Chinmo is the larval member of the molecular trinity that directs Drosophila metamorphosis.

The molecular control of insect metamorphosis from larva to pupa to adult has long been a mystery. The Broad and E93 transcription factors, which can modify chromatin domains, are known to direct the production of the pupa and the adult, respectively. We now show that chinmo, a gene related to broad, is essential for the repression of these metamorphic genes. Chinmo is strongly expressed during the formation and growth of the larva and its removal results in the precocious expression of broad and E93 in the first stage larva, causing a shift from larval to premetamorphic functions. This trinity of Chinmo, Broad, and E93 regulatory factors is mutually inhibitory. The interaction of this network with regulatory hormones likely ensures the orderly progression through insect metamorphosis.

A Life's Journey Through Insect Metamorphosis.

This autobiographical article describes the research career of Lynn M. Riddiford from its early beginnings in a summer program for high school students at Jackson Laboratory to the present "retirement" at the Friday Harbor Laboratories. The emphasis is on her forays into many areas of insect endocrinology, supported by her graduate students and postdoctoral associates. The main theme is the hormonal regulation of metamorphosis, especially the roles of juvenile hormone (JH). The article describes the work of her laboratory first in the elucidation of the endocrinology of the tobacco hornworm, Manduca sexta, and later in the molecular aspects of the regulation of cuticular and pigment proteins and of the ecdysone-induced transcription factor cascade during molting and metamorphosis. Later studies utilized Drosophila melanogaster to answer further questions about the actions of JH.

Juvenile hormone regulates body size and perturbs insulin signaling in Drosophila.

The role of juvenile hormone (JH) in regulating the timing and nature of insect molts is well-established. Increasing evidence suggests that JH is also involved in regulating final insect size. Here we elucidate the developmental mechanism through which JH regulates body size in developing Drosophila larvae by genetically ablating the JH-producing organ, the corpora allata (CA). We found that larvae that lack CA pupariated at smaller sizes than control larvae due to a reduced larval growth rate. Neither the timing of the metamorphic molt nor the duration of larval growth was affected by the loss of JH. Further, we show that the effects of JH on growth rate are dependent on the forkhead box O transcription factor (FOXO), which is negatively regulated by the insulin-signaling pathway. Larvae that lacked the CA had elevated levels of FOXO activity, whereas a loss-of-function mutation of FOXO rescued the effects of CA ablation on final body size. Finally, the effect of JH on growth appears to be mediated, at least in part, via ecdysone synthesis in the prothoracic gland. These results indicate a role of JH in regulating growth rate via the ecdysone- and insulin-signaling pathways.

Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster.

Juvenile hormone (JH) coordinates timing of female reproductive maturation in most insects. In Drosophila melanogaster, JH plays roles in both mating and egg maturation. However, very little is known about the molecular pathways associated with mating. Our behavioral analysis of females genetically lacking the corpora allata, the glands that produce JH, showed that they were courted less by males and mated later than control females. Application of the JH mimic, methoprene, to the allatectomized females just after eclosion rescued both the male courtship and the mating delay. Our studies of the null mutants of the JH receptors, Methoprene tolerant (Met) and germ cell-expressed (gce), showed that lack of Met in Met(27) females delayed the onset of mating, whereas lack of Gce had little effect. The Met(27) females were shown to be more attractive but less behaviorally receptive to copulation attempts. The behavioral but not the attractiveness phenotype was rescued by the Met genomic transgene. Analysis of the female cuticular hydrocarbon profiles showed that corpora allata ablation caused a delay in production of the major female-specific sex pheromones (the 7,11-C27 and -C29 dienes) and a change in the cuticular hydrocarbon blend. In the Met(27) null mutant, by 48 h, the major C27 diene was greatly increased relative to wild type. In contrast, the gce(2.5k) null mutant females were courted similarly to control females despite changes in certain cuticular hydrocarbons. Our findings indicate that JH acts primarily via Met to modulate the timing of onset of female sex pheromone production and mating.

A molt timer is involved in the metamorphic molt in Manduca sexta larvae.

