Toxocara vitulorum (Ascaridida: Nematoda): Mitochondrial gene content, arrangement and composition compared with other Toxocara species

Molecular & Biochemical Parasitology 166 (2009) 89–92

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Molecular & Biochemical Parasitology

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Toxocara vitulorum (Ascaridida: Nematoda): Mitochondrial gene content, arrangement and composition compared with other Toxocara species夽 Susiji Wickramasinghe a , Lalani Yatawara a , R.P.V.J. Rajapakse b , Takeshi Agatsuma a,∗ a b

Department of Environmental Health Sciences, Kochi Medical School, Oko, Nankoku City, Kochi 783-8505, Japan Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka

a r t i c l e

i n f o

Article history: Received 29 January 2009 Received in revised form 23 February 2009 Accepted 23 February 2009 Available online 5 March 2009 Keywords: Toxocara vitulorum Mitochondrial genome Gene arrangement Ribosomal Protein-coding Phylogeny

a b s t r a c t Partial mitochondrial (mt) genome sequence (10,486 bp) from the parasitic nematode Toxocara vitulorum was determined and its organization and structure compared with those of T. cati, T. canis and T. malaysiensis. The obtained mt genome sequence of T. vitulorum contains 10 protein-coding genes (cytochrome c oxidase subunits 1–3, Nicotinamide adenine dinucleotide dehydrogenase subunits 1–5, ATP synthase subunit 6 and cytochrome b), 14 transfer RNA genes and the large ribosomal RNA gene (rrnL), non-coding regions. ORF encoding for ATPase subunit 8 is not found in this partial mtDNA sequence. Five translation initiation codons were inferred, ATT, ATG, GTG, GTT and TTG. Most of the genes used TAG or TAA as a stop codon and two genes ended with a T. The gene arrangement and composition of the T. vitulorum mt genome is very similar to that of other Toxocara species mitochondrial genomes sequenced thus far. All genes are transcribed in the same direction, as other Toxocara species. This genome has a high A + T content (67.5%) and low G + C content (32.5%). Phylogenetic reconstruction based on aligned nucleotide sequences of seven taxa provided strong support that Toxocara vitulorum is more closely related to T. malaysiensis than to T. canis and T. cati. © 2009 Elsevier B.V. All rights reserved.

Most metazoan mitochondrial (mt) genomes are circular and small in size (14–20 kb) [1] and contain 12–13 protein-coding genes, 22 tRNAs, 2 ribosomal RNAs and the non-coding regions. The genes of mt genome involve in cellular energy metabolism and provide energy for their maintenance, reproduction and survival. Toxocara canis, T. cati, T. malaysiensis and T. vitulorum are the most important nematodes of the genus Toxocara, causing significant health problems in animals as well as in humans (accidental/opportunistic host). The complete mt genomes of T. canis (14,332 bp), T. cati (14,029 bp) and T. malaysiensis (14,266 bp) have been determined recently [2]. Further, the complete mt genomes of two more species (Ascaris suum and Anisakis simplex) of the order Ascaridida have been sequenced [3,4]. The mt genomes of the three Toxocara species are circular, and contain 12 proteinencoding genes (nad1, atp6, nad2, cytb, cox3, nad4, cox1, cox2, nad3, nad5, nad6, and nad4L), two ribosomal RNA genes (rrnL and rrnS),

Abbreviations: atp6, ATP synthase subunit 6; cytb, cytochrome b; cox1, cox2 and cox3, cytochrome c oxidase subunits 1–3; nad1, nad2, nad3, nad4 and nad5, nicotinamide adenine dinucleotide dehydrogenase subunits 1–5; rrnL, large ribosomal RNA; rrnS, small ribosomal RNA; trn, transfer RNA; Tcan, Toxocara canis; Tcat, Toxocara cati; Tmal, Toxocara malaysiensis; Tvit, Toxocara vitulorum; mt, mitochondrial. 夽 Note: Nucleotide data reported in this paper is available in the GenBankTM , EMBL and DDBJ databases under the accession number FJ664617. ∗ Corresponding author. Tel.: +81 88 880 2535; fax: +81 88 880 2535. E-mail address: [email protected] (T. Agatsuma). 0166-6851/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2009.02.012

