Fat body lipolysis connects poor nutrition to hypopharyngeal gland degradation in Apis mellifera.

The hypopharyngeal glands (HGs) of honey bee nurse workers secrete the major protein fraction of jelly, a protein and lipid rich substance fed to developing larvae, other worker bees, and queens. A hallmark of poorly nourished nurses is their small HGs, which actively degrade due to hormone-induced autophagy. To better connect nutritional stress with HG degradation, we looked to honey bees and other insect systems, where nutrient stress is often accompanied by fat body degradation. The fat body contains stored lipids that are likely a substrate for ecdysteroid synthesis, so we tested whether starvation caused increased fat body lipolysis. Ecdysteroid signaling and response pathways and IIS/TOR are tied to nutrient-dependent autophagy in honey bees and other insects, and so we also tested whether and where genes in these pathways were differentially regulated in the head and fat body. Last, we injected nurse-aged bees with the honey bee ecdysteroid makisterone A to determine whether this hormone influenced HG size and autophagy. We find that starved nurse aged bees exhibited increased fat body lipolysis and increased expression of ecdysteroid production and response genes in the head. Genes in the IIS/TOR pathway were not impacted by starvation in either the head or fat body. Additionally, bees injected with makisterone A had smaller HGs and increased expression of autophagy genes. These data support the hypothesis that nutritional stress induces fat body lipolysis, which may liberate the sterols important for ecdysteroid production, and that increased ecdysteroid levels induce autophagic HG degradation.

De novo characterization of Phenacoccus solenopsis transcriptome and analysis of gene expression profiling during development and hormone biosynthesis.

The cotton mealybug Phenacoccus solenopsis is a devastating pest of cotton causing tremendous loss in the yield of crops each year. Widespread physiological and biological studies on P. solenopsis have been carried out, but the lack of genetic information has constrained our understanding of the molecular mechanisms behind its growth and development. To understand and characterize the different developmental stages, RNA-Seq platform was used to execute de-novo transcriptome assembly and differential gene expression profiling for the eggs, first, second, third instar and adult female stages. About 182.67 million reads were assembled into 93,781 unigenes with an average length of 871.4 bp and an N50 length of 1899 bp. These unigenes sequences were annotated and classified by performing NCBI non-redundant (Nr) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG), Gene ontology (GO), the Swiss-Prot protein database (Swiss-Prot), and nearest related organism Acyrthosiphon pisum (pea aphid) database. To get more information regarding the process of metamorphosis, we performed a pairwise comparison of four developmental stages and obtained 29,415 differentially expressed genes. Some of the differentially expressed genes were associated with functional protein synthesis, anti-microbial protection, development and hormone biosynthesis. Functional pathway enrichment analysis of differentially expressed genes showed the positive correlation with specific physiological activities of each stage, and these results were confirmed by qRT-PCR experiments. This study gives a valuable genomics resource of P. solenopsis covering all its developmental stages and will promote future studies on biological processes at the molecular level.

The adipokinetic hormone of the coleopteran suborder Adephaga: Structure, function, and comparison of distribution in other insects.

The aim of the current study is to identify the adipokinetic hormone(s) (AKHs) of a basal suborder of the species-rich Coleoptera, the Adephaga, and possibly learn more about the ancestral AKH of beetles. Moreover, we wanted to compare the ancestral AKH with AKHs of more advanced beetles, of which a number are pest insects. This would allow us to assess whether AKH mimetics would be suitable as insecticides, that is, be harmful to the pest species but not to the beneficial species. Nine species of the Adephaga were investigated and all synthesize only one octapeptide in the corpus cardiacum, as revealed by Edman degradation sequencing techniques or by mass spectrometry. The amino acid sequence pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp corresponds to Schgr-AKH-II that was first identified in the desert locust. It is assumed that Schgr-AKH-II-the peptide of a basal beetle clade-is the ancestral AKH for beetles. Some other beetle families, as well as some Hymenoptera (including honey bees) also contain this peptide, whereas most of the pest beetle species have different AKHs. This argues that those peptides and their receptors should be explored for developing mimetics with insecticidal properties. A scenario where Schgr-AKH-II (the only AKH of Adephaga) is used as basic molecular structure to derive almost all other known beetle AKHs via single step mutations is very likely, and supports the interpretation that Schgr-AKH-II is the ancestral AKH of Coleoptera.

