Induced alterations during in vitro development ... - Wiley Online Library

Archives of Insect Biochemistry and Physiology 5:201-209 (1987)

Diflubenzuron-Induced Alterations During In Vitro Development of 'Tenebrio molitor Pupal integument N. Soltani, A. Quennedey, J.P. Delbecque, and J. Delachambre D6partment de Biologie Animale, Universite' d 'Annaba, Algkrie (N. S.}; Laborafoire de Zoologie, Unit6 associe'e au CNRS No. 674, Universite' de Bourgogne, Dijon, France (A.Q., J.P.D., J.D.} The effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20-hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants i n vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture i n the presence of 20hydroxyecdysone, DFB at high dose ( 2 20 pg/ml) was able t o inhibit cuticle secretion, but lower doses (< 10 pglml) resulted in epicuticle deposition. These observations confirm i n vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells. In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after i n vivo DFB treatment in Tenebrio pupae. Key words: Coleoptera, DFB, ecdysteroids, cuticle, in vitro culture, ultrastructure

INTRODUCTION

It is now well established that diflubenzuron (DFB), a substituted urea growth regulator, inhibits cuticle deposition in insects and some other arthro-

Acknowledgments: We thank Professor C. Noirot, Dr. C. Green, and Dr. B. Mauchamp for helpful suggestions and we are grateful to Dr. A.C. Grosscurt (Philips-Duphar BV) for kindly supplying the diflubenzuron. Received December 9,1986; accepted March 17,1987. Address reprint requests to Dr. J. Delachambre, Laboratoire de Zoologie, Universiti. de Bourgogne, 6 Bd Gabriel, 21100 Dijon, France.

0 1987 Alan R. Liss, Inc.

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pods: several studies, indeed, have shown a reduction in thickness and architectural alterations of the cuticle secreted after DFB treatment in vivo [l81. Though it is generally admitted that DFB acts at the epidermal level, DFBinduced disturbances on the ecdysteroid levels or on the 20-hydroxyecdysone metabolism have been also described in several insect species [9-111, thus leading to the suggestion that DFB could inhibit cuticle deposition via the control of molting hormone secretion. This hypothesis was strengthened by observations on the centipede Lithobius [lo] and on the beetle Tenebrio [ll] showing that, after inhibition of cuticle secretion by high DFB doses, injection of 20-hydroxyecdysone was able to reinduce cuticle secretion. The present investigation with Tenebrio rnolitor was thus designed to determine in vitro whether there is a relationship between DFB and ecdysteroids, as suggested by previous in vivo studies [8,11]. For this purpose, two different kinds of experiments were considered: (1)the effects of DFB were determined on cuticle secretion induced by exogenous 20-hydroxyecdysone in vitro, the epidermal explants being treated by DFB either before or during culture: (2) taking into account that the pupal integument of Tenebrio is also able to secrete small but significant amounts of ecdysteroids in vitro 1121, the effects of DFB were analyzed on this endogenous hormone secretion. MATERIALS AND METHODS Insects

Tenebrio molitor pupae were collected from a stock colony and kept at 27°C and 70% relative humidity. Under these conditions, the interval between pupal and adult ecdyses was 6.3 rt 0.3 days (mean 2 SEM). Tissue Culture Culture conditions for Tenebrio integument were those described in Quennedey et al. [13]. Each individual explant consisted of four abdominal sternites, sterilely excised, cleaned from fat body and muscle, and washed twice for 5 min in Landureau culture medium [14]. Then sternites were generally cultured in sterile dishes (Nunclon) from 1to 10 days at 27"C, floating on 1 ml Landureau medium supplemented with 2% fetal calf serum (Gibco) and 1pglml20-hydroxyecdysone (Simes, Milano, Italy). When experiments were designed for ecdysteroid secretion measurements, neither calf serum nor exogenous hormone was added.

DFB Treatment Treatment with DFB (Philips-Duphar BV, The Netherlands) was made either before or during culture. For treatment before culture, newly ecdysed pupae were dipped for 3-4 s in a 10 mglml(32 mM) acetone solution of DFB. Controls were dipped in acetone alone or not treated at all. Three days after treatment, abdominal sternites from individuals exhibiting complete developmental inhibition, ie, blocked pupae as previously defined 181, were cultured without addition of DFB in the medium. In the series of experiments performed for ecdysteroid secretion measurements, sternites from newly

