A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling

Authors: VATANPARAST, MOHAMMAD - Andong National University; ESMACILY, MOJTABA - Andong National University; Stanley, David; KIM, YONGGYUN - Andong National University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/2024
Publication Date: 7/17/2024
Citation: Vatanparast, M., Esmacily, M., Stanley, D.W., Kim, Y. 2024. A PLA2 deletion mutant using CRISPRCas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling. PLOS ONE. https://journals.plos.org/plosone/article/authors?id=10.1371/journal.pone.0304958.
DOI: https://doi.org/10.1371/journal.pone.0304958

Interpretive Summary: During the early 1990s, Stanley's research group discovered a group of molecules that signal insect immune responses to infection. In the absence of these signals, most insects die within hours of infections. Subsequent research identified specific immune actions that are signaled by these signals, however a serious shortcoming in our knowledge about insect immunology was that we had no idea of the number of genes that act in insect immunity and how many of them rely on these signal molecules. In this paper we report on the number of genes that act in insect immunology (over 12,000) and how many of them are related to the signal molecules we discovered years ago (about 69%). This new information will be used by scientists around the world who are working on developing novel insect pest management systems based on severely weakening insect immune responses to infections, occurs in virtually all insects. Research in this area may lead to novel pest management technologies which will help increase global production of foods with high qualities while minimizing some of the harm agriculture exerts on environments.

Technical Abstract: Eicosanoids are the oxygenated C20 polyunsaturated fatty acids that mediate various physiological processes in metazoans. They are synthesized from C20 precursors, which are released from phospholipids by the catalytic activity of phospholipase A2 (PLA2). The inhibition of PLA2 activity leads to various physiological alterations, including immunosuppression. Considering the complexity of insect immunity, it remains unknown which immune-associated factors are influenced by eicosanoids. Group III PLA2s are common in insects. A uniquely encoded secretory PLA2 (sPLA2) is associated with cellular and humoral immune responses in a lepidopteran insect, Spodoptera exigua. A deletion mutant of this gene was generated using a CRISPR/Cas9 genome editing technology. Purifying selection through two generations produced a homozygous deletion mutant line. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are under the control of eicosanoids, recodorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.