Results Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue8-9.Sept2000:

776 N.E. Gruntenko et al. Insect Biochemistry and Molecular Biology 30 2000 775–783 In this work, we analysed the mutations ts403, Met, T bh and ebony e with respect to the response of JH- degradation system to stress. The recessive temperature sensitive lethal mutation llts403 results in the failure of heat shock protein HSP83 and HSP35 to be expressed, and a number of HSP70 proteins are only par- tially expressed Evgen’ev and Denisenko, 1990. Met 27 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 receptionWilson and Fabian, 1986; Shemshedini and Wilson, 1990. Met 27 completely lacks Met transcript and is clearly a null allele Wilson and Ashok, 1998. T bh nM18 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. e is 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, n Met 27 , T bh nM18 and Ste strains carring llts403, Met 27 , T bh 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 young v Met 27 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 Met 27 females under stress; 4 that T bh nM18 females show a significantly higher JH-hydrolysis level and different stress-reactivity than does the wild type; and 5 that young Ste females demonstrate significantly lower JH-hydrolysis and stress-reactivity, compared to the wild type.

2. Materials and methods

2.1. Drosophila strains The following D. melanogaster strains were used: the wild type laboratory strain Canton S; wild type iso- female strain 921500 from a natural population of Gorno-Altaisk; laboratory balancer strain First Multiple Seven FM7; vermilion n strain from which the n Met 27 strain was derived; laboratory balancer strain In2LRCyL ; In3LRDSb , carrying morphological mutations with recessive lethal action Curly, Lobe chromosome 2 and Dichaete, Stubble chromosome 3; hereafter termed CyLDSb; strain ts403 carrying the recessive temperature sensitive lethal mutation llts403 Arking, 1975; strain n Met 27 carrying a null allele of the Methoprene-tolerant gene Wilson and Ashok, 1998; strain T bh nM18 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 at 220 ° 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 30 µ l 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 10 µ l were utilized for the reaction. A mix- ture consisting of 0.1 µ g unlabeled JH-III Sigma and 12,500 dpm 3 H labeled JH-III 17.4 Cimmol at C-10, NEN Research Products, Germany was used as sub- strate. The reaction was carried out in siliconized tubes in 100 µ l of incubation mixture for 3 h, and it was stopped by the addition of 250 µ l heptane and 50 µ l of a solution containing 5 ammonia and 50 methanol VV. The tubes were shaken vigorously and centri- fuged at 12,000 rpm for 10 min. Samples 100 µ l 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 and ts403 under normal and stress conditions are shown in Fig. 1. The data indicate that under normal conditions, JH-hydrolysing activity in ts403 females 777 N.E. Gruntenko et al. Insect Biochemistry and Molecular Biology 30 2000 775–783 Fig. 1. Hydrolysis of [ 3 H]JH-III in 1-day-old females of Canton S and ts403 strains of D. melanogaster under normal and stress 38 ° C, 3 h conditions. Means ± SE. does not differ from that in Canton S ones. The data of Fig. 1 also demonstrate that ts403 females respond to stress as well as Canton S do: 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 Met 27 and n females under normal conditions and under heat stress JH-hydrolysis in 1-day old females of both n and n Met 27 strains under normal and stress conditions are shown in Fig. 2. They indicate that under normal con- ditions n Met 27 females show a significantly P,0.01 Fig. 2. Hydrolysis of [ 3 H]JH-III in 1-day-old females of n and n Met 27 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 Met 27 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 T bh nM18 and Ste females under normal conditions and under heat stress The levels of JH-hydrolysing activity in 1-day-old females of T bh nM18 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 of T bh nM18 strain is significantly higher than that in Canton S P,0.001. In contrast, Ste females 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 T bh nM18 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, T bh nM18 and Ste strains of D. melanogaster under normal and stress 38 ° C 3 h conditions. Means ± SE. 778 N.E. Gruntenko et al. Insect Biochemistry and Molecular Biology 30 2000 775–783 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 27 and n females Under normal conditions, the level of JH degradation in 5-day-old n Met 27 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 of ts403 and Canton S strains of D. mel- anogaster . Means ± SE. females. After heat stress, mature n Met 27 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 bh 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 bh nM18 strain is significantly higher 779 N.E. Gruntenko et al. Insect Biochemistry and Molecular Biology 30 2000 775–783 Fig. 6. Effect of short term heat stress 38 ° C 3 h on JH-hydrolyzing activity in 5-day-old females of n and n Met 27 strains of D. melanogas- ter . Means ± SE. P,0.001 than that of Canton S Fig. 7. It is clear that mature T bh 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 27 , T bh nM18 and Ste have lower levels of stress-reactivity P,0.05 for ts403, P,0.01 for n Met 27 and P,0.001 for Tbh nM18 and Ste. We further analysed the stress-reactivity in mature females 6-day-old Canton S, ts403 and T bh nM18 strains and 5-day-old FM7, n and n Met 27 strains. It is clear from the data in Fig. 9 that the stress-reactivity of mature T bh 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 Tbh nM18 . The stress-reactivity of n Met 27 females is insignificant. Fig. 7. Effect of short term heat stress 38 ° C, 3 h on JH-hydrolysing activity in 6-day-old females of T bh nM18 and Canton S strains of D. melanogaster . Means ± SE.

4. Discussion