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Protein synthesis in the fat body of Tenebrio molitor (L.) during oocyte maturation: Effect of diflubenzuron, cycloheximide and starvation
J. stored
Pergamon
0022474X(94)OOO46g
Prod.
Rex
Vol.
31, No.
2, pp.
117-122,
1995
Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-474X/95 $9.50 + 0.00
Protein Synthesis in the Fat Body of Tenebriu molitor (L.) During Oocyte Maturation: Effect of Diflubenzuron, Cycloheximide and Starvation N. SOLTANI-MAZOUNI* DPpartement
de Biologie
Animale,
and N. SOLTANI
Universitk de Annaba, Algeria
BP 12 23000,
Annaba,
(Received for publication 3 November 1994)
Abstract-Diflubenzuron (DFB), incorporated into the diet (5 and 10 mg gg’) was tested on protein synthesis in the fat body of adult females of TeueMo mofitor (L.) during the first 6 days of aduit life. DFB had no significant effect on fat body protein during the period examined, as evidenced both by the electropboretic pattern and the incorporation of tritiated leucine. In contrast, cycloheximide (2.5cg per insect) injected into newly emerged females siguikantly affected both the level of protein and the incorporation of tritiated leucine into proteins, as did starvation. Key
words-Tenebrio
molitor,
ditlubenzuron, cycloheximide, starvation, reproduction,
fat body, proteins.
INTRODUCTION
Diflubenzuron (DFB) is an inhibitor of chitin synthesis in several insect species (Cohen, 1987; Ishaya, 1990; Soltani et al., 1993). Adult treatment disturbs the secretion of post-ecdysial cuticle and the production of peritrophic membrane (Clarke et al., 1977; Soltani, 1984). In addition, this compound affects reproduction in adult females and a decrease in fecundity has been observed in several insect species (Holst, 1974; Fytizas, 1976; Lim and Lee, 1981, 1982; Soltani, 1987; Soltani and Soltani-Mazouni, 1992). Very little is known about the action of DFB on reproduction, and the mechanism by which this decrease of fecundity occurs remains to be determined. Previous studies performed with Tenebrio molitor (L.) have shown that DFB affects both the longevity and weight of adults (Soltani, 1984), the size of basal oocytes, the number of oocytes per ovary and the fecundity (Soltani, 1987). In addition, it disturbs the ovarian synthesis of DNA during oocyte maturation (Soltani-Mazouni and Soltani, 1994). However, it had no significant effect on the fine structure of follicular cells and basal oocytes (Soltani-Mazouni, 1994). The evidence accumulated suggests that DFB affects the reproduction via the vitellogenesis process. The increase of excrement weight, alterations of the peritrophic membrane in DFB-treated adults (Soltani, 1984) and the decrease of lipid and trehalose concentrations in haemolymph observed in T. mofitor (Soltani-Mazouni and Soltani, 1992) suggest that DFB interferes with the vitellogenesis *Author
for correspondence.
N. Soltani-Mazouni and N. Soltani
118
process probably via the food transport process. In order to extend the previous results and to investigate this hypothesis, the present study with T. molitor was designed to evaluate the effect of DFB on protein synthesis in the fat body during sexual maturation. Since DFB and starvation caused similar effects, not only on adult weight, but also on longevity in T. molitor (Soltani, 1984), the results of DFB-treated females were compared to those of controls and starved batches. Moreover, cycloheximide, an inhibitor of protein synthesis (Abu-Hakima and Davey, 1977) which acts mainly through blocking translation (Sass and Kovacs, 1980) and by stabilization of polyribosomes (Goldberg, 1965; Tscherne and Pestka, 1975) was also evaluated on the in viuo incorporation of tritiated leucine into fat body proteins. MATERIALS
AND METHODS
Insects and treatments
Pupae from a stock colony were sexed and kept separate until adult emergence. Adults were collected O-4 h following emergence and reared on wheat flour at 27°C and 70% r.h. in almost continuous darkness (20 pairs of insects per rearing box). Adult mealworms were treated from their emergence with DFB (Philips Duphar B.V., The Netherlands) incorporated into the diet at two doses (5 and 10 mg g-‘). Control insects were provided with wheat flour alone. Cycloheximide (Sigma) was dissolved in distilled water and injected (1~1 per female) with a Hamilton syringe into the body of newly emerged females at 2.5 pg ~1~’ per insect. Control insects were injected with 1~1 distilled water alone. Incorporation of tritiated leucine
