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The Apis mellifera pupal melanization program is affected by treatment with a juvenile hormone analogue
Journal of Insect Physiology 44 (1998) 499–507
The Apis mellifera pupal melanization program is affected by treatment with a juvenile hormone analogue M.M.G. Bitondi *, I.M. Mora, Z.L.P. Simo˜es, V.L.C. Figueiredo Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Departamento de Biologia, Av. Bandeirantes 3900, 14040-901 Ribeira˜o Preto SP, Brazil Received 3 March 1997; received in revised form 21 July 1997
Abstract Apis mellifera treated during different developmental phases with pyriproxyfen, a juvenile hormone analogue, show profound alterations in cuticular pigmentation and sclerotization. When the treatment is effected during the feeding phase of the fifth larval instar (LF5), the pupal development is blocked and pigmentation does not occur. Treatment of older larvae, at the spinning phase of the fifth larval instar (LS5), of prepupae (PP) or pupae at the beginning of the pupal period (Pw, white-eyed, unpigmented cuticle pupae) does not impair pigmentation, but, instead, this process is accelerated, intensified and abnormal. Hormonal treatment during these developmental phases (LS5, PP and Pw) induces earlier activity of phenoloxidase, an enzyme of the reaction chain leading to melanin synthesis. Treated pupae have significantly higher enzymatic levels and show a graded response in phenoloxidase activity after treatment with 0.1, 1 or 5 g pyriproxyfen. Besides pigmentation, other developmental events were also altered in treated bees: pupal development was shortened, and the expression of esterase-6 activity, the onset of which coincides with the beginning of pigmentation, was shifted with the precocious initiation of this process in treated pupae. The significance of these results is discussed in relation to the mode of hormonal action on cuticular pigmentation in insects. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Apis mellifera; Cuticular melanization; Pyriproxyfen; Phenoloxidase; Hymenoptera
1. Introduction Melanin synthesis and deposition in the cuticle occur at the proper time during insect development. An important enzyme in the biosynthesis of melanin is phenoloxidase. When activated, this enzyme catalyzes the hydroxylation of tyrosine to dopa and oxidizes dopa to the corresponding quinone, giving rise to melanin (Andersen, 1985). This same enzymatic activity is also important in cuticle sclerotization (Hiruma and Riddiford, 1988). In several insect species, phenoloxidase exists as an inactive precursor, prophenoloxidase, which is activated by a cascade of serine proteases (Yoshida and Ashida, 1986). Cuticular melanization is a developmental process hormonally regulated. Until now, most of the studies concerning hormonal control of phenoloxidase synthesis
* Corresponding author. Fax: 55 (16) 633-6482; E-mail: [email protected] 0022–1910 /98 /$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 7 ) 0 0 1 1 3 - 3
and consequent cuticular melanization have been performed in lepidopteran larvae. Juvenile hormone, ecdysteroids as well as neuroendocrine factors have been implicated in these processes (Curtis et al., 1984; Hiruma et al., 1984, 1985, 1993; Hiruma and Riddiford, 1988; Matsumoto et al., 1981, 1986, 1990; Ohashi et al., 1983). In Manduca sexta, the cellular basis of the action of juvenile hormone on phenoloxidase synthesis and consequent melanization is best known (Riddiford, 1994). In this lepidopteran, the decline in juvenile hormone during head capsule slippage is an essential condition for prophenoloxidase synthesis in the epidermis. Under this condition, this enzyme, specific for melanization, is incorporated into the newly synthesized cuticle and is subsequently activated to phenoloxidase at the time of the decline in ecdysteroid titre. Exogenous juvenile hormone prevents the synthesis of prophenoloxidase and consequent melanization if given at the time of head capsule slippage which occurs before the peak of ecdysteroid titre (Riddiford, 1994). In the hymenopteran A. mellifera, the process of
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cuticular melanization begins at the pupal phase. This developmental period is characterized by basal levels of juvenile hormone in the hemolymph (Rembold, 1987) and by a peak of ecdysteroid (Feldlaufer et al., 1985) occurring approximately in the middle of the pupal period, before the initiation of cuticular melanization. As observed for M. sexta this low level of juvenile hormone seems to be required for the normal cuticular melanization of A. mellifera because disturbances in this developmental event were observed after topical treatment with juvenile hormone or analogues. Hrdys (1973, in Rembold et al., 1974) reported that feeding of honey bee colonies with juvenile hormone analogues leads to development of black brood or, according to our interpretation, of brood with intense cuticular pigmentation. Zdarek and Haragsim (1974) also observed alterations in cuticular tanning after topical applications of juvenile hormone analogues to developing honey bees. But, in contrast to what was observed in M. sexta, these data on honey bees pointed to an intensification of cuticular pigmentation following topical application of juvenile hormone, and not inhibition. The above considerations led us to undertake the present study in order to characterize the juvenile hormone action on phenoloxidase activity and consequent cuticular melanization of A. mellifera. Pyriproxyfen, a juvenile hormone analogue, was topically applied to larvae of fifth instar, prepupae and pupae of various ages. Subsequently, the onset and timing of the pigmentation process and the developmental phase sensitivity to this hormone in terms of pigmentation were then investigated. An esterase (Est-6), whose onset of activity coincides with the beginning of pigmentation (Figueiredo et al., 1996), was used as a molecular marker of this event. Since pyriproxyfen was applied to different developmental phases, its effects on other critical events, in addition to cuticle pigmentation, such as timing of larval–pupal transition, pupal development and adult emergence were also described in order to characterize the juvenile hormone mimicking action of this analogue in A. mellifera.
2. Material and methods 2.1. Apis mellifera Larvae, prepupae and pupae of Africanized A. mellifera workers were collected from hives of the experimental apiary of the Department of Genetics, Faculty of Medicine of Ribeira˜o Preto, Sa˜o Paulo State, Brazil. To obtain immatures of uniform age, queens were periodically confined for 6 h on combs without brood, where the eggs were laid. The limitation of the period of oviposition allowed us to use groups of larvae, prepupae or pupae varying by a maximum of 6 h in age. These
developmental phases were treated with pyriproxyfen (2[1-methyl-2(4-phenoxyphenoxy) ethoxyl] pyridine, a juvenile hormone analogue, product of Sumitomo Chemical Co. Ltd, Osaka, Japan, and their development was monitored from treatment to emergence. The larval instars and prepupal or pupal phases of Africanized A. mellifera were distinguished according to Michelette and Soares (1993). 2.2. Treatment LF5 larvae (feeding phase of the fifth larval instar), aged 4–4.5 days a.h. were topically treated with 1 g pyriproxyfen dissolved in 1 l acetone, in their own comb cell, i.e., in situ. Controls were treated with 1 l acetone or left untreated. The solutions of pyriproxyfen or acetone were applied to the cuticles of LF5 larvae using a micropipetter. For each experiment, three areas of a comb containing larvae of the same age were delimited for pyriproxyfen or acetone treatment and for controls, respectively. A map delimiting each area was drawn on a cellophane sheet placed on the comb with the aid of a special pen. After treatment, the combs were returned to the hives of origin where they were maintained until reaching the Pw phase (white-eyed, unpigmented cuticle pupae) at the beginning of the pupal period (about 8.5–9 days a.h.) when the pupae were removed from the combs and placed in an incubator at 34°C and 80% relative humidity. Such conditions permit the normal progress of development until emergence. During removal of the pupae from the combs, the maps were used to identify the areas containing pupae treated or not during the larval period. In several experiments the larvae treated with pyriproxyfen and subsequently reintroduced into the colonies were preferentially removed from their cells by the workers. The results of these experiments were not computed. We only considered the experiments in which larval removal was negligible or approximately equal for the treated and untreated groups. LS5 larvae (spinning phase of the fifth larval instar), aged 5–6 days a.h., prepupal (PP, 7–8 days a.h) and pupal stages, Pw (8.5–9 days a.h.), Pp (pink-eyed, unpigmented cuticle pupae, 10 days a.h.), Pdp (dark-pink eyed, unpigmented cuticle pupae, 11 days a.h.), and Pb (brown-eyed, unpigmented cuticle pupae, 12–13 days a.h.) were collected from the hives for treatment with 1 g pyriproxyfen. Pw pupae were also treated with other concentrations of pyriproxyfen (0.1 and 5 g per l acetone).These non-feeding developmental phases were not returned to the hives, but were maintained in an incubator at 34°C and 80% relative humidity. All controls were treated with 1 l acetone or left untreated. The developmental ages at which the bees were treated and some developmental events pertaining to the end of the
