The citrus spiny whitefly Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae), is a pest of citrus (Citrus spp.; Rutaceae) and tea (Camelia sinensis (L.) Kuntze; Ericales: Theaceae), and several serious outbreaks of a tea-infesting whitefly in China and Japan have been attributed to this species over the last 20 yr (Kanmiya et al. 2011). The citrus spiny whitefly affects host plants by removal of plant sap, but whiteflies also cause indirect damage by producing honeydew, which promotes sooty mold. In addition to citrus and tea, this whitefly infests many other host plants, as noted in Evans (2008). Furthermore, A. spiniferus is an EPPO (European and Mediterranean Plant Protection Organization) quarantine species that has recently been moved from the A1 to the A2 list (Anonymous 2011). It is worth mentioning that commonly A. spiniferus reproduces sexually and infrequently by arrhenotoky (Huang et al. 1999), which may influence the Hardy-Weinberg equilibrium (see Discussion).

Because A. spiniferus is a well-known insect pest, some aspects of its biology, behavior, ecology, and management have been thoroughly investigated (Van den Berg et al. 2000; Han and Cui. 2002; Guo et al. 2006; Muniappan et al. 2006; Peng et al. 2010; Niu et al. 2014). In contrast, little is known about the genetic diversity and population structure of A. spiniferus, and no microsatellite markers have been developed for this species. Fu & Han (2007) preliminarily analyzed the genetic diversity of A. spiniferus from 7 populations in eastern China using random amplified polymorphic DNA (RAPD). Kanmiya et al (2011) proposed new specific status for tea- infesting populations of the nominal citrus spiny whitefly A. spiniferus using molecular marker (mtCOI gene), morphological and acoustic analysis to distinguish it from A. spiniferus that constitutes the citrus-infesting population. However, genetic diversity for A. spiniferus has not been comprehensively investigated, and it is poorly understood. Such gaps in our knowledge highlight the need for the development of highly polymorphic and informative molecular markers in order to accurately assess and monitor genetic variation in and population structure of A. spiniferus populations.

Microsatellite DNA, also known as simple sequence repeats (SSRs), is a technique that has been widely used in many areas of research (Carleton et al. 2001; Peterlunger et al. 2003). In the field of entomology, it has been used for gene mapping, population genetic structure analysis, identification of genetic relationships, and genetic map constructions (Gorman 1997; Zenger et al. 2005). In this study, we developed 10 high polymorphic microsatellite markers for A. spiniferus, and crossspecies amplifications were also conducted of the 5 closely related whitefly species (Aleurodicus disperses Russell, Pealius mori Takahashi, Aleuroclava aucubae Kuwana, Bemisia tabaci (Gennadius) MEAM1 and B. tabaci MED). These markers are expected to be used widely in studies on the genetic diversity and population genetic structure of whitefly species in their native and invasive ranges and in tracing their global invasion history.

Materials and Methods

Aleurocanthus spiniferus samples were collected from tea plants in 6 provinces: Shandong (N 34.73° E 117.29°), Jiangsu (N 32.41° E 119.42°), Zhejiang (N 30.19° E 120.09°), Hunan (N 28.20° E 113.09°), Guizhou (N 27.66° E 107.60°), and Yunnan (N 25.13° E 102.75°) in China. At first, we did not identify the gender of the 60 A. spiniferus individuals, until we noticed that all 10 study loci deviated from Hardy-Weinberg equilibrium. To ensure the accuracy of our results, another 48 female individuals from Jiangsu (N 32.41° E 119.42°) and Sichuan (N 30.57° È 104.07°) provinces were tested later. Gender was determined as per Dubey & Ko (2012). All specimens (including those from the 5 closely related whitefly species) were preserved in 100% ethanol and stored at -20 °C until DNA extractions were performed.

