Methoprene-Tolerant (Met) Acts as Methyl Farnesoate Receptor to Regulate Larva Metamorphosis in Mud Crab, Scylla paramamosain

The conserved role of juvenile hormone (JH) signals in preventing larvae from precocious metamorphosis has been confirmed in insects. Crustaceans have different metamorphosis types from insects; we previously proved that methyl farnesoate (MF) can prohibit larvae metamorphosis in mud crabs, but the molecular signal of this process still needs to be elucidated. In this study, <i>methoprene-tolerant</i> (<i>Met</i>) of <i>Scylla paramamosain</i> was obtained and characterized, which we named <i>Sp-Met</i>. <i>Sp-Met</i> contains a 3360 bp ORF that encodes 1119 amino acids; the predicted protein sequences of Sp-Met include one bHLH, two PAS domains, one PAC domain, and several long unusual Gln repeats at the C-terminal. AlphaFold2 was used to predict the 3D structure of Sp-Met and the JH binding domain of Met. Furthermore, the binding properties between Sp-Met and MF were analyzed using CD-DOCK2, revealing a putative high affinity between the receptor and ligand. In silico site-directed mutagenesis suggested that insect Mets may have evolved to exhibit a higher affinity for both MF or JH III compared to the Mets of crustaceans. In addition, we found that the expression of <i>Sp-Met</i> was significantly higher in female reproductive tissues than in males but lower in most of the other examined tissues. During larval development, the expression variation in <i>Sp-Met</i> and <i>Sp-Kr-h1</i> was consistent with the immersion effect of MF. The most interesting finding is that knockdown of <i>Sp-Met</i> blocked the inhibitory effect of MF on metamorphosis in the fifth zoea stage and induced pre-metamorphosis phenotypes in the fourth zoea stage. The knockdown of <i>Sp-Met</i> significantly reduced the expression of <i>Sp-Kr-h1</i> and two ecdysone signaling genes, <i>Sp-EcR</i> and <i>Sp-E93</i>. However, only the reduction in <i>Sp-Kr-h1</i> could be rescued by MF treatment. In summary, this study provides the first evidence that MF inhibits crustacean larval metamorphosis through Met and that the MF-Met→<i>Kr-h1</i> signal pathway is conserved in mud crabs. Additionally, the crosstalk between MF and ecdysteroid signaling may have evolved differently in mud crabs compared to insects.