A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

Abstract

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Primary cell culture method for the honeybee Apis mellifera

“…The honeybee cells examined herein showed non-adherent characteristics even in coated cell culture dishes, and thus differed from the adherent honeybee cells isolated in previous studies (Bergem et al 2006;Hunter 2010;Goblirsch et al 2013). In a previous study, we isolated adherent cells from honeybee eggs by using different culturing methods and observed them as fibroblast-like cells on coated cell culture dishes.…”

“…This may reflect the lack of a good cell culture system for honeybees. Several honeybee cell culture methods have been reported (Bergem et al 2006;Hunter 2010;Goblirsch et al 2013), but these methods have varied widely in their utilized target tissues, growth media, and isolation methods.…”

“…1b). Sterilization buffer (0.525% sodium hypochlorite, 0.1% Tween 20, 0.5% benzalkonium chloride, and 70% ethanol) (Goblirsch et al 2013) was added to each well, and the eggs were surface-sterilized for 2 min with gentle agitation. The sterilized eggs were rinsed twice (5 min each) with sterilized phosphate-buffered saline (PBS), 10-15 eggs were transferred to microcentrifuge tubes, and 1 ml of L-15 insect medium-based honeybee cell (LHB) growth medium [L-15 insect basal medium containing 20% fetal bovine serum, 10 mM HEPES, 1 mM NaHCO 3 , 1 mM sodium pyruvate, 1% D(+) trehalose dehydrate, 1× yeast extract, 1× amino acid, 1× vitamins, and 0.5% gentamycin] was added to each tube.…”

C3larvin Toxin, an ADP-ribosyltransferase from Paenibacillus larvae

“…A logical next step would be to test C3larvin for cell entry into an insect cell line. Until recently, there have been no available honey bee cell lines for testing; however, a honey bee cell line has recently been developed (34). This cell line should be tested to see whether C3larvin can enter and cause morphological changes.…”

Challenges associated with research on RNA viruses of insects

“…Although some picornavirads infect these groups of insects [1,3], extra efforts are needed to obtain cell lines from other insects where more picornavirads can be studied [36]. A new stable cell line derived from honey bee embryos has been reported [37 ] and will benefit investigation of dicistrovirids and iflavirids infecting the honey bee.…”