Bulletin of Entomological Research, Page 1 of 7 doi:10.1017/S0007485315000899 © Cambridge University Press 2015

Microsatellite markers for ferrugineus (Coleoptera: ) and other Cryptolestes species

Y. Wu1,2,F.Li1,Z.Li2, V. Stejskal3,Z.Kučerová3, G. Opit4, R. Aulicky3, T. Zhang1,P.He1 and Y. Cao1* 1Academy of State Administration of Grain, No. 11 Baiwanzhuang Street, Beijing, China: 2Department of Entomology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, China: 3Department of Pest Control of Stored Products and Food Safety, Crop Research Institute, Drnovská 507, Prague, Czech Republic: 4Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, Oklahoma, USA

Abstract

Cryptolestes ferrugineus (Stephens, 1831) is an important pest of stored pro- ducts. 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. In this study, we isolated 18 novel polymorphic microsatellite loci from an en- riched genomic library based on a biotin/streptavidin capture protocol. These loci will be useful tool to better understand the genetic structure and migration patterns of C. ferrugineus throughout the world. The genetic parameters were estimated based on 80 individual C. ferrugineus from two natural populations. The results revealed that 18 loci were different polymorphic levels. The numbers of alleles ranged from 3 to 12, and eleven loci demonstrated polymorphic information contents greater than 0.5. The observed (HO) and expected (HE) heterozygosities ranged from 0.051 to 0.883 and 0.173 to 0.815, respectively. Five locus/population combinations signifi- cantly deviated from Hardy–Weinberg equilibrium. We also demonstrated the po- tential utility of the C. ferrugineus microsatellites as population and species markers for four additional Cryptolestes species.

Keywords: , Cryptolestes microsatellites, primers, population genetics

(Accepted 7 October 2015)

