Fig. 2

Constitutive expression of D. immitis Pgp-11 in the mf life-stage of the ML-susceptible Missouri and Berkeley isolates, the putative susceptible Georgia III and Big Head isolates and the ML-resistant JYD-34 isolates was determined using droplet digital PCR. The changes in constitutive expression were calculated relative to the ML-susceptible Missouri isolate, set as the onefold control. The copy numbers of DimPgp-11 were normalized to the copy numbers of the three reference genes: DimGAPDH, DimActin and Dimpmp-3. All data are reported as mean fold change, with error bars corresponding to the standard deviation of three individual replicates. The three biological replicates of each of the five USA laboratory-maintained isolates were prepared at a concentration of 100,000 mf. The significance of the constitutive expression fold changes relative to the Missouri isolate was analyzed by the parametric unpaired t-test with Welch correction, two-tailed P-value and 95% confidence interval using Prism 9.3.0c (GraphPad Software, Inc.). Asterisk indicates a significant difference (P = 0.03) in the constitutive expression of DimPgp-11 between the JYD-34 and Missouri isolates