Rapid discrimination of the common species of the stored product pest Liposcelis (Psocoptera: Liposcelididae) from China and the Czech Republic, based on PCR-RFLP analysis

Psocids of the genus Liposcelis (Psocoptera: Liposcelididae) are stored product pests that are difficult to identify morphologically. A molecular method based on Restriction Fragment Length Polymorphism (RFLP) of the PCR-amplified 16S rDNA gene was developed for the rapid discrimination of four common species (L. bostrychophila, L. entomophila, L. decolor, and L. paeta). Different developmental stages and populations (P.R. China and Czech Republic) were tested. One DNA fragment of about 500 bp in length was amplified from genomic DNA and the fragment was then digested using the restriction endonuclease DraI. Identification of the relevant banding pattern allowed all the developmental stages and both sexes to be discriminated in the species tested. The banding patterns of L. entomophila from all populations were identical, while the relevant restriction digests and sequence analysis confirmed that the Chinese and Czech populations of L. bostrychophila, L. decolor, and L. paeta differed. In conclusion, PCR-RFLP with one pair of primers (16Sar and 16Sbr) and one restrictive endonuclease, DraI, proved a reliable method for rapidly discriminating the Liposcelis species tested. 713 * Corresponding authors; e-mails: lizh@cau.edu.cn; kucerova@vurv.cz (12 geographical populations – Table 1). Adult identification was confirmed by C. Lienhard (Museum of Natural History, Geneva, Switzerland). The rearing diet consisted of wholewheat flour: skimmed milk powder: yeast powder in a ratio of 10 : 1 : 1 (Ding et al., 2001). The insects were kept at 27–28.5°C and 75–80% RH. Males, females, and nymphs of each species and geographical population (20 individuals of each species and developmental stage) were subjected to PCRRFLP. In the parthenogenetic L. bostrychophila, females and nymphs were tested. The PCR products from a single individual of each of the species L. bostrychophila, L. decolor, and L. paeta was additionally analyzed by sequencing. All psocids were killed in 100% ethanol and their DNA extracted immediately.

Correct identification of psocid species is a prerequisite for the establishment of an effective pest management program.Traditional morphological identification of Liposcelis adults (Lienhard, 1990;Bai & Cao, 1997;Li et al., 1999;Lienhard & Smithers, 2002) or of the immature stages (Ku erová, 2002) is difficult and requires specialists.Methods for rapidly identifying booklice are unavailable.Molecular techniques based on DNA analysis offer a viable and economical alternative method of identification (Muraji & Nakahara, 2001).Different DNA and Polymerase Chain Reaction (PCR)-based molecular markers have been developed for insect diagnostic pur-poses, including Random Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphisms (RFLP), satellite DNA, and Inter-Simple Sequence Repeats (ISSR) (Li, 2007).The nucleotide sequences of Liposcelis have been analyzed in order to infer phylogenetic relationships, geographic origin, population genetics or gene flow (Cruickshank et al., 2001;Barker et al., 2003;Johnson & Mockford, 2003;Yoshizawa & Johnson, 2003;Mikac, 2006Mikac, , 2007;;Mikac & Clarke, 2006).As a result, more than 60 sequences have been submitted to international DNA sequence databases, such as the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank.Using these Liposcelis DNA sequences, we have analyzed a rapid discrimination method for Liposcelis from China based primarily on PCR-RFLP (Qin et al., 2007).
In this study, the above discrimination investigations are extended by extracting genetic DNA from single individuals and using PCR-RFLP to discriminate between four species of Liposcelis originating from the People's Republic of China (P.R. China) and Czech Republic (CZ), L. bostrychophila, L. entomophila, L. decolor, and L. paeta.

