Complete mitochondrial genome reveals genetic diversity of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae)

Acta Tropica 152 (2015) 157–164

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Complete mitochondrial genome reveals genetic diversity of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) Hoi-Sen Yong a,b , Sze-Looi Song b , Praphathip Eamsobhana c,∗ , Share-Yuan Goh a,b , Phaik-Eem Lim d a

Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia Chancellory High Impact Research, University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand d Institute of Ocean and Earth Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia b

a r t i c l e

i n f o

Article history: Received 26 June 2015 Received in revised form 31 August 2015 Accepted 1 September 2015 Available online 5 September 2015 Keywords: Angiostrongylus cantonensis Rat lungworm Mitochondrial genome Transfer RNA Genetic diversity Phylogenetics

a b s t r a c t Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. Earlier work on its mitochondrial genome was based on long polymerase chain reaction method. To date, only the mitogenome of the isolates from China has been studied. We report here the complete mitogenome of the Thailand isolate based on next generation sequencing and compare the genetic diversity with other isolates. The mitogenome of the Thailand isolate (13,519 bp) is longer than those of the China isolates (13,497–13,502 bp). Five protein-coding genes (atp6, cox1, cox2, cob, nad2) show variations in length among the isolates. The stop codon of the Thailand isolate differs from the China and Taiwan isolates in 4 genes (atp6, cob, nad2, nad6). Additionally, the Thailand isolate has 4 incomplete T stop codon compared to 3 in the China and Taiwan isolates. The control region is longer in the Thailand isolate (258 bp) than the China (230–236 bp) and Taiwan (237 bp) isolates. The intergenic sequence between nad4 and cox1 genes in the Thailand isolate lacks 2 bp (indels) at the 5’-end of the sequence as well as differs at 7 other sites compared to the China and Taiwan isolates. In the Thailand isolate, 18 tRNAs lack the entire TC-arm, compared to 17 in the China isolate and 16 in the Taiwan isolate. Phylogenetic analyses based on 36 mt-genes, 12 PCGs, 2 rRNA genes, 22 tRNA genes and control region all indicate closer genetic affinity between the China and Taiwan isolates compared to the Thailand isolate. Based on 36 mt-genes, the inter-isolate genetic distance varies from p = 3.2% between China and Taiwan isolates to p = 11.6% between Thailand and China isolates. The mitogenome will be useful for population, phylogenetics and phylogeography studies. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The rat lungworm Angiostrongylus cantonensis is a food-borne zoonotic parasite of public health importance in the tropics and subtropics. It causes eosinophilic meningitis or meningoencephalitis (Alicata, 1962; Rosen et al., 1962; Eamsobhana, 2014). It is endemic to Asia and the Pacific Basin but has spread to other parts of the world, including Caribbean islands, North and South America, and Africa (Eamsobhana, 2014). It was first observed in Canton (now Guangzhou), China in the lungs of Rattus norvegicus and Rattus rattus (Chen, 1935). Humans are non-permissive accidental host, acquiring the infection by consuming raw or inadequately cooked food harboring the infective larvae of the parasite (Cross, 1987).

∗ Corresponding author. E-mail address: [email protected] (P. Eamsobhana). http://dx.doi.org/10.1016/j.actatropica.2015.09.001 0001-706X/© 2015 Elsevier B.V. All rights reserved.

Because of public health importance, immunodiagnosis of human angiostrongyliasis has been investigated extensively (Eamsobhana and Yong, 2009). Additionally, the parasite has received much attention in clinical and laboratory studies (GraeffTeixeira et al., 2009). Mitochondrial genes in particular have been applied for systematics, population and phylogenetic studies (Dusitsittipon et al., 2014; Eamsobhana et al., 2010; Foronda et al., 2010; Lee et al., 2014; Monte et al., 2012; Tokiwa et al., 2012; Yong et al., 2015a). The Angiostrongylus genus is represented by some 22 species in the world (Spratt, 2015). To date, the complete mitochondrial genome (mitogenome) of A. cantonensis from China has been studied based on long PCR method (Lv et al., 2012, 2014). Five mitogenome haplotypes with 745 variable sites have been reported from three provinces in China (Lv et al., 2014). We report here the complete mitogenome of A. cantonensis from Thailand and Taiwan based on next-generation sequencing, and comparison of the genetic diversity in different geographical isolates.

