Abstract
Silkmoth, Bombyx mori, is one of the important model insects in which transgenic techniques and the GAL4/UAS system are applicable. However, due to cytotoxicity and low transactivation activity of GAL4, effectiveness of the GAL4/UAS system and its application in B. mori are still limited. In the present study, we refined the previously reported UAS vector by exploiting transcriptional and translational enhancers, and achieved 200-fold enhancement of reporter GFP fluorescence in the GAL4/UAS system. Enhanced protein expression of membrane-targeted GFP and calcium indicator protein (GCaMP5G) drastically improved visualization of fine neurite structures and neural activity, respectively. Also, with the refined system, we generated a transgenic strain that expresses tetanus toxin light chain (TeTxLC), which blocks synaptic transmission, under the control of GAL4. Ectopic TeTxLC expression in the sex pheromone receptor neurons inhibited male courtship behavior, proving effectiveness of TeTxLC on loss-of-function analyses of neural circuits. In addition, suppression of prothoracicotropic hormone (PTTH) or insulin-like peptide (bombyxin) secretion impaired developmental timing and growth rate, respectively. Furthermore, we revealed that larval growth is sex-differentially regulated by these peptide hormones. The present study provides important technical underpinnings of transgenic approaches in silkmoths and insights into mechanisms of postembryonic development in insects.
Highlights
Transgenic techniques that enable expression of endogenous or exogenous genes in desired cells or tissues are essential for current biological researches
Based on the refinements reported in D. melanogaster[12,13], we increased upstream activating sequence (UAS) copy number and inserted intervening sequence (IVS), synthetic translational enhancer (Syn21), and terminator of Autographa californica nuclear polyhedrosis virus (AcNPV) p10 gene (p10T), as transcriptional and translational enhancers (Fig. 1A)
It is possible that cytotoxicity of GAL4 partly disturbed prothoracicotropic hormone (PTTH) neural functions. These results indicate that PTTH neural transmission plays important roles in regulation of developmental timing and body weight through control of 20E biosynthesis, but is not necessary for molting, metamorphosis, and survival
Summary
Transgenic techniques that enable expression of endogenous or exogenous genes in desired cells or tissues are essential for current biological researches. Comprehensive optimizations of targeted gene expression systems, including the GAL4/UAS system, were conducted in D. melanogaster[12,13] In these reports, GAL4 optimization and addition of transcriptional and translational enhancers to UAS vectors significantly improved transgene expression. GAL4 optimization and addition of transcriptional and translational enhancers to UAS vectors significantly improved transgene expression These results prompted us to examine the possibility that refinement of UAS vector improves transgene expression while avoiding GAL4 cytotoxicity in B. mori, and further explore its effectiveness in studies of sexual behavior and development. PTTH and bombyxin play important roles in determining the timing of molting and metamorphosis (growth period) and the rate of gaining weight (growth rate), respectively These two peptide hormones are produced in distinct neurosecretory cells in the brain and secreted from corpora allata to hemolymph in B. mori[30,31]. We unexpectedly found that actions of PTTH and bombyxin on larval development are sex-differential
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