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larval phase or occurring during the pupal period are shown in Fig. 1. 2.3. Morphogenetic action of pyriproxyfen The action of pyriproxyfen on cuticular and eye pigmentation, on developmental timing and on the larva– pupa and pupa–adult transition was examined by comparing treated and control bees every day after treatment until emergence. The bees were examined under a stereoscopic microscope for cuticle and eye pigmentation. 2.4. Sample preparation for phenoloxidase quantification Pools of two Pbm (brown-eyed, medium pigmented cuticle) pupae aged 14–15 days a.h., treated or not during the Pw phase, were homogenized in 400 l of 0.01 M sodium cacodylate buffer with 5 mM calcium chloride, pH 7.0. After centrifugation at 14 000g for 15 min at a temperature of 4–10°C, the supernatants were collected, filtered through glass wool and immediately used for quantification of phenoloxidase. The material was kept on ice throughout the procedure. 2.5. Phenoloxidase quantification Phenoloxidase activity was assayed according to Azambuja et al. (1991) using L-DOPA as substrate. The
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reaction mixture used for enzyme quantification in treated and control pupae was prepared with 450 l of the same cacodylate buffer used for homogenization of the pupae, 200 l of saturated solution of L-DOPA (2 mg/ml in cacodylate buffer) and 50 l of the filtered supernatant obtained from the pupal extract. After 15 min at room temperature, the absorbance of the reaction mixture was recorded at 490 nm in a spectrophotometer against a blank prepared with 500 l cacodylate buffer and 200 l L-DOPA. The determinations were made in the range in which the reaction rate was proportional to enzyme concentration. Phenylthiourea was added to some reaction mixtures for the characterization of the phenoloxidase specifically inhibited by this thiol reagent. The activity of the enzyme was expressed as percentage in the change in absorbance, considering as 100% the activity obtained from the reaction mixtures to which trypsin (Sigma, 1 mg/ml of cacodylate buffer), an activator of prophenoloxidase, the inactive precursor of phenoloxidase, was added. The supernatants added to the reaction mixture were usually clear and adequate for spectrophotometric quantification. In addition, possible opacity of the reaction mixture or spontaneous phenoloxidase activation affecting the results were carefully tested by measuring the absorbance of 50 l of each supernatant in 650 l of cacodylate buffer against the blank. ANOVA was used to compare the data related to quantification of phenoloxidase activity in pyriproxyfentreated and control pupae. Twenty sample pools, each
Fig. 1. Developmental timing of A. mellifera workers and phases at which they were treated with pyriproxyfen: LF5 (feeding phase of the fifth larval instar); LS5 (spinning phase of the fifth larval instar); PP (prepupae); Pw (white-eyed, unpigmented cuticle pupae); Pp (pink-eyed, unpigmented cuticle pupae); Pdp (dark pink-eyed, unpigmented cuticle pupae); Pb (brown-eyed, unpigmented cuticle pupae); Pbl (brown-eyed, light pigmented cuticle pupae); Pbm (brown-eyed, medium pigmented cuticle pupae); Pbd (brown-eyed, dark pigmented cuticle pupae). Some developmental events occurring at the end of the larval phase or during the pupal period are also indicated.
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prepared with two pupae treated with pyriproxyfen or acetone and 16 pools, each containing two control pupae, were analysed. 2.6. Sample preparation for electrophoretic analysis of Est-6 Pdp and Pb pupae aged 10.5 and 11.5 days a.h., respectively, treated with pyriproxyfen or only with acetone at an early developmental phase, (Pw, aged 8.5– 9 days a.h.), and untreated controls were homogenized in 200 l of 0.02 M Tris-HCl buffer, pH 7.5 and centrifuged at 12 400g for 20 min at 5°C. The supernatants were immediately absorbed onto Whatman No. 3 filter paper (5 × 6 mm) and inserted into the starch gel for electrophoretic analysis. 2.7. Electrophoresis Esterase-6 was detected by the methods described by Bitondi and Mestriner (1983) and Figueiredo et al. (1996). The starch gels were prepared at 10% in 0.02 M Tris-HCl buffer, pH 7.5. Esterase migration occurred for 4–5 h at 7°C, at 5 V/cm. The same buffer, at 0.3 M, was used in the electrode compartments. A solution of 1% 4-methylumbelliferyl butyrate in acetone (w/v) diluted 10% (v/v) in 0.1 M acetate buffer, pH 5.4, was used as substrate, poured on the internal surfaces of the horizontally cut gels for visualization of Est-6.
3. Results 3.1. Morphogenetic action of pyriproxyfen Pyriproxyfen, the juvenile hormone analogue used, acts on cuticular and eye pigmentation, on pupal–adult transition and on pupal developmental timing. 3.1.1. Cuticular and eye pigmentation When pyriproxyfen was topically applied to worker larvae (LS5) or prepupae (PP) or even at the beginning of the pupal period (Pw) an anomalous pigmentation was observed in pupae. The pigmentation of the thorax and head was precocious and intense. In the dorsal thoracic cuticle of treated pupae, the pigments were arrayed in a characteristic design (Fig. 2(a)) formed by points and circles (Fig. 2(b)), which was never seen in the controls. Thus, the juvenile hormone analogue induced not only an early cuticular pigmentation but also caused an alteration in this process. As the pigmentation process initiated earlier in treated pupae, they could easily be distinguished from controls at the Pb phase. During this developmental period, the beginning of pigmentation could be seen in the articulations and extremities of the
appendages of the treated pupae, whereas the controls still did not have pigments in the cuticle. Treatment at an earlier phase, LF5, caused an interruption of development at the beginning of the pupal phase. Treated pupae died before reaching the stage of pigmentation. Treatment of older developmental phases, from Pp to Pb, did not impair the normal pigmentation program of the cuticle. Eye pigmentation did not occur in pupae treated with pyriproxyfen during the LF5 phase. When this juvenile hormone analogue was applied later during development, from the LS5 to the Pw phase, the anterior edges of the eyes did not darken (Fig. 2(c) and (d)). Also the proper color of the pigments seen in the pigmented stripe of the eye was affected. In this way, the changes in the color of the eyes, which normally characterize pupal development and define the different steps of pupal development (see Rembold et al., 1980; Michelette and Soares, 1993), were not seen in the treated pupae. As was the case for the cuticle, the pigmentation of the eyes was normal when the pyriproxyfen treatment was applied later, from Pp to Pb pupae. The cuticle and eye sensitivities to pyriproxyfen treatment during the different developmental phases are shown in Fig. 3. 3.1.2. Larval–pupal transition and emergence Pyriproxyfen treatment before or during the larval– pupal transition, at the LF5, LS5 or PP phase, did not affect this developmental event, which occurred simultaneously in treated and control larvae and prepupae. However, the pupal–adult transition, i.e., the emergence of the adult, did not occur. Even when the treatment was applied later, at the Pw phase, emergence was impaired. After these developmental periods (from the Pp to the Pb phase), the treatment did not impair adult emergence, but this event was anticipated (Fig. 3). 3.1.3. Pupal development Pyriproxyfen caused an acceleration of pupal development. Treated Pp, Pdp or Pb pupae emerged before controls. Pupal development was faster even when the treatment was effected earlier, during the LS5, PP or Pw phase. However, in this case, as mentioned above, the altered pigmentation and sclerotization of the cuticle impaired the pupal–adult transition and, consequently, these treated larvae or pupae died at the end of the pupal period. When a younger phase (LF5) was submitted to treatment, pupal development was blocked at the beginning of the pupal phase and the immatures died (Fig. 3). 3.2. Phenoloxidase activity In this study, we determined phenoloxidase activity in A. mellifera Pbm pupae (14–15 days a.h.) treated or not
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Fig. 2. Precocious and anomalous pigmentation in A. mellifera pupae treated with pyriproxyfen. (a) Left: aspect of the cuticle of 13-day a.h. pupae (Pbl), treated during the LS5 phase (5–6 days a.h.) or during the PP phase (7–8 days a.h.) or at the beginning of the pupal period, during the Pw phase (8.5–9 days a.h.) with 1 g pyriproxyfen. In these pupae, the pigmentation of the thorax and head was precocious, intense and anomalous. Right: acetone-treated (control) Pbl pupae (13 days a.h.) showing normal pigmentation (27 × ). (b) Detail of the altered pigmentation of the thoracic cuticle of A. mellifera pupae aged 13 days a.h., treated at the LS5 phase (5–6 days a.h.), during the PP phase (7–8 days a.h.), or during the Pw phase (8.5–9 days a.h.) with 1 g pyriproxyfen. The pigments form points and circles characteristically arrayed (57 × ). (c) Effect of pyriproxyfen on A. mellifera eye pigmentation. Left: typical eye of Pbl pupae aged 13 days a.h., treated during the LS5 phase (5–6 days a.h.), during the PP phase (7–8 days a.h.), or at the beginning of the pupal period (Pw, 8.5–9 days a.h.) with 1g pyriproxyfen. The anterior edge of the eyes did not undergo pigmentation. Also, the eye color is altered. Right: acetone-treated control showing normal eye pigmentation (27 × ). (d) Detail of the unpigmented eye stripe (63 × ).
