et al., 1993).

Here we asked whether these mutations would affect the decrease in JH degradation occurring in D. mel-anogaster when stressed. In order to answer this ques-tion, we studied the JH degradation in individuals of ts403,nMet27_{, T}_b_hnM18_and_Ste_{strains (carring}_l(l)ts403, Met27_{, T}_b_h_and_e_{mutations, respectively), under normal} and stress conditions, and compared their stress-reac-tivity (calculated as percent change in JH hydrolysis under stress compared to hydrolysis under normal conditions) with that in a number of wild type and lab-oratory strains.

We demonstrated (1) that ts403 females respond to stress by a decrease in JH degradation, as occurs in wild type females, but that their stress-reactivity significantly differs from that of wild type; (2) that in youngv Met27 females, similar to wild type flies, JH hydrolysis is decreased upon stress, but their stress-reactivity is sig-nificantly lower than in wild type; (3) that JH degra-dation is unaffected in older v Met27 _{females under} stress; (4) that TbhnM18 _{females show a significantly} higher JH-hydrolysis level and different stress-reactivity than does the wild type; and (5) that youngSte females demonstrate significantly lower JH-hydrolysis and stress-reactivity, compared to the wild type.

2. Materials and methods

2.1. Drosophila strains

The followingD. melanogasterstrains were used: the wild type laboratory strain Canton S; wild type iso-female strain 921500 from a natural population of Gorno-Altaisk; laboratory balancer strainFirst Multiple Seven (FM7); vermilion (n) strain from which the n

Met27 _{strain was derived; laboratory balancer strain}

In(2LR)Cy/L; In(3LR)D/Sb, carrying morphological mutations with recessive lethal action Curly, Lobe (chromosome 2) andDichaete, Stubble(chromosome 3; hereafter termed CyLDSb); strain ts403 carrying the recessive temperature sensitive lethal mutationl(l)ts403 (Arking, 1975); strain n Met27 _{carrying a null allele of} the Methoprene-tolerant gene (Wilson and Ashok, 1998); strainTbhnM18_{carrying a null mutation at the} Tyr-amine b-hydroxylase locus (Monastirioti et al., 1996); and the laboratory Ste strain carrying the e mutation. Cultures were raised on standard medium (Rauschenbach et al., 1987) at 25°C, and adults were synchronized by eclosion. Flies were subjected to stress at 38°C for 3 h, and were subsequently frozen in liquid nitrogen 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 ice in 30µl 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 the supernatant (10µl) were utilized for the reaction. A mix-ture consisting of 0.1 µg unlabeled JH-III (Sigma) and 12,500 dpm 3_{H 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 tubes in 100 µl of incubation mixture for 3 h, and it was stopped by the addition of 250µl heptane and 50 µl of a 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 (100 µl) of both aqueous and heptane phases were placed in vials containing dioxane scintillation fluid and counted. Con-trol experiments have shown a linear substrate–reaction relationship (Gruntenko et al., 1999), as well as the fact that measured activity is proportional to homogenate (i.e. enzyme) concentration (Rauschenbach, 1991; unpub-lished data).

The significance of the differences between the data sets was tested by Student’s t-test. Sample size varied from 12 to 28 individuals for each measurement in all experiments.

3. Results

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

JH-hydrolysing activity in 1-day old females of strains Canton S andts403under normal and stress conditions are shown in Fig. 1. The data indicate that under normal conditions, JH-hydrolysing activity in ts403 females

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Fig. 1. Hydrolysis of [3_{H]JH-III in 1-day-old females of}_{Canton S}

andts403strains ofD. melanogasterunder normal and stress (38°C, 3 h) conditions. Means±SE.

does not differ from that in Canton Sones. The data of Fig. 1 also demonstrate that ts403 females respond to stress as well as Canton Sdo: exposure to 38°C evokes in females of both strains a significant (P_,0.001) decrease in JH-hydrolysing activity compared to control females maintained at 25°C.

3.2. JH degradation in 1-day old n Met27 _and_n females under normal conditions and under heat stress

JH-hydrolysis in 1-day old females of both n and n

Met27 _{strains under normal and stress conditions are} shown in Fig. 2. They indicate that under normal con-ditions n Met27 _{females show a significantly (}_P

,0.01)

Fig. 2. Hydrolysis of [3_{H]JH-III in 1-day-old females of} _n _and _n Met27_{strains of}_{D. melanogaster}_{under normal and stress (38}°_{C 3 h)}

conditions. Means±SE.

lower JH-hydrolysing activity than do n females. Exposure to 38°C causes females of both n and Met27 strains to show a significant (P,0.001) decrease in JH-hydrolysis level, compared to control females kept at 25°C.

