Introduction
The rusty grain beetle Cryptolestes ferrugineus (Stephens, 1831) is a major secondary stored-product pest that feeds on cereal, wheat, legume and oil seeds, as well as their processed products (Throne et al., Reference Throne, Doehlert and McMullen2002; Hagstrum & Subramanyam, Reference Hagstrum and Subramanyam2009). C. ferrugineus causes major economic losses by reducing the amount of grain available for sale, lowering the quality of grain, and creating favorable conditions for mold and fungus growth. Moreover, the presence of excrement and insect fragments contaminate the grain and food products (Trematerra et al., Reference Trematerra, Stejskal and Hubert2011). Due to its broad host range, short life cycle, and high reproductive capacity, this species has rapidly colonized temperate and tropical regions around the world (Freeman, Reference Freeman1952; Sinha, Reference Sinha1975; Banks, Reference Banks1979; Thomas & Zimmerman, Reference Thomas and Zimmerman1989; Kučerová & Stejskal, Reference Kučerová and Stejskal2002; Hagstrum & Subramanyam, Reference Hagstrum and Subramanyam2009). The C. ferrugineus population of China is the largest and most widespread and has caused serious economic losses in several southern provinces, including Hainan, Yunnan, Guangxi, Guangdong, Fujian, Zhejiang, Jiangxi, Sichuan and Chongqing. Recently, increases in the import and export trades of grain have enhanced the dispersal and invasion of this pest. Once this insect pest is established and begins to spread in a new area, eradication and control become important but difficult. Nevertheless, established population genetic techniques (using markers such as microsatellites) are now routinely used in the management of pests (Wares et al., Reference Wares, Hughes, Grosberg, Sax, Stachowicz and Gaines2005; Ascunce et al., Reference Ascunce, Yang, Oakey, Calcaterra, Wu, Shih, Goudet, Ross and Shoemaker2011).
Nuclear microsatellite makers are tandem repeats of nucleotide sequences that are distributed throughout the genome. These genetic markers offer an advantage over other classes of molecular markers because high mutation rates lead to high levels of allelic variability within populations (Selkoe & Toonen, Reference Selkoe and Toonen2006). These markers have become one of the most popular types of molecular markers for investigations of population structures, colonization processes, temporal and spatial population dynamics, and evolutionary trends (Ascunce et al., Reference Ascunce, Yang, Oakey, Calcaterra, Wu, Shih, Goudet, Ross and Shoemaker2011; Wu et al., Reference Wu, Li, Ruiz-Arce, McPheron, Wu and Li2011). Microsatellite makers are especially useful in studies of species invasions in which they can help to distinguish the magnitude, location and frequency of colonization events as well as differences in the levels of diversity and adaptive potential in the introduced populations relative to the native populations (Davies et al., Reference Davies, Villablanca and Roderick1999; Wares et al., Reference Wares, Hughes, Grosberg, Sax, Stachowicz and Gaines2005).
The key to applying this technology is to obtain the microsatellite loci and design primers according to the flanking sequences of the loci. Microsatellite loci have been isolated and characterized for important insect pests, such as fruit flies in the genus Bactrocera (Wu et al., Reference Wu, Li and Wu2009; Buahom et al., Reference Buahom, Du, Wu, Deng, Jiang, Fu and Li2013), thrips (Brunner & Frey, Reference Brunner and Frey2004; Wu et al., Reference Wu, Liu, Qiu, Li and Cao2014), Leptinotarsa decemlineata Say (Grapputo, Reference Grapputo2006), and Oedaleus decorus Gevm (Berthier et al., Reference Berthier, Loiseau, Streiff and Rarlettaz2008). Regarding common stored-product pests, microsatellite loci have been reported for three species of Bruchidae (Sembene et al., Reference Sembene, Vautrin, Silvain, Rasplus and Delobel2003; Alvarez et al., Reference Alvarez, Aebi, Risterucci, Hossaert-Mckey and Benrey2003, Reference Alvarez, Born, Risterucci, Sourrouille, Benrey and Hossaert-Mckey2004, Reference Alvarez, McKey, Hossaert-McKey, Born, Mercier and Benrey2005; Aebi et al., Reference Aebi, Shani, Butcher, Alvarez, Risterucci and Benrey2004), three species of Liposcelididae (Mikac, Reference Mikac2006; Mikac & Fitzstimmons, Reference Mikac and Fitzstimmons2010; Wei et al., Reference Wei, Yuan, Wang, Zhu and Wang2011), and Tribolium castaneum Herbst (Pai et al., Reference Pai, Sharakhov, Braginets, Costa and Yan2003). To date, there are no reports of microsatellite markers isolated from C. ferrugineus. In the present study, we describe the development and characterization of polymorphic microsatellite markers in C. ferrugineus and assess their utilities as genetic markers for four Cryptolestes species.