Adult females sampled at 0, 3 and 6 days after emergence from control and treated batches were injected with 3 PCi L-(4,5-H) leucine (sp.act. 140 Ci/mM, Amersham) in 3 ~1 aqueous solution containing 2% ethanol. The radiolabelled precursor was injected into the body with a Hamilton syringe between abdominal sternites 3 and 4. Controls were injected with 3 ~1 distilled water containing 2% ethanol. After treatment, all insects were replaced under the same rearing conditions (27”C, 70% r.h.) for 1 h. Then, the females were frozen at -20°C before fat body extraction. Extraction of proteins from fat body
The abdominal fat body was removed, rinsed with saline solution and then trichloracetic acid (20%). Immediately after blotting on filter paper, samples were transferred into tared tubes and weighed. After weighing, the tissue was homogenized in 1 ml of trichloracetic acid (20%) and centrifuged at 4200 rpm for 10 min. The precipitate obtained was washed with 1 ml of methanol-chloroform (1: 1 by volume) to remove lipids and then recentrifuged as before. The final residue was suspended in 1 ml of NaOH (0.05 N) and divided in two. Determination of protein amounts and radioactivity
One aliquot (0.1 ml) was used for determination according to Bradford (1976). The incorporation measured in the remaining volume (0.9 ml). After the radioactivity was counted in a Beckman LS
of protein levels by the Coomassie blue method of labelled leucine into protein of fat body was addition of scintillation fluid BCS (Amersham), 6000 SC spectrometer.
Polyacrylamide gel electrophoresis
Proteins were analyzed by sodium dodecylsulfate polyacrylamide (99%) slab gels (SDS-PAGE) by the method of Laemmli (1970). Six-day-old females (5-6 females per treatment) were collected from control and 10 mg g-’ DFB-treated cultures and used for isolation of fat body. Pooled fat bodies were homogenized in lysis buffer (Tris-HCl50 mM pH 7; Nonidet 0.5%; P-mercaptoethanol 1%; phenyl methyl sulfonyl fluoride 0.1%) and centrifuged (4200 rpm, 15 min). The supernatant was removed and treated overnight at -20°C by 10 vol of cooled acetone and then recentrifuged (6000 rpm, 15 min). The pellet was resuspended in Laemmli sample buffer. The protein content in each sample was determined before lyophilization according to Bradford (1976) using bovine serum albumin as the standard. Fifteen pg of fat body protein were applied to each lane of the gel. The
Protein synthesis in the fat body of T.
119
molitor
gels were stained in 0.025% Coomassie brilliant blue R 250 (Merck), 10% acetic acid, 25% propanol 2, and destained in 10% acetic acid in water. Statistical analysis Results are represented as the mean & standard deviation (SD) established on measurements from individual females of the same age for each treatment. A comparison of mean values was made with the Student t-test. The level of significance was set at 5%. The number of females tested are given with the results. RESULTS
Effect on the protein level in the fat body In controls, the level of protein in the fat body reached its highest point at day 3 following adult emergence (Fig. 1). This initial increase of protein in the fat body was also observed in starved females and after DFB-treatment or cycloheximide injection, again with a maximum value being recorded at day 3. DFB at 5 mg g-’ dose had no significant effect on the protein level attained. On the contrary, DFB-treatment with the 10 mg g-’ dose resulted in a slight but significant (P = 0.016) decrease in protein levels at day 3, though not at day 6. Starvation significantly decreased the protein level in the fat body at day 3 (P = 0.046) and 6 (P = 0.009) respectively, compared to controls of the same age. Cycloheximide injection (2.5 fig per insect) of the newly emerged female also caused a significant decrease of protein content at day 3 (P = 0.012) and 6 (P = 0.048). Eflect on protein pattern of fat body Proteins of fat bodies shown in Fig. 2. More orally to newly emerged body proteins compared
from 6-day-old females were separated by SDS-PAGE and results are than 20 bands were detected. DFB-treatment (10mg gg’) administered mealworms had no significant effect on the electrophoretic pattern of fat to controls.