Manduca sexta larvae are a model for growth control in insects, particularly for the demonstration of critical weight, a threshold weight that the larva must surpass before it can enter metamorphosis on a normal schedule, and the inhibitory action of juvenile hormone on this checkpoint. We examined the effects of nutrition on allatectomized (CAX) larvae that lack juvenile hormone to impose the critical weight checkpoint. Normal larvae respond to prolonged starvation at the start of the last larval stage, by extending their subsequent feeding period to ensure that they begin metamorphosis above critical weight. CAX larvae, by contrast, show no homeostatic adjustment to starvation but start metamorphosis 4 d after feeding onset, regardless of larval size or the state of development of their imaginal discs. By feeding starved CAX larvae for various durations, we found that feeding for only 12-24 h was sufficient to result in metamorphosis on day 4, regardless of further feeding or body size. Manipulation of diet composition showed that protein was the critical macronutrient to initiate this timing. This constant period between the start of feeding and the onset of metamorphosis suggests that larvae possess a molt timer that establishes a minimal time to metamorphosis. Ligation experiments indicate that a portion of the timing may occur in the prothoracic glands. This positive system that promotes molting and the negative control via the critical weight checkpoint provide antagonistic pathways that evolution can modify to adapt growth to the ecological needs of different insects.

Nancy E. Beckage (1950-2012): pioneer in insect host-parasite interactions.

Nancy E. Beckage is widely recognized for her pioneering work in the field of insect host-parasitoid interactions beginning with endocrine influences of the tobacco hornworm, Manduca sexta, host and its parasitoid wasp Apanteles congregatus (now Cotesia congregata) on each other's development. Moreover, her studies show that the polydnavirus carried by the parasitoid wasp not only protects the parasitoid from the host's immune defenses, but also is responsible for some of the developmental effects of parasitism. Nancy was a highly regarded mentor of both undergraduate and graduate students and more widely of women students and colleagues in entomology. Her service both to her particular area and to entomology in general through participation on federal grant review panels and in the governance of the Entomological Society of America, organization of symposia at both national and international meetings, and editorship of several different journal issues and of several books is legendary. She has left behind a lasting legacy of increased understanding of multilevel endocrine and physiological interactions among insects and other organisms and a strong network of interacting scientists and colleagues in her area of entomology.

The role of juvenile hormone in dominance behavior, reproduction and cuticular pheromone signaling in the caste-flexible epiponine wasp, Synoeca surinama.

BACKGROUND: The popular view on insect sociality is that of a harmonious division of labor among two morphologically distinct and functionally non-overlapping castes. But this is a highly derived state and not a prerequisite for a functional society. Rather, caste-flexibility is a central feature in many eusocial wasps, where adult females have the potential to become queens or workers, depending on the social environment. In non-swarming paper wasps (e.g., Polistes), prospective queens fight one another to assert their dominance, with losers becoming workers if they remain on the nest. This aggression is fueled by juvenile hormone (JH) and ecdysteroids, major factors involved in caste differentiation in most eusocial insects. We tested whether these hormones have conserved aggression-promoting functions in Synoeca surinama, a caste-flexible swarm-founding wasp (Epiponini) where reproductive competition is high and aggressive displays are common. RESULTS: We observed the behavioral interactions of S. surinama females in field nests before and after we had removed the egg-laying queen(s). We measured the ovarian reproductive status, hemolymph JH and ecdysteroid titers, ovarian ecdysteroid content, and analyzed the cuticular hydrocarbon (CHC) composition of females engaged in competitive interactions in both queenright and queenless contexts. These data, in combination with hormone manipulation experiments, revealed that neither JH nor ecdysteroids are necessary for the expression of dominance behaviors in S. surinama. Instead, we show that JH likely functions as a gonadotropin and directly modifies the cuticular hydrocarbon blend of young workers to match that of a reproductive. Hemolymph ecdysteroids, in contrast, are not different between queens and workers despite great differences in ovarian ecdysteroid content. CONCLUSIONS: The endocrine profile of S. surinama shows surprising differences from those of other caste-flexible wasps, although a rise in JH titers in replacement queens is a common theme. Extensive remodeling of hormone functions is also evident in the highly eusocial bees, which has been attributed to the evolution of morphologically defined castes. Our results show that hormones which regulate caste-plasticity can lose these roles even while caste-plasticity is preserved.

Reproductive status, endocrine physiology and chemical signaling in the Neotropical, swarm-founding eusocial wasp Polybia micans.

