Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids

Abstract

Ecdysteroid hormones regulate several aspects of insect development and reproduction. The predominant ecdysteroids produced by insects including mosquitoes are ecdysone (E) and 20-hydroxyecdysone (20E). The ability to measure E and 20E titers is essential for many studies, but few sensitive, low cost options are currently available for doing so. To address this deficiency, we developed a new enzyme-linked immunoassay (EIA). In the first part of the study, we compared the affinity of two new antisera named EAB25 and EAB27 to other available ecdysteroid antisera. EAB25 had a 27-fold higher affinity for 20E than E, while EAB27 had a four-fold higher affinity for 20E. In the second part of the study, EIA protocols were developed for analyzing E and 20E produced by the mosquito Aedes aegypti. Results indicated that pelts from fourth instar larvae and ovaries from blood-fed, adult females produced E and 20E. Methanol extraction in the presence of magnesium from whole body samples altered antibody recognition of E and 20E by EIA. However, extraction with 1-butanol and two organic/water phase separations eliminated this problem and improved assay performance. We conclude the new antisera used in the EIA provide a low-cost, flexible, and sensitive method for measuring E and 20E in insects.

Whole blood and blood components from vertebrates differentially affect egg formation in three species of anautogenous mosquitoes

“…Following this incubation period, medium supernatant containing secreted ecdysteroids was collected and frozen at – 20 °C. Ecdysteroid titer was then determined by EIA using the EAB27 primary antibody and 20-hydroxyecdysone (Sigma) as a standard [ 78 ].…”

CasEcR and CasMIH Genes in the Blue Crab, Callinectes sapidus: A Temporal Evaluation and Melatonin Effects

“…Hemolymph ecdysteroid levels were determined by Enzyme-Linked Immunosorbent Assay (ELISA) adapted from McKinney et al (2017) . Melatonin quantification was also determined by ELISA according to the kit manufacturer’s instruction (IBL International, Flughafenstr, Hamburg, Germany).…”

Ad libitum consumption of protein- or peptide-sucrose solutions stimulates egg formation by prolonging the vitellogenic phase of oogenesis in anautogenous mosquitoes

“…Ecdysteroid production by ovaries was measured as previously described [ 50 ]. Briefly, ovary pairs from two mosquitoes were explanted and incubated in 60 µl Beyenbach saline at 28 °C and 70% RH for 6 h. The resulting supernatant containing secreted ecdysteroids was then collected and frozen at –20 °C until quantification by enzyme-linked immunosorbent assay (EIA) using the primary antibody EAB27, which detects ecdysone and 20E equally and 20E (Sigma) as a standard [ 50 ]. Trypsin-like serine protease activity in mosquito midguts was quantified using fresh midgut homogenates from individual females that were incubated with Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA) [ 51 ].…”