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Table 1 Gene order of mitochondrial genome of Angiostrongylus cantonensis (Thailand isolate, KT186242). Gene

Location

Strand

trnP(tgg) trnV(tac) nad6 nad4l trnW(tca) trnE(ttc) rrnS trnS2(tga) trnN(gtt) trnY(gta) nad1 atp6 trnK(ttt) trnL2(taa) trnS1(tct) nad2 trnI(gat) trnR(acg) trnQ(ttg) trnF(gaa) cob trnL1(tag) cox3 trnT(tgt) nad4 cox1 trnC(gca) trnM(cat) trnD(gtc) trnG(tcc) cox2 trnH(gtg) rrnL nad3 nad5 trnA(tgc) Control region

1–55 64–117 127–552 553–784 785–839 842–902 901–1598 1598–1650 1650–1708 1714–1771 1772–2647 2650–3247 3248–3308 3309–3365 3366–3415 3416–4264 4274–4328 4328–4381 4384–4438 4439–4496 4497–5606 5606–5660 5661–6426 6427–6484 6485–7714 7778–9355 9355–9414 9415–9473 9476–9531 9532–9587 9588–10280 10279–10332 10333–11288 11287–11622 11624–13205 13206–13261 13262–13519

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

2. Materials and methods The whole genome of the Thailand isolate of A. cantonensis (Bioproduct PRJNA 260338; Yong et al., 2015b) and that of the Taiwan isolate (BioProject PRJEB493; WormBase ParaSite release 2–March 2015) were used to obtain the respective mitogenome. The mitogenome of the China isolate was from GenBank (NC 013065). Angiostrongylus costaricensis NC 013067 and Angiostrongylus vasorum NC 018602 were used as outgroups. 2.1. Sequence and genome analysis Raw sequences were extracted in FASTQ format and the quality of sequences was evaluated using the FastQC software (Andrews, 2010). All the ambiguous nucleotides and reads with an average quality value lower than Q20 were excluded from further analysis. De novo assembly was performed using the CLC Genomic Workbench v.7.0.4 (Qiagen, Germany) and contigs greater than 13 kbp were subjected to BLAST (Altschul et al., 1990) alignment against the nucleotide database at national center for biotechnology information (NCBI). Contigs with hits to mitochondrial genes or genomes were identified and extracted from CLC genomic workbench. 2.2. Mitogenome identification, annotation and visualization A contig identified as mitogenome was manually examined for repeats at the beginning and end of the sequence to establish a circular mtDNA. It was then annotated with MITOS (Bernt

Size(bp) 55 54 426 232 55 61 698 53 59 58 876 598 61 57 50 849 55 54 55 58 1110 55 766 58 1230 1578 60 59 56 56 693 54 956 336 1582 56 258

Intergenicsequence

Start/stopcodon

8 9 ATG/TAA ATT/T 2 −2 −1 −1 5 2

TTG/TAG ATT/T

9 −1 2

TTG/TAA

−1

TTG/TAG TTG/T

63 −1

TTG/TAG ATT/TAA

2

−2 −2 1

TTG/TAG

TTG/TAA ATA/T

et al., 2012) followed by manual validation of the coding regions using the NCBI ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). The sequin file for the Thailand isolate generated from MITOS was edited and submitted to NCBI according to ORF Finder result (NCBI GenBank accession number KT186242). The circular mitogenome of A. cantonensis was visualized with Blast Ring Image Generator (BRIG) (Alikhan et al., 2011). 2.3. Phylogenetic analysis The total length of the aligned sequences of 36 mt-genes, 12 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and control region as well as the selected models used for maximum likelihood (ML) and Bayesian Inference (BI) analyses are summarized in Table A1 (Appendix A: Supplementary data). The sequences of the 12 PCGs and control region were separately aligned using ClustalX (Thompson et al., 1997) program and subsequently edited and trimmed using BioEdit v.7.0.5.3 (Hall, 1999). The sequences of rrnS, rrnL and 22 mt-tRNA genes were aligned by MAFFT v.7 (Katoh and Standley, 2013). Kakusan v.3 (Tanabe, 2007) was used to determine the best-fit nucleotide substitution models for maximum likelihood (ML) and Bayesian (BI) analyses selected using the corrected Akaike Information Criterion (Akaike, 1973) and the Bayesian information criterion (Schwarz, 1978), respectively (Table A1, Appendix A: Supplementary data). Phylograms of 12 concatenated PCGs, 36 mt-genes, 2 rRNA genes, 22 mt-tRNA genes and control region were constructed using TreeFinder (Jobb et al., 2004). Bootstrap values (BP) were generated via 1000 ML bootstrap replicates. Bayesian analyses were

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Table 2 Nucleotide composition of mitochondrial whole genome, protein-coding genes, rRNA genes and control region of Angiostrongylus cantonensis. (a) Thailand isolate, KT186242; (b) China isolate NC 013065; and (c) Taiwan isolate PRJEB493. Region