3.3. JH degradation in 1-day-old TbhnM18 _{and Ste} females under normal conditions and under heat stress

The levels of JH-hydrolysing activity in 1-day-old females of TbhnM18 _and _Ste _{strains under normal and} stress conditions are shown in Fig. 3, together with that of Canton S. The data reveal that under normal con-ditions, the level of JH degradation in females ofTbhnM18 strain is significantly higher than that in Canton S (P,0.001). In contrast,Stefemales are distinguished by a lower level of JH-hydrolysing activity compared to Canton S (P,0.001). The data in Fig. 3 also show that TbhnM18 _and

Ste females respond to heat stress as do Canton S: exposure to 38°C elicits in females of all three strains a decrease in the level of JH degradation com-pared to control females (P_,0.001).

Fig. 3. Hydrolysis of [3_{H]JH-III in 1-day-old females of}_{Canton S}_, TbhnM18_and_Ste _{strains of}_{D. melanogaster}_{under normal and stress}

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3.4. JH degradation under normal and stress conditions in l-day-old females of wild type (both Canton S and strain 921500) and laboratory (FM7, n

and CyLDSb) strains

It can be seen in Fig. 4 that 1-day-old females of Can-ton S strain are characterized by a level of JH degra-dation similar to that of the iso-female strain921500and of laboratory strains FM7, n and CyLDSb, which have no mutations relating with any components of the stress reaction (differences betweenCanton Sand other strains are insignificant). Heat treatment of females of all these strains results in a significant (P_,0.001) lowering of the level of JH degradation (compared to control females kept at 25°C).

3.5. JH degradation in 6-day-old Canton S and ts403 females under normal conditions and under heat stress

Since JH degradation can control Drosophila repro-duction under normal and heat stress conditions (Rauschenbach et al., 1996), we further measured the level of JH-hydrolysing activity in 6-day-old females of Canton S and ts403 strains. As seen in Fig. 5, under normal conditions the JH-hydrolysing activity in mature ts403 females does not differ from that of Canton 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 stress conditions in 5-day-old n Met27_and _n_females

Under normal conditions, the level of JH degradation in 5-day-old n Met27 _{females is the same as that in} _n

Fig. 4. Hydrolysis of [3_{H]JH-III in l-day-old females of}₉₂₁₅₀₀_,_{Canton S}_,_FM7_,_n_and_CyLDSb_{strains of}_{D. melanogaster}_{under normal and}

stress (38°C, 3 h) conditions. Means±SE.

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

females. After heat stress, maturenMet27_{females show} no changes in the level of JH metabolism compared with mature nfemales (Fig. 6) which respond to stress with a significant decrease in JH-hydrolysing activity (P,0.001).

3.7. JH degradation in 6-day old TbhnM18 _{and Canton} S females under normal conditions and under heat stress

Under normal conditions, the level of JH degradation in females of the TbhnM18 _{strain is significantly higher}

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Fig. 6. Effect of short term heat stress (38°C 3 h) on JH-hydrolyzing activity in 5-day-old females ofnandnMet27_{strains of}_D. melanogas-ter. Means±SE.

(P_,0.001) than that ofCanton S(Fig. 7). It is clear that matureTbhnM18_{females respond to heat stress by a sharp} decrease in JH-hydrolysing activity (P_,0.001).

3.8. The stress-reactivity of the JH degradation system in 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 to the value under normal conditions (every experiment value was related to the average value for the control group, since it is impossible to determine the JH-hydrolysing activity of the same individual under both control and stress conditions). As seen in Fig. 8, 1-day-old females of wild type (921500 and Canton S) and laboratory (FM7, n and CyLDSb) strains have similar stress-reactivity (the differences between strains are not significant). On the other hand, it is also apparent from the data of Fig. 8, that 1-day-old females having stress-related mutations (ts403,nMet27_,_T_b_hnM18_and_Ste_{) have} lower levels of stress-reactivity (P,0.05 for ts403, P,0.01 fornMet27_and_P_{,0.001 for}_T_b_hnM18_and_Ste_). We further analysed the stress-reactivity in mature females (6-day-old Canton S, ts403andTbhnM18_strains and 5-day-old FM7, n and n Met27 _{strains). It is clear} from the data in Fig. 9 that the stress-reactivity of mature TbhnM18 _and _ts403 _{females is significantly higher than} that of wild type (Canton S) and laboratory (FM7 and n) strains (P,0.001 forts403andP,0.05 forTbhnM18_). The stress-reactivity of n Met27_{females is insignificant.}

Fig. 7. Effect of short term heat stress (38°C, 3 h) on JH-hydrolysing activity in 6-day-old females ofTbhnM18_and _{Canton S}_{strains of}_D. melanogaster. Means±SE.

