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Hormone-dependent protein patterns in integument and cuticular pigmentation in Apis mellifera during pharate adult development
Journal of Insect Physiology 47 (2001) 1275–1282 www.elsevier.com/locate/jinsphys
Hormone-dependent protein patterns in integument and cuticular pigmentation in Apis mellifera during pharate adult development A.E. Santos, M.M.G. Bitondi *, Z.L.P. Simo˜es Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras; Departamento de Gene´tica, Faculdade de Medicina. Universidade de Sa˜o Paulo, Av Bandeirantes 3900, 14040-901 Ribeirao Preto, SP, Brazil Received 6 February 2001; accepted 11 June 2001
Abstract The epidermal proteins from staged Apis mellifera pupae and pharate adults and the progress of cuticular pigmentation until adult eclosion were used as parameters to study integument differentiation under hormonal treatment. Groups of bees were treated at the beginning of the pupal stage with the juvenile hormone analog pyriproxyfen (PPN) or as pharate adults with 20-hydroxyecdysone (20E). Another group was treated with both hormones applied successively at these same developmental periods. Controls were maintained without treatment. The epidermal proteins, separated by SDS-PAGE and identified by silver staining, were studied at seven intervals during the pupal and pharate adult stages. The initiation and progress of cuticular pigmentation was also monitored and compared to controls. The results showed that PPN reduced the interval of expression of some epidermal proteins, whereas 20E had an antagonistic effect, promoting a prolongation in the time of expression of the same proteins. In PPN-treated bees, cuticular pigmentation started precociously, whereas in 20E-treated individuals this developmental event was postponed. The double hormonal treatment restored the normal progress of cuticular pigmentation and, to a large extent, the temporal epidermal protein pattern. These results are discussed in relation to the 20E titer modulation and morphogenetic hormone interaction. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Apis mellifera; Cuticular pigmentation; Integument; Pyriproxyfen; Ecdysteroids
1. Introduction In Apis mellifera (Hymenoptera, Apidae) workers, the pupal and pharate adult stages have been conveniently subdivided into seven phases and the morphological identification of each phase, based on the pigmentation of the eyes and cuticle, is available for European (Rembold et al., 1980) as well as Africanized honey bees (Michelette and Soares, 1993). Thus, the younger phases showing yet unpigmented cuticle, have been classified as Pw, Pp, Pdp or Pb according to the eye coloration, i.e. white, pink, dark-pink and brown, respectively. The initiation and progress of cuticle pigmentation characterizes the subsequent brown-eyed phases, Pbl, Pbm and Pbd, which can be distinguished by their light, intermediate or dark body pigmentation, respectively.
* Corresponding author. Tel.: +55-16-602-3805; fax: 55-16-6336482. E-mail address: [email protected] (M.M.G. Bitondi).
The initiation of cuticle pigmentation, as well as its progressive hardening are coordinated by ecdysteroid hormones. A peak of makisterone A, the ecdysteroid mainly present in honey bees, was detected by Feldlaufer et al. (1985) at the Pb phase. The beginning of cuticle pigmentation during the next phase (Pbl) occurs when the ecdysteroid titer is declining (Zufelato et al., 2000; Pinto et al., 2000). Throughout the pupal and pharate adult stages, the level of juvenile hormone (JH) remains low in honey bees (Rembold, 1987), and this condition seems essential for the initiation of cuticular pigmentation at the right time because topical application of pyriproxyfen (PPN), a JH-analog, to young, still unpigmented pupae causes precocious and anomalous pigmentation (Bitondi et al., 1998; Zufelato et al., 2000). This effect was attributed to hormonal interactions since a delay in the increase of endogenous ecdysteroid levels in the hemolymph was observed after PPN-treatment (Zufelato et al., 2000). Thus, the maintenance of a low rather than an increasing level of ecdysteroids in the Pb phase, as a consequence
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of PPN-treatment, permits the precocious initiation of pigmentation. In Pb individuals not subjected to PPNtreatment, the high ecdysteroid titer temporarily prevents the initiation of cuticular pigmentation, which then occurs in the next phase, i.e. Pbl, when the level of ecdysteroids declines (Zufelato et al., 2000). In holometabolous insects, the variation in the epidermal protein pattern during development reflects differential gene transcription that leads to the construction of the adult exoskeleton. In this respect, the insect epidermis constitutes a useful model to study the temporal and hormonal regulation of gene expression since different proteins are synthesized as development proceeds. Moreover, the pupal/pharate adult stages, characterized by a well-defined increase and subsequent decrease in hemolymph ecdysteroids, are useful for the study of the expression of hormonally regulated proteins. Identification of these proteins contributes to the understanding of the morphogenetic actions of hormones on gene regulation during development. The expression of proteins susceptible to ecdysteroids as well as the time of initiation and progress of cuticular pigmentation were used here as parameters to study the interaction of an ecdysteroid — 20-hydroxyecdysone (20E) — and a JH-analog (PPN) during differentiation of the honey bee pupal integument.