Ouija board: A transcription factor evolved for only one target in steroid hormone biosynthesis in the fruit fly Drosophila melanogaster.

Transcription factors generally regulate gene expression of multiple targets. In contrast, our recent finding suggests that the zinc finger protein Ouija board controls steroid hormone biosynthesis through specific regulation of only one gene spookier in Drosophila. It sheds light on a specialized but essential factor that evolved for one target.

Energy Homeostasis Control in Drosophila Adipokinetic Hormone Mutants.

Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based on AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone [termed "adipokinetic hormone precursor-related peptide" (APRP)] is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. Here we employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, we provide evidence for a negative autoregulatory loop in Akh gene regulation.

The Obesity-Linked Gene Nudt3 Drosophila Homolog Aps Is Associated With Insulin Signaling.

Several genome-wide association studies have linked the Nudix hydrolase family member nucleoside diphosphate-linked moiety X motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown, and NUDT3 expression, regulation, and signaling in the central nervous system has not been studied. We performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system, to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward- and feeding-related regions of the hypothalamus and amygdala, whereas Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brainstem of food-deprived mice. Knocking down Aps in the Drosophila central nervous system, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype while inducing hyperphagia. Finally, the loss of IPC Aps lowered the expression of Akh, Ilp6, and Ilp3, genes known to be inhibited by insulin signaling. These results point toward a role for this gene in the regulation of insulin signaling, which could explain the robust association with obesity in humans.

Initiation of metamorphosis and control of ecdysteroid biosynthesis in insects: The interplay of absence of Juvenile hormone, PTTH, and Ca(2+)-homeostasis.

The paradigm saying that release of the brain neuropeptide big prothoracicotropic hormone (PTTH) initiates metamorphosis by activating the Torso-receptor/ERK pathway in larval prothoracic glands (PGs) is widely accepted nowadays. Upon ligand-receptor interaction Ca(2+) enters the PG cells and acts as a secondary messenger. Ecdysteroidogenesis results, later followed by apoptosis. Yet, some data do not fit in this model. In some species decapitated animals can still molt, even repeatedly, and metamorphose. PTTH does not universally occur in all insect species. PGs may also have other functions; PGs as counterpart of the vertebrate thymus? There are also small PTTHs. Finally, PTTH remains abundantly present in adults and plays a role in control of ecdysteroidogenesis (=sex steroid production) in gonads. This is currently documented only in males. This urges a rethinking of the PTTH-PG paradigm. The key question is: Why does PTTH-induced Ca(2+) entry only result in ecdysteroidogenesis and apoptosis in specific cells/tissues, namely the PGs and gonads? Indeed, numerous other neuropeptides also use Ca(2+) as secondary messenger. The recent rediscovery that in both invertebrates and vertebrates at least some isoforms of Ca(2+)-ATPase need the presence of an endogenous farnesol/juvenile hormone(JH)-like sesquiterpenoid for keeping cytosolic [Ca(2+)]i below the limit of apoptosis-induction, triggered the idea that it is not primarily PTTH, but rather the drop to zero of the JH titer that acts as the primordial initiator of metamorphosis by increasing [Ca(2+)]i. PTTH likely potentiates this effect but only in cells expressing Torso. PTTH: an evolutionarily ancient gonadotropin?

Expression of stress-related genes in the parthenogenetic forms of the pea aphid, Acyrthosiphon pisum.

Aphids are an economically important group of insects that have an intricate life cycle with seasonal polyphenism. This study aimed to explore the physiological background of aphid migration from unfavorable nutritional conditions to a new, intact host plant. Specifically, the relative expression of stress/metabolism-related genes and changes in metabolic reserves were determined for the winged and wingless forms of female pea aphids, Acyrthosiphon pisum, under two different nutritional conditions. The expression level was determined for the following sets of genes: the adipokinetic hormone (AKH) and its receptor, enzymes involved in carbohydrate and lipid metabolism, detoxifying enzymes, and genes encoding exoskeleton/cuticular proteins and cytoskeleton proteins. In both forms, the transcription of the adipokinetic hormone was upregulated during nutritional stress, whereas its receptor mRNA levels remained unchanged. Similarly, the expression of genes engaged in glycogen and triglyceride degradation was elevated. Glycogen reserves and phospholipids appeared to be used during stress. In comparison, nutrient rich reproductively active females of both forms appeared to use triglycerides. Moreover, we revealed changes in the mRNA level of the detoxifying genes delta-class glutathione S-transferase (GST-δ) and cytochrome P450 monooxygenase (CYP450), as well as the CP gene (which encodes exoskeleton/cuticular proteins) and the cofilin gene (the products of which influence cytoskeleton organization). These results indicate the possible correlation between nutritional stress, energy content, AKH, and the stress-related enzymes of different metabolic pathways in winged and wingless forms of A. pisum.

