P1.135 Monday, Jan. 4 RNAi mediated disruption of ecdysteroid signaling during limb regeneration in the fiddler crab, Uca pugilator. DAS, S.*; HOPKINS, P.M.; KHAMBADAKONE, D.; DURICA, D.S.; Univ. of Oklahoma; Univ. of Oklahoma; Univ. of Oklahoma; Univ. of Oklahoma email@example.com
Growth, molting, limb regeneration and reproduction in crustaceans are regulated by arthropod steroid hormones, ecdysteroids. To study the role of ecdysteroid signaling during limb regeneration in fiddler crabs, we have applied RNAi (dsRNA) mediated silencing of UpEcR and UpRXR, which encode the functional heterodimer ecdysteroid receptor. The silencing of receptors should disrupt ecdysteroid signaling. Silencing was evaluated by receptor knockdown, and disruption was evaluated by changes in growth rate and morphology of limb regenerates. In these experiments, both EcR and RXR dsRNA (200ng/200nl), were injected into the blastemal chamber on days four and seven following autotomy. RT-PCR results indicated a two-fold knockdown of both UpEcR (p= 0.001) and UpRXR (p=0.003) in day nine blastemas. Previously, we have shown that injections with either dsEcR or dsRXR into the blastema led to a significant drop in circulating ecdysteroid titers. Lowered ecdysteroids were also observed in crabs injected with both dsEcR and dsRXR. Taken together with the previously reported effects of RNAi on contralateral uninjected limb buds, these results suggest that RNAi has a systemic effect. In addition, 62% of dsRNA injected limb blastemas failed to emerge and progress towards blastemal growth as compared to 5% of control blastemas. Preliminary hematoxylin and eosin staining of the arrested blastema have shown absence of cuticular ingrowths that may be due to lack of cellular proliferation and differentiation. Although some of the dsRNA injected blastema progressed towards basal and proecdysial limb growth, the crabs failed to molt and died whereas control crabs molted and regenerated limbs. The failure to molt after dsRNA injections in the crab implies a sustained systemic effect.