Characterization and expression profiling of microRNAs in response to plant feeding in two host-plant strains of the lepidopteran pest Spodoptera frugiperda

Abstract

Background: A change in the environment may impair development or survival of living organisms leading them to adapt to the change. The resulting adaptation trait may reverse, or become fixed in the population leading to evolution of species. Deciphering the molecular basis of adaptive traits can thus give evolutionary clues. In phytophagous insects, a change in host-plant range can lead to emergence of new species. Among them, Spodoptera frugiperda is a major agricultural lepidopteran pest consisting of two host-plant strains having diverged 3 MA, based on mitochondrial markers. In this paper, we address the role of microRNAs, important gene expression regulators, in response to host-plant change and in adaptive evolution.

Results: Using small RNA sequencing, we characterized miRNA repertoires of the corn (C) and rice (R) strains of S. frugiperda, expressed during larval development on two different host-plants, corn and rice, in the frame of reciprocal transplant experiments. We provide evidence for 76 and 68 known miRNAs in C and R strains and 139 and 171 novel miRNAs. Based on read counts analysis, 34 of the microRNAs were differentially expressed in the C strain larvae fed on rice as compared to the C strain larvae fed on corn. Twenty one were differentially expressed on rice compared to corn in R strain. Nine were differentially expressed in the R strain compared to C strain when reared on corn. A similar ratio of microRNAs was differentially expressed between strains on rice. We could validate experimentally by QPCR, variation in expression of the most differentially expressed candidates. We used bioinformatics methods to determine the target mRNAs of known microRNAs. Comparison with the mRNA expression profile during similar reciprocal transplant experiment revealed potential mRNA targets of these host-plant regulated miRNAs.

Conclusions: In the current study, we performed the first systematic analysis of miRNAs in Lepidopteran pests feeding on host-plants. We identified a set of the differentially expressed miRNAs that respond to the plant diet, or differ constitutively between the two host plant strains. Among the latter, the ones that are also deregulated in response to host-plant are molecular candidates underlying a complex adaptive trait.

Keywords: Adaptation; Insect plant interaction; Phenotypic plasticity; Regulation of gene expression; microRNAs.

Fig. 1

Size profiling of small non coding RNAs and their homology to different RNA classes or to Transposable Elements (TE). The percentage of sncRNA reads is plotted as a function of their size (between 15 nt to 40 nt corresponding to the size range that has been selected from the gel for library construction), a and c SfC, c and d SfR, in green on corn, in red on rice. CC: SfC on corn, CR: SfC on rice, RC: SfR on rice, RR: SfR on rice. a and b total reads, c and d unique reads. e and f Pie charts representing the average % of reads (total counts from 2 replicates on corn for SfC (e) or SfR (f)) mapping either to SfC or plant miR precursors, or TE (SfC TE copies) as expected for putative endo-siRNA or piRNAs, or mRNA (SfC OGS2.2), or SfC tRNA, SfC rRNA (18S and 28S RNAs)

Fig. 2

Base composition of known or novel mature miRNAs

Fig. 3

Differential expression of miRNAs genes on rice compared to corn in Spodoptera frugiperda larvae (L4 instar), after rearing for 3 generations on whole plants. The miRNAs showing a significant differential expression after DESeq2 analysis (log2foldchange > 1 or < 1 and FDR < 0.05) are shown. a In SfC b In SfR. In red, miRNAs up-regulated on rice, in green miRNAs up-regulated on corn

Fig. 4

Differential expression of miRNAs genes according to the genetic background. The relative expression of miRNAs in SfR compared to SfC is shown, either on corn (a) or on rice (b). In red, miRNAs up-regulated in SfR, in green miRNAs up-regulated in SfC

Fig. 5

Are the constitutive expression differences between strains involved in phenotypic plasticity within strains? This Venn diagram highlights the miRNAs that are differentially expressed (FDR < 0.05) both between strains on the same plant and within strain (SfC: (a), SfR: (b)) on rice compared to corn

Fig. 6

Examples of complementarity between miRNAs seed sequences and the secondary structure of their putative targets. Using mfold [25] to predict and draw secondary structures of 3’UTR of the predicted targets, we could show that miR-34 and miR-190 map to loops rather than stems in the secondary structures of their targets, takeout (GSSPFG00021718001-RA) in (a) and acyl-CoA desaturase (GSSPFG00006314001-RA) in (b), their most differentially expressed candidates, respectively