GLAS, P.S.; TUBERTY, S.R.; The Citadel; Appalachian State University: Glucose Production Precedes Hatching in Grass Shrimp Embryos

Grass shrimp embryos undergo extreme size changes immediately before hatching, increasing from 0.7 mm to 1.0 mm in length with the shedding of the outermost 3 embryonic envelopes (Glas et al., 1997). During this time, ecdysteriod levels also increase dramatically (Glas and Tuberty, 2002). Chung et al. (1999) suggest “a surge in CHH (crustacean hyperglycemic hormone) may signal the completion of ecdysis via rapid uptake of water” and Spanings-Pierrot (2000) found CHH to “regulate ion and water balance in crustaceans” (see Chang et al., 2001, for review). Since it is suggested that CHH may drive glucose production in order to initiate the water uptake needed for size increase associated with molting, we decided to look for glucose in grass shrimp embryos in relation to the shedding of the outer embryonic envelopes and subsequent hatching. Approximately 100 embryos at each developmental time were homogenized in 500 ul of 80% methanol. The homogenate was centrifuged for 5 min at 600 X g, and the supernatant was transferred to a clean tube. Methanol (1.0 ml) was added and the solution was vortexed. Two samples of 250 ul each were removed and place in clean dry glass tubes and dried on a Centri-Vap system. The dry samples were covered and stored in the cold until all the samples were collected. To each sample, 500 ul of assay buffer was added and glucose was assayed according to the protocol supplied with the Amplex Red Glucose/Glucose Oxidase Assay kit (A-22189 Molecular Probes). The results showed an increase in glucose immediately before shedding of the outer embryonic envelopes with earlier embryos showing little to no glucose. This suggests that glucose production may be the mechanism that drives embryonic size increase, and potentially, this may be directed by CHH. Further studies will seek the initiation and production of CHH in the embryos from six to twelve hours before hatching.