TY - JOUR

T1 - Ecdysone quantification from whole body samples of drosophila melanogaster larvae

AU - Koyama, Takashi

AU - Mirth, Christen K.

N1 - Funding Information: We thank for our funding sources, Fundação Calouste Gulbenkian (Portugal) and Australian Research Council (FT170100259) to CKM and the Fundação para a Ciênica e a Tecnologia (Portugal) to TK (SFRH/BPD/74313/2010). Publisher Copyright: © 2021 Bio-protocol LLC. All Rights Reserved.

PY - 2021/2/5

Y1 - 2021/2/5

N2 - Steroid hormones strictly control the timing of sexual maturation and final body size both in vertebrates and invertebrates. In insects, the steroid hormone ecdysone controls the timing of the molts between larval instars as well as the transition to metamorphosis. Growth during the final instar accounts for over 80% of the increase in final mass in insects, and the duration of this growth period is driven by a sequence of small ecdysone pulses that ultimately induce metamorphosis. Historically the biologically active form of ecdysone, 20-hydroxyecdysone (20E), was quantified using radio-immunoassays, bioassays, or chromatography assays. However, these assays are methodologically complicated and often time consuming. Furthermore, collecting samples for precise measurements of ecdysone concentrations using these assays is limited in small insects like Drosophila melanogaster. Here, we describe an accurate and sensitive method to collect carefully-staged third instar larvae suitable for preparing samples for ecdysone quantification using a commercially-available 20E enzyme immunoassay (EIA). Because we resynchronize larval development at the molt to the final instar, collect large samples, and weigh each sample, we are able to detect a small ecdysone peak early in the final instar known as the critical weight ecdysone peak. This method detects peaks as low as 6 pg 20E/mg larval sample, allowing us to quantify other small ecdysone peaks in flies - the necessary prerequisite for eventually determining their regulation and function.

AB - Steroid hormones strictly control the timing of sexual maturation and final body size both in vertebrates and invertebrates. In insects, the steroid hormone ecdysone controls the timing of the molts between larval instars as well as the transition to metamorphosis. Growth during the final instar accounts for over 80% of the increase in final mass in insects, and the duration of this growth period is driven by a sequence of small ecdysone pulses that ultimately induce metamorphosis. Historically the biologically active form of ecdysone, 20-hydroxyecdysone (20E), was quantified using radio-immunoassays, bioassays, or chromatography assays. However, these assays are methodologically complicated and often time consuming. Furthermore, collecting samples for precise measurements of ecdysone concentrations using these assays is limited in small insects like Drosophila melanogaster. Here, we describe an accurate and sensitive method to collect carefully-staged third instar larvae suitable for preparing samples for ecdysone quantification using a commercially-available 20E enzyme immunoassay (EIA). Because we resynchronize larval development at the molt to the final instar, collect large samples, and weigh each sample, we are able to detect a small ecdysone peak early in the final instar known as the critical weight ecdysone peak. This method detects peaks as low as 6 pg 20E/mg larval sample, allowing us to quantify other small ecdysone peaks in flies - the necessary prerequisite for eventually determining their regulation and function.

KW - 20-hydroxyecdysone (20E)

KW - Critical weight

KW - Drosophila melanogaster

KW - Enzyme immunoassay (EIA)

KW - Larval staging

KW - Larval synchronization

KW - Whole body ecdysone quantification

U2 - 10.21769/BioProtoc.3915

DO - 10.21769/BioProtoc.3915

M3 - Journal article

C2 - 33732802

AN - SCOPUS:85116209267

VL - 11

JO - Bio-protocol

JF - Bio-protocol

SN - 2331-8325

IS - 3

M1 - e3915

ER -