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How does juvenile hormone control insect metamorphosis and reproduction?
General and Comparative Endocrinology 179 (2012) 477–484
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2012 Howard Bern Lecture
How does juvenile hormone control insect metamorphosis and reproduction? Lynn M. Riddiford ⇑ Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, United States
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Article history: Received 24 January 2012 Revised 22 May 2012 Accepted 1 June 2012 Available online 20 June 2012 Keywords: Juvenile hormone Methoprene-tolerant Insect metamorphosis Insulin-like peptide Vitellogenesis Female receptivity
a b s t r a c t In insects juvenile hormone (JH) regulates both metamorphosis and reproduction. This lecture focuses on our current understanding of JH action at the molecular level in both of these processes based primarily on studies in the tobacco hornworm Manduca sexta, the flour beetle Tribolium castaneum, the mosquito Aedes aegypti, and the fruit fly Drosophila melanogaster. The roles of the JH receptor complex and the transcription factors that it regulates during larval molting and metamorphosis are summarized. Also highlighted are the intriguing interactions of the JH and insulin signaling pathways in both imaginal disc development and vitellogenesis. Critical actions of JH and its receptor in the timing of maturation of the adult optic lobe and of female receptivity in Drosophila are also discussed. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Juvenile hormone (JH) is a hormone unique to insects although its immediate precursor methyl farnesoate is found in the Crustacea where it is known to mediate reproduction in some species [47]. In all insects JH has primary roles in the regulation of metamorphosis and reproduction. In some species, it has also been co-opted to regulate other developmental processes such as various polyphenisms and caste determination in social insects [25,48]. This article is based on my Howard Bern Lecture at the 2012 Society for Integrative and Comparative Biology meeting. It is an overview of my work on the study of JH action together with my perspective of the recent studies of others which are greatly enriching our current understanding of the action of JH in insect metamorphosis and reproduction. It is not a comprehensive review as there are several of these available [10,13,23,28,51]. 2. Regulation of larval molting and metamorphosis In insects all growth occurs prior to metamorphosis and is accompanied by the periodic shedding or molting of the exoskeleton. This growth and molting is orchestrated by two hormones, ecdysone1 and JH [55,57]. Alpha-ecdysone is a steroid hormone secreted by the prothoracic gland that is converted in the periphery to 20-hydroxyecdysone (20E) (Fig. 1A), which initiates and orches⇑ Fax: +1 571 209 4928. E-mail address: [email protected] Ecdysone is used throughout in the generic sense of the hormone that controls molting and metamorphosis with a-ecdysone and 20-hydroxyecdysone (20E) referring to the actual chemical molecules involved [57]. 1
0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2012.06.001
trates the molting process. Juvenile hormone is a sesquiterpenoid with JH III being the primary hormone produced by the corpora allata (CA) of most insects (Fig. 1B) [23,55]. Methyl farnesoate, the precursor of JH III, is released by nymphal cockroach CA and by the larval ring gland of higher Diptera [8,23,29]. JH bisepoxide is also secreted by higher dipteran CA [23]. In Lepidoptera the larval CA produce JH I and JH II, whereas the adult CA may release various combinations of JH I, II, and III as well as the respective JH acids depending on the species [23]. The JH analogs, methoprene and pyriproxifen (Fig. 1B), mimic the action of JH. In this paper I will use the generic terms ecdysone and JH except for discussions of experimental use of a particular hormonal compound. JH is present in the larva and allows ecdysone action to initiate the molt but prevents its actions in switching genetic programs necessary for metamorphosis [26,52,53,55]. During the intermolt feeding period, JH is also necessary for the maintenance of isomorphic proliferation of the imaginal discs and primordia [67]. The tobacco hornworm, Manduca sexta, was the ‘‘white rat’’ of insect endocrinology that allowed us to begin to elucidate how JH directs ecdysone action [48,55]. During the final (5th) larval instar the JH titer declines to undetectable levels due to the cessation of secretion from the CA coupled with the appearance of a JH-specific esterase in both the hemolymph and the tissues that degrades existing JH. This loss of JH allows prothoracicotropic hormone (PTTH) release that stimulates a small amount of ecdysone release. Ecdysone in the absence of JH causes the cessation of feeding and the onset of wandering to find a pupation site. It also causes pupal commitment so that the larva can no longer undergo a larval molt but rather only a pupal molt when challenged with ecdysone. In vitro experiments with the abdominal epidermis from feeding
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Fig. 1. Chemical structures of the active insect ecdysteroid 20-hydroxyecdysone (A) and of the major juvenile hormones (JH) and JH mimics methoprene and pyriproxifen (B). Structure in (A) based on [57] and in (B) on [23]. JHB3, JH bisepoxide.
larvae showed that exposure to 20E in the absence of JH was sufficient to cause pupal commitment, whereas exposure to 20E and JH maintained the larval state [52,53]. During the prepupal period JH reappears at the time of the ecdysteroid rise for the pupal molt and is necessary for prevention of precocious metamorphosis in some tissues such as the eye, the optic lobe, and the ventral diaphragm [15–17,34]. JH again disappears by the time of pupal ecdysis so that the subsequent ecdysteroid rise that causes adult commitment and subsequent differentiation occurs in the absence of JH. Exogenous JH at the onset of this rise causes the formation of a second pupa [56] just as Williams [71] first showed for the Cecropia silkmoth. 3. JH receptor The JH receptor has been long sought, and two major candidates have been put forward: (1) Ultraspiracle (USP), a member of the nuclear hormone receptor family [12,64], that forms a heterodimer with the ecdysone receptor (EcR) to mediate ecdysone action [57]; (2) Methoprene-tolerant (Met), a protein in Drosophila that confers resistance to JH [72,73]. Ultraspiracle from Drosophila melanogaster binds JH III with intermediate affinity (Kd = 2 10 7 M), whereas it binds methyl farnesoate with the high nanomolar affinity expected for a hormone receptor [31]. Methyl farnesoate is found in larvae, but is 100 times less active than JH III in preventing metamorphosis when applied at the time of pupariation [32]. When synthesis of methyl farnesoate and thus of all JHs is prevented in Drosophila by expression of HMGCoA reductase RNAi, most larvae die during the molt to the 3rd larval stage [30]. When these larvae were fed farnesol, they could ecdyse to the 3rd instar and at higher concentrations of farnesol could pupariate, but neither methyl farnesoate nor JH III alone had this effect [30,32]. Importantly, USP null mutants die during the molt to the 2nd instar as pharate 2nd instar larvae showing double mouthhooks and spiracles [57].
Interestingly, mutants lacking Met progress normally through larval life and metamorphosis, showing only delayed and severely reduced fecundity [72], calling into question the role of Met as the JH receptor in the regulation of metamorphosis. This problem has recently been resolved by the finding of a second gene, germ cells expressed (gce), in Drosophila that is highly similar to Met with Met likely arising from gce by gene duplication [5,6]. Although mutants lacking either Met or Gce showed normal progression through metamorphosis, mutants lacking both pupariate but die at pupal head eversion [1] (which signals the completion of the formation of the pupa in the higher Diptera), similar to animals that have been genetically allatectomized (CAX) [41,59]. Recent studies in other insects have provided convincing evidence that Met is the primary JH receptor. Konopova and Jindra [36] used double stranded Met RNA (Met RNAi) to knock down the level of Met in early stage (3rd or 4th) Tribolium castaneum (flour beetle) larvae and obtained precocious metamorphosis after the 5th or 6th larval stage respectively, showing that Met was essential for larval molting. Also, loss of Met by RNAi leads to precocious metamorphosis in the linden bug, Pyrrhocoris apterus [38], indicating that Met likely mediates JH action in all insects. Met is a member of the basic helix-loop-helix (bHLH) family of transcription factors and recently Tribolium Met has been shown to bind JH via its Pas B domain [18]. Drosophila Met can form a homodimer with itself or a heterodimer with Gce, but only in the absence of JH [22]. Binding of JH causes dissociation of the dimer and the association of Met with a steroid hormone receptor coactivator known as Taiman in Drosophila [4], bFtz-F1 interacting steroid receptor coactivator (FISC) in the mosquito Aedes aegypti [79], and steroid receptor coactivator (SRC) in Tribolium [75], all of which interact with EcR during ecdysone action. The JH-Met-FISC complex binds to the JH response element (JHRE) in the promoter of the early trypsin gene in adult female mosquito midgut and causes up-regulation of this gene [40]. Similarly, the JH-Met-SRC
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complex is necessary for the up-regulation of Krüppel homolog (Kr-h1) in Tribolium larvae and in a mosquito cell line [75]. Both Met and Gce can also interact with the orphan nuclear receptor b-FTZ-F1, and Gce is necessary for the up-regulation of E75A in Drosophila S2 cells by JH in the absence of 20E [9]. The nature of the 20E/JH interaction is elusive although recently Jones et al. [33] have developed a model system using a hexamerin core promoter and a DR1 ecdysone response element that is responsive to both hormones and may yield the answer. 4. Larval molting In Tribolium larvae the loss of Kr-h1 by RNAi leads to precocious metamorphosis that cannot be rescued by exogenous JH [46]. In both Tribolium [46] and Drosophila [50], Kr-h1 mRNA is present throughout larval life falling to low levels in the mid-final instar, then transiently reappearing during the onset of the pupal molt when it is necessary for normal prepupal development. In Tribolium mid-final instar larvae Kr-h1 mRNA can be up-regulated by exogenous JH, but not when Met levels have been reduced by RNAi [75]. In Drosophila the loss of Kr-h1 function leads to the precocious appearance of Broad, the pupal specifier (see section 5.1 below), in the fat body during the molt to the 3rd instar [27]. Also, Kr-h1 mRNA is present in the penultimate nymphal instar of both Pyrrhocoris [38] and the cockroach Blattella germanica [42], and disappears in the final instar; its reduction by RNAi in the penultimate instar leads to precocious metamorphosis. In these hemimetabolous nymphs, Broad is present in the penultimate instar, then disappears in the final instar, a necessity for metamorphosis to the adult [28,38]. Thus, Kr-h1 is necessary for both larval and nymphal molting, in the former preventing the turning-on of Broad expression (Fig. 2), in the latter preventing the turning-off of Broad. The presence of JH maintains Kr-h1 expression and thereby prevents metamorphosis. 5. Pupal commitment and the onset of metamorphosis 5.1. Epidermis Twenty-hydroxyecdysone is known to act on tissues by causing a cascade of gene activation first worked out by Ashburner et al. [3] studying its effects on the ‘‘puffing’’ activity in D. melanogaster salivary gland chromosomes. Later molecular studies showed that most of the 20E-induced genes in this cascade were transcription factors [35,64]. A similar cascade of transcription factors was found to be induced by 20E in Manduca abdominal epidermis in the larval, pupal and adult molts (reviewed in [26]). A comparison of the cascades induced during the larval molt and at the onset of metamorphosis revealed one major difference. In the absence of JH at the time of pupal commitment, ecdysone and 20E induced the appearance of Broad (Br) [76], a transcription factor containing the Broad-Tramtrack-Bric-a-brac (BTB) protein–protein interaction
Fig. 2. Model for the molecular basis of the control of larval molting and metamorphosis by JH based on studies by [26,38,46,78]. JH, juvenile hormone; Kr-h1, Krüppel homolog 1; Met, Methoprene-tolerant; Tai, Taiman. See text for details. Dashed lines signify reduced effect.
