Metabolic analysis of Drosophila larval hemocytes

(a) Oxygen consumption rates (OCR; pmol/min) and (b) extracellular acidification rates (ECAR; mpH/min) of hemocytes (HmlΔ-Gal4, UAS-EGFP) with different numbers (0.43 x 106 cell/ml, 1.13 x 106 cell/ml, and 1.45 x 106 cell/ml). OCR and ECAR increase in proportion to the number of hemocytes. (c) Schematic overview of metabolic inhibitors used in this study. 2-deoxy-glucose (2-DG), a competitive inhibitor of hexokinase, is a glucose analog that lacks the 2-hydroxyl group. Oligomycin inhibits mitochondria ATP synthase, Complex V. Rotenone or antimycin A inhibit electron transport chain complex I or complex II, respectively. Carbonyl cyanine-4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling chemical that decreases the proton gradient between mitochondria intermembrane space and mitochondrial matrix. (d) Representative Seahorse glycolytic rate assay profile. At baseline, ECAR reflects both glycolytic proton efflux and mitochondrial-derived CO2 hydration. Inhibition of mitochondrial respiration with rotenone and antimycin A (Rot/AA) induces ECAR via elevated compensatory glycolysis. Subsequent 2-DG injection suppresses ECAR sustained by glycolysis. The assay medium for Seahorse glycolytic rate assay contains glucose, pyruvate, and glutamine allowing cells to utilize both glycolysis and mitochondrial respiration. (e) Seahorse glycolytic rate assay using embryonically derived hemocytes. Rot and AA were injected after the 6th measurement and 2-DG, after the 9th measurement (gray). No glycolytic compensation was observed following Rot/AA treatment. The control group (black) received assay medium at volumes matched to the Rot/AA or 2-DG injections. (f) Representative Seahorse glycolysis stress test profile. Glucose was injected to determine glycolysis-induced ECAR levels. Subsequent oligomycin injection inhibits mitochondrial ATP synthase and forces ATP production through glycolysis, indicating glycolytic capacity. Final 2-DG blocks glycolysis. The drop from pre-2-DG ECAR represents the amount of ECAR produced by glycolysis. The remaining ECAR reflects non-glycolytic acidification. The assay medium contains glutamine only (no glucose or pyruvate). (g) Seahorse XF glycolysis stress test using embryonically derived hemocytes. Glucose was injected after the 6th measurement, oligomycin after the 9th measurement, and 2-DG after 12th measurement (gray). Glucose injection marginally increased ECAR, which is subsequently blocked by oligomycin treatment. The final 2-DG injection did not change ECAR in hemocytes. The control group (black) received assay medium lacking glucose and pyruvate at volumes matched to the glucose or oligomycin injections. (h) ATP production rates of larval hemocytes and S2 cells calculated using the Seahorse real-time ATP rate assay. Hemocytes generated 19.9 % of ATP through glycolysis (glycoATP) and 80.1 % by mitochondrial respiration (mitoATP). S2 cells recapitulated this ratio, producing 21.7 % of glycoATP and 78.3 % via mitoATP. (i) Representative Seahorse cell mito stress test profile. The mito stress test measures the key parameters of mitochondrial function, including basal and maximal respiration rates and mitochondrial spare capacities. The oligomycin-induced drop in OCR reflects the ATP-linked mitochondrial respiration and the residual OCR, proton peak. Carbonyl cyanine-4 (trifluoromethoxy) phenylhydrazone (FCCP) injection uncouples the proton gradient across intermembrane and ATP synthesis that promotes maximal respiration of mitochondria. (j) Seahorse XF mito stress test using embryonically derived hemocytes. Oligomycin, FCCP, or Rot/AA was serially injected after the 6th, 9th, or 12th measurement, respectively (gray). Oligomycin injection reduced OCR, which was recovered by FCCP. Subsequent Rot/AA injection further reduced OCR. Controls (black) were injected with the same volume of mito stress test medium. In (h), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.