A%

C%

G%

T%

A + T%

G + C%

AT skew

GC skew

Whole genome

(a) 26.8 (b) 24.5 (c) 24.5 (a) 23.4 (b) 22.5 (c) 22.7 (a) 23.8 (b) 20.8 (c) 20.9 (a) 25.1 (b) 24.4 (c) 22.9 (a) 23.2 (b) 20.4 (c) 20.8 (a) 24.9 (b) 22.1 (c) 21.6 (a) 21.7 (b) 20.5 (c) 19.5 (a) 26.4 (b) 23.4 (c) 22.3 (a) 23.2 (b) 19.6 (c) 19.9 (a) 25.7 (b) 23.0 (c) 23.4 (a) 20.7 (b) 17.2 (c) 17.2 (a) 26.5 (b) 23.1 (c) 23.6 (a) 22.3 (b) 17.6 (c) 16.7 (a) 34.4 (b) 32.2 (c) 32.6 (a) 33.5 (b) 32.3 (c) 33.2 (a) 43.8 (b) 43.7 (c) 45.2

(a) 6.2 (b) 6.2 (c) 6.2 (a) 5.0 (b) 5.3 (c) 5.1 (a) 9.2 (b) 9.3 (c) 9.3 (a) 7.8 (b) 7.3 (c) 7.8 (a) 6.3 (b) 6.4 (c) 6.7 (a) 7.0 (b) 7.5 (c) 6.7 (a) 7.0 (b) 7.0 (c) 6.6 (a) 3.3 (b) 3.4 (c) 4.0 (a) 2.7 (b) 3.0 (c) 3.0 (a) 6.7 (b) 6.7 (c) 6.6 (a) 2.6 (b) 2.6 (c) 2.6 (a) 5.1 (b) 4.8 (c) 5.0 (a) 4.7 (b) 4.0 (c) 4.2 (a) 7.3 (b) 7.0 (c) 7.2 (a) 6.0 (b) 5.5 (c) 5.5 (a) 3.9 (b) 4.8 (c) 4.6

(a) 18.6 (b) 20.6 (c) 20.6 (a) 20.4 (b) 21.2 (c) 21.0 (a) 20.9 (b) 23.2 (c) 23.0 (a) 22.5 (b) 23.1 (c) 24.4 (a) 18.7 (b) 22.4 (c) 22.0 (a) 18.8 (b) 21.3 (c) 21.6 (a) 21.1 (b) 22.4 (c) 23.2 (a) 17.7 (b) 19.6 (c) 20.6 (a) 20.5 (b) 23.5 (c) 23.2 (a) 16.9 (b) 19.3 (c) 18.8 (a) 20.7 (b) 25.0 (c) 25.0 (a) 17.5 (b) 20.6 (c) 20.4 (a) 16.7 (b) 21.1 (c) 21.8 (a) 17.6 (b) 18.7 (c) 18.5 (a) 16.2 (b) 16.0 (c) 15.3 (a) 6.6 (b) 10.8 (c) 10.1

(a) 48.4 (b) 48.7 (c) 48.7 (a) 51.2 (b) 51.0 (c) 51.2 (a) 46.1 (b) 46.7 (c) 46.8 (a) 44.6 (b) 45.2 (c) 44.9 (a) 51.8 (b) 50.8 (c) 50.5 (a) 49.3 (b) 49.1 (c) 50.1 (a) 50.2 (b) 50.1 (c) 50.7 (a) 52.6 (b) 53.6 (c) 53.1 (a) 53.6 (b) 53.9 (c) 53.9 (a) 50.7 (b) 51.0 (c) 51.2 (a) 56.0 (b) 55.2 (c) 55.2 (a) 50.9 (b) 51.5 (c) 51.0 (a) 56.3 (b) 57.3 (c) 57.3 (a) 40.7 (b) 42.1 (c) 41.7 (a) 44.3 (b) 46.2 (c) 46.0 (a) 45.7 (b) 40.7 (c) 40.1

(a) 75.2 (b) 73.3 (c) 73.2 (a) 74.6 (b) 73.5 (c) 73.8 (a) 69.9 (b) 67.6 (c) 67.7 (a) 69.7 (b) 69.6 (c) 67.8 (a) 75.1 (b) 71.1 (c) 71.3 (a) 74.1 (b) 71.2 (c) 71.7 (a) 71.9 (b) 70.7 (c) 70.2 (a) 79.0 (b) 77.0 (c) 75.4 (a) 76.8 (b) 73.5 (c) 73.8 (a) 76.3 (b) 74.0 (c) 74.6 (a) 76.7 (b) 72.4 (c) 72.4 (a) 77.4 (b) 74.6 (c) 74.6 (a) 78.6 (b) 74.9 (c) 73.9 (a) 75.1 (b) 74.3 (c) 74.3 (a) 77.8 (b) 78.5 (c) 79.1 (a) 89.5 (b) 84.4 (c) 85.2

