| In recent years, with global warming and the adjustment of planting structure in China, the cut flowers pest species increase gradually and more and more damage seriously, resistant type and level increases. At present, the beet armyworm, Spodoptera exigua (Hiibner) is an important Noctuidae pest, thus how to carry on the effective prevention and control is an important work on pest control.In order to effectively monitor and manage this pest, it is necessary to identify the larva species of the flowers cutworm pest, the population dynamic of the beet armyworm, investigate its genetic variation and intraspecific phylogeographic structure in China. In this paper using sex pheromone traps in shenyang area of the beet armyworm population dynamics for a four years system monitoring (2012-2015); Determine the main pest species flower cutworm field in shenyang area, discusses the DNA Barcoding technology in flower cutworm insect larvae to identify the effectiveness of the application; Use of mtDNA and SSR molecular marker technology to study the genetic diversity and genetic structure of the beet armyworm; Finally, in order to make clear infection rate and strain system development status, we also investigated the distribution and infection characteristics of Wolbachia in the different geographical populations of the beet armyworm. The aims was to explore relationship of Wolbachia and host species evolution, thus to provide theoretical basis for the biological control of pests. The mainly results from the following several aspects:1. During 2012-2015, we collected 46,274,503 and 772 individuals of S. exigua by using of the sex pheromone traps in the whole years, respectively. On July 06 2012, the beet armyworm occurred, and only one peak in the whole year. On June 24 2013, the beet armyworm occurred, and three peak occured throughout the year. In the late of October, the numbers of the beet armyworm decreased rapidly; On 9 June 2014, the beet armyworm also occurred. Before 25 July, the numbers of the beet armyworm was low, two peak occurs throughout the year. Compared with the three years before, in 2015, the beet armyworm occurred large, early spring occurred earlier, at the end of May (May 25) the beet armyworm occurrence. Three peaks occurs throughout the whole year.2. Determine the five species of Noctuidae latter larvae damages on the flowers in China, namely Spodoptera exigua, Helicoverpa armigera, Mamestra brassicae, Spodoptera litura, and Mythimna separate. Additionally, we also used the universal primers of DNA barcode, COI and Cytb genes to identify the insect larvae. Five flower cutworm insect larvae COI gene sequences of interspecific average genetic distance was 0.093, average intraspecific genetic distance was 0.0018 Among them, the genetic distance between S. litura and S. exigua was the smallest (0.066). The genetic distance between S. litura and H. armigera was the largest (0.107). In contrast, five flower cutworm insect larvae interspecific average genetic distance was 0.110 base on Cytb gene sequences, the average intraspecific genetic distance was 0.002,. Among them, the genetic distance of S. exigua and S. litura was 0.084. S. exigua and M. brassicae was 0.138.3. We used mtDNA COI sequences (578 bp), and mtDNA Cytb (724 bp) to investigate its genetic diversity and genetic structure. Species samples were collected from 44 and 47 geographic locations, respectively. Overall, low levels of genetic diversity(COI:0.276 ± 0.020, Pi=0.00115 ± 0.00014; Cytb:Hd= 0.654 ± 0.017, Pi= 0.00238 ± 0.00020) for the total populations in these study regions were obtained.41 and 112 haplotypes were identified. Phylogenetic and median-joining network analyses indicated that there was no distinct geographic distribution pattern among haplotypes. The results also indicated that the mean difference distance between the populations was 0.001 and 0.003 base on COI and Cytb gene sequences, respectively. There was largest population genetic distance between HH and the other 48 geographic populations based on COI. There was largest population genetic distance between TL, NJ, BD and the other geographic populations based on Cytb. In addition, the beet armyworm Mantel correlation test indicated that there was no significant relationship between geographical distance and genetic distance. The analysis of molecular variance (global AMOVA) showed that the percentage of variation within a population (COI:77.14%; Cytb:84.86%) was greater than that between the populations. In order to clear the population genetic structure, we also take into account the various influence factors of population distribution, such as the geographic area (northeast, north China, east China, south China, central China, northwest, southwest), administrative divisions (20 provinces) and climatic regionalization (tropical, subtropical, warm temperate zone, temperate and cool), the results showed that the different geographical conditions and climatic conditions may not the main factors which influent the population genetic structure and species distribution pattern. Neutral testing results showed that neutral inspection parameters (Tajima's D and Fu's FS) are negative, with an obvious peak of unimodal type curve revealed the beet army worm had experience population expansion in China.4. To more clarify issues such as migration and demographic history of S. exigua in China, the genetic structure and diversity of S. exigua populations at 44 sampling sites,20 provinces were analyzed by using 8 nuclear microsatellite loci. Relatively low levels of genetic diversity for the total populations in these study regions were detected (microsatellites:Na=5.909, Ne=3.351). Among them, KM population was the highest population genetic diversity. Through Nei's genetic distance build of beet army worm in different geographic populations of UPGMA cluster analysis tree result showed that there was large genetic distance between LY, HH, GL, DA and other geographic populations. There was also significant genetic differentiation among some populations, such as TA, WH and DA population and other populations. Bayesian clustering analysis shows that when K= 2, beet armyworm population obviously divided into two, and a neighbor joining unweighted tree, considering the 916 individual showed two distinct groups, the ocean (S group) and the inland (L group). Mantel Test analysis showed that there is no certain genetic differentiation and geographic distance. Global AMOVA indicated that the percentage of variation within a population (95.53%) was greater than that between the populations (5.47%). Moreover, hierarchical AMOVA indicated that 0.16% genetic variation is derived from the groups (S and L of regional groups),3.91% comes from between the two groups of species.5. Using wsp gene, we investigated the infection rate of intracellular symbiotic bacteria Wolbachia in a total of 765 S. exigua collected from 44 locations,20 provinces. However, the infection rate of symbiotic bacteria in different geographic populations of beet army worm is extremely low, only one samples, which come from yancheng city, jiangsu province was infected Wolbachia. Additionally, the results of phylogenetic analyses indicated that the type of Wolbachia infected was Supergroup B. |
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