Genetic diversity analysis of an isolate of microsporidia infesting Athetis lepigone

In this study, PCR amplification, cloning and sequencing analysis were adopted to explore genetic diversity of microsporidia (LEP9557) infecting Athetis lepigone. The small subunit ribosomal DNA (SSU rDNA), internal transcribed spacer (ITS) and intergenic spacers (IGS) of ribosomal RNA (rRNA) were cloned from the strain and sequenced. By means of multiple sequence alignment, we found that the three gene regions had different levels of polymorphism. There was greater polymorphism in ITS (74 variable sites) and IGS (55.59%) regions than in the SSU rDNA (17 variable sites). Phylogenetic analysis was performed using Kimura 2-parameter with neighbor joining and the results showed that LEP9557 had a close relationship with Nosema bombycis. Sequences of each clone were submitted to Genbank (Accession number: MF150254-MF150258). All of the results indicated the presence of genetic diversity in LEP9557, which established the foundation for identifying the phylogeny and relationships with other microsporidian strains, and had significant biological meaning for maintaining the survival and population continuity of the strain.

larvae of A. Lepigone [4,7], which had the closest relationship with Nosema bombycis and was considered to be potentially applied to control A. lepigone because of its high infectivity in larvae of A. lepigone. After being infected, A. lepigone larvae did not die directly, but showed series of symptoms such as anorexia, growth restriction, incomplete molting, pupation deformity, eclosion deformity and decreasing fecundity. Thus, this microsporidia strain is worthy of further study in sustainable A. lepigone control.
Ribosomal DNA is usually used for examining relationships among all known life forms, because it contains highly variable regions including many of the nucleotide positions in internal transcribed spacer (ITS), intergenic spacers (IGS) and highly conserved regions such as portions of the SSU rDNA and LSU rDNA. SSU rDNA is often used for the analysis of relationships between genera, and therefore can be used to determine the taxonomic status of microsporidia at generic level. Having a greater variation in biological species and subspecies, rDNA ITS could be used for the species level study [8].
In this study, we selected an isolate of Nosema (LEP9557) infecting A. lepigone and obtained its rDNA sequences including SSU, ITS and IGS regions to evaluate its genetic diversity. We also isolated and sequenced multiple clones (from 5 to 16) for each of the three rDNA regions from this population. Considering the variations in the ITS and IGS regions, this information might be very useful for the research in continuation and evolution of microsporidian species and for the biocontrol of A. lepigone by microsporidia.

Spore isolation and purification
To ensure the same source of infection, we obtained the microsporidian spores from an 5 infected A. lepigone larva collected from Handan, Hebei province, China. The third-instar larvae of A. lepigone in captivity were fed on the spores at a concentration of 1.0×10 7 spores/mL for 40 h, and then reared on artificial diet for 6-7 d. Subsequently, the midguts of infected larvae were separated, filtered and centrifuged to obtain the crude extracts of the microsporidia. Spores were purified by a discontinuous Percoll density gradient (25%, 50%, 75%, 100%, v/v), centrifuged at 17,500 rpm for 20 min, rinsed several times, and stored at 4°C [9].

Amplification, cloning and sequencing of rDNA sequence
Genomic DNA was extracted using CTAB kit (Aidlab Biotechnologies Co., Ltd Beijing China). Three pairs of primers corresponding to ITS, SSU, IGS rDNA (Table 1) were designed and synthesized according to the previous report [10]. The composition of the PCR reactions: 12.5 μL 2 × EasyTaq Supermix; 0.5μL each of the forward and reverse primers (10 μM); 1 μL template DNA; 10.5 μL ddH 2 O. The amplification was carried out using the following conditions: an initial DNA denaturation step at 94°C for 5min followed by 30 cycles of denaturation at 94°C for 1 min; annealing for 30 s (SSU at 53°C, ITS at 58°C, IGS at 50°C), and elongation at 72°C for 45 s. The last step was a 8 min extension at 72°C. Then the PCR products were purified using the Agarose Gel Extraction kit (TIANGEN, Beijing, China), and transformed into Escherichia coli DH5α. Three replicates of each clone were sent to the Sangon Biotech Company for sequencing.

Sequence analysis
Sequences obtained from each clone were identified by BLAST of the NCBI GenBank.
Phylogenetic trees of SSU, ITS and IGS rDNA were constructed using the neighbor-joining 6 method (Saitou and Nei, 1987) in MEGA 5.2 program. Bootstrap support was evaluated based on 1,000 replicates.

SSU rDNA sequences
We obtained five clones of SSU rDNA (S1-S5). The length of SSU rDNA clones had little variations, except S4 which was 1,209 bp, the other four clones were all 1,208 bp. The sequences of the five clones were deposited in GenBank, with the GenBank accession numbers of MF150254-MF150258 (S1-S5) ( Table 2). The sequence similarity was 99% among these five clones. There were only 17 polymorphic sites including 16 transitions / transversions and one deletion at position 738 (Table 3).
Phylogenetic analysis (Fig 1) showed that these five clones clustered separately and then gathered together to form a subgroup which shared 59% similarity with AB097401.2 Nosema bombycis but had a relatively distant phylogenetic relationship with FJ772435.1 Nosema heliothidis and AY747307.1 Nosema spodopterae. This result preliminarily indicated that LEP9557 belonged to Nosema genus.