with pyriproxyfen during the Pw phase. Fig. 4 compares the phenoloxidase activity detected in pupae treated with 1 g of pyriproxyfen and controls. The enzyme activity was significantly higher in pupae treated with this juvenile hormone analogue than in controls treated with acetone or untreated (p ⬍ 0.001). The values obtained for acetone-treated versus untreated pupae were not significantly different (p > 0.05).
The effect of pyriproxyfen on pupal cuticle pigmentation was dose dependent. Fig. 5 shows a graded response in phenoloxidase activity in Pbm pupae treated at the beginning of the pupal phase (Pw) with different doses of pyriproxyfen. Pupae treated with 5 g showed higher phenoloxidase activity than pupae treated with 1 g, and when treatment was carried out with 0.1 g these values were still lower. The phenoloxidase activity
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Fig. 3. Effects of 1 g pyriproxyfen on cuticle and eye pigmentation, on larva–pupa transition, on pupal development and emergence, depending on the A. mellifera phase treated.
observed in acetone-treated and untreated pupae was very low when compared to pyriproxyfen-treated pupae. Phenylthiourea (PTU), a common phenoloxidase inhibitor, inhibited L-DOPA oxidation when added to the reaction mixture. When PTU was added to the supernatant prepared with 5 or 1 g-pyriproxyfen-treated pupae, no increase in absorbance was detected. 3.3. Esterase-6
the initiation of cuticular pigmentation at the Pbl (browneyed, light pigmented cuticle pupae) phase (Figueiredo et al., 1996). In pyriproxyfen-treated pupae, which show precocious pigmentation, Est-6 activity was also detected earlier than in controls. This result reinforces our previous data (Figueiredo et al., 1996) indicating a function of this esterase in cuticular pigmentation. Thus, the hormonal treatment causing precocious melanization also shifts the Est-6 expression to younger developmental phases.
An electrophoretic pattern of Est-6 is shown in Fig. 6. The onset of activity of this esterase coincides with 4. Discussion
Fig. 4. Phenoloxidase activity in A. mellifera Pbm pupae, aged 14– 15 days a.h., treated during the Pw phase with 1 g pyriproxyfen, with acetone or left untreated. The enzyme activity was expressed as a percentage of the change in absorbance at 490 nm, considering the amount activated by trypsin as 100% of activity. The samples are pools of pyriproxyfen- or acetone-treated or untreated pupae.
Pupal cuticle showed profound alterations in pigmentation after pyriproxyfen treatment. Even when this analogue was applied after pupae formation during the Pw phase, the pigmentation pattern was considerably disturbed and the onset of cuticular tanning occurred earlier than in control pupae. Furthermore, cuticular sclerotization also did not proceed correctly, with the cuticle being less hard than in control bees. These effects impair adult emergence. When the analogue was applied earlier, during the LF5 phase, pupal development was blocked at the beginning of the pupal phase and pigmentation did not occur. The analogue utilized also had a general effect on pupal developmental timing. When applied to Pp, Pdp or Pb pupae, developmental periods during which the cuticle is no longer affected, pyriproxyfen promoted an acceleration in pupal development and, consequently, precocious emergence. Even when the analogue was applied earlier, and caused a severe alteration of the cuticle making emergence inviable, the period of pupal
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Fig. 5. Dose-response of A. mellifera phenoloxidase activity to pyriproxyfen. Time course of L-DOPA oxidation activity in extracts of worker Pbm pupae treated at the beginning of the pupal phase (Pw) with 0.1, 1 or 5 g pyriproxyfen. Controls were treated with acetone, or were not treated.
development was shortened. Thus, although treated PP or Pw pupae did not emerge, they showed the spontaneous appendage movements, which characterize the end of the pupal period and indicate the proximity of emergence, earlier than the normal controls. The shortening of the pupal period by 1–2 days with consequent impairment of pupal–adult emergence after synthetic Cecropia juvenile hormone treatment has already been described for A. mellifera carnica by Rembold et al. (1974) who treated younger worker larvae aged 2–3 days (L3 to L4 instar larvae according to Rembold et al., 1980). These larvae developed into pupae with malformed eyes like those obtained after treatment with pyr-
Fig. 6. Electrophoretic pattern of A. mellifera Est-6. Starch gel electrophoresis of extracts of Pb pupae, aged 11.5 days a h., treated with 1 g pyriproxyfen (samples 1–5) at the beginning of the pupal phase (Pw, 8.5–9 days a.h.) and untreated controls (samples 6–9). The onset of Est-6 activity occurred earlier in pyriproxyfen-treated pupae than in controls.