Effect on the protein synthesis in the fat body The course of the in vivo incorporation (dpm pg-’ of fat body proteins) of tritiated leucine into fat body proteins during the 6 days following emergence was determined respectively in controls, DFB-treated, cycloheximide-injected and starved females (Fig. 3). In controls, the incorporation increased during the period examined. The incorporation of tritiated leucine into fat body proteins is similar in the treated samples since the values recorded increased during the time following adult emergence. There was no significant (P > 0.05) difference in the amount of incorporation between control and DFB-treated batches at each of the different times evaluated. Finally both starvation
Control DFB 5mg g-’ DFB 1Omg g-’ Starvation Cycloheximide
2Spg
0 0
3
6
Age (days)
Fig. 1. The protein level (pg protein mg-’ of fat body) in the fat body during the sexual maturation of T. molitor (m + SD, n = 3-5 females, for each age asterisks indicate a significant difference between control and treated series).
120
N. Soltani-Mazouni
KDa
and N. Soltani
A
B
Fig. 2. Electrophoretic separation of fat body proteins from 6-day old untreated (A) and 10 mg g-’ DFB-treated (B) females of T. molitor on a 9% SDS-PAGE. Numbers on the left show positions of molecular weight markers (kDa).
and cycloheximide caused a significant decline in the amount of tritiated leucine incorporation at days 3 (starvation: P = 0.011; cycloheximide: P = 0.001) and 6 (starvation: P = 0.046; cycloheximide: P = 0.021) compared to controls of the same age. DISCUSSION The insect fat body is the principal site for intermediary metabolism, storage of reserve material, and in adult females, production of vitellogenin. Vitellogenin is secreted by the fat body into haemolymph and incorporated into oocytes by receptor-mediated endocytosis (Kunkel and Nordin, 1985). Comprehensive reviews of various aspects of the biochemistry of fat body have already appeared (Keeley, 1985; Dean et al., 1985; Raikhel and Dhadialla, 1992).
DFB 5mg g-’ DFB IOmg g“
Starvation Cycloheximide
2.w
Age (days) Fig. 3. The in uiuo incorporation (dpm pg- ’ of fat body proteins) of tritiated leucine into fat body protein during the sexual maturation of T. molitor (m f SD, n = 3-6 females, for each age asterisks indicate a significant difference between control and treated series).
Protein synthesis in the fat body of T. molitor
121
The experiments conducted on mealworm females show clearly that DFB had no significant effects on proteins of the fat body during the period examined, as evidenced both by the electrophoretic pattern and the incorporation of tritiated leucine. This is consistent with the absence of a significant effect on haemolymph protein concentrations in DFB-treated insects (Soltani-Mazouni and Soltani, 1992). On the contrary, cycloheximide injected to newly emerged females and starvation significantly affected both the amount of protein and the incorporation of tritiated leucine into fat body proteins. The effects of cycloheximide were more marked than those caused by starvation in the fat body of T. molitor females. It has been shown in T. molitor that DFB affects the numbers of both oocytes and vitellogenic follicles but not the fine structure of follicular cells and basal oocytes (Soltani-Mazouni, 1994). The results accumulated suggest that the reduction in fecundity observed in T. molitor (Soltani, 1987) and other species (Lim and Lee, 1982; Soltani and Soltani-Mazouni, 1992), is probably due to interference of DFB with the vitellogenesis process particularly protein synthesis in the ovaries. This hypothesis is supported by biochemical evidence. It has been demonstrated that DFB inhibits the in vitro synthesis of ovarian DNA (Soltani-Mazouni and Soltani, 1994) and protein (SoltaniMazouni et al., 1993). In order to establish this theory further studies on the fine structure of both germarium and chorion, the pattern of ovarian protein and the in uiuo incorporation of labelled precursors into ovaries are needed. thank Professor C. Noirot, Bourgogne University, for helpful suggestions and Philips Duphar B.V. for the diflubenzuron. This work was supported by Grant No. 87 MES 69.
