Fig 1.
Method for culturing hemocyte cells.
Table 1.
List of primers.
Fig 2.
Manipulation timeline of the ex vivo cell culture.
Days after incubation were referred to as Day 0, Day 1, Day 2, and thereafter. The cells on each day after the incubation were referred to as Day 1 cells, Day 2 cells, Day 3 cells, and thereafter. Experimental procedures for the induction or inhibition of gene expression are described below the time course. To induce gene expression, the expression vector plasmid was transfected into cells on Day 1. To inhibit gene expression (RNAi, procedure A), a mixture of the expression vector plasmid and dsRNA was transfected into cells on Day 1. In another procedure to inhibit gene expression (RNAi, procedure B), cells were incubated with dsRNA-containing culture medium on Day 1, incubated for one day, and the expression vector plasmid was then transfected into cells on Day 2.
Fig 3.
Morphology of honey bee cells prepared from each tissue.
A. Embryonic cells derived from whole embryos. B. Pupal fat body cells were aggregated and irregular in shape and size. C. Hemolymph-derived cells were adherent and relatively uniform in shape and size. D. A magnified image of larval hemocytes. The arrow and arrowhead indicate a plasmatocyte and granulocyte, respectively. Bars indicate 100 μm.
Fig 4.
Ex vivo cultured hemocyte cells and viability after incubation.
A. Hemocyte cells obtained from the hemolymph of 15 larval equivalents were fully confluent (approximately 4.45 x 105 cells/well) in a well of a 96-well plate. The bar indicates 100 μm. B. Viability of cells after the incubation. Viability is the ratio of viable cells among all cells counted using a hemocytometer (three replications for each incubation period). Error bars indicate the standard error of the mean.
Fig 5.
Alignment of nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene.
A partial sequence of the Apis mellifera COI gene (658 bp excluding the primer sites) is shown on the top. Twenty-eight out of the 29 clones obtained from ex vivo cultured cells had the same sequences to A. mellifera COI (middle line). One clone (bottom line) had a single base substitution (arrow). Asterisks indicate identical bases.
Fig 6.
Induction of egfp gene expression by the introduction of expression vector plasmids into ex vivo cultured cells.
Fluorescent micrographs of Day 4 cells transfected with expression vectors of A. BmA3::EGFP, B. BmA3::EGFP/Dmhsp70::EGFP, and C. IE2::EGFP. The bar indicates 100 μm. D. EGFP fluorescence was predominantly detected in cells transfected with the IE2::EGFP vector plasmid. Error bars indicate the standard error of the mean in three replications.
Fig 7.
Selection of optimal conditions for the induction of gene expression.
A. Comparison of transfection reagents. B. The optimal amount of vector plasmid DNA to be transfected was 5.0 μg/well when 0.4 μl/well of the TransIT-Insect reagent was applied. The graph on the left shows transfection efficiency when the conditions recommended by the supplier were employed. C. The optimal dose of the transfection reagent was 0.8 μl/well when 5.0 μg/well of plasmid DNA was applied. D. Effects of medium exchange after transfection on cell viability. Error bars indicate the standard error of the mean of three replications.
Fig 8.
Comparison of transcripts levels by RT-PCR.
Short fragments of the egfp gene (656 bp, top panel) and A. mellifera elongation factor-1α gene (315 bp, bottom panel) were amplified using gene-specific primer sets and total RNA extracted from Day 5 cells as the templates. egfp gene transcripts were detected in cells transfected with the IE2::EGFP vector, but not in cells to which dsRNA corresponding to the egfp gene was applied. Each lane indicates the treatments that ex vivo cells received: no treatment (Control), transfection of the IE2::EGFP vector (IE2::EGFP), transfection of the IE2::EGFP vector associated with egfp dsRNA (IE2::EGFP + egfp dsRNA), and transfection of the IE2::EGFP vector associating with dsRNA corresponding to an unrelated A. mellifera kynurenine 3-monooxygenase (kmo) gene (IE2::EGFP + kmo dsRNA).
Fig 9.
Fluorescent micrographs showing a decrease in EGFP emissions by RNAi-based gene silencing in ex vivo cultured cells.
Top panels (A-C) show the effects of the application of dsRNA using the transfection reagent (RNAi, procedure A), and bottom panels (E-G) show the effects of soaking with dsRNA without transfection reagent (RNAi, procedure B). Ex vivo cells were transfected with the IE2::EGFP vector (A and E, IE2::EGFP), transfected with the IE2::EGFP vector and the application of egfp dsRNA (B and F, +egfp dsRNA), and transfected with the IE2::EGFP vector and application of kmo dsRNA (C and G, +kmo dsRNA). D, H. The transfection of and soaking with egfp dsRNA both decreased the proportion of EGFP-positive cells. Bars indicate the standard error of the mean of three replications.
Fig 10.
Procedure for inducing and inhibiting gene expression in ex vivo cultured hemocyte cells under optimal conditions.