2. Material and methods 2.1. Collection and treatment of A. mellifera Pw pupae were carefully collected from combs in free-flying hives maintained by the Experimental Apiary at the Medical School of Ribeira˜ o Preto, Sa˜ o Paulo University. After removal from the combs, the Pw pupae were divided into four groups that were respectively left untreated (n=315), treated with the JH analog pyriproxyfen (2-[1-methyl-2(4-phenoxyphenoxy) ethoxyl] pyridine, PPN, Sumitomo) (n=96), with 20-hydroxyecdysone (20E, Fluka Chemie) (n=147), or with both hormones (n=70). All groups were maintained in the same incubator at 34°C and 80% RH where development progressed until the time of adult emergence of untreated controls. The developing pupal and pharate adult phases (Pw, Pp, Pdp, Pb, Pbl, Pbm and Pbd) were staged according to eye color and absence or presence and intensity of cuticle pigmentation. Both criteria permit an estimate of age in days after larval eclosion (Michelette and Soares, 1993). PPN-treatment was applied during the Pw phase, within the window of sensitivity for PPN-treatment (Bitondi et al., 1998), whereas 20E was injected into Pdp or Pb bees before the increase of ecdysteroids in the hemolymph. In double-treatment experiments, a group of Pw pupae were first treated with PPN, and some days
after, when they reach the Pdp or Pb phase, they were injected with 20E. Pw pupae were treated with 1 µg of PPN in acetone (1 µg/µl), topically applied to the dorsum of the abdomen. Solutions of 20E were prepared by first dissolving this hormone in ethanol (5 mg/250 µl) and diluting it in Ringer for insects (1:3 v/v) immediately before treatment. Pdp or Pb bees were injected with 5 µg 20E in ethanol/Ringer (5 µg/µl). Controls treated only with acetone or ethanol diluted in Ringer, used as solvent for PPN and 20E, respectively, were not prepared because these substances do not interfere with cuticular pigmentation or pupal development as we have shown in previous studies (Bitondi et al., 1998; Zufelato et al., 2000). The developing protein pattern also did not differ between solvent-treated and untreated pupae, as noted in pilot experiments. Fig. 1 shows the pupal and pharate adult phases treated with PPN or 20E, the sequence of developmental phases showing unpigmented or pigmented eye and cuticle, as well as the levels of endogenous JH and ecdysteroids. 2.2. Integument sample preparation After PPN and/or 20E treatment, the abdominal integument was dissected, cleared of fat body, washed in Ringer’s solution, homogenized in water (two abdominal integuments per sample) and centrifuged at 12,000g for 5 min at 7°C. The supernatant containing the epidermal soluble proteins was stored at ⫺20°C for protein quantification and polyacrylamide gel electrophoresis (SDSPAGE). The same was done with the integument of untreated controls. 2.3. Protein determination Aliquots of the integument extracts were used for total protein quantification by the method of Bradford (1976) using bovine serum albumin as a standard. 2.4. SDS-PAGE Separation of epidermal proteins was routinely carried out by the method of Laemmli (1970) on a denaturing 4–12% polyacrylamide slab gel. SDS was not added to the separation and stacking gels but only to the sample and electrode buffers. Samples from integument extracts containing 0.5 µg protein were diluted in Laemmli’s SDS-sample buffer, boiled for 2 min, spun at 12,000g for 5 min at 7°C, and loaded on each lane of the polyacrylamide gels. Electrophoretic analyses were done with 83 samples from untreated bees, 40 samples from 20Etreated bees, 24 samples from PPN-treated bees, and 16 samples from double-treated bees. Electrophoresis was run at 15–20 mA and the gels were silver-stained
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Fig. 1. Sequence of developmental phases during pupal and pharate adult stages, and level of endogenous juvenile hormone (according to Rembold, 1987) and ecdysteroids (according Feldlaufer et al., 1985; Zufelato et al., 2000). The arrow indicates the phase treated with PPN and arrowheads indicate phases treated with 20E.
(Caetano-Anolle´ s and Gresshoff, 1994). Molecular weight markers were used as standards to determine the relative molecular weights of the epidermal proteins.
3. Results 3.1. Epidermal protein expression is arrested by 20Etreatment Some proteins from the epidermis showed differential expression from the Pdp phase until the end of the pharate adult stage. These proteins (or polypeptides) are identified by letters (a–g) in Fig. 2A. The expression of protein a was limited to the Pb phase and beginning of the Pbl phase. Protein b was expressed during Pdp, Pb and beginning of the Pbl phases. The c, d, e and f proteins also started to be expressed in the Pb phase and their activities peaked during the next phase, Pbl. The Pbl phase marks the initiation of cuticular pigmentation, an important event of adult cuticle differentiation. From this phase onwards, the cuticle progressively acquires the hardness and dark brown coloration that characterizes the adult cuticle. The g protein showed decreasing expression as development progressed. With the exception of protein a which migrated at approximately 45 kDa, all these epidermal proteins had a small molecular size, ranging from below 10 to 30 kDa. The low molecular weight is a characteristic of epidermal (or cuticular) proteins from other insects. In Manduca sexta, the molecular weight of proteins expressed during cuticular development, ranged from below 10 to
100 kDa (Hopkins et al., 2000). In Antheraea polyphemus the major proteins from the cuticle are in the molecular weight range of 15–45 kDa (Sridhara 1983, 1994). The expression of all these proteins appeared to be dependent on variation in the levels of ecdysteroids, which peaked in the hemolymph during the Pb phase (Feldlaufer et al., 1985; Zufelato et al., 2000), and this aspect was studied here by injecting 20E during the Pdp or the early Pb phase. In general, a prolongation in the time of expression of these proteins was observed in bees treated with 20E (Fig. 2C). Proteins a, b, c, d, e, f and g maintained their activity for a longer period in 20Etreated bees. The prolongation in the expression of these proteins was accompanied by a delay in the initiation of cuticular pigmentation. In 20E-treated bees, the thoracic pigmentation was initiated 24 h later than in control ones (see asterisks in Fig. 2A and C). Table 1 shows the time of onset and intensification of cuticular pigmentation in untreated controls and in 20E-treated bees until the time of emergence of controls. 3.2. PPN reduces the time of expression of some proteins Topical application of PPN to Pw bees accelerated the transition of a typical protein pattern from one phase to the next. Thus, treated bees reached the protein pattern typical of the end of the pharate adult stage earlier than controls. Fig. 2B shows that the protein pattern 96 h after PPN-treatment was similar to that observed much later (192 h) in controls. The effect of PPN was the opposite of the effect of 20E (Fig. 2C). Whereas 20E caused an
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Fig. 2. SDS-PAGE of the developmental profile of epidermal proteins during successive phases (Pdp, Pb, Pbl and Pbd) of Apis mellifera. At left of the gels, the letters and arrows indicate protein expression in: (A) control; (B) PPN-treated; (C) 20E-treated and (D) double-treated bees (PPN and 20E). The numbers below the gel figures indicate hours of development from the time of PPN application at Pw, considered to be time 0. Asterisks mark the initiation of the thoracic pigmentation (see Table 1). Molecular weight markers are given in kDa, at the right of the gels.