(a) 24.8 (b) 26.7 (c) 26.8 (a) 25.4 (b) 26.5 (c) 26.2 (a) 30.1 (b) 32.4 (c) 32.3 (a) 30.3 (b) 30.4 (c) 32.2 (a) 24.9 (b) 28.9 (c) 28.7 (a) 25.9 (b) 28.8 (c) 28.3 (a) 28.1 (b) 29.3 (c) 29.8 (a) 21.0 (b) 23.0 (c) 24.6 (a) 23.2 (b) 26.5 (c) 26.2 (a) 23.7 (b) 26.0 (c) 25.4 (a) 23.3 (b) 27.6 (c) 27.6 (a) 22.6 (b) 25.4 (c) 25.4 (a) 21.4 (b) 25.1 (c) 26.1 (a) 24.9 (b) 25.7 (c) 25.7 (a) 22.2 (b) 21.5 (c) 20.9 (a) 10.5 (b) 15.6 (c) 14.8

(a) −0.287 (b) −0.330 (c) −0.331 (a) −0.373 (b) −0.388 (c) −0.386 (a) −0.319 (b) −0.383 (c) −0.383 (a) −0.280 (b) −0.299 (c) −0.324 (a) −0.381 (b) −0.428 (c) −0.417 (a) −0.329 (b) −0.379 (c) −0.397 (a) −0.396 (b) −0.419 (c) −0.444 (a) −0.332 (b) −0.392 (c) −0.408 (a) −0.396 (b) −0.467 (c) −0.461 (a) −0.328 (b) −0.378 (c) −0.373 (a) −0.460 (b) −0.525 (c) −0.525 (a) −0.315 (b) −0.381 (c) −0.367 (a) −0.433 (b) −0.530 (c) −0.549 (a) −0.084 (b) −0.133 (c) −0.122 (a) −0.139 (b) −0.177 (c) −0.162 (a) −0.021 (b) 0.036 (c) 0.060

(a) 0.500 (b) 0.539 (c) 0.537 (a) 0.606 (b) 0.600 (c) 0.607 (a) 0.389 (b) 0.429 (c) 0.424 (a) 0.485 (b) 0.520 (c) 0.516 (a) 0.498 (b) 0.554 (c) 0.533 (a) 0.456 (b) 0.479 (c) 0.527 (a) 0.502 (b) 0.526 (c) 0.557 (a) 0.686 (b) 0.704 (c) 0.675 (a) 0.767 (b) 0.774 (c) 0.771 (a) 0.430 (b) 0.485 (c) 0.480 (a) 0.777 (b) 0.812 (c) 0.812 (a) 0.549 (b) 0.622 (c) 0.606 (a) 0.561 (b) 0.681 (c) 0.674 (a) 0.414 (b) 0.455 (c) 0.440 (a) 0.459 (b) 0.488 (c) 0.469 (a) 0.257 (b) 0.385 (c) 0.372

atp6

cox1

cox2

cox3

cob

nad1

nad2

nad3

nad4

nad4l

nad5

nad6

rrnS

rrnL

Control region

conducted using the Markov chain Monte Carlo (MCMC) method via Mr. Bayes v.3.1.2 (Huelsenbeck and Ronquist, 2001), with two independent runs of 2 × 106 generations with four chains, and with trees sampled every 200th generation. Likelihood values for all post-analysis trees and parameters were evaluated for convergence and burn-in using the “sump” command in MrBayes and the computer program Tracer v.1.5 (http://tree.bio.ed.ac.uk/software/ tracer/). The first 200 trees from each run were discarded as burn-in (where the likelihood values were stabilized prior to the burn-in), and the remaining trees were used for the construction of a 50% majority-rule consensus tree. Phylogenetic trees were viewed and edited by FigTree v.1.4 (Rambaut, 2012).

2.4. Genetic divergence Uncorrected (p) pairwise genetic distances were estimated using PAUP* 4.0b10 software (Swofford, 2002). The haplotype/gene diversity and nucleotide diversity were analyzed using Arlequin v.3.5 software (Excoffier and Lischer, 2010).

3. Accession number The whole mitogenome of A. cantonensis (Thailand isolate) has been deposited at GenBank under the accession KT186242.