Materials and methods
Specimen rearing and collection
Cryptolestes ferrugineus adults from a laboratory strain that was established in 2014 from specimens collected from a granary in Hebei were used to create an enriched DNA library. A laboratory colony was maintained on whole wheat at 24°C and 65–70% relative humidity. The adult specimens of five Cryptolestes species, i.e., C. ferrugineus (Stephens), Cryptolestes capensis (Waltl), Cryptolestes pusilloides (Steel & Howe), Cryptolestes pusillus (Schönherr) and Cryptolestes turcicus (Grouvelle) were acquired from China, the Czech Republic, and the USA. Altogether, 11 adult strains were used in this study, and the number of individual was shown in table 1. Two strains of C. ferrugineus were used to test the polymorphisms which collected from Shandong, 35°3′N/118°20′E and Hainan, 18°15′N/109°31′E, 40 individuals each area. Cross-species amplifications were performed on one strain each of C. capensis and C. pusilloides and three strains each of C. pusillus and C. turcicus. The samples were laboratory strains or were collected from grain storage facilities and were preserved in 95% ethanol and stored at −80°C prior to DNA extraction. These insects were identified to the species levels by morphology (Lefkovitch, Reference Lefkovitch1962; Halstead, Reference Halstead1993) and mitochondrial cytochrome c oxidase subunit I (COI) sequence as described by Wang et al. (Reference Wang, Li, Zhang, Varadínová, Jiang, Kučerová, Stejskal, Opit, Cao and Li2014).
Table 1. List of samples used in the isolation and evaluation of the C. ferrugineus microsatellite markers.
Isolation and screening of microsatellites
The enrichment method used to establish the microsatellite-rich genomic libraries was modified from the FIASCO method (Zane et al., Reference Zane, Bargelloni and Patarnello2002). This method was based on biotinylated oligonucleotide sequences bound to streptavidin-coated magnetic particles. Genomic DNA was extracted from ten whole C. ferrugineus adults (a pooled samples) from the Qingyuan granary in Hebei according to the protocol of the DNeasy Blood & Tissue Kit (Qiagen). Following Sau3AI (TaKaRa) digestion of the genomic DNA for 3.5 h, restriction fragments of 300–1200 bp were recovered and purified using the QIAquick Gel Extraction Kit (Qiagen). The purified fragments were then ligated to two adaptor oligonucleotides (Adaptor A: 5′-GGCCAGAGACCCCAAGCTTCG-3′; and Adaptor B: 5′-phosphate-GATCCGAAGCTTGGGGTCTCTGGCC-3′) with T4 DNA ligase (TaKaRa) overnight at 16°C. The ligation products were amplified using the adapter A sequence as the forward and reverse primers. Polymerase chain reaction (PCR) amplification in final reaction volume of 25 µl consisted of 12.5 µl MasterMix with dye (TIANGEN, China), 9.5 µl ddH2O, 1 µl (10pm) of primer (adapter A), and 2 µl of ligation products as template DNA. PCR cycler conditions were initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s with the final extension at 72°C for 10 min. The recovered PCR products were denatured and hybridized to biotinylated (AG)15 and (TCA)10 probes (Sangon). These heteroduplexes were then captured using Dynabeads M-280 Streptavidin (Invitrogen) and eluted. The DNA was subsequently enriched by PCR using adaptor A as the primer (the same as above). The microsatellite-enriched DNA fragments were ligated into pGEM-T Easy vectors (Promega) and transformed into DH5α-competent cells (TAKARA BIO INC). The positive recombinant clones were screened by PCR using the adapter A and M13 + /M 13 – as the primers, single clone as template, PCR reaction mixture components and cycling conditions was the same as above. PCR products were confirmed by 1% agarose gel electrophoresis. The clone amplified at least two bands was sequenced on an ABI 3730xl DNA Analyser (Microread Company, China). Based on the sequence data, the clones that yielded suitable flanking sequences were selected for primer design by software. The primers were first screened in 10 C. ferrugineus.