iproxyfen (our data). This effect was also described by Zdarek and Haragsim (1974) and the patterns of malformation induced in compound eyes by the different analogues used are very similar. Alteration in eye pigmentation was also described for lepidopterans (Hiruma, 1980) but in this case malformed eyes were observed in allatectomized larvae. This effect of juvenile hormone (or of its absence) on insect eye differentiation has not been clarified yet. Experimental maintenance of high hormonal levels during the larval or prepupal phases by administration of pyriproxyfen did not impair the larva–pupa transition, and had no effect on the timing of this developmental event. This result is consistent with the data obtained by Zdarek and Haragsim (1974) for A. mellifera carnica. These authors observed that application of other juvenile hormone analogues to larvae of the last instar does not prevent larva–pupa transition. A known effect of juvenile hormone on A. mellifera is a shift in caste formation from workers to intercastes after treatment of larvae. The highest degrees of intercaste traits were obtained by Wirtz (1973) after treatment of 3–3.5-day-old worker larvae with juvenile hormone. Thus, the most sensitive period for the action of juvenile hormone on queen-worker decision occurs in developmental phases younger than that used by us. However it is possible that the precocious pigmentation in pyrip-
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roxyfen-treated workers could be a queen-like trait brought about by this analogue. Queens have a higher juvenile hormone titer when compared to workers (Rembold et al., 1974; Rachinsky et al., 1990) and pigmentation starts earlier in queen pupae than in workers. Early pigmentation, hormonally induced, could lead to a more rapid differentiation of the pupal cuticle, with consequent shortening of pupal development. In this sense, treated workers are closer to queens than untreated ones. In general, the effects provoked by pyriproxyfen on eye development, larval–pupal and pupal–adult transition mimic those described for other juvenile hormone analogues (Rembold et al., 1974; Zdarek and Haragsim, 1974), thus making this recently developed analogue very attractive for studies on hormonal control of pigmentation, metamorphosis and other developmental processes, as well as for studies on caste determination in bees. The alteration in the pigmentation and sclerotization processes caused by pyriproxyfen in A. mellifera was related to an increase in phenoloxidase activity. In Drosophila, there is genetic evidence suggesting that this enzyme is responsible for both processes (Pentz et al., 1986). Treated bee workers have significantly higher phenoloxidase levels than controls and this effect is dose-dependent. The analogue can either directly induce phenoloxidase synthesis or activation, or may act on some of the several steps of the reaction cascade leading to melanin synthesis. Interestingly, the onset of Est-6 activity, an esterase previously related to pigmentation (Figueiredo et al., 1996) occurs early in treated pupae, as shown in the present study. Thus, the early induction of pigmentation by hormonal treatment is accompanied by the precocious onset of Est-6 activity. We do not know if this esterase is a serine-protease and, if so, if it has a function in the activation of prophenoloxidase, the inactive form of phenoloxidase. The juvenile hormone analogue used may be acting on Est-6 synthesis and/or activation and, in this way, may cause the precocious activation of phenoloxidase in treated bees. This hypothesis needs further investigation. In Manduca sexta, the cuticular phenoloxidase responsible for melanization is synthesized by the epidermis in the absence of juvenile hormone. The application of this hormone at the time of head capsule slippage inhibited synthesis of phenoloxidase by the epidermis and thus prevented melanization. Hormonal treatment after phenoloxidase synthesis prevented its subsequent synthesis, causing partial melanization (Hiruma and Riddiford, 1988). In hormonally treated A. mellifera, on the contrary, the phenoloxidase synthesis or activity was intensified, an effect differing from that observed in M. sexta. Even among lepidopterans, where the control of melanization is best known, the effects of juvenile hormone cannot be generalized. For instance, the sequence of
endocrinologic events leading to melanization in M. sexta seems to be rather similar to that observed in Spodoptera litura (Morita et al., 1988). However, the melanization mechanism of these species differs from that of Leucania separata (Ogura, 1975) and Mamestra brassicae (Hiruma et al., 1984), in which juvenile hormone does not inhibit melanization. Thus, although melanization is hormonally controlled, different mechanisms could be selected by evolution. There are differences in hormone profiles between M. sexta (Riddiford, 1994) and A. mellifera (Rembold, 1987; Rachinsky et al., 1990). Also, pigmentation occurs in different developmental phases in these insect orders. But, for both, pigmentation occurs under conditions of low juvenile hormone titer and is preceded by an increase in ecdysteroids. Moreover, for M. sexta as well as for A. mellifera, this period of low juvenile hormone titer is critical, and very sensitive to doses of exogenous juvenile hormone. Studies on different insect species will definitely contribute to the establishment of a more encompassing model of hormonal control of pigmentation and to the determination of the specific function of juvenile hormone on this developmental event. How pyriproxyfen acts on phenoloxidase activation is not yet known. We do not exclude the possibility of this juvenile hormone analogue interacting with ecdysteroids, affecting their hemolymph titer profiles and, consequently the chain of cellular events leading to pigmentation in A. mellifera. In this case, the hormonal equilibrium strictly necessary for normal pigmentation was disrupted by the exogenous juvenile hormone analogue applied during a developmental moment when the basal level of juvenile hormone is absolutely necessary for the occurrence of normal pigmentation and cuticular development. Rachinsky and Engels (1995) showed that juvenile hormone application to early fifth-instar honeybee larvae results in an increased ecdysteroid titer in the early prepupal phase. Thus, a high juvenile hormone titer may turn on an enhanced ecdysteroid production leading to an alteration in the pigmentation process dependent on ecdysteroid. Dopa decarboxylase, another enzyme involved in pigmentation is also hormonally regulated. During a larval molt 20-hydroxyecdysone is necessary to program the later expression of this enzyme, but the subsequent decline of this hormone is required for the occurrence of enzyme activity. It appears that the rise of 20-hydroxyecdysone selects the dopa decarboxylase gene for later expression when the hormone declines. If the hormone is maintained above a threshold level, the selected gene will not be expressed. Moreover, juvenile hormone can modulate the later level of dopa decarboxylase expression (Hiruma et al., 1985; Hiruma and Riddiford, 1990). It can be hypothesized that in A. mellifera a selection of the phenoloxidase gene also occurs in the absence of
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juvenile hormone. The application of pyriproxyfen during the LF5 phase can block the selection of this gene for later expression, which will impair pigmentation. When the analogue is applied later, the selection has already occurred and the enzyme is produced, but the treatment still affects phenoloxidase activity, as seen by the precocious and altered melanin production. The present study showed an effect of a juvenile hormone analogue on cuticular pigmentation of A. mellifera. As demonstrated here, this effect was correlated with phenoloxidase and Est-6 activation. Further studies on this subject may contribute to the clarification of some steps of the complex, hormonally regulated reaction cascade leading to insect cuticular pigmentation.
Acknowledgements The authors are grateful to L.R. Aguiar and P.R. Epifaˆnio for assistance in the apiary and laboratory, respectively. We also thank Dr D. De Jong for correcting the English. This research was supported by FAPESP and CNPq.
References Andersen, S.O., 1985. Sclerotization and tanning of the cuticle. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 3. Pergamon Press, Oxford, pp. 59–74. Azambuja, P., Garcia, E.S., Ratcliffe, N.A., Warten Jr, J.D., 1991. Immune-depression in Rhodnius prolixus induced by the growth inhibitor azadirachtin. Journal of Insect Physiology 36, 771–777. Bitondi, M.M.G., Mestriner, M.A., 1983. Esterase isozymes of Apis mellifera: substrate and inhibition characteristics, developmental ontogeny, and electrophoretic variability. Biochemical Genetics 21, 985–1002. Curtis, A.T., Hori, M., Green, J.M., Wolfgang, W.J., Hiruma, K., Riddiford, L.M., 1984. Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and black mutant Manduca sexta larvae. Journal of Insect Physiology 8, 597–606. Feldlaufer, M.F., Herbert Jr, E.W., Svoboda, J.A., Thompson, M.J., Lusby, W.R., 1985. Makisterone A: the major ecdysteroid from the pupa of the honey bee, Apis mellifera. Insect Biochemistry 15, 597–600. Figueiredo, V.L.C., Paulino-Simo˜es, Z.L., Bitondi, M.M.G., 1996. Developmental pattern of esterases in Apis mellifera honey bees. I. Stage-dependent changes of esterase isozymes in Africanized worker. Apidologie 27, 47–54. Hiruma, K., 1980. Possible roles of juvenile hormone in the prepupal stage of Mamestra brassicae. General and Comparative Endocrinology 41, 392–399. Hiruma, K., Matsumoto, S., Isogai, A., Susuki, A., 1984. Control of ommochrome synthesis by both juvenile hormone and melanization hormone in the cabbage armyworm, Mamestra brassicae. Journal of Comparative Physiology 154, 13–21. Hiruma, K., Norman, A., Riddiford, L.M., 1993. A neuroendocrine factor essential for cuticular melanization in the tobacco hornworm, Manduca sexta. Journal of Insect Physiology 39, 353–360. Hiruma, K., Riddiford, L.M., 1988. Granular phenoloxidase involved in cuticular melanization in the tobacco hornworm: regulation of