4. Results and discussion 4.1. Mitogenome features The mitogenome of A. cantonensis Thailand isolate is 13,519 bp long, with base composition of 26.8% A, 48.4% T, 18.6% G, and 6.2% C (Tables 1 and 2). It is larger than that of A. cantonensis China isolates (13,497–13,502 bp) (Lv et al., 2012, 2014) and similar to other species of the Angiostrongylus genus (range of 13,422–13,585 bp) and other Metastrongyloid genera (range of 13,414–13,793 bp) that are available in the GenBank. The mitogenome features of the China and Taiwan isolates are summarized in Tables A2 and A3 (Appendix A: Supplementary data). As in other Angiostrongylus species—A. cantonensis China isolate and A. costaricensis Brazil isolate (Lv et al., 2012) and A. vasorum

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Table 3 Start and stop codons and size of mitochondrial protein-coding genes of geographical isolates of Angiostrongylus cantonensis. Thailand (KT186242); Taiwan (PRJEB493, ID: 261570); China (Lv et al., 2012, 2014). Gene

Thailand start/stop codon

atp6 cox1 cox2 cox3 cob nad1 nad2 nad3 nad4 nad4l nad5 nad6

ATT/T ATT/TAA TTG/TAG TTG/T TTG/TAG TTG/TAG TTG/TAA TTG/TAA TTG/TAG ATT/T ATA/T ATG/TAA

Thailand size (bp) 598 1578 693 766 1110 876 849 336 1230 232 1582 426

China start/stop codon

China size (bp)

Taiwan start/stop codon

Taiwan size (bp)

ATT/TAG ATT/TAG; ATT/TA TTG/TAG; TTG/T TTG/T TTG/TAA; TTG/TA TTG/TAG TTG/TAG TTG/TAG; TTG/TAA TTG/TAG ATT/T ATA/T ATG/TAG

600 1578/1577 693/691 766 1110/1109 876 849/846/852 336 1230 232 1582 426

ATT/TAG ATT/TAA TTG/TAG TTG/T TTG/TAA TTG/TAG TTG/TAG TTG/TAA TTG/TAG ATT/T ATA/T ATG/TAG

600 1578 693 766 1110 876 849 336 1230 232 1490* 426

*possible incomplete sequence or deletion.

(Gasser et al., 2012)—the mitogenome of A. cantonensis Thailand isolate has 36 genes (12 protein-coding genes—PCGs, 2 rRNA genes, and 22 tRNA genes) and two non-coding regions (258-bp control region and 63-bp minor region) (Table 1, Fig. 1). All genes are transcribed from the same strand (Table 1). The nucleotide composition of the Thailand isolate of A. cantonensis is similar to those of China and Taiwan isolates (Table 2). The whole mitogenome, 12 PCGs, 2 rRNA genes and control region all

exhibit positive GC skew and negative AT skew, except the control region of the China and Taiwan isolates with marginally positive AT skew. 4.2. Protein-coding genes Of the 12 mt-PCGs, 5 (atp6, cox1, cox2, cob, nad2) show variations in length among the isolates, excepting the length of nad5 in

Fig. 1. Complete mitochondrial genome of Angiostrongylus cantonensis (Thailand isolate) with BRIG visualization showing the protein coding genes, rRNAs, tRNAs and non-coding regions. GC skew is shown on the outer surface of the ring whereas GC content is shown on the inner surface.

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the Taiwan isolate with a gap (indels) of 92 bp (Table 3). Deletion in nad5 gene has been reported in natural populations of Caenorhabditis briggsae which may adversely affect fitness and mitochondrial functioning while promoting aging (Estes et al., 2011). The start codon for each PCG is invariant. The commonest start codon is TTG (in 7 PCGs—cox2, cox3, cob, nad1, nad2, nad3, nad4), followed by three for ATT (atp6, cox1, nad4l), and one each for ATA (nad5) and ATG (nad6) (Table 3). However, there is considerable variation in the stop codon. The Thailand isolate differs from the China and Taiwan isolates in 4 genes (atp6, cob, nad2, nad6), while the China isolate has different stop codon for cox1 from the other two isolates. Among the China isolates some have different anticodon in individual genes (cox1, cox2, cob). Additionally, the Thailand isolate has 4 incomplete T stop codon compared to 3 in the China and Taiwan isolates (Table 3). 4.3. Ribosomal RNA genes There is slight variation in the length of each rRNA gene among the isolates of A. cantonensis (Table 4). The length of rrnS gene ranges from 694 bp to 702 bp, while that of rrnL gene ranges from 954 bp to 961 bp.