In the evolution of caste-based societies in Hymenoptera, the classical insect hormones juvenile hormone (JH) and ecdysteroids were co-opted into new functions. Social wasps, which show all levels of sociality and lifestyles, are an ideal group in which to study such functional changes. Virtually all studies on the physiological mechanisms underlying reproductive division of labor and caste functions in wasps have been done on independent-founding paper wasps, and the majority of these studies have focused on species specially adapted for overwintering. The relatively little-studied tropical swarm-founding wasps of the Epiponini (Vespidae) are a diverse group of permanently social wasps, with some species maintaining caste flexibility well into the adult phase. We investigated the behavior, reproductive status, JH and ecdysteroid titers in hemolymph, ecdysteroid content of the ovary and cuticular hydrocarbon (CHC) profiles in the caste-monomorphic, epiponine wasp Polybia micans Ducke. We found that the JH titer was not elevated in competing queens from established multiple-queen nests, but increased in lone queens that lack direct competition. In queenless colonies, JH titer rose transiently in young potential reproductives upon challenge by nestmates, suggesting that JH may prime the ovaries for further development. Ovarian ecdysteroids were very low in workers but higher and correlated with the number of vitellogenic oocytes in the queens. Hemolymph ecdysteroid levels were low and variable in both workers and queens. Profiles of P. micans CHCs reflected caste, age and reproductive status, but were not tightly linked to either hormone. These findings show a significant divergence in hormone function in swarm-founding wasps compared with independently founding ones.

The juvenile hormone signaling pathway in insect development.

The molecular action of juvenile hormone (JH), a regulator of vital importance to insects, was until recently regarded as a mystery. The past few years have seen an explosion of studies of JH signaling, sparked by a finding that a JH-resistance gene, Methoprene-tolerant (Met), plays a critical role in insect metamorphosis. Here, we summarize the recently acquired knowledge on the capacity of Met to bind JH, which has been mapped to a particular ligand-binding domain, thus establishing this bHLH-PAS protein as a novel type of an intracellular hormone receptor. Next, we consider the significance of JH-dependent interactions of Met with other transcription factors and signaling pathways. We examine the regulation and biological roles of genes acting downstream of JH and Met in insect metamorphosis. Finally, we discuss the current gaps in our understanding of JH action and outline directions for future research.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

A role for juvenile hormone in the prepupal development of Drosophila melanogaster.

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met(27) larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis.

Drosophila postembryonic nervous system development: a model for the endocrine control of development.

During postembryonic life, hormones, including ecdysteroids, juvenile hormones, insulin-like peptides, and activin/TGFß ligands act to transform the larval nervous system into an adult version, which is a fine-grained mosaic of recycled larval neurons and adult-specific neurons. Hormones provide both instructional signals that make cells competent to undergo developmental change and timing cues to evoke these changes across the nervous system. While touching on all the above hormones, our emphasis is on the ecdysteroids, ecdysone and 20-hydroxyecdysone (20E). These are the prime movers of insect molting and metamorphosis and are involved in all phases of nervous system development, including neurogenesis, pruning, arbor outgrowth, and cell death. Ecdysteroids appear as a series of steroid peaks that coordinate the larval molts and the different phases of metamorphosis. Each peak directs a stereotyped cascade of transcription factor expression. The cascade components then direct temporal programs of effector gene expression, but the latter vary markedly according to tissue and life stage. The neurons read the ecdysteroid titer through various isoforms of the ecdysone receptor, a nuclear hormone receptor. For example, at metamorphosis the pruning of larval neurons is mediated through the B isoforms, which have strong activation functions, whereas subsequent outgrowth is mediated through the A isoform through which ecdysteroids play a permissive role to allow local tissue interactions to direct outgrowth. The major circulating ecdysteroid can also change through development. During adult development ecdysone promotes early adult patterning and differentiation while its metabolite, 20E, later evokes terminal adult differentiation.

How does juvenile hormone control insect metamorphosis and reproduction?

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.

Larval leg integrity is maintained by Distal-less and is required for proper timing of metamorphosis in the flour beetle, Tribolium castaneum.

