90i compound microscope

Five‐day‐old adults were injected with live E. coli, and 1 hr later, the hemocytes were collected by perfusion. Hemocytes were allowed to adhere to the slide for 20 min at room temperature, fixed for 5 min by adding 50 μl of 4% formaldehyde and 0.03 mM Hoechst 33,342 (nuclear stain) in PBS, and coverslips were mounted with Aqua‐Poly/Mount. Hemocytes were examined under 100X magnification using simultaneous DIC and fluorescence illumination on a Nikon 90i compound microscope. For each mosquito, the number of bacteria that had been phagocytosed by each of the first 50 hemocytes viewed was recorded. These values were used to calculate two parameters: (a) the phagocytic index, which is defined as the percentage of cells that engage in phagocytosis; and (b) the phagocytic capacity, which is defined as the average number of phagocytosed bacteria per hemocyte (Coggins et al., 2012; Hillyer et al., 2005; League et al., 2017). Three independent trials consisting of five individuals per treatment group and sex were conducted, and the data were pooled for analysis.

For each mosquito, an incision was made across the lateral and ventral portions of the suture that joins the 7th and 8th abdominal segment of naïve, injured (LB-injected), and E. coli-infected larvae, 1-day-old adults and 5-day-old adults at 24 h post-treatment. A finely pulled glass capillary needle was then inserted into the thorax, approximately 200 μl of Schneider’s Drosophila medium was perfused through the mosquito, and the diluted hemolymph was collected within two 1 cm diameter etched rings on a Rite-On glass slide (Gold Seal; Portsmouth, NH, USA) [46 (link)]. After allowing the hemocytes to adhere to the slide for 20 min, cells were fixed and stained using Hema 3 (Fisher Scientific, Pittsburgh, PA, USA), and, after drying, mounted with coverslips using Poly-Mount (Polysciences, Warrington, PA, USA) [45 (link), 47 (link)]. The total number of hemocytes was then counted under bright-field conditions at 40× magnification using either a Nikon 90i compound microscope (Nikon, Tokyo, Japan) or an Olympus BH-2 microscope (Olympus, Tokyo, Japan). Three independent trials consisting of approximately 5 individuals per treatment group were conducted (n = 14–16 per group). Data were analyzed by two-way ANOVA, using age (including stage) and treatment as variables, followed by Šidák’s post-hoc test.

Circulating hemocytes were collected by perfusion from 6‐day‐old adults at 24‐hr post‐treatment with E. coli as previously described (League et al., 2017). An additional group that did not receive an E. coli injection as adults was also analyzed. Briefly, an incision was made across the last abdominal segment, a microinjection needle was inserted into the neck membrane, and approximately 200 μl of Grace's insect medium was injected. The diluted hemolymph that exited through the abdominal incision was collected within a 1‐cm‐diameter etched ring on a Rite‐On glass slide (Gold Seal). After allowing the hemocytes to adhere to the slide for 20 min at room temperature, cells were fixed and stained using Hema 3 (Fisher Scientific), dried, and mounted under a coverslip using Poly‐Mount (Polysciences) (Hillyer, Schmidt, Fuchs, Boyle, & Christensen, 2005). The total number of hemocytes was then counted under bright‐field illumination at 40X magnification using a Nikon 90i compound microscope (Nikon, Tokyo, Japan). Three independent trials consisting of 15 individuals per treatment group and sex were conducted, and the data were pooled for analysis.