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domain and a DNA binding domain containing a pair of cysteine– histidine zinc fingers [7]. Its induction within an epidermal segment correlated with the loss of cellular JH sensitivity for induction of another larval molt, indicating that its appearance signified pupal commitment of the cells [77]. Broad remained in the epidermis peaking during the prepupal surge of ecdysteroid, then fell to trace levels and disappeared by pupal day 4 as the ecdysteroid titer for the adult molt rose [26,78]. Importantly, when the pupa was given exogenous JH I, Br did not disappear, but rather increased again with the increase of the ecdysteroid titer and the epidermis formed a second pupal cuticle [78]. Thus, in Manduca 20E acting in the absence of JH is sufficient to activate broad (br) expression at pupal commitment and also is sufficient to turn off its expression during the adult molt (Figs. 2 and 3). In Tribolium br RNA is present at low levels during most of larval life, then its levels increase late in the final larval instar, remain high during the prepupal period, then decline to undetectable in the early pupa [37,49,63]. These studies also showed that br RNAi caused the formation of a larval-pupal-adult mosaic at the time of the pupal molt, indicating that Broad was required for normal pupal development. Exogenous JH (the JH mimics hydroprene or methoprene) given to the pupa at the outset of adult development caused the re-expression of br during the onset of the adult molt and a second pupa was formed as seen in Manduca [37,49,63]. In this case JH was found to act via Met by up-regulating Kr-h1 which then acted to prevent turning off of br expression by 20E [46] by some as yet unknown mechanism. In the higher Diptera such as Drosophila in contrast to most holometabolous insects, the final instar larval cuticle becomes the puparium within which the head, thoracic and genital discs evert and form pupal structures such as legs and wings, whereas the larval abdominal cells make the pupal cuticle of the abdomen [21]. The abdominal histoblasts are quiescent during larval life, then start dividing shortly after pupariation and spread out over the pupal abdomen to form the imaginal (adult) epidermis as the ecdysteroid titer rises for adult development. In Drosophila Broad also appears during the final larval instar and the complete loss of function of br causes lethality at the time of pupariation [7]. Exogenous JH (JH mimic pyriproxifen), however, only delayed its appearance in the larval abdominal epidermis and also delayed
Fig. 3. Summary of the essential role of the Broad transcription factor in mediating the endocrine control of metamorphosis in the imaginal discs and in the abdominal epidermis. JH, juvenile hormone; 20E, 20-hydroxyecdysone. See text for details.
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the onset of wandering [58]. By contrast, JH given at pupariation prevented the normal cessation of br expression in the abdominal imaginal epidermis as the ecdysteroid titer rises for the adult molt [78]. The exogenous JH also caused the re-expression of Kr-h1 mRNA in the abdomen during the adult molt and misexpression of Kr-h1 mRNA in this epidermis was found to prolong br expression in some cells [45]. These cells then made a second pupal cuticle rather than adult cuticle. The adult head and thorax arising from imaginal discs were unaffected by the treatment with exogenous JH; their levels of br mRNA declined on schedule and they formed normal adult cuticular structures [78]. When br mRNA was over-expressed, particularly the Z1 isoform, during the time of bristle formation and during the onset of cuticle formation during the adult molt, a second pupal cuticle formed in both the abdomen and the disc-derived head and thorax [78]. This action of Broad in suppressing adult cuticle formation is at least partly through its inhibition of the expression of another transcription factor DHR38 [19], which is needed for the transcription of at least one of the adult cuticle genes, Acp65A [14]. The basis for the different responsiveness of lepidopteran and higher dipteran epidermis and imaginal discs to JH remains a mystery. 5.2. Lepidopteran imaginal discs In Manduca the wing discs grow isomorphically during larval life, then in the final 5th instar undergo patterning and morphogenetic growth to form the pupal wing, whereas the pupal eye and leg primordia remain as quiescent diploid cells until the final instar when they begin to divide and undergo morphogenetic growth to form the pupal structures [43,67]. Normally feeding in the final instar initiates the appearance of Broad in the discs and the primordia followed some hours later by the onset of morphogenesis. Feeding sucrose alone was sufficient for the appearance of br mRNA, but the addition of amino acids was necessary for the subsequent growth [43]. The up-regulation of br mRNA in the wing disc correlated with the pupal commitment of the disc [39]. Starvation of the freshly ecdysed larvae prevented the appearance of Broad and the subsequent morphogenetic growth, but surprisingly both occurred in starved larvae that had been allatectomized during the molt to the final instar and so lacked JH [67]. JH (methoprene) applied to the allatectomized starved larvae suppressed both the appearance of Broad and the subsequent disc and primordia morphogenesis. This suppressive effect of JH did not require exposure to ecdysteroids since it occurred in metathoracic leg primordia isolated from the prothoracic glands that produce ecdysone. Subsequent studies showed that either Manduca bombyxin [an insulin-like peptide (ILP)] or bovine insulin could substitute for feeding in starved larvae by inducing Broad expression and pupal commitment in the wing discs [39]. When wing discs from freshly ecdysed final instar larvae were cultured in hormone-free medium, br mRNA was up-regulated. The addition of bombyxin or insulin did not affect this upregulation. The addition of methoprene, by contrast, prevented the increase in br mRNA. When both hormones were present, insulin suppressed the repressive effects of JH so that the br mRNA level was similar to that seen in hormone-free medium. Down-regulation of the insulin receptor in the disc by RNAi abolished this effect of insulin, suggesting that some component of the insulin signaling pathway is interfering with the action of JH. Nothing further is known about this intriguing interaction of JH and insulin signaling. Thus, the appearance or up-regulation of the transcription factor Broad signals the onset of metamorphosis and its presence is required through pupal differentiation. It must then disappear in order for adult differentiation to occur. As summarized in Fig. 3, in the epidermis of the basal holometabolous insects JH prevents the turning on of br mRNA by 20E; whereas in the final stage imaginal discs of lepidopterans, the JH suppression of br expression is
overcome by increased insulin signaling due to feeding. During the pupal-adult transition JH is normally not present and 20E causes the cessation of br transcription and adult development proceeds. When exogenous JH is present, Broad levels remain high both in pupal structures formed from imaginal discs and primordia (except in the higher Diptera) and in the abdominal epidermis, and a second pupa is formed. In the derived higher Diptera such as Drosophila, JH application has no effect on the imaginal discs so br mRNA is down-regulated by 20E resulting in a mosaic animal with an adult head and thorax but a pupal abdomen. Considering the recent evidence mainly from Tribolium and Drosophila described above, in all these actions JH likely acts via the Met receptor modulating the synthesis of Kr-h1 (see Fig. 2). 6. Prepupal role of JH In holometabolous insects JH reappears during the prepupal period at the time of the ecdysteroid rise for pupation and in Lepidoptera it acts to prevent premature adult development of imaginal structures such as the eye, optic lobe and the ventral diaphragm [15–17,71]. Recent studies in Drosophila have begun to reveal some of the mechanisms involved in this action of JH. When Drosophila larvae were genetically allatectomized by expressing the cell death gene grim in the CA, traces of the CA could still be seen in 2nd instar larvae but were undetectable in the final 3rd stage larva. These larvae grew more slowly and usually pupariated at a smaller size 12–24 h later than intact larvae [41,59]. Their discs everted normally and they formed pupal cuticle, but the prepupae could not complete the process of pupal head eversion and died soon afterwards. Internally the fat body which normally dissociates during adult development showed premature dissociation and a large increase in caspase-dependent apoptotic cell death [41]. All of these effects were prevented by the feeding the larvae either of the JH mimics, methoprene or pyriproxifen. Similar effects are seen in mutants lacking both Met and Gce but not in mutants lacking only one of these proteins [1]. Normally during wandering and the prepupal period the photoreceptors from the developing eye disc grow into the optic lobe, induce proliferation and aid in coordinating its development [65]. By 12 h after pupariation (AP), proliferation declines sharply and by about 18 h AP differentiation of the adult optic lobe begins as the ecdysteroid titer rises for the adult molt (Fig. 4). In allatectomized prepupae, proliferation levels in the medulla of the optic lobe were similar at pupariation but declined prematurely by 6–8 h AP at which time the R7 photoreceptor showed the precocious onset of adult differentiation normally seen between 18 and 24 h AP [59]. At pupariation the optic lobe normally has high levels of the A isoform of the EcR, but none of the B1 isoform [66]. EcR-B1 appears in the optic lobe beginning about the time of head eversion 10–12 h AP and reaches high levels by 24 h AP. In allatectomized animals low levels of EcR-B1 were already present at pupariation and increased to high levels by 6–8 h AP [59]. Feeding the JH mimic pyriproxifen during the third instar or application of JH III or pyriproxifen at the onset of wandering prevented all the effects of allatectomy on optic lobe development as well as the death at head eversion. Thus, JH prevents precocious adult differentiation of the optic lobe in the forming pupa in response to the pupariation peak of ecdysteroid. The Met null mutant Met27 shows a similar precocious adult differentiation of the optic lobe as seen in the allatectomized prepupa, and this precocious differentiation cannot be prevented by exogenous JH [59]. This premature differentiation results in anomalous morphogenesis of the lobula neuropil of the optic lobe (Fig. 5) and abnormal patterning of neuronal connections in that region. In preliminary assays of visual function, these animals were unresponsive, indicating that they were likely functionally blind (J. Tuthill and L.M.
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Fig. 4. Summary of the regulation of the timing of adult optic lobe development in Drosophila melanogaster by JH as shown by studies on genetically allatectomized (CAX) animals that die at the time of head eversion to the pupa ( ) [59]. See text for details. The hormone titers are redrawn from [54]. E, ecdysis; HE, head eversion; W, wandering.
Fig. 5. Frontal sections of the adult optic lobe of wild type (wt) (Canton S) (left), Met27 null mutant (middle), and gce2.5k null mutant of Drosophila melanogaster. Arrows point to abnormal lobes of the lobula (lo) that project towards the medulla (m) in the Met27mutant. Staining is by N-cadherin (aqua) and neuroglian (red). Bar = 50 lm. The left and center pictures are from [59].