161

Table 4 Length (bp) of rrnS, rrnL and control region in geographical isolates of Angiostrongylus cantonensis. Thailand isolate (KT186242); China isolates (Lv et al., 2012, 2014) ; Taiwan isolate (PRJEB493). Minor non-coding region (intergenic space between nad4 and cox1 genes). Isolate

rrnS

rrnL

ControlRegion

Minornon-coding region

Thailand China Taiwan

698 694–702 697

956 954–961 959

258 230–236 237

63 64–65 65

bp poly-T stretch and two 6-bp poly-T stretches in addition to other shorter poly-T stretches present in all isolates (Table A4, Appendix A: Supplementary data). In the minor non-coding region (intergenic sequence between nad4 and cox1 genes), the Thailand isolate lacks 2 bp (indels) at the 5 -end of the sequence compared to the China and Taiwan isolates (Table 5). Additionally, it differs from the other isolates in 6 sites due to base substitution (5 tranversion and 1 transition) and one site due to inversion (or 2 base substitution). The Taiwan isolate differs from the China isolate at one site (due to transition). In view of its variability, this minor non-coding region may prove useful for barcoding purpose and molecular phylogeography as well as population study.

4.4. Non-coding regions

4.5. Transfer RNAs

The control region, flanked by trnA and trnP (Fig. 1), is longer in the Thailand isolate (258 bp) than the China (230–236 bp; Lv et al., 2012, 2014) and Taiwan (237 bp) isolates (Table 4). The Thailand isolate also differs from the other isolates by possession of one 11-

Of the tRNAs, tryptophan (W) had ACU anticodon (opal suppressor) in all three isolates of A. cantonensis instead of the canonical Trp tRNA ACC anticodon (Table 6, Fig. 2). In the Thailand isolate, 18 tRNAs lacked the entire TC-arm, compared to 17 in the

Table 5 Spacing sequence between nad4 and cox1 genes in mitogenome of Angiostrongylus cantonensis. AcThai, Thailand isolate KT186242; AcChina, NC 013065; AcTaiwan, PRJEB493. transition;  tranversion; inversion; 䊉 deletion/insertion.

Table 6 Anticodon and length of tRNAs of Thailand (KT186242), China (Lv et al., 2012, 2014) and Taiwan (PRJEB493) isolates of Angiostrongylus cantonensis. W(Trp) had UCA anticodon (opal nonsense suppressor) instead of canonical CCA anticodon. tRNA

Thailandanticodon

Thailandlength

Chinaanticodon

Chinalength

Taiwananticodon

Taiwanlength

P(Pro) V(Val) W(Trp) E(Glu) S2(Ser) N(Asn) Y(Tyr) K(Lys) L2(Leu) S1(Ser) I(Ile) R(Arg) Q(Gln) F(Phe) L1(Leu) T(Thr) C(Cys) M(Met) D(Asp) G(Gly) H(His) A(Ala)

UGG UAC UCA UUC UGA GUU GUA UUU UAA UCU GAU ACG UUG GAA UAG UGU GCA CAU GUC UCC GUG UGC

55 54 55 61 53 59 58 61 57 50 55 54 55 58 55 58 60 59 56 56 54 56

UGG UAC UCA UUC UGA GUU GUA UUU UAA UCU GAU ACG UUG GAA UAG UGU GCA CAU GUC UCC GUG UGC

55 54 57 59/57/54 53/55 56 60 61/60 56/55 50 57/59 54 56/57/55 56 56 54/57 57 59 53/54 57/56 55 55

UGG UAC UCA UUC UGA GUU GUA UUU UAA UCU GAU ACG UUG GAA UAG UGU GCA CAU GUC UCC GUG UGC

55 63 57 57 55 57 60 61 56 50 57 54 56 56 56 53 58 60 53 56 55 55

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Fig. 2. Cloverleaf structure of the 22 inferred tRNAs in the mitochondrial genome of Angiostrongylus cantonensis (Thailand isolate). Note atypical secondary structure of 18 tRNAs lacking entire TC arm, lysine and methionine with the TC arm lacking the TC-stem, and serine S1 and serine S2 without the entire DHU arm.

China isolate and 16 in the Taiwan isolate (Table A5, Appendix A: Supplementary data). In all three isolates, two tRNAs (lysine and methionine) lacked TC-stem, while two (serine S1 and S2) lacked DHU-arm (Table A5, Appendix A: Supplementary data; Fig. 2). The number of base pairs in the DHU-stem and TC-stem of each tRNA

is similar in the three isolates, except the TC-stem of China and Taiwan isolates with 1 bp in glutamine (without TC-loop) and 2 bp in valine (Table A5, Appendix A: Supplementary data). The anticodon is invariant for the respective tRNAs in the three isolates. However, the length of individual tRNA may differ in the

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163

Fig. 3. Bayesian inference and maximum likelihood tree based on 36 mitochondrial genes of the whole mitochondrial genomes of Angiostrongylus cantonensis isolates with Angiostrongylus costaricensis and Angiostrongylus vasorum as outgroups. Numeric values at the nodes are Bayesian posterior probabilities/ML bootstrap.

