Fat body tissue is the main metabolic system during honeybee larvae development (Martins & Ramalho-Ortigão, 2012) and is made up of mainly oenocytes and trophocytes (Cousin et al., 2013). These cells are mainly located below the larval body wall and around the gastrointestinal tract (Makki et al., 2014). Both oenocytes and trophocytes can be isolated or in clusters surrounded by connective tissue in the hemocoel contacting the hemolymph and enhancing the exchange of metabolites (Roma et al., 2010). They are responsible for the production of hemolymph proteins (Ruvolo & da Cruz, 1993), resembling to mammalian hepatocytes, with detoxification and excretion functions, synthesis of proteins, hormones, storage of lipids and carbohydrates (Paes de Oliveira & Da Cruz Landim, 2003; Gutierrez et al., 2007; Landim, 2009). Oenocytes are large, round acidophilic cells derived from the ectoderm and distributed among the trophocytes (Ruvolo & da Cruz, 1993) and produce cuticulin, the precursor for both the larva and the reproductive adult (Roma et al., 2010). Trophocytes are round oval shaped cells that contain intracellular lipids, proteins, and carbohydrates droplets (Paes de Olivaira & Da Cruz Landim, 2003).
Previous studies determined that oenocytes are morphologically affected by larvae chlorophenol herbicide exposure (Cousin et al., 2013). Furthermore, the starvation of the
Apoptosis is a normal process in insects and mammals during development, regulated by the activation of cysteinyl aspartate proteases called “caspases” (Gregorc & Bowen, 1997). The starting caspase proteins trigger an enzymatic cascade that activates effectors caspases, including caspase-3. The acaricide amitraz triggers apoptosis in the midgut epithelial cells of the honeybee larvae (Gregorc & Bowen, 1997). Adding to that, recent studies determined that glyphosate increases apoptosis in gut epithelial cells during larvae development (Vázquez, et al., 2018).
A recent, massive death of bee larvae, called “River disease”, was caused by the intake of the toxic secretions of the
The study was performed on an experimental farm “Campo Experimental Nº2” (Faculty of Veterinary, Universidad de la República, Libertad, San José, Uruguay).
Honeybee larvae were fixed and immersed in a fixative solution (ethanol 96% 27 ml, formalin 5% 11 ml, glacial acetic acid 7 ml, and distilled water 55 ml). Samples were then dehydrated in increasing concentrations of ethanol (50%, 70%, 95%, 100%), immersed in chloroform and paraffin embedded. Subsequently, 5 μm sections were obtained using a microtome (Leica Reichert Jung Biocut 2030, Wetzlar, Germany) for staining techniques and immunohistochemistry.
The Haematoxylin-Eosin (H&E) technique was used to stain sections with yellow eosin and Mayer's Haematoxylin for histomorphometrical analyses.
The immunohistochemistry technique was applied to analyse the caspase-3 in oenocytes and trophocytes. Briefly, the technique included dewaxing of the larvae sections in an oven at 60ºC, antigenic recovery and hydration in 0.01 M citrate buffer pH 8.0 and 4 ml of Tween at a high-temperature microwave for three minutes. Subsequently, endogenous peroxidases were blocked with 3% hydrogen peroxide (H2O2). Afterwards, the slides were incubated for eighteen hours at 4°C with anti-caspase-3 (Polyclonal Rabbit IgG, AF835, 0.2 mg/ml, R & D Systems, USA, dilution 1: 500). Subsequently, biotinylated secondary antibody (anti rabbit secondary IgG-HRP / DAB kit, ab 64261 Abcam) were incubated for thirty minutes. The slides were then incubated for thirty minutes with horseradish-peroxidase-enzymatic-complex (HRP), and finally diaminobenzidine (DAB) chromogen solution (DAB+ hydrogen peroxide) was added for one minute. The slides were then washed with distilled water and counterstained with Harris Hematoxylin. To verify the specificity of the technique, negative controls were performed, replacing the primary antibody with phosphate buffer solution (PBS) pH 7.4. After each step, a rinse in PBS was performed.
Histological H&E and immunohistochemistry images were retrieved with software (Dino-Capture 2.0 software, AnMo Electronics Corporation, Taiwan) and a digital camera (Dino-Eyepiece, AM-423X, AnMo Electronics Corporation, Taiwan) connected to a binocular microscope (Professional Premiere®, model MRP-5000, Manassas, USA).