22 transfer RNA (trn) genes, an AT-rich region and the non-coding regions. The ATPase subunit 8 gene is absent in Toxocara species and other nematode mt genomes reported thus far except in Trichinella spiralis [5]. All genes are transcribed in the same direction in the mt genomes of Toxocara species and are high in A and T, but low in G and C. The AT bias has significant effect on both the codon usage pattern and amino acid composition of proteins [2]. T. vitulorum is a large (20–30 cm) round worm found in the small intestine of cattle and buffalo world-wide, but prevalence is highest in the tropical countries [6–11]. It is mainly transmitted through colostrums and milk, causing disease (severe anemia, diarrhea, weight loss, and anorexia) particularly in buffalo calves between 1 and 3 months of age. In having bovine definitive hosts, T. vitulorum differs markedly from other members of the genus that infect canids and felids. The pattern of larval migration of this parasite is very similar to that of T. canis in the mouse model. Therefore, the possibility of human infection by this parasite should be investigated. The objectives of the present study were to determine the gene order, composition, codon usage pattern, translation initiation/termination codons of the mt genome of T. vitulorum and to compare these findings with those of T. canis, T. cati and T. malaysiensis. Further, mt genome sequences provide useful molecular markers for the identification of closely related species and enhance knowledge of their genetic diversity to use in comparative genomics and phylogenetics.

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Fig. 1. Schematic representation of the genomic organization of a large portion of the T. vitulorum mt genome. This region contains 10 protein-encoding genes (nad1, atp6, nad2, cytb, cox3, nad4, cox1, cox2, nad3 and nad5), 14 tRNAs, 2 non-coding regions (underlined) and the rrnL gene. The symbol | above the sequence indicates the beginning of each gene. The start and stop codons are shown in bold. * indicates a stop codon.

T. vitulorum samples were obtained from an infected buffalo calf in Sri Lanka and samples were fixed in 70% (v/v) ethanol and stored at −20 ◦ C until use. The total genomic DNA was extracted from part of a single worm using an Easy-DNATM Kit (Invitrogen Corporation, Carlsbad, CA). Briefly, a piece of worm was vacuum dried to evaporate ethanol, then incubated with TE buffer (320 ␮l), Solution A (20 ␮l), Solution B (10 ␮l) and proteinase K (5 mg/ml) at 60 ◦ C overnight. Next day, Solution A (300 ␮l), Solution B (120 ␮l) and chloroform (750 ␮l) were added. DNA in the final aqueous solution was precipitated using ethanol (100%). Finally, the DNA pellet was re-suspended in TE buffer (49 ␮l) and 1 ␮l of 2 mg/ml Rnase added. The specific identity of T. vitulorum DNA was verified from other Toxocara species using the sequence of the nuclear ribosomal internal transcribed spacers 2 (ITS-2) and small subunit ribosomal RNA (rrnS). Primers were designed to sequence the mt genome of T. vitulorum based on an alignment of the conserved regions of A. suum (X54253), C. elegans (X54252), N. americanus (AJ417719), and A. duodenale (AJ417718) mitochondrial sequences [3,12]. The names and primer sequences are given in Supplementary Table 1. All the polymerase chain reactions (PCRs) were carried out in a final reaction volume of 25 ␮l. Amplifications were performed with

1 ␮l of genomic DNA extract using 1 U Ex Taq Polymerase (TaKaRa), 10× Ex Taq buffer, 0.2 mM each dNTP and 10 pmol of each primer. PCR conditions used were 3 min denaturation at 94 ◦ C; 30–35 cycles of 30 s at 94 ◦ C; 30 s at 50 ◦ C and 1 min at 72 ◦ C; followed by a final 5 min extension at 72 ◦ C. All the PCR reactions were carried out in a MyCyclerTM Thermal cycler (BioRad, USA). Amplicons were detected on 1% agarose gels stained with ethidium-bromide. Amplified PCR products were purified in agarose gel using GENECLEAN II Kit. Purified PCR products were sub-cloned in pGEMR T-vector system (Promega, USA). Ligated products were transformed into E. coli JM109 cells. Positive clones were obtained and plasmid DNA extraction was performed using the alkaline SDS method. Nucleotide sequences were determined with an ABI PRISM 3100 Avant DNA sequencer using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem, CA, USA). Sequences were assembled manually and aligned against the published complete mt genome sequences of T. canis, T. cati, and T. malaysiensis [2] using the Clustal W programme. The proteincoding genes of T. vitulorum were determined using DNAsis and GENETYXMAC (ver. 6.0). The translation initiation and termination codons were identified based on comparison with those of T. canis,