Metabolic variations of hemocytes during larval development

(a) ATP production rate of Drosophila larval hemocytes at different developmental timepoints. Hemocytes at 72 h after egg laying (AEL) produce 33.5% ATP by glycolysis and 66.5 % by mitochondrial respiration. At 96 h AEL, hemocytes exhibit 16.1 % of glycoATP and 83.9% of mitoATP production rates and generate larger amount of ATP than 72 h or 120 h. Hemocytes at 120 h AEL express 15.7 % of glycoATP and 84.3 % of mitoATP production rates. The ratio of glycoATP reduces over development. (b) ATP-linked respiration levels of hemocytes at 72 h, 96 h, and 120 h AEL. OCR is the highest at 96 h AEL. (c) Proton leak levels of larval hemocytes at different developmental timepoints. Proton leak gradually decreases during development. (d) ATP production rates of circulating or sessile hemocytes. Circulating hemocytes display 18.6 % of glycoATP and 81.4 % of mito ATP production rates. Sessile hemocytes show 10.0 % of glycoATP and 90.0 % of mitoATP production rates. (e) ATP-linked respiration levels of circulating or sessile hemocytes. ATP-linked respiration levels are indistinguishable in circulating or sessile hemocytes. (f) Proton leak levels of circulating or sessile hemocytes. Circulating or sessile hemocytes exhibit similar proton leak levels. In (a) to (f) statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.

Metabolic changes of hemocytes in multiple genetic backgrounds or upon wasp infestation

(a) ATP Production Rate of wild-type (HmlΔ-Gal4, UAS-EGFP/+) or Rasv12-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-RasV12) calculated by Seahorse XF real-time ATP rate assay. Wild-type hemocytes display 22.1 % of glycoATP and 77.9 % of mito ATP production rates while hemocytes expressing Rasv12 exhibit 13.4 % of glycoATP and 96.6 % of mitoATP. Rasv12-expressing hemocytes express higher ATP production rates than wildtype. (b) ATP-linked respiration levels of wild-type hemocytes (HmlΔ-Gal4, UAS-EGFP/+) or Rasv12-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-RasV12). (c) Proton leak levels of hemocytes of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or Rasv12-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-RasV12). (d) Mito stress test measuring OCR levels of wildtype (HmlΔ-Gal4, UAS-EGFP/+; blue) or Rasv12-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP;UAS-RasV12; red). (e) ATP production rate of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or caspase-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-hid,rpr) calculated by Seahorse XF real-time ATP rate assay. Wild-type hemocytes express 22.1% of glycoATP and 77.9% of mito ATP production rates. Genetic ablation of Hml+ hemocytes leads to 13.4% of glycoATP and 13.4% of mitoATP production rates with higher ATP production rates than wild type. (f) ATP-linked respiration levels of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or caspase-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-hid,rpr). (g) Proton leak levels of hemocytes from wildtype (HmlΔ-Gal4, UAS-EGFP/+) or caspase-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-hid,rpr). (h) Mito stress test measuring OCR levels of wildtype (HmlΔ-Gal4, UAS-EGFP/+; blue) or caspase-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-hid,rpr; red). (i) ATP production rate of wild-type (HmlΔ-Gal4, UAS-EGFP/+) or hemocytes expressing hopTum-l (HmlΔ-Gal4, UAS-EGFP; UAS-hopTum-l) measured by Seahorse XF real-time ATP rate assay. Wild type hemocytes express 19.7 % of glycoATP and 80.3 % of mito ATP production rates. Overexpression of hopTum-lin hemocytes results in 14.0 % of glycoATP and 86.0 % of mitoATP production rate and higher ATP production rates. (j) ATP-linked respiration of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or hopTum-l-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-hopTum-l). (k) Proton leak levels of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or hopTum-l-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-hopTum-l). (l) Mito stress test measuring OCR levels of wildtype (HmlΔ-Gal4, UAS-EGFP/+; blue) or hopTum-l -expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-hopTum-l; red). (m) ATP production rate of hemocytes (HmlΔ-Gal4, UAS-EGFP) at 120 h AEL or 48 h post-infestation (48 h PI; equivalent to 120 h AEL) measured by Seahorse XF real-time ATP rate assay. Wild-type hemocytes express 12.2 % of glycoATP and 87.8 % of mito ATP production rates. Immune-activated hemocytes show 14.6 % of glycoATP and 85.4 % of mitoATP production rates with higher ATP production rates than unchallenged controls. (n) ATP-linked respiration levels of control hemocytes at 120 h AEL or 48 h PI. (o) Proton leak levels of hemocytes at 120 h AEL or 48 h PI. (p) Mito stress test measuring OCR levels of hemocytes at 120 h AEL (blue) and 48 h PI (equivalent to 120 h AEL; red). In (d), (h), (l), and (p), oligomycin, FCCP, and Rot/AA were injected after 6th, 9th, or 12th measurement, respectively. Blue or red dashed line indicates average OCR levels from 1st to 6th cycle measurement. In (a)-(c), (e)-(g), (i)-(k), and (m)-(o), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean.Error bars: standard deviation.