ITS rDNA sequences
The sequence lengths of the sixteen clones of rDNA ITS ranged from 180 to 188 bp (Table 4), suggesting obvious length polymorphism. Polymorphism analysis revealed that, each sequence of the sixteen clones was unique with 96%~99% similarity to N. bombycis rDNA ITS sequence from GenBank. Sequence alignment showed that conversions / transversions and insertions / deletions existed on mononucleotide of ITS gene. There were 74 variation sites including 25 insertion/deletion sites and most of the variation sites were within the ITS sequence (Fig 2).
We performed phylogenetic analysis of the ITS sequence of LEP9557 shown above (Fig   3). T1 and T6 were in the same branch with 90% similarity and they had close relationship with JF443603.1 N. bombycis. Similarly, T16, T5 and T9 also had high similarity with N.
bombycis. In addition, it can be observed from the phylogenetic tree that microsporidian species in the Nosema genus had obvious genetic diversity (Fig 3). For example, GQ334400.1 N. bombycis and FJ443628.1 N. heliothidis were in the same branch with only 91% similarity concluding that, LEP9557 also had a certain degree of genetic diversity because the similarities between any two ITS clones were low. This phenomenon may be due to the short size and high mutation rate of ITS sequence.

Analysis of IGS rDNA sequences
The fifteen IGS sequences of the isolate varied in length from 269 bp to 286 bp (Table 5), showing obvious length polymorphism, with about 55.59% polymorphic positions. Among these clones, G4 and G5 had same sequence and their sequence similarity with N. bombycis rDNA sequence from GenBank was between 93%~99%. Sequence alignment showed that the IGS region was highly variable (Fig 4). Base sites were changeable including conversions/transversions of mononucleotides and insertions/deletions of bases, and most of the polymorphic positions were located within the IGS sequences.
Based on the phylogenetic tree of LEP9557 IGS rDNA, the similarities between G10 and G13 and between G4 and G5 were up to 99% and 100%, respectively (Fig 5). This result together with the result described above indicated that, G4 and G5 were the copy of same gene. This result was similar in both the IGS and the ITS region, which, again, indicated that 8 this isolate had intraspecific genetic diversity and its IGS region also had high mutation rate.

Discussion
At present, most of the studies on molecular biology of microsporida are focused on rDNA genes that are often present as multiple copies of tandem repeat units in all of the microsporidian genomes [11]. Each of the repeat units of ribosomal genes was composed of transcription units and intergenic spacer regions. For LEP9557, the pattern of the transcript unit was LSU-ITS-SSU-IGS-5S. This arrangement is similar to that of ribosomal genes of N.
Cunning et al. [13] indicated that the SSU rDNA and other highly conserved genes may be useful in the classification between genera rather than between species. Tsai et al. [14] suggested that ITS rDNA should have great potential applications in the classification between closely related species. The result of our study revealed that, there was only 1 bp difference between the sequences of the SSU rDNA gene. Polymorphism of mononucleotides mainly existed among SSU rDNA clones, while the mutation frequencies of regions such as rDNA-ITS and rDNA-IGS were higher than that of SSU rDNA. This might because SSU rDNA is extremely conservative and restricted when the close systematic relationship was measuring.
ITS sequences have more differential positions owing to a faster evolutionary rate. This polymorphism could be used for fine classification of species and subspecies and as a specific-fingerprinting to study variations in species [15]. The high variability of ITS has unique advantage in the study of biological classification, identification, phylogenetic and structural changes, and has been widely used in the field of clinical medicine [16], applied microbiology [17,18] and ecology [19,20]. In this study, ITS sequences, with high variability, were different between these clones not only in length but also in sequence. The difference in length and sequence of ITS would play an important role in the identification of PCR products. Considering that, if the ITS sequences were less than 300 bp, the information we obtained from those sequences were limited. With short sequences, different clones were easily clustered in phylogenetic analysis. If full-length sequences of rDNA ITS and IGS were used in the genetic polymorphic analysis of microsporidia, the possibility of deviation could be reduced.
In this study, LEP9557 represent significant intraspecific genetic differentiation, which may be a result of genetic variation during passage or the specificity of host tissues in which population individual parasite dwells. Sequence polymorphism showed the diversity and complexity of the genetic background of the microsporidian isolate. This result may be an adaptive reaction to resist adverse environment during evolution, and may have biological significance for maintaining the survival and continuity of the microsporidian population.
T G a S1-S5 indicated respectively small subunit rDNA for isolate LEP9557 b indicated start and end sites, respectively. c "-" indicated a missing nucleotide