Modulatory effects of bombyxin on ecdysteroidogenesis in Bombyx mori prothoracic glands.

In the present study, we investigated the modulatory effects of ecdysteroidogenesis of prothoracic glands (PGs) by bombyxin, an endogenous insulin-like peptide in the silkworm, Bombyx mori. The results showed that bombyxin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. Moreover, the injection of bombyxin into day 4-last instar larvae increased ecdysteroidogenesis 24h after the injection, indicating its possible in vivo function. Phosphorylation of the insulin receptor and Akt, and the target of rapamycin (TOR) signaling were stimulated by bombyxin, and stimulation of Akt phosphorylation and TOR signaling appeared to be dependent on phosphatidylinositol 3-kinase (PI3K). Bombyxin inhibited the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and the inhibition appeared to be PI3K-independent. Bombyxin-stimulated ecdysteroidogenesis was blocked by either an inhibitor of PI3K (LY294002) or a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, AICAR), indicating involvement of the PI3K/Akt and AMPK signaling pathway. Bombyxin did not stimulate extracellular signal-regulated kinase (ERK) signaling of PGs. Bombyxin, but not prothoracicotropic hormone (PTTH) stimulated cell viability of PGs. In addition, bombyxin treatment also affected mRNA expression levels of insulin receptor, Akt, AMPKα, -ß, and -γ in time-dependent manners. These results suggest that bombyxin modulates ecdysteroidogenesis in B. mori PGs during development.

Un primer análisis de la investigación en México sobre los determinantes sociales de la salud: 2005-2012 / A first analysis of research on social determinants of health in Mexico: 2005-2012

Objetivo. Examinar la investigación hecha en México sobre los determinantes sociales de la salud (DSS) durante el periodo 2005-2012 con base en la caracterización del sistema nacional de investigación en salud y la producción científica sobre este tema. Material y métodos. Análisis en dos etapas: revisión documental de fuentes oficiales sobre investigación en salud en México y búsqueda sistemática de literatura sobre DSS. Resultados. Los DSS fueron mencionados en el Programa de Acción Específico de Investigación en Salud 2007-2012, pero no figuran en las estrategias y objetivos; en su lugar, se enfatizan primordialmente aspectos de infraestructura y administrativos. En el periodo se publicaron 145 artículos sobre DSS, cuyas temáticas más abordadas fueron "condiciones de salud", "sistemas de salud" y "nutrición y obesidad". Conclusiones. A pesar de que existe investigación en México sobre DSS, la instrumentación de esos hallazgos en políticas de salud no se ha implementado. El Programa Sectorial de Salud 2013-2018 representa una ventana de oportunidad para posicionar resultados de investigación que promuevan políticas de equidad en salud.

Objective. To examine the research on social determinants of health (SDH) produced in Mexico during the period 2005-2012, based on the characterization of the national health research system and the scientific production on this topic. Materials and methods. Two-stage analyses: Review of Mexican documents and official sources on health research and systematic bibliographic review of the literature on SDH. Results. Although SDH were mentioned in the Specific Action Plan for Health Research 2007-2012, they are not implemented in strategies and goals, as the emphasis is put mostly in infrastructure and administrative aspects of research. In the period studied, 145 articles were published on SDH topics such as health conditions, health systems and nutrition and obesity. Conclusions. In spite of the availability of research on SDH in Mexico, the operationalization of such findings into health policies has not been possible. The current Sectorial Program on Health 2013-2018 represents a window of opportunity to position research findings that promote health equity policies.

Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps.

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

Suppression of allatotropin simulates reproductive diapause in the mosquito Culex pipiens.

