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Characterization of Limb Autotomy Factor-Proecdysis (LAF_pro), Isolated From Limb Regenerates, That Suspends Molting in the Land Crab Gecarcinus lateralis

¹ Department of Biology, Cell and Molecular Biology Program, and Program in Molecular, Cellular and Integrative Neurosciences, Colorado State University, Fort Collins, Colorado 80523
² University of California, Davis, Bodega Marine Laboratory, Bodega Bay, California 94923

* To whom correspondence should be addressed. E-mail: don{at}lamar.ColoState.edu

Molting and limb regeneration are tightly coupled processes, both of which are regulated by ecdysteroid hormone synthesized and secreted by the Y-organs. Regeneration of lost appendages can affect the timing and duration of the proecdysial, or premolt, stage of the molt cycle. Autotomy of all eight walking legs induces precocious molts in various decapod crustacean species. In the land crab Gecarcinus lateralis, autotomy of a partially regenerated limb bud before a critical period during proecdysis (regeneration index <17) delays molting so that a secondary limb bud (2° LB) forms and the animal molts with a complete set of walking legs. It is hypothesized that 2° LBs secrete a factor, termed limb autotomy factor-proecdysis (LAF_pro), that inhibits molting by suppressing the Y-organs from secreting ecdysone. Molting was induced by autotomy of eight walking legs; autotomy of primary (1°) LBs reduced the level of ecdysteroid hormone in the hemolymph 73% by one week after limb bud autotomy (LBA). Injection of extracts from 2° LBs, but not 1° LBs, inhibited 1° LB growth in proecdysial animals, thus having the same effect on molting as LBA. The inhibitory activity in 2° LB extracts was stable after boiling in water for 15 min, but was destroyed by boiling 15 min in 0.1 N acetic acid or incubation with proteinase K. These results support the hypothesis that LAF_pro is a peptide that resembles a molt-inhibiting hormone.

Abbreviations: CHH, crustacean hyperglycemic hormone • 20E, 20-hydroxyecdysone • LAF_an, limb autotomy factor-anecdysis • LAF_pro, limb autotomy factor-proecdysis • LB, limb bud • LBA, limb bud autotomy • LGIF, limb growth inhibitory factor • MIH, molt-inhibiting hormone • MLA, multiple limb autotomy

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