Abstract
Genes expressed in response to increased neuronal activity are widely used as activity markers in recent behavioral neuroscience. In the present study, we established transgenic reporter system for whole-brain activity mapping in the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect used in neuroethology and behavioral ecology. In the cricket brain, a homolog of early growth response-1 (Gryllus egr-B) was rapidly induced as an immediate-early gene (IEG) in response to neuronal hyperexcitability. The upstream genomic fragment of Gryllus egr-B contains potential binding sites for transcription factors regulated by various intracellular signaling pathways, as well as core promoter elements conserved across insect/crustacean egr-B homologs. Using the upstream genomic fragment of Gryllus egr-B, we established an IEG promoter-driven transgenic reporter system in the cricket. In the brain of transgenic crickets, the reporter gene (a nuclear-targeted destabilized EYFP) was induced in response to neuronal hyperexcitability. Inducible expression of reporter protein was detected in almost all neurons after neuronal hyperexcitability. Using our novel reporter system, we successfully detected neuronal activation evoked by feeding in the cricket brain. Our IEG promoter-driven activity reporting system allows us to visualize behaviorally relevant neural circuits at cellular resolution in the cricket brain.
Significance Statement Insects are the largest and most diverse group of organisms, and show a wide variety of behaviors. Despite the importance of a comparative approach, recent insect neuroethology mostly relies on the fruit fly Drosophila melanogaster, a versatile model insect with particularly powerful genetic tools, and a relatively small number of researchers use non-Drosophila insects. In the present study, a novel genetic reporting system for whole-brain activity mapping was established in the two-spotted cricket Gryllus bimaculatus by using the newly identified and evolutionary conserved gene regulatory region of an immediate-early gene, early growth response. This reporting system allowed us to analyze the behaviorally-evoked neural activity patterns at cellular resolution in the cricket brain.
Footnotes
The authors declare that they have no conflicts of interest.
This work was supported, in part, by the Grant-in-Aid for Japanese Society for the Promotion of Science (JSPS) Research Fellow (24•3065) to TW; the JSPS KAKENHI (16K20875) to TW; and Akiyama Life Science Foundation research grant 2014 to TW. The white-eye mutant of G. bimaculatus (gwhite mutant) was kindly gifted by Prof. Sumihare Noji in the University of Tokushima. The pXL-BacII, pBSII-IFP2-orf, and pBSII-ITR1.1k-EYFP plasmids were kindly gifted by Prof. Malcolm J. Fraser, Jr. in the University of Notre Dame.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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