Selection and Evaluation of Tissue Specific Reference Genes in Lucilia sericata during an Immune Challenge

Abstract

The larvae of the common green bottle fly Lucilia sericata (Diptera: Calliphoridae) have been used for centuries to promote wound healing, but the molecular basis of their antimicrobial, debridement and healing functions remains largely unknown. The analysis of differential gene expression in specific larval tissues before and after immune challenge could be used to identify key molecular factors, but the most sensitive and reproducible method qRT-PCR requires validated reference genes. We therefore selected 10 candidate reference genes encoding products from different functional classes (18S rRNA, 28S rRNA, actin, β-tubulin, RPS3, RPLP0, EF1α, PKA, GAPDH and GST1). Two widely applied algorithms (GeNorm and Normfinder) were used to analyze reference gene candidates in different larval tissues associated with secretion, digestion, and antimicrobial activity (midgut, hindgut, salivary glands, crop and fat body). The Gram-negative bacterium Pseudomonas aeruginosa was then used to boost the larval immune system and the stability of reference gene expression was tested in comparison to three immune genes (lucimycin, defensin-1 and attacin-2), which target different pathogen classes. We observed no differential expression of the antifungal peptide lucimycin, whereas the representative targeting Gram-positive bacteria (defensin-1) was upregulated in salivary glands, crop, nerve ganglion and reached its maximum in fat body (up to 300-fold). The strongest upregulation in all immune challenged tissues (over 50,000-fold induction in the fat body) was monitored for attacin-2, the representative targeting Gram-negative bacteria. Here we identified and validated a set of reference genes that allows the accurate normalization of gene expression in specific tissues of L. sericata after immune challenge.

Fig 1. Distribution of quantification cycle (Cq) values for all L. sericata candidate genes obtained by qRT-PCR.

Data for all naïve and immune-challenged samples were pooled (n = 42). Boxplots show first to third quartile of values in the box, the center line indicates the median, vertical dotted bars extend to the highest and lowest value.

Fig 2. Validation of Pseudomonas aeruginosa immune challenge.

A: Samples of hemolymph from naïve, wounded and immune-challenged larvae were tested in the E. coli zone of inhibition assay, with 100 mg/ml ampicillin as a positive control. Only immune-challenged larvae generated a zone of inhibition. B: Distribution of quantification cycle (Cq) values for immune gene attacin-2 in all naïve and immune-challenged samples. Raw values for all three biological replicates of the six tissue and the larvae samples are displayed separately.

Fig 3. Determination of the optimal number of control genes for normalization.

Pairwise variation (V_n/n+1) analysis between the normalization factors NF_n and NF_n+1 to determine the number of reference genes required for accurate normalization in every individual sample group. The dashed line at 0.15 represents the set threshold below which the number of reference genes is optimal.

Fig 4. Quantitative RT-PCR analysis of lucimycin, defensin-1 and attacin-2 upon immune challenge.

The mRNA expression of lucimycin (A), defensin-1 (B) and attacin-2 (C) was determined in different L. sericata tissues. Relative mRNA expression levels of individual immune genes were compared in between immune-challenged and naïve samples and normalized with two, three or six reference genes.