Riddiford, unpublished). To probe further the cellular basis of JH action, we expressed Met RNAi in various neurons in the eye and the developing optic lobe and found that loss of Met in either the photoreceptor neurons or in the lamina neurons (the outermost layer of neurons of the optic lobe) led to precocious appearance of EcR-B1 in those neurons [59]. Only loss of Met in the photoreceptor neurons, however, led to precocious differentiation of the R7 photoreceptor within the medulla. Yet overexpression of EcR-B1 in the photoreceptors was insufficient to cause this precocious differentiation. Thus, other unknown effects of the loss of JH or of Met in the photoreceptors must also be necessary for the precocious differentiation that was observed. Interestingly, the gce2.5k null mutant showed neither precocious expression of EcR-B1 in the prepupal optic lobe nor abnormal morphogenesis of the adult lobula (L.M. Riddiford, unpublished; Fig. 5). Thus, in its regulation of prepupal optic lobe development in Drosophila, JH appears to be acting only via Met. 7. Roles of JH in reproductive maturation 7.1. Vitellogenin synthesis JH has long been known to regulate various aspects of reproductive maturation after adult emergence with the specific effects varying depending on the insect studied (see [13,51,74] for
comprehensive reviews). In many feeding adult females, JH regulates vitellogenin (Vg) (yolk protein) synthesis, but the molecular mechanisms have remained elusive. Recent studies in Tribolium have shown that Vg synthesis in the fat body requires both feeding and JH [61]. Normally these females begin feeding 2.5 days after adult eclosion and Vg2 mRNA levels begin to increase at day 4 and reach high levels by day 5. Feeding was unable to stimulate Vg2 mRNA expression in the absence of JH, but injection of bovine insulin restored normal Vg2 levels. Furthermore, RNAi-mediated suppression of FOXO expression, a repressive transcription factor whose loss from the nucleus is mediated by the insulin signaling pathway [24], caused precocious appearance of Vg2 mRNA on day 2. Further in vivo and in vitro experiments showed that JH stimulated an increase in mRNA expression in the fat body and brain of one of the 4 ILPs (ILP2) that normally was up-regulated by day 2 after ecdysis. This up-regulation was prevented by Met RNAi, indicating that JH is working through the Met receptor. Feeding caused the appearance of ILP3 mRNA in the fat body followed later by Vg2 mRNA. Knockdown of ILP2 or ILP3 by RNAi caused partial or complete loss of Vg2 mRNA respectively. Thus, Sheng et al. [61] hypothesize that JH acts via Met to increase production of ILP2 and feeding stimulates the production of ILP3. The two together then act through the insulin signaling pathway to cause the phosphorylation of FOXO and its loss from the nucleus, thus relieving the repression of Vg2 mRNA transcription. Thus, JH is
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indirectly stimulating Vg production in this beetle. Whether its simulation of Vg production in other insects is mediated similarly remains to be determined. In Drosophila JH primarily regulates Vg uptake into the eggs [62] but the mechanisms involved are unknown. The Met null mutant shows both a delay in egg maturation and greatly reduced fecundity [72]. Loss of Gce has little effect on the timing of egg maturation and only slightly reduces fecundity [1]. 7.2. Female receptivity Among the insects, JH is known to play various roles in female reproductive behavior, ranging from stimulating pheromone production in some cockroaches and beetles to regulating virgin female receptivity to courting males in a range of insects from grasshoppers to flies [60]. In D. melanogaster the female at the time of eclosion is unreceptive to male courtship attempts, but nearly all mate by 48 h after eclosion [20,44]. Implantation of active CA into female abdomens shortly before eclosion caused the recipients to mate precociously on day 1 as compared to only 30% of the controls implanted with a piece of aorta [44]. Thus, Manning [44] suggested that JH was involved in the maturation of female receptivity. To explore the action of JH in the maturation of female receptivity, we destroyed the CA late in adult development by using the temperature-sensitive GAL80ts to prevent earlier GAL4-driven expression of UAS-diphtheria toxin in the CA [11]. These allatectomized females were unreceptive to males at 24 h after eclosion, but by 48 h nearly all mated in group assays similarly to controls. Application of methoprene (ED50 = 2 ng) to the allatectomized females at eclosion resulted in over 50% mating at 24 h as compared to about 30% mating for the parental controls. These results confirm those of Manning [44] that JH is involved in the maturation of female receptivity. We further found that Met27 null mutants showed a similar delay in the onset of female receptivity to that seen in allatectomized females. Reduction of Met expression in the central nervous system using Met RNAi driven by the neuronal-specific driver elav-GAL4 caused a similar delay in the onset of female receptivity as found in the Met27 mutant. Therefore, JH acting via Met in the CNS is somehow fostering the maturation of neuronal circuits necessary for female acceptance of male courtship attempts so that she mates. However, other factors are also clearly involved in this maturation since it still occurs, albeit delayed, in the absence of JH. Interestingly, when assayed as individual pairs at 48 h after eclosion, the time to copulation was similar for Met27 females and controls whereas allatectomized females took nearly 3 times as long (J. Bilen and L.M. Riddiford, unpublished). Preliminary experiments show that the longer time to copulation for allatectomized females is at least partly due to their decreased attractiveness to males as measured by male courtship of headless females. Surprisingly, though, in this assay Met27 females were found to be more attractive than controls. These findings may indicate that JH has a role in the synthesis of the female-specific pheromones that appear beginning about 18–24 h after eclosion [2,68]. 8. Conclusions Studies in the past few years have greatly enhanced our knowledge of the action of JH in insect metamorphosis and reproduction at the molecular level. Along with these new insights have come surprises such as the interaction of the JH-Met receptor complex with the EcR coactivator to regulate specific genes and the interplay between JH and the insulin-signaling pathway in controlling the onset of metamorphosis in imaginal discs and in the regulation
of vitellogenesis. Mysteries still remain such as why are Drosophila imaginal discs refractory to JH in its maintenance of Broad expression? Is Kr-h1 the critical transcription factor specifying the larval state and how is it regulating Broad? Over the years studies on the action of JH in many different insect species beginning with the bloodsucking bug Rhodnius prolixus [69,70] have been important to our understanding of how JH is intimately involved in the regulation of both metamorphosis and reproduction. The tobacco hornworm M. sexta provided a manipulatable endocrine system to study its action at the cellular and molecular level but lacked the genetics necessary to probe these molecular actions. Drosophila then became the leader in the search for mechanisms of action but with the disadvantage of a highly derived developmental pattern and the lack of a well-defined role for JH in metamorphosis. With the advent of RNAi and genomic approaches, studies on other insect species such as the flour beetle Tribolium and the linden bug Pyrrhocoris are again contributing critical new insights into JH action. This comparative approach is clearly in the spirit of Howard Bern, one of the founders of comparative endocrinology whose wisdom, insights, and mentoring will be sorely missed in the field. Acknowledgments I thank Dr. James Truman for help in the imaging of the gce2.5k mutant and for critical comments on the manuscript, Dr. Julide Bilen for critical comments on the manuscript, Dr. Jian Wang for the gce2.5k mutant, and the Howard Hughes Medical Institute for support of unpublished work cited here. References [1] M.A. Abdou, Q. Heb, D. Wen, O. Zyaan, J. Wang, J. Xu, A.A. Baumann, J. Joseph, T.G. Wilson, S. Li, J. Wang, Drosophila Met and Gce are partially redundant in transducing juvenile hormone action, Insect Biochem. Mol. Biol. 41 (2011) 938–945. [2] M. Arienti, C. Antony, C. Wicker-Thomas, J.-P. Delbecque, J.-M. Jallon, Ontogeny of Drosophila melanogaster female sex-appeal and cuticular hydrocarbons, Integr. Zool. 5 (2010) 272–282. [3] M. Ashburner, C. Chihara, P. Meltzer, G. Richards, Temporal control of puffing activity in polytene chromosomes, Cold Spring Harb. Symp. Quant. Biol. 38 (1974) 655–662. [4] J. Bai, Y. Uehara, D.J. Montell, Regulation of invasive cell behavior by Taiman, a Drosophila protein related to AIB1, a steroid receptor coactivator amplified in breast cancer, Cell 103 (2000) 1047–1058. [5] A. Baumann, Y. Fujiwara, T.G. Wilson, Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila, J. Insect Physiol. 56 (2010) 1445–1455. [6] A. Baumann, J. Barry, S. Wang, Y. Fujiwara, T.G. Wilson, Paralogous genes involved in juvenile hormone action in Drosophila melanogaster, Genetics 185 (2010) 1327–1336. [7] C. Bayer, L. von Kalm, J.W. Fristrom, Gene regulation in imaginal disc and salivary gland development during Drosophila metamorphosis, in: L.I. Gilbert, J.R. Tata, B.G. Atkinson (Eds.), Metamorphosis, Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells, Academic Press, San Diego, 1996, pp. 321–361. [8] W.G. Bendena, J. Zhang, S.M. Burtenshaw, S.S. Tobe, Evidence for differential biosynthesis of juvenile hormone (and related) sesquiterpenoids in Drosophila melanogaster, Gen. Compar. Endocrinol. (2011) 56–61. [9] T.J. Bernardo, E.B. Dubrovsky, The Drosophila juvenile hormone receptor candidates Methoprene-tolerant (Met) and germ cell-expressed (gce) utilize a conserved LIXXL motif to bind the FTZ-F1 nuclear receptor, J. Biol. Chem. 287 (2012) 7821–7833. [10] T.J. Bernardo, E.B. Dubrovsky, Molecular mechanisms of transcription activation by juvenile hormone: a critical role for bHLH-PAS and nuclear receptor proteins, Insects 3 (2012) 324–338. [11] J. Bilen, L. Riddiford, Juvenile hormone modulates the maturation of female receptivity in Drosophila melanogaster, Abstract 646A, Drosophila Research Conference, Gen. Soc. America (2011) 267–268. [12] F. Bonneton, V. Laudet, Evolution of nuclear receptors in insects, in: L.I. Gilbert (Ed.), Insect Endocrinology, Elsevier, Amsterdam, 2012, pp. 310–365. [13] M. Bownes, The regulation of yolk protein gene expression and vitellogenesis in higher Diptera, in: A.S. Raikhel, T.W. Sappington (Eds.), Reproductive Biology of Invertebrates, vol. 12, Part B Progress in Vitellogenesis, Science Publishers, Enfield, USA/Plymouth, UK, 2004, pp. 95–128.