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its synthesis in the epidermis by juvenile hormone. Developmental Biology 130, 87–97. Hiruma, K., Riddiford, L.M., 1990. Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone. Developmental Biology 138, 214–224. Hiruma, K., Riddiford, L.M., Hopkins, T.L., Morgan, T.D., 1985. Roles of dopa carboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone. Journal of Comparative Physiology B 155, 659–669. Matsumoto, S., Isogai, A., Susuki, A., 1986. Isolation and amino terminal sequence of melanization and reddish coloration hormone (MRCH) from the silkworm, Bombyx mori. Insect Biochemistry 16, 775–779. Matsumoto, S., Isogai, A., Susuki, A., Ogura, N., Sonobe, H., 1981. Purification and properties of the melanization and reddish colouration hormone (MRCH) in the armyworm, Leucania separata (Lepdoptera). Insect Biochemistry 6, 725–733. Matsumoto, S., Kitamura, A., Nagasawa, H., Kataoka, H., Orikasa, C., Mitsui, T., Susuki, A., 1990. Functional diversity of a neurohormone produced by the suboesophageal ganglion: molecular identity of melanization and reddish colouration hormone and pheromone biosynthesis activating neuropeptide. Journal of Insect Physiology 36, 427–432. Michelette, E.R.F., Soares, A.E.E., 1993. Characterization of preimaginal developmental stages in Africanized honey bee workers (Apis mellifera L). Apidologie 24, 431–440. Morita, M., Hatakoshi, M., Tojo, S., 1988. Hormonal control of cuticular melanization in the common cutworm, Spodoptera litura. Journal of Insect Physiology 8, 751–758. Ogura, N., 1975. Hormonal control of larval coloration in the armyworm, Leucania separata. Journal of Insect Physiology 21, 559–576. Ohashi, M., Tsusue, M., Kiguchi, K., 1983. Juvenile hormone control of larval colouration in the silkworm, Bombyx mori: characterization and determination of epidermal brown colour induced by the hormone. Insect Biochemistry 13, 123–127. Pentz, E.S., Black, B.C., Wright, T.R.F., 1986. A diphenol oxidase gene is part of a cluster of genes involved in catecholamine metabolism and sclerotization in Drosophila. I. Identification of the biochemical defect in Dox-A2 [1(2)37 Bf] mutants. Genetics 112, 823–841. Rachinsky, A., Engels, W., 1995. Caste development in honeybees (Apis mellifera): juvenile hormone turns on ecdysteroids. Naturwissenschaften 82, 378–379. Rachinsky, A., Strambi, C., Strambi, A., Hartfelder, K., 1990. Caste and metamorphosis: hemolymph titers of juvenile hormone and ecdysteroids in last instar honeybee larvae. General and Comparative Endocrinology 79, 31–38. Rembold, H., 1987. Caste specific modulation of juvenile hormone titers in Apis mellifera. Insect Biochemistry 17, 1003–1006. Rembold, H., Czoppelt, Ch., Rao, P.J., 1974. Effect of juvenile hormone treatment on caste differentiation in the honeybee Apis mellifera. Journal of Insect Physiology 20, 1193–1202. Rembold, H., Kremer, J.P., Ulrich, G.M., 1980. Characterization of postembryonic developmental stages of the female castes of the honey bee, Apis mellifera. L. Apidologie 11, 29–38. Riddiford, L.M., 1994. Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions. Advances in Insect Physiology 24, 213–274. Wirtz, P., 1973. Differentiation in the honey bee larvae. Med Landbouwhogeschool Wageningen 73, 1–155. Yoshida, H., Ashida, M., 1986. Microbial activation of two serine enzymes and prophenoloxidase in the plasma fraction of hemolymph of the silkworm, Bombyx mori. Insect Biochemistry 16, 539–545. Zdarek, J., Haragsim, O., 1974. Action of juvenoids on metamorphosis of the honey-bee, Apis mellifera. Journal of Insect Physiology 20, 209–221.
The Apis mellifera pupal melanization program is affected by treatment with a juvenile hormone analogue M.M.G. Bitondi *, I.M. Mora, Z.L.P. Simo˜es, V.L.C. Figueiredo Faculdade de Filosofia, Cieˆncias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Departamento de Biologia, Av. Bandeirantes 3900, 14040-901 Ribeira˜o Preto SP, Brazil Received 3 March 1997; received in revised form 21 July 1997
Abstract Apis mellifera treated during different developmental phases with pyriproxyfen, a juvenile hormone analogue, show profound alterations in cuticular pigmentation and sclerotization. When the treatment is effected during the feeding phase of the fifth larval instar (LF5), the pupal development is blocked and pigmentation does not occur. Treatment of older larvae, at the spinning phase of the fifth larval instar (LS5), of prepupae (PP) or pupae at the beginning of the pupal period (Pw, white-eyed, unpigmented cuticle pupae) does not impair pigmentation, but, instead, this process is accelerated, intensified and abnormal. Hormonal treatment during these developmental phases (LS5, PP and Pw) induces earlier activity of phenoloxidase, an enzyme of the reaction chain leading to melanin synthesis. Treated pupae have significantly higher enzymatic levels and show a graded response in phenoloxidase activity after treatment with 0.1, 1 or 5 g pyriproxyfen. Besides pigmentation, other developmental events were also altered in treated bees: pupal development was shortened, and the expression of esterase-6 activity, the onset of which coincides with the beginning of pigmentation, was shifted with the precocious initiation of this process in treated pupae. The significance of these results is discussed in relation to the mode of hormonal action on cuticular pigmentation in insects. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Apis mellifera; Cuticular melanization; Pyriproxyfen; Phenoloxidase; Hymenoptera
1. Introduction Melanin synthesis and deposition in the cuticle occur at the proper time during insect development. An important enzyme in the biosynthesis of melanin is phenoloxidase. When activated, this enzyme catalyzes the hydroxylation of tyrosine to dopa and oxidizes dopa to the corresponding quinone, giving rise to melanin (Andersen, 1985). This same enzymatic activity is also important in cuticle sclerotization (Hiruma and Riddiford, 1988). In several insect species, phenoloxidase exists as an inactive precursor, prophenoloxidase, which is activated by a cascade of serine proteases (Yoshida and Ashida, 1986). Cuticular melanization is a developmental process hormonally regulated. Until now, most of the studies concerning hormonal control of phenoloxidase synthesis
* Corresponding author. Fax: 55 (16) 633-6482; E-mail: [email protected] 0022–1910 /98 /$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 7 ) 0 0 1 1 3 - 3
and consequent cuticular melanization have been performed in lepidopteran larvae. Juvenile hormone, ecdysteroids as well as neuroendocrine factors have been implicated in these processes (Curtis et al., 1984; Hiruma et al., 1984, 1985, 1993; Hiruma and Riddiford, 1988; Matsumoto et al., 1981, 1986, 1990; Ohashi et al., 1983). In Manduca sexta, the cellular basis of the action of juvenile hormone on phenoloxidase synthesis and consequent melanization is best known (Riddiford, 1994). In this lepidopteran, the decline in juvenile hormone during head capsule slippage is an essential condition for prophenoloxidase synthesis in the epidermis. Under this condition, this enzyme, specific for melanization, is incorporated into the newly synthesized cuticle and is subsequently activated to phenoloxidase at the time of the decline in ecdysteroid titre. Exogenous juvenile hormone prevents the synthesis of prophenoloxidase and consequent melanization if given at the time of head capsule slippage which occurs before the peak of ecdysteroid titre (Riddiford, 1994). In the hymenopteran A. mellifera, the process of
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cuticular melanization begins at the pupal phase. This developmental period is characterized by basal levels of juvenile hormone in the hemolymph (Rembold, 1987) and by a peak of ecdysteroid (Feldlaufer et al., 1985) occurring approximately in the middle of the pupal period, before the initiation of cuticular melanization. As observed for M. sexta this low level of juvenile hormone seems to be required for the normal cuticular melanization of A. mellifera because disturbances in this developmental event were observed after topical treatment with juvenile hormone or analogues. Hrdys (1973, in Rembold et al., 1974) reported that feeding of honey bee colonies with juvenile hormone analogues leads to development of black brood or, according to our interpretation, of brood with intense cuticular pigmentation. Zdarek and Haragsim (1974) also observed alterations in cuticular tanning after topical applications of juvenile hormone analogues to developing honey bees. But, in contrast to what was observed in M. sexta, these data on honey bees pointed to an intensification of cuticular pigmentation following topical application of juvenile hormone, and not inhibition. The above considerations led us to undertake the present study in order to characterize the juvenile hormone action on phenoloxidase activity and consequent cuticular melanization of A. mellifera. Pyriproxyfen, a juvenile hormone analogue, was topically applied to larvae of fifth instar, prepupae and pupae of various ages. Subsequently, the onset and timing of the pigmentation process and the developmental phase sensitivity to this hormone in terms of pigmentation were then investigated. An esterase (Est-6), whose onset of activity coincides with the beginning of pigmentation (Figueiredo et al., 1996), was used as a molecular marker of this event. Since pyriproxyfen was applied to different developmental phases, its effects on other critical events, in addition to cuticle pigmentation, such as timing of larval–pupal transition, pupal development and adult emergence were also described in order to characterize the juvenile hormone mimicking action of this analogue in A. mellifera.