arrest in the protein pattern development, PPN promoted its acceleration. The same opposite effect was observed in relation to pigmentation which was anticipated and intensified in PPN-treated bees and postponed in 20Etreated bees when compared to controls (see asterisks in Fig. 2A–C and Table 1). 3.3. Double hormonal treatment reconstitutes to a large extent the normal temporal expression of epidermal proteins Treatment with 20E during the Pdp phase or at the beginning of the Pb phase overcame the effect caused
by PPN treatment during the younger phase (Pw) (Fig. 2D). After double treatment, the expression of proteins b, c, d, f and g continued to be intense by the 96th h, and all of them were still detectable by the 144th h (Fig. 2D), whereas in bees treated with PPN only, the expression of these proteins stopped earlier: they were no longer detected at the 96th h (Fig. 2B). In PPNtreated bees (Fig. 2B) proteins a and e were not detected, suggesting inhibition of their expression by the JH analog. Alternatively, the onset of expression of these proteins could have been anticipated and their temporal interval of expression abbreviated due to PPN appli-
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Table 1 Onset and progress of pigmentation in pupae treated with PPN and/or 20E compared to untreated controls
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bees never showed the dark brown color of the cuticle characteristic of the end of the pharate adult stage. Bees treated with PPN, on the contrary, pigmented earlier than untreated controls and their integument acquired a very dark brown color and hardness earlier. In these bees, the normal pigmentation was restored after injecting 20E (Fig. 3).
4. Discussion 4.1. Exogenous 20E causes prolongation in the time of expression of specific epidermal proteins and arrests cuticular pigmentation The activation of certain proteins or initiation of their expression when the levels of endogenous ecdysteroids are high in hemolymph argues for a dependence of these proteins on the ecdysteroid titer. The injection of 20E into the honey bees during the Pdp or Pb phases and the consequent prolongation in the time of expression of the cation. This also might explain why the proteins a and e were not more detectable by the 72th h. Comparisons between double-treated and control pupae (Fig. 2D and A, respectively) showed that proteins a–g were expressed at the 96th h in both groups. But, although all of these proteins were evidenced in both groups at the 120th and 144th h, their quantity seemed to be diminished in double-treated individuals. Also, the 168th h protein pattern in double-treated bees was more similar to the control at the 192th h than at the corresponding 168th h. These results denote that double treatment reconstitutes the timing of progression of the protein pattern, although not completely. Double treatment completely restored the normal intensity of the cuticular pigmentation and at the time corresponding to the end of the pharate adult period, double-treated bees were indistinguishable from controls. As observed in bees treated only with PPN, the onset of pigment formation in double-treated bees occurred approximately 24 h earlier than in controls (Table 1). But, in contrast to treatment with PPN alone, the normal pigmentation pattern soon was temporally reconstituted in bees subjected to both hormonal treatments. 3.4. Pigmentation of the integument and hormonal treatment The onset of cuticle pigmentation marks the transition from the Pb to the Pbl phases. Treatment of younger phases, Pdp or Pb, with 20E arrested the onset of pigmentation, and therefore maintained the bee morphologically like Pb for a longer time than observed in untreated controls. Thus, the 20E-treated bees started to produce pigments later and this process progressed slowly. These
Fig. 3. Cuticular pigmentation in pharate adults: (A) treated with PPN; (B) treated with 20E; (C) untreated and (D) treated with PPN and subsequently with 20E. PPN was topically applied at the beginning of the pupal stage (Pw phase) and 20E was injected in pharate adults (Pdp or early Pb phases). All individuals have the same chronological age.
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a, b, c, d, e, f and g proteins strongly reinforces this argument. Thus, the maintenance of a protein pattern characteristic of a pharate adult stage for a longer time indicates that integument differentiation was arrested by treatment with 20E (compare Fig. 2A and C). The alteration in the timing of epidermal protein expression in 20E-treated bees was accompanied by the arrest of cuticular pigmentation (or melanin synthesis). The onset and intensification of cuticular melanization is indicative of the progress of integument differentiation toward the adult stage. In 20E treated bees, this developmental event was initiated later, probably when the hemolymph ecdysteroid titer increased by the injected exogenous 20E, declined as a consequence of hormone degradation. There are a number of examples in insects (Schwartz and Truman, 1983; Hiruma et al., 1991; Apple and Fristrom, 1991) showing that the decline of ecdysteroid titer is required for the late events of differentiation and adult eclosion. Treatment with 20E delayed adult eclosion in M. sexta (Truman, 1981; Truman et al., 1983) and in the coleopteran Tenebrio molitor (Sla´ ma, 1980 in Truman et al., 1983). By reducing ecdysteroid levels in M. sexta through abdomen ligation, Schwartz and Truman (1983) observed acceleration in abdominal tissue differentiation. Injection of 20E reversed this effect, showing that the rate of development is under ecdysteroid regulation. All these examples show that decline of the ecdysteroid titer is required for adult differentiation and are in accordance with the model proposed by Schwartz and Truman (1983) for control of the rate of development in M. sexta. These authors pointed out that during the earlier phases of pupal development, the ecdysteroids act positively to promote development, whereas during the later stages they switch to being inhibitory. This endocrine control seems to ensure that the different tissues complete development in a coordinated fashion. 4.2. PPN reduces the temporal interval of expression of 20E-dependent proteins and anticipates and intensifies cuticular pigmentation JH is undetectable in A. mellifera hemolymph along the pupal and pharate adult stages (Rembold, 1987). This condition is essential for the normal rate of differentiation of the cuticle as indicated by the fact that topical application of the JH analog PPN to young pupae causes precocious pigmentation and acceleration of development (Bitondi et al., 1998; Zufelato et al., 2000; this work). The temporal interval of expression of the 20E-dependent proteins was reduced in PPN-treated honey bees that soon showed the epidermal protein pattern typical of pharate adults ready to emerge. PPN also suppressed or at least caused a considerable reduction in the tem-
