Molecular characterization and gene expression pattern of two putative molt-inhibiting hormones from Litopenaeus vannamei

Two cDNA sequences (Liv-MIH1 and Liv-MIH2) were cloned from the eyestalk ganglia of the white shrimp Litopenaeus vannamei. The conceptually translated peptide precursors consist of a mature peptide (77 residues for Liv-MIH1, 75 residues for Liv-MIH2), preceded by a 28-residue signal peptide. Both mature peptides share highest sequence identity with other known MIHs, and contain several conserved residues that have been proposed to be functionally critical for MIH activity. Analysis of genomic sequences reveals that both genes are organized in a 3 exon/2 intron manner, with the same sites of intron insertion. The transcripts of Liv-MIH1 and Liv-MIH2 were detected exclusively in the eyestalk, but not in other neural and non-neural tissues examined. Phylogenetic analysis indicates that Liv-MIH1 and Liv-MIH2 cluster with the type II peptides that are considered as penaeid MIH. In addition, a quantitative real-time polymerase chain reaction (PCR) assay was developed and validated for the quantification of gene expression of Liv-MIH1 and Liv-MIH2. Transcript levels for both genes remained constant through stages A - D_1^′ (ranges of relative expression levels are 97.9 ± 2.9 to 104.5 ± 8.9% for Liv-MIH1, and 85.6 ± 6.7 to 104.7 ± 10.8% for Liv-MIH2), and declined afterwards, reaching a lowest level during stage D₂D₃ (40.6 ± 0.4% for Liv-MIH1, and 48.5 ± 3.2% for Liv-MIH2). These significant decreases in the transcript levels correspond to a significant increase in hemolymph ecdysteroid titers at stage D₂D₃. These results clearly indicate that Liv-MIH1 and Liv-MIH2 are type II peptides of the crustacean hyperglycemic hormone family and most likely function as MIHs in the white shrimp. They are discussed with regard to the presence of multiple MIHs and possible functional divergence of type II peptides in Penaeidae, as well as endocrine regulation of crustacean molting.