Mitochondrial modification in hemocytes upon wasp infestation

(a) Mitochondria morphology of plasmatocytes or lamellocytes at different developmental time points and upon wasp infestation (srp-Gal4 UAS-mito-GFP). Larvae were infected by wasps at 72 h AEL; thus, no corresponding lamellocyte image at this timepoint. 96 h AEL corresponds to 24 h post-infection (PI). 120 h AEL, 48 h PI. Phalloidin, red; mito-GFP; green, DAPI, blue. Scale bar: 5 μm (white), 20 μm (yellow) (b) The number and (c) size of mitochondria per cell at different developmental time points or upon wasp infection conditions (d) Dot plot expression of significantly changed metabolic genes across cell clusters. PM: plasmatocytes under conventional conditions, PM (inf.): plasmatocytes under wasp infestation, CC: crystal cells under conventional conditions, CC (inf.): crystal cells under wasp infestation, LM (inf.): lamellocytes under wasp infestation. Dot size represents the percentage of cells expressing each gene. Dot color shows average expression levels of metabolic genes. (e) Encapsulation of wasp eggs in Drosophila larvae at 60 h post-infestation (60 h PI). Control larvae (HmlΔ-Gal4, UAS-EGFP/+) containing melanized wasp eggs (red arrowhead) (left). Hemocyte-specific Drp1 RNAi (HmlΔ-Gal4, UAS-EGF, Drp1 RNAi) reduces the rate of wasp egg encapsulation (right). Yellow arrowhead marks the wasp ovipositor injection site. Scale bar: 1 mm (black) (f) Quantification of encapsulation rates shown in (e). Each dot represents the encapsulation ratio from each trial (n=147, 230 respectively). (g) A schematic diagram correlating metabolic gene expressions, mitochondrial morphology, and metabolic modification in plasmatocytes and lamellocytes. After wasp infestation hemocytes differentiate into lamellocytes and increase the expression of sugar transporters (CG1208, CG4607 and Tret1-1) and trehalase (Treh) to enhance sugar uptake. In addition, lamellocytes increase both the number and size of mitochondria, leading to an increased capacity for oxidative phosphorylation (OXPHOS). (h) Quantification of ECAR following glucose or trehalose injection corresponding to Supplementary Figure 5(b) and (c). Pink shade indicates glucose-injected samples; purple indicates trehalose. Black bar denotes hemocytes upon 48 h post-infection (corresponding to 120 h AEL). (i) Glycemic levels of larval hemolymph at different developmental time points (96 h AEL and 120 h AEL) and infestation condition (24 h PI and 48 h PI). Green shading indicates wasp infested conditions. (j) Encapsulation of wasp eggs in Drosophila larvae at 60 h post-infestation (60 h PI). Wild-type larvae containing melanized wasp eggs (red arrowhead) (left). Hemocyte-specific Hex-A RNAi (srp-Gal4 UAS-Treh RNAi) or Treh RNAi (srp-Gal4 UAS-Hex-A RNAi) reduced the rate of wasp egg encapsulation (middle and right). Yellow arrowhead marks the wasp ovipositor injection site. Scale bar: 1 mm (black) (k) Quantification of encapsulation rates shown in (h). Each dot represents the encapsulation ratio from 40 animals (n=200, 160, 120, respectively). In (b), (c), (f), (h), (i) and (k), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.