Insect collection and culture
Liposcelis bostrychophila, L. entomophila, L. decolor, and L. paeta were collected in storehouses in the P.R.China and CZ (12 geographical populations -Table 1).Adult identification was confirmed by C. Lienhard (Museum of Natural History, Geneva, Switzerland).The rearing diet consisted of wholewheat flour: skimmed milk powder: yeast powder in a ratio of 10 : 1 : 1 (Ding et al., 2001).The insects were kept at 27-28.5°C and 75-80% RH.Males, females, and nymphs of each species and geographical population (20 individuals of each species and developmental stage) were subjected to PCR-RFLP.In the parthenogenetic L. bostrychophila, females and nymphs were tested.The PCR products from a single individual of each of the species L. bostrychophila, L. decolor, and L. paeta was additionally analyzed by sequencing.All psocids were killed in 100% ethanol and their DNA extracted immediately.

DNA extraction
Genomic DNA was extracted from each geographical population of each species using the method of Gong et al. (2002), which is a slight modification based on the method of cetyltrimethylammonium bromide (CTAB).

Restriction endonuclease digestion and sequencing
Restriction endonuclease digestion was performed in a 20 µl reaction volume containing 10 µl of the target DNA (PCR product), 2 µl restriction buffer, 1 µl restriction endonuclease (DraI) and 7 µl ddH2O.The digest was incubated at 37°C for 6 h.A 3.5%-4.0%(w/v) agarose gel was made from 1 × TBE buffer, and 8 µl of the digested PCR product was subjected to electrophoresis alongside a DNA marker.
This sequencing method was used on the following species.Genomic DNA of Liposcelis bostrychophila, L. decolor, and L. paeta was extracted from a single individual of each species and the 16S rDNA gene amplified using the 16Sar and 16Sbr primers.The amplified products were then separated on a 1.0% (w/v) agarose gel (1 × TAE), and the bands (stained with ethidium bromide) were excised.The agarose gel slice containing the DNA band of interest was placed in a centrifuge tube.The DNA was then purified and cloned, then the bacterial liquid culture was used as a template for nucleotide sequencing (AuGCT Biotechnology Synthesis Lab, Beijing, China).DNAMAN software was used for restriction analysis of the six sequences.

PCR analysis
All of the PCR products were nearly the same size (~500 bp).The PCR products were not influenced by species, geographical population, stage of development or sex.

RFLP and sequencing analysis
The PCR products could be digested at specific regions.Though different Liposcelis species could be visually distinguished according to their electrophoresis patterns, digestion result of Liposcelis of the same species was not influenced by individuality, sex or developmental stage.
Samples from the P.R.China 16S rDNA of four species of Liposcelis were successfully cut into distinct visible bands.Four bands (~300 bp, 120 bp, 70 bp, and 50 bp) were visible in the 16S rDNA of L. entomophila; two clear bands of different sizes (~350 bp and 100 bp; ~350 bp, and 200 bp; ~300 bp and 220 bp) were seen in the 16S rDNA of L. bostrychophila, L. decolor, and L. paeta, respectively.These results demonstrate that restriction endonuclease digestion was not affected by the different geographical populations, stages of development or sex of the four common species of the stored product pest Liposcelis from the P.R.China (Fig. 1).
Samples from the CZ and comparison with those from the P.R.China The repeated experiments on the CZ samples produced identical results, and were not influenced by stage of development or sex.16S rDNA of L. entomophila was excised into four visible bands (~300 bp, 120 bp, 70 bp and 50 bp), which were identical with those result obtained for the P.R.China samples.For the other three Liposcelis species, i.e.L. bostrychophila, L. decolor, and L. paeta, the sizes of these fragments differed from those the samples from the P.R.China (Fig. 2).Therefore, the different visible bands observed after restriction digestion of 16S rDNA were further analyzed and confirmed by sequencing.The results showed that the sequences from the same species exhibited a high level similarity, with limited genetic mutations.Fig. 3 shows the sequence lengths and digestion sites.For L. bostrychophila, the 16S rDNA of the sample from the P.R.China had 3 digestion sites and 4 fragments of 326 bp, 100 bp, 34 bp, and 33 bp, L. entomophila_HB-P.R.China

