Anciently duplicated <Emphasis Type="Italic">Broad Complex</Emphasis> exons have distinct temporal functions during tissue morphogenesis

Broad Complex (BRC) is an essential ecdysone-pathway gene required for entry into and progression through metamorphosis in Drosophila melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. These defects are phenocopied by the juvenile hormone mimic methoprene. Four BRC isoforms are produced by alternative splicing of a protein-binding BTB/POZ-encoding exon (BTB _BRC ) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1 _BRC , Z2 _BRC , Z3 _BRC , Z4 _BRC ). Highly conserved orthologs of BTB _BRC and all four Z _BRC were found among published cDNA sequences or genome databases from Diptera, Lepidoptera, Hymenoptera, and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometabolous orders. Tramtrack subfamily members, abrupt, tramtrack, fruitless, longitudinals lacking (lola), and CG31666 were characterized throughout Holometabola and used to root phylogenetic analyses of Z _BRC exons, which revealed that the Z _BRC clade includes Z _abrupt . All four Z _BRC domains, including Z4 _BRC , which has no known essential function, are evolving in a manner consistent with selective constraint. We used transgenic rescue to explore how different BRC isoforms contribute to shared tissue-morphogenesis functions. As predicted from earlier studies, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two different developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. The sequential action of BRC isoforms indicates subfunctionalization of duplicated Z _BRC exons even when they contribute to common developmental processes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene

Parasitism of the tobacco hornworm, Manducasexta, by the braconid wasp Cotesiacongregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid’s emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 μg. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

Ecdysone differentially regulates metamorphic timing relative to 20-hydroxyecdysone by antagonizing juvenile hormone in Drosophila melanogaster

In insects, a steroid hormone, 20-hydroxyecdysone (20E), plays important roles in the regulation of developmental transitions by initiating signaling cascades via the ecdysone receptor (EcR). Although 20E has been well characterized as the molting hormone, its precursor ecdysone (E) has been considered to be a relatively inactive compound because it has little or no effect on classic EcR mediated responses. I found that feeding E to wild-type third instar larvae of Drosophila melanogaster accelerates the metamorphic timing, which results in elevation of lethality during metamorphosis and reduced body size, while 20E has only a minor effect. The addition of a juvenile hormone analog (JHA) to E impeded their precocious pupariation and thereby rescued the reduced body size. The ability of JHA impeding the effect of E was not observed in the Methoprene-tolerant (Met) and germ-cell expressed (gce) double mutant animals lacking JH signaling, indicating that antagonistic action of JH against E is transduced via a primary JH receptor, Met, or a product of its homolog, Gce. I also found that L3 larvae are susceptible to E around the time when they reach their minimum viable weight. These results indicate that E, and not just 20E, is also essential for proper regulation of developmental timing and body size. Furthermore, the precocious pupariation triggered by E is impeded by the action of JH to ensure that animals attain body size to survive metamorphosis.     

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.     

Methoprene does not affect juvenile hormone titers in honey bee (Apis mellifera) workers

Methoprene, a juvenile hormone (JH) analog, is a widely used insecticide that also accelerates behavioral development in honey bees (Apis mellifera). JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers of methoprene‐treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene‐treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal‐aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers’ JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.     

3S-(+)-3,7-Dimethyl-1,5-Octadiene-3,7-Diol and Ionone Derivatives from Tea

1-Isobutyl-5-(4-phenoxyphenyl)imidazole (KK-98), an inhibitor of juvenile hormone (JH) biosynthesis in the cockroach, and related imidazole compounds were evaluated against silkworm, Bombyx mori, for their activity to induce precocious metamorphosis. KK-98 induced precocious metamorphosis in the 4th instar larvae at high doses. Replacement of the 4-phenoxy group by a 3-phenoxy or 3-benzyloxy group on the benzene ring increased the activity. Among this series of compounds, 5-(3-benzyloxyphenyl)-1-isopropylimidazole (8) showed the highest activity. The induction of precocious metamorphosis by compound 8 was rescued by the simultaneous application of methoprene, a JH minie. When newly molted 3rd instar larvae were treated with a high dose of compound 8, a few larvae formed larval-pupal intermediates in the 3rd instar stage, which has not been formed by treating of any other imidazoles so far.     

Soldier determination in Pheidole bicarinata: Effect of methoprene on caste and size within castes

Topical application of methoprene to final-instar larvae of the ant Pheidole bicarinata can induce soldier development. Soldier induction takes place if methoprene levels are above a soldier-determining threshold during a critical period of juvenile hormone-sensitivity that occurs during about days 4–6 of the final instar. Furthermore, the amount of exogenous methoprene applied affects the timing of metamorphosis and the adult size in both the minor worker and soldier castes. When larvae that receive methoprene treatment become minor workers these are always larger than acetone-treated controls. In larvae that become soldiers, growth and timing of metamorphosis vary with the dose of methoprene, but in a more complex way. A high dose of methoprene produces a metamorphic delay and large soldiers. However, the lowest effective dose for soldier induction produces early metamorphosis and small adults. On the basis of these results, we have expanded our model of a mechanism by which juvenile hormone could control determination of worker castes in Pheidole bicarinata.     

