Genome Determination Of Mitochondria In Four Locusts And Analysis Of Orthoptera System

In this study, the strategies of combined Long-PCR and Sub-PCR amplification are used to sequence the whole mitochondrial genomes of four Orthoptera species, S. prasiniferum sinense, C. davidiana, B. miramae miramae and M. japonicus. After sequence assembling, annotation and structural analysis, the four mitochondrial genomes combined with that of other Orthoptera species deposited in GenBank, as well as other insects which complete mitochondrial genome were sequenced in our laboratory, total 94 Orthopter species mitochodrial genomic sequences were used to reconstruct Orthoptera phylogeny using Maximum likelihood and Bayesian inference. Finally, in order to test whether mitochondrial protein-encoding genes of longipennate and brachypterous underwent different selective pressures, the 94 insects were divided into the longipennate and brachypterous in accordance with the wing type, and the non-synonymous/synonymous substitution ratio (ω=dN/dS) were calculated and compared. The main conclusions are summarized as following:1. The complete mitogenomes of S. prasiniferum sinense, M. japonicus, C. davidiana and B. miramae miramae arel5591bp,16046bp,16085,15919bp in size, respectivly. All four mitogenomes contain a standard set of 22 transfer RNA genes (tRNAs),13 protein-coding genes,2 ribosomal RNA genes (rRNAs) and an A+T control region, in the same order as those of the other caeliferan species, including the rearrangement of tRNAAsp and tRNALys. The nucleotide compositions of the four insect mitogenomes are significantly biased toward A+T. The strongest A+T biase is found in C. davidiana, B. miramae miramae and M. japonicus which were higher than 75%, but S. prasiniferum sinense is only 74%.2. The A+T content of protein-coding genes, excluding stop codons, is 73%,75%, 74.3% and 74.5% in S. prasiniferum sinense, M. japonicus, C. davidiana and B. miramae miramae, respectively. This significant AT-bias affects codon usage in proteins, with ATN and TAN being the most frequently used start condons and stop codons, respectively. The incomplete termination codon TA was found nad5 in S. prasiniferum sinense and C. davidiana, CO1 has an incomplete stop codon T in B. miramae miramae. In four mitogenomes, when the three codon positions of protein-coding genes were considered separately, the highest A+T content was found in the third codon positions (87.8% in S. prasiniferum sinense,90.1% in M. Japonicus,87.5% in C. Davidiana and 88.1% in B. miramae miramae).3. All twenty-two tRNA of the four species can be folded into the typical cloverleaf structure with the exception of tRNASer(AGN), in which the dihydrouridine(DHU) arm simply forms a loop. Non-canonical base pairs have been observed in stems of tRNA secondary structures. In S. prasiniferum sinense, there are 31 non-canonical base pairs, consisting of 24 G-U pairs,4 U-U,1 A-C,1 A-A,1 A-G mismatches. In M. Japonicus, there are 24 non-canonical base pairs, consisting of 21 G-U,1A-A,1 A-G,1 U-U mismatches, and in C. davidiana,21 G-U,1 U-U,1 A-A mismatches, whereas in the case of B. miramae miramae,22 G-U,3 U-U,3 A-C,2 A-A, 1 C-U mismatches have been identified.4. The two genes encoding the large and small ribosomal RNA subunits (16S rRNA and 12S rRNA) are located between tRNALeu(CUN) and tRNAVal, and between tRNAVal and the A+T-rich region, respectively. The length of 12S rRNA is 788bp in S. prasiniferum sinense,847bp in M. Japonicus,834bp in C. Davidiana and 836bp in B. miramae miramae; The 16S rRNA is 1315bp in S. prasiniferum sinense,1310bp in M. Japonicus,1316bp in C. Davidiana and 1319bp in B. miramae miramae. The A+T-rich region known for the initiation of replication in invertebrates is located in the conserved position between the 12S rRNA and trnIle genes. This region has an A+T content of 84.7% in B. miramae miramae,83.9% in M. Japonicus,84.5% in C. Davidiana and 86.5% in S. prasiniferum sinense, which is the highest A+T content of different regions in the four species mitogenomes.5. The phylogenetic relationships of 94 Orthoptera species were determined using different databases from the complete mitochondrial genomes, using Maximum likelihood and Bayesian analysis. The analysis of mitogenomes suggested the following results:the ML and BI methods generated phylogenetic trees with the same topology. Based on the tree topologies by both 37genes and 13PCGs datasets, two suborders of Orthoptera, Cealifera and Ensifera, were both recovered as monophletic. Within Ensifera, we found Raphidophoroidea form a clade with Tettigonioidea and that this clade was sister to Grylloidea. Within Caelifera, Oedipodidae, Pamphagidae and Catantopidae formed a monophyletic group. B. miramae miramae and C. Davidiana belong to the family Oedipodidae in Xia’s system, and both species were assigned to the family Oedipodidae in our analysis. Within the family Catantopidae, S. prasiniferum sinense had a closer relationship with C. Elegans and O. Chinesis. The monophyly of Eumastacoidea, Tetridoidea, Tridactyloidea and Acrididae, Gomphoceridae, Arcypteridae of Acridoidea was not supported in the mtgenome analysis, for example, M. Japonicus of Acrididae formed a clade with L. litangensis of Arcypteridae.6. When we calculated ω values for terminal branches to measure the strength of selection between different winged insects (long winged vs. short winged grasshoppers), it was not found any significant difference in ω values between groups of insects. But the analyses of each mtDNA protein-encoding genes showed that the ω of ATP8 gene greater than 1 for longipennate insect, suggesting that the gene is considered under positive selection; the ω values for other genes were significantly less than 1 showed that the function of these genes are existent and they have experience constrained selective pressure to maintain the function and ensure energy supply.