Comparative Analyses Of Mitochondrial Genomes Between Two Gynaephora Species(Lepidoptera:Lymantriidae)

Grassland caterpillars (Insecta:Lepidoptera:Lymantriidae:Gynaephora). also known as red head and black caterpillar or grassland lymantrid moth, comprise15nominated species in the world, eight of which are endemic to the Qinghai-Tibetan Plateau (QTP) and are among the most important insect pests damaging alpine meadow of the QTP. In the long evolutionary history, grassland caterpillars have well adapted to the high-altitude environment (e.g. hypoxia and severe cold), and are the dominant groups of alpine meadow pests. Therefore, it is necessary to explore the genetic basis of high-altitude adaptive evolution of grassland caterpillars, which will be helpful to understand the ecological adaptation mechanism and outbreak of grassland caterpillars. The dissertation closely focus on the adaptive evolution of grassland caterpillars to high-altitude of the QTP. Two grassland caterpillars, Gynaephora alpherakii and Gynaephora menynanensis were collected from Naqu County of Tibet (asl4800m) and Menyuan County of Qinghai Province (asl3000m), respectively. Supercooling points, complete mitochondrial genomes, and expression patterns of nine mitochondrial genes of two Gynaephora spp. were studied. in order to clarify differential response of mitochondrial genomes of grassland caterpillars to different high-altitude environment, and provide invaluable information on the ecologically evolutionary mechanism and disaster rule of the two grassland caterpillars at molecular level. The main results are as follows:1. The supercooling point and freezing point of larvae (1-6instar), pupae, adults and eggs of G. alpherakii and G. menyuanensis have been investigated, respectively. The results show that supercooling points of first, fifth, sixth instar larvae, pupae and adults of G. alpherakii are significantly lower than that of G. menyuanensis, and fourth instar is opposite. Freezing point of larvae and adult of G. alpherakii is generally higher than that of G. menyuanensis, pupae and eggs are opposite. These results suggest that the cold resistance of G. alpherakii from higher altitude is better than that of G. menyuanensis from lower altitude.2. The complete mitochondrial genomes of G. alpherakii (GenBank accession number:KJ957168) and G. menyuanensis (GenBank accession number:KC185412) have been determined using12pairs of universal primers and specific primers and PCR walking approach. The two mitochondrial genomes are highly similar, and are typical of circular DNA molecules. The size of mitochondrial genome of G. alpherakii and G. menyuanensis were15755bp and15740bp. respectively. They encode37classic metazoan mitochondrial genes, i.e.13protein-coding genes, two ribosomal RNA genes (rrnL and rrnS) and22transfer RNA (tRNA) genes. In addition, the two mitochondrial genomes also contain a long non-coding regions with the length of449bp. i.e. A+T-rich region. The order and orientation of the mitochondrial genes are identical to the inferred ancestral arrangement of insects except for tniAI. which moves to the upstream of the transfer RNA (tRNA) cluster trnI-trnQ-trnM as reported in other lepidopteran mitogenomes. The nucleotide composition of the two mitochondrial genomes are significantly biased toward A and T. The total A+T content of the major strand of G. alpherakii and G. menyuanensis is81.31%and81.53%, respetively, which are highest among all sequenced noctuids. All of the22tRNAs found in the two mitochondrial genomes have the typical cloverleaf secondary structures.Comparative analyses of two Gynaephora mitochondrial genome reveal that only atp8of G. alpherakii is6bp (two codons) longer than that of G. menyuanensis, and the remaining12protein-coding genes have no length variation. The ω values (dN/dS) of all protein-coding genes are less than1, indicating that these genes are evolving under strongly purifying selection, and the mitochondrial genome plays a key role for adaptive evolution of grassland caterpillars.3. In order to further study the effects of different altitude environment on mitochondrial gene expression, gene expression patterns of nine protein-coding genes (atp6, cox1, cox2, cox3, cob, nadl, nad2. nad4and nad5) of egg, larva (1-6instar). pupa and adult of G. alpherakii and G. menyuanensis using qPCR. Compared with G. menyuanensis from the lower-altitude environment, one gene of the third instar (cox2). two genes of the fourth instar (atp6and cox2), three genes of pupal (atp6. cox2and nad1), and six genes of adult (atp6, cox2, cob, nad2, nad4and nad5) are increased significantly in G. alpherakii:for the remaining genes, significant reduce or no significant difference of expression level are found. These results suggest that the mitochondrial gene of grassland caterpillars in different development stages are differently expressed. and different altitude environment have an important influence on the expression level of mitochondrial genes of grassland caterpillars.