2.7. Expression of melanization and anti-microbial peptide genes

Invertebrates lack an adaptive immune system and hence, innate immune system plays an important role in host defense against pathogens. Antimicrobial peptides (AMPs) represent the front lines in the coevolutionary struggle between host and pathogen as they are the main effector molecules of innate immunity in many organisms (Hanson et al., 2019). Drosophila larvae compete with fungi and bacteria for nutrients. We isolated RNA from whole body of 10 late L3 female larvae per sample and measured mRNA transcript levels of anti-microbial peptide genes grouped into three families based on their main biological targets, gram-positive bacteria (Def), gram-negative bacteria (CecA1, Dro, AttA, Dpt), or fungi (Drs, Mtk) (Imler and Bulet, 2005). Further we quantified the transcript level of genes involved in melanization (PPO1 and PPO2). Similarly, we also measured mRNA expression level of eater and NimC1-genes involved in adherence of hemocyte to sessile chambers; and Tep3 which along with eater and NimC1 is involved in phagocytosis.

Total RNA was extracted from each sample using TRI Reagent (Sigma, T9424), and the quality of the RNA was determined using a NanoDrop 2000 spectrophotometer at 260 and 280 nm absorbance; a 260/280 ratio close to 2 was considered as pure RNA. To remove any genomic DNA contamination, RNA aliquot was processed with RQ1 DNase (M6101; Promega, USA) and reverse transcribed for cDNA synthesis using the first strand cDNA synthesis kit (Cat no. K1622; Thermo Fisher Scientific). Real-time quantitative polymerase chain reaction (qPCR) with SYBR Green chemical MasterMix (Cat no. A25742, Applied Biosystems by Thermo Fischer Scientific) was used to quantify the mRNA expression level of the gene of interest on a ViiA7 thermal cycler (Applied Biosystems, Foster City, CA, USA). We used gene specific primer designed using Primer3 and validated in silico on NCBI blast (Table 1). Total reaction mixture of 6 μl had 3 μl SYBR Green PCR MasterMix, 1 μl forward primer (1 μM), 1 μl reverse primer (1 μM), and 1 μl (10 ng/l) cDNA. Both, genes of interest and the reference (Rp49) gene were run in duplicates. The fold change in 2-(Ct) value was used to calculate mRNA expression (Livak and Schmittgen, 2001). In a nutshell, fluorescence exceeding background level gave cycle threshold (Ct). ΔCt was calculated (Ct target gene - Ct reference gene). Ct values were normalized using Ct value of pool sample that consisted of a mix of cDNA of all samples (ΔΔCt) from both populations. Fold change was calculated by negative value of ΔΔCt powered to 2 (2^−ΔΔCt) and was plotted.

Primers used in the study.