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ecdysed pupae were explanted either immediately or 3 days after DFB treatment. In experiments in which tissues were treated with DFB in vitro, sternites from newly ecdysed pupae were cultured in medium that contained 5-25 pg DFB per ml medium (1.6-8 nM). These concentrations were higher than the theoretical limit of solubility of DFB into water (0.2 pg per ml). However, we have verified, using high-performance liquid chromatography as previously described [15], that such doses added into the culture medium with 5 pl acetone in a reliably sonicated solution produced relatively stable suspensions, even after several days (more than one-third of the initial dose remained in suspension 3 days after the start of experiment). In controls, the medium contained either 5 pl acetone or none. Electron Microscopy Sternal explants cultured for 1-10 days in 20-hydroxyecdysone-supplemented medium were fixed according to Friend and Farquhar [16], dehydrated, and embedded in an Epon-Araldite mixture. Sections stained with uranyl acetate and lead citrate were examined with a Hitachi HUllE electron microscope at 75 kV. Ecdysteroid Measurements In the experiments in which the explants were cultured without addition of exogenous hormone, aliquots from the medium after a 5-day incubation were extracted with 500 pl ethanol, sonicated, and centrifuged at 10,OOOg for 4 min. The supernatants were evaporated under nitrogen and the extracts were suitably diluted in 0.1 M citrate buffer (pH 6.2) for duplicate ecdysteroid measurements via RIA [ l q with antibodies having a 2.5-fold-higher affinity for ecdysone than for 20-hydroxyecdysone. RESULTS DFB Effect on Cuticle Secretion

Insect tissues treated with DFB before culture. The cuticle of a blocked pupa 3 days after DFB treatment, ie, at the start of the incubation, consisted mainly of preecdysial cuticle and a 1-3-pm postecdysial cuticle with few irregular lamellae, as already described [8]. Controls (Fig. 1)were explants from DFB-treated pupae (Fig. 2), cultured in the presence of 20-hydroxyecdysone, were able to undergo apolysis (after approximately 1day in culture) and then to secrete a new cuticle. However, instead of the typical helicoidal lamellae seen in controls (Fig. 3), the endocuticle of treated pupae presented many abnormalities: after a 5-day culture in the presence of 20-hydroxyecdysone, the thickness was reduced (1-2 pm vs 3-4 pm) and only electron-dense droplets were observed beneath the epicuticle (Fig. 4). After a 10-day culture, a lamellate endocuticle with cuticular canals was deposited in controls (Fig. 5), whereas the dense droplets of treated explants seemed to coalesce into large globular masses between epicuticle and epidermis (Fig. 6). Moreover, the epidermal thickness was considerably reduced.

Fig. 1. A control sternite cultured for 5 days in the presence of 20-hydroxyecdysone. Beneath the apolysed pupal cuticle (PC), a new cuticle has been secreted by the epidermis (E). Scale bar = 2 pm. ~3,500. Fig. 2. A sternite from a diflubenzuron (DFBI-blocked pupa (ie, from a pupa 3 days after dipping, at ecdysis, in a IO-mg/ml acetone solution of DFB), cultured for 5 days in the presence of 20-hydroxyecdysone. As in the control, a new cuticle (arrow) has been secreted by the epidermis (E) under the pupal cuticle (PC). Scale bar = 2 pm. x 3,500. Fig. 3. Enlarged view of the new cuticle secreted by a control epidermis after a 5-day culture in the presence of 20-hydroxyecdysone. Lamellate endocuticle (ENDO) can be seen under the epicuticle (EPI). Scale bar = 1 pm. x 20,000.

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The cuticle secreted by control explants from acetone-treated pupae was similar to that of the untreated ones. Insect tissues treated with DFB during culture. In the case of in vitro DFB treatment, the ability of 20-hydroxyecdysone to induce new cuticle secretion seemed to depend on the DFB concentration in the medium. For 5-10-pglml concentrations, 20-hydroxyecdysone induced only a thin epicuticle within 5 days of culture (Fig. 7). Higher DFB concentrations (20 and 25 pglml) prevented any cuticle induction by 20-hydroxyecdysone (Fig. 8).

Fig. 7. A sternite from a newly ecdysed pupa cultured for 5 days in a medium containing 10 pg of DFB per ml. A newly secreted epicuticle (arrow) is present under the apolysed pupal

cuticle (PC). Scale bar = 1 pm. x 10,000. Fig. 8. A sternite from a newly ecdysed pupa cultured for 5 days in a medium containing 20 pg of DFB per ml. The epidermis (E) has been unable to secrete any cuticular component under the apolysed pupal cuticle (PC). Scale bar = 1 pm. x 10,000.

Fig. 4. A sternite from a DFB-blocked pupa (same treatment as Fig. 2) after 5 days of culture in the presence of 20-hydroxyecdysone: enlarged view of the new cuticle secreted by the epidermis. Under the epicuticle (EPI), only amorphous electron-dense droplets (arrows) have been secreted. Scale bar = 1 pm. x 20,000. Fig. 5. A control sternite cultured for 10 days in the presence of 20-hydroxyecdysone. Under epicuticle (arrow), ten endocuticular lamellae (ENDO) have been secreted by the epidermis (E). Scale bar = 1pm. ~10,000. Fig. 6. A sternite from a DFB-blocked pupa (same treatment as Fig. 2) cultured for 10 days. Under the epicuticle (white arrow), large coalescent droplets (black arrows) secreted by the epidermis (E) are seen. Scale bar = 1 pm. x 10,000.