poral interval of expression of some honey bee 20Edependent proteins (a and e proteins). An inhibitory effect of a JH-analog on epidermal proteins was also observed in T. molitor. The treatment prevented the accumulation of the adult-specific ACP-22 transcript (Bouhin et al., 1992) and impaired the appearance of the ACP-20 transcript (Charles et al., 1992). 4.3. PPN-ecdysteroid interaction It is largely accepted that the presence of JH during a JH-sensitive period maintains the current developmental state of holometabolous insect, whereas absence of JH can entail a switch in developmental pathways (Nijhout, 1994). During the end of the last larval instar, JH drops to an undetectable level and remains undetectable throughout the pupal and pharate adult stages. This is true for M. sexta, which has one of the best understood JH titer profiles for the period prior to and during metamorphosis (Riddiford 1985, 1994), as well as for other holometabola, among them A. mellifera honey bees (Rembold, 1987; Rachinsky et al., 1991). Thus, the differentiation of adult epidermis occurs in the absence of JH, and exposure of pupae and pharate adults to exogenous JH could interfere with this process. In Hyalophora cecropia, application or injection of exogenous JH early during the pupal stage caused development of a normal or nearly normal second pupal cuticle and consequent maintenance of the status quo (Williams, 1959 in Nijhout, 1994). Also, JH provided experimentally just after pupal ecdysis of A. polyphemus (Sridhara, 1985) and T. molitor (Bouhin et al., 1992), suppressed the appearance of new cuticular mRNAs. In T. molitor, one of these mRNAs is expressed in the adult cuticle. The mRNAs expressed after JH-treatment were similar to those found in the pupal epidermis. This is consistent with the formation of a second pupal cuticle instead of an adult one (Riddiford, 1994). Application of JH to Diptera at the time of head eversion also prevented normal differentiation of the abdominal histoblasts (Bhaskaran, 1972; Postlethwait, 1974). When a JH analog, PPN, was applied to honey bees at the beginning of the pupal stage, a precocious and intense pigmentation developed in the cuticle. Also, the epidermal protein pattern typical of pharate adults ready to emerge appeared earlier than in controls. These results and also the observed acceleration in development caused by PPN suggested a rapid switch to adult exoskeleton differentiation. At first sight, these results do not seem to match the general developmental function of JH in maintaining the epidermal status quo. At least for the characters studied here (cuticular pigmentation, epidermal protein pattern and duration of pupal/pharate adult stages), the JH analog seemed to stimulate differentiation to the adult stage. However, despite the appar-
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ent advance in development, PPN-treated bees did not emerge as adults but died. These results were interpreted in the light of another study from our laboratory (Zufelato et al., 2000), important in the context discussed above. Using a radioimmunoassay we showed that the ecdysteroid peak normally occurring in honey bees at the Pb stage was arrested after treatment with PPN. As a consequence, at the Pb stage, PPN-treated bees had a low rather than a high ecdysteroid level in the hemolymph. This low ecdysteroid level permitted the expression of characteristics typical of older individuals, which normally develop in the presence of a low ecdysteroid titer. Thus, synthesis of pigments and an epidermal protein pattern characteristic of older individuals were detected earlier in PPN-treated bees. The injection of 20E into developing bees previously treated with PPN, completely rescued the normal pigmentation pattern and, to a large extent, the epidermal protein pattern. These results are attributed to the reconstitution of the ecdysteroid peak at the correct developmental time. The JH-analog PPN could be altering the ecdysteroid pattern by affecting the prothoracic glands, the site of ecdysteroid synthesis. The results of an earlier study on the JH-20E interaction using injections of crude JH extracts or implantation of active corpora allata, the JH source, into brainless pupae of Lepidoptera (where there is no prothoracicotropic hormone to induce ecdysteroid synthesis) suggested a function for JH in stimulating ecdysteroid secretion in prothoracic glands (Smith, 1985 in Nijhout, 1994). Recently, Dai and Gilbert (1998) measured the ability of the prothoracic glands to synthesize ecdysteroids in vitro after dissecting them from M. sexta pupae injected with JH at the beginning of the pupal stage. They showed that JH prolongs the period of activity of these glands by preventing the onset of programmed cell death (Dai and Gilbert 1997, 1998). This result possibly explains why JH-treated M. sexta pupae (or JH-analog-treated A. mellifera pupae) did not emerge as adults: eclosion was impaired because glandular integrity and activity were maintained. The specific molecular mechanisms through which JH interferes with developmental programs regulated by ecdysteroids remain obscure (Zhou et al., 1998). Reports on the action of JH and ecdysteroids in different insects, specific tissues, cells, or biochemical and genetic events are useful for understanding the hormonal control of developmental processes, differentiation and metamorphosis. Some reports have brought to light variations in the general model of hormonal action on pupal and pharate adult development. In T. molitor, for example, secretion of adult cuticle was detected even when a high 20E level was experimentally maintained. This is contrary to the general ‘rule’ commented upon above according to which the late events of adult differentiation
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require a decline of the ecdysteroid titer (Schwartz and Truman, 1983; Hiruma et al., 1991; Apple and Fristrom, 1991). Also, the expression of an adult specific gene — ACP-20 — was stimulated by 20E. The activity of this gene was not dependent on the decay of the ecdysteroid peak, pointing to a mode of regulation different from that of other adult cuticle genes (Braquart et al., 1996). Thus, exposure to a high ecdysteroid titer at the end of the pharate adult period may not inhibit the expression of certain adult characters. By identifying specific honey bee epidermal proteins dependent on 20E, and also by showing that cuticular pigmentation occurring precociously in PPN-treated honey bees could be rescued to the normal pattern after treatment with 20E, our work makes a contribution to studies on processes of development dependent on hormonal modulation.
