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Genetic tools as enabling technology for the control of behavior and recovery of neural function Back


Controlling insect behavior is not only of interest in basic research, where it can help elucidating the basis of behavioral strategies and their neural control mechanisms but is projected to have applications in important areas such as pest management, including the fine-tuning in the use of beneficial insects and other arthropods. The understanding of neural circuits and behavior has traditionally been based on the synthesis of the pillars of experimental analysis and modeling. Models and be implemented in simulations and these can be used control robots to perform, in turn, experiments on the models themselves. This approach is used for example in the effort to elucidate the mechanism of phermone source localization behavior in the silkmoth. Controlling insect behavior is a technology that opens completely new vistas because detailed manipulations cannot only be done on models but on the real living organsisms. Currently, fundamental technology is being developed using genetic methods to allow fine-grained behavioral control in the silkmoth.


Fig. 1 Development of fundamental technology for controling insect behavior.


For the generation of transgenic silkmoths, the piggyBac transposon (Tamura et al., 2000; Tamura et al., 2007), and the GAL4-UAS gene expression control system (Imamura et al., 2003) are a well-established approach. The Silkworm genome project has been completed (International Silkworm Genome Consortium, 2008), provinding the necessary sequence information for targeted manipulations. Therefore, it is now possible to transfer and express genes of interest in defined target neurons in the silkmoth.

Thus far, we generated a silkmoth transgenic line that in which GAL4 has been integrated downstream of the promoters for two neuropeptide hormones. Identified neurosecretory cells could be specifically labeled with GFP and observed in the next generation obtained by crossing these GAL4 lines with a UAS-GFP line (Yamagata et al., 2008; Fig. 2). This experiment demonstrates that GAL4-UAS system can be used in the silkmoth for expressing genes of interest in neurons of interest (Fig. 2). Based on these results, we currently conduct a series of experiments that focus on sensory receptor neurons responding to bombycol as targets for functional alteration. We have generated a transgenic silkmoth in which GAL4 is inserted downstream of the promotor sequence for the gene encoding BmOR1, the receptor for bombykol, the main sex pheromone component in the silkmoth. In a second step, other arbitrary olfactory receptor genes were introduced into UAS lines, thereby making it possible to confer the ability of orienting towards odors detected by these receptors in the same way as towards conspecific females (or their pheromone). Using such transgenic silkmoths, it is possible to develop odor sensors for any odor, incorporating the silkmoths capability of odor source localization. We are also generating transgenic silkmoths in which the properties of sensory neuron and central neuron are altered in specific, well-defined ways. For this porpose, we have generated a transgenic silkmoth UAS line that contains the channelrodpshin 2 (ChR2) (Nagel et al., 2003) gene in its genome.



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Fig. 2 Specific expression of green fluorecent protein (GFP) in neurosecretory cells in the sikmoth brain using the GAL4-UAS system containig neuropeptide promotor. (A) brains of Bombyxin-GAL4/UAS-GFP silkmoth. Larval brain (left) and adult brain (right). (B) Brains of PTTH-GAL4/UAS-GFP silkmoth. Larval brain (left) and adult brain (right). D: dordsal. V: ventral, A: anterior, P: posterior. Scale bar: 200 um.



Fig. 3 Conceptual diagram of odor senor silkmoth.



Reference

Imamura M, Nakai J, Inoue S, Quan GX, Kanda T, Tamura T.: Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori, Genetics 165, 1329-1340 (2003).

International Silkworm Consortium. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem. Mol. Biol. 38, 1036-1045 (2008).

Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, Hegemann P. and Bamberg E. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 13940-13945 (2003).

Tamura T, Kuwabara N, Uchino K, Kobayashi I, Kanda T. An improved DNA injection method for silkworm eggs drastically increases the efficiency of producing transgenic silkworms. J. Insect Biotechnol. Sericol, 76, 155-159 (2007).

Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P.: Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector, Nat. Biotechnol. 18, 81-84 (2000).

Yamagata, T., Sakurai, T., Uchino, K., Sezutsu, H., Tamura, T. and Kanzaki, R. GFP labeling of neurosecretory cells with the GAL4/UAS system in the silkmoth brain enables selective intracellular staining of neurons. Zool. Sci. 25, 509-516 (2008).
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