Developmental stage- and sex-specific expression of immune-related genes in N. lugens Total RNA was extracted from eggs, 2nd instar nymphs, 5th instar nymphs, female adults and male adults, individually. First-strand cDNA (20 ng) was analyzed in each qRT-PCR reaction. The reactions were performed with specific primers for amplifying (A) PGRP/GRP genes; (B) Toll genes; (C) CLIP genes; and (D) immune effector genes. The relative expression levels of each gene in each developmental stage or sex were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values that were obtained from reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set at one. Three technical replication (n=3) was conducted and the ΔΔCt method was used to measure the relative transcript levels in each treated sample.