Table 7 Percentage of uncorrected “p” distance matrix between different isolates of Angiostrongylus cantonensis and related taxa based on (a) 36 mt-genes, (b) 12 protein-coding genes, (c) 2 rRNA genes, (d) 22 tRNA genes, and (e) control region. Control region is not available for A. vasorum. Taxon

1

2

3

4

1. Thailand isolate KT186242 2. China isolate NC 013065

– (a) 11.6 (b) 12.2 (c) 9.1 (d) 9.6 (e) 18.2 (a) 11.4 (b) 12.0 (c) 9.0 (d) 9.8 (e) 17.3 (a) 17.6 (b) 18.4 (c) 15.5 (d) 14.0 (e) 28.6 (a) 17.7 (b) 18.2 (c) 16.3 (d) 15.3 (e) –

(a) 3.2 (b) 3.5 (c) 2.7 (d) 1.8 (e) 3.5 (a) 17.4 (b) 18.1 (c) 15.3 (d) 14.3 (e) 29.5 (a) 16.3 (b) 16.7 (c) 15.8 (d) 13.5 (e) –

(a) 17.4 (b) 18.0 (c) 15.6 (d) 14.9 (e) 30.3 (a) 16.1 (b) 16.5 (c) 15.9 (d) 13.1 (e) –

(a) 17.0 (b) 17.5 (c) 16.8 (d) 13.1 (e)–

3. Taiwan isolate PRJEB493

4. A. costaricensis NC 013067

5. A. vasorum NC 018602

three isolates. For example, 7 tRNAs (E, N, L2, F, C, D, A) have longer and 5 (W, Y, I, L1, H) have shorter length in the Thailand isolate (Table 6). 4.6. Intergenic spacers The Thailand isolate has fewer regions (10 regions) with intergenic sequence compared to the China (12 regions) and Taiwan (11 regions) isolates (Table 1; Tables A2 and A3, Appendix A: Supplementary data). In contrast, it has a larger number of overlapping regions ranging from 1 to 2 bp: 8 for Thailand, 5 for China and 6 for Taiwan. 4.7. Phylogenetic relationships and genetic divergence Fig. 3 depicts the molecular phylogeny of A. cantonesis isolates in relation to other taxa of the Angiostrongylidae family based on 36 mt-genes (12 PCGs, 2 rRNA genes and 22 tRNA genes). The phylograms based on 12 PCGs, 2 rRNA genes and 22 tRNA genes as well as the control region are congruent with that based on 36 mtgenes (Fig. A1, Appendix A: Supplementary data). Of the three A. cantonensis isolates, the China and Taiwan isolates form a sister group. A. costaricensis and A. vasorum form a sister group which is concordant with findings based on cytochrome b (Yong et al., 2015) and cytochrome c oxidase subunit 1 (Eamsobhana et al., 2010) nucleotide sequences. In a study of 7 complete mitogenomes in China isolates of A. cantonensis, 5 mt-haplotypes were found to form two clades, one consisting of isolates from Fujian, Hainan and Hunan, the other consisting of isolates from Hainan and Yunnan (Lv et al., 2014). The haplotype/gene diversity for the three isolates (Thailand, China and Taiwan) of A. cantonensis is 1.00 ± 0.27, and the