Acknowledgements-We
REFERENCES Abu-Hakima R. and Davey K. G. (1977) Effects of hormones and inhibitors of macromolecular synthesis on the follicle cells of Rhodnius. J. Insect Physiol. 23, 913-917. Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254. Clarke L., Temple G. H. and Vincent J. F. V. (1977) The effects of a chitin inhibitor Dimilin on the production of peritrophic membrane in the locust, Locusta migratoria. J. Inseci Physiol. 23, 241-246. Cohen E. (1987) Chitin biochemistry: synthesis and inhibition. A. Rev. Ent. 322, 71-93. Dean R. L., Locke M. and Collins J. V. (1985) Structure of the fat body. In Comprehensive Insect Biochemistry, Physiology and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 3, pp. 155-210. Pergamon Press, Oxford. Fytizas E. (1976) L’action du TH 6040 sur la metamorphose de Dacus oleae Gmel. (Diptera, Trypetidae). Z. angew. En?. 81,440444. Goldberg I. H. (1965) Mode of action of antibiotics-II. Drugs affecting nucleic acid and protein synthesis. Am. J. Med. 29, 722-752.
Holst H. (1974) Die fertilitatsbeinflussend Wirkung des neuen Insectizids PDD 60-40 bei Epilachna varivestis Muls. (Col., Coccinellidae) und Leptniotarsa decemlineata Say. (Col., Chrysomelidae). Z. Pjkrankh. PJ%churz 81, 1-7. Ishaya I. (1990). Benzoylphenyl ureas and other selective control agents-mechanism and application. In Pesticides and Ahernatives (Edited by Casida. J. E.), pp. 365-376. Elsevier Science, Amsterdam. Keeley L. L. (1985) Physiology and biochemistry of fat body. In Comprehensive Insect Biochemistry, Physiology and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 3, pp. 211-248. Pergamon Press, Oxford. Kunkel J. G. and Nordin J. H. (1985) Yolk proteins. In Comprehensive Insect Biochemistry, Physiology and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 1, pp. 83-111. Pergamon Press, Oxford. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Narure 227, 680685.
Lim S. J. and Lee S. S. (1981) The effect of starvation on hemolymph metabolites, fat body and ovarian development in Oxya japonica (Acrididae: Orthoptera). J. Insect Physiol. 27, 93-96. Lim S. 1. and Lee S. S. (1982) Toxicity of diflubenzuron to the grasshopper Oxya japonica: effects on reproduction. Ent. exp. appi. 31(2), 154-158.
Raikhel A. S. and Dhadialla T. S. (1992) Accumulation of yolk proteins in insect oocytes. A. Rev. Em. 37, 217-251. Sass M. and Kovacs J. (1980) The effects of actinomycin D, cycloheximide and puromicyn on the 20-hydroxyecdysone induced autophagocytosis in larval fat body cells of Pieris brassicae. J. Insecf Physiol. 26, 569-577. Soltani N. (1984) Effects of ingested diflubenzuron on the longevity and the peritrophic membrane of adult mealworms (Tenebrio molitor L.). Pestic. Sci. 15, 221-225. Soltani N. (1987) Effects du diflubenzuron sur la reproduction chez Tenebrio molitor L. Annales ANPP 111(6), 471475. Soltani N. and Soltani-Mazouni N. (1992) Diflubenzuron and oogenesis in codling moth, Cydia pomonella (L.) Pestic. Sci. 34, 257-26 1.
Soltani N., Chebira S., Delbecque J. P. and Delachambre J. (1993) Biological activity of flucycloxuron, a novel benzoylphenylurea derivative, on Tenebrio moliror: comparison with diflubenzuron and triflumuron. Experientia 45(12), 1088-1091.
Soltani-Mazouni N. (1994) Effects of ingested diflubenzuron on ovarian development during the sexual maturation of mealworms. Tissue Cell 26(3), 439445. Soltani-Mazouni N. and Soltani N. (1992) Effets du diflubenzuron sur les metabolites hemolymphatiques et ovariens chez Tenebrio mohtor L. au tours de la maturation sexuelle. Anna/s Sot. em. Roy. Be/g. 35, 743-747.