L.M. Riddiford / General and Comparative Endocrinology 179 (2012) 477–484 [14] N. Bruey-Sedano, J. Alabouvette, M. Lestradet, L. Hong, A. Girard, E. Gervasio, B. Quennedey, J.P. Charles, The Drosophila ACP65A cuticle gene: deletion scanning analysis of cis-regulatory sequences and regulation by DHR38, Genesis 43 (2005) 17–27. [15] D.T. Champlin, J.W. Truman, Ecdysteroid control of cell proliferation during optic lobe neurogenesis in the moth, Manduca sexta, Development 125 (1998) 269–277. [16] D.T. Champlin, J.W. Truman, Ecdysteroids govern two phases of eye development during metamorphosis of the moth, Manduca sexta, Development 125 (1998) 2009–2018. [17] D.T. Champlin, S.E. Reiss, J.W. Truman, Truman, Hormonal control of ventral diaphragm myogenesis during metamorphosis of the moth, Manduca sexta, Dev. Genes Evol. 209 (1999) 265–274. [18] J.-P. Charles, T. Iwema, V.C. Epa, K. Takaki, J. Rynes, M. Jindra, Ligand-binding properties of a juvenile hormone receptor, Methoprene–tolerant, Proc. Natl. Acad. Sci. USA 108 (2011) 21128–21133. [19] H.-Y. Cui, M. Lestradet, N. Bruey-Sedano, J.-P. Charles, L.M. Riddiford, Elucidation of the regulation of an adult cuticle gene Acp65A by the transcription factor Broad, Insect Mol. Biol. 18 (2009) 421–429. [20] J.-F. Ferveur, Drosophila female courtship and mating behaviors: sensory signals, genes, neural structures and evolution, Curr. Opin. Neurobiol. 20 (2010) 764–769. [21] D. Fristrom, J. Fristrom, The metamorphic development of the adult epidermis, in: M. Bate, A. Martinez-Arias (Eds.), The Development of Drosophila melanogaster, vol II, Cold Spring Harbor Laboratory Press, Plainview, New York, 1993, pp. 843–897. [22] J. Godlewski, S. Wang, T.G. Wilson, Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila, Biochem. Biophys. Res. Commun. 342 (2006) 1305–1311. [23] W.G. Goodman, M. Cusson, The juvenile hormones, in: L.I. Gilbert (Ed.), Insect Endocrinology, Elsevier, Amsterdam, 2012, pp. 310–365. [24] S.S. Grewal, Insulin/TOR signaling in growth and homeostasis: a view from the fly world, Int. J. Biochem. Cell Biol. 41 (2009) 1006–1010. [25] K. Hartfelder, D.J. Emlen, Endocrine control of insect polyphenism, in: L.I. Gilbert (Ed.), Insect Endocrinology, Elsevier, Amsterdam, 2012, pp. 464–522. [26] K. Hiruma, L.M. Riddiford, Developmental expression of mRNAs for epidermal and fat body proteins and hormonally regulated transcription factors in the tobacco hornworm, Manduca sexta, J. Insect Physiol. 56 (2010) 1390–1395. [27] J. Huang, L. Tian, C. Peng, M. Abdou, D. Wen, Y. Wang, S. Li, J. Wang, DPPmediated TGFbeta signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase, Development 138 (2011) 2283–2291. [28] M. Jindra, S.R. Palli, L.M. Riddiford, The juvenile hormone signaling pathway in insect development, Annu. Rev. Entomol. 58 (2013), http://dx.doi.org/10.1146/ annurev-ento-120811-153700. [29] D. Jones, G. Jones, Farnesoid secretions of dipteran ring glands: what we do know and what we can know, Insect Biochem. Mol. Biol. 37 (2007) 771–798. [30] D. Jones, G. Jones, P. Teal, C. Hammac, L. Messmer, K. Osborne, Y.H. Belgace, J.R. Martin, Suppressed production of methyl farnesoid hormones yields developmental defects and lethality in Drosophila larvae, Gen. Comp. Endocrin. 165 (2010) 244–254. [31] G. Jones, D. Jones, P. Teal, A. Sapa, M. Wozniak, The retinoid-X receptor ortholog, Ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand, FEBS J. 273 (2006) 4983–4996. [32] G. Jones, D. Jones, X. Li, L. Tang, L. Ye, P. Teal, L. Riddiford, C. Sandifer, D. Borovsky, J-R. Martin, Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster, J. Insect Physiol. 56 (2010) 1456–1464. [33] G. Jones, D. Jones, F. Fang, Y. Xu, D. New, W.-H. Wu, Juvenile hormone action through a defined enhancer motif to modulate ecdysteroid-activation of natural core promoters, Comp. Biochem. Physiol. B 161 (2012) 219–225. [34] K. Kiguchi, L.M. Riddiford, The role of juvenile hormone in pupal development of the tobacco hornworm, Manduca sexta, J. Insect Physiol. 24 (1978) 673– 680. [35] K. King-Jones, C.S. Thummel, Nuclear receptors – a perspective from Drosophila, Nat. Rev. Genet. 6 (2005) 311–323. [36] B. Konopova, M. Jindra, Juvenile hormone resistance gene Methoprenetolerant controls entry into metamorphosis in the beetle Tribolium castaneum, Proc. Natl. Acad. Sci. USA 104 (2007) 10488–10493. [37] B. Konopova, M. Jindra, Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis, Development 135 (2008) 559–568. [38] B. Konopova, V. Smykal, M. Jindra, Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects, PLoS One 6 (2011) e28728. [39] T. Koyama, M.O. Syropyatova, L.M. Riddiford, Insulin/IGF signaling regulates the change in commitment in imaginal discs and primordia by overriding the effect of juvenile hormone, Dev. Biol. 324 (2008) 258–265. [40] M. Li, E.A. Mead, J. Zhu, Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression, Proc. Natl. Acad. Sci. USA 108 (2011) 638–643. [41] Y. Liu, Z. Sheng, H. Liu, D. Wen, Q. He, S. Wang, W. Shao, R.-J. Jiang, S. An, Y. Sun, W.G. Bendena, J. Wang, L.I. Gilbert, T.G. Wilson, Q. Song, S. Li, Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila, Development 136 (2009) 2015–2025.
483
[42] J. Lozano, X. Belles, Conserved repressive function of Krüppel homolog 1 on insect metamorphosis in hemimetabolous and holometabolous species, Sci. Rep. 1 (2011): doi:10.1038/srep00163. [43] S.G.B. MacWhinnie, J.P. Allee, C.A. Nelson, L.M. Riddiford, J.W. Truman, D.T. Champlin, The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta, Dev. Biol. 285 (2005) 285–297. [44] A. Manning, Corpus allatum and sexual receptivity in female Drosophila melanogaster, Nature 211 (1966) 1321–1322. [45] C. Minakuchi, X. Zhou, L.M. Riddiford, Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster, Mech. Dev. 125 (2008) 91–105. [46] C. Minakuchi, T. Namiki, T. Shinoda, Krüppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum, Dev. Biol. 325 (2009) 341–350. [47] G.P.C. Nagaraju, Reproductive regulators in decapod crustaceans: an overview, J. Exp. Biol. 214 (2011) 3–16. [48] H.F. Nijhout, Insect Hormones, Princeton University Press, Princeton, 1995. [49] R. Parthasarathy, A. Tan, H. Bai, S.R. Palli, Transcription factor broad suppresses precocious development of adult structures during larval–pupal metamorphosis in the red flour beetle, Tribolium castaneum, Mech. Dev. 125 (2008) 299–313. [50] F. Pecasse, Y. Beck, C. Ruiz, G. Richards, Krüppel-homolog, a stage-specific modulator of the prepupal ecdysone response, is essential for Drosophila metamorphosis, Dev. Biol. 221 (2000) 53–67. [51] A.S. Raikhel, M.R. Brown, X. Belles, Hormonal control of reproductive processes In: L.I. Gilbert, K. Iatrou, S.S. Gill, (Eds.), Comprehensive Insect Science vol. 3, Elsevier, Pergamon, Amsterdam pp. 433-491 [52] L.M. Riddiford, Hormonal control of insect epidermal cell commitment in vitro, Nature 259 (1976) 115–117. [53] L.M. Riddiford, Ecdysone-induced change in cellular commitment of the epidermis of the tobacco hornworm, Manduca sexta, at the initiation of metamorphosis, Gen. Comp. Endocrin. 34 (1978) 438–446. [54] L.M. Riddiford, Hormones and Drosophila development, in: M. Bate, A. Martinez-Arias (Eds.), The Development of Drosophila, Cold Spring Harbor Laboratory Press, Plainview, 1993, pp. 899–939. [55] L.M. Riddiford, Cellular and molecular actions of juvenile hormone. I. General considerations and premetamorphic actions, Adv. Insect Physiol. 24 (1994) 213–274. [56] L.M. Riddiford, A.M. Ajami, Juvenile hormone: its assay and effects on pupae of Manduca sexta, J. Insect Physiol. 19 (1973) 749–762. [57] L.M. Riddiford, P.T. Cherbas, J.W. Truman, Ecdysone receptors and their biological actions, Vit. Horm. 60 (2001) 1–73. [58] L.M. Riddiford, K. Hiruma, X. Zhou, C.A. Nelson, Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster, Insect Biochem. Mol. Biol. 33 (2003) 1327– 1338. [59] L.M. Riddiford, J.W. Truman, C.K. Mirth, Y. Shen, A role for juvenile hormone in the prepupal development of Drosophila melanogaster, Development 137 (2010) 1117–1126. [60] J.M. Ringo, Hormonal regulation of sexual behavior in insects, in: D.W. Pfaff, A.P. Arnold, A.M. Eigen, S.E. Fahrbach, R.T. Rubin (Eds.), Hormones, Brain and Behavior, vol. 3, Academic Press, Amsterdam, 2002, pp. 93–114. [61] Z. Sheng, J. Xu, H. Bai, F. Zhu, S.R. Palli, Juvenile hormone regulates vitellogenin gene expression through insulin-like peptide signaling pathway in the red flour beetle, Tribolium castaneum, J. Biol. Chem. 286 (2011) 41924–41936. [62] M. Soller, M. Bownes, E. Kubli, Control of oocyte maturation in sexually mature Drosophila females, Dev. Biol. 208 (1999) 337–351. [63] Y. Suzuki, J.W. Truman, L.M. Riddiford, The role of the transcription factor Broad in the development and evolution of the holometabolous insect pupa, Tribolium castaneum, Development 135 (2008) 569–577. [64] C.S. Thummel, Flies on steroids-Drosophila metamorphosis and the mechanism of steroid hormone action, Trends Genetics 12 (1996) 306–310. [65] C.-Y. Ting, C.-H. Lee, Visual circuit development in Drosophila, Curr. Opin. Neurobiol. 17 (2007) 65–72. [66] J.W. Truman, W.S. Talbot, S.E. Fahrbach, D.S. Hogness, Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development, Development 120 (1994) 219– 234. [67] J.W. Truman, K. Hiruma, J.P. Allee, S.G.B. MacWhinnie, D.T. Champlin, L.M. Riddiford, Juvenile hormone is required to couple imaginal disc formation with nutrition in insects, Science 312 (2006) 1385–1388. [68] C. Wicker-Thomas, T. Chertemps, Molecular biology and genetics of hydrocarbon production, in: G.J. Blomquist, A.-G. Bagneres (Eds.), Insect Hydrocarbons: Biology, Biochemistry and Chemical Ecology, Cambridge University Press, Cambridge, 2010, pp. 53–74. [69] V.B. Wigglesworth, The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling moulting and ‘metamorphosis’, Quart. J. Micr. Sci. 77 (1934) 191–222. [70] V.B. Wigglesworth, The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus (Hemiptera), Quart. J. Micr. Sci. 79 (1936) 91–121. [71] C.M. Williams, The juvenile hormone. II. Its role in the endocrine control of molting, pupation, and adult development in the Cecropia silkworm, Biol. Bull. 121 (1961) 572–585.
484
L.M. Riddiford / General and Comparative Endocrinology 179 (2012) 477–484
[72] T.G. Wilson, M. Ashok, Insecticide resistance resulting from an absence of target-site gene product, Proc. Natl. Acad. Sci. USA 95 (1998) 14040– 14044. [73] T.G. Wilson, J. Fabian, A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone, Dev. Biol. 118 (1986) 190–201. [74] G.R. Wyatt, K.G. Davey, Cellular and molecular actions of juvenile hormone. II. Roles of juvenile hormone in adult insects, Adv. Insect Physiol. 26 (1996) 1– 155. [75] Z. Zhang, J. Xu, Z. Sheng, Y. Sui, S.R. Palli, Steroid receptor co-activator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, Methoprene tolerant, J. Biol. Chem. 286 (2011) 8437– 8447.
[76] B. Zhou, K. Hiruma, T. Shinoda, L.M. Riddiford, Juvenile hormone prevents ecdysteroid-induced expression of Broad Complex RNAs in the epidermis of the tobacco hornworm, Manduca sexta, Dev. Biol. 203 (1998) 233–244. [77] B. Zhou, L.M. Riddiford, Hormonal regulation and patterning of the Broad Complex in the epidermis and wing discs of the tobacco hornworm, Manduca sexta, Dev. Biol. 231 (2001) 125–137. [78] X. Zhou, L.M. Riddiford, Broad-Complex specifies pupal development and mediates the prevention of the pupal-adult transformation by juvenile hormone in Drosophila and Manduca, Development 129 (2002) 2259–2269. [79] J. Zhu, L. Chen, G. Sun, A.S. Raikhel, The competence factor bFtz-F1 potentiates ecdysone receptor activity via recruiting a p160/SRC coactivator, Mol. Cell. Biol. 26 (2006) 9402–9412.
