PGE2 binding affinity of hemocyte membrane preparations of Manduca sexta and identification of the receptor-associated G proteins in two lepidopteran species

Authors: KHAN, FALGUNI - Andong National University; TUNAZ, HASAN - Kahramanmaras Sutcu University; KIM, YONGGYUN - Andong National University; HAAS, ERIC - Creighton University; Stanley, David

Submitted to: Archives of Insect Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Insects express very effective immune systems that protect them from infections by a wide range of lethal invaders. Their immune systems are very effective, which makes insect immunity an important target for generating advanced pest management technologies. In this paper report on identifying a specific element of insect immunity in two pest insect species and show that blocking this element renders insects unable to defend themselves from infections. This new information will be used by scientists around the world who work to develop advanced insect pest control tools. This work will lead to substantially improved insect pest management programs, which will contribute to improved food security for people at the global level.

Technical Abstract: Prostaglandin E2 (PGE2) is an eicosanoid that mediates a range of insect physiological actions, including reproduction and immunity. Its receptor was identified and functionally assessed in two lepidopteran insects, Manduca sexta and Spodoptera exigua. However, its binding affinity to the receptor has not been reported. The PGE2 receptor is a G-protein coupled receptor (GPCR) although its corresponding G protein is not identified. PGE2 binding assays were performed with membrane preparations from hemocytes of M. sexta larvae. We recorded an optimal binding in 4 h reactions conducted at pH 7.5 with 12 nM tritium-labelled PGE2. We found that hemocytes express a single population of high affinity (Kd = 35 pmol/mg protein) PGE2 binding sites, which are specific and saturable. The outcomes of experiments on the influence of purine nucleotides suggested these are functional GPCRs. A bioinformatics analysis led to a proposed trimeric G protein in the S. exigua transcriptome, in which the Ga subunit is classified into five different types: Ga(o), Ga(q), Ga(s), Ga(12), and Ga(f). After confirming expressions of these five types in S. exigua, individual RNA interference (RNAi) treatments were applied to the larvae using gene-specific double-stranded RNAs. RNAi treatments specific to Ga(s) or Ga(12) gene expression significantly suppressed the cellular immune responses although the RNAi treatments specific to other three Ga components did not. While PGE2 treatments led to elevated hemocyte cAMP or Ca2+ levels, the RNAi treatments specific to Ga(s) or Ga(12) genes led to significantly reduced second messenger levels under PGE2, the RNAi treatments specific to the other three Ga components did not. These results showed that the PGE2 receptor has high PGE2 affinity in the nano-molar range and binds G proteins containing Ga(s) or Ga(12) trimeric component in S. exigua and M. sexta, and likely all lepidopteran insects.