Ecdysteroid measurements using an enzyme immunoassay (EIA)

Ecdysteroid titers in S. gregaria hemolymph and ecdysteroid levels in the female gonads were measured using an enzyme immunoassay, modified from Porcheron et al.¹² and discussed by Pascual et al.¹³ and Lafont et al.¹⁴. In this protocol a peroxidase conjugate of 20-hydroxy-ecdysone (20E) was used as tracer together with rabbit L2 polyclonal antibodies. This L2 antiserum has a strong affinity for ecdysone (E), 3-deoxyecdysone and 2-deoxyecdysone and a 6- to 8-fold lower affinity for 20E. Both serum and tracer were very kindly given by Prof. J.P. Delbecque (Université de Bordeaux, France). Hemolymph samples were collected from 12-day-old adult female locusts by piercing the insect’s cuticle behind its hind leg and holding a capillary to the wound. 10 µL of hemolymph was collected from each animal, which was immediately transferred to 90 µL of cold ethanol (100%) and stored at −20 °C until further processing, as described by Marchal et al.¹⁵. For the sampling of the gonads, one ovarium was dissected from each 12-day-old adult female locust and transferred to 500 µL of cold ethanol (100%) and stored at −20 °C until further processing as described by Van Wielendaele et al.¹⁶. Both ‘free’ and total ecdysteroid levels were determined. ‘Free’ ecdysteroids are the non-conjugated ecdysteroids. The total ecdysteroid levels were determined after enzymatic conversion of the conjugated ecdysteroids in ‘free’ ecdysteroids. The standard curve used in all measurements was obtained with 20E, and therefore results are expressed as 20E equivalents. GraphPad Prism 6 (GraphPad Software Inc.) was used to test the statistical significance of the observed differences.