Acknowledgements The authors thank Luı´s Roberto Aguiar for technical assistance in the apiary. This research was supported by FAPESP.
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Hormone-dependent protein patterns in integument and cuticular pigmentation in Apis mellifera during pharate adult development A.E. Santos, M.M.G. Bitondi *, Z.L.P. Simo˜es Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras; Departamento de Gene´tica, Faculdade de Medicina. Universidade de Sa˜o Paulo, Av Bandeirantes 3900, 14040-901 Ribeirao Preto, SP, Brazil Received 6 February 2001; accepted 11 June 2001
Abstract The epidermal proteins from staged Apis mellifera pupae and pharate adults and the progress of cuticular pigmentation until adult eclosion were used as parameters to study integument differentiation under hormonal treatment. Groups of bees were treated at the beginning of the pupal stage with the juvenile hormone analog pyriproxyfen (PPN) or as pharate adults with 20-hydroxyecdysone (20E). Another group was treated with both hormones applied successively at these same developmental periods. Controls were maintained without treatment. The epidermal proteins, separated by SDS-PAGE and identified by silver staining, were studied at seven intervals during the pupal and pharate adult stages. The initiation and progress of cuticular pigmentation was also monitored and compared to controls. The results showed that PPN reduced the interval of expression of some epidermal proteins, whereas 20E had an antagonistic effect, promoting a prolongation in the time of expression of the same proteins. In PPN-treated bees, cuticular pigmentation started precociously, whereas in 20E-treated individuals this developmental event was postponed. The double hormonal treatment restored the normal progress of cuticular pigmentation and, to a large extent, the temporal epidermal protein pattern. These results are discussed in relation to the 20E titer modulation and morphogenetic hormone interaction. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Apis mellifera; Cuticular pigmentation; Integument; Pyriproxyfen; Ecdysteroids
1. Introduction In Apis mellifera (Hymenoptera, Apidae) workers, the pupal and pharate adult stages have been conveniently subdivided into seven phases and the morphological identification of each phase, based on the pigmentation of the eyes and cuticle, is available for European (Rembold et al., 1980) as well as Africanized honey bees (Michelette and Soares, 1993). Thus, the younger phases showing yet unpigmented cuticle, have been classified as Pw, Pp, Pdp or Pb according to the eye coloration, i.e. white, pink, dark-pink and brown, respectively. The initiation and progress of cuticle pigmentation characterizes the subsequent brown-eyed phases, Pbl, Pbm and Pbd, which can be distinguished by their light, intermediate or dark body pigmentation, respectively.
* Corresponding author. Tel.: +55-16-602-3805; fax: 55-16-6336482. E-mail address: [email protected] (M.M.G. Bitondi).
The initiation of cuticle pigmentation, as well as its progressive hardening are coordinated by ecdysteroid hormones. A peak of makisterone A, the ecdysteroid mainly present in honey bees, was detected by Feldlaufer et al. (1985) at the Pb phase. The beginning of cuticle pigmentation during the next phase (Pbl) occurs when the ecdysteroid titer is declining (Zufelato et al., 2000; Pinto et al., 2000). Throughout the pupal and pharate adult stages, the level of juvenile hormone (JH) remains low in honey bees (Rembold, 1987), and this condition seems essential for the initiation of cuticular pigmentation at the right time because topical application of pyriproxyfen (PPN), a JH-analog, to young, still unpigmented pupae causes precocious and anomalous pigmentation (Bitondi et al., 1998; Zufelato et al., 2000). This effect was attributed to hormonal interactions since a delay in the increase of endogenous ecdysteroid levels in the hemolymph was observed after PPN-treatment (Zufelato et al., 2000). Thus, the maintenance of a low rather than an increasing level of ecdysteroids in the Pb phase, as a consequence
0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 1 1 4 - 7
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of PPN-treatment, permits the precocious initiation of pigmentation. In Pb individuals not subjected to PPNtreatment, the high ecdysteroid titer temporarily prevents the initiation of cuticular pigmentation, which then occurs in the next phase, i.e. Pbl, when the level of ecdysteroids declines (Zufelato et al., 2000). In holometabolous insects, the variation in the epidermal protein pattern during development reflects differential gene transcription that leads to the construction of the adult exoskeleton. In this respect, the insect epidermis constitutes a useful model to study the temporal and hormonal regulation of gene expression since different proteins are synthesized as development proceeds. Moreover, the pupal/pharate adult stages, characterized by a well-defined increase and subsequent decrease in hemolymph ecdysteroids, are useful for the study of the expression of hormonally regulated proteins. Identification of these proteins contributes to the understanding of the morphogenetic actions of hormones on gene regulation during development. The expression of proteins susceptible to ecdysteroids as well as the time of initiation and progress of cuticular pigmentation were used here as parameters to study the interaction of an ecdysteroid — 20-hydroxyecdysone (20E) — and a JH-analog (PPN) during differentiation of the honey bee pupal integument.