Seahorse XF assays using embryonically derived hemocytes.

(a) Glycolysis stress test of Drosophila larval brain. Glucose, oligomycin, or 2-DG was injected after the 6th, 9th, or 12th measurement, respectively (gray). Controls (black) received assay medium only at volumes matched to glucose, oligomycin, or 2-DG injections. (b) Basal OCR levels of embryonically derived hemocytes (HmlΔ-Gal4, UAS-EGFP) with different cell density (0.43 x 106 cells/ml, green; 1.13 x 106 cells/ml, red; 1.45 x 106 cells/ml, blue). Measurement was performed up to 15 cycles (90 minutes). (c) Basal ECAR levels of embryonically derived hemocytes (HmlΔ-Gal4, UAS-EGFP) with different cell density (0.43 x 106 cells/ml, green; 1.13 x 106 cells/ml, red; 1.45 x 106 cells/ml, blue). Measurement was performed up to 15 cycles (90 minutes). (d) Glycolytic rate assay (OCR) using embryonically derived hemocytes (HmlΔ-Gal4, UAS-EGFP). Rot/AA complex was injected after the 6th cycle and 2-deoxy-glucose (2-DG), after the 9th cycle (gray). Controls (black) received assay medium only at volumes matched. (e) Real-time ATP rate assay (OCR) using embryonically derived hemocytes (HmlΔ-Gal4, UAS-EGFP). Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle (gray). Controls (black) received assay medium only at volumes matched. (f) Real-time ATP rate assay (ECAR) using embryonically derived hemocytes (HmlΔ-Gal4, UAS-EGFP). Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle (gray). Controls (black) received assay medium only at volumes matched. (g) ATP production rate of hemocytes from two genetic backgrounds, Oregon R (OreR) and HmlΔ-Gal4 UAS-EGFP. OreR displays 15.3 % glycoATP and 84.7 % mitoATP. HmlΔ-Gal4 UAS-EGFP shows 14.6 % glycoATP and 85.3% mitoATP. (h) Real-time ATP rate assay (OCR) using hemocytes from OreR (gray) or HmlΔ-Gal4 UAS-EGFP (black). Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle. (i) Real-time ATP rate assay (ECAR) using hemocytes from OreR (gray) or HmlΔ-Gal4 UAS-EGFP (black). Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle. (j) Real-time ATP rate assay (OCR) using hemocytes (HmlΔ-Gal4 UAS-EGFP; black) or S2 cells (gray). Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle. (k) Real-time ATP rate assay (ECAR) using hemocytes (HmlΔ-Gal4 UAS-EGFP; black) or S2 cells (gray). Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle. In (g), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.

Real-time ATP rate assay using Drosophila larval hemocytes.

(a) Real-time ATP rate assay (OCR) using hemocytes at 72 h (blue), 96 h (red), or 120 h (green) AEL. Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle. (b) ATP levels of Drosophila hemocytes at 96 h (left) or 120 h AEL (right). It was unfeasible to measure ATP levels of hemocytes at 72 h AEL. (c) Real-time ATP rate assay (ECAR) using hemocytes at 72 h (blue), 96 h (red), or 120 h (green) AEL. Oligomycin was injected after the 6th cycle and Rot/AA, after the 9th cycle. (d) Real-time ATP rate assay (OCR) using larval circulating (blue) or sessile (red) hemocytes. Oligomycin was injected after 6th cycle and Rot/AA, after 9th cycle. (e) Real-time ATP rate assay (ECAR) using larval circulating (blue) or sessile (red) hemocytes. Oligomycin was injected after 6th cycle; Rot/AA, after 9th cycle; and 2-DG, after 12th cycle. In (b), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.

