JIA-2018-09

2067 ZHANG Shuai et al. Journal of Integrative Agriculture 2018, 17(9): 2066–2073 (Blackman and Eastop 2000). Different genetic diversity has been observed in A . gossypii (Vanlerberghe-Masutti et al. 1999; Brevault et al. 2008; Charaabi et al. 2008; Carletto et al. 2009b; Razmjou et al. 2010; Komazaki et al. 2011). Genetic diversity is correlated with host type, and the existence of A . gossypii biotypes was determined by host transference experiments (Carletto et al. 2009b). Moreover, RAPD bands can be considered as diagnostic loci since they were fixed in A . gossypii populations collected on cucurbits and absent in those collected on noncucurbit host plants (Vanlerberghe-Masutti and Chavigny 1998). Using A . gossypii microsatellite loci (Vanlerberghe-Masutti et al. 1999), Carletto et al. (2009b) found that genotypes were associated with host plants, and 5 host-races including cucurbits, cotton, eggplant, potato, and chili or sweet pepper were identified at a large geographical scale. We found that 31 A . gossypii populations in northern China could be classified as 3 host biotypes (Luo et al. 2016). Mitochondrial genomes have been frequently used to explore insect biotype (Frohlich et al. 1999); a previous study found that there was high divergence in cytochrome b sequences between cucurbit and cotton host-races of A . gossypii (Carletto et al. 2009a). Based on complete mitochondrial sequences, we developed a molecular marker with 5 single- nucleotide polymorphisms to distinguish the biotypes (Wang et al. 2016). Previous research has demonstrated that A . gossypii can be holocyclic in northern China. Generally, A . gossypii eggs hatch in March on primary hosts, then alate adults appear and migrate to cotton fields, where seedlings emerge in late April to mid-May. In autumn, alate adults return to the primary hosts to mate and oviposit (Xia 1997). More than 10 of these primary hosts are extremely abundant in China, including hibiscus ( Hibiscus syriacus ), Chinese prickly ash ( Zanthoxylum bungeanum ), and pomegranate ( Punica granatum ) (Ebert and Cartwright 1997; Xia 1997; Wu et al. 2004). Populations of A . gossypii in northern China exhibit greater genotypic diversity and broad host range, but the details of life cycles of different biotypes remain unclear. The objective of the current study was to determine the host range of different host biotypes and the life cycle. To achieve this goal, A . gossypii collected from northern China in 2014 were analyzed using a portion of the mitochondrial gene. 2. Materials and methods 2.1. Plants and insects In 2014, 5 common summer host species (cotton, cucumber, zucchini, muskmelon, and kidney bean) were planted in a field in the same site in Anyang (36°5´34.8´´N, 114°31´47.19´´E), Henan Province, China, samples were collected during different seasons from those summer hosts (Wang et al. 2016). Populations from the urban ornamental plants (hibiscus, pomegranate, Chinese prickly ash, and Sophora japonica ) in the village of Anyang were also collected, and combined with collected data previously reported (Wang et al. 2016). Samples of wingless A . gossypii aphids were collected from cotton fields in northern China in late August 2014 at a total of 20 sites, including 6 populations in Hebei Province, 6 in Henan Province (includingAnyang), and 8 in Shandong Province, as done previously reported (Luo et al. 2016). Only 1 individual per plant from 8–48 cotton plants per site was collected to avoid sampling the offspring of a single female. 2.2. Sample sequencing Genomic DNA from single individuals was extracted using a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) following the protocol described by the manufacturer, and all extractions were stored at −20°C. Cytb and 16S gene regions were amplified with CytbF and 16SR primers (Wang et al. 2016; Zhang et al. 2016). The amplified products were sequenced at Shanghai Sangon Biotech, following trimming of the fragment to remove DNA data with poor quality. 2.3. Data analyses The number of polymorphic sites, haplotype diversity, nucleotide diversity, average number of nucleotide differences, and the number of haplotypes were calculated using DnaSP ver. 5.0 (Librado and Rozas 2009). Samples with haplotypes representing more than 2 individuals were analyzed. The alignment of the haplotype sequences was done manually in MEGA ver. 6.0 (Tamura et al. 2013), and 1 000 neighbor-joining (NJ) bootstrap replicates were performed. Then a NJ tree was constructed, and built using the iTOL webpage (http://itol.embl.de/ ) (Letunic and Bork 2016). Venny ver. 2.0 (http://bioinfogp.cnb.csic.es/ tools/venny) was used to identify overlapping (and unique) haplotypes and generate Venn diagrams. 3. Results 3.1. Identification of haplotypes All sequences were trimmed with ContigExpress Software, and sequences with lengths of 577 nt were kept. A total of 1 046 individual A . gossypii sequences were selected. Using DnaSP Software, 57 haplotypes were obtained from 1046 individual A . gossypii sequences. All sequences were