2. Material and methods 2.1. Apis mellifera Larvae, prepupae and pupae of Africanized A. mellifera workers were collected from hives of the experimental apiary of the Department of Genetics, Faculty of Medicine of Ribeira˜o Preto, Sa˜o Paulo State, Brazil. To obtain immatures of uniform age, queens were periodically confined for 6 h on combs without brood, where the eggs were laid. The limitation of the period of oviposition allowed us to use groups of larvae, prepupae or pupae varying by a maximum of 6 h in age. These
developmental phases were treated with pyriproxyfen (2[1-methyl-2(4-phenoxyphenoxy) ethoxyl] pyridine, a juvenile hormone analogue, product of Sumitomo Chemical Co. Ltd, Osaka, Japan, and their development was monitored from treatment to emergence. The larval instars and prepupal or pupal phases of Africanized A. mellifera were distinguished according to Michelette and Soares (1993). 2.2. Treatment LF5 larvae (feeding phase of the fifth larval instar), aged 4–4.5 days a.h. were topically treated with 1 g pyriproxyfen dissolved in 1 l acetone, in their own comb cell, i.e., in situ. Controls were treated with 1 l acetone or left untreated. The solutions of pyriproxyfen or acetone were applied to the cuticles of LF5 larvae using a micropipetter. For each experiment, three areas of a comb containing larvae of the same age were delimited for pyriproxyfen or acetone treatment and for controls, respectively. A map delimiting each area was drawn on a cellophane sheet placed on the comb with the aid of a special pen. After treatment, the combs were returned to the hives of origin where they were maintained until reaching the Pw phase (white-eyed, unpigmented cuticle pupae) at the beginning of the pupal period (about 8.5–9 days a.h.) when the pupae were removed from the combs and placed in an incubator at 34°C and 80% relative humidity. Such conditions permit the normal progress of development until emergence. During removal of the pupae from the combs, the maps were used to identify the areas containing pupae treated or not during the larval period. In several experiments the larvae treated with pyriproxyfen and subsequently reintroduced into the colonies were preferentially removed from their cells by the workers. The results of these experiments were not computed. We only considered the experiments in which larval removal was negligible or approximately equal for the treated and untreated groups. LS5 larvae (spinning phase of the fifth larval instar), aged 5–6 days a.h., prepupal (PP, 7–8 days a.h) and pupal stages, Pw (8.5–9 days a.h.), Pp (pink-eyed, unpigmented cuticle pupae, 10 days a.h.), Pdp (dark-pink eyed, unpigmented cuticle pupae, 11 days a.h.), and Pb (brown-eyed, unpigmented cuticle pupae, 12–13 days a.h.) were collected from the hives for treatment with 1 g pyriproxyfen. Pw pupae were also treated with other concentrations of pyriproxyfen (0.1 and 5 g per l acetone).These non-feeding developmental phases were not returned to the hives, but were maintained in an incubator at 34°C and 80% relative humidity. All controls were treated with 1 l acetone or left untreated. The developmental ages at which the bees were treated and some developmental events pertaining to the end of the
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larval phase or occurring during the pupal period are shown in Fig. 1. 2.3. Morphogenetic action of pyriproxyfen The action of pyriproxyfen on cuticular and eye pigmentation, on developmental timing and on the larva– pupa and pupa–adult transition was examined by comparing treated and control bees every day after treatment until emergence. The bees were examined under a stereoscopic microscope for cuticle and eye pigmentation. 2.4. Sample preparation for phenoloxidase quantification Pools of two Pbm (brown-eyed, medium pigmented cuticle) pupae aged 14–15 days a.h., treated or not during the Pw phase, were homogenized in 400 l of 0.01 M sodium cacodylate buffer with 5 mM calcium chloride, pH 7.0. After centrifugation at 14 000g for 15 min at a temperature of 4–10°C, the supernatants were collected, filtered through glass wool and immediately used for quantification of phenoloxidase. The material was kept on ice throughout the procedure. 2.5. Phenoloxidase quantification Phenoloxidase activity was assayed according to Azambuja et al. (1991) using L-DOPA as substrate. The
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reaction mixture used for enzyme quantification in treated and control pupae was prepared with 450 l of the same cacodylate buffer used for homogenization of the pupae, 200 l of saturated solution of L-DOPA (2 mg/ml in cacodylate buffer) and 50 l of the filtered supernatant obtained from the pupal extract. After 15 min at room temperature, the absorbance of the reaction mixture was recorded at 490 nm in a spectrophotometer against a blank prepared with 500 l cacodylate buffer and 200 l L-DOPA. The determinations were made in the range in which the reaction rate was proportional to enzyme concentration. Phenylthiourea was added to some reaction mixtures for the characterization of the phenoloxidase specifically inhibited by this thiol reagent. The activity of the enzyme was expressed as percentage in the change in absorbance, considering as 100% the activity obtained from the reaction mixtures to which trypsin (Sigma, 1 mg/ml of cacodylate buffer), an activator of prophenoloxidase, the inactive precursor of phenoloxidase, was added. The supernatants added to the reaction mixture were usually clear and adequate for spectrophotometric quantification. In addition, possible opacity of the reaction mixture or spontaneous phenoloxidase activation affecting the results were carefully tested by measuring the absorbance of 50 l of each supernatant in 650 l of cacodylate buffer against the blank. ANOVA was used to compare the data related to quantification of phenoloxidase activity in pyriproxyfentreated and control pupae. Twenty sample pools, each
Fig. 1. Developmental timing of A. mellifera workers and phases at which they were treated with pyriproxyfen: LF5 (feeding phase of the fifth larval instar); LS5 (spinning phase of the fifth larval instar); PP (prepupae); Pw (white-eyed, unpigmented cuticle pupae); Pp (pink-eyed, unpigmented cuticle pupae); Pdp (dark pink-eyed, unpigmented cuticle pupae); Pb (brown-eyed, unpigmented cuticle pupae); Pbl (brown-eyed, light pigmented cuticle pupae); Pbm (brown-eyed, medium pigmented cuticle pupae); Pbd (brown-eyed, dark pigmented cuticle pupae). Some developmental events occurring at the end of the larval phase or during the pupal period are also indicated.
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prepared with two pupae treated with pyriproxyfen or acetone and 16 pools, each containing two control pupae, were analysed. 2.6. Sample preparation for electrophoretic analysis of Est-6 Pdp and Pb pupae aged 10.5 and 11.5 days a.h., respectively, treated with pyriproxyfen or only with acetone at an early developmental phase, (Pw, aged 8.5– 9 days a.h.), and untreated controls were homogenized in 200 l of 0.02 M Tris-HCl buffer, pH 7.5 and centrifuged at 12 400g for 20 min at 5°C. The supernatants were immediately absorbed onto Whatman No. 3 filter paper (5 × 6 mm) and inserted into the starch gel for electrophoretic analysis. 2.7. Electrophoresis Esterase-6 was detected by the methods described by Bitondi and Mestriner (1983) and Figueiredo et al. (1996). The starch gels were prepared at 10% in 0.02 M Tris-HCl buffer, pH 7.5. Esterase migration occurred for 4–5 h at 7°C, at 5 V/cm. The same buffer, at 0.3 M, was used in the electrode compartments. A solution of 1% 4-methylumbelliferyl butyrate in acetone (w/v) diluted 10% (v/v) in 0.1 M acetate buffer, pH 5.4, was used as substrate, poured on the internal surfaces of the horizontally cut gels for visualization of Est-6.
