Comparative transcriptome analysis of lufenuron-resistant and susceptible strains of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Abstract

Background: The evolution of insecticide resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) has resulted in large economic losses and disturbances to the environment and agroecosystems. Resistance to lufenuron, a chitin biosynthesis inhibitor insecticide, was recently documented in Brazilian populations of S. frugiperda. Thus, we utilized large-scale cDNA sequencing (RNA-Seq analysis) to compare the pattern of gene expression between lufenuron-resistant (LUF-R) and susceptible (LUF-S) S. larvae in an attempt to identify the molecular basis behind the resistance mechanism(s) of S. frugiperda to this insecticide.

Results: A transcriptome was assembled using approximately 19.6 million 100 bp-long single-end reads, which generated 18,506 transcripts with a N50 of 996 bp. A search against the NCBI non-redundant database generated 51.1% (9,457) functionally annotated transcripts. A large portion of the alignments were homologous to insects, with the majority (45%) being similar to sequences of Bombyx mori (Lepidoptera: Bombycidae). Moreover, 10% of the alignments were similar to sequences of various species of Spodoptera (Lepidoptera: Noctuidae), with 3% of them being similar to sequences of S. frugiperda. A comparative analysis of the gene expression between LUF-R and LUF-S S. frugiperda larvae identified 940 differentially expressed transcripts (p ≤ 0.05, t-test; fold change ≥ 4). Six of them were associated with cuticle metabolism. Of those, four were overexpressed in LUF-R larvae. The machinery involved with the detoxification process was represented by 35 differentially expressed transcripts; 24 of them belonging to P450 monooxygenases, four to glutathione-S-transferases, six to carboxylases and one to sulfotransferases. RNA-Seq analysis was validated for a number of selected candidate transcripts by using quantitative real time PCR (qPCR).

Conclusions: The gene expression profile of LUF-R larvae of S. frugiperda differs from LUF-S larvae. In general, gene expression is much higher in resistant larvae when compared to the susceptible ones, particularly for those genes involved with pathways for xenobiotic detoxification, mainly represented by P450 monooxygenases transcripts. Our data indicate that enzymes involved with the detoxification process, and mostly the P450, are one of the resistance mechanisms employed by the LUF-R S. frugiperda larvae against lufenuron.

Fig. 1

Distribution of differentially expressed transcripts from an RNA-seq analysis of susceptible (LUF-S) and lufenuron-resistant (LUF-R) strains of S. frugiperda, induced or non-induced by lufenuron

Fig. 2

Distribution of the transcripts of the S. frugiperda transcriptome based on the comparative analysis of the gene expression of susceptible (LUF-S) and lufenuron-resistant (LUF-R) strains, induced or non-induced by lufenuron. Marked in green are the transcripts with a significant difference in the expression level, based on the discriminative significance values (t-test, p < 0.05) and relative expression (>4) adopted here

Fig. 3

Distribution of gene ontology (GO) attributed to differentially expressed transcripts in susceptible (LUF-S) and lufenuron-resistant (LUF-R) S. frugiperda strains

Fig. 4

Comparative distribution of functionally annotated transcripts of lufenuron-susceptible (LUF-S) and lufenuron-resistant (LUF-R) S. frugiperda strains that showed changes in the expression level. RPKM values were represented as a scale of colors ranging from green to red, which will encompass values from the lowest (green) to the highest (red) RPKM values

Fig. 5

Comparative distribution of transcripts associated with cuticle metabolism of lufenuron-susceptible (LUF-S) and lufenuron-resistant (LUF-R) S. frugiperda strains that showed changes in the expression level. RPKM values were represented as a scale of colors ranging from green to red, which will encompass values from the lowest (green) to the highest (red) RPKM values

Fig. 6

Comparative distribution of transcripts associated with detoxification enzymes of lufenuron-susceptible (LUF-S) and lufenuron-resistant (LUF-R) S. frugiperda strains that showed changes in the expression level. RPKM values were represented as a scale of colors ranging from green to red, which will encompass values from the lowest (green) to the highest (red) RPKM values. The title of each transcript consists of the identification of genes and code of the transcript. In P450’s transcribed capital letter and number is the class ID and group associated with gene superfamily

Fig. 7

qPCR analysis of selected CYP transcripts identified as differentially expressed in a broad RNA-Seq analysis of the larval transcriptome of susceptible (LUF-S) and resistant (LUF-R) strains of S. frugiperda to lufenuron, exposed (induced) or not (non-induced) to lufenuron treatment. Expression of the selected genes is provided as fold change (ΔΔCt) using their expression at the LUF-S, non-induced larvae as a reference