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2012 Howard Bern Lecture
How does juvenile hormone control insect metamorphosis and reproduction? Lynn M. Riddiford ⇑ Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, United States
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Article history: Received 24 January 2012 Revised 22 May 2012 Accepted 1 June 2012 Available online 20 June 2012 Keywords: Juvenile hormone Methoprene-tolerant Insect metamorphosis Insulin-like peptide Vitellogenesis Female receptivity
a b s t r a c t In insects juvenile hormone (JH) regulates both metamorphosis and reproduction. This lecture focuses on our current understanding of JH action at the molecular level in both of these processes based primarily on studies in the tobacco hornworm Manduca sexta, the flour beetle Tribolium castaneum, the mosquito Aedes aegypti, and the fruit fly Drosophila melanogaster. The roles of the JH receptor complex and the transcription factors that it regulates during larval molting and metamorphosis are summarized. Also highlighted are the intriguing interactions of the JH and insulin signaling pathways in both imaginal disc development and vitellogenesis. Critical actions of JH and its receptor in the timing of maturation of the adult optic lobe and of female receptivity in Drosophila are also discussed. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Juvenile hormone (JH) is a hormone unique to insects although its immediate precursor methyl farnesoate is found in the Crustacea where it is known to mediate reproduction in some species [47]. In all insects JH has primary roles in the regulation of metamorphosis and reproduction. In some species, it has also been co-opted to regulate other developmental processes such as various polyphenisms and caste determination in social insects [25,48]. This article is based on my Howard Bern Lecture at the 2012 Society for Integrative and Comparative Biology meeting. It is an overview of my work on the study of JH action together with my perspective of the recent studies of others which are greatly enriching our current understanding of the action of JH in insect metamorphosis and reproduction. It is not a comprehensive review as there are several of these available [10,13,23,28,51]. 2. Regulation of larval molting and metamorphosis In insects all growth occurs prior to metamorphosis and is accompanied by the periodic shedding or molting of the exoskeleton. This growth and molting is orchestrated by two hormones, ecdysone1 and JH [55,57]. Alpha-ecdysone is a steroid hormone secreted by the prothoracic gland that is converted in the periphery to 20-hydroxyecdysone (20E) (Fig. 1A), which initiates and orches⇑ Fax: +1 571 209 4928. E-mail address: [email protected] Ecdysone is used throughout in the generic sense of the hormone that controls molting and metamorphosis with a-ecdysone and 20-hydroxyecdysone (20E) referring to the actual chemical molecules involved [57]. 1
0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2012.06.001
trates the molting process. Juvenile hormone is a sesquiterpenoid with JH III being the primary hormone produced by the corpora allata (CA) of most insects (Fig. 1B) [23,55]. Methyl farnesoate, the precursor of JH III, is released by nymphal cockroach CA and by the larval ring gland of higher Diptera [8,23,29]. JH bisepoxide is also secreted by higher dipteran CA [23]. In Lepidoptera the larval CA produce JH I and JH II, whereas the adult CA may release various combinations of JH I, II, and III as well as the respective JH acids depending on the species [23]. The JH analogs, methoprene and pyriproxifen (Fig. 1B), mimic the action of JH. In this paper I will use the generic terms ecdysone and JH except for discussions of experimental use of a particular hormonal compound. JH is present in the larva and allows ecdysone action to initiate the molt but prevents its actions in switching genetic programs necessary for metamorphosis [26,52,53,55]. During the intermolt feeding period, JH is also necessary for the maintenance of isomorphic proliferation of the imaginal discs and primordia [67]. The tobacco hornworm, Manduca sexta, was the ‘‘white rat’’ of insect endocrinology that allowed us to begin to elucidate how JH directs ecdysone action [48,55]. During the final (5th) larval instar the JH titer declines to undetectable levels due to the cessation of secretion from the CA coupled with the appearance of a JH-specific esterase in both the hemolymph and the tissues that degrades existing JH. This loss of JH allows prothoracicotropic hormone (PTTH) release that stimulates a small amount of ecdysone release. Ecdysone in the absence of JH causes the cessation of feeding and the onset of wandering to find a pupation site. It also causes pupal commitment so that the larva can no longer undergo a larval molt but rather only a pupal molt when challenged with ecdysone. In vitro experiments with the abdominal epidermis from feeding
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Fig. 1. Chemical structures of the active insect ecdysteroid 20-hydroxyecdysone (A) and of the major juvenile hormones (JH) and JH mimics methoprene and pyriproxifen (B). Structure in (A) based on [57] and in (B) on [23]. JHB3, JH bisepoxide.
larvae showed that exposure to 20E in the absence of JH was sufficient to cause pupal commitment, whereas exposure to 20E and JH maintained the larval state [52,53]. During the prepupal period JH reappears at the time of the ecdysteroid rise for the pupal molt and is necessary for prevention of precocious metamorphosis in some tissues such as the eye, the optic lobe, and the ventral diaphragm [15–17,34]. JH again disappears by the time of pupal ecdysis so that the subsequent ecdysteroid rise that causes adult commitment and subsequent differentiation occurs in the absence of JH. Exogenous JH at the onset of this rise causes the formation of a second pupa [56] just as Williams [71] first showed for the Cecropia silkmoth. 3. JH receptor The JH receptor has been long sought, and two major candidates have been put forward: (1) Ultraspiracle (USP), a member of the nuclear hormone receptor family [12,64], that forms a heterodimer with the ecdysone receptor (EcR) to mediate ecdysone action [57]; (2) Methoprene-tolerant (Met), a protein in Drosophila that confers resistance to JH [72,73]. Ultraspiracle from Drosophila melanogaster binds JH III with intermediate affinity (Kd = 2 10 7 M), whereas it binds methyl farnesoate with the high nanomolar affinity expected for a hormone receptor [31]. Methyl farnesoate is found in larvae, but is 100 times less active than JH III in preventing metamorphosis when applied at the time of pupariation [32]. When synthesis of methyl farnesoate and thus of all JHs is prevented in Drosophila by expression of HMGCoA reductase RNAi, most larvae die during the molt to the 3rd larval stage [30]. When these larvae were fed farnesol, they could ecdyse to the 3rd instar and at higher concentrations of farnesol could pupariate, but neither methyl farnesoate nor JH III alone had this effect [30,32]. Importantly, USP null mutants die during the molt to the 2nd instar as pharate 2nd instar larvae showing double mouthhooks and spiracles [57].
Interestingly, mutants lacking Met progress normally through larval life and metamorphosis, showing only delayed and severely reduced fecundity [72], calling into question the role of Met as the JH receptor in the regulation of metamorphosis. This problem has recently been resolved by the finding of a second gene, germ cells expressed (gce), in Drosophila that is highly similar to Met with Met likely arising from gce by gene duplication [5,6]. Although mutants lacking either Met or Gce showed normal progression through metamorphosis, mutants lacking both pupariate but die at pupal head eversion [1] (which signals the completion of the formation of the pupa in the higher Diptera), similar to animals that have been genetically allatectomized (CAX) [41,59]. Recent studies in other insects have provided convincing evidence that Met is the primary JH receptor. Konopova and Jindra [36] used double stranded Met RNA (Met RNAi) to knock down the level of Met in early stage (3rd or 4th) Tribolium castaneum (flour beetle) larvae and obtained precocious metamorphosis after the 5th or 6th larval stage respectively, showing that Met was essential for larval molting. Also, loss of Met by RNAi leads to precocious metamorphosis in the linden bug, Pyrrhocoris apterus [38], indicating that Met likely mediates JH action in all insects. Met is a member of the basic helix-loop-helix (bHLH) family of transcription factors and recently Tribolium Met has been shown to bind JH via its Pas B domain [18]. Drosophila Met can form a homodimer with itself or a heterodimer with Gce, but only in the absence of JH [22]. Binding of JH causes dissociation of the dimer and the association of Met with a steroid hormone receptor coactivator known as Taiman in Drosophila [4], bFtz-F1 interacting steroid receptor coactivator (FISC) in the mosquito Aedes aegypti [79], and steroid receptor coactivator (SRC) in Tribolium [75], all of which interact with EcR during ecdysone action. The JH-Met-FISC complex binds to the JH response element (JHRE) in the promoter of the early trypsin gene in adult female mosquito midgut and causes up-regulation of this gene [40]. Similarly, the JH-Met-SRC
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complex is necessary for the up-regulation of Krüppel homolog (Kr-h1) in Tribolium larvae and in a mosquito cell line [75]. Both Met and Gce can also interact with the orphan nuclear receptor b-FTZ-F1, and Gce is necessary for the up-regulation of E75A in Drosophila S2 cells by JH in the absence of 20E [9]. The nature of the 20E/JH interaction is elusive although recently Jones et al. [33] have developed a model system using a hexamerin core promoter and a DR1 ecdysone response element that is responsive to both hormones and may yield the answer. 4. Larval molting In Tribolium larvae the loss of Kr-h1 by RNAi leads to precocious metamorphosis that cannot be rescued by exogenous JH [46]. In both Tribolium [46] and Drosophila [50], Kr-h1 mRNA is present throughout larval life falling to low levels in the mid-final instar, then transiently reappearing during the onset of the pupal molt when it is necessary for normal prepupal development. In Tribolium mid-final instar larvae Kr-h1 mRNA can be up-regulated by exogenous JH, but not when Met levels have been reduced by RNAi [75]. In Drosophila the loss of Kr-h1 function leads to the precocious appearance of Broad, the pupal specifier (see section 5.1 below), in the fat body during the molt to the 3rd instar [27]. Also, Kr-h1 mRNA is present in the penultimate nymphal instar of both Pyrrhocoris [38] and the cockroach Blattella germanica [42], and disappears in the final instar; its reduction by RNAi in the penultimate instar leads to precocious metamorphosis. In these hemimetabolous nymphs, Broad is present in the penultimate instar, then disappears in the final instar, a necessity for metamorphosis to the adult [28,38]. Thus, Kr-h1 is necessary for both larval and nymphal molting, in the former preventing the turning-on of Broad expression (Fig. 2), in the latter preventing the turning-off of Broad. The presence of JH maintains Kr-h1 expression and thereby prevents metamorphosis. 5. Pupal commitment and the onset of metamorphosis 5.1. Epidermis Twenty-hydroxyecdysone is known to act on tissues by causing a cascade of gene activation first worked out by Ashburner et al. [3] studying its effects on the ‘‘puffing’’ activity in D. melanogaster salivary gland chromosomes. Later molecular studies showed that most of the 20E-induced genes in this cascade were transcription factors [35,64]. A similar cascade of transcription factors was found to be induced by 20E in Manduca abdominal epidermis in the larval, pupal and adult molts (reviewed in [26]). A comparison of the cascades induced during the larval molt and at the onset of metamorphosis revealed one major difference. In the absence of JH at the time of pupal commitment, ecdysone and 20E induced the appearance of Broad (Br) [76], a transcription factor containing the Broad-Tramtrack-Bric-a-brac (BTB) protein–protein interaction
Fig. 2. Model for the molecular basis of the control of larval molting and metamorphosis by JH based on studies by [26,38,46,78]. JH, juvenile hormone; Kr-h1, Krüppel homolog 1; Met, Methoprene-tolerant; Tai, Taiman. See text for details. Dashed lines signify reduced effect.
