Spodoptera frugiperda transcriptional response to infestation by Steinernema carpocapsae

Abstract

Steinernema carpocapsae is an entomopathogenic nematode (EPN) used in biological control of agricultural pest insects. It enters the hemocoel of its host via the intestinal tract and releases its symbiotic bacterium Xenorhabdus nematophila. In order to improve our knowledge about the physiological responses of its different hosts, we examined the transcriptional responses to EPN infestation of the fat body, the hemocytes and the midgut in the lepidopteran pest Spodoptera frugiperda. The tissues poorly respond to the infestation at an early time post-infestation of 8 h with only 5 genes differentially expressed in the fat body of the caterpillars. Strong transcriptional responses are observed at a later time point of 15 h post-infestation in all three tissues. Few genes are differentially expressed in the midgut but tissue-specific panels of induced metalloprotease inhibitors, immune receptors and antimicrobial peptides together with several uncharacterized genes are up-regulated in the fat body and the hemocytes. Among the most up-regulated genes, we identified new potential immune effectors, unique to Lepidoptera, which show homology with bacterial genes of unknown function. Altogether, these results pave the way for further functional studies of the responsive genes' involvement in the interaction with the EPN.

Figure 1

Tissue specific transcriptional response time series of Spodoptera frugiperda larvae to EPN infestation. (A) Overview of the experimental design. In three independent experiments, 9 infested and 9 control larvae from culture plates were dissected at 8 hpi and 15 hpi. Hemocytes, fat bodies and midguts were extracted and pooled by organ for each time and condition. Polyadenylated RNAs were purified from these pools and corresponding cDNA libraries were built. cDNA were sequenced on a single end by Illumina and RNAseq data were analyzed to identify the genes that are differentially expressed during Steinernema carpocapsae infestation. (B) Growth of Xenorhabdus nematophila following S. frugiperda infestation with 150 symbiotic S. carpocapsae. At 8 hpi and 15 hpi, the number of CFU per mL of hemolymph was estimated from three independent experiments with three technical replicates (three larvae per technical replicate). Error bars indicate standard errors of the means. (C) Survival curve. Larvae were infested with 150 nematobacterial IJ. Data represent means ± SEs of four independent experiments, each containing 12 larvae. (D) Significantly differentially expressed genes in response to EPN. This heatmap shows z-score of expression variation across all RNAseq samples (red being overexpressed and blue under-expressed) for 271 genes with significant variations to EPN infestation. (E) Venn diagram showing the tissue specificity of the EPN responsive genes at 15 hpi. The response can be overexpression (Up:U) or under-expression (Down:D). For example, there are 71 genes varying significantly upon EPN infestation in both the fat body (FB) and the hemocytes (HC). Of these, 66 are up-regulated in both tissues (UU), 3 are down-regulated in both tissues (DD) and 2 are down-regulated in FB and up-regulated in HC (DU). By convention, the order of the U and D letters represent respectively MG, FB and HC tissues.

Figure 2

Midgut Associated Response. Heatmaps of differential expression (in log2FoldChange - green, under-expression, red, overexpression according to values in Supplementary Data S1) across all experimental conditions of genes found significantly differentially expressed (A) specifically in the MG at 15 hpi (MG15 - 4 genes) (B) common to MG15 and FB15 (9 genes) and (C) common to MG15 and HC15 (10 genes).

Figure 3

Fat body and hemocytes associated responses. As in Fig. 2, heatmaps of differential expression for genes (A) specific to FB15 (14 genes), (B) specific to HC15 (77 genes) and (C) common to FB15 and HC15 (71 genes).

Figure 4

Inducible MetalloProtease Inhibitor. (A) WebApollo viewer showing the annotation of inducible metalloprotease inhibitors genes in cluster on the scaffold_1741 in the genome of Spodoptera frugiperda. (B) As in Fig. 2, heatmap of differential expression for the identified IMPI genes.

Figure 5

Expression for the 31 genes common to midgut, the fat body and the hemocytes. (A) As in Figs 2 and 3, heatmap of differential expression for the 31 genes common to MG15, FB15 and HC15. (B) WebApollo viewer showing the annotation of unknown genes in cluster on the scaffold_520. (C) WebApollo viewer showing the annotation of clustered genes of bacterial origin within a defensin cluster.