Polymorphism testing and cross-species amplification
The polymorphisms of the microsatellite loci were further tested in 80 C. ferrugineus individuals. Genomic DNA was prepared from whole individual according to the protocol of the Tissue/Cell DNA Mini Kit (Tiangen). Amplification was performed in a 20 µl volume containing 100 ng of genomic DNA, 2 µl of 10 × Taq DNA polymerase buffer [100 mM Tris-HCl (pH 8.3), 15 mM MgCl2], 40 µM dNTP, 0.5 U Taq polymerase (Tiangen) and 6 µM of each primer (Sangon), one of which was labeled with a fluorescent dye (6-FAM or 5-HEX). The PCR profile included an initial denaturing step at 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 54°C for 35 s and 72°C for 40 s, and final extension step at 72°C for 3 min. Fluorescently labeled fragments were detected on an ABI PRISM 377 Genetic Analyzer, with a ROX-500 size standard (Microread). The allele sizes were analyzed with GeneMarker V2.2.0 (SoftGenetics, USA).
The loci were cross-species amplified with the DNA from 50 individuals from four additional pest species of the genus Cryptolestes Ganglbauer 1899 (Coleoptera: Laemophloeidae), i.e., C. capensis, C. pusilloides, C. pusillus and C. turcicus, using the conditions detailed for C. ferrugineus. When no products were observed after two rounds of PCR, a result of no amplification was recorded.
Statistical analysis
The primer pairs were designed using PRIMER PREMIER 3.0 (Rozen & Skaletsky, Reference Rozen and Skaletsky2000). Micro-Checker V.2.2.3 (Van Oosterhout et al., Reference Van Oosterhout, Hutchinson, Wills and Shipley2004) was used to detect the presence of null alleles. The program Genepop on the Web (http://www.genepop.curtin.edu.au/) was used to test for linkage disequilibrium between the pairs of loci in each population, deviations from Hardy–Weinberg equilibrium (HWE) at each locus/population combination were tested using Fisher's exact tests, which were also used to estimate the observed (H O) and expected heterozygosity (H E). The polymorphic information content (PIC) was calculated using the CERVUS 2.0 program (Marshall et al., Reference Marshall, Slate, Kruuk and Pemberton1998). The genetic differentiation index, i.e., F ST was calculated with Arlequin version 3.0 (Excoffier et al., Reference Excoffier, Laval and Schneider2005).
Results
Microsatellites
Overall, the sequence analyses of 200 randomly picked positive colonies indicated that 165 (82.5% of clones) of the colonies contained microsatellites, and 96 of these colonies had microsatellite sequences with more than six perfect repeat units. In 25 clones, the microsatellite sequences were too close to the linker for primer design, and two clones were found to be identical. Primers were designed for 69 sequences, and 45 of these exhibited successful amplification in ten C. ferrugineus. Fifteen primers were monomorphic, and 12 produced nonspecific amplicons. A total of 18 primer pairs produced well-defined robust products of the expected size and had at least two alleles at every locus.
These 18 microsatellite loci were further tested for polymorphisms on 80 individuals from two populations of C. ferrugineus, and the summary data are presented in table 2. Across all 80 individuals, the numbers of alleles per locus ranged from 3 to 12. For locus CFer16, which contains a tri-nucleotide repeat, the allele size range is 114–128 bp. Since this range is not divisible by 3, these data indicate either an impure repeat, and/or size variation in the flanking region of some alleles at this locus. The observed heterozygosities (H O) ranged from 0.051 to 0.883 (mean = 0.473), and the expected heterozygosities (H E) ranged from 0.173 to 0.815 (mean = 0.569). Eleven loci exhibited PIC > 0.5. Five of these markers (i.e., CFer1, CFer6, CFer7, CFer9 and CFer13) exhibited significant deviations from HWE after Bonferroni correction due to deficits of heterozygotes. Micro-Checker revealed the presence of null alleles in four of these loci (i.e., CFer1 CFer6, CFer7 and CFer13), potentially resulting from inbreeding in the populations genotyped. Significant genotypic linkage disequilibrium was not observed between any pair of loci. The F ST value between the two populations was 0.0015 (P > 0.05), indicating little genetic differentiation and no significant between the two populations.
Table 2. Characterization of 18 microsatellite loci isolated from C. ferrugineus.
N a, number of alleles per locus; H O, observed heterozygosity; H E, expected heterozygosity; PIC, polymorphic information content.
