Primary cell culture method for the honeybee Apis mellifera

Abstract

Honeybees are among the most important pollinators in nature, and honeybee-associated products are useful in various areas, including the food industry. However, honeybees may be infected by various types of pathogens. The study of honeybee-associated diseases would greatly benefit from a successful cell culture system, but although some honeybee cell culture techniques have been introduced, these methods have not yet been fully established. Here, we describe a primary cell culture method for the honeybee, Apis mellifera. We isolated, sterilized, and seeded egg cells into non-coated cell culture dishes to generate cell aggregates. After approximately 10 d, aggregates were dissociated and seeded to cell culture dishes. Cell passages were continuously performed, with sub-culturing every 3-4 d. The cells expressed non-adherent phenotypes. Their growth increased with the passage number when they were cultured in growth medium based on L-15 insect medium but not Schneider's insect medium. Finally, polymerase chain reaction confirmed that the cells originated from A. mellifera. Our results suggest that the culturing methods described herein are appropriate for isolating primary cells from honeybee eggs. These methods could thus facilitate the study of honeybee-associated pathogenesis, development, and toxicology.

Cell Lines for Honey Bee Virus Research

“…Importantly, the cultured neurons showed surface properties and a transmitter phenotype similar to those of their in vivo counterparts [41], indicating the potential for primary cell cultures in the study of cell biology. Additional honey bee primary cell lines were established from eggs [42][43][44][45], guts [41,46] and larval or pupal tissues (Table 1) [27,35,41,[47][48][49].…”

“…(4) The identity of the cells was confirmed by polymerase chain reaction (PCR) amplification of a specific gene sequence from DNA extracted from cultured cells, and sequencing of the PCR product. The target genes included actin and laminin for confirmation of honey bee cell lines [43,47]. Mitochondrial cytochrome c oxidase subunit I (COI) is also commonly employed for this purpose.…”

“…Ju and Ghil used L-15 medium-based honey bee cell (LHB) growth medium and Schneider's insect medium-based honey bee cell (SHB) growth medium, with more cells produced in the LHB medium than in SHB medium after six passages. The doubling time in LHB medium was only about eight days [43]. Clearly, identification of a suitable cell culture medium is critical for the maintenance of primary cell cultures.…”

Cell Lines for Honey Bee Virus Research

“…Importantly, the cultured neurons showed surface properties and a transmitter phenotype similar to those of their in vivo counterparts [41], indicating the potential for primary cell cultures in the study of cell biology. Additional honey bee primary cell lines were established from eggs [42][43][44][45], guts [41,46] and larval or pupal tissues (Table 1) [27,35,41,[47][48][49].…”

“…4) The identity of the cells was confirmed by polymerase chain reaction (PCR) amplification of a specific gene sequence from DNA extracted from cultured cells, and sequencing of the PCR product. Target genes included actin and laminin for confirmation of honey bee cell lines [43,47]. Mitochondrial cytochrome c oxidase subunit I (COI) is also commonly employed for this purpose.…”