2. Material and methods 2.1. Collection and treatment of A. mellifera Pw pupae were carefully collected from combs in free-flying hives maintained by the Experimental Apiary at the Medical School of Ribeira˜ o Preto, Sa˜ o Paulo University. After removal from the combs, the Pw pupae were divided into four groups that were respectively left untreated (n=315), treated with the JH analog pyriproxyfen (2-[1-methyl-2(4-phenoxyphenoxy) ethoxyl] pyridine, PPN, Sumitomo) (n=96), with 20-hydroxyecdysone (20E, Fluka Chemie) (n=147), or with both hormones (n=70). All groups were maintained in the same incubator at 34°C and 80% RH where development progressed until the time of adult emergence of untreated controls. The developing pupal and pharate adult phases (Pw, Pp, Pdp, Pb, Pbl, Pbm and Pbd) were staged according to eye color and absence or presence and intensity of cuticle pigmentation. Both criteria permit an estimate of age in days after larval eclosion (Michelette and Soares, 1993). PPN-treatment was applied during the Pw phase, within the window of sensitivity for PPN-treatment (Bitondi et al., 1998), whereas 20E was injected into Pdp or Pb bees before the increase of ecdysteroids in the hemolymph. In double-treatment experiments, a group of Pw pupae were first treated with PPN, and some days
after, when they reach the Pdp or Pb phase, they were injected with 20E. Pw pupae were treated with 1 µg of PPN in acetone (1 µg/µl), topically applied to the dorsum of the abdomen. Solutions of 20E were prepared by first dissolving this hormone in ethanol (5 mg/250 µl) and diluting it in Ringer for insects (1:3 v/v) immediately before treatment. Pdp or Pb bees were injected with 5 µg 20E in ethanol/Ringer (5 µg/µl). Controls treated only with acetone or ethanol diluted in Ringer, used as solvent for PPN and 20E, respectively, were not prepared because these substances do not interfere with cuticular pigmentation or pupal development as we have shown in previous studies (Bitondi et al., 1998; Zufelato et al., 2000). The developing protein pattern also did not differ between solvent-treated and untreated pupae, as noted in pilot experiments. Fig. 1 shows the pupal and pharate adult phases treated with PPN or 20E, the sequence of developmental phases showing unpigmented or pigmented eye and cuticle, as well as the levels of endogenous JH and ecdysteroids. 2.2. Integument sample preparation After PPN and/or 20E treatment, the abdominal integument was dissected, cleared of fat body, washed in Ringer’s solution, homogenized in water (two abdominal integuments per sample) and centrifuged at 12,000g for 5 min at 7°C. The supernatant containing the epidermal soluble proteins was stored at ⫺20°C for protein quantification and polyacrylamide gel electrophoresis (SDSPAGE). The same was done with the integument of untreated controls. 2.3. Protein determination Aliquots of the integument extracts were used for total protein quantification by the method of Bradford (1976) using bovine serum albumin as a standard. 2.4. SDS-PAGE Separation of epidermal proteins was routinely carried out by the method of Laemmli (1970) on a denaturing 4–12% polyacrylamide slab gel. SDS was not added to the separation and stacking gels but only to the sample and electrode buffers. Samples from integument extracts containing 0.5 µg protein were diluted in Laemmli’s SDS-sample buffer, boiled for 2 min, spun at 12,000g for 5 min at 7°C, and loaded on each lane of the polyacrylamide gels. Electrophoretic analyses were done with 83 samples from untreated bees, 40 samples from 20Etreated bees, 24 samples from PPN-treated bees, and 16 samples from double-treated bees. Electrophoresis was run at 15–20 mA and the gels were silver-stained
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Fig. 1. Sequence of developmental phases during pupal and pharate adult stages, and level of endogenous juvenile hormone (according to Rembold, 1987) and ecdysteroids (according Feldlaufer et al., 1985; Zufelato et al., 2000). The arrow indicates the phase treated with PPN and arrowheads indicate phases treated with 20E.
(Caetano-Anolle´ s and Gresshoff, 1994). Molecular weight markers were used as standards to determine the relative molecular weights of the epidermal proteins.
3. Results 3.1. Epidermal protein expression is arrested by 20Etreatment Some proteins from the epidermis showed differential expression from the Pdp phase until the end of the pharate adult stage. These proteins (or polypeptides) are identified by letters (a–g) in Fig. 2A. The expression of protein a was limited to the Pb phase and beginning of the Pbl phase. Protein b was expressed during Pdp, Pb and beginning of the Pbl phases. The c, d, e and f proteins also started to be expressed in the Pb phase and their activities peaked during the next phase, Pbl. The Pbl phase marks the initiation of cuticular pigmentation, an important event of adult cuticle differentiation. From this phase onwards, the cuticle progressively acquires the hardness and dark brown coloration that characterizes the adult cuticle. The g protein showed decreasing expression as development progressed. With the exception of protein a which migrated at approximately 45 kDa, all these epidermal proteins had a small molecular size, ranging from below 10 to 30 kDa. The low molecular weight is a characteristic of epidermal (or cuticular) proteins from other insects. In Manduca sexta, the molecular weight of proteins expressed during cuticular development, ranged from below 10 to
100 kDa (Hopkins et al., 2000). In Antheraea polyphemus the major proteins from the cuticle are in the molecular weight range of 15–45 kDa (Sridhara 1983, 1994). The expression of all these proteins appeared to be dependent on variation in the levels of ecdysteroids, which peaked in the hemolymph during the Pb phase (Feldlaufer et al., 1985; Zufelato et al., 2000), and this aspect was studied here by injecting 20E during the Pdp or the early Pb phase. In general, a prolongation in the time of expression of these proteins was observed in bees treated with 20E (Fig. 2C). Proteins a, b, c, d, e, f and g maintained their activity for a longer period in 20Etreated bees. The prolongation in the expression of these proteins was accompanied by a delay in the initiation of cuticular pigmentation. In 20E-treated bees, the thoracic pigmentation was initiated 24 h later than in control ones (see asterisks in Fig. 2A and C). Table 1 shows the time of onset and intensification of cuticular pigmentation in untreated controls and in 20E-treated bees until the time of emergence of controls. 3.2. PPN reduces the time of expression of some proteins Topical application of PPN to Pw bees accelerated the transition of a typical protein pattern from one phase to the next. Thus, treated bees reached the protein pattern typical of the end of the pharate adult stage earlier than controls. Fig. 2B shows that the protein pattern 96 h after PPN-treatment was similar to that observed much later (192 h) in controls. The effect of PPN was the opposite of the effect of 20E (Fig. 2C). Whereas 20E caused an
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Fig. 2. SDS-PAGE of the developmental profile of epidermal proteins during successive phases (Pdp, Pb, Pbl and Pbd) of Apis mellifera. At left of the gels, the letters and arrows indicate protein expression in: (A) control; (B) PPN-treated; (C) 20E-treated and (D) double-treated bees (PPN and 20E). The numbers below the gel figures indicate hours of development from the time of PPN application at Pw, considered to be time 0. Asterisks mark the initiation of the thoracic pigmentation (see Table 1). Molecular weight markers are given in kDa, at the right of the gels.