The dramatic transformation from a larva to an adult must be accompanied by a coordinated activity of genes and hormones that enable an orchestrated transformation from larval to pupal/adult tissues. The maintenance of larval appendages and their subsequent transformation to appendages in holometabolous insects remains elusive at the developmental genetic level. Here the role of a key appendage patterning gene Distal-less (Dll) was examined in mid- to late-larval stages of the flour beetle, Tribolium castaneum. During late larval development, Dll was expressed in appendages in a similar manner as previously reported for the tobacco hornworm, Manduca sexta. Removal of this late Dll expression resulted in disruption of adult appendage patterning. Intriguingly, earlier removal resulted in dramatic loss of structural integrity and identity of larval appendages. A large amount of variability in appendage morphology was observed following Dll dsRNA injection, unlike larvae injected with dachshund dsRNA. These Dll dsRNA-injected larvae underwent numerous supernumerary molts, which could be terminated with injection of either JH methyltransferase or Methoprene-tolerant dsRNA. Apparently, the partial dedifferentiation of the appendages in these larvae acts to maintain high JH and, hence, prevents metamorphosis.

The nuclear receptor E75A has a novel pair-rule-like function in patterning the milkweed bug, Oncopeltus fasciatus.

Genetic studies of the fruit fly Drosophila have revealed a hierarchy of segmentation genes (maternal, gap, pair-rule and HOX) that subdivide the syncytial blastoderm into sequentially finer-scale coordinates. Within this hierarchy, the pair-rule genes translate gradients of information into periodic stripes of expression. How pair-rule genes function during the progressive mode of segmentation seen in short and intermediate-germ insects is an ongoing question. Here we report that the nuclear receptor Of'E75A is expressed with double segment periodicity in the head and thorax. In the abdomen, Of'E75A is expressed in a unique pattern during posterior elongation, and briefly resembles a sequence that is typical of pair-rule genes. Depletion of Of'E75A mRNA caused loss of a subset of odd-numbered parasegments, as well as parasegment 6. Because these parasegments straddle segment boundaries, we observe fusions between adjacent segments. Finally, expression of Of'E75A in the blastoderm requires even-skipped, which is a gap gene in Oncopeltus. These data show that the function of Of'E75A during embryogenesis shares many properties with canonical pair-rule genes in other insects. They further suggest that parasegment specification may occur through irregular and episodic pair-rule-like activity.

Rhodnius, Golden Oil, and Met: A History of Juvenile Hormone Research.

Juvenile hormone (JH) is a unique sesquiterpenoid hormone which regulates both insect metamorphosis and insect reproduction. It also may be utilized by some insects to mediate polyphenisms and other life history events that are environmentally regulated. This article details the history of the research on this versatile hormone that began with studies by V. B. Wigglesworth on the "kissing bug" Rhodnius prolixus in 1934, through the discovery of a natural source of JH in the abdomen of male Hyalophora cecropia moths by C. M. Williams that allowed its isolation ("golden oil") and identification, to the recent research on its receptor, termed Methoprene-tolerant (Met). Our present knowledge of cellular actions of JH in metamorphosis springs primarily from studies on Rhodnius and the tobacco hornworm Manduca sexta, with recent studies on the flour beetle Tribolium castaneum, the silkworm Bombyx mori, and the fruit fly Drosophila melanogaster contributing to the molecular understanding of these actions. Many questions still need to be resolved including the molecular basis of competence to metamorphose, differential tissue responses to JH, and the interaction of nutrition and other environmental signals regulating JH synthesis and degradation.

Insulin/IGF signaling regulates the change in commitment in imaginal discs and primordia by overriding the effect of juvenile hormone.

At the beginning of the final larval (fifth) instar of Manduca sexta, imaginal precursors including wing discs and eye primordia initiate metamorphic changes, such as pupal commitment, patterning and cell proliferation. Juvenile hormone (JH) prevents these changes in earlier instars and in starved final instar larvae, but nutrient intake overcomes this effect of JH in the latter. In this study, we show that a molecular marker of pupal commitment, broad, is up-regulated in the wing discs by feeding on sucrose or by bovine insulin or Manduca bombyxin in starved final instar larvae. This effect of insulin could not be prevented by JH. In vitro insulin had no effect on broad expression but relieved the suppression of broad expression by JH. This effect of insulin was directly on the disc as shown by its reduction in the presence of insulin receptor dsRNA. In starved penultimate fourth instar larvae, broad expression in the wing disc was not up-regulated by insulin. The discs became responsive to this action of insulin during the molt to the fifth instar together with the ability to become pupally committed in response to 20-hydroxyecdysone. Thus, the Manduca bombyxin acts as a metamorphosis-initiating factor in the imaginal precursors.