122
N. Soltani-Mazouni and N. Soltani
Soltani-Mazouni N. and Soltani N. (1994) Diflubenzuron affected DNA synthesis in the ovaries of Tenebrio mofitor. J. Inoert. Reprod. Dew U, 19-21. Soltani-Mazouni N., Delachambre J. and Soltani N. (1993) Le diflubenzuron et la reproduction chez Tenebrio molitor: effet sur l’incorporation ovarienne de precurseurs tritiks. Med. Fat. Lana’bouww. Univ. Gent 58/2b, 625-633. Tscheme J. S. and Pestka S. (1975) Inhibition of protein synthesis in intact Hela cells. Antimicrob. Agents Chemother. 8, 479-487.
Pergamon
0022474X(94)OOO46g
Prod.
Rex
Vol.
31, No.
2, pp.
117-122,
1995
Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-474X/95 $9.50 + 0.00
Protein Synthesis in the Fat Body of Tenebriu molitor (L.) During Oocyte Maturation: Effect of Diflubenzuron, Cycloheximide and Starvation N. SOLTANI-MAZOUNI* DPpartement
de Biologie
Animale,
and N. SOLTANI
Universitk de Annaba, Algeria
BP 12 23000,
Annaba,
(Received for publication 3 November 1994)
Abstract-Diflubenzuron (DFB), incorporated into the diet (5 and 10 mg gg’) was tested on protein synthesis in the fat body of adult females of TeueMo mofitor (L.) during the first 6 days of aduit life. DFB had no significant effect on fat body protein during the period examined, as evidenced both by the electropboretic pattern and the incorporation of tritiated leucine. In contrast, cycloheximide (2.5cg per insect) injected into newly emerged females siguikantly affected both the level of protein and the incorporation of tritiated leucine into proteins, as did starvation. Key
words-Tenebrio
molitor,
ditlubenzuron, cycloheximide, starvation, reproduction,
fat body, proteins.
INTRODUCTION
Diflubenzuron (DFB) is an inhibitor of chitin synthesis in several insect species (Cohen, 1987; Ishaya, 1990; Soltani et al., 1993). Adult treatment disturbs the secretion of post-ecdysial cuticle and the production of peritrophic membrane (Clarke et al., 1977; Soltani, 1984). In addition, this compound affects reproduction in adult females and a decrease in fecundity has been observed in several insect species (Holst, 1974; Fytizas, 1976; Lim and Lee, 1981, 1982; Soltani, 1987; Soltani and Soltani-Mazouni, 1992). Very little is known about the action of DFB on reproduction, and the mechanism by which this decrease of fecundity occurs remains to be determined. Previous studies performed with Tenebrio molitor (L.) have shown that DFB affects both the longevity and weight of adults (Soltani, 1984), the size of basal oocytes, the number of oocytes per ovary and the fecundity (Soltani, 1987). In addition, it disturbs the ovarian synthesis of DNA during oocyte maturation (Soltani-Mazouni and Soltani, 1994). However, it had no significant effect on the fine structure of follicular cells and basal oocytes (Soltani-Mazouni, 1994). The evidence accumulated suggests that DFB affects the reproduction via the vitellogenesis process. The increase of excrement weight, alterations of the peritrophic membrane in DFB-treated adults (Soltani, 1984) and the decrease of lipid and trehalose concentrations in haemolymph observed in T. mofitor (Soltani-Mazouni and Soltani, 1992) suggest that DFB interferes with the vitellogenesis *Author
for correspondence.