Metabolic measurement of larval hemocytes from different genetic backgrounds

(a) Quantification of the number of total hemocytes per one larva in each genotype. HmlΔ-Gal4, UAS-EGFP/+ and HmlΔ-Gal4, UAS-EGFP; UAS-RasV12. (b) Quantification of the number of lamellocytes per one larva in each genotype. HmlΔ-Gal4, UAS-EGFP/+ and HmlΔ-Gal4, UAS-EGFP; UAS-RasV12. (c) Real-time ATP rate assay (OCR) using hemocytes in HmlΔ-Gal4, UAS-EGFP/+ (blue) and HmlΔ-Gal4, UAS-EGFP; UAS-RasV12 (red). (d) Real-time ATP rate assay (ECAR) using hemocytes in HmlΔ-Gal4, UAS-EGFP/+ (blue) and HmlΔ-Gal4, UAS-EGFP; UAS-RasV12 (red). (e) Real-time ATP rate assay (OCR) using hemocytes in HmlΔ-Gal4, UAS-EGFP/+ (blue) and HmlΔ-Gal4, UAS-EGFP; UAS-hid,rpr (red). (f) Real-time ATP rate assay (ECAR) using hemocytes in HmlΔ-Gal4, UAS-EGFP/+ (blue) and HmlΔ-Gal4, UAS-EGFP; UAS-hid,rpr (red). (g) Quantification of the number of crystal cells per imaging area (a square imaging area of 638.9 µm × 638.9 µm) in each genotype. HmlΔ-Gal4, UAS-EGFP/+ (left) and HmlΔ-Gal4, UAS-NICD (right). Each dot represents the average of four imaging spots from one larva. (h) ATP production rate of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or NICD-expressing hemocytes (HmlΔ-Gal4, UAS-NICD). (i) ATP-linked respiration levels of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or NICD-expressing hemocytes (HmlΔ-Gal4, UAS-NICD). (j) Proton leak levels of wildtype (HmlΔ-Gal4, UAS-EGFP/+) or NICD-expressing hemocytes (HmlΔ-Gal4, UAS-NICD). (k) Mito stress test measuring OCR levels of wild type (HmlΔ-Gal4, UAS-EGFP/+; blue) or NICD-expressing hemocytes (HmlΔ-Gal4, UAS-NICD; red). Due to the fragile nature of crystal cells, control incubation time was reduced. Oligomycin was injected after 3rd cycle; FCCP after 6th cycle; Rot/AA after 9th cycle. In (c), (d), (e), and (f), oligomycin was injected after 6th cycle measurement. Rot/AA were injected after 9th cycle measurement. In (a), (b), and (g)-(j) statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.

Metabolic measurement of larval hemocytes upon immune activation.