Location collected Population
TABLE 1. Liposcelis species and populations used in this study (populations were coded by combining species names with acronyms of collection countries and sites).and the 16S rDNA of the sample from CZ had 2 digestion sites and 3 fragments of 225 bp, 192 bp, and 72 bp.The 16S rDNA of L. decolor from the P.R.China and CZ displayed 1 digestion site and 2 fragments (338 bp and 167 bp; 337 bp and 168 bp, respectively).For L. paeta, the 16S rDNA of the sample from the P.R.China had only 1 digestion site, and 2 fragments of 279 bp, and 201 bp, but that of the sample from the CZ had 3 digestion sites and 4 fragments of 200 bp, 184 bp, 62 bp and 33 bp.The sequencing results showed that the mutation points in 16S rDNA were consistent with the banding patterns of the electrophoresis map, except for some small fragments that could not be detected by agarose gel electrophoresis.1. Electrophoresis of DraI-digested PCR products from the 16S rDNA of four common species of stored Liposcelis from the P.R. China.Lanes: 1-3 -female, male, and nymphs of L. entomophila_HB-P.R.China; 4-6 -female, male, and nymphs of L. entomophila_CQ-P.R.China; 7-8 -female and nymphs of L. bostrychophila_GX-P.R.China; 9-10 -female and nymphs of L. bostrychophila_CQ-P.R.China; 11-13 -female, male, and nymphs of L. decolor_CQ-P.R.China; 14-16 -female, male, and nymphs of L. decolor_HN-P.R.China; 17-19 -female, male, and nymphs of L. paeta_ZJ-P.R.China; 20-22 -female, male, and nymphs of L. paeta_JX-P.R.China; M -molecular size marker (bp).

DISCUSSION
In this report, we show that PCR-RFLP based on 16S rDNA is an efficient way of rapidly discriminating four storage pest psocids from the P.R.China and CZ.One pair of primers 16Sar/Sbr and one restrictive endonuclease, DraI, are sufficient to discriminate between the Liposcelis species tested.The whole process from extraction of genomic DNA to the formation of a restriction map takes only 12h.This rapid molecular method can be used by plant quarantine offices to identify the most commonly occurring species of Liposcelis infesting stored products.
The results of this study indicate that the PCR-RFLP method is able to identify L. entomophila using the same digestion pattern of 16S rDNA, regardless of differences between geographical populations, stages of development or sex of the individuals in the samples.In comparison, L. bostrychophila, L. decolor, and L. paeta were well discriminated, but the numbers or sizes of the 16S rDNA band differed, even for the same species.For each species of these three psocids, the 16S rDNA of the P.R.China and CZ populations showed different stable electrophoresis patterns.Further, the sequence analysis indicated that the points of mutation in the 16S rDNA are consistent with the electrophoresis patterns.This enabled us to conclude that there are intraspecific genetic differences between the P.R.China and CZ populations of L. bostrychophila, L. decolor, and L. paeta.Further worldwide geographical studies are required in order to draw general conclusions regarding intra-specific genetic differences in Liposcelis.
Recently, molecular methods have been used to detect natural populations of Diptera, Coleoptera, Hymenoptera, and Lepidoptera (Ratcliffe et al., 2002;Loxdale & Mac-Donald, 2004;Gómez-Zurita et al., 2005;Li, 2007).The common methods used are RAPD analysis, RFLP patterns, Amplified Fragment Length Polymorphism (AFLP) molecular markers, species-specific primers, and ISSR.Of these methods, PCR-RFLP analysis of molecular markers is regarded as an easy, fast and reliable method (Muraji & Nakahara, 2002) for identifying closely related species that have an ambiguous taxonomic status, similar insect species that are difficult to identify using morphological characteristics and the immature stages of insects (Sperling et al., 1994;Tuda et al., 1995;Litjens et al., 2001;Zapata et al., 2007).Since different genetic mutations occur in DNA sequences in species during evolution, it is possible to find different points of discrimination using restriction endonucleases.Based on this study, it may be concluded that the PCR-RFLP method with one pair of primers, 16Sar/Sbr, and one restrictive endonuclease, DraI, is a convenient and reliable way to rapidly discriminate between the four common species of stored product psocids.This method can detect the species of Liposcelis studied not only the adults, but also the immature stages.Moreover it confirmed that this method is able to reveal some intra-specific differences associated with the geographical distribution of these species.Further molecular research on other psocid species and their geographical strains on a worldwide scale is needed in the future.