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. 2014 Aug 25;15:714.
doi: 10.1186/1471-2164-15-714.

Olive Fly Transcriptomics Analysis Implicates Energy Metabolism Genes in Spinosad Resistance

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Free PMC article

Olive Fly Transcriptomics Analysis Implicates Energy Metabolism Genes in Spinosad Resistance

Efthimia Sagri et al. BMC Genomics. .
Free PMC article

Abstract

Background: The olive fly, Bactrocera oleae, is the most devastating pest of cultivated olives. Its control has been traditionally based on insecticides, mainly organophosphates and pyrethroids. In recent years, the naturalyte spinosad is used against the olive fly. As with other insecticides, spinosad is subject to selection pressures that have led to resistance development. Mutations in the α6 subunit of the nicotinic acetylcholine receptor (nAChR) have been implicated in spinosad resistance in several species (e.g., Drosophila melanogaster) but excluded in others (e.g., Musca domestica). Yet, additional mechanisms involving enhanced metabolism of detoxification enzymes (such as P450 monooxygenases or mixed function oxidases) have also been reported. In order to clarify the spinosad resistance mechanisms in the olive fly, we searched for mutations in the α6-subunit of the nAChR and for up-regulated genes in the entire transcriptome of spinosad resistant olive flies.

Results: The olive fly α6-subunit of the nAChR was cloned from the laboratory sensitive strain and a spinosad selected resistant line. The differences reflected silent nucleotide substitutions or conserved amino acid changes. Additionally, whole transcriptome analysis was performed in the two strains in order to reveal any underlying resistance mechanisms. Comparison of over 13,000 genes showed that in spinosad resistant flies nine genes were significantly over-expressed, whereas ~40 were under-expressed. Further functional analyses of the nine over-expressed and eleven under-expressed loci were performed. Four of these loci (Yolk protein 2, ATP Synthase FO subunit 6, Low affinity cationic amino acid transporter 2 and Serine protease 6) showed consistently higher expression both in the spinosad resistant strain and in wild flies from a resistant California population. On the other side, two storage protein genes (HexL1 and Lsp1) and two heat-shock protein genes (Hsp70 and Hsp23) were unfailingly under-expressed in resistant flies.

Conclusion: The observed nucleotide differences in the nAChR-α6 subunit between the sensitive and spinosad resistant olive fly strains did not advocate for the involvement of receptor mutations in spinosad resistance. Instead, the transcriptome comparison between the two strains indicated that several immune system loci as well as elevated energy requirements of the resistant flies might be necessary to lever the detoxification process.

Figures

Figure 1
Figure 1
Basic characteristics of the Bactrocera oleae nAChR α6 subunit. N-terminal site is presented in dashed line and it is consisted of 20 amino acids. There are four transmembrane domains (TM1-4) (bold italic letters) and three glycosylation sites (blue boxes). The YxCC motif of alpha subunits is shown in orange box and the Cystein residues in green ovals. Six ligand binding loops are underlined. The three mutations are indicated by vertical arrows.
Figure 2
Figure 2
Functional annotation of differentially expressed genes. Gene expression levels of the differentially expressed genes (Log2, fold change), as resulted from the RNA-seq analysis, is shown at the left part of the Figure. Gene Ontology (GO) classification of the same genes for the ontologies: Biological Process (BP), Molecular Function (MF), and Interpro (IP) protein domains, are listed at the right part of the Figure. In crimson red are the up-regulated genes. The non-statistically significantly up-regulated Cytochrome P450 6a23-like (Cyp6α23) is shown in lighter color. In green are the down-regulated genes.
Figure 3
Figure 3
M-A plot of the differential expression between sensitive and resistant flies. The red color spots are the statistical significant differentially expressed genes with q-value < 0.05.
Figure 4
Figure 4
Relative expression profiles of genes potentially associated with spinosad resistance. The red color bars represent the up-regulated genes, Yolk protein 2 (Yp2, Panel A), ATP synthase F O subunit 6 (ATP synthase, Panel B), Low affinity cationic amino acid transporter 2 (CAT-2, Panel C), Serine protease 6 (SP6, Panel D), 4-nitrophenylphophatase (pNPPase, Panel E), Salivary Cys-rich secreted peptide-vWF (SalCys, Panel F), Cytochrome P450 6a23-like (Cyp6α23, Panel G) and Antigen 5 precursor (Ant5, Panel H), for the mean of three male and three female individual flies, after functional analysis by qRT-PCR. Only for the Yolk protein the evaluation was based on female expression, since males show zero expression values. The green color bars represent the down-regulated genes Heat-shock protein 70 (Hsp70, Panel I), Heat-shock protein 23 (Hsp23, Panel J), Larval serum protein 1 (LSP1, Panel K), Hexamerin L1 (HexL1, Panel L), Chitinase 5 (Cht5, Panel M), Oxidase/peroxidase (oxidase, Panel N), Macrophage mannose receptor 1 (mmr1, Panel O), Cell division cycle-associated protein 7 (Cdc, Panel P), for the mean of three male and three female individual flies, after functional analysis by qRT-PCR. The five RNA viral genes are not included. Standard error is also depicted in the bars. Small letters next to the error bars indicate significantly different mean values estimated by pairwise comparisons (either Tukey’s or Kruskal-Wallis tests). All comparisons were performed on Ln transformed data except for macrophage mannose receptor 1.
Figure 5
Figure 5
Relative expression of Cyp6α23 and Yolk protein in individual flies. Relative expression of Cyp6α23 (Panel A) and Yolk protein 2 (Panel B) gene loci in the heads of individual olive flies of the w-GR (brown color bars), LAB (blue bars), w-CAL (orange bars) and SPIN (green bars) populations after functional analysis by qRT-PCR.
Figure 6
Figure 6
STRING analysis. The network displays the predictions of protein interaction and association with experimentally determined interactions plus those from the literature of the selected gene list of up-regulated as well as down-regulated gene-products. The input gene list included the following genes: CAT-2 (CG5535), Serine protease 6 (CG2071), Yolk protein 2 (Yp2, CG2979), pNPPase (CG5567), Lsp1 (CG6821), oxidase (CG10211), MMR (CG9134), Cytochrome P450 6a23-like (Cyp6a23, CG10242), vWF domain (CG32667) and chitinase (CG9307). Network was enlarged based on Drosophila protein interactions. The ATP synthase gene (CG8189) and the Hsps were withdrawn from the list, because the resulting network was very dense and uninterpretable. Interestingly, the gene Ag5r2 (Antigen 5) even if it was also absent from the input list, it appears to be correlated with the other genes, supporting our hypothesis of interacting pathways. STRING Version 9.O was used for this analysis. Different colored edges indicate the types of evidence used in predicting the associations1. Up-regulated genes are indicated by red arrows, whereas down-regulated ones by green arrows. 1A red line indicates the presence of fusion evidence; a green line - neighborhood evidence; a blue line - coocurrence evidence; a purple line - experimental evidence; a yellow line - textmining evidence; a light blue line - database evidence; a black line - coexpression evidence.

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