N. Soltani-Mazouni and N. Soltani
118
process probably via the food transport process. In order to extend the previous results and to investigate this hypothesis, the present study with T. molitor was designed to evaluate the effect of DFB on protein synthesis in the fat body during sexual maturation. Since DFB and starvation caused similar effects, not only on adult weight, but also on longevity in T. molitor (Soltani, 1984), the results of DFB-treated females were compared to those of controls and starved batches. Moreover, cycloheximide, an inhibitor of protein synthesis (Abu-Hakima and Davey, 1977) which acts mainly through blocking translation (Sass and Kovacs, 1980) and by stabilization of polyribosomes (Goldberg, 1965; Tscherne and Pestka, 1975) was also evaluated on the in viuo incorporation of tritiated leucine into fat body proteins. MATERIALS
AND METHODS
Insects and treatments
Pupae from a stock colony were sexed and kept separate until adult emergence. Adults were collected O-4 h following emergence and reared on wheat flour at 27°C and 70% r.h. in almost continuous darkness (20 pairs of insects per rearing box). Adult mealworms were treated from their emergence with DFB (Philips Duphar B.V., The Netherlands) incorporated into the diet at two doses (5 and 10 mg g-‘). Control insects were provided with wheat flour alone. Cycloheximide (Sigma) was dissolved in distilled water and injected (1~1 per female) with a Hamilton syringe into the body of newly emerged females at 2.5 pg ~1~’ per insect. Control insects were injected with 1~1 distilled water alone. Incorporation of tritiated leucine
Adult females sampled at 0, 3 and 6 days after emergence from control and treated batches were injected with 3 PCi L-(4,5-H) leucine (sp.act. 140 Ci/mM, Amersham) in 3 ~1 aqueous solution containing 2% ethanol. The radiolabelled precursor was injected into the body with a Hamilton syringe between abdominal sternites 3 and 4. Controls were injected with 3 ~1 distilled water containing 2% ethanol. After treatment, all insects were replaced under the same rearing conditions (27”C, 70% r.h.) for 1 h. Then, the females were frozen at -20°C before fat body extraction. Extraction of proteins from fat body
The abdominal fat body was removed, rinsed with saline solution and then trichloracetic acid (20%). Immediately after blotting on filter paper, samples were transferred into tared tubes and weighed. After weighing, the tissue was homogenized in 1 ml of trichloracetic acid (20%) and centrifuged at 4200 rpm for 10 min. The precipitate obtained was washed with 1 ml of methanol-chloroform (1: 1 by volume) to remove lipids and then recentrifuged as before. The final residue was suspended in 1 ml of NaOH (0.05 N) and divided in two. Determination of protein amounts and radioactivity
One aliquot (0.1 ml) was used for determination according to Bradford (1976). The incorporation measured in the remaining volume (0.9 ml). After the radioactivity was counted in a Beckman LS
of protein levels by the Coomassie blue method of labelled leucine into protein of fat body was addition of scintillation fluid BCS (Amersham), 6000 SC spectrometer.
Polyacrylamide gel electrophoresis
Proteins were analyzed by sodium dodecylsulfate polyacrylamide (99%) slab gels (SDS-PAGE) by the method of Laemmli (1970). Six-day-old females (5-6 females per treatment) were collected from control and 10 mg g-’ DFB-treated cultures and used for isolation of fat body. Pooled fat bodies were homogenized in lysis buffer (Tris-HCl50 mM pH 7; Nonidet 0.5%; P-mercaptoethanol 1%; phenyl methyl sulfonyl fluoride 0.1%) and centrifuged (4200 rpm, 15 min). The supernatant was removed and treated overnight at -20°C by 10 vol of cooled acetone and then recentrifuged (6000 rpm, 15 min). The pellet was resuspended in Laemmli sample buffer. The protein content in each sample was determined before lyophilization according to Bradford (1976) using bovine serum albumin as the standard. Fifteen pg of fat body protein were applied to each lane of the gel. The
Protein synthesis in the fat body of T.
119
molitor
gels were stained in 0.025% Coomassie brilliant blue R 250 (Merck), 10% acetic acid, 25% propanol 2, and destained in 10% acetic acid in water. Statistical analysis Results are represented as the mean & standard deviation (SD) established on measurements from individual females of the same age for each treatment. A comparison of mean values was made with the Student t-test. The level of significance was set at 5%. The number of females tested are given with the results. RESULTS
Effect on the protein level in the fat body In controls, the level of protein in the fat body reached its highest point at day 3 following adult emergence (Fig. 1). This initial increase of protein in the fat body was also observed in starved females and after DFB-treatment or cycloheximide injection, again with a maximum value being recorded at day 3. DFB at 5 mg g-’ dose had no significant effect on the protein level attained. On the contrary, DFB-treatment with the 10 mg g-’ dose resulted in a slight but significant (P = 0.016) decrease in protein levels at day 3, though not at day 6. Starvation significantly decreased the protein level in the fat body at day 3 (P = 0.046) and 6 (P = 0.009) respectively, compared to controls of the same age. Cycloheximide injection (2.5 fig per insect) of the newly emerged female also caused a significant decrease of protein content at day 3 (P = 0.012) and 6 (P = 0.048). Eflect on protein pattern of fat body Proteins of fat bodies shown in Fig. 2. More orally to newly emerged body proteins compared
from 6-day-old females were separated by SDS-PAGE and results are than 20 bands were detected. DFB-treatment (10mg gg’) administered mealworms had no significant effect on the electrophoretic pattern of fat to controls.