Introduction grain, and creating favorable conditions for mold and fungus growth. Moreover, the presence of excrement and insect The rusty grain Cryptolestes ferrugineus (Stephens, fragments contaminate the grain and food products 1831) is a major secondary stored-product pest that feeds on (Trematerra et al., 2011). Due to its broad host range, short cereal, wheat, legume and oil seeds, as well as their processed life cycle, and high reproductive capacity, this species has rap- products (Throne et al., 2002; Hagstrum & Subramanyam, idly colonized temperate and tropical regions around the 2009). C. ferrugineus causes major economic losses by reducing world (Freeman, 1952; Sinha, 1975; Banks, 1979; Thomas & the amount of grain available for sale, lowering the quality of Zimmerman, 1989;Kučerová & Stejskal, 2002; Hagstrum & Subramanyam, 2009). The C. ferrugineus population of China is the largest and most widespread and has caused serious eco- *Author for correspondence nomic losses in several southern provinces, including Hainan, Phone: + 86-10-58523665 Yunnan, Guangxi, Guangdong, Fujian, Zhejiang, Jiangxi, Fax: + 86-10-58523700 Sichuan and Chongqing. Recently, increases in the import Email: [email protected] and export trades of grain have enhanced the dispersal and 2 Y. Wu et al. invasion of this pest. Once this insect pest is established and These were identified to the species levels by morph- begins to spread in a new area, eradication and control become ology (Lefkovitch, 1962; Halstead, 1993) and mitochondrial important but difficult. Nevertheless, established population cytochrome c oxidase subunit I (COI) sequence as described genetic techniques (using markers such as microsatellites) by Wang et al. (2014). are now routinely used in the management of pests (Wares et al., 2005; Ascunce et al., 2011). Isolation and screening of microsatellites Nuclear microsatellite makers are tandem repeats of nu- cleotide sequences that are distributed throughout the gen- The enrichment method used to establish the micro- ome. These genetic markers offer an advantage over other satellite-rich genomic libraries was modified from the classes of molecular markers because high mutation rates FIASCO method (Zane et al., 2002). This method was based lead to high levels of allelic variability within populations on biotinylated oligonucleotide sequences bound to (Selkoe & Toonen, 2006). These markers have become one of streptavidin-coated magnetic particles. Genomic DNA was ex- the most popular types of molecular markers for investiga- tracted from ten whole C. ferrugineus adults (a pooled samples) tions of population structures, colonization processes, tem- from the Qingyuan granary in Hebei according to the protocol poral and spatial population dynamics, and evolutionary of the DNeasy Blood & Tissue Kit (Qiagen). Following Sau3AI trends (Ascunce et al., 2011;Wuet al., 2011). Microsatellite ma- (TaKaRa) digestion of the genomic DNA for 3.5 h, restriction kers are especially useful in studies of species invasions in fragments of 300–1200 bp were recovered and purified using which they can help to distinguish the magnitude, location the QIAquick Gel Extraction Kit (Qiagen). The purified frag- and frequency of colonization events as well as differences ments were then ligated to two adaptor oligonucleotides in the levels of diversity and adaptive potential in the intro- (Adaptor A: 5′-GGCCAGAGACCCCAAGCTTCG-3′; and duced populations relative to the native populations (Davies Adaptor B: 5′-phosphate-GATCCGAAGCTTGGGGTCTCT ′ et al., 1999; Wares et al., 2005). GGCC-3 ) with T4 DNA ligase (TaKaRa) overnight at 16°C. The key to applying this technology is to obtain the micro- The ligation products were amplified using the adapter A se- satellite loci and design primers according to the flanking se- quence as the forward and reverse primers. Polymerase chain quences of the loci. Microsatellite loci have been isolated and reaction (PCR) amplification in final reaction volume of 25 µl characterized for important insect pests, such