(a) Quantification of DAPI-positive embryonic hemocytes in HmlΔ-Gal4, UAS-EGFP/+ and HmlΔ-Gal4, UAS-EGFP; UAS-HopTuml. (b) Quantification of Hml-positive embryonic hemocytes HmlΔ-Gal4, UAS-EGFP/+ and HmlΔ-Gal4, UAS-EGFP; UAS-HopTuml. (c) Quantification of lamellocytes among embryonic hemocyte in HmlΔ-Gal4, UAS-EGFP/+ and HmlΔ-Gal4, UAS-EGFP; UAS-HopTuml. (d) Real-time ATP rate assay (OCR) using wild-type hemocytes (HmlΔ-Gal4, UAS-EGFP/+) or HopTuml-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-HopTuml). (e) Real-time ATP rate assay (ECAR) using wild-type hemocytes (HmlΔ-Gal4, UAS-EGFP/+) or HopTuml-expressing hemocytes (HmlΔ-Gal4, UAS-EGFP; UAS-HopTuml). (f) Real-time ATP rate assay (OCR) using (HmlΔ-Gal4, UAS-EGFP) at 96 h AEL or 24 h post-infestation (24 h PI; equivalent to 96 h AEL). (g) Real-time ATP rate assay (ECAR) using (HmlΔ-Gal4, UAS-EGFP) at 96 h AEL or 24 h post-infestation (24 h PI; equivalent to 96 h AEL). (h) Real-time ATP rate assay (OCR) using (HmlΔ-Gal4, UAS-EGFP) at 120 h AEL or 48 h post-infestation (48 h PI; equivalent to 120 h AEL). (i) Real-time ATP rate assay (ECAR) using (HmlΔ-Gal4, UAS-EGFP) at 120 h AEL or 48 h post-infestation (48 h PI; equivalent to 120 h AEL). In (d), (e), (f), (g), (h), and (i), oligomycin was injected after 6th cycle measurement. Rot/AA were injected after 9th cycle measurement. In (a)-(c), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.

Metabolic modification in hemocytes upon wasp infestation.

(a) Dot plot expression of Marf and Drp1 across cell clusters. PM: plasmatocytes under conventional conditions, PM (inf.): plasmatocytes under wasp infestation, CC: crystal cells under conventional conditions, CC (inf.): crystal cells under wasp infestation, LM (inf.): lamellocytes under wasp infestation. Dot size represents the percentage of cells expressing each gene. Dot color shows average expression levels of each gene. (b) Quantification of lamellocytes among circulating hemocytes in HmlΔ-Gal4 UAS-GFP/+ or HmlΔ-Gal4 UAS-GFP UAS-Drp1 RNAi at 48 h post-infestation (48 h PI; equivalent to 120 h AEL). (c) Seahorse XF glycolysis stress test using wild-type hemocytes. Glucose injection (blue) marginally increased ECAR, which was subsequently blocked by oligomycin. The final 2-DG injection did not alter ECAR in hemocytes. This phenotype was not recapitulated by trehalose (red) or the control group (black). Controls received assay medium lacking substrates at volumes matched to the substrate, oligomycin, or 2-DG injections. (d) Seahorse XF glycolysis stress test using hemocytes at 48 h post-infestation (PI). Both glucose (blue) and trehalose (red) marginally increased ECAR, which was subsequently blocked by oligomycin treatment. The final 2-DG injection did not change ECAR in hemocytes. Controls (black) received assay medium lacking substrates at volumes matched to the substrate, oligomycin, or 2-DG injections. In (c) and (d), metabolic substrate was injected after 3rd cycle measurement. Oligomycin was injected after 9th cycle measurement. 2-DG was injected after 12th cycle measurement. In (b), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). Bars in graphs: the mean. Error bars: standard deviation.

Knockdown of hex-A and treh shows no significant effect on hemocyte differentiation.

(a) Quantification of DAPI-positive embryonic hemocytes in Srp-Gal4/+, Srp-Gal4 UAS-Hex-A RNAi, and Srp-Gal4 UAS-Treh RNAi at 48 h post-infestation (48 h PI; equivalent to 120 h AEL). (b) Quantification of Pxn-positive embryonic hemocytes in Srp-Gal4/+, Srp-Gal4 UAS-Hex-A RNAi, and Srp-Gal4 UAS-Treh RNAi at 48 h post-infestation (48 h PI; equivalent to 120 h AEL). (c) Quantification of lamellocytes among embryonic hemocyte in Srp-Gal4/+, Srp-Gal4 UAS-Hex-A RNAi, and Srp-Gal4 UAS-Treh RNAi at 48 h post-infestation (48 h PI; equivalent to 120 h AEL). In (a)-(c), statistical analysis was performed using unpaired t-test. n.s: not significant (p > 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. Bars in graphs: the mean. Error bars: standard deviation.