Figure 1.
Honey bee hemolymph analysis by flow cytometry.
Hemolymph samples from three individual bees were collected and probed with WGA and PI as described in the Methods. Both fluorescence (A) and light scatter (B) data are displayed for hemolymph obtained from a single bee. A) The dot-plot showing fluorescence of WGA-FITC vs PI identifies recognizable groups of particulates in quadrants Q1-Q4, with resulting particle percentages as shown. B) The FSC vs SSC plot provides information on physical parameters for the same particles (in matching color) as those shown in Figure A. Predominance of permeabilized cells (86% of total events from combined Q1 and Q2 quadrants) is shown for a single bee in C, while one bee with 89% plasmatocytes (from Q4) is shown in D.
Figure 2.
Association of flow cytometry light scatter profiles with particle types observed in Wright-stained smears.
A) Analysis of flow cytometry data from a total of 634 individual bees, including several common bee strains, resulted in a FSC vs SSC summary plot that provides information about size and complexity in particles observed in hemolymph. Colors of regions used in the summary plot match those shown in Figure 1. B) Photomicrographs of Wright-stained particles correspond to regions Q1-Q4 of the summary plot include Q1: image of a permeabilized cell (blue arrow) lacking an obvious plasma membrane (and plasmatocyte with plasma membrane for comparison), Q2: permeabilized cell surrounded by a plasma membrane, Q3: microparticles, and Q4: plasmatocyte with rounded termini (and pointed termini, inset). The scale bar represents 5 µm for all images.
Figure 3.
Honey bee hemocyte staining by annexin V.
Hemolymph from five bees was pooled and probed with both PI and annexin V, then examined by flow cytometry. A) Events displayed in the FSC vs SSC plot were used to locate regions corresponding to permeabilized cells (red dots) and plasmatocytes (blue dots), where overlap of cell types was minimal. Green dots represent microparticles and cells lying outside the selected regions. B) Fluorescence measurements obtained on particle subsets shown in A (using the same coloring scheme) indicate permeabilized cells are bound by annexin V and PI, while plasmatocytes were not labeled by either fluorochrome. The data are representative of 12 separate examinations involving hemolymph from 60 bees tested on three separate days.
Figure 4.
JC-1 staining to identify mitochondrial membrane activity in honey bee hemocytes.
Hemolymph samples from five bees were pooled and split into two equal volumes, then exposed to either the JC-1 probe alone, or JC-1 in the presence of the mitochondrial membrane potential disruptor, CCCP. Permeabilized cells and plasmatocytes were identified in FSC vs SSC plots, which were selected to determine the fluorescence intensity for the respective cell type. Bars represent the mean fluorescence intensity ratio (red/green) for replicates, both with (open bars) and without (filled bars) CCCP. Error bars define SEM, n = 7 samples; where each sample represents a pool of five unique bees. The murine SP2/0 cell line was used as a positive control. Data are representative of results obtained on two separate days.
Figure 5.
Aggregation of honey bee hemocytes involving permeabilized cells and plasmatocytes.
Hemocyte aggregates were occasionally found in Wright-stained smears of hemolymph samples inadvertently containing setae from the surface of the bee (white arrow, inset). Resulting aggregates included both plasmatocytes (plas.) and permeabilized cells (perm.). Plasma membranes were seen surrounding plasmatocytes. This observation contrasted with permeabilized cell nuclei that appeared to be associated with variable amounts of cytoplasm, with little evidence of plasma membranes. The total magnification for each image is 1000X, and the scale bar represents 10 µm for the primary image and 20 µm for the inset.
Figure 6.
Hemocyte profiling of bees from colonies differing in numbers of Varroa destructor mites.
A) Varroa mites collected from hives 1-3 are represented by the number collected per day on adhesive paper. Hemocyte profiling was performed for all three hives on day 88 following the peak noted in hive 2. B) Hemolymph particles in bees from each hive were quantified and reported in number per µl of hemolymph, according to their localization in specific quadrants (Q1-Q4) of the fluorescence plots. Significant differences between hives were noted for Q1 (permeabilized cells: P = 0.02 comparing hive 1 to 3), and for Q4 (plasmatocytes: P<0.001 comparing hive 1 to hive 2). Other comparisons within quadrant types were not found to be significantly different.
Figure 7.
Correlation of plasmatocyte WGA-FITC fluorescence to FSC in bees exposed to variable Varroa mite numbers.
A) Plasmatocyte populations were selected from FSC vs SSC plots for bees represented in Figure 6B, then evaluated for both WGA-FITC fluorescence and FSC with respect to each hive. Plasmatocytes from bees in hive 1 showed significantly greater (P<0.001, n = 16) WGA-FITC labeling intensity and FSC values when compared to either hives 2 or 3. By contrast, plasmatocytes from hives 2 and 3 were not significantly different (P>0.05) for either parameter. B) Linear regression analysis for plasmatocytes from all hives collectively shows correlation (R2 = 0.33, P<0.001) of WGA-FITC labeling to FSC values, where color labeling of individual bees identifies the hive number.