The Complete Mitogenome of Toxocara vitulorum: Novel In-Sights into the Phylogenetics in Toxocaridae

Abstract

Toxocara vitulorum (Ascaridida: Nematoda) is one of the most common intestinal nematodes of cattle and buffalos and, therefore, represents a serious threat to their populations worldwide. Despite its significance in veterinary health the epidemiology, population genetics, and molecular ecology of this nematode remain poorly understood. The mitogenome can yield a foundation for studying these areas and assist in the surveillance and control of T. vitulorum. Herein, the first whole mitogenome of T. vitulorum was sequenced utilizing Illumina technology and characterized with bioinformatic pipeline analyses. The entire genome of T. vitulorum was 15,045 bp in length and contained 12 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs). The gene arrangement (GA) of T. vitulorum was similar to those of other Toxocara species under GA3. The whole genome showed significant levels of AT and GC skew. Comparative mitogenomics including sequence identities, Ka/Ks, and sliding window analysis, indicated a purifying selection of 12 PCGs with cox1 and nad6 having the lowest and highest evolutionary rate, respectively. Whole amino acid sequence-based phylogenetic analysis supported a novel sister-species relationship of T. vitulorum with the congeneric species Toxocara canis, Toxocara cati, and Toxocara malaysiensis in the family Toxocaridae. Further, 12 (PCGs) single gene-based phylogenies suggested that nad4 and nad6 genes shared same topological trees with that of the whole genome, suggesting that these genes were suitable as novel genetic markers for phylogenetic and evolutionary studies of Ascaridida species. This complete mitogenome of T. vitulorum refined phylogenetic relationships in Toxocaridae and provided the resource of markers for population genetics, systematics, and epidemiology of this bovine nematode.

Keywords: Toxocara; mitochondrial genome; phylogenetic relationships; roundworms.

Figure 1

Circular map for T. vitulorum mitogenome. 36 genes follow standard nomenclature, including 22 tRNAs, which are denoted by one letter in accordance with the IPUC-IUB single-letter amino acid codes. The two leucine genes were differed by L1 and L2, and the two serine genes by S1 and S2. All the genes are present on the same strand and transcribed in the same direction (5′–3′), as indicated by the arrow. NCR indicates the non-coding region. AT indicates the AT-rich region.

Figure 2

AT-skew vs. GC-skew of the different genetic positions of 29 mitogenomes. The X axis provides the A + T/G + C values, while the Y axis provides the skew values. For each species, the entire mitogenome, concatenated PCGs, concatenated tRNAs, concatenated rRNAs, the 1st, 2nd, 3rd, and 12 PCGs were separate calculated.

Figure 3

The RSCU and codon numbers in T. vitulorum mitogenome. Different color bars represent the RSCU of each codon; the codon numbers are indicated by the orange line graph (right Y axis scale).

Figure 4

Predicted secondary structures of 22 tRNAs in T. vitulorum mitogenome. The tRNAs are labeled with their corresponding IPUC-IUB single-letter amino acid codes. Dashes (−) denote Watson–Crick bonds, dots (·) indicate mispaired nucleotide bonds, and (‿) indicate tRNA anticodons.

Figure 5

Pair-wise comparison of the nucleotide (left) and amino acid (right) sequence identities for PCGs. Colors from purple (high identity) to white (low identity) indicate the different identities of nucleotide sequences, and colors from green (high identity) to white (low identity) indicate the different identities of amino acid sequences.

Figure 6

Sliding window analysis of the concatenated alignments of PCGs. The black line represents the value of the nucleotide diversity and are calculated using the parameters: window size = 200 bp and step size = 20 bp. Gene names, boundaries, and average nucleotide diversity values are indicated above the graph. Colors from yellow (high diversity) to white (low diversity) indicate the different nucleotide diversities.

Figure 7

Evolutionary rates of PCGs between T. vitulorum and 28 other Rhabditida mitogenomes. The rates of non-synonymous substitutions (Ka) and synonymous substitutions (Ks) and the ratio of Ka/Ks are calculated for each PCG.

Figure 8

Phylogenetic relationships among Ascaridoidea nematodes. The phylogeny is inferred using the MP, ML, and BI methods of 27 Ascaridida species for which complete mitogenome sequences are available, using the Seuratoidea species (C. robustus) as the outgroup. (A) Phylogenetic tree is based on concatenated amino acid sequences of 12 PCGs. (B) Twelve topological phenograms are based on each PCG, and the tree topology consistent with the trees in A are marked with red line boxes. The black circle sign represents the species sequenced in this study. The numbers along the branches in A and B indicate bootstrap values/posterior probabilities (<95% or 0.95 supports not shown) resulting from different analyses in the order MP/ML/BI.