@article {6383, title = {A molt timer is involved in the metamorphic molt in Manduca sexta larvae.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {2013 Jul 30}, pages = {12518-25}, abstract = {

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.

}, issn = {1091-6490}, doi = {10.1073/pnas.1311405110}, author = {Suzuki, Yuichiro and Koyama, Takashi and Hiruma, Kiyoshi and Riddiford, Lynn M and Truman, James W} } @article {2493, title = {Developmental expression of mRNAs for epidermal and fat body proteins and hormonally regulated transcription factors in the tobacco hornworm, Manduca sexta.}, journal = {Journal of Insect Physiology}, volume = {56}, year = {2010}, month = {2010 Oct}, pages = {1390-5}, abstract = {

This paper provides a compilation of diagrammatic representations of the expression profiles of epidermal and fat body mRNAs during the last two larval instars and metamorphosis of the tobacco hornworm, Manduca sexta. Included are those encoding insecticyanin, three larval cuticular proteins, dopa decarboxylase, moling, and the juvenile hormone-binding protein JP29 produced by the dorsal abdominal epidermis, and arylphorin and the methionine-rich storage proteins made by the fat body. The mRNA profiles of the ecdysteroid-regulated cascade of transcription factors in the epidermis during the larval molt and the onset of metamorphosis and in the pupal wing during the onset of adult development are also shown. These profiles are accompanied by a brief summary of the current knowledge about the regulation of these mRNAs by ecdysteroids and juvenile hormone based on experimental manipulations, both in vivo and in vitro.

}, keywords = {Animals, Developmental, Epidermis, Fat Body, Gene Expression Regulation, Insect Hormones, Insect Proteins, Manduca, Messenger, RNA, Transcription Factors}, issn = {1879-1611}, doi = {10.1016/j.jinsphys.2010.03.029}, author = {Hiruma, Kiyoshi and Riddiford, Lynn M} } @article {2497, title = {The molecular mechanisms of cuticular melanization: the ecdysone cascade leading to dopa decarboxylase expression in Manduca sexta.}, journal = {Insect Biochemistry and Molecular Biology}, volume = {39}, year = {2009}, month = {2009 Apr}, pages = {245-53}, abstract = {

Many insect developmental color changes are known to be regulated by both ecdysone and juvenile hormone. Yet the molecular mechanisms underlying this regulation have not been well understood. This review highlights the hormonal mechanisms involved in the regulation of two key enzymes [dopa decarboxylase (DDC) and phenoloxidase] necessary for insect cuticular melanization, and the molecular action of 20-hydroxyecdysone on various transcription factors leading to DDC expression at the end of a larval molt in Manduca sexta. In addition, the ecdysone cascade found in M. sexta is compared with that of other organisms.

}, keywords = {Animals, Developmental, Dopa Decarboxylase, Ecdysone, Gene Expression Regulation, Insect Proteins, Manduca, Melanins}, issn = {1879-0240}, doi = {10.1016/j.ibmb.2009.01.008}, author = {Hiruma, Kiyoshi and Riddiford, Lynn M} } @article {2506, title = {The coordination of the sequential appearance of MHR4 and dopa decarboxylase during the decline of the ecdysteroid titer at the end of the molt.}, journal = {Molecular and Cellular Endocrinology}, volume = {276}, year = {2007}, month = {2007 Sep 30}, pages = {71-9}, abstract = {

During the last larval molt in Manduca sexta, in response to an increasing, then decreasing ecdysteroid titer, a number of transcription factors such as E75B, MHR3, MHR4, and betaFTZ-F1 appear and disappear in the abdominal epidermis leading to dopa decarboxylase (DDC) expression. Messenger RNAs for both the 20E-induced transcription factors, MHR3 and E75B, are maximal near the peak of the ecdysteroid titer with MHR4 mRNA appearing as the titer declines followed by betaFTZ-F1 and DDC mRNAs. E75B and MHR4 mRNA were not expressed in Manduca GV1 cells, either during exposure to 20E or after its removal. When either MHR3 dsRNA was transfected or E75B was constitutively expressed in these cells, MHR4 mRNA appeared in response to 20E by 6h. E75B was found to form a heterodimer with MHR3 using the BacterioMatch II two-hybrid assay. We conclude that MHR3 apparently suppresses MHR4 expression in the presence of 20E; the appearance of E75B then removes MHR3 by dimerization, allowing MHR4 to be expressed. Because of significant basal activity of the ddc promoter in the GV1 cells, we could perform rescue experiments by adding various factors. Constitutive expression of either E75B or MHR4 in the cells suppressed the significant basal activity of the 3.2kb ddc promoter in the GV1 cells, but 20E had no effect on this activity. Thus, E75B and MHR4 are 20E-induced inhibitory factors that suppress ddc expression and therefore act as ecdysteroid-regulated timers to coordinate the onset of ddc expression at the end of the molt.