1 Deviation from HWE at P < 0.05 after Bonferroni correction.
Cross-species amplification
The 18 C. ferrugineus markers were also tested in other four Cryptolestes species to examine their potential applicability to other Cryptolestes pests (table 3). Six loci (i.e., CFer2, CFer5, CFer6, CFer11, CFer14, and CFer16) exhibited amplified bands in all four Cryptolestes species. Four primers pairs (CFer1, CFer3, CFer7, and CFer8) exhibited cross-amplified products in one to three of the other species. Eight loci exhibited no amplification in any of other Cryptolestes species. Some loci exhibited different allele sizes in the other species, such as CFer2, CFer6, CFer8, CFer14, and CFer16, which indicates that some of the microsatellite loci exhibited species-specific allele sizes.
Table 3. Results of the testing of C. ferrugineus microsatellite primers in four Cryptolestes species.
2(), two different alleles observed.
1 Several bands.
2 Products seen in most but not in all samples.
3 Faint but distinguishable bands.
4 No amplification products observed.
A total of nine markers that yielded two or more alleles were identified in the four Cryptolestes species, including six loci for C. turcicus, seven loci for C. pusillus, and five loci each for C. pusillus and C. pusilloides. Several markers (marked2 in table 3) produced product but did not amplify in all individuals, suggesting the possibility of nulls or suboptimal PCR conditions. The 18 primer pairs utilized in produced amplicons in these four cross-species, seven were completely monomorphic, and 14 had only two alleles. Therefore, although we showed some cross-amplification for our primer pairs, further screening is required to determine whether there is sufficient variation to make these markers useful in these other species.
Discussion
C. ferrugineus and several Cryptolestes pests are geographically widespread and achieve maximum population densities in grain stores in which abundant food resources and micro-habitats are present. Understanding the population genetic structures of Cryptolestes species is essential for formulating effective flat grain beetle control strategies because this knowledge aids the estimation of the temporal stabilities and spatial connectivities of populations.
The cross-species applications of microsatellite primers are difficult. For example, only 13 of 42 Bactrocera species primer pairs have polymorphisms in Bactrocera cucurbitae (Coquillett) (Shearman et al., Reference Shearman, Gilchrist, Crisafulli, Graham, Lange and Frommer2006). In the present study, approximately one-third of the microsatellite markers of C. ferrugineus could be used in the Cryptolestes species, but the level of polymorphism is lower than in the species from which the microsatellite have been isolated. Therefore, in this case the best method of obtaining microsatellite markers is to screen from genomic DNA libraries.
Novel-use monitoring techniques such as genetics can be used to provide valuable information on variation within and among populations. For the western corn rootworm (WCR) which is an important pest of maize in southern Europe and the USA, microsatellite genetic monitoring was conducted during the introduction and establishment/spread phases of WCRs invasion of this region, and found that Serbia was the geographic source to Croatia (Lemic et al., Reference Lemic, Mikac, Kozina, Benitez, Mclean and Bažok2015; Lvkošić et al., Reference Lvkošić, Gorman, Lemić and Mikac2014). Regarding as stored-product pest, microsatellite molecular monitoring was conducted on Acanthoscelides obtectus Say, showing that the origin of this species is probably further south than Mesoamerica, and a second more recent migration event from Andean America to Mexico (Alvarez et al., Reference Alvarez, McKey, Hossaert-McKey, Born, Mercier and Benrey2005). Also microsatellite markers were used to investigate the genetic structure among invasive Liposcelis decolor populations from Australia, and found long distance dispersal by L. decolor at moderate to potentially high levels (Mikac & Fitzstimmons, Reference Mikac and Fitzstimmons2010). To predict adult population abundance and variability of stored-product pest, traditional and genetic of populations should be combined with each other.
Understanding the genetic structures of populations and the mechanisms that control those structures is a major objective of evolutionary biology. The microsatellite markers reported in this study represented efficient tools for evaluating the genetic diversity and structure of C. ferrugineus populations. We are using these 18 loci to investigate the population genetics of this species, and the present study provides the first tool to try to understand the population dynamics and origin and dispersal trends with the ultimate aim of improving the prevention and control measures for this pest insect.
Acknowledgements
We thank Ms. Fengzhuo Liu for the C. ferrugineus sample collection and culture. Financial support for this research was provided by the International Science and Technology Cooperation Program of China (2013DFG32350), the Czech government (KONTAKT – LH12160), Grain Industry Research Special Funds for Public Welfare Projects (201513002), and a project of the National Natural Science Foundation of China (No. 31201519).
