Identification And Horizontal Transfer Of Non-Autonomous Helitrions HaLep1 In Helicoverpa Armigera

Transposable elements （TEs） are prevalent in the genomes of almost all eukaryotes, and are increasingly recognized as an important force in the evolution of eukaryotic genomes by affecting the structure, function and expression of the host genes. Horizontal transfer （HT） can be defined as the exchange of genetic material between species by nonvertical inheritance without the aid of any form of sexual mechanism. While over 200 solid cases of horizontal transfers of TEs （horizontal transposon transfer or HTT） have been described so far in multicellular eukaryotes the unequivocal confirmation of any specific mechanism acting to shuttle DNA among eukaryotes remaining poorly appreciated. Helitrons is a recently discovered superfamily of transposons. Unlike traditional DNA TEs, Helitrons are unique in that they do not produce target site duplications on their integration into the host genome and do not contain terminal repeats. In this study, by using bioinformatic methods and molecular techniques, the distribution of the non-autonomous Helitrons, Lepl, in lepidopteran insects as well as it’s horizontal transfer among lepidopteran insects and non-lepidopteran species were studied. The results might not only assist in understanding the evolutionary molecular mechanism of Helitrons but also lay the foundation for the phylogenetic study of lepidopteran insect species.The HaLep1₁ element was previously obtained by genome walking and located at 756 bp upstream of the translation start codon of the CYP6AE12 gene in Helicoverpa armigera. The HaLep1₁ sequence was compared with NCBI H. armigera nucleotide collection （nr/nt） databases with Blastn （www.ncbi.nlm.gov/cgibin/BLAST）, and a total of 21 full length sequences with high homology to HaLep1₁ were identified from non-redundant database, and named HaLep1₂-HaLep1₂2. Multiple sequence alignment revealed that almost all HaLepl copies have characteristic 5’-TC and 3’-CTRY nucleotide termini as well as CTRR motif at the 3’end of acquired sequence, and the integration occurs precisely between the host A and T nucleotides. Phylogenetic analysis demonstrated the presence of three clear major lineages, designated Lineage A （HaLeplA）, Lineage B （HaLeplB）, and Lineage C （HaLep1C）, among which,6 elements form lineage HaLep1A, while HaLep1B and HaLep1C were represented by 6 and 9 elements respectively. Notably, HaLepl elements from Lineage A and Lineage B showed relatively high identity with 134 bp Lepl consensus sequence （83%-89%）, while HaLepl elements from Lineage C showed only 68% to 78% identity with Lepl consensus sequence. These results suggested that HaLepl lineages might transfer independently into the genome of H. armigera.The HaLepl₁ sequence was used as a query to search against lepidoptera nucleotide （nr/nt） and EST （est_others） collections to detect sequences with high identity with HaLepl₁ in lepidopteran species other than H. armigera. The result showed that HaLepl 1 sequence shared the highest similarity with two species of Heliothinae including Helicoverpa zea and Heliothis virescens. Remarkably, the acquired sequence at 3’end was only found in H. zea and H. virescens, suggesting that the acquired sequence was unique to H. armigera and its closely related species. The insertion polymorphism of HaLepl elements was further assessed by PCR and homology searches. The results of PCR and subsequent sequencing of DNA products showed that in samples of 12 individuals, the percentage of individuals with the band for HaLep1₁ insertion was 25%. No Lepl-like sequence was found in paralogous sites of HaLep1₂0 （accession number:FP340435） in H. armigera as well as in orthologous site of HaLepl1₈ in H. zea （accession number:DQ788839）. The result of intra-species insertion polymorphism of HaLep1₁ suggested a very recent transposition, and the insertion polymorphism of HaLep1₈ m two different but closely related species suggested that HaLepl 8 might horizontally transfer into a common ancestor of H. armigera and H. zea, and the absence of orthologous copy in H. zea was due to the loss of ancestrally polymorphic insertions in these two species.The 134-bp Lep1 concensus sequence was used as query to search with Blastn against non-lepidopteran insect genome sequences as well as NCBI non-lepidopteran databases, including the whole genome shotgun, nucleotide collection （nr/nt）, genome survey sequences, high throughput genomic sequences, and expressed sequence tag databases. Multiple Lep1-like elements were found in several non-lepidopteran species, including two aphids, Acyrthosiphon pisum and Aphis gossypii, two parasitoid wasps, Cotesia vestalis, and Copidosoma floridanum, one beetle, Anoplophora glabripennis, as well as two bracoviruses in parasitoid wasps, and one intracellular microsporidia parasite, Nosema bombycis. The patchy distribution and high sequence similarity of Lepl-like elements among distantly related lineages as well as incongruence of Lepl-like elements and host phylogeny suggest the occurrence of HT. Remarkably, the acquired sequences of both NbLepl from N. bombycis and CfLepl from C. floridanum showed over 90% identity with their lepidopteran host Lepl. Thus, our study provides evidence of HT facilitated by host-parasite interactions. Furthermore, in the context of these data, we discussed the putative directions and vectors of HT of Lepl Helitrons.