Heavy metals inhibit limb regeneration in horseshoe crab larvae

We studied the effects of heavy metals on the regeneration of walking legs in horseshoe crabs (Limulus polyphemus). The second walking leg was amputated in embryos (stage 20 and 21) and first instar (trilobite) larvae, and the length and morphology of the regenerated appendage was observed after molting to the second instar stage. Regeneration following continuous exposure to TBT (0. 001-100 mg/L), mercury (0.001-100 mg/L), cadmium (0.01-100 mg/L), chromium (0.1-100 mg/L), lead (0.1-100 mg/L), and copper (1-100 mg/L) was measured relative to regeneration in seawater. Although regeneration was incomplete in controls, treatment with heavy metals led to smaller and/or malformed legs. The impacts of heavy metals on survival, molting, and regeneration of horseshoe crab larvae were ranked as follows: organotin > Hg > Cd > Cr > Zn > Pb >== Cu. Cu and Pb did not inhibit regeneration, even at 100 mg/L. TBT, Hg, Cd, Cr, and Zn inhibited the regeneration of appendages, although first instar larvae successfully molted into second instars even after treatment. Regeneration was comparable to seawater controls in less than 2.5 mg/L Zn. In 5.0 and 10.0 mg/L Zn, regeneration was inhibited and the length of regenerated appendages remained shorter in all second instars. Larvae treated with 10 mg/L Zn for 1-week intervals during the molt cycle showed similar patterns of regeneration. The regeneration of claws was not all or none, and formation of the claw was proportional to the length of regenerated appendages. Limb regeneration in horseshoe crab larvae may be a useful model system for the study of pollutant impacts.