In vivo gene transfer into the adult honeybee brain by using electroporation Takekazu Kunieda and Takeo Kubo * Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received 14 February 2004 Abstract The honeybee, Apis mellifera L., is a social insect and they show wide variety of exquisite social behaviors to maintain colony activity. To enable the elucidation of those social behaviors at a molecular level and gene function in the nervous system, we de- veloped an in vivo method to perform gene transfer in the adult brain of living honeybee by electroporation. When green fluorescent protein-expressing plasmid was transferred to the brain with this system, green fluorescence was observed near the anode location. The expression of transfected genes was confirmed at both transcriptional and translational levels by reverse transcription-poly- merase chain reaction and immunoblot analyses. This system will facilitate the analysis of gene function and the regulatory mechanisms of gene networks in the nervous system and provide clues to clarify the relation between those genes and the complex behaviors of the honeybee. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Honeybee; Brain; Electroporation; Gene transfer; Behavior; Eusocial insect The honeybee (Apis mellifera L.) is a eusocial insect with a colony that is composed of three types of adults: the queen, workers, and drones. While queens and drones are specialized for a reproductive role, workers are engaged in various social labors, such as nursing the brood, guarding the colony from natural enemies, and foraging for nectar and pollen in an age-dependent manner [1]. Another prominent ability of the honeybee is their use of the dance language. The workers can communicate the location of a food source by the waggle-dance, known as dance language [1,2]. This be- havior is observed only in the honeybee species, sug- gesting that during evolution they acquired a unique brain function to use the dance language. The molecular basis underlying the highly advanced social behaviors of the honeybee, however, remains unclear. Recently, many candidate genes involved in honeybee social behaviors were identified using differential display and/or cDNA microarray technology, and their expres- sion was demonstrated to be behavior-associated [3–6], age-dependent [7], or brain region-selective [8–12]. The biochemical properties were also characterized for some of the genes [13,14]. The in vivo functions of these genes, however, were analyzed in only a few cases and in an indirect manner using chemical compounds [6]. Quantitative trait loci analyses have identified some genetic loci associated with some of the honeybee social behaviors [15–18]. The genes responsible for these social behaviors, however, have not been identified. To gain a better understanding of the honeybee social behaviors at the molecular level, technology is needed that allows for the introduction of exogenous genes into living individuals. Few gene manipulation methods, however, are available for the honeybee [19]. In eusocial insects like the honeybee, the embryos and larvae are cared for by adult workers. Once injury or abnormality is detected in the larvae, they are destroyed and/or re- moved from the colony and die thereafter. Thus, avoidance of this rejection is critical for successful gene manipulation in the honeybee and it would be advan- tageous to develop a technique that enables direct gene transfer into the adult honeybee. We focused on elec- troporation, as it is applicable for differentiated adult post-mitotic cells and is easily adapted to non-model animals, like the honeybee. Electroporation has been * Corresponding author. Fax: +81-3-5800-3553. E-mail address: stkubo@mail.ecc.u-tokyo.ac.jp (T. Kubo). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.178 Biochemical and Biophysical Research Communications 318 (2004) 25–31 BBRC www.elsevier.com/locate/ybbrc