DGAT1, but not DGAT2, is required for ovarian lipid storage and egg hatchability in Rhodnius prolixus

Triacylglycerol (TAG) is the main lipid reserve in insect eggs and represents a critical energy source for embryonic development. In the hematophagous vector Rhodnius prolixus, oocyte growth relies heavily on lipids transported from the fat body through the hemolymph, which are delivered mainly as diacylglycerol and free fatty acids. Therefore, the ovary must catalyze the final esterification step converting these substrates into TAG to ensure proper lipid storage in developing oocytes. This reaction is mediated by diacylglycerol acyltransferases (DGATs), yet the specific roles of DGAT1 and DGAT2 in insect reproduction remain poorly understood. Here, we investigated the functions of RpDGAT1 and RpDGAT2 in ovarian physiology and reproduction in R. prolixus. RNAi gene knockdown efficiently reduced transcript levels of both enzymes without affecting blood digestion, survival, or oviposition rates. However, RpDGAT1 knockdown caused pronounced reproductive defects. Females produced morphologically abnormal eggs with collapsed appearance and reduced size, leading to an approximately 70% reduction in hatching rates. Ultrastructural analyses revealed alterations in eggshell organization as seen by SEM, but no changes in the hydrocarbon contents of the eggshell were detected. Lipid analyses showed that RpDGAT1 knockdown reduced ovarian TAG levels at day 10 after blood feeding, but not at day 5, while increasing ovarian phosphatidylethanolamine at day 10. Ovarian free fatty acid levels remained unchanged at both time points. In phenotypic eggs, TAG levels were reduced and free fatty acids were increased. Consistently, oocytes from RpDGAT1-knocked down females exhibited smaller lipid droplets, reflecting impaired neutral lipid storage. Hemolymph lipid composition and ovarian incorporation of radiolabeled palmitate were unchanged, indicating that reduced ovarian TAG was not associated with detectable changes in circulating lipid availability or overall ovarian fatty acid uptake. In contrast, RpDGAT2 knockdown produced no detectable reproductive phenotype. Together, our findings demonstrate that RpDGAT1, but not RpDGAT2, is essential for TAG accumulation, proper oocyte development, and egg hatchability in R. prolixus, highlighting the importance of lipid metabolism in insect reproductive physiology.