as fruit flies in consisted of 12.5 µl MasterMix with dye (TIANGEN, China), the Bactrocera (Wu et al., 2009; Buahom et al., 2013), 9.5 µl ddH2O, 1 µl (10pm) of primer (adapter A), and 2 µl of thrips (Brunner & Frey, 2004;Wuet al., 2014), Leptinotarsa de- ligation products as template DNA. PCR cycler conditions cemlineata Say (Grapputo, 2006), and Oedaleus decorus Gevm were initial denaturation at 94°C for 3 min, followed by 30 cy- (Berthier et al., 2008). Regarding common stored-product cles of 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s with the pests, microsatellite loci have been reported for three species final extension at 72°C for 10 min. The recovered PCR pro- of Bruchidae (Sembene et al., 2003; Alvarez et al., 2003, 2004, ducts were denatured and hybridized to biotinylated (AG)15 2005; Aebi et al., 2004), three species of Liposcelididae and (TCA)10 probes (Sangon). These heteroduplexes were (Mikac, 2006; Mikac & Fitzstimmons, 2010; Wei et al., 2011), then captured using Dynabeads M-280 Streptavidin and Tribolium castaneum Herbst (Pai et al., 2003). To date, (Invitrogen) and eluted. The DNA was subsequently enriched there are no reports of microsatellite markers isolated from by PCR using adaptor A as the primer (the same as above). The C. ferrugineus. In the present study, we describe the develop- microsatellite-enriched DNA fragments were ligated into ment and characterization of polymorphic microsatellite mar- pGEM-T Easy vectors (Promega) and transformed into kers in C. ferrugineus and assess their utilities as genetic DH5α-competent cells (TAKARA BIO INC). The positive re- markers for four Cryptolestes species. combinant 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 Materials and methods the same as above. PCR products were confirmed by 1% agar- ose gel electrophoresis. The clone amplified at least two bands Specimen rearing and collection was sequenced on an ABI 3730xl DNA Analyser (Microread Cryptolestes ferrugineus adults from a laboratory strain that Company, China). Based on the sequence data, the clones was established in 2014 from specimens collected from a gran- that yielded suitable flanking sequences were selected for pri- ary in Hebei were used to create an enriched DNA library. A mer design by software. The primers were first screened in 10 laboratory colony was maintained on whole wheat at 24°C C. ferrugineus. and 65–70% relative humidity. The adult specimens of five Cryptolestes species, i.e., C. ferrugineus (Stephens), Cryptolestes Polymorphism testing and cross-species amplification capensis (Waltl), Cryptolestes pusilloides (Steel & Howe), (Schönherr) and The polymorphisms of the microsatellite loci were further (Grouvelle) were acquired from China, the Czech Republic, tested in 80 C. ferrugineus individuals. Genomic DNA was pre- and the USA. Altogether, 11 adult strains were used in this pared from whole individual according to the protocol of the study, and the number of individual was shown in table 1. Tissue/Cell DNA Mini Kit (Tiangen). Amplification was per- Two strains of C. ferrugineus were used to test the polymorph- formed in a 20 µl volume containing 100 ng of genomic DNA, isms which collected from Shandong, 35°3′N/118°20′E and 2µlof10×Taq DNA polymerase buffer [100 mM Tris-HCl ′ ′ Hainan, 18°15 N/109°31 E, 40 individuals each area. Cross- (pH 8.3), 15 mM MgCl2], 40 µM dNTP, 0.5 U Taq polymerase species amplifications were performed on one strain each of (Tiangen) and 6 µM of each primer (Sangon), one of which was C. capensis and C. pusilloides and three strains each of C. pusillus labeled with a fluorescent dye (6-FAM or 5-HEX). The PCR and C. turcicus. The samples were laboratory strains or were profile included an initial denaturing step at 95°C for 5 min, collected from grain storage facilities and were preserved in followed by 35 cycles of 94°C for 30 s, 54°C for 35 s and 95% ethanol and stored at −80°C prior to DNA extraction. 72°C for 40 s, and final extension step at 72°C for 3 min. Microsatellite markers in Cryptolestes ferrugineus 3

