The physiological importance of sesquiterpenoid signaling has been recognized in both insects and crustaceans, particularly in the regulation of reproduction. Crustaceans mainly use methyl farnesoate (MF) as the innate ligand whereas insects use an epoxide form of MF called JH III. However, the reception and molecular signaling downstream of MF in crustaceans is much less explored than JH signaling in insects, where the Methoprene-tolerant (Met) protein has been characterized as a JH receptor. The Met gene belongs to the bHLH –PAS family of transcription factors, and has been characterized in a number of insects. In vitro-synthesized Drosophila Met protein has been shown to bind JH III with high affinity. Subsequent experiments have shown that Met heterodimerizes with steroid receptor coactivator (SRC). SRC in turn may function as co-activator for ecdysone signaling in insects. A comparable situation might exist in crustaceans. A Met homolog was identified in the cladoceran crustaceans such as Daphnia pulex and D. magna, and like insects, Met was found to interact with the crustacean homolog of SRC in the presence of JH III.  Interestingly, the Met/SRC interaction was found to be 10 times more sensitive to MF than to JH III, suggesting that MF could be the natural ligand for Met in crustaceans.

We are interested in characterizing the molecular basis of MF signalling in brachyuran crabs.  We have identified a Met ortholog in Uca pugilator which is expressed during limb regeneration. Transcriptome analysis data showed hits in all the stages of the limb bud library throughout the molt cycle; a detailed analysis of met expression is in progress. BlastP results revealed conserved regional similarities to that of the cladoceran crustacean Daphnia pulex met (34%), as well as higher percentage similarity to insects Thermobia domestica (38%) and Zootermopsis nevadensis (35%). A SRC homolog, the heterodimer partner of Met-tolerant, was also observed in the limb bud transcriptome library. We are currently producing expression vector constructs to examine protein/protein and protein/ligand binding.

Phylogenetic tree of Met and SRC amino acid sequences from U. pugilator , G. lateralis with other arthropods, based on the 50% majority tree rule from the Bayesian analysis.

Representation of conservation within the ligand binding domain of Met. Are the circled amino acid residues critical to the change in JHIII/MF affinity between the insect and crustacean receptors?