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Table 1 Positions and characteristics of mitochondrial genes and non-coding sequences of mt genome of T. vitulorum and comparison of that with other Toxocara species. Genes

nad1 atp6 trnK trnL (UUR) trnS (AGY) nad2 trnI trnR trnQ trnF cytb trnL (CUN) cox3 Intergenic region trnT nad4 Non-coding region cox1 trnC trnM trnD trnG cox2 trnH rrnL nad3 nad5

Position (5 -3 )

Sequence length

Tvit

Nucleotide (bp)

1–873 875–1472 1473–1533 1534–1588 1589–1641 1642–2486 2486–2547 2548–2602 2604–2658 2658–2718 2719–3825 3832–3886 3887–4654 4655–4670 4671–4726 4727–5956 5957–6064 6065–7645 7648–7702 7711–7770 7772–7827 7827–7882 7883–8596 8587–8642 8643–9596 9597–9932 9934–10,486

Initiation and termination codons No. of amino acid

Tvit

Tcan

Tcat

Tmal

291 200

TTG/TAA ATT/T

TTG/TAG ATT/T

TTG/TAA ATT/T

TTG/TAG ATT/T

282

282

ATT/T

ATT/T

GTT/T

ATT/T

369

369

369

TTG/TAA

GTG/TAG

ATG/TAA

GTG/TAG

256

256

256

256

TTG/TAG

ATG/TAG

ATG/TAA

GTG/TAG

410

410

410

410

GTG/TAG

ATA/TAG

ATA/TAG

ATA/TAG

527

526

526

527

TTG/TAG

TTG/TAG

TTG/TAG

TTG/TAG

238

238

237

237

GTT/TAG

GTT/TAG

GTG/TAA

GTA/TAG

112 184 (partial)

112 528

112 528

112 528

TTG/TAA ATG/?

TTG/TAG ATA/T

TTG/TAG ATA/T

TTG/TAG ATG/T

Tvit

Tcan

Tcat

Tmal

Tvit

Tcan

Tcat

Tmal

873 598 61 56 52 844 62 55 55 60 1107 55 768 16 56 1230 108 1581 55 60 56 56 714 56 953 336 553 (partial)

873 598 62 55 52 844 61 54 55 58 1107 56 768

873 598 63 55 51 844 61 56 55 59 1107 55 768

873 598 61 56 53 844 63 55 55 57 1107 57 768

291 200

291 200

291 200

282

282

369

57 1230 111 1578 55 60 59 56 714 56 958 336 1582

57 1230 116 1578 54 60 56 58 711 58 955 336 1582

55 1230 112 1581 56 60 59 57 711 59 955 336 1582

T. cati, and T. malaysiensis. Transfer RNA genes were identified using DNAsis, or by recognizing potential secondary structures and anticodon sequences by aligning sequences with those of T. canis, T. cati, and T. malaysiensis. Phylogenetic analyses were performed using MEGA4 [13] and the aligned sequences. For distance analyses, the maximum composite likelihood method was used to construct the distance matrix and the trees were inferred from this using the neighbourjoining (NJ) approach and Maximum Parsimony (MP) method. All sites were included in all analyses. Bootstrap resampling (1000 pseudoreplicates) was done and a bootstrap consensus tree produced. 1. General features of the mt genome of T. vitulorum The partial sequence obtained for the mt genome of T. vitulorum was 10,486 bp in length. The obtained mt genome sequence of T. vitulorum contains 10 protein-coding genes (cox1-3, nad1-5, atp6 and cytb), 14 transfer RNA genes, rrnL gene and non-coding regions. The sequence was deposited in the GenBank under the accession number FJ664617. Sequence data encompassing the nad6, nad4L, rrnS, AT-region and 8 transfer RNA genes were not obtained in this study. This mt genome is very similar to the mt genomes of other Toxocara species sequenced thus far. All genes are transcribed in the same direction as other Toxocara species. A schematic presentation of the genomic organization of this partial 10.5 kb mt genome of T. vitulorum determined is given in Fig. 1. The arrangement of the genes of the mt genome of T. vitulorum is the same as T. canis, T. cati, T. malaysiensis, A. suum and A. simplex but differ significantly from those of Dirofilaria immitis, Onchocerca volvulus, Strongyloids stercoralis and Trichinella spiralis [5,14–16]. There are two non-coding regions, large is located between the trnS2 and trnN, small or second longest non-coding region is located between genes cox1 and nad4 [2]. In our study, we found third non-coding region (16 bp), located between cox3 and trnT in the mt genome of T. vitulorum (Table 1).