Table 1. List of samples used in the isolation and evaluation of the C. ferrugineus microsatellite markers.

Species Locality Number Strain C. ferrugineus China, Hebei province, Qingyuan 10 laboratory cultures C. ferrugineus China, Shandong province, Linyi 40 field collection C. ferrugineus China, Hainan province, Sanya 40 field collection C. turcicus China, Guangdong province, Guangzhou 5 field collection C. turcicus China, Shandong province, Dezhou 5 field collection C. turcicus Czech Republic, Prague 5 laboratory cultures C. pusillus China, Hainan province, Haikou 5 field collection C. pusillus Czech Republic, Prague 5 laboratory cultures C. pusillus USA, Kansas 5 laboratory cultures C. capensis Czech Republic, Prague 10 laboratory cultures C. pusilloides China, Yunnan province, Luxi 10 laboratory cultures

Fluorescently labeled fragments were detected on an ABI Across all 80 individuals, the numbers of alleles per locus ran- PRISM 377 Genetic Analyzer, with a ROX-500 size standard ged from 3 to 12. For locus CFer16, which contains a tri- (Microread). The allele sizes were analyzed with nucleotide repeat, the allele size range is 114–128 bp. Since GeneMarker V2.2.0 (SoftGenetics, USA). this range is not divisible by 3, these data indicate either an im- The loci were cross-species amplified with the DNA from pure repeat, and/or size variation in the flanking region of 50 individuals from four additional pest species of the genus some alleles at this locus. The observed heterozygosities Cryptolestes Ganglbauer 1899 (Coleoptera: Laemophloeidae), (HO) ranged from 0.051 to 0.883 (mean = 0.473), and the ex- i.e., C. capensis, C. pusilloides, C. pusillus and C. turcicus, using pected heterozygosities (HE) ranged from 0.173 to 0.815 (mean the conditions detailed for C. ferrugineus. When no products = 0.569). Eleven loci exhibited PIC > 0.5. Five of these markers were observed after two rounds of PCR, a result of no ampli- (i.e., CFer1, CFer6, CFer7, CFer9 and CFer13) exhibited signifi- fication was recorded. cant 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 Statistical analysis CFer13), potentially resulting from inbreeding in the popula- The primer pairs were designed using PRIMER PREMIER tions genotyped. Significant genotypic linkage disequilibrium 3.0 (Rozen & Skaletsky, 2000). Micro-Checker V.2.2.3 (Van was not observed between any pair of loci. The FST value be- Oosterhout et al., 2004) was used to detect the presence of tween the two populations was 0.0015 (P > 0.05), indicating lit- null alleles. The program Genepop on the Web (http:// tle genetic differentiation and no significant between the two www.genepop.curtin.edu.au/) was used to test for linkage populations. disequilibrium between the pairs of loci in each population, deviations from Hardy–Weinberg equilibrium (HWE) at each locus/population combination were tested using Cross-species amplification Fisher’s exact tests, which were also used to estimate the ob- The 18 C. ferrugineus markers were also tested in other four served (H ) and expected heterozygosity (H ). The poly- O E Cryptolestes species to examine their potential applicability to morphic information content (PIC) was calculated using the other Cryptolestes pests (table 3). Six loci (i.e., CFer2, CFer5, CERVUS 2.0 program (Marshall et al., 1998). The genetic differ- CFer6, CFer11, CFer14, and CFer16) exhibited amplified entiation index, i.e., F was calculated with Arlequin version ST bands in all four Cryptolestes species. Four primers pairs 3.0 (Excoffier et al., 2005). (CFer1, CFer3, CFer7, and CFer8) exhibited cross-amplified products in one to three of the other species. Eight loci exhib- Results ited no amplification in any of other Cryptolestes species. Some loci exhibited different allele sizes in the other species, such as Microsatellites CFer2, CFer6, CFer8, CFer14, and CFer16, which indicates that Overall, the sequence analyses of 200 randomly picked some of the microsatellite loci exhibited species-specific allele positive colonies indicated that 165 (82.5% of clones) of the col- sizes. onies contained microsatellites, and 96 of these colonies had A total of nine markers that yielded two or more alleles microsatellite sequences with more than six perfect repeat were identified in the four Cryptolestes species, including six units. In 25 clones, the microsatellite sequences were too loci for C. turcicus, seven loci for C. pusillus, and five loci close to the linker for primer design, and two clones were each for C. pusillus and C. pusilloides. Several markers (marked2 found to be identical. Primers were designed for 69 sequences, in table 3) produced product but did not amplify in all indi- and 45 of these exhibited successful amplification in ten C. fer- viduals, suggesting the possibility of nulls or suboptimal rugineus. Fifteen primers were monomorphic, and 12 pro- PCR conditions. The 18 primer pairs utilized in produced am- duced nonspecific amplicons. A total of 18 primer pairs plicons in these four cross-species, seven were completely produced well-defined robust products of the expected size monomorphic, and 14 had only two alleles. Therefore, al- and had at least two alleles at every locus. though we showed some cross-amplification for our primer These 18 microsatellite loci were further tested for poly- pairs, further screening is required to determine whether morphisms on 80 individuals from two populations of C. fer- there is sufficient variation to make these markers useful in rugineus, and the summary data are presented in table 2. these other species. 4

Table 2. Characterization of 18 microsatellite loci isolated from C. ferrugineus.

Locus/GenBank No. Repeat Primer sequence (5′−3′) Allele size Total Na PIC Shandong (n = 40) Hainan (n =40) motif range (bp) Na HO HE Na HO HE – 1 CFer1/KR699605 (GT)6 F: ATTTTCTCTTCTGATTCGTATT 202 208 4 0.166 2 0.033 0.097 4 0.067 0.247 R: FAM-TTGTTACCTATATCGAGGATTT – 1 1 CFer2/KR699606 (TG)9 F: GTACCAGCCGTCGGGACGCAAC 195 209 7 0.731 6 0.500 0.763 7 0.800 0.786 R: HEX- GAACGCCCTCGTATGTAAAAAA – CFer3/KR699607 (TG)9 F: TAGAGCAATAAGCAAAAGCCCC 106 124 8 0.759 7 0.714 0.782 7 0.760 0.818 R: FAM-TATCATGCGTAAATGAACCAGA – 1 CFer4/KR699608 (TG)10 F: CACAAAACAAATAAAAACCAGC 153 167 6 0.516 6 0.567 0.588 6 0.367 0.527 R: HEX- AAAAAGAAAACAAATCGACCAG – CFer5/KR699609 (TG)9 F: GGTTAGACAGGATAAACAAAAC 277 295 9 0.764 9 0.633 0.781 9 0.800 0.824 R: FAM-AGTATGTGAACAAGCAAGAAAG – 1 1 CFer6/KR699610 (GT)5 F: CTCGTTTTTCTCCTATCCCCCA 241 255 5 0.201 5 0.133 0.190 4 0.107 0.230 R: HEX-CCACCGACCGCCCCTTCTTTAT – 1 1 CFer7/KR699611 (ATG)6 F: ATCAGCACTACGACAAAAATGG 103 120 3 0.403 3 0.067 0.513 3 0.133 0.391 R: HEX-TGGTGTTGAAGTATGACTACGA