nucleotide diversity is 0.09 ± 0.06 for 36 mt-genes, 0.09 ± 0.07 for 12 PCGs, 0.07 ± 0.05 for 2 rRNA genes, and 0.06 ± 0.05 for 22 tRNA genes. It is evident that A. cantonensis exhibits considerable genetic diversity. The Ka /Ks (nonsynonymous and synonymous substitution rates) ratio is 0.80 between Thailand and China isolates, 0.83 between Thailand and Taiwan isolates, and 0.72 between China and Taiwan isolates, implying purifying or stabilizing selection. Table 7 summarizes the genetic divergence of the A. cantonensis isolates and congeners. The Thailand isolate of A. cantonensis is genetically very different from those of China (p = 11.6% for 36 mt-genes and ranging from p = 9.1% for rRNA genes to p = 18.2% for control region) and Taiwan (p = 11.4% for 36 mt-genes and ranging from p = 9.0% for rRNA genes to p = 17.3% for control region). In Taiwan, based on cox1 sequences the Hualien isolate differs from four other isolates by 10–11% (Lee et al., 2014). Based on cob gene the genetic distance between A. cantonensis and its closely related congener A. malaysiensis is p = 8.3–9.2% (Yong et al., 2015a). Similarly, the genetic distance based on cox1 gene is p = 11.1–11.7% between A. cantonensis and A. malaysiensis, and p = 11.4% between the Costa Rica and Brazil isolates of A. costaricensis (Eamsobhana et al., 2010). Further studies with better taxon sampling are needed to address the diverse genetic lianeages of A. cantonensis. The genetic distance between the China and Taiwan isolates is p = 3.2% for 36 mt-genes and ranging from p = 1.8% for tRNA genes to p = 3.5% for 12 PCGs as well as control region. Based on cob gene, the intraspecific divergence for A. cantonensis is p = 0–2.9% and for A. malaysiensis p = 0-0.1% (Yong et al., 2015a). Based on cox1 sequences, the genetic distance among Thailand, China and Hawaii isolates is p = 0.83–3.61% (Eamsobhana et al., 2010), and among Japan, China, Taiwan and Thailand isolates is p = 0.8–5.0% (Tokiwa et al., 2012).

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In the present study, all the five datasets (36 mt-genes, 12 PCGs, 2 rRNA genes, 22 tRNA genes and control region) yield concordant results on the genetic diversity among A. cantonensis isolates as well as interspecific diversity among the congeners (Table 7). 5. Conclusion In this study, we report the complete mitogenome of A. cantonensis Thailand isolate based on NGS using the Illumina Hi-Seq platform (Yong et al., 2015b). The results are consistent with reported Angiostrongylus mitogenomes based on long-PCR (Lv et al., 2012, 2014; Gasser et al., 2012). All five datasets (36 mt-genes, 12 PCGs, 2 rRNA genes, 22 tRNA genes and control region) reveal close affinity between the China and Taiwan isolates which are genetically very distant from the Thailand isolate. The mitogenome will prove useful for studies on population genetics, phylogenetics, phylogeography and systematics. Acknowledgements This study was funded in part by MoHE-HIR Grant (H-50001-00A000025) and the University of Malaya (H-5620009). We thank the University of Malaya and Faculty of Medicine Siriraj Hospital, Mahidol University for providing various research facilities and other support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica.2015. 09.001. References Akaike, H., 1973. Information theory and an extension of the maximum likelihood principle. In: Petrov, B.N., Csaki, F. (Eds.), Proceedings of the 2nd International Symposium on Information Theory. Budapest, Akademia Kiado, pp. 267–281. Alicata, J.E., 1962. Angiostrongylus cantonensis (Nematoda: Metastrongylidae) as a causative agent of eosininophilic meningitis in man in Hawaii and Tahiti. Can. J. Zool. 40, 5–8. Alikhan, N.F., Petty, N.K., Ben Zakour, N.L., Beatson, S.A., 2011. BLAST ring image generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12, 402. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215 (3), 403–410. Andrews, S., 2010. FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/ fastqc. Bernt, M., Donath, A., Juhling, F., Externbrink, F., Florentz, C., Fritzsch, G., Putz, J., Middendorf, M., Stadler, P.F., 2012. MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313–319. Chen, H.T., 1935. Un nouveau nematode pulmonaire, Pulmonema cantonensis n.g., n.sp. des rats de. Canton. Ann. Parasit. Hum. Comp. 13, 312–317 (in French). Cross, J.H., 1987. Public health importance of Angiostrongylus cantonensis and its relatives. Parasitol. Today 3, 367–369. Dusitsittipon, S., Thaenkham, U., Watthanakulpanich, D., Adisakwattana, P., Komalamisra, C., 2014. Genetic differences in the rat lungworm, Angiostrongylus cantonensis (Nematoda: Angiostrongylidae), in Thailand. J. Helminthol., http://dx.doi.org/10.1017/S0022149x14000388. Eamsobhana, P., 2014. The Rat Lungworm Angiostrongylus cantonensis: Parasitology, Genetics and Molecular Phylogeny, second ed. Akson Graphic and Design Publishing House, Bangkok. Eamsobhana, P., Lim, P.E., Solano, G., Zhang, H., Gan, X., Yong, H.S., 2010. Molecular differentiation of Angiostrongylus taxa (Nematoda: Angiostrongylidae) by cytochrome c oxidase subunit I (COI) gene sequences. Acta Trop. 116, 152–156.

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