arrest in the protein pattern development, PPN promoted its acceleration. The same opposite effect was observed in relation to pigmentation which was anticipated and intensified in PPN-treated bees and postponed in 20Etreated bees when compared to controls (see asterisks in Fig. 2A–C and Table 1). 3.3. Double hormonal treatment reconstitutes to a large extent the normal temporal expression of epidermal proteins Treatment with 20E during the Pdp phase or at the beginning of the Pb phase overcame the effect caused
by PPN treatment during the younger phase (Pw) (Fig. 2D). After double treatment, the expression of proteins b, c, d, f and g continued to be intense by the 96th h, and all of them were still detectable by the 144th h (Fig. 2D), whereas in bees treated with PPN only, the expression of these proteins stopped earlier: they were no longer detected at the 96th h (Fig. 2B). In PPNtreated bees (Fig. 2B) proteins a and e were not detected, suggesting inhibition of their expression by the JH analog. Alternatively, the onset of expression of these proteins could have been anticipated and their temporal interval of expression abbreviated due to PPN appli-
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Table 1 Onset and progress of pigmentation in pupae treated with PPN and/or 20E compared to untreated controls
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bees never showed the dark brown color of the cuticle characteristic of the end of the pharate adult stage. Bees treated with PPN, on the contrary, pigmented earlier than untreated controls and their integument acquired a very dark brown color and hardness earlier. In these bees, the normal pigmentation was restored after injecting 20E (Fig. 3).
4. Discussion 4.1. Exogenous 20E causes prolongation in the time of expression of specific epidermal proteins and arrests cuticular pigmentation The activation of certain proteins or initiation of their expression when the levels of endogenous ecdysteroids are high in hemolymph argues for a dependence of these proteins on the ecdysteroid titer. The injection of 20E into the honey bees during the Pdp or Pb phases and the consequent prolongation in the time of expression of the cation. This also might explain why the proteins a and e were not more detectable by the 72th h. Comparisons between double-treated and control pupae (Fig. 2D and A, respectively) showed that proteins a–g were expressed at the 96th h in both groups. But, although all of these proteins were evidenced in both groups at the 120th and 144th h, their quantity seemed to be diminished in double-treated individuals. Also, the 168th h protein pattern in double-treated bees was more similar to the control at the 192th h than at the corresponding 168th h. These results denote that double treatment reconstitutes the timing of progression of the protein pattern, although not completely. Double treatment completely restored the normal intensity of the cuticular pigmentation and at the time corresponding to the end of the pharate adult period, double-treated bees were indistinguishable from controls. As observed in bees treated only with PPN, the onset of pigment formation in double-treated bees occurred approximately 24 h earlier than in controls (Table 1). But, in contrast to treatment with PPN alone, the normal pigmentation pattern soon was temporally reconstituted in bees subjected to both hormonal treatments. 3.4. Pigmentation of the integument and hormonal treatment The onset of cuticle pigmentation marks the transition from the Pb to the Pbl phases. Treatment of younger phases, Pdp or Pb, with 20E arrested the onset of pigmentation, and therefore maintained the bee morphologically like Pb for a longer time than observed in untreated controls. Thus, the 20E-treated bees started to produce pigments later and this process progressed slowly. These
Fig. 3. Cuticular pigmentation in pharate adults: (A) treated with PPN; (B) treated with 20E; (C) untreated and (D) treated with PPN and subsequently with 20E. PPN was topically applied at the beginning of the pupal stage (Pw phase) and 20E was injected in pharate adults (Pdp or early Pb phases). All individuals have the same chronological age.
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a, b, c, d, e, f and g proteins strongly reinforces this argument. Thus, the maintenance of a protein pattern characteristic of a pharate adult stage for a longer time indicates that integument differentiation was arrested by treatment with 20E (compare Fig. 2A and C). The alteration in the timing of epidermal protein expression in 20E-treated bees was accompanied by the arrest of cuticular pigmentation (or melanin synthesis). The onset and intensification of cuticular melanization is indicative of the progress of integument differentiation toward the adult stage. In 20E treated bees, this developmental event was initiated later, probably when the hemolymph ecdysteroid titer increased by the injected exogenous 20E, declined as a consequence of hormone degradation. There are a number of examples in insects (Schwartz and Truman, 1983; Hiruma et al., 1991; Apple and Fristrom, 1991) showing that the decline of ecdysteroid titer is required for the late events of differentiation and adult eclosion. Treatment with 20E delayed adult eclosion in M. sexta (Truman, 1981; Truman et al., 1983) and in the coleopteran Tenebrio molitor (Sla´ ma, 1980 in Truman et al., 1983). By reducing ecdysteroid levels in M. sexta through abdomen ligation, Schwartz and Truman (1983) observed acceleration in abdominal tissue differentiation. Injection of 20E reversed this effect, showing that the rate of development is under ecdysteroid regulation. All these examples show that decline of the ecdysteroid titer is required for adult differentiation and are in accordance with the model proposed by Schwartz and Truman (1983) for control of the rate of development in M. sexta. These authors pointed out that during the earlier phases of pupal development, the ecdysteroids act positively to promote development, whereas during the later stages they switch to being inhibitory. This endocrine control seems to ensure that the different tissues complete development in a coordinated fashion. 4.2. PPN reduces the temporal interval of expression of 20E-dependent proteins and anticipates and intensifies cuticular pigmentation JH is undetectable in A. mellifera hemolymph along the pupal and pharate adult stages (Rembold, 1987). This condition is essential for the normal rate of differentiation of the cuticle as indicated by the fact that topical application of the JH analog PPN to young pupae causes precocious pigmentation and acceleration of development (Bitondi et al., 1998; Zufelato et al., 2000; this work). The temporal interval of expression of the 20E-dependent proteins was reduced in PPN-treated honey bees that soon showed the epidermal protein pattern typical of pharate adults ready to emerge. PPN also suppressed or at least caused a considerable reduction in the tem-
