Hormonal and nutritional regulation of insect fat body development and function.

The insect fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. Similar to other larval organs, fat body undergoes a developmental "remodeling" process during the period of insect metamorphosis, with the massive destruction of obsolete larval tissues by programmed cell death and the simultaneous growth and differentiation of adult tissues from small clusters of progenitor cells. Genetic ablation of Drosophila fat body cells during larval-pupal transition results in lethality at the late pupal stage and changes sizes of other larval organs indicating that fat body is the center for pupal development and adult formation. Fat body development and function are largely regulated by several hormonal (i.e. insulin and ecdysteroids) and nutritional signals, including oncogenes and tumor suppressors in these pathways. Combining silkworm physiology with fruitfly genetics might provide a valuable system to understand the mystery of hormonal regulation of insect fat body development and function.

Juvenile hormone acid methyl transferase is a key regulatory enzyme for juvenile hormone synthesis in the Eri silkworm, Samia cynthica ricini.

During the period of adult emergence in the Eri silkworm, Samia cynthia ricini, the corpora allata (CA) are apparently reactivated in females, but not males. This creates a significant sexual dimorphism in juvenile hormone (JH) synthesis by CA. To determine the underlying molecular mechanisms in this process, we cloned cDNAs of two enzymes involved in the JH synthesis pathway: 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and juvenile hormone acid methyl transferase (JHAMT). Both Samcri-HMGR and -JHAMT mRNAs were detected in CA almost exclusively. However, their expression patterns were different from each other. During the period of adult emergence, Samcri-HMGR was expressed in CA at a constantly high level suggesting it plays little role for the regulation of JH synthesis. In contrast, the patterns of both Samcri-JHAMT mRNA level and enzyme activity were closely correlated with the patterns of JH synthesis, CA reactivation, and sexual dimorphism of JH synthesis. In addition, JHAMT mRNA levels were paralleled JH synthesis in the fifth-instar larvae of S. cynthia ricini and the pharate adults of the silkworm Bombyx mori. We infer from these results that JHAMT is a key regulatory enzyme for JH synthesis in the Eri silkworm.

Knockdown of ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system leads to lethal ecdysis deficiency in silkworm.

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.

Biochemical and molecular characterization of allatotropin and allatostatin from the Eri silkworm, Samia cynthia ricini.

In a previous study, allatotropic and allatostatic activities were observed in brain extract from the Eri silkworm, Samia cynthia ricini (Samcri) [Li, S., Jiang, R.-J., Cao, M.-X., 2002b. Allatotropic and allatostatic activities in brain extracts of the Eri silkworm, S. cynthia ricini, and the effects of Manduca sexta allatotropin and M. sexta allatostatin on juvenile hormone in vitro. Physiol. Entomol. 27, 322-329]. In the present study, the HPLC purified Samcri-allatotropin (AT) and -allatostatin (AST) factors were shown to have the same retention time as those of M. sexta (Manse)-AT and -AST, respectively. Moreover, the amino acid sequences of mature Samcri-AT and -AST deduced from their encoding cDNAs are identical to the Manse-AT and -AST amino acid sequences. Both Samcri-AT and -AST genes were expressed in brain, nerve cord, and midgut, with Samcri-AT also detected in gonads and epidermis, suggesting their pleiotropic physiological functions. The expression levels of Samcri-AT and -AST genes correlated well with the allatoregulatory activities during the period of adult emergence indicating the two peptides tightly control JH synthesis, in a contradictive and cooperative manner. Our biochemical and molecular data of Samcri-AT and -AST and other studies demonstrate that these two peptides regulate JH synthesis by corpora allata in Lepidoptera and have pleiotropic physiological effects.

Development of a heat shock inducible and inheritable RNAi system in silkworm.

A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42 degrees C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal-adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages.

The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpa armigera encodes multiple peptides that break, rather than induce, diapause.

FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera.

Ras1(CA) overexpression in the posterior silk gland improves silk yield.

Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.

Transcriptional regulation of the insulin signaling pathway genes by starvation and 20-hydroxyecdysone in the Bombyx fat body.

Genetic studies in the fruitfly, Drosophila melanogaster, have uncovered a conserved insulin/insulin growth factor signaling (IIS) pathway that regulates nutrition-dependent growth rates of insects. From the silkworm, Bombyx mori, we have identified and characterized several key genes involved in the IIS pathway, including InR, IRS, PI3K110, PI3K60, PTEN, PDK, and Akt. Tissue distribution analysis showed that most of these genes were highly expressed in the fat body implying that the IIS pathway is functionally important within insect adipose tissue. Developmental profile studies revealed that the expression levels of InR, IRS, PI3K110, and PDK were elevated in the fat body during molting and pupation, periods when animals ceased feeding and hemolymph levels of 20-hydroxyecdysone (20E) were high. Starvation rapidly up-regulated the mRNA levels of these same genes in the fat body, while 20E slowly induced their transcription. We conclude that 20E slowly reduces food consumption and then indirectly induces a state of starvation resulting in the elevation of the mRNA levels of InR, IRS, PI3K110, and PDK in the Bombyx fat body during molting and pupation.

Developmental regulation of glycolysis by 20-hydroxyecdysone and juvenile hormone in fat body tissues of the silkworm, Bombyx mori.

20-Hydroxyecdysone (20E) and juvenile hormone (JH) control a variety of physiological events during insect development and metamorphosis. To understand how 20E and JH developmentally regulate energy metabolism in insects, we performed a genome-wide microarray analysis of fat body tissues isolated from the silkworm, Bombyx mori. Many genes involved in energy metabolism, including genes in the glycolytic pathway, were down-regulated during molting and pupation, when 20E levels are high. Notably, 20E treatment exhibited inhibitory effects on key glycolytic enzyme mRNA levels and activities, and RNA interference of the 20E receptor EcR-USP had the opposite effects to 20E treatment. Meanwhile, JH treatment stimulated both mRNA levels and activities of the key glycolytic enzymes, presumably via antagonizing the 20E action. Taken together, we conclude that 20E acts as a general blocker for glycolysis in the Bombyx fat body during molting and pupation, whereas the physiological role of JH is contrast with 20E during molting.

20-hydroxyecdysone reduces insect food consumption resulting in fat body lipolysis during molting and pupation.

The insect steroid hormone 20-hydroxyecdysone (20E) acts through a specific nuclear receptor complex, ecdysone receptor (EcR) and ultraspiracle (USP). EcR and USP are FXR/LXR and RXR orthologs, respectively, which play critical roles in the regulation of lipid metabolism in mammals. Lipid concentration in Bombyx hemolymph and lipase activity in fat body peaked during molting and pupation, suggesting that 20E induces lipolysis at these stages. Differing from their mammalian orthologs, the 20E-bound EcR-USP was not able to directly stimulate fat body lipolysis in both Bombyx and Drosophila. Instead in Bombyx, 20E slowly reduced food consumption and then induced starvation, resulting in fat body lipolysis. Molecular analysis revealed that the evolutionarily conserved adipose triacylglycerol lipase gene Brummer was transcriptionally up-regulated by 20E-induced starvation during molting and pupation. To our knowledge, this is the first report demonstrating that the steroid hormone 20E is a critical regulator of lipolysis in insects.

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila.

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.