4. Discussion

In adult female insects, JH controls reproduction by regulation of the growth of previtellogenic and/or vitel-logenic follicles, maturation of ovaries, stimulation and maintenance of vitellogenesis, uptake of vitellogenins from hemolymph to oocytes, and oviposition (Shapiro et al., 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 be present at high levels to initiate maturation of ovaries and stimulate vitellogenesis, and then at lower levels to maintain 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 for the onset of oviposition, the JH titer must be decreased in some insects (Riddiford, 1970; Temin et al., 1986;

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Fig. 8. Stress-reactivity of JH-degradation system in young females of 921500, Canton S,FM7,n, CyLDSb, ts403,nMet27_,_T_b_hnM18_and_Ste

strains ofD. melanogaster. Means±SE.

Fig. 9. Stress-reactivity of JH degradation system in matured females of Canton S,FM7,n,ts403,nMet27_and_T_b_hnM18_{strains of}_D. mel-anogaster. Means±SE.

Shapiro et al., 1986; Khlebodarova et al., 1996; Rausch-enbach et al., 1996; Soller et al., 1999). The n Met27 characteristics revealed earlier and in the present study are consistent with this requirement. Indeed,nMet27_flies have reduced oogenesis (Wilson and Ashok, 1998) and decreased fertility (Gruntenko et al., 2000) under normal

conditions. It is possible that n Met27 _{females have an} elevated JH level resulting from decreased JH-hydrolys-ing activity (see Figs. 2 and 6) or that the impaired JH reception in this strain may prevent JH-titre-mediated regulation of both the JH degradation system and oogen-esis. In both cases fertility would be disturbed.

We believe that our data obtained onTbhnM18_females in this study also agree with the idea that a high JH titre impedes ovipositon. Earlier it was shown that TbhnM18 females are sterile: although they mate, they retain fully developed eggs (Monastirioti et al., 1996). It was sug-gested that this phenotype is connected with the fact that in the absence of OA the function of oviductal muscle is impaired (Monastirioti et al., 1996) based on the find-ing that OA modulates activity of the oviductal muscle in two orthopteran species (Kalogianni and Theophilidis, 1993). The experiments of Thompson et al. (1990) dem-onstrated that OA inhibits JH biosynthesis in the adult female cockroach,Diploptera punctata. If a similar situ-ation exists in D. melanogaster, the absence of OA in TbhnM18 _{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 propro-duction, which would elicit an increase in JH-hydrolysing activity for maintenance of the JH titre. Indeed,TbhnM18_females were shown to have levels of JH degradation almost twice those in Canton S (see Figs. 3 and 7), although the increased JH-hydrolysing activity is not enough to lower 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 unidentified phenotype/process.

Evidence supporting the regulation of JH metabolism by biogenic amines in Drosophila comes from our data on Ste strain. The flies carrying e mutation are known to 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 and Belles, 1989; Granger et al., 1996) we might expect the JH titre to be affected, with consequent changes in JH-hydrolysing activity in females ofStestrain. Indeed,Ste females have a level of JH degradation almost a half that of Canton S(see Fig. 3).

We cannot exclude the possibility that the differences between Ste,TbhnM18_and_{Canton S}_{females in the level} of JH-hydrolysis are the result of strain polymorphism. However, four strains without any stress-related mutations (921500, FM7, n and CyLDSb) were exam-ined and were found to have JH-hydrolysing activity similar to that of Canton S, under normal conditions. Hence, the high JH-hydrolysis level in TbhnM18 _females and low level in Ste apparently result from the corre-sponding mutations.