Genomic DNA was extracted using the saltingout method as described by Teng et al. (2009). The enriched library was constructed by FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) protocol according to Zane et al. (2002) with slight modifications in terms of PCR amplification conditions. The genomic DNA was first digested with the restriction enzyme Msel (BioLabs, Beijing, China) and approximately 250 ng of DNA was ligated to lpg Msel adaptor ( 5′-TACTCAGGACTCAT-3′ /5′-GACGATGAGTCCTGAG - 3′). The digestionligation products was diluted (1:10) and amplified with adaptor-specific primers (5′-GATGAGTCCTGAGTAAN-3′, Msel-N) in 20 µL reactions containing 1 × PCR buffer (10 mM Tris-HCl, pH 9.0, 25 °C; 50 mM KCl), MgCl₂ 1.5 mM, dNTPs 250 µM, MseI-N 0.5 µM, 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 5 µL diluted digestionligation DNA. The PCR conditions were 5 min at 94 °C followed by 20 cycles of 30 s at 94 °C, 1 min at 53 °C, 1 min at 72 °C with a final extension of 10 min at 72 °C. The PCR products were hybridized with biotinylated (AG)₁₂ and (GT)₁₂for 1 h at 68 °C, respectively, after denaturation for 5 min at 95 °C. The DNA fragments that hybridized to biotinylated probes were selectively captured by streptavidin-coated magnetic beads (Streptavidin Magnesphere Paramagnetic Particles, Promega, Shanghai, China). Microsatellite-rich fragments were amplified by MseI-N primers for 35 cycles and cloned into an Escherichia coli strain (DH5α) using pGEM-T Easy vectors (Promega). Insertpositive bacterial clones identified by blue-white selection were amplified using M13 primers and visualized by agarose gel electrophoresis. A total of 132 positive clones were chosen for sequencing.

Because the flanking regions of some sequences were too short, and only 41 pairs of primers were designed and synthesized in the end. In addition, we attached FAM, HEX and TAMRA fluorophores at the 5′ ends of each forward primer. PCR was conducted in 25 µL volumes containing PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 50 ng genomic DNA, 0.75 U Taq polymerase, and 4 pmol of primer. Amplification included an initial denaturation step at 94 °C for 4 min, followed by 42 cycles of 50 s at 94 °C, 50 s at 54–63 °C depending on the primer pair (Table 1), 1 min at 72 °C, and a final extension for 10 min at 72 °C. The PCR products of 3 fluorophores (FAM, HEX and TAMRA) were mixed in a ratio that was based on the brightness of bands visualized by agarose gel electrophoresis. Parameters relevant to multiplex analysis of 10 loci are included in Table 1. PCR products were analyzed using an ABI 3730XL DNA sequencer. Electropherograms were derived using Gene Scan 4.0 and used to deduce DNA fragment sizes using Gene Mapper 4.0 (Sangon Biotech, Shanghai).

Cross-species amplification was tested in 5 closely related whitefly species (Aleurodicus dispersus, Pealius mori, Aleuroclava aucubae, Bemisia tabaci MEAM1 and B. tabaci MED). DNA samples from 10 individuals of each species were tested.

The number of alleles at each polymorphic locus, size range, and heterozygosities (both observed and expected) were calculated using Cervus 2.0 software (Marshall et al. 1998). Deviation from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium at each locus were calculated using GenePop 3.4 software (Rousset 2008). The null allele frequency was calculated using Micro-Checker (Van Oosterhout et al. 2004).

Results

The 41 pairs of primers described above were used for screening microsatellite polymorphism. Twenty-eight loci successfully amplified the target regions, while only 10 loci revealed high microsatellite polymorphisms. All of the polymorphic information contents (PIC) of these 10 primers were greater than 0.5. The number of alleles ranged from 12 to 27 across the 60 individuals tested. The expected heterozygosity (H_E) and the observed heterozygosity (H_O) ranged from 0.851 to 0.958 and from 0.621 to 0.850, respectively (Table 1). Null allele frequencies at those loci ranged from 0.0352 to 0.1619 and all loci exhibited no significant evidence of null alleles, but deviated from Hardy-Weinberg equilibrium based on testing of 60 unsexed individuals. Table 2 provided characteristics of the loci for A. spiniferus from 48 female individuals in which all loci also deviated from the Hardy-Weinberg equilibrium, and no loci exhibited significant evidence of null alleles. In addition, loci C19-2 and D14, and also I20 and F12 manifested linkage disequilibrium after sequential Bonferroni correction.