}, keywords = {Animals, Biological, Cell Line, Cytoplasmic and Nuclear, Developmental, Dopa Decarboxylase, Ecdysteroids, Gene Expression Regulation, Genetic, Insect Proteins, Manduca, Models, Molting, Promoter Regions, Protein Binding, Receptors}, issn = {0303-7207}, doi = {10.1016/j.mce.2007.07.002}, author = {Hiruma, Kiyoshi and Riddiford, Lynn M} } @article {2512, title = {Differential control of MHR3 promoter activity by isoforms of the ecdysone receptor and inhibitory effects of E75A and MHR3.}, journal = {Developmental Biology}, volume = {272}, year = {2004}, month = {2004 Aug 15}, pages = {510-21}, abstract = {

MHR3 is an ecdysone-inducible transcription factor whose expression in both Manduca sexta epidermis and the Manduca GV1 cell line is induced by 20-hydroxyecdysone (20E) in vitro. There are four putative ecdysone response elements (EcRE) in the 2.6-kb flanking region of the MHR3 promoter. The most proximal, EcRE1, is necessary for activation of the promoter by 20E in the GV1 cells because the mutation of EcRE1 caused the loss of responsiveness to 20E. Previous studies showed that EcR-B1/USP-1 bound only to EcRE1 and high levels of this complex increased the 20E-induced activation, whereas the presence of high USP-2 prevented this increased activation. When we expressed EcR-A alone or in combination with USP-1 under the control of Autographa californica baculovirus promoter (pIE1hr), the activation of the 2.6-kb promoter by 20E was reduced by about 50\%. Moreover, when EcR-A was expressed together with both EcR-B1 and USP-1, it reduced the normal activation caused by EcR-B1 and USP-1 by 50\%. Gel mobility shift assays showed no binding of EcR-A/USP-1 to EcRE1. The presence of EcR-A, however, reduced the binding of EcR-B1/USP-1 by about 50\%. These findings suggest that EcR-A competes with EcR-B1 for binding of USP-1, leading to a decline in activity of the promoter. In addition, E75A, another ecdysone-induced transcription factor, and MHR3 itself suppressed MHR3 promoter activity by binding to the monomeric response element (MRE2). Therefore, MHR3 can be down-regulated both by itself and by E75A.

}, keywords = {Animals, Base Sequence, Binding Sites, Cell Line, DNA-Binding Proteins, Ecdysterone, Epidermis, Gene Expression Regulation, Genetic, Glycoproteins, Insect Proteins, Manduca, Molecular Sequence Data, Mutation, Promoter Regions, Protein Isoforms, Receptors, Response Elements, Serpins, Steroid, Transcription Factors}, issn = {0012-1606}, doi = {10.1016/j.ydbio.2004.04.028}, author = {Hiruma, Kiyoshi and Riddiford, Lynn M} } @article {2514, title = {E74 exhibits stage-specific hormonal regulation in the epidermis of the tobacco hornworm, manduca sexta.}, journal = {Developmental Biology}, volume = {258}, year = {2003}, month = {2003 Jun 1}, pages = {76-90}, abstract = {

The transcription factor E74 is one of the early genes induced by ecdysteroids during metamorphosis of Drosophila melanogaster. Here, we report the cloning and hormonal regulation of E74 from the tobacco hornworm, Manduca sexta (MsE74). MsE74 is 98\% identical to that of D. melanogaster within the DNA-binding ETS domain of the protein. The 5{\textquoteright}-isoform-specific regions of MsE74A and MsE74B share significantly lower sequence similarity (30-40\%). Developmental expression by Northern blot analysis reveals that, during the 5th larval instar, MsE74B expression correlates with pupal commitment on day 3 and is induced to maximal levels within 12h by low levels of 20-hydroxyecdysone (20E) and repressed by physiologically relevant levels of juvenile hormone I (JH I). Immunocytochemical analysis shows that MsE74B appears in the epidermis before the 20E-induced Broad transcription factor that is correlated with pupal commitment (Zhou and Riddiford, 2001). In contrast, MsE74A is expressed late in the larval and the pupal molts when the ecdysteroid titer has declined to low levels and in the adult molt just as the ecdysteroid titer begins to decline. This change in timing during the adult molt appears not to be due to the absence of JH as there was no change during the pupal molt of allatectomized animals. When either 4th or 5th instar larval epidermis was explanted and subjected to hormonal manipulations, MsE74A induction occurred only after exposure to 20E followed by its removal. Thus, MsE74B appears to have a similar role at the onset of metamorphosis in Manduca as it does in Drosophila, whereas MsE74A is regulated differently at pupation in Manduca than at pupariation in Drosophila.

}, keywords = {Amino Acid, Amino Acid Sequence, Animals, Base Sequence, Biological, Cells, Cultured, Developmental, Drosophila, Ecdysterone, Epidermis, Gene Expression Regulation, Genes, Insect, Insect Proteins, Juvenile Hormones, Larva, Manduca, Messenger, Metamorphosis, Molecular Sequence Data, Open Reading Frames, Protein Isoforms, Protein Structure, Pupa, RNA, Sequence Homology, Tertiary, Transcription Factors}, issn = {0012-1606}, author = {Stilwell, Geoffrey E and Nelson, Charles A and Weller, John and Cui, Haiyang and Hiruma, Kiyoshi and Truman, James W and Riddiford, Lynn M} }