3. Results 3.1. Morphogenetic action of pyriproxyfen Pyriproxyfen, the juvenile hormone analogue used, acts on cuticular and eye pigmentation, on pupal–adult transition and on pupal developmental timing. 3.1.1. Cuticular and eye pigmentation When pyriproxyfen was topically applied to worker larvae (LS5) or prepupae (PP) or even at the beginning of the pupal period (Pw) an anomalous pigmentation was observed in pupae. The pigmentation of the thorax and head was precocious and intense. In the dorsal thoracic cuticle of treated pupae, the pigments were arrayed in a characteristic design (Fig. 2(a)) formed by points and circles (Fig. 2(b)), which was never seen in the controls. Thus, the juvenile hormone analogue induced not only an early cuticular pigmentation but also caused an alteration in this process. As the pigmentation process initiated earlier in treated pupae, they could easily be distinguished from controls at the Pb phase. During this developmental period, the beginning of pigmentation could be seen in the articulations and extremities of the
appendages of the treated pupae, whereas the controls still did not have pigments in the cuticle. Treatment at an earlier phase, LF5, caused an interruption of development at the beginning of the pupal phase. Treated pupae died before reaching the stage of pigmentation. Treatment of older developmental phases, from Pp to Pb, did not impair the normal pigmentation program of the cuticle. Eye pigmentation did not occur in pupae treated with pyriproxyfen during the LF5 phase. When this juvenile hormone analogue was applied later during development, from the LS5 to the Pw phase, the anterior edges of the eyes did not darken (Fig. 2(c) and (d)). Also the proper color of the pigments seen in the pigmented stripe of the eye was affected. In this way, the changes in the color of the eyes, which normally characterize pupal development and define the different steps of pupal development (see Rembold et al., 1980; Michelette and Soares, 1993), were not seen in the treated pupae. As was the case for the cuticle, the pigmentation of the eyes was normal when the pyriproxyfen treatment was applied later, from Pp to Pb pupae. The cuticle and eye sensitivities to pyriproxyfen treatment during the different developmental phases are shown in Fig. 3. 3.1.2. Larval–pupal transition and emergence Pyriproxyfen treatment before or during the larval– pupal transition, at the LF5, LS5 or PP phase, did not affect this developmental event, which occurred simultaneously in treated and control larvae and prepupae. However, the pupal–adult transition, i.e., the emergence of the adult, did not occur. Even when the treatment was applied later, at the Pw phase, emergence was impaired. After these developmental periods (from the Pp to the Pb phase), the treatment did not impair adult emergence, but this event was anticipated (Fig. 3). 3.1.3. Pupal development Pyriproxyfen caused an acceleration of pupal development. Treated Pp, Pdp or Pb pupae emerged before controls. Pupal development was faster even when the treatment was effected earlier, during the LS5, PP or Pw phase. However, in this case, as mentioned above, the altered pigmentation and sclerotization of the cuticle impaired the pupal–adult transition and, consequently, these treated larvae or pupae died at the end of the pupal period. When a younger phase (LF5) was submitted to treatment, pupal development was blocked at the beginning of the pupal phase and the immatures died (Fig. 3). 3.2. Phenoloxidase activity In this study, we determined phenoloxidase activity in A. mellifera Pbm pupae (14–15 days a.h.) treated or not
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Fig. 2. Precocious and anomalous pigmentation in A. mellifera pupae treated with pyriproxyfen. (a) Left: aspect of the cuticle of 13-day a.h. pupae (Pbl), treated during the LS5 phase (5–6 days a.h.) or during the PP phase (7–8 days a.h.) or at the beginning of the pupal period, during the Pw phase (8.5–9 days a.h.) with 1 g pyriproxyfen. In these pupae, the pigmentation of the thorax and head was precocious, intense and anomalous. Right: acetone-treated (control) Pbl pupae (13 days a.h.) showing normal pigmentation (27 × ). (b) Detail of the altered pigmentation of the thoracic cuticle of A. mellifera pupae aged 13 days a.h., treated at the LS5 phase (5–6 days a.h.), during the PP phase (7–8 days a.h.), or during the Pw phase (8.5–9 days a.h.) with 1 g pyriproxyfen. The pigments form points and circles characteristically arrayed (57 × ). (c) Effect of pyriproxyfen on A. mellifera eye pigmentation. Left: typical eye of Pbl pupae aged 13 days a.h., treated during the LS5 phase (5–6 days a.h.), during the PP phase (7–8 days a.h.), or at the beginning of the pupal period (Pw, 8.5–9 days a.h.) with 1g pyriproxyfen. The anterior edge of the eyes did not undergo pigmentation. Also, the eye color is altered. Right: acetone-treated control showing normal eye pigmentation (27 × ). (d) Detail of the unpigmented eye stripe (63 × ).
with pyriproxyfen during the Pw phase. Fig. 4 compares the phenoloxidase activity detected in pupae treated with 1 g of pyriproxyfen and controls. The enzyme activity was significantly higher in pupae treated with this juvenile hormone analogue than in controls treated with acetone or untreated (p ⬍ 0.001). The values obtained for acetone-treated versus untreated pupae were not significantly different (p > 0.05).
The effect of pyriproxyfen on pupal cuticle pigmentation was dose dependent. Fig. 5 shows a graded response in phenoloxidase activity in Pbm pupae treated at the beginning of the pupal phase (Pw) with different doses of pyriproxyfen. Pupae treated with 5 g showed higher phenoloxidase activity than pupae treated with 1 g, and when treatment was carried out with 0.1 g these values were still lower. The phenoloxidase activity
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Fig. 3. Effects of 1 g pyriproxyfen on cuticle and eye pigmentation, on larva–pupa transition, on pupal development and emergence, depending on the A. mellifera phase treated.
observed in acetone-treated and untreated pupae was very low when compared to pyriproxyfen-treated pupae. Phenylthiourea (PTU), a common phenoloxidase inhibitor, inhibited L-DOPA oxidation when added to the reaction mixture. When PTU was added to the supernatant prepared with 5 or 1 g-pyriproxyfen-treated pupae, no increase in absorbance was detected. 3.3. Esterase-6
the initiation of cuticular pigmentation at the Pbl (browneyed, light pigmented cuticle pupae) phase (Figueiredo et al., 1996). In pyriproxyfen-treated pupae, which show precocious pigmentation, Est-6 activity was also detected earlier than in controls. This result reinforces our previous data (Figueiredo et al., 1996) indicating a function of this esterase in cuticular pigmentation. Thus, the hormonal treatment causing precocious melanization also shifts the Est-6 expression to younger developmental phases.
An electrophoretic pattern of Est-6 is shown in Fig. 6. The onset of activity of this esterase coincides with 4. Discussion
Fig. 4. Phenoloxidase activity in A. mellifera Pbm pupae, aged 14– 15 days a.h., treated during the Pw phase with 1 g pyriproxyfen, with acetone or left untreated. The enzyme activity was expressed as a percentage of the change in absorbance at 490 nm, considering the amount activated by trypsin as 100% of activity. The samples are pools of pyriproxyfen- or acetone-treated or untreated pupae.