Comparing thyroid and insect hormone signaling

Transitions between different states of development, physiology,and life history are typically mediated by hormones. In insects,metamorphosis and reproductive maturation are regulated by aninteraction between the sesquiterpenoid juvenile hormone (JH)and the steroid 20-hydroxy-ecdysone (20E). In vertebrates andsome marine invertebrates, the lipophilic thyroid hormones (THs)affect metamorphosis and other life history transitions. Interestingly,when applied to insects, THs can physiologically mimic manyfacets of JH action, suggesting that the molecular actions ofTHs and JH/20E might be similar. Here we discuss functionalparallels between TH and JH/20E signaling in insects, with aparticular focus on the fruit fly, Drosophila melanogaster,a genetically and physiologically tractable model system. Comparingthe effects of THs with the well defined physiological rolesof insect hormones such as JH and 20E in Drosophila might provideimportant insights into hormone function and the evolution ofendocrine signaling.     

CHITIN SYNTHASE B: A MIDGUT‐SPECIFIC GENE INDUCED BY INSECT HORMONES AND INVOLVED IN FOOD INTAKE IN Bombyx mori LARVAE

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third‐instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20‐hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry‐leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin‐synthesis‐dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.     

Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti

Female Aedes aegypti that were fed blood and immediately abdominally ligated did not deposit yolk. Injection of 20-hydroxyecdysone (1.5–5.0 ng) or topical application of juvenile hormone (JH) analogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood-gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated in 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar to that in intact controls. When ligated abdomens were first treated with methoprene and immediately injected with a crude head extract of egg development neurosecretory hormone, vitellogenin synthesis was induced at a rate similar to that in blood-fed controls. Methoprene at this concentration (25 pg), did not cause an increase in whole-body ecdysteroid titers. Larger amounts of methoprene (1.65 ng) were needed to stimulate egg development and ecdysteroid production. Implantation of ecdysone-secreting ovaries into ligated abdomens did not stimulate vitellogenesis in the recipients. However, in recipients that were first treated with methoprene (25 pg), implantation of ecdysone-secreting ovaries resulted in normal egg development. These experiments indicate that the appearance of JH precedes 20-hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti.     

Spiny lobster development: mechanisms inducing metamorphosis to the puerulus: a review

This review outlines current knowledge of mechanisms effecting metamorphosis in decapod crustaceans and insects. The comparative approach demonstrates some of the complexities that need resolving to find an answer to the question raised frequently by ecologists: “What triggers metamorphosis in spiny lobsters?” It is evident that crustacean moulting and metamorphosis are genetically controlled through endocrine systems that mediate gene expression. The molecular mechanisms underlying these developmental processes have been studied intensively in insects, particularly in the fruitfly, Drosophila melanogaster (Diptera), and some lepidopteran species. Comparatively, there is minimal information available for a few decapod crustacean species, but none for spiny lobsters (Palinuridae). Nothing was known of hormone signalling transduction pathways, via nuclear receptors (NRs) and gene activation during larval moults in palinurids—until a recent, ground-breaking study of early phyllosomal development of Panulirus ornatus by Wilson et al. (Rock Lobster Enhancement and Aquaculture Subprogram. FRDC Project 2000/263, Australian Govt, Fisheries Research and Development Corporation and Australian Institute of Marine Science, Nov 2005). Their study not only identified homologues of five hormone NRs of D. melanogaster, but also patterns of gene regulation showing strong similarities to those of gene expression found in insect larval development. Their results indicated that control of moulting and metamorphosis in palinurids closely parallels that in insects, suggesting that insects can serve as model systems for elucidating molecular mechanisms in larval decapods. In insects and crustaceans, the steroid hormone, ecdysone, (20E) initiates moulting. In insects, juvenile hormone (JH) mediates the type of larval moult that occurs, either anamorphic or metamorphic. The latter results when the level of JH in the haemolymph drops in the final larval instar. High levels of JH inhibit the metamorphic moult during insect larval development. The interaction of 20E and JH is not fully understood, and the operative molecular mechanisms are still being elucidated. No nuclear receptor for JH has been identified, and alternative JH signalling pathways await identification. In decapod crustaceans, methyl farnesoate (MF), a precursor of JH, replaces the latter in other functions mediated by JH in insects; but there is little evidence indicating that MF plays a similar ‘antimetamorphic’ role in decapod larval moults.     

Juvenile hormone analog and injection effects on locust hemolymph protein synthesis

In adult female Locusta migratoria, at about day 8 after eclosion, when vitellogenin (Vg) is first produced as a result of induction by juvenile hormone (JH), the intensity of hemolymph protein electrophoretic bands at about 75 kDa and 20 kDa increases sharply, suggesting that JH may induce additional proteins. A major component of the elevated protein is persistent storage protein (PSP; subunit 74 kDa). Administration of the JH analog, methoprene, to precocene-treated adult locusts was followed by a rise in hemolymph levels of PSP but not in apolipophorin III (19 kDa), identified immunochemically and electrophoretically. The synthesis of PSP in adult fat body was confirmed by incorporation of [³H]leucine. At 48 h after treatment with methoprene, Vg synthesis was induced in females (as previously observed) and synthesis of PSP in both sexes was elevated above controls, while synthesis of apolipophorin III was not stimulated. We conclude that in adult locust fat body the synthesis of several proteins responds in different ways to the JH analog: Vg (and a 21 kDa protein described elsewhere) is induced de novo solely in females; PSP (and a 19 kDa protein described elsewhere) is stimulated in both sexes but is not fully JH-dependent; apolipophorin III is not stimulated. In these experiments, methoprene was administered both by injection in mineral oil and topically in acetone. After injection of mineral oil as a vector control, incorporation into secreted proteins was stimulated at 24 h, presumably due to a wound effect; topical application of acetone avoids this effect and is a preferred route for administration of JH analog. © 1992 Wiley-Liss, Inc.