The reason to investigate JH metabolism in the ts403 strain, with its impairment of HSR, was the existing evi-dence on the regulation of the expression of heat shock proteins (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 the conditionally ecdysteroid-deficient D. melanogaster strain ecd-1 results in a block of hsp26 RNA and a decline in hsp83 RNA level; subsequent addition of exogenous 20-OH-ecdysone restores expression of both genes (Thomas and Lengyel, 1986). JH was reported to inhibit in a dose-dependent manner the ecdysterone induction of the small hsp genes of Drosophila, expressed in cultured cells (Berger et al., 1992). Our data revealed that the block in HSR expression does not affect JH degradation under normal conditions (see Figs. 1 and 5).

How do the mutations in the different components of the D. melanogaster reaction effect the stress-reactivity of the JH degradation system? We have pre-viously demonstrated that exposure of Drosophila females to stress results in a sharp decrease of the JH-hydrolysing activity and as a consequence, the onset of oviposition by young females is delayed 24 h, while mature females cease oviposition for two days (Rauschenbach et al. 1995, 1996). We have also shown that in certainD. virilisandD. melanogasterstrains that do not respond to stress, the level of JH-hydrolysis in females was significantly lower compared to that of wild type. 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 the JH degradation system. Mature females of wild type and laboratory strains without any disturbances in their 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 response differs from that in wild type: in mature females it is higher (see Fig. 9) and in young ones, lower (see Fig. 8). The stress-reactivity in ts403 females does not change with age in contrast to wild-type (see Figs. 8 and 9). YoungStefemales demonstrate the lowest stress-reactivity (see Fig. 8). YoungnMet27_{females also have} decreased stress-reactivity compared toMet27_{flies of the} same age (see Fig. 8). MaturenMet27_{females show the} most essential differencies from wild type: their stress-reactivtity is insignificant (see Fig. 9).

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

Acknowledgements

This study was supported by grants from the Russian Fundamental Research Foundation and the Siberian Branch of the Russian Academy of Sciences for Young Prominent Scientists. Dr. Gruntenko was the recipient of a travel award from the Organizing Committee of the Seventh International Conference on the Juvenile Hor-mones.

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Rauschenbach, I.Y., Lukashina, N.S., Maksimovsky, L.F., Korochkin, L.I., 1987. Stress-like reaction ofDrosophilato 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 of Drosophila virilisin response to heat stress. 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 of Dro-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 regulation of dopamine content inDrosophila virilisunder normal and heat stress 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 in larval 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 metabolism during 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 hormone and in insect growth regulator inDrosophilais associated with an altered cytosolic juvenile hormone binding protein. Proceedings of the National Academy of Science USA 87, 2072–2076.

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3.4. JH degradation under normal and stress

conditions in l-day-old females of wild type (both

Canton S and strain 921500) and laboratory (FM7,

n

and CyLDSb) strains

It can be seen in Fig. 4 that 1-day-old females of

Can-ton S

strain are characterized by a level of JH

degra-dation similar to that of the iso-female strain

921500

and

of laboratory strains

FM7

,

n

and

CyLDSb

, which have

no mutations relating with any components of the stress

reaction (differences between

Canton S

and other strains

are insignificant). Heat treatment of females of all these

strains results in a significant (

P

_,

0.001) lowering of the

level of JH degradation (compared to control females

kept at 25

°

C).

3.5. JH degradation in 6-day-old Canton S and ts403

females under normal conditions and under heat stress

Since JH degradation can control

Drosophila

repro-duction under normal and heat stress conditions

(Rauschenbach et al., 1996), we further measured the

level of JH-hydrolysing activity in 6-day-old females of

Canton S

and

ts403

strains. As seen in Fig. 5, under

normal conditions the JH-hydrolysing activity in mature

ts403

females does not differ from that of

Canton 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 stress

conditions in 5-day-old

n

Met

_and

_n

_females

Under normal conditions, the level of JH degradation

in 5-day-old

n

Met

_{females is the same as that in}

_n

Fig. 4. Hydrolysis of [3_{H]JH-III in l-day-old females of}₉₂₁₅₀₀_,_{Canton S}_,_FM7_,_n_and_CyLDSb_{strains of}_{D. melanogaster}_{under normal and}

stress (38°C, 3 h) conditions. Means±SE.

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

females. After heat stress, mature

n

Met

_{females show}

no changes in the level of JH metabolism compared with

mature

n

females (Fig. 6) which respond to stress with

a

significant

decrease

in

JH-hydrolysing

activity

(

P

,

0.001).