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domain and a DNA binding domain containing a pair of cysteine– histidine zinc fingers [7]. Its induction within an epidermal segment correlated with the loss of cellular JH sensitivity for induction of another larval molt, indicating that its appearance signified pupal commitment of the cells [77]. Broad remained in the epidermis peaking during the prepupal surge of ecdysteroid, then fell to trace levels and disappeared by pupal day 4 as the ecdysteroid titer for the adult molt rose [26,78]. Importantly, when the pupa was given exogenous JH I, Br did not disappear, but rather increased again with the increase of the ecdysteroid titer and the epidermis formed a second pupal cuticle [78]. Thus, in Manduca 20E acting in the absence of JH is sufficient to activate broad (br) expression at pupal commitment and also is sufficient to turn off its expression during the adult molt (Figs. 2 and 3). In Tribolium br RNA is present at low levels during most of larval life, then its levels increase late in the final larval instar, remain high during the prepupal period, then decline to undetectable in the early pupa [37,49,63]. These studies also showed that br RNAi caused the formation of a larval-pupal-adult mosaic at the time of the pupal molt, indicating that Broad was required for normal pupal development. Exogenous JH (the JH mimics hydroprene or methoprene) given to the pupa at the outset of adult development caused the re-expression of br during the onset of the adult molt and a second pupa was formed as seen in Manduca [37,49,63]. In this case JH was found to act via Met by up-regulating Kr-h1 which then acted to prevent turning off of br expression by 20E [46] by some as yet unknown mechanism. In the higher Diptera such as Drosophila in contrast to most holometabolous insects, the final instar larval cuticle becomes the puparium within which the head, thoracic and genital discs evert and form pupal structures such as legs and wings, whereas the larval abdominal cells make the pupal cuticle of the abdomen [21]. The abdominal histoblasts are quiescent during larval life, then start dividing shortly after pupariation and spread out over the pupal abdomen to form the imaginal (adult) epidermis as the ecdysteroid titer rises for adult development. In Drosophila Broad also appears during the final larval instar and the complete loss of function of br causes lethality at the time of pupariation [7]. Exogenous JH (JH mimic pyriproxifen), however, only delayed its appearance in the larval abdominal epidermis and also delayed
Fig. 3. Summary of the essential role of the Broad transcription factor in mediating the endocrine control of metamorphosis in the imaginal discs and in the abdominal epidermis. JH, juvenile hormone; 20E, 20-hydroxyecdysone. See text for details.
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the onset of wandering [58]. By contrast, JH given at pupariation prevented the normal cessation of br expression in the abdominal imaginal epidermis as the ecdysteroid titer rises for the adult molt [78]. The exogenous JH also caused the re-expression of Kr-h1 mRNA in the abdomen during the adult molt and misexpression of Kr-h1 mRNA in this epidermis was found to prolong br expression in some cells [45]. These cells then made a second pupal cuticle rather than adult cuticle. The adult head and thorax arising from imaginal discs were unaffected by the treatment with exogenous JH; their levels of br mRNA declined on schedule and they formed normal adult cuticular structures [78]. When br mRNA was over-expressed, particularly the Z1 isoform, during the time of bristle formation and during the onset of cuticle formation during the adult molt, a second pupal cuticle formed in both the abdomen and the disc-derived head and thorax [78]. This action of Broad in suppressing adult cuticle formation is at least partly through its inhibition of the expression of another transcription factor DHR38 [19], which is needed for the transcription of at least one of the adult cuticle genes, Acp65A [14]. The basis for the different responsiveness of lepidopteran and higher dipteran epidermis and imaginal discs to JH remains a mystery. 5.2. Lepidopteran imaginal discs In Manduca the wing discs grow isomorphically during larval life, then in the final 5th instar undergo patterning and morphogenetic growth to form the pupal wing, whereas the pupal eye and leg primordia remain as quiescent diploid cells until the final instar when they begin to divide and undergo morphogenetic growth to form the pupal structures [43,67]. Normally feeding in the final instar initiates the appearance of Broad in the discs and the primordia followed some hours later by the onset of morphogenesis. Feeding sucrose alone was sufficient for the appearance of br mRNA, but the addition of amino acids was necessary for the subsequent growth [43]. The up-regulation of br mRNA in the wing disc correlated with the pupal commitment of the disc [39]. Starvation of the freshly ecdysed larvae prevented the appearance of Broad and the subsequent morphogenetic growth, but surprisingly both occurred in starved larvae that had been allatectomized during the molt to the final instar and so lacked JH [67]. JH (methoprene) applied to the allatectomized starved larvae suppressed both the appearance of Broad and the subsequent disc and primordia morphogenesis. This suppressive effect of JH did not require exposure to ecdysteroids since it occurred in metathoracic leg primordia isolated from the prothoracic glands that produce ecdysone. Subsequent studies showed that either Manduca bombyxin [an insulin-like peptide (ILP)] or bovine insulin could substitute for feeding in starved larvae by inducing Broad expression and pupal commitment in the wing discs [39]. When wing discs from freshly ecdysed final instar larvae were cultured in hormone-free medium, br mRNA was up-regulated. The addition of bombyxin or insulin did not affect this upregulation. The addition of methoprene, by contrast, prevented the increase in br mRNA. When both hormones were present, insulin suppressed the repressive effects of JH so that the br mRNA level was similar to that seen in hormone-free medium. Down-regulation of the insulin receptor in the disc by RNAi abolished this effect of insulin, suggesting that some component of the insulin signaling pathway is interfering with the action of JH. Nothing further is known about this intriguing interaction of JH and insulin signaling. Thus, the appearance or up-regulation of the transcription factor Broad signals the onset of metamorphosis and its presence is required through pupal differentiation. It must then disappear in order for adult differentiation to occur. As summarized in Fig. 3, in the epidermis of the basal holometabolous insects JH prevents the turning on of br mRNA by 20E; whereas in the final stage imaginal discs of lepidopterans, the JH suppression of br expression is
overcome by increased insulin signaling due to feeding. During the pupal-adult transition JH is normally not present and 20E causes the cessation of br transcription and adult development proceeds. When exogenous JH is present, Broad levels remain high both in pupal structures formed from imaginal discs and primordia (except in the higher Diptera) and in the abdominal epidermis, and a second pupa is formed. In the derived higher Diptera such as Drosophila, JH application has no effect on the imaginal discs so br mRNA is down-regulated by 20E resulting in a mosaic animal with an adult head and thorax but a pupal abdomen. Considering the recent evidence mainly from Tribolium and Drosophila described above, in all these actions JH likely acts via the Met receptor modulating the synthesis of Kr-h1 (see Fig. 2). 6. Prepupal role of JH In holometabolous insects JH reappears during the prepupal period at the time of the ecdysteroid rise for pupation and in Lepidoptera it acts to prevent premature adult development of imaginal structures such as the eye, optic lobe and the ventral diaphragm [15–17,71]. Recent studies in Drosophila have begun to reveal some of the mechanisms involved in this action of JH. When Drosophila larvae were genetically allatectomized by expressing the cell death gene grim in the CA, traces of the CA could still be seen in 2nd instar larvae but were undetectable in the final 3rd stage larva. These larvae grew more slowly and usually pupariated at a smaller size 12–24 h later than intact larvae [41,59]. Their discs everted normally and they formed pupal cuticle, but the prepupae could not complete the process of pupal head eversion and died soon afterwards. Internally the fat body which normally dissociates during adult development showed premature dissociation and a large increase in caspase-dependent apoptotic cell death [41]. All of these effects were prevented by the feeding the larvae either of the JH mimics, methoprene or pyriproxifen. Similar effects are seen in mutants lacking both Met and Gce but not in mutants lacking only one of these proteins [1]. Normally during wandering and the prepupal period the photoreceptors from the developing eye disc grow into the optic lobe, induce proliferation and aid in coordinating its development [65]. By 12 h after pupariation (AP), proliferation declines sharply and by about 18 h AP differentiation of the adult optic lobe begins as the ecdysteroid titer rises for the adult molt (Fig. 4). In allatectomized prepupae, proliferation levels in the medulla of the optic lobe were similar at pupariation but declined prematurely by 6–8 h AP at which time the R7 photoreceptor showed the precocious onset of adult differentiation normally seen between 18 and 24 h AP [59]. At pupariation the optic lobe normally has high levels of the A isoform of the EcR, but none of the B1 isoform [66]. EcR-B1 appears in the optic lobe beginning about the time of head eversion 10–12 h AP and reaches high levels by 24 h AP. In allatectomized animals low levels of EcR-B1 were already present at pupariation and increased to high levels by 6–8 h AP [59]. Feeding the JH mimic pyriproxifen during the third instar or application of JH III or pyriproxifen at the onset of wandering prevented all the effects of allatectomy on optic lobe development as well as the death at head eversion. Thus, JH prevents precocious adult differentiation of the optic lobe in the forming pupa in response to the pupariation peak of ecdysteroid. The Met null mutant Met27 shows a similar precocious adult differentiation of the optic lobe as seen in the allatectomized prepupa, and this precocious differentiation cannot be prevented by exogenous JH [59]. This premature differentiation results in anomalous morphogenesis of the lobula neuropil of the optic lobe (Fig. 5) and abnormal patterning of neuronal connections in that region. In preliminary assays of visual function, these animals were unresponsive, indicating that they were likely functionally blind (J. Tuthill and L.M.
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Fig. 4. Summary of the regulation of the timing of adult optic lobe development in Drosophila melanogaster by JH as shown by studies on genetically allatectomized (CAX) animals that die at the time of head eversion to the pupa ( ) [59]. See text for details. The hormone titers are redrawn from [54]. E, ecdysis; HE, head eversion; W, wandering.
Fig. 5. Frontal sections of the adult optic lobe of wild type (wt) (Canton S) (left), Met27 null mutant (middle), and gce2.5k null mutant of Drosophila melanogaster. Arrows point to abnormal lobes of the lobula (lo) that project towards the medulla (m) in the Met27mutant. Staining is by N-cadherin (aqua) and neuroglian (red). Bar = 50 lm. The left and center pictures are from [59].
