Gnetic Diversity And Cold Hardiness Of Multiple Eiytral Color Morphs Of The Overwintering Harmonia Axyrudis(Pailas)

Based on the gene sequences of mtDNA, CO I and 16 S rRNA as well as the analysis method, the molecular differentiation of 15 different stain morphs of Harmonia axyridis was studied. The variable nucleotide sites were calculated and sequence differences among different strain morphotypes and individuals were compared. Molecular phylogenetic trees were constructed to better explore and research the potential determinant of the stain morphs of H. axyridis. Meanwhile, the early and depth of diapause of H. axyridis were devided into four groups according to background and gender: black male(BM), black female(BF), yellow male(YM)and yellow female( YF). Then, as the change of following five parameters: supercooling points and freezing points, weight and water content, trehalose and glycogen concentration, Treh1 and Treh2 activity, Treh and TPS relative expression, 4 sets of samples were measured or calculated in order to explore whether there are differences in certain laws of the cold hardiness among different spot morphs of H. axyridis.The diversity results showed that there were 152 and 234 variable sites in the gene sequences of mitochondrial DNA(mtDNA) in the CO I and 16 S r RNA genes, respectively. On those sites, the main form of base change was transversion. A seven-base insert segment(CGACTAC) was found in COI-B1 B and COI-Y4 D, whereas no insert fragment was found in the sequence of 16 S r RNA. To find exogenous species, the adjacency method(NJ: neighbourhood joining) was used to construct molecular phylogenetic growth trees. In addition, ClustalW and DAMBEXP(software) were used to calculate similarities and genetic distances. The results revealed that in mtDNA, CO I stain morphotypes of COI-YIB, COI-Y3 D, COI-Y8 F and COI-Y2 B were not in the same branch as most of the other stain morphotypes and that significant differences existed between the two groups in terms of similarity and genetic distance. Similarly, in 16 S rRNA, 16S-Y1 C and 16S-Y2 B were found in the same branch of an exogenous species whereas these two morphs were quite different from the other morphs with regard to their molecular phylogenetic evolution, similarity and genetic distance. These findings suggest that the genetic differentiation among most individuals within each stain group is small, that H. axyridis with the same or similar elytral color morph do not cluster in the same branch and that individuals of the same stain morph differentiate in terms of their genetic evolution.Meanwhile, the cold hardiness of overwintering H. axyridis were evaluated. By determining their super-cooling point(SCP), which is an important parameter of insect cold hardiness, it is found that BM decreased from-10.25 ℃ to-16.73 ℃, YM decreased from-11.74 ℃ to-16.51 ℃, BF decreased from-12.76 ℃ to-18.54 ℃, and YF from-12.06 ℃ to-17.33 ℃. It is indicated that with the continued cold acclimation, ladybirds gradually increase in tolerance to cold; no matter how deep the diapause turned, females? FCP were lower than males?, which meant that females owned more adaptability to the cold condition. The weight and moisture content of four groups were found both reduced, and changes in the water content of each group varied: BF fell from 59.48% to 54.06%, YF increased from 47.47% to 54.38%, BM increased from 53.20% to 54.26%, and YM increased from 53.53% to 54.39%. In overall, the females? change extent of water content was greater than males, indicating females in response to cold could make more substantial changes to adjust themselves to get a better adaptation to adversity. As to the measurement of trehalose content, yellow groups significantly increased with the deepening of the depth of diapause, thus providing more energy to withstand the cold stress; black groups, in contrast, might fail to overwinter. Determining the enzyme activity of Trehlase in Treh1 found that all groups of them increased with the deepening of the depth of diapaus, and groups of YF, YM and BM were significant, including the range of males was extremely higher than females. It is indicated the cold tolerance of females was higher than males, which needed more energy to resist the trouble. The conclusion also meets above super-cooling point and water content. In summary, cold hardiness of the yellow female groups tend to be strongest.In addition, the use of Real-time fluorescence quantitative PCR relative expression of TPS and Treh determination discovered that, TPS and Treh genes expressed in various diapause of H. axyridis. With the diapause deepen, HaTreh1-2 changed extremely obvious. It rose obviously in the four groups, which may obtain that HaTreh1-2 played an important role in the adversity protection of H. axyridis.