The usage of T (48.7%) and G (23.5%) is higher than A (18.8%) and C (9%) in mt genome of T. vitulorum. The nucleotide identity is 87% between T. vitulorum and T. canis and T. malaysiensis, 86% between T. vitulorum and T. cati, 82% between T. vitulorum and A. suum, and 80% between T. vitulorum and A. simplex. Comparison of nucleotide composition of the protein-coding regions of four Toxocara species is shown in Table 1. T is the predominant nucleotide and C is the least favored. 2. Protein-coding genes, trn, rrnL and non-coding regions Most protein-coding genes of T. vitulorum (atp6, nad2, cytb, cox3, nad4, and nad3) are same as those in other Toxocara species. The cox1 gene ranges in length from 1578 to 1581 bp in the genus (Table 1) and cox2 ranges from 711 to 714 bp (Table 1). As shown in Table 1, the predicted initiation and termination codons for the protein-coding genes of T. vitulorum were compared with those of three other Toxocara species. The start codons we inferred in T. vitulorum were TTG, ATT, GTG, GTT, ATG. Stop codons were TAA, TAG and T. The most common start codon was TTG (5 of 10 protein coding genes). Four of the 9 protein genes used TAG as a translation termination codon and the other genes (nad1, cytb, nad3, atp6 and nad2) used TAA and T respectively. Four Toxocara species used similar start codons in nad1, atp6, cox1, and nad3 (Table 1). Twenty-two transfer RNA genes occur in the mt genomes of the three Toxocara species [2]. In our study, we have determined 14 of these ranging from 52 to 61 bp in length (Table 1) and 8 more remain to be sequenced and identified. The rrnL gene of T. vitulorum was identified by sequence comparison with other Toxocara species. The rrnL is located between trnH and nad3. The rrnL gene ranges in lengths from 953 bp to 958 bp (Table 1). Two prominent non-coding regions have been identified in T. cati, T. canis and T. malaysiensis [2]. First, an AT- rich region, ranging from 711 to 936 bp is located between trnS2 and trnN. This region was not sequenced in our study. The second non-coding region is located between nad4 and cox1. The length of this region is 108 bp for T. vitulorum, 111 bp

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similar to those of T. canis, T. cati and T. malaysiensis. Phylogenetic analysis revealed that T. vitulorum is more closely related to T. malaysiensis than T. canis and T. cati. Our findings provide a rich resource of molecular markers for comparative genomics and mitochondrial systematics of socio-economic important parasites. Acknowledgement We extend our thanks to Prof. David Blair of James Cook University, Australia, for invaluable review of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2009.02.012. References Fig. 2. The phylogenetic trees constructed using aligned 10, 486 bp mitochondrial nucleotide sequence of six species of ascaridoids (including protein-coding, intergenic, trn and non-coding sequence). A: Neighbor-joining tree, B: maximum parsimony tree. Sequence of Onchocerca volvulus has been used as an outgroup.

for T. canis, 112 bp for T. malaysiensis and 116 bp for T. cati. In addition to these two regions, we have identified another short non-coding region (16 bp in length) between cox3 and trnT in the mt genome of T. vitulorum. This may be an inter-genic sequence between the genes as we have not been obtained the complete mt genome of T. vitulorum. The comparison of base composition of the protein-coding genes of four Toxocara species is presented in Supplementary Table 2. Usage of T is the highest in protein-coding genes (41–55%), followed by G (21–25%), A (15–23%) and C (3–11%) respectively. The high A, T and G content of the protein-coding genes is reflected by the very frequent presence of codons consisting of T, A and G such as TTT (Phe), TTG (Leu), GTT (Val), GGT (Gly), AAT (Asn), ATT (Ile) and TAT (Tyr) in the protein-coding genes of T. vitulorum. 3. Phylogenetic analysis The aligned nucleotide sequences (10,486 bp) of seven taxa were used in the phylogenetic reconstruction. Fig. 2. shows the trees inferred by neighbor joining (NJ) and maximum parsimony (MP) methods. Interestingly, T. vitulorum clustered with T. malaysiensis and T. cati branched with T. canis in both methods. This result was consistent with our previous study which used nuclear ribosomal internal transcribed spacer region 2 (ITS-2) to determine relationship between T. vitulorum and other ascaridoids. It seems that T. vitulorum is more closely related to T. malaysiensis than to T. cati and T. canis. The data described in this study shows that the arrangement, composition and content of the mt genes of T. vitulorum are very

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