The results of cross-species amplification showed that all loci could be used for amplification at different levels except F12 (Table 3). In particular, loci A21-2, D14, F13-2, F42-2, and I20 were readily amplified in all 5 additional whitefly species.

Discussion

Traditionally, microsatellite markers are developed by extensive screening for microsatellite containing clones through repetitive hybridization of a repeat motif probe to a large number of random clones (Rassmann et al. 1991). Such an isolation strategy resulted in low rate of the number of positive clones (containing microsatellites) detection (Zane et al. 2002). Using modified protocols of Hamilton et al (1999) and Glenn et al (2000) to construct and clone genomic libraries increased proportions of inserts that contained tandem repeat arrays. A great number of microsatellite repeat regions have been detected, sequenced and used to design specific flanking primers for microdatellite amplification.

Our studied loci indicated a high degree of polymorphism (PIC > 0.5), and can be successfully used to study the population genetic structures of A. spiniferus and other closely related whitefly species. The 10 loci still deviated from HardyWeinberg equilibrium when another 48 female whiteflies were tested, although we avoided the influence factor of reproduction (Table 2), which was consistent with the findings of Kobmoo et al. (2009), who found that 4 out of 10 loci in Ceratosolen fusciceps Mayr (Hymenoptera: Agaonidae) deviated from the Hardy-Weinberg equilibrium (HWE), and the global test of deviation from the HWE equilibrium was significant (P < 0.01) even when only females were tested. This deviation occurred because the involved individuals were indeed inbreeding. It is important to note that the HWE test is a low power statistical test to identify genotyping errors (Leal 2005). Thus, the reasons for deviating from the HWE by A. spiniferus may be due to other factors such as mutation, natural selection, small population size (genetic drift and population bottle necks) or presence of population substructures including Wahlund's effect ( http://www.dorak.info/genetics/popgen.html). Specifically, Martins et al. (2012) reported that 11 of the 15 microsatellite loci in Planococcus citri (Hemiptera: Pseudococcidae) significantly deviated from the HWE, probably due to data structuring, since individuals from 2 significantly distant geographic areas were used. Probably this is a potential factor affecting the HWE for A. spiniferus, since we used 48 females from Jiangsu (N 32.41° E 119.42°) and Sichuan (N 30.57° E 104.07°) provinces for testing. Moreover, small population size results in random sampling errors as well as unpredictable genotype frequencies; and in small populations, departure from expected genotype frequencies occurs more easily. Probably this is another factor because we used 60 samples from 6 provinces (10 from each location) in the beginning. Furthermore, as mentioned above, a very high mutation rate in the population (typical mutation rates are < 10–4 per generation) and selection of one or a combination of genotypes (Ineichen & Batschelet 1975) also could violate the HWE. Clearly, further population genetic structure studies are urgently needed to assess these hypotheses.

Table 1.

Characteristics of polymorphic microsatellite loci of Aleurocanthus Spiniferus from 60 individuals collected from tea plants in 6 Provinces (Shandong, Jiangsu, Zhejiang, Hunan, Guizhou, and Yunnan) in China.

Table 2.

Characteristics of polymorphic micresatellite loci for Aleurocanthus spiniferus from 48 female individuals from tea plant s in Jiangsu and Sichuan provinces.

Table 3.

Results of cross-species amplification tests involving DNA samples from 10 individuals of each of 5 closely related whitefly species.

Our results may also provide new insights about the reproductive mechanism of A. spiniferus. Since few individuals engaged in arrhenotokous reproduction, the reproductive strategies of this insect may not significantly affect HardyWeinberg equilibrium of the loci studied.

Microsatellites are particularly reliable for studying recent biological invasions (Zygouridis et al. 2009) and are a powerful means to detect the origin and invasion route of exotic insect species (Fonseca et al. 2010). Our cross-species amplification results indicated that the 10 microsatellite polymorphic loci may possess high versatility, and can be used to study gene flow, genetic diversity and population genetic structure of related whitefly species. Meanwhile, this information will facilitate the selection of molecular markers for B. tabaci, a controversial invasive species worldwide. Overall, these microsatellite markers show suitable resolution to meet our needs in subsequent studies and to give us insights into the genetic variability, gene flow and the mating system of whitefly species.