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DFB Effect on In Vitro Ecdysteroid Secretion The amounts of ecdysteroids released into the culture medium by the epidermis were determined by using RIA after 5-day incubations. Of course in these experiments no exogenous 20-hydroxyecdysone was added at the start of the culture. Results obtained with DFB treatments (either before or during culture) were compared to those of acetone-treated and untreated controls. In the case of DFB treatment of newly ecdysed pupae before culture, DFB reduced the amounts of ecdysteroids secreted in the culture medium by sternites explanted either immediately or 3 days after treatment (Table 1). Indeed, the titer in DFB-treated experiments was significantly different from the titer in untreated or acetone-treated experiments (P < .01 in the t-test for sternites explanted immediately after DFB treatment and P < .001 for sternites explanted after 3 days). Acetone treatments had no significant effect (P = .2) on ecdysteroid secretion. When explanted tissues were exposed to DFB in vitro (Table 2), no significant differences were observed between controls, acetone-treated, and 5-15pglml DFB-treated explants (P > .05). However, at higher DFB concentrations (20-25 pglml), the ecdysteroid secretion was significantly reduced (P < .OOl) . DISCUSSION Compensation of DFB Effects by 20-Hydroxyecdysone The present in vitro study confirms previous in vivo results, observed in Tenebrio pupae [ll] and in the centipede Lithobius [lo], about the interactions between DFB, an inhibitor of cuticle synthesis, and molting hormone 20hydroxyecdysone. When cultured in vitro, epidermal organs can be induced to secrete a new cuticle by 20-hydroxyecdysone [18-201. In Tenebrio, our in vitro experiments, using pupal abdominal sternites, demonstrated that such an induction is also possible in spite of DFB treatment before culture: 20-hydroxyecdysone can TABLE 1. Ecdysteroid Titers in Culture Medium After a 5-day Incubation of Sternites Explanted Either Immediately or 3 Days After Dipping Into Acetone Solutions of DFB (10 mglml) or Pure Acetone or not Treated (Controls)*

Treatment Control Acetone DFB

Ecdysteroid titers (pg) from sternites explanted Immediately after 3 days after treatment treatment

1,740 k 92 1,727 rf: 69 1,360 k 72

2,089 rf: 164 2,151 I11 173 1,163 rf: 62a

*Each value is the mean rf: SEM of five to nine cultures measured in duplicate by RIA and expressed as pgisternite. DFB, diflubenzuron. aExplants from blocked pupae.


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TABLE 2. Ecdysteroid Titers in Culture Medium Supplemented With 5-25 pg DFBlml, After a 5day Incubation of Explants From Newly Ecdysed Puvaet Treatment

Titers (pg)*

Control Acetone DFB 5 p g DFB 10 fig DFB 15 p g DFB 20 p g DFB 25 p g

1,942 f 139 a 1,731 k 68 a 1,707 k 227 a 1,929 k 218a 1,542 236 a 785 rt 123 b 928 f 91 b

Qach value is the mean t SEM of five to ten cultures measured in duplicate by RIA and expressed as pgisternite. *Values followed by the same letter are not significantly different (P > .05) in the leastsignificative difference test, after variance analysis.

thus suppress the inhibition of cuticle synthesis due to DFB. The new cuticular cycle observed in vitro begins with the depositon of a well-formed epicuticle by the DFB-treated explants. However, the newly induced endocuticle is thinner than in controls and has many structural abnormalities. Comparable architectural alterations have also been observed in Pieris larvae [4,21], Thuumetopoeu larvae [5], Luciliu larvae [22], and in Leptinotuvsu embryos [6] in which dense amorphous droplets, probably proteinaceaous [23], replace the normal fibrillar structure. These abnormalities are generally related to chitin biosynthesis alterations [2-41. In Tenebrio, it has been demonstrated by wheat germ agglutin histochemical binding that the cuticle, induced by 20-hydroxyecdysone after DFB treatment in vivo, contains N-acetyl-aminosugars, thus suggesting the presence of incompletely polymerized compounds [ll] Moreover, in this species, adult differentiations (columnar cuticle, pit-glands, dermal glands) are also inhibited in vivo, suggesting that DFB also disturbs the switchover from pupal to adult genetic programs, probably by interfering with differential mitoses and DNA syntheses [8]. When the DFB treatment is applied in vitro, more pronounced alterations were observed in Tenebrio pupal sternites. It is possible to completely prevent the cuticle secretion induced by 20-hydroxyecdysone with DFB concentrations greater than or equal to 20 pglml. However, some cuticle synthesis (essentially epicuticule) is possible at lower DFB doses. Such results agree well with studies on Chilo [24], which demonstrate an inverse relationship between the DFB concentration in the culture medium and the thickness of the newly secreted cuticle. Although DFB concentrations used in this study were higher than its theoretical limit of solubility into water, dose differences were observed: this may be due to the fact that DFB could act not only under its soluble form but also in suspension or contact. Moreover, it has been shown that such molecules are efficaciously concentrated by tissues [25]. e