3.7. JH degradation in 6-day old T

b

h

nM18

_{and Canton}

S females under normal conditions and under heat

stress

Under normal conditions, the level of JH degradation

in females of the

T

b

h

nM18

_{strain is significantly higher}

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Fig. 6. Effect of short term heat stress (38°C 3 h) on JH-hydrolyzing activity in 5-day-old females ofnandnMet27_{strains of}_D.

melanogas-ter. Means±SE.

(

P

_,

0.001) than that of

Canton S

(Fig. 7). It is clear that

mature

T

b

h

nM18

_{females respond to heat stress by a sharp}

decrease in JH-hydrolysing activity (

P

_,

0.001).

3.8. The stress-reactivity of the JH degradation system

in 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 to

the value under normal conditions (every experiment

value was related to the average value for the control

group, since it is impossible to determine the

JH-hydrolysing activity of the same individual under both

control and stress conditions). As seen in Fig. 8,

1-day-old females of wild type (

921500

and

Canton S

) and

laboratory (

FM7

,

n

and

CyLDSb

) strains have similar

stress-reactivity (the differences between strains are not

significant). On the other hand, it is also apparent from

the data of Fig. 8, that 1-day-old females having

stress-related mutations (

ts403

,

n

Met

_,

_T

_b

_h

nM18

_and

_Ste

_{) have}

lower levels of stress-reactivity (

P

,

0.05 for

ts403

,

P

,

0.01 for

n

Met

_and

_P

_,

_{0.001 for}

_T

_b

_h

nM18

_and

_Ste

_).

We further analysed the stress-reactivity in mature

females (6-day-old

Canton S

,

ts403

and

T

b

h

nM18

_strains

and 5-day-old

FM7

,

n

and

n

Met

_{strains). It is clear}

from the data in Fig. 9 that the stress-reactivity of mature

T

b

h

nM18

_and

_ts403

_{females is significantly higher than}

that of wild type (

Canton S

) and laboratory (

FM7

and

n

) strains (

P

,

0.001 for

ts403

and

P

,

0.05 for

T

b

h

nM18

_).

The stress-reactivity of

n

Met

_{females is insignificant.}

Fig. 7. Effect of short term heat stress (38°C, 3 h) on JH-hydrolysing activity in 6-day-old females ofTbhnM18_and _{Canton S}_{strains of}_D.

melanogaster. Means±SE.

4. Discussion

In adult female insects, JH controls reproduction by

regulation of the growth of previtellogenic and/or

vitel-logenic follicles, maturation of ovaries, stimulation and

maintenance of vitellogenesis, uptake of vitellogenins

from hemolymph to oocytes, and oviposition (Shapiro et

al., 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 be

present at high levels to initiate maturation of ovaries

and stimulate vitellogenesis, and then at lower levels to

maintain 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 for

the onset of oviposition, the JH titer must be decreased

in some insects (Riddiford, 1970; Temin et al., 1986;

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Fig. 8. Stress-reactivity of JH-degradation system in young females of 921500, Canton S,FM7,n, CyLDSb, ts403,nMet27_,_TbhnM18_and_Ste

strains ofD. melanogaster. Means±SE.

Fig. 9. Stress-reactivity of JH degradation system in matured females of Canton S,FM7,n,ts403,nMet27_and_TbhnM18_{strains of}_D.

mel-anogaster. Means±SE.

Shapiro et al., 1986; Khlebodarova et al., 1996;

Rausch-enbach et al., 1996; Soller et al., 1999). The

n

Met

characteristics revealed earlier and in the present study

are consistent with this requirement. Indeed,

n

Met

_flies

have reduced oogenesis (Wilson and Ashok, 1998) and

decreased fertility (Gruntenko et al., 2000) under normal

conditions. It is possible that

n

Met

_{females have an}

elevated JH level resulting from decreased

JH-hydrolys-ing activity (see Figs. 2 and 6) or that the impaired JH

reception in this strain may prevent JH-titre-mediated

regulation of both the JH degradation system and

oogen-esis. In both cases fertility would be disturbed.