Effect on the protein synthesis in the fat body The course of the in vivo incorporation (dpm pg-’ of fat body proteins) of tritiated leucine into fat body proteins during the 6 days following emergence was determined respectively in controls, DFB-treated, cycloheximide-injected and starved females (Fig. 3). In controls, the incorporation increased during the period examined. The incorporation of tritiated leucine into fat body proteins is similar in the treated samples since the values recorded increased during the time following adult emergence. There was no significant (P > 0.05) difference in the amount of incorporation between control and DFB-treated batches at each of the different times evaluated. Finally both starvation
Control DFB 5mg g-’ DFB 1Omg g-’ Starvation Cycloheximide
2Spg
0 0
3
6
Age (days)
Fig. 1. The protein level (pg protein mg-’ of fat body) in the fat body during the sexual maturation of T. molitor (m + SD, n = 3-5 females, for each age asterisks indicate a significant difference between control and treated series).
120
N. Soltani-Mazouni
KDa
and N. Soltani
A
B
Fig. 2. Electrophoretic separation of fat body proteins from 6-day old untreated (A) and 10 mg g-’ DFB-treated (B) females of T. molitor on a 9% SDS-PAGE. Numbers on the left show positions of molecular weight markers (kDa).
and cycloheximide caused a significant decline in the amount of tritiated leucine incorporation at days 3 (starvation: P = 0.011; cycloheximide: P = 0.001) and 6 (starvation: P = 0.046; cycloheximide: P = 0.021) compared to controls of the same age. DISCUSSION The insect fat body is the principal site for intermediary metabolism, storage of reserve material, and in adult females, production of vitellogenin. Vitellogenin is secreted by the fat body into haemolymph and incorporated into oocytes by receptor-mediated endocytosis (Kunkel and Nordin, 1985). Comprehensive reviews of various aspects of the biochemistry of fat body have already appeared (Keeley, 1985; Dean et al., 1985; Raikhel and Dhadialla, 1992).
DFB 5mg g-’ DFB IOmg g“
Starvation Cycloheximide
2.w
Age (days) Fig. 3. The in uiuo incorporation (dpm pg- ’ of fat body proteins) of tritiated leucine into fat body protein during the sexual maturation of T. molitor (m f SD, n = 3-6 females, for each age asterisks indicate a significant difference between control and treated series).
Protein synthesis in the fat body of T. molitor
121
The experiments conducted on mealworm females show clearly that DFB had no significant effects on proteins of the fat body during the period examined, as evidenced both by the electrophoretic pattern and the incorporation of tritiated leucine. This is consistent with the absence of a significant effect on haemolymph protein concentrations in DFB-treated insects (Soltani-Mazouni and Soltani, 1992). On the contrary, cycloheximide injected to newly emerged females and starvation significantly affected both the amount of protein and the incorporation of tritiated leucine into fat body proteins. The effects of cycloheximide were more marked than those caused by starvation in the fat body of T. molitor females. It has been shown in T. molitor that DFB affects the numbers of both oocytes and vitellogenic follicles but not the fine structure of follicular cells and basal oocytes (Soltani-Mazouni, 1994). The results accumulated suggest that the reduction in fecundity observed in T. molitor (Soltani, 1987) and other species (Lim and Lee, 1982; Soltani and Soltani-Mazouni, 1992), is probably due to interference of DFB with the vitellogenesis process particularly protein synthesis in the ovaries. This hypothesis is supported by biochemical evidence. It has been demonstrated that DFB inhibits the in vitro synthesis of ovarian DNA (Soltani-Mazouni and Soltani, 1994) and protein (SoltaniMazouni et al., 1993). In order to establish this theory further studies on the fine structure of both germarium and chorion, the pattern of ovarian protein and the in uiuo incorporation of labelled precursors into ovaries are needed. thank Professor C. Noirot, Bourgogne University, for helpful suggestions and Philips Duphar B.V. for the diflubenzuron. This work was supported by Grant No. 87 MES 69.
Acknowledgements-We
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