Characterization of the first insect prostaglandin (PGE 2 ) receptor: MansePGE 2 R is expressed in oenocytoids and lipoteichoic acid (LTA) increases transcript expression

In arthropods, eicosanoids derived from the oxygenated metabolism of arachidonic acid are significant in mediating immune responses. However, the lack of information about insect eicosanoid receptors is an obstacle to completely decipher immune mechanisms underlying both eicosanoid downstream signal cascades and their relationship to immune pathogen-associated molecular patterns (PAMPs). Here, we cloned and sequenced a G protein-coupled receptor (MW 46.16 kDa) from the model lepidopteran, Manduca sexta (Sphingidae). The receptor shares similarity of amino acid motifs to human prostaglandin E (PGE ) receptors, and phylogenetic analysis supports its classification as a prostaglandin receptor. In agreement, the recombinant receptor was activated by PGE resulting in intracellular cAMP increase, and therefore designated MansePGE R. Expression of MansePGE R in Sf9 cells in which the endogenous orthologous receptor had been silenced showed similar cAMP increase upon PGE challenge. Receptor transcript expression was identified in various tissues in larvae and female adults, including Malpighian tubules, fat body, gut and hemocytes, and in female ovaries. In addition to the cDNA cloned that encodes the functional receptor, an mRNA was found featuring the poly-A tail but lacking the predicted transmembrane (TM) regions 2 and 3, suggesting the possibility that internally deleted receptor proteins exist in insects. Immunocytochemistry and in situ hybridization revealed that among hemocytes, the receptor was exclusively localized in the oenocytoids. Larval immune challenges injecting bacterial components showed that lipoteichoic acid (LTA) increased MansePGE R expression in hemocytes. In contrast, injection of LPS or peptidoglycan did not increase MansePGE R transcript levels in hemocytes, suggesting the LTA-associated increase in receptor transcript is regulated through a distinct pathway. This study provides the first characterization of an eicosanoid receptor in insects, and paves the way for establishing the hierarchy in signaling steps required for establishing insect immune responses to infections.