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Antimicrobial peptides (AMPs) and phenoloxidase (PO) are key effectors of the innate immune response of insects. AMPs are synthesized by the fat body of insects (Drosophila, Lepidoptera...) and are found in the hemolymph a few hours after a bacterial injection. Resistance to AMPs is recognized as an important virulence phenotype in human pathogenic bacteria but remains to be investigated for insect pathogenic bacteria. We have previously shown that recognition of Xenorhabdus and Photorhabdus by the Drosophila immune system is controlled by the Imd signaling pathway (Aymeric et al, 2010), the activation of which leads to the induction of AMPs synthesis. In collaboration with Egyptian colleagues, we also showed that the Imd pathway regulates the expression of lysozyme-like proteins (LLPs) in silkworm (Satyavathi et al, 2018). Over the past decades, numerous bacterial virulence effectors with immunosuppressive properties have been identified (Nuñez-Valdez et al, 2019). Nevertheless, only a few studies have aimed to describe the immune response produced by insect hosts after infestation with the entomopathogenic nematode complex. Having resources such as the genome (Gouin et al, 2017) and a reference transcriptome (Legeai et al, 2014) of S. frugiperda, we performed RNAseq analysis to decipher the tissue-specific transcriptional responses to infestation with an entomopathogenic nematode-bacterial complex in our model crop pest lepidoptera (Huot et al, 2019). As expected, the fat body and hemocytes produce a strong, stable immune response. The bacterium induces expression of genes involved in a classical antibacterial response (overexpression of the AMPs attacin, cecropin, gloverin and lebocin), the nematode induces expression of lectins and genes involved in melanization and encapsulation (Figure 1) (Huot et al, 2020).

Figure 1. Hypothetical schematic of the structure of the immune response of the Spodoptera frugiperda larva to the nematobacterial complex. In green, responses mainly induced by the nematode partner and in orange, main responses induced by the bacterial symbiont. Line thickness and letter size symbolize the relative strength of the induced transcriptional responses.

Finally, this study identified 2 gene clusters (Unk and GBH) highly overexpressed in a tissue- and pathogen-specific manner (Figure 2) whose functional characterization is currently underway.

Figure 2. Transcriptional expression profiles of potential novel immune effectors in Spodoptera frugiperda.

Despite the strong immune response, the symbiotic Photorhabdus bacterium grows rapidly (48 h) in the hemolymph, leading to death by sepsis. We have shown that Photorhabdus modify their bacterial envelope by reducing the net charge, which allows a better resistance to cationic AMPs produced by the insect. Indeed, we observed in the culture medium that the major part of the population of the wild-type strain is susceptible to AMPs, but a resistant minor subpopulation (about 5 out of 1000 bacteria) is still present in the bacterial culture (Figure 3A) (Mouammine et al, 2017).

Figure 3. A polymyxin-resistant subpopulation of TT01 is responsible for sepsis causing insect death. (A) Bacteria are plated on agar in the presence of polymyxin-containing filters. Red circles indicate colonies present in bacterial growth inhibition halos. (B) Bacterial growth in the presence (gray columns) or absence (black columns) of polymyxin and mortality of Spodoptera littoralis insect larvae after injection of P. luminescens TT01. (C) Bacterial levels in the insect cadaver seven days after injection of TT01 (same legend as in B).

The resistant subpopulation rapidly reverses, suggesting that the phenomenon is not genetic. Comparison of the genomes of the majority population and the resistant subpopulation (Single Molecule Real-Time sequencing, PacBio) did not reveal any genetic rearrangement or mutation explaining this phenotypic change, which confirms the epigenetic hypothesis. Our RNA-Seq analysis of the transcriptome of the resistant subpopulation reveals an overexpression of PhoP-dependent resistance genes involved in the modification of lipid A of LPS. Moreover, upon infection of the insect, we observed that the AMP-sensitive subpopulation disappears 6 h post-injection (time corresponding to the synthesis of AMPs by the host) and that the resistant subpopulation is responsible for the sepsis causing the death of the insect (Figure 3B). Then, the system reverses to return to the initial equilibrium in the insect cadaver (Figure 3C). This original strategy based on a mixture of pre-existing subpopulations can illustrate a phenomenon called "risk minimization" or "bet hedging".

Bibliography

Aymeric, J.-L., Givaudan, A., Duvic, B. 2010. Imd pathway is involved in the interaction of Drosophila melanogaster with the entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus luminescens. Mol Immunol 47, 2342-2348. DOI : 10.1016/j.molimm.2010.05.012.

Gouin, A., Bretaudeau, A., Nam, K., Gimenez, S., Aury, J.-M., Duvic, B., et al. 2017. Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges. Sci Rep 7, 1-12. DOI : 10.1038/s41598-017-10461-4.

Huot, L., Bigourdan, A., Pages, S., Ogier, J.C., Girard, P.A., Negre, N., Duvic, B. 2020. Partner-specific induction of Spodoptera frugiperda immune genes in response to the entomopathogenic nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophila. Dev Comp Immunol 108, 103676. DOI : 10.1016/j.dci.2020.103676.

Huot, L., George, S., Girard, P.-A., Severac, D., Nègre, N., Duvic, B. 2019. Spodoptera frugiperda transcriptional response to infestation by Steinernema carpocapsae. Sci Rep 9, 12879. DOI : 10.1038/s41598-019-49410-8.

Legeai, F., Gimenez, S., Duvic, B., Escoubas, J.-M., Gosselin-Grenet, A.-S., Blanc, F., et al. 2014. Establishment and analysis of a reference transcriptome for Spodoptera frugiperda. BMC Genomics 15, 704. DOI : 10.1186/1471-2164-15-704.

Mouammine, A., Pages, S., Lanois Nouri, A., Gaudriault, S., Jubelin, G., Bonabaud, M. et al. 2017. An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence. Sci Rep 7, 43670. DOI : 10.1038/srep43670.

Nuñez-Valdez, M.E., Lanois, A., Pages, S., Duvic, B., Gaudriault, S. 2019. Inhibition of Spodoptera frugiperda phenoloxidase activity by the products of the Xenorhabdus rhabduscin gene cluster. PLoS One 14, e0212809. DOI : 10.1371/journal.pone.0212809.

Satyavathi, V.V., Mohamed, A.A., Kumari, S., Mamatha, D.M., Duvic, B. 2018. The IMD pathway regulates lysozyme-like proteins (LLPs) in the silkmoth Antheraea mylitta. J Invertebr Pathol 154, 102-108. DOI : 10.1016/j.jip.2018.04.006.