– 1 Wu Y. CFer8/KR699612 (TG)10 F: AATATCATAAATAAGCCAGGGT 256 288 12 0.604 9 0.400 0.633 9 0.600 0.641 R: FAM-CAAGGAGTGTAAATTACAACAA – 1 CFer9/KR699613 (TG)10 F: GTAATATACTGTGGGTACTGGGT 174 214 6 0.336 5 0.233 0.377 3 0.172 0.331 R: FAM-TGTTTCTGTCTTGTTTATCTGTG al. et – 1 CFer10/KR699614 (GT)10 F: ATGTACAGCTTCATATACTTTC 171 181 6 0.599 5 0.900 0.654 6 0.867 0.657 R: HEX-ACTTGTTATTTTTCATAGATCC – CFer11/KR699615 (AC)11 F: TTAAGTCATTTTTGCAGCCAAGA 104 120 8 0.784 8 0.767 0.822 7 0.733 0.817 R: HEX-GCCGTACATATTTAGAAAGCGTG – CFer12/KR699616 (CA)8 F: CGCGTCGATCCCCTCTATAACA 154 174 11 0.726 11 0.700 0.778 8 0.600 0.759 R: HEX-CCAATCAGAAAGCGGTCCCAAT – 1 CFer13/KR699617 (AC)7 F: ATAAACTCCTATCCAGAAATGC 204 218 5 0.476 5 0.300 0.504 3 0.400 0.558 R: FAM-CGTGTACAATGAAATGTAAACG – CFer14/KR699618 (TG)6 F: AACTGAGTTGCGATGGGGGAAAAC 182 198 6 0.560 6 0.667 0.681 4 0.433 0.577 R: FAM-TCCTGCGGAATGACACACGTATAA – CFer15/KR699619 (TG)8 F: ACCTTGCTTCCTTTTTTATTCA 244 260 7 0.557 7 0.533 0.632 5 0.433 0.603 R: HEX-ATTTACCCCTATGTCCTCGTTT – 1 CFer16/KR699620 (TCA)7_(AC)7 F: TGGCGTGTTATGCGCGGCTTTTT 114 128 8 0.468 8 0.567 0.511 7 0.300 0.475 R: HEX-CCCTCACCGTTTCTTAGCTTTCT – 1 CFer17/KR699621 (TCA)5 F: AGCTCTTCCTGCTGAGTGTTGT 120 135 4 0.233 4 0.267 0.298 3 0.067 0.188 R: HEX-CGGTTGCCATTCCTTCTTCTTT – CFer18/KR699622 (TCA)9 (TTA) 5 F: TGATGTTTGGTTTATCTCCTTT 159 174 6 0.690 6 0.667 0.759 6 0.733 0.732 R: FAM-TTAGCATCCACATTGCACTATT

Na, number of alleles per locus; HO, observed heterozygosity; HE, expected heterozygosity; PIC, polymorphic information content. 1Deviation from HWE at P < 0.05 after Bonferroni correction. Microsatellite markers in Cryptolestes ferrugineus 5

Table 3. Results of the testing of C. ferrugineus microsatellite primers in four Cryptolestes species.