poral interval of expression of some honey bee 20Edependent proteins (a and e proteins). An inhibitory effect of a JH-analog on epidermal proteins was also observed in T. molitor. The treatment prevented the accumulation of the adult-specific ACP-22 transcript (Bouhin et al., 1992) and impaired the appearance of the ACP-20 transcript (Charles et al., 1992). 4.3. PPN-ecdysteroid interaction It is largely accepted that the presence of JH during a JH-sensitive period maintains the current developmental state of holometabolous insect, whereas absence of JH can entail a switch in developmental pathways (Nijhout, 1994). During the end of the last larval instar, JH drops to an undetectable level and remains undetectable throughout the pupal and pharate adult stages. This is true for M. sexta, which has one of the best understood JH titer profiles for the period prior to and during metamorphosis (Riddiford 1985, 1994), as well as for other holometabola, among them A. mellifera honey bees (Rembold, 1987; Rachinsky et al., 1991). Thus, the differentiation of adult epidermis occurs in the absence of JH, and exposure of pupae and pharate adults to exogenous JH could interfere with this process. In Hyalophora cecropia, application or injection of exogenous JH early during the pupal stage caused development of a normal or nearly normal second pupal cuticle and consequent maintenance of the status quo (Williams, 1959 in Nijhout, 1994). Also, JH provided experimentally just after pupal ecdysis of A. polyphemus (Sridhara, 1985) and T. molitor (Bouhin et al., 1992), suppressed the appearance of new cuticular mRNAs. In T. molitor, one of these mRNAs is expressed in the adult cuticle. The mRNAs expressed after JH-treatment were similar to those found in the pupal epidermis. This is consistent with the formation of a second pupal cuticle instead of an adult one (Riddiford, 1994). Application of JH to Diptera at the time of head eversion also prevented normal differentiation of the abdominal histoblasts (Bhaskaran, 1972; Postlethwait, 1974). When a JH analog, PPN, was applied to honey bees at the beginning of the pupal stage, a precocious and intense pigmentation developed in the cuticle. Also, the epidermal protein pattern typical of pharate adults ready to emerge appeared earlier than in controls. These results and also the observed acceleration in development caused by PPN suggested a rapid switch to adult exoskeleton differentiation. At first sight, these results do not seem to match the general developmental function of JH in maintaining the epidermal status quo. At least for the characters studied here (cuticular pigmentation, epidermal protein pattern and duration of pupal/pharate adult stages), the JH analog seemed to stimulate differentiation to the adult stage. However, despite the appar-
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ent advance in development, PPN-treated bees did not emerge as adults but died. These results were interpreted in the light of another study from our laboratory (Zufelato et al., 2000), important in the context discussed above. Using a radioimmunoassay we showed that the ecdysteroid peak normally occurring in honey bees at the Pb stage was arrested after treatment with PPN. As a consequence, at the Pb stage, PPN-treated bees had a low rather than a high ecdysteroid level in the hemolymph. This low ecdysteroid level permitted the expression of characteristics typical of older individuals, which normally develop in the presence of a low ecdysteroid titer. Thus, synthesis of pigments and an epidermal protein pattern characteristic of older individuals were detected earlier in PPN-treated bees. The injection of 20E into developing bees previously treated with PPN, completely rescued the normal pigmentation pattern and, to a large extent, the epidermal protein pattern. These results are attributed to the reconstitution of the ecdysteroid peak at the correct developmental time. The JH-analog PPN could be altering the ecdysteroid pattern by affecting the prothoracic glands, the site of ecdysteroid synthesis. The results of an earlier study on the JH-20E interaction using injections of crude JH extracts or implantation of active corpora allata, the JH source, into brainless pupae of Lepidoptera (where there is no prothoracicotropic hormone to induce ecdysteroid synthesis) suggested a function for JH in stimulating ecdysteroid secretion in prothoracic glands (Smith, 1985 in Nijhout, 1994). Recently, Dai and Gilbert (1998) measured the ability of the prothoracic glands to synthesize ecdysteroids in vitro after dissecting them from M. sexta pupae injected with JH at the beginning of the pupal stage. They showed that JH prolongs the period of activity of these glands by preventing the onset of programmed cell death (Dai and Gilbert 1997, 1998). This result possibly explains why JH-treated M. sexta pupae (or JH-analog-treated A. mellifera pupae) did not emerge as adults: eclosion was impaired because glandular integrity and activity were maintained. The specific molecular mechanisms through which JH interferes with developmental programs regulated by ecdysteroids remain obscure (Zhou et al., 1998). Reports on the action of JH and ecdysteroids in different insects, specific tissues, cells, or biochemical and genetic events are useful for understanding the hormonal control of developmental processes, differentiation and metamorphosis. Some reports have brought to light variations in the general model of hormonal action on pupal and pharate adult development. In T. molitor, for example, secretion of adult cuticle was detected even when a high 20E level was experimentally maintained. This is contrary to the general ‘rule’ commented upon above according to which the late events of adult differentiation
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require a decline of the ecdysteroid titer (Schwartz and Truman, 1983; Hiruma et al., 1991; Apple and Fristrom, 1991). Also, the expression of an adult specific gene — ACP-20 — was stimulated by 20E. The activity of this gene was not dependent on the decay of the ecdysteroid peak, pointing to a mode of regulation different from that of other adult cuticle genes (Braquart et al., 1996). Thus, exposure to a high ecdysteroid titer at the end of the pharate adult period may not inhibit the expression of certain adult characters. By identifying specific honey bee epidermal proteins dependent on 20E, and also by showing that cuticular pigmentation occurring precociously in PPN-treated honey bees could be rescued to the normal pattern after treatment with 20E, our work makes a contribution to studies on processes of development dependent on hormonal modulation.
Acknowledgements The authors thank Luı´s Roberto Aguiar for technical assistance in the apiary. This research was supported by FAPESP.
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