Pupal cuticle showed profound alterations in pigmentation after pyriproxyfen treatment. Even when this analogue was applied after pupae formation during the Pw phase, the pigmentation pattern was considerably disturbed and the onset of cuticular tanning occurred earlier than in control pupae. Furthermore, cuticular sclerotization also did not proceed correctly, with the cuticle being less hard than in control bees. These effects impair adult emergence. When the analogue was applied earlier, during the LF5 phase, pupal development was blocked at the beginning of the pupal phase and pigmentation did not occur. The analogue utilized also had a general effect on pupal developmental timing. When applied to Pp, Pdp or Pb pupae, developmental periods during which the cuticle is no longer affected, pyriproxyfen promoted an acceleration in pupal development and, consequently, precocious emergence. Even when the analogue was applied earlier, and caused a severe alteration of the cuticle making emergence inviable, the period of pupal
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Fig. 5. Dose-response of A. mellifera phenoloxidase activity to pyriproxyfen. Time course of L-DOPA oxidation activity in extracts of worker Pbm pupae treated at the beginning of the pupal phase (Pw) with 0.1, 1 or 5 g pyriproxyfen. Controls were treated with acetone, or were not treated.
development was shortened. Thus, although treated PP or Pw pupae did not emerge, they showed the spontaneous appendage movements, which characterize the end of the pupal period and indicate the proximity of emergence, earlier than the normal controls. The shortening of the pupal period by 1–2 days with consequent impairment of pupal–adult emergence after synthetic Cecropia juvenile hormone treatment has already been described for A. mellifera carnica by Rembold et al. (1974) who treated younger worker larvae aged 2–3 days (L3 to L4 instar larvae according to Rembold et al., 1980). These larvae developed into pupae with malformed eyes like those obtained after treatment with pyr-
Fig. 6. Electrophoretic pattern of A. mellifera Est-6. Starch gel electrophoresis of extracts of Pb pupae, aged 11.5 days a h., treated with 1 g pyriproxyfen (samples 1–5) at the beginning of the pupal phase (Pw, 8.5–9 days a.h.) and untreated controls (samples 6–9). The onset of Est-6 activity occurred earlier in pyriproxyfen-treated pupae than in controls.
iproxyfen (our data). This effect was also described by Zdarek and Haragsim (1974) and the patterns of malformation induced in compound eyes by the different analogues used are very similar. Alteration in eye pigmentation was also described for lepidopterans (Hiruma, 1980) but in this case malformed eyes were observed in allatectomized larvae. This effect of juvenile hormone (or of its absence) on insect eye differentiation has not been clarified yet. Experimental maintenance of high hormonal levels during the larval or prepupal phases by administration of pyriproxyfen did not impair the larva–pupa transition, and had no effect on the timing of this developmental event. This result is consistent with the data obtained by Zdarek and Haragsim (1974) for A. mellifera carnica. These authors observed that application of other juvenile hormone analogues to larvae of the last instar does not prevent larva–pupa transition. A known effect of juvenile hormone on A. mellifera is a shift in caste formation from workers to intercastes after treatment of larvae. The highest degrees of intercaste traits were obtained by Wirtz (1973) after treatment of 3–3.5-day-old worker larvae with juvenile hormone. Thus, the most sensitive period for the action of juvenile hormone on queen-worker decision occurs in developmental phases younger than that used by us. However it is possible that the precocious pigmentation in pyrip-
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roxyfen-treated workers could be a queen-like trait brought about by this analogue. Queens have a higher juvenile hormone titer when compared to workers (Rembold et al., 1974; Rachinsky et al., 1990) and pigmentation starts earlier in queen pupae than in workers. Early pigmentation, hormonally induced, could lead to a more rapid differentiation of the pupal cuticle, with consequent shortening of pupal development. In this sense, treated workers are closer to queens than untreated ones. In general, the effects provoked by pyriproxyfen on eye development, larval–pupal and pupal–adult transition mimic those described for other juvenile hormone analogues (Rembold et al., 1974; Zdarek and Haragsim, 1974), thus making this recently developed analogue very attractive for studies on hormonal control of pigmentation, metamorphosis and other developmental processes, as well as for studies on caste determination in bees. The alteration in the pigmentation and sclerotization processes caused by pyriproxyfen in A. mellifera was related to an increase in phenoloxidase activity. In Drosophila, there is genetic evidence suggesting that this enzyme is responsible for both processes (Pentz et al., 1986). Treated bee workers have significantly higher phenoloxidase levels than controls and this effect is dose-dependent. The analogue can either directly induce phenoloxidase synthesis or activation, or may act on some of the several steps of the reaction cascade leading to melanin synthesis. Interestingly, the onset of Est-6 activity, an esterase previously related to pigmentation (Figueiredo et al., 1996) occurs early in treated pupae, as shown in the present study. Thus, the early induction of pigmentation by hormonal treatment is accompanied by the precocious onset of Est-6 activity. We do not know if this esterase is a serine-protease and, if so, if it has a function in the activation of prophenoloxidase, the inactive form of phenoloxidase. The juvenile hormone analogue used may be acting on Est-6 synthesis and/or activation and, in this way, may cause the precocious activation of phenoloxidase in treated bees. This hypothesis needs further investigation. In Manduca sexta, the cuticular phenoloxidase responsible for melanization is synthesized by the epidermis in the absence of juvenile hormone. The application of this hormone at the time of head capsule slippage inhibited synthesis of phenoloxidase by the epidermis and thus prevented melanization. Hormonal treatment after phenoloxidase synthesis prevented its subsequent synthesis, causing partial melanization (Hiruma and Riddiford, 1988). In hormonally treated A. mellifera, on the contrary, the phenoloxidase synthesis or activity was intensified, an effect differing from that observed in M. sexta. Even among lepidopterans, where the control of melanization is best known, the effects of juvenile hormone cannot be generalized. For instance, the sequence of
endocrinologic events leading to melanization in M. sexta seems to be rather similar to that observed in Spodoptera litura (Morita et al., 1988). However, the melanization mechanism of these species differs from that of Leucania separata (Ogura, 1975) and Mamestra brassicae (Hiruma et al., 1984), in which juvenile hormone does not inhibit melanization. Thus, although melanization is hormonally controlled, different mechanisms could be selected by evolution. There are differences in hormone profiles between M. sexta (Riddiford, 1994) and A. mellifera (Rembold, 1987; Rachinsky et al., 1990). Also, pigmentation occurs in different developmental phases in these insect orders. But, for both, pigmentation occurs under conditions of low juvenile hormone titer and is preceded by an increase in ecdysteroids. Moreover, for M. sexta as well as for A. mellifera, this period of low juvenile hormone titer is critical, and very sensitive to doses of exogenous juvenile hormone. Studies on different insect species will definitely contribute to the establishment of a more encompassing model of hormonal control of pigmentation and to the determination of the specific function of juvenile hormone on this developmental event. How pyriproxyfen acts on phenoloxidase activation is not yet known. We do not exclude the possibility of this juvenile hormone analogue interacting with ecdysteroids, affecting their hemolymph titer profiles and, consequently the chain of cellular events leading to pigmentation in A. mellifera. In this case, the hormonal equilibrium strictly necessary for normal pigmentation was disrupted by the exogenous juvenile hormone analogue applied during a developmental moment when the basal level of juvenile hormone is absolutely necessary for the occurrence of normal pigmentation and cuticular development. Rachinsky and Engels (1995) showed that juvenile hormone application to early fifth-instar honeybee larvae results in an increased ecdysteroid titer in the early prepupal phase. Thus, a high juvenile hormone titer may turn on an enhanced ecdysteroid production leading to an alteration in the pigmentation process dependent on ecdysteroid. Dopa decarboxylase, another enzyme involved in pigmentation is also hormonally regulated. During a larval molt 20-hydroxyecdysone is necessary to program the later expression of this enzyme, but the subsequent decline of this hormone is required for the occurrence of enzyme activity. It appears that the rise of 20-hydroxyecdysone selects the dopa decarboxylase gene for later expression when the hormone declines. If the hormone is maintained above a threshold level, the selected gene will not be expressed. Moreover, juvenile hormone can modulate the later level of dopa decarboxylase expression (Hiruma et al., 1985; Hiruma and Riddiford, 1990). It can be hypothesized that in A. mellifera a selection of the phenoloxidase gene also occurs in the absence of
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juvenile hormone. The application of pyriproxyfen during the LF5 phase can block the selection of this gene for later expression, which will impair pigmentation. When the analogue is applied later, the selection has already occurred and the enzyme is produced, but the treatment still affects phenoloxidase activity, as seen by the precocious and altered melanin production. The present study showed an effect of a juvenile hormone analogue on cuticular pigmentation of A. mellifera. As demonstrated here, this effect was correlated with phenoloxidase and Est-6 activation. Further studies on this subject may contribute to the clarification of some steps of the complex, hormonally regulated reaction cascade leading to insect cuticular pigmentation.
Acknowledgements The authors are grateful to L.R. Aguiar and P.R. Epifaˆnio for assistance in the apiary and laboratory, respectively. We also thank Dr D. De Jong for correcting the English. This research was supported by FAPESP and CNPq.
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