Locus Species (no. of individuals) C. turcicus (15) C. pusillus (15) C. capensis (10) C. pusilloides (10) CFer1 44 1 (172) 4 CFer2 11 (197) 6 (193,195,197,201,203,207) 2 (237,239) 22 (227,251) CFer3 53 (98,100,104,108,114) 1 (100) 4 232 (102,112) CFer4 44 44 CFer5 2 (293,297) 4 (279,281,283,285) 1 (293) 1 (293) CFer6 2 (255,257) 4 (241,243,245,247) 12 (247) 2 (253,255) CFer7 4 2 (118,121,8) 44 CFer8 4 3 (257,259,263) 3 (280,282,284) 1 (273) CFer9 44 44 CFer10 44 44 CFer11 22 (110,112) 22 (110,112) 22 (110,112) 22 (110,112) CFer12 44 44 CFer13 44 44 CFer14 31 (186,188,190) 2 (154,182) 4 (168,180,194,196) 3 (160,168,174) CFer15 44 44 CFer16 43 (112,126,130,156) 1 (154) 2 (142,168) 2 (160,168) CFer17 44 44 CFer18 44 44

2(), two different alleles observed. 1Several bands. 2Products seen in most but not in all samples. 3Faint but distinguishable bands. 4No amplification products observed.

Discussion dispersal by L. decolor at moderate to potentially high levels (Mikac & Fitzstimmons, 2010). To predict adult population C. ferrugineus and several Cryptolestes pests are geographic- abundance and variability of stored-product pest, traditional ally widespread and achieve maximum population densities and genetic of populations should be combined with each in grain stores in which abundant food resources and micro- other. habitats are present. Understanding the population genetic Understanding the genetic structures of populations and structures of Cryptolestes species is essential for formulating ef- the mechanisms that control those structures is a major object- fective flat grain beetle control strategies because this knowl- ive of evolutionary biology. The microsatellite markers re- edge aids the estimation of the temporal stabilities and ported in this study represented efficient tools for evaluating spatial connectivities of populations. the genetic diversity and structure of C. ferrugineus popula- The cross-species applications of microsatellite primers are tions. We are using these 18 loci to investigate the population difficult. For example, only 13 of 42 Bactrocera species primer genetics of this species, and the present study provides the first pairs have polymorphisms in Bactrocera cucurbitae (Coquillett) tool to try to understand the population dynamics and origin (Shearman et al., 2006). In the present study, approximately and dispersal trends with the ultimate aim of improving the one-third of the microsatellite markers of C. ferrugineus could prevention and control measures for this pest insect. be used in the Cryptolestes species, but the level of polymorph- ism 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 Acknowledgements DNA libraries. We thank Ms. Fengzhuo Liu for the C. ferrugineus sample Novel-use monitoring techniques such as genetics can be collection and culture. Financial support for this research used to provide valuable information on variation within was provided by the International Science and Technology and among populations. For the western corn rootworm Cooperation Program of China (2013DFG32350), the (WCR) which is an important pest of maize in southern Czech government (KONTAKT – LH12160), Grain Industry Europe and the USA, microsatellite genetic monitoring was Research Special Funds for Public Welfare Projects conducted during the introduction and establishment/spread (201513002), and a project of the National Natural Science phases of WCRs invasion of this region, and found that Serbia Foundation of China (No. 31201519). was the geographic source to Croatia (Lemic et al., 2015; Lvkošić et al., 2014). Regarding as stored-product pest, microsatellite molecular monitoring was conducted on References Acanthoscelides obtectus Say, showing that the origin of this spe- cies is probably further south than Mesoamerica, and a second Aebi, A., Shani, T., Butcher, R.D.J., Alvarez, N., Risterucci, A.M. more recent migration event from Andean America to Mexico & Benrey, B. (2004) Isolation and characterization of poly- (Alvarez et al., 2005). Also microsatellite markers were used to morphic microsatellites markers in Zabrotes subfasciatus investigate the genetic structure among invasive Liposcelis de- Boheman (Coleoptera: Bruchidae). Molecular Ecology Notes 4, color populations from Australia, and found long distance 752–754. 6 Y. Wu et al.

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