Riddiford, unpublished). To probe further the cellular basis of JH action, we expressed Met RNAi in various neurons in the eye and the developing optic lobe and found that loss of Met in either the photoreceptor neurons or in the lamina neurons (the outermost layer of neurons of the optic lobe) led to precocious appearance of EcR-B1 in those neurons [59]. Only loss of Met in the photoreceptor neurons, however, led to precocious differentiation of the R7 photoreceptor within the medulla. Yet overexpression of EcR-B1 in the photoreceptors was insufficient to cause this precocious differentiation. Thus, other unknown effects of the loss of JH or of Met in the photoreceptors must also be necessary for the precocious differentiation that was observed. Interestingly, the gce2.5k null mutant showed neither precocious expression of EcR-B1 in the prepupal optic lobe nor abnormal morphogenesis of the adult lobula (L.M. Riddiford, unpublished; Fig. 5). Thus, in its regulation of prepupal optic lobe development in Drosophila, JH appears to be acting only via Met. 7. Roles of JH in reproductive maturation 7.1. Vitellogenin synthesis JH has long been known to regulate various aspects of reproductive maturation after adult emergence with the specific effects varying depending on the insect studied (see [13,51,74] for
comprehensive reviews). In many feeding adult females, JH regulates vitellogenin (Vg) (yolk protein) synthesis, but the molecular mechanisms have remained elusive. Recent studies in Tribolium have shown that Vg synthesis in the fat body requires both feeding and JH [61]. Normally these females begin feeding 2.5 days after adult eclosion and Vg2 mRNA levels begin to increase at day 4 and reach high levels by day 5. Feeding was unable to stimulate Vg2 mRNA expression in the absence of JH, but injection of bovine insulin restored normal Vg2 levels. Furthermore, RNAi-mediated suppression of FOXO expression, a repressive transcription factor whose loss from the nucleus is mediated by the insulin signaling pathway [24], caused precocious appearance of Vg2 mRNA on day 2. Further in vivo and in vitro experiments showed that JH stimulated an increase in mRNA expression in the fat body and brain of one of the 4 ILPs (ILP2) that normally was up-regulated by day 2 after ecdysis. This up-regulation was prevented by Met RNAi, indicating that JH is working through the Met receptor. Feeding caused the appearance of ILP3 mRNA in the fat body followed later by Vg2 mRNA. Knockdown of ILP2 or ILP3 by RNAi caused partial or complete loss of Vg2 mRNA respectively. Thus, Sheng et al. [61] hypothesize that JH acts via Met to increase production of ILP2 and feeding stimulates the production of ILP3. The two together then act through the insulin signaling pathway to cause the phosphorylation of FOXO and its loss from the nucleus, thus relieving the repression of Vg2 mRNA transcription. Thus, JH is
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indirectly stimulating Vg production in this beetle. Whether its simulation of Vg production in other insects is mediated similarly remains to be determined. In Drosophila JH primarily regulates Vg uptake into the eggs [62] but the mechanisms involved are unknown. The Met null mutant shows both a delay in egg maturation and greatly reduced fecundity [72]. Loss of Gce has little effect on the timing of egg maturation and only slightly reduces fecundity [1]. 7.2. Female receptivity Among the insects, JH is known to play various roles in female reproductive behavior, ranging from stimulating pheromone production in some cockroaches and beetles to regulating virgin female receptivity to courting males in a range of insects from grasshoppers to flies [60]. In D. melanogaster the female at the time of eclosion is unreceptive to male courtship attempts, but nearly all mate by 48 h after eclosion [20,44]. Implantation of active CA into female abdomens shortly before eclosion caused the recipients to mate precociously on day 1 as compared to only 30% of the controls implanted with a piece of aorta [44]. Thus, Manning [44] suggested that JH was involved in the maturation of female receptivity. To explore the action of JH in the maturation of female receptivity, we destroyed the CA late in adult development by using the temperature-sensitive GAL80ts to prevent earlier GAL4-driven expression of UAS-diphtheria toxin in the CA [11]. These allatectomized females were unreceptive to males at 24 h after eclosion, but by 48 h nearly all mated in group assays similarly to controls. Application of methoprene (ED50 = 2 ng) to the allatectomized females at eclosion resulted in over 50% mating at 24 h as compared to about 30% mating for the parental controls. These results confirm those of Manning [44] that JH is involved in the maturation of female receptivity. We further found that Met27 null mutants showed a similar delay in the onset of female receptivity to that seen in allatectomized females. Reduction of Met expression in the central nervous system using Met RNAi driven by the neuronal-specific driver elav-GAL4 caused a similar delay in the onset of female receptivity as found in the Met27 mutant. Therefore, JH acting via Met in the CNS is somehow fostering the maturation of neuronal circuits necessary for female acceptance of male courtship attempts so that she mates. However, other factors are also clearly involved in this maturation since it still occurs, albeit delayed, in the absence of JH. Interestingly, when assayed as individual pairs at 48 h after eclosion, the time to copulation was similar for Met27 females and controls whereas allatectomized females took nearly 3 times as long (J. Bilen and L.M. Riddiford, unpublished). Preliminary experiments show that the longer time to copulation for allatectomized females is at least partly due to their decreased attractiveness to males as measured by male courtship of headless females. Surprisingly, though, in this assay Met27 females were found to be more attractive than controls. These findings may indicate that JH has a role in the synthesis of the female-specific pheromones that appear beginning about 18–24 h after eclosion [2,68]. 8. Conclusions Studies in the past few years have greatly enhanced our knowledge of the action of JH in insect metamorphosis and reproduction at the molecular level. Along with these new insights have come surprises such as the interaction of the JH-Met receptor complex with the EcR coactivator to regulate specific genes and the interplay between JH and the insulin-signaling pathway in controlling the onset of metamorphosis in imaginal discs and in the regulation
of vitellogenesis. Mysteries still remain such as why are Drosophila imaginal discs refractory to JH in its maintenance of Broad expression? Is Kr-h1 the critical transcription factor specifying the larval state and how is it regulating Broad? Over the years studies on the action of JH in many different insect species beginning with the bloodsucking bug Rhodnius prolixus [69,70] have been important to our understanding of how JH is intimately involved in the regulation of both metamorphosis and reproduction. The tobacco hornworm M. sexta provided a manipulatable endocrine system to study its action at the cellular and molecular level but lacked the genetics necessary to probe these molecular actions. Drosophila then became the leader in the search for mechanisms of action but with the disadvantage of a highly derived developmental pattern and the lack of a well-defined role for JH in metamorphosis. With the advent of RNAi and genomic approaches, studies on other insect species such as the flour beetle Tribolium and the linden bug Pyrrhocoris are again contributing critical new insights into JH action. This comparative approach is clearly in the spirit of Howard Bern, one of the founders of comparative endocrinology whose wisdom, insights, and mentoring will be sorely missed in the field. Acknowledgments I thank Dr. James Truman for help in the imaging of the gce2.5k mutant and for critical comments on the manuscript, Dr. Julide Bilen for critical comments on the manuscript, Dr. Jian Wang for the gce2.5k mutant, and the Howard Hughes Medical Institute for support of unpublished work cited here. References [1] M.A. Abdou, Q. Heb, D. Wen, O. Zyaan, J. Wang, J. Xu, A.A. Baumann, J. Joseph, T.G. Wilson, S. Li, J. Wang, Drosophila Met and Gce are partially redundant in transducing juvenile hormone action, Insect Biochem. Mol. Biol. 41 (2011) 938–945. [2] M. Arienti, C. Antony, C. Wicker-Thomas, J.-P. Delbecque, J.-M. Jallon, Ontogeny of Drosophila melanogaster female sex-appeal and cuticular hydrocarbons, Integr. Zool. 5 (2010) 272–282. [3] M. Ashburner, C. Chihara, P. Meltzer, G. Richards, Temporal control of puffing activity in polytene chromosomes, Cold Spring Harb. Symp. Quant. Biol. 38 (1974) 655–662. [4] J. Bai, Y. Uehara, D.J. Montell, Regulation of invasive cell behavior by Taiman, a Drosophila protein related to AIB1, a steroid receptor coactivator amplified in breast cancer, Cell 103 (2000) 1047–1058. [5] A. Baumann, Y. Fujiwara, T.G. Wilson, Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila, J. Insect Physiol. 56 (2010) 1445–1455. [6] A. Baumann, J. Barry, S. Wang, Y. Fujiwara, T.G. Wilson, Paralogous genes involved in juvenile hormone action in Drosophila melanogaster, Genetics 185 (2010) 1327–1336. [7] C. Bayer, L. von Kalm, J.W. Fristrom, Gene regulation in imaginal disc and salivary gland development during Drosophila metamorphosis, in: L.I. Gilbert, J.R. Tata, B.G. Atkinson (Eds.), Metamorphosis, Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells, Academic Press, San Diego, 1996, pp. 321–361. [8] W.G. Bendena, J. Zhang, S.M. Burtenshaw, S.S. Tobe, Evidence for differential biosynthesis of juvenile hormone (and related) sesquiterpenoids in Drosophila melanogaster, Gen. Compar. Endocrinol. (2011) 56–61. [9] T.J. Bernardo, E.B. Dubrovsky, The Drosophila juvenile hormone receptor candidates Methoprene-tolerant (Met) and germ cell-expressed (gce) utilize a conserved LIXXL motif to bind the FTZ-F1 nuclear receptor, J. Biol. Chem. 287 (2012) 7821–7833. [10] T.J. Bernardo, E.B. Dubrovsky, Molecular mechanisms of transcription activation by juvenile hormone: a critical role for bHLH-PAS and nuclear receptor proteins, Insects 3 (2012) 324–338. [11] J. Bilen, L. Riddiford, Juvenile hormone modulates the maturation of female receptivity in Drosophila melanogaster, Abstract 646A, Drosophila Research Conference, Gen. Soc. America (2011) 267–268. [12] F. Bonneton, V. Laudet, Evolution of nuclear receptors in insects, in: L.I. Gilbert (Ed.), Insect Endocrinology, Elsevier, Amsterdam, 2012, pp. 310–365. [13] M. Bownes, The regulation of yolk protein gene expression and vitellogenesis in higher Diptera, in: A.S. Raikhel, T.W. Sappington (Eds.), Reproductive Biology of Invertebrates, vol. 12, Part B Progress in Vitellogenesis, Science Publishers, Enfield, USA/Plymouth, UK, 2004, pp. 95–128.
