Insertion of miniature subterminal inverted repeat-like elements in diapause-regulated genes in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Determining the genomic structure of diapause-associated transcripts (DAT) -2 and -3 led to the isolation of four novel miniature subterminal inverted repeat-like elements (MSITE): Mild-1, -2, -3 and -4. Mild-1 a is inserted within the first intron of diapause protein-1. Mild-1a is 284 bp in length, has a 14 bp target site duplication and three sets of subterminal inverted repeats. The second element, Mild-2a, is inserted within the 3' terminus of Mild-1a. Mild-2a is 29 bp long with a 3 bp target site duplication and one set of subterminal inverted repeats. Using primers based on Mild-1, genomic clones were developed leading to the isolation of Mild-3a. Mild-3a shares 60% identity with Mild-1a, is 253 bp long, has a 9 bp target site duplication and has one set of subterminal inverted repeats. Mild-4a is inserted within the first intron of DAT-2 and is 227 bp in length with a 12 bp target site duplication. Mild-4a appears to be an intermediate form between a miniature inverted repeat transposable element (MITE) and a MSITE because the 5' inverted repeat is terminal (i.e., adjacent to the target site duplication) as in MITEs, but the 3' inverted repeat is separated (in this case, by 33 bp) from the 3' target site duplication as in MSITEs. The target site duplications of Mild-1, -3 and -4 families share a common conserved core of AATTT. All of the transposable elements are AT rich and are able to form hairpin structures. Within the promoter legion of DAT-3 is a 163 bp sequence (Mild-1b) that shares 77% identity to the 3 terminus of Mild-1a. Mild-4a has identity to 25 and 53 bp regions within the promoter of the juvenile hormone esterase B gene. Southern blot analysis revealed the presence of Mild-1 and -3 elements in both Leptinotarsa decemlineata and Leptinotarsa juncta indicating that these elements are ancestral to the L. decemlineata, L. juncta separation.