Insect Molecular Biology (2000) 9(6), 625–634 © 2000 Blackwell Science Ltd 625 Blackwell Science, Ltd Sperm-mediated transformation of the honey bee, Apis mellifera K. O. Robinson, 1 H. J. Ferguson, 2, * S. Cobey, 2 H. Vaessin 3 and B. H. Smith 2 1 Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH 43210, 2 Department of Entomology, The Ohio State University, Columbus, OH 43210, 3 Department of Molecular Genetics and Neurobiotechnology Center, The Ohio State University, Columbus, OH 43210, USA Abstract Our primary objective was to identify techniques to transform the genome of the honey bee ( Apis mellifera ) with foreign DNA constructs. The strategy we adopted was to linearize foreign DNA and introduce it with sperm during the instrumental insemination of virgin queen honey bees. We analysed extracts from larvae within the same cohort and isolated the predicted fragment by means of PCR amplification of genomic DNA. Larvae that carried the construct also expressed the introduced DNA. We propagated several transgenic lines for up to three generations, which demonstrates its heritability. Once carried by a queen, the construct can be detected in that queen’s larvae over several months. However, there was no evidence of integration of the construct, at least as determined by genomic Southern analysis. Nevertheless, this demonstrates the general viability of the technique for introduction of DNA, and it should be augmented by further use of transposable elements that enhance integration. Keywords: honey bee, sperm, transformation, GFP. Introduction The ability to manipulate an animal’s genome through trans- genic approaches is crucial for understanding the relationship between genes and behaviour (Miklos, 1993; Dubnau & Tully, 1998). However, appropriate sets of techniques have not yet been fully developed for a wide array of organisms. Yet there are compelling reasons to develop methods for the transformation of animals such as the honey bee (Robinson et al. , 1997). Honey bees have a rich history as an experi- mental organism for basic research in social behaviour (Seeley, 1995), neuroethology (Menzel, 1990), and beha- vioural genetics (Page & Robinson, 1991; Page et al. , 1998). They exhibit complex social interactions, including, among other behaviours, dance communication to indicate food resources (von Frisch, 1967), hormonally based behavioural castes of worker bees (Robinson et al. , 1997), and well developed learning capabilities (Menzel, 1990). Quantitative genetic analyses have shown that behaviours such as pollen and nectar collection (Robinson & Page, 1989), colony guarding (Page & Robinson, 1991), colony hygienic behaviour (Robinson & Page, 1988), and learning (Brandes, 1988; Brandes et al. , 1988; Brandes & Menzel, 1990; Bhagavan et al. , 1994; Benatar et al. , 1995; Chandra et al ., 2000) all have heritable components that influence the behaviour of individual worker bees. From an economic standpoint, the demands of increased crop production, coupled with stresses of disease and pest infestation, have increased the potential value of directly transforming the honey bee genome (Rothenbuhler, 1979). The standard technique for the introduction of plasmid DNA into an organism involves microinjection into an early stage pronucleus (mice; Palmiter & Brinster, 1986), into a one- to two-cell developmental stage (zebrafish; Fadool et al. , 1998), or into early stage embryos in eggs (fruit flies; Rubin & Spradling, 1982). Initial efforts to transform honey bee eggs with plasmid DNA by means of microinjection or liposome-mediated transfer (Bachiller et al. , 1991) revealed evidence of transient expression (H.J. Ferguson, unpub- lished data). However, even if these techniques were perfected, investigators would still be faced with the difficult task of rearing manipulated honey bee embryos into adults, given the high rejection rate that workers show toward manipulated embryos. For honey bees these techniques require manipulation of embryos in ways that may limit their reintroduction into the colony. This arises because social insects progressively feed and/or care for embryos and Received 10 May 2000; accepted 16 August 2000. Correspondence: Prof. B. H. Smith, Department of Entomology, 1735 Neil Ave., The Ohio State University, Columbus, OH 43210–1220, USA. Tel.: (614) 292 0465; fax: (614) 292 5237; e-mail: smith.210@osu.edu *Present address: USDA-ARS, 5230 Konnowac Pass Road, Wapato, WA 98951, USA.