The morphometry of oenocytes and trophocytes was determined by image analysis with ImageJ software (ImageJ 1.51 g, Wayne Rasband open source, National Institutes of Health, USA,
Morphometric analyses were performed in thirty microscopic images of bee larvae oenocytes at high-power field (HPF = 400×). The oenocyte area (μm2) and diameter (μm) were measured with a
Trophocytes were analysed as described above for oenocytes. The trophocyte area (μm2) and
Caspase-3 immunostaining area (%) and intensity (mean gray value of pixels) were measured in oenocytes and trophocytes with ImageJ software. A selection of each cell type was done, afterwards brown areas were selected, using a
All results for cellular area (μm2), cellular diameter (μm), immunostaining area (%) and intensity of immunostaining (mean gray value of pixels) for caspase-3 in oenocytes and trophocytes were expressed as means ± standard error of the mean (S.E.M.). Differences were compared by Student's t-test (Two-Sample t-test) using PROC TTEST for each dependent variable and SAS statistical analysis (SAS, v. 9.1, SAS Institute Inc., Cary, NC, USA) considering the level of significance to be p<0.05.
Larvae from the healthy group showed defined contours, acidophilus cytoplasm and euchromatic nucleus (Fig. 1a). Oenocytes from the toxic group showed a heterochromatic nucleus and loss of nucleus roundness (Fig. 1b). The morphometric parameters, cellular area and diameter that were normally distributed, decreased in the toxic group compared to the healthy group (area 3274±147 μm2
The trophocytes of the healthy group larvae had a round shape, lipid vacuoles and euchromatic nuclei (Fig. 2a). In contrast, the toxic group trophocytes lacked net cell boundaries and the number of lipid vacuoles was lower (Fig. 2b). The morphometric parameters, cellular area and diameter that were normally distributed, decreased in the toxic group compared to the healthy group (area 1348±85 μm2
The immunoexpression of caspase-3 in oenocytes was clearly distributed along the cytoplasm in the toxic group larvae, whereas there was less intense immunoexpression in the healthy group larvae (Fig. 3a–b). The immunohistochemistry variables caspase-3 immunostaining area (%) and intensity (mean gray value of pixels) were normally distributed. The immunostaining area (%) for caspase-3 in oenocytes of the toxic group larvae was higher compared to healthy group larvae (0.85±0.25
The immunoexpression of caspase-3 in trophocytes was intense in both the cytoplasm and nucleus of the toxic group larvae in contrast to the healthy group larvae where the immunoexpression was observed to be light brown at the cytoplasm, more intense in the perinuclear region and clearly negative in the nucleus (Fig. 4a–b). The immunohistochemestry variables caspase-3 immunostaining area (%) and intensity (mean gray value of pixels) were normally distributed. The immunostaining area (%) for caspase-3 of the toxic group larvae was higher compared to that of the healthy larvae (22±0.62
In this study we determined that fat body cell's morphology and apoptotic protein immunoexpression was altered in
The toxic honeydew ingestion impaired the metabolic action and morphological aspects of the oenocytes and trophocytes. Furthermore, toxic honeydew increased caspase-3 immunoexpression, an executor enzyme involved in cellular apoptosis. These changes match with previous reports on the paraquat intoxication of honeybee larvae, in which oenocytes reduce in size as a consequence of this herbicide (Cousin et al., 2013). Because of morphologically decreased fat body cells in the cross-section area, a possible loss of integrity in the cellular membranes and pyknotic nucleus in the cells affected by the toxic group, we suggest an increase of the apoptosis process.
The increase observed in the caspase-3 immunostaining of oenocytes due to toxic honeydew ingestion is one of the major alterations in larvae as oenocytes secrete ecdysteroid hormones involved in larval development, metamorphosis and remodelling (Cousin et al., 2013). Moreover, the nuclear immunolocalization of active caspase-3 from the cytoplasm compartment to the nucleus suggests that it occurs in the cells of the toxic group larvae; this translocation had been previously detected in culture cells during the progression of apoptosis (Kamada et al., 2005). The observation of caspase-3 at nuclear level could explain the beginning of cell proteolysis process as previously described in the apoptosis process (Ramuz et al., 2003). During the larval development of honeybees, apoptosis was observed to occur in the immunoexpression of caspase-3 in oenocytes and trophocytes in the healthy group, while the immunoexpression area occupied by caspase-3 increased in the toxic group.
Moreover, the translocation of caspase-3 to the nuclei in the fat body cells of the toxic group indicates the onset of proteolysis, the disassembly of the nuclear envelope, the intensification of the signal in membranes and proteolysis damage. The immunoexpression of active caspase-3 determined an onset of apoptosis in the nucleus as well as the cytoplasm in the toxic group. The nuclear immunostaining is indicative of apoptosis, which precedes the disruption of cell membranes in this process.
The results contribute to clarifying the cellular mechanisms involved in “