The cessation of juvenile hormone (JH) production is a key endocrine event that halts ovarian development and hence initiates diapause in females of the mosquito, Culex pipiens. The shutdown in endocrine activity of the corpora allata (CA), the source of JH, was manifested in the smaller size of CA in females reared under short daylengths (diapause) compared to those reared under long daylengths (nondiapause), as well as in low expression of the mRNA encoding allatotropin, the neuropeptide that promotes JH biosynthesis in the CA. Genes encoding both allatotropin and allatostatin were identified in C. pipiens, but only expression levels of allatotropin differed in the two types of females. Knockdown of allatotropin mRNA using RNA interference in females programmed for nondiapause resulted in a cessation of ovarian development akin to diapause. This arrest in development could be reversed with an application of JH. Our results thus suggest that suppression of allatotropin is a critical link in regulating the shutdown of the CA during diapause.

Serotonin receptor B may lock the gate of PTTH release/synthesis in the Chinese silk moth, Antheraea pernyi; a diapause initiation/maintenance mechanism?

The release of prothoracicotropic hormone, PTTH, or its blockade is the major endocrine switch regulating the developmental channel either to metamorphosis or to pupal diapause in the Chinese silk moth, Antheraea pernyi. We have cloned cDNAs encoding two types of serotonin receptors (5HTRA and B). 5HTRA-, and 5HTRB-like immunohistochemical reactivities (-ir) were colocalized with PTTH-ir in two pairs of neurosecretory cells at the dorsolateral region of the protocerebrum (DL). Therefore, the causal involvement of these receptors was suspected in PTTH release/synthesis. The level of mRNA(5HTRB) responded to 10 cycles of long-day activation, falling to 40% of the original level before activation, while that of 5HTRA was not affected by long-day activation. Under LD 16:8 and 12:12, the injection of dsRNA(5HTRB) resulted in early diapause termination, whereas that of dsRNA(5HTRA) did not affect the rate of diapause termination. The injection of dsRNA(5HTRB) induced PTTH accumulation, indicating that 5HTRB binding suppresses PTTH synthesis also. This conclusion was supported pharmacologically; the injection of luzindole, a melatonin receptor antagonist, plus 5th inhibited photoperiodic activation under LD 16:8, while that of 5,7-DHT, induced emergence in a dose dependent fashion under LD 12:12. The results suggest that 5HTRB may lock the PTTH release/synthesis, maintaining diapause. This could also work as diapause induction mechanism.

Polycomb repressive complex 2 (PRC2) protein ESC regulates insect developmental timing by mediating H3K27me3 and activating prothoracicotropic hormone gene expression.

The decision made by insects to develop into adults or halt development (enter diapause and prolong lifespan) is commonly based on environmental signals that provide reliable predictors of future seasons of adversity. For example, the short day lengths of early autumn accurately foretell the advent of winter, but little is known about the molecular mechanisms that preside over the hormonal events dictating whether the insect proceeds with development or enters diapause. In Helicoverpa armigera we show that day length affects H3K27me3 by affecting polycomb repressive complex 2 (PRC2) protein extra sex comb (ESC) and regulates the prothoracicotropic hormone (PTTH) gene, thus directly influencing developmental timing. ESC expression in brains of developing (nondiapause) pupae is higher than in brains from diapausing pupae. High ESC expression is localized in two pairs of PTTH neurosecretory cells, and H3K27me3 recruits on the PTTH promoter. Double strand ESC and PRC2 inhibitor (DzNep) treatment in vitro show that ESC triggers PTTH promoter activity, which in turn depends on PRC2 methyltransferase activity. Injection of DzNep into pupae programmed for development reduces the H3K27me3 mark and PTTH gene expression, thereby delaying development. Although ESC is best known as a transcriptional repressor, our results show that ESC prompts development and metamorphosis. We believe this is the first report showing that the PRC2 complex functions as an activator and that a low level of H3K27me3 can prolong lifespan (i.e. induce diapause) by controlling PTTH gene expression in insects.

Drosophila EHBP1 regulates Scabrous secretion during Notch-mediated lateral inhibition.

Notch signaling is an evolutionarily conserved pathway that plays a central role in numerous developmental and disease processes. The versatility of the Notch pathway relies on the activity of context-dependent regulators. These include rab11, sec15, arp3 and Drosophila EHBP1 (dEHBP1), which control Notch signaling and cell fate acquisition in asymmetrically dividing mechanosensory lineages by regulating the trafficking of the ligand Delta. Here, we show that dEHBP1 also controls the specification of R8 photoreceptors, as its loss results in the emergence of supernumerary R8 photoreceptors. Given the requirements for Notch signaling during lateral inhibition, we propose that dEHBP1 regulates distinct aspects of Notch signaling in different developmental contexts. We show that dEHBP1 regulates the exocytosis of Scabrous, a positive regulator of Notch signaling. In conclusion, dEHBP1 provides developmental versatility of intercellular signaling by regulating the trafficking of distinct Notch signaling components.

Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori.

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.

Insulin-like and testis ecdysiotropin neuropeptides are regulated by the circadian timing system in the brain during larval-adult development in the insect Rhodnius prolixus (Hemiptera).

Insulin-like peptides (ILPs) regulate numerous functions in insects including growth, development, carbohydrate metabolism and female reproduction. This paper reports the immunohistochemical localization of ILPs in brain neurons of Rhodnius prolixus and their intimate associations with the brain circadian clock system. In larvae, three groups of neurons in the protocerebrum are ILP-positive, and testis ecdysiotropin (TE) is co-localized in two of them. During adult development, the number of ILP groups increased to four. A blood meal initiates transport and release of ILPs, indicating that release is nutrient dependent. Both production and axonal transport of ILPs continue during adult development with clear cytological evidence of a daily rhythm that closely correlates with the daily rhythm of ILPs release from brains in vitro. The same phenomena were observed with TE previously. Double labeling for ILPs and pigment dispersing factor (PDF) (contained in the brain lateral clock cells, LNs) revealed intimate associations between axons of the ILP/TE cells and PDF-positive axons in both central brain and retrocerebral complex, revealing potential neuronal pathways for circadian regulation of ILPs and TE. Similar close associations were found previously between LN axons and axons of the brain neurons producing the neuropeptide prothoracicotropic hormone. Thus, the brain clock system controls rhythmicity in multiple brain neurohormones. It is suggested that rhythms in circulating ILPs and TE act in concert with known rhythms of circulating ecdysteroids in both larvae and adults to orchestrate the timing of cellular responses in diverse tissues of the animal, thereby generating internal temporal order within it.

In search for non-steroidogenic functions of the prothoracic glands of the desert locust, Schistocerca gregaria: a peptidomic and proteomic approach.

The only well established function of the prothoracic glands (PGs) of insects is the production of ecdysteroids. In gregarious locusts, like in most insect species, the PGs degenerate soon after the adult molt. In this way they resemble the thymus of mammals, a gland with an important role in the build up of the immune system in young animals. In adult solitarious locusts the PGs persist much longer, however without producing substantial amounts of ecdysteroids. In the literature the existence of a well developed rough endoplasmic reticulum and Golgi complex system has been repeatedly reported, suggesting an active role in peptide or/and protein synthesis and release. The nature of the secreted products remained unknown. Our pepdidomic analysis of an acidified methanolic extract of PGs of last instar gregarious nymphs did not yield any indication for the presence of known locust or other peptides. The peptide release assay was also negative. For our proteomic analysis, we developed an EST-based identification strategy. We successfully identified 50 protein spots on a two dimensional map. In addition to typical protein synthesis-related proteins, a number of proteins with a role in detoxification processes were found, suggesting some role of the PGs in the defense system.

An FXPRLamide neuropeptide induces seasonal reproductive polyphenism underlying a life-history tradeoff in the tussock moth.

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH(2) (FXPRLa) neuropeptides: DH, PBAN, and α-, ß-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.

Identification of a cis-regulatory element that directs prothoracicotropic hormone gene expression in the silkworm Bombyx mori.

In the silkworm Bombyx mori and other insects, prothoracicotropic hormone (PTTH) plays a central role in controlling molting and metamorphosis by stimulating the prothoracic glands to synthesize and release the molting hormone ecdysone. Using an AcNPV (Autographa californica nucleopolyhedrovirus)-mediated transient gene transfer system, we identified a cis-regulatory element that participates in the decision to switch expression of PTTH on or off in PTTH-producing neurosecretory cells (PTPCs). The nucleotide sequence of this cis-regulatory element is similar to a cis-regulatory element that participates in direction of expression of diapause hormone-pheromone biosynthesis activating neuropeptide gene (DH-PBAN) (Shiomi et al., 2007). Furthermore, we found that B. mori Pitx (BmPitx), a bicoid-like homeobox transcription factor, binds the element and activates PTTH expression. Therefore, we propose that the cell-specific expression of two neuropeptide hormone genes, PTTH and DH-PBAN, is activated by the Pitx transcription factor, which may act as a pan-activator in the insect neuroendocrine system and in vertebrate pituitary cells.