Effect of DFB on Ecdysteroid Secretion In Tenebrio pupae, the large ecdystroid peak [26] does not originate from the prothoracic glands, which have already degenerated [27], but from sev-

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era1 other tissues, and particularly epidermis as demonstrated in vivo and in vitro [12,26,28]. As reported earlier [ll], DFB applied to newly ecdysed Tenebrio pupae inhibits this ecdysteroid increase in vivo. In the present work, such an inhibition was also observed at the epidermal level in vitro either for explants from DFB-blocked pupae or for cultures added with high DFB doses. These experiments suggest that the hormonal inhibition is at least partly due to a direct effect of DFB on the epidermis in vitro and possibly also in vivo. Thus, DFB inhibits the secretion of molting hormone, at least in the peculiar case of Tenebrio pupal-adult development, in the absence of prothoracic gland, when the hormonal peak is under the control of epidermis, However, it appears that in other species or even in Tenebrio larvae, when the molting hormone peak is under the control of prothoracic glands only, the DFB effect on the epidermis does not affect ecdysteroid titers (unreported data). In conclusion, DFB and 20-hydroxyecdysone exert antagonistic and at least partly reversible effects on epidermal cells for the synthesis of cuticle. However, our experiments suggest that DFB also has endocrine consequences on the secretion of 20-hydroxyecdysone controlled by epidermis. All these results and other recent reports thus strongly suggest that DFB probably has a complex mode of action, not only at the chitin synthesis level 129-311 but also at the hormonal and DNA level [8,11,32-541. LITERATURE CITED 1. Daalen JJ, Meltzer J, Mulder R, Wellinga K: A selective insecticide with a novel mode of action. Naturwissenschaften 59, 312 (1972). 2. Post LC, Vincent JFV: A new insecticide inhibits chitin synthesis. Naturwissenschaften 60, 431 (1973). 3. Hunter E, Vincent JFV: The effects of a novel insecticide on insect cuticle. Experientia 30, 1432 (1974). 4. Mulder R, Gijswijt MJ: The laboratory evaluation of two promising new insecticides which interfere with cuticle deposition. Pestic Sci 4, 737 (1973). 5. Denneulin JC: Les sterols de la processionnaire du pin Thaumetopoea pityocampa Schiff. Quelques aspects de leur metabolisme. Effet d’un insecticide de synthPse. ThPse, Bordeaux (1976). 6. Grosscurt AC: Diflubenzuron: Some aspects of its ovicidal and larvicidal mode of action and an evaluation of its practical possibilities. Pestic Sci 9, 373 (1978). 7. Soltani N: Effects of ingested Diflubenzuron on the longevity and the peritrophic membrane of adult mealworms (Tenebriomolitor). Pestic Sci 15, 221 (1984). 8. Soltani N, Besson MT, Delachambre J: Effects of Diflubenzuron on the pupal-adult development of Tenebrio molifor (Coleoptera, Tenebrionidae): Growth and development, cuticle secretion, epidermal cell density and DNA synthesis. Pestic Biochem Physiol 21, 256 (1984). 9. Yu SJ, Terriere LC: Ecdysone metabolism by soluble enzymes from three species of Diptem and its inhibition by the insect growth regulator TH 6040. Pestic Sci 7, 48 (1977). 10. Scheffel H, Kuchenmeister J: Influence of Diflubenzuron on moult initiation and chitin biosynthesis in the centipede Lithobius fotfzcatus L. In: Regulation of Insect Development and Behaviour. Sehnal F, Zabza A, Men JJ, Cymborowski B, eds. Technical University Wroclaw, p 138 (1981). 11. Soltani N, Delbecque JP, Delachambre J, Mauchamp B: Inhibition of ecdysteroid increase by diflubenzuron in Tenebrio molitor pupae and compensation of diflubenzuron effect on cuticle secretion by 20-hydroxyecdysone.Int J Reprod Dev 7, 323 (1984). 12. Delachambre J, Besson MT, Quennedey A, Delbecque JP: Relationships between hormones and epidermal cell cycles during the metamorphosis of Tenebrio molitor. In: Biosyn-


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