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The wingless-related integration site(WNT)proteins are a family of secreted glycoproteins that are evolutionarily conserved and are believed to be involved in evolution in vertebrates and invertebrates.WNT signaling pathways may be associated with limb regeneration and development in crustaceans.However,the detail mechanisms remain unclear.Therefore,the distribution of WNT4 in the hepatopancreas,muscle,hemocyte,ganglion,heart,eyestalk,gill tissue,and different larvae development stages of the swimming crab(Portunus trituberculatus)were characterized using immunofluorescence,real-time PCR,and Western blotting.Significant PtWNT4 expression was detected in heart and eyestalk.In addition,PtWNT4 was expressed in all larval stages ofP.trituberculatus with a dynamic expression pattern,especially in the eyestalk and other organs in the carapace area.The injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels in the eyestalk,heart,and muscle,resulting in 1.9-fold,2.2-fold,and 2.7-fold decreases compared with those detected in the group injected with crab saline(P<0.05),respectively,indicating successful gene silencing.Overall,expression analysis on the WNT4 using RNAi provides an insight to its functional mechanism during limb regeneration in P.trituberculatus.The results not only demonstrated the requirement for WNT4 in limb regeneration of Crustaceans,but also suggested its ability to promote larval development at specific stages.