L.M. Riddiford / General and Comparative Endocrinology 179 (2012) 477–484 [14] N. Bruey-Sedano, J. Alabouvette, M. Lestradet, L. Hong, A. Girard, E. Gervasio, B. Quennedey, J.P. Charles, The Drosophila ACP65A cuticle gene: deletion scanning analysis of cis-regulatory sequences and regulation by DHR38, Genesis 43 (2005) 17–27. [15] D.T. Champlin, J.W. Truman, Ecdysteroid control of cell proliferation during optic lobe neurogenesis in the moth, Manduca sexta, Development 125 (1998) 269–277. [16] D.T. Champlin, J.W. Truman, Ecdysteroids govern two phases of eye development during metamorphosis of the moth, Manduca sexta, Development 125 (1998) 2009–2018. [17] D.T. Champlin, S.E. Reiss, J.W. Truman, Truman, Hormonal control of ventral diaphragm myogenesis during metamorphosis of the moth, Manduca sexta, Dev. Genes Evol. 209 (1999) 265–274. [18] J.-P. Charles, T. Iwema, V.C. Epa, K. Takaki, J. Rynes, M. Jindra, Ligand-binding properties of a juvenile hormone receptor, Methoprene–tolerant, Proc. Natl. Acad. Sci. USA 108 (2011) 21128–21133. [19] H.-Y. Cui, M. Lestradet, N. Bruey-Sedano, J.-P. Charles, L.M. Riddiford, Elucidation of the regulation of an adult cuticle gene Acp65A by the transcription factor Broad, Insect Mol. Biol. 18 (2009) 421–429. [20] J.-F. Ferveur, Drosophila female courtship and mating behaviors: sensory signals, genes, neural structures and evolution, Curr. Opin. Neurobiol. 20 (2010) 764–769. [21] D. Fristrom, J. Fristrom, The metamorphic development of the adult epidermis, in: M. Bate, A. Martinez-Arias (Eds.), The Development of Drosophila melanogaster, vol II, Cold Spring Harbor Laboratory Press, Plainview, New York, 1993, pp. 843–897. [22] J. Godlewski, S. Wang, T.G. Wilson, Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila, Biochem. Biophys. Res. Commun. 342 (2006) 1305–1311. [23] W.G. Goodman, M. Cusson, The juvenile hormones, in: L.I. Gilbert (Ed.), Insect Endocrinology, Elsevier, Amsterdam, 2012, pp. 310–365. [24] S.S. Grewal, Insulin/TOR signaling in growth and homeostasis: a view from the fly world, Int. J. Biochem. Cell Biol. 41 (2009) 1006–1010. [25] K. Hartfelder, D.J. Emlen, Endocrine control of insect polyphenism, in: L.I. Gilbert (Ed.), Insect Endocrinology, Elsevier, Amsterdam, 2012, pp. 464–522. [26] K. Hiruma, L.M. Riddiford, Developmental expression of mRNAs for epidermal and fat body proteins and hormonally regulated transcription factors in the tobacco hornworm, Manduca sexta, J. Insect Physiol. 56 (2010) 1390–1395. [27] J. Huang, L. Tian, C. Peng, M. Abdou, D. Wen, Y. Wang, S. Li, J. Wang, DPPmediated TGFbeta signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase, Development 138 (2011) 2283–2291. [28] M. Jindra, S.R. Palli, L.M. Riddiford, The juvenile hormone signaling pathway in insect development, Annu. Rev. Entomol. 58 (2013), http://dx.doi.org/10.1146/ annurev-ento-120811-153700. [29] D. Jones, G. Jones, Farnesoid secretions of dipteran ring glands: what we do know and what we can know, Insect Biochem. Mol. Biol. 37 (2007) 771–798. [30] D. Jones, G. Jones, P. Teal, C. Hammac, L. Messmer, K. Osborne, Y.H. Belgace, J.R. Martin, Suppressed production of methyl farnesoid hormones yields developmental defects and lethality in Drosophila larvae, Gen. Comp. Endocrin. 165 (2010) 244–254. [31] G. Jones, D. Jones, P. Teal, A. Sapa, M. Wozniak, The retinoid-X receptor ortholog, Ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand, FEBS J. 273 (2006) 4983–4996. [32] G. Jones, D. Jones, X. Li, L. Tang, L. Ye, P. Teal, L. Riddiford, C. Sandifer, D. Borovsky, J-R. Martin, Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster, J. Insect Physiol. 56 (2010) 1456–1464. [33] G. Jones, D. Jones, F. Fang, Y. Xu, D. New, W.-H. Wu, Juvenile hormone action through a defined enhancer motif to modulate ecdysteroid-activation of natural core promoters, Comp. Biochem. Physiol. B 161 (2012) 219–225. [34] K. Kiguchi, L.M. Riddiford, The role of juvenile hormone in pupal development of the tobacco hornworm, Manduca sexta, J. Insect Physiol. 24 (1978) 673– 680. [35] K. King-Jones, C.S. Thummel, Nuclear receptors – a perspective from Drosophila, Nat. Rev. Genet. 6 (2005) 311–323. [36] B. Konopova, M. Jindra, Juvenile hormone resistance gene Methoprenetolerant controls entry into metamorphosis in the beetle Tribolium castaneum, Proc. Natl. Acad. Sci. USA 104 (2007) 10488–10493. [37] B. Konopova, M. Jindra, Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis, Development 135 (2008) 559–568. [38] B. Konopova, V. Smykal, M. Jindra, Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects, PLoS One 6 (2011) e28728. [39] T. Koyama, M.O. Syropyatova, L.M. Riddiford, Insulin/IGF signaling regulates the change in commitment in imaginal discs and primordia by overriding the effect of juvenile hormone, Dev. Biol. 324 (2008) 258–265. [40] M. Li, E.A. Mead, J. Zhu, Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression, Proc. Natl. Acad. Sci. USA 108 (2011) 638–643. [41] Y. Liu, Z. Sheng, H. Liu, D. Wen, Q. He, S. Wang, W. Shao, R.-J. Jiang, S. An, Y. Sun, W.G. Bendena, J. Wang, L.I. Gilbert, T.G. Wilson, Q. Song, S. Li, Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila, Development 136 (2009) 2015–2025.
483
[42] J. Lozano, X. Belles, Conserved repressive function of Krüppel homolog 1 on insect metamorphosis in hemimetabolous and holometabolous species, Sci. Rep. 1 (2011): doi:10.1038/srep00163. [43] S.G.B. MacWhinnie, J.P. Allee, C.A. Nelson, L.M. Riddiford, J.W. Truman, D.T. Champlin, The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta, Dev. Biol. 285 (2005) 285–297. [44] A. Manning, Corpus allatum and sexual receptivity in female Drosophila melanogaster, Nature 211 (1966) 1321–1322. [45] C. Minakuchi, X. Zhou, L.M. Riddiford, Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster, Mech. Dev. 125 (2008) 91–105. [46] C. Minakuchi, T. Namiki, T. Shinoda, Krüppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum, Dev. Biol. 325 (2009) 341–350. [47] G.P.C. Nagaraju, Reproductive regulators in decapod crustaceans: an overview, J. Exp. Biol. 214 (2011) 3–16. [48] H.F. Nijhout, Insect Hormones, Princeton University Press, Princeton, 1995. [49] R. Parthasarathy, A. Tan, H. Bai, S.R. Palli, Transcription factor broad suppresses precocious development of adult structures during larval–pupal metamorphosis in the red flour beetle, Tribolium castaneum, Mech. Dev. 125 (2008) 299–313. [50] F. Pecasse, Y. Beck, C. Ruiz, G. Richards, Krüppel-homolog, a stage-specific modulator of the prepupal ecdysone response, is essential for Drosophila metamorphosis, Dev. Biol. 221 (2000) 53–67. [51] A.S. Raikhel, M.R. Brown, X. Belles, Hormonal control of reproductive processes In: L.I. Gilbert, K. Iatrou, S.S. Gill, (Eds.), Comprehensive Insect Science vol. 3, Elsevier, Pergamon, Amsterdam pp. 433-491 [52] L.M. Riddiford, Hormonal control of insect epidermal cell commitment in vitro, Nature 259 (1976) 115–117. [53] L.M. Riddiford, Ecdysone-induced change in cellular commitment of the epidermis of the tobacco hornworm, Manduca sexta, at the initiation of metamorphosis, Gen. Comp. Endocrin. 34 (1978) 438–446. [54] L.M. Riddiford, Hormones and Drosophila development, in: M. Bate, A. Martinez-Arias (Eds.), The Development of Drosophila, Cold Spring Harbor Laboratory Press, Plainview, 1993, pp. 899–939. [55] L.M. Riddiford, Cellular and molecular actions of juvenile hormone. I. General considerations and premetamorphic actions, Adv. Insect Physiol. 24 (1994) 213–274. [56] L.M. Riddiford, A.M. Ajami, Juvenile hormone: its assay and effects on pupae of Manduca sexta, J. Insect Physiol. 19 (1973) 749–762. [57] L.M. Riddiford, P.T. Cherbas, J.W. Truman, Ecdysone receptors and their biological actions, Vit. Horm. 60 (2001) 1–73. [58] L.M. Riddiford, K. Hiruma, X. Zhou, C.A. Nelson, Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster, Insect Biochem. Mol. Biol. 33 (2003) 1327– 1338. [59] L.M. Riddiford, J.W. Truman, C.K. Mirth, Y. Shen, A role for juvenile hormone in the prepupal development of Drosophila melanogaster, Development 137 (2010) 1117–1126. [60] J.M. Ringo, Hormonal regulation of sexual behavior in insects, in: D.W. Pfaff, A.P. Arnold, A.M. Eigen, S.E. Fahrbach, R.T. Rubin (Eds.), Hormones, Brain and Behavior, vol. 3, Academic Press, Amsterdam, 2002, pp. 93–114. [61] Z. Sheng, J. Xu, H. Bai, F. Zhu, S.R. Palli, Juvenile hormone regulates vitellogenin gene expression through insulin-like peptide signaling pathway in the red flour beetle, Tribolium castaneum, J. Biol. Chem. 286 (2011) 41924–41936. [62] M. Soller, M. Bownes, E. Kubli, Control of oocyte maturation in sexually mature Drosophila females, Dev. Biol. 208 (1999) 337–351. [63] Y. Suzuki, J.W. Truman, L.M. Riddiford, The role of the transcription factor Broad in the development and evolution of the holometabolous insect pupa, Tribolium castaneum, Development 135 (2008) 569–577. [64] C.S. Thummel, Flies on steroids-Drosophila metamorphosis and the mechanism of steroid hormone action, Trends Genetics 12 (1996) 306–310. [65] C.-Y. Ting, C.-H. Lee, Visual circuit development in Drosophila, Curr. Opin. Neurobiol. 17 (2007) 65–72. [66] J.W. Truman, W.S. Talbot, S.E. Fahrbach, D.S. Hogness, Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development, Development 120 (1994) 219– 234. [67] J.W. Truman, K. Hiruma, J.P. Allee, S.G.B. MacWhinnie, D.T. Champlin, L.M. Riddiford, Juvenile hormone is required to couple imaginal disc formation with nutrition in insects, Science 312 (2006) 1385–1388. [68] C. Wicker-Thomas, T. Chertemps, Molecular biology and genetics of hydrocarbon production, in: G.J. Blomquist, A.-G. Bagneres (Eds.), Insect Hydrocarbons: Biology, Biochemistry and Chemical Ecology, Cambridge University Press, Cambridge, 2010, pp. 53–74. [69] V.B. Wigglesworth, The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling moulting and ‘metamorphosis’, Quart. J. Micr. Sci. 77 (1934) 191–222. [70] V.B. Wigglesworth, The function of the corpus allatum in the growth and reproduction of Rhodnius prolixus (Hemiptera), Quart. J. Micr. Sci. 79 (1936) 91–121. [71] C.M. Williams, The juvenile hormone. II. Its role in the endocrine control of molting, pupation, and adult development in the Cecropia silkworm, Biol. Bull. 121 (1961) 572–585.
484
L.M. Riddiford / General and Comparative Endocrinology 179 (2012) 477–484
[72] T.G. Wilson, M. Ashok, Insecticide resistance resulting from an absence of target-site gene product, Proc. Natl. Acad. Sci. USA 95 (1998) 14040– 14044. [73] T.G. Wilson, J. Fabian, A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone, Dev. Biol. 118 (1986) 190–201. [74] G.R. Wyatt, K.G. Davey, Cellular and molecular actions of juvenile hormone. II. Roles of juvenile hormone in adult insects, Adv. Insect Physiol. 26 (1996) 1– 155. [75] Z. Zhang, J. Xu, Z. Sheng, Y. Sui, S.R. Palli, Steroid receptor co-activator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, Methoprene tolerant, J. Biol. Chem. 286 (2011) 8437– 8447.
[76] B. Zhou, K. Hiruma, T. Shinoda, L.M. Riddiford, Juvenile hormone prevents ecdysteroid-induced expression of Broad Complex RNAs in the epidermis of the tobacco hornworm, Manduca sexta, Dev. Biol. 203 (1998) 233–244. [77] B. Zhou, L.M. Riddiford, Hormonal regulation and patterning of the Broad Complex in the epidermis and wing discs of the tobacco hornworm, Manduca sexta, Dev. Biol. 231 (2001) 125–137. [78] X. Zhou, L.M. Riddiford, Broad-Complex specifies pupal development and mediates the prevention of the pupal-adult transformation by juvenile hormone in Drosophila and Manduca, Development 129 (2002) 2259–2269. [79] J. Zhu, L. Chen, G. Sun, A.S. Raikhel, The competence factor bFtz-F1 potentiates ecdysone receptor activity via recruiting a p160/SRC coactivator, Mol. Cell. Biol. 26 (2006) 9402–9412.