We believe that our data obtained on

T

b

h

nM18

_females

in this study also agree with the idea that a high JH titre

impedes ovipositon. Earlier it was shown that

T

b

h

nM18

females are sterile: although they mate, they retain fully

developed eggs (Monastirioti et al., 1996). It was

sug-gested that this phenotype is connected with the fact that

in the absence of OA the function of oviductal muscle

is impaired (Monastirioti et al., 1996) based on the

find-ing that OA modulates activity of the oviductal muscle

in two orthopteran species (Kalogianni and Theophilidis,

1993). The experiments of Thompson et al. (1990)

dem-onstrated that OA inhibits JH biosynthesis in the adult

female cockroach,

Diploptera punctata

. If a similar

situ-ation exists in

D. melanogaster

, the absence of OA in

T

b

h

nM18

_{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 propro-duction,

which would elicit an increase in JH-hydrolysing activity

for maintenance of the JH titre. Indeed,

T

b

h

nM18

_females

were shown to have levels of JH degradation almost

twice those in

Canton S

(see Figs. 3 and 7), although

the increased JH-hydrolysing activity is not enough to

lower 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 unidentified

phenotype/process.

Evidence supporting the regulation of JH metabolism

by biogenic amines in

Drosophila

comes from our data

on

Ste

strain. The flies carrying

e

mutation are known

to 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 and

Belles, 1989; Granger et al., 1996) we might expect the

JH titre to be affected, with consequent changes in

JH-hydrolysing activity in females of

Ste

strain. Indeed,

Ste

females have a level of JH degradation almost a half that

of

Canton S

(see Fig. 3).

We cannot exclude the possibility that the differences

between

Ste

,

T

b

h

nM18

_and

_{Canton S}

_{females in the level}

of JH-hydrolysis are the result of strain polymorphism.

However,

four

strains

without

any

stress-related

mutations (

921500

,

FM7

,

n

and

CyLDSb

) were

exam-ined and were found to have JH-hydrolysing activity

similar to that of Canton S, under normal conditions.

Hence, the high JH-hydrolysis level in

T

b

h

nM18

_females

and low level in

Ste

apparently result from the

corre-sponding mutations.

The reason to investigate JH metabolism in the

ts403

strain, with its impairment of HSR, was the existing

evi-dence on the regulation of the expression of heat shock

proteins (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 the

conditionally ecdysteroid-deficient

D. melanogaster

strain

ecd-1

results in a block of hsp26 RNA and a

decline in hsp83 RNA level; subsequent addition of

exogenous 20-OH-ecdysone restores expression of both

genes (Thomas and Lengyel, 1986). JH was reported to

inhibit in a dose-dependent manner the ecdysterone

induction of the small hsp genes of

Drosophila

,

expressed in cultured cells (Berger et al., 1992). Our data

revealed that the block in HSR expression does not affect

JH degradation under normal conditions (see Figs. 1

and 5).

How do the mutations in the different components of

the

D. melanogaster

reaction effect the

stress-reactivity of the JH degradation system? We have

pre-viously demonstrated that exposure of

Drosophila

females to stress results in a sharp decrease of the

JH-hydrolysing activity and as a consequence, the onset of

oviposition by young females is delayed 24 h, while

mature

females

cease

oviposition

for

two

days

(Rauschenbach et al. 1995, 1996). We have also shown

that in certain

D. virilis

and

D. melanogaster

strains that

do not respond to stress, the level of JH-hydrolysis in

females was significantly lower compared to that of wild

type. 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 the

JH degradation system. Mature females of wild type and

laboratory strains without any disturbances in their

reaction demonstrate significantly lower

stress-reactivity than younger ones (see Figs. 8 and 9). In

con-trast, mature

T

b

h

nM18

_{females demonstrate higher}

stress-reactivity than younger ones. Moreover, this response

differs from that in wild type: in mature females it is

higher (see Fig. 9) and in young ones, lower (see Fig.

8). The stress-reactivity in

ts403

females does not

change with age in contrast to wild-type (see Figs. 8

and 9). Young

Ste

females demonstrate the lowest

stress-reactivity (see Fig. 8). Young

n

Met

_{females also have}

decreased stress-reactivity compared to

Met

_{flies of the}

same age (see Fig. 8). Mature

n

Met

_{females show the}

most essential differencies from wild type: their

stress-reactivtity is insignificant (see Fig. 9).

In summary this work suggests that JH may play the

key role in the development of the insect stress-reaction.

Acknowledgements

This study was supported by grants from the Russian

Fundamental Research Foundation and the Siberian

Branch of the Russian Academy of Sciences for Young

Prominent Scientists. Dr. Gruntenko was the recipient of

a travel award from the Organizing Committee of the

Seventh International Conference on the Juvenile

Hor-mones.

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