BACKGROUND
mitochondria are sub-cellular organelles involved in oxidative phosphorylation, offering energy to organisms.
they play important roles in cellular metabolism, living and apoptosis.
within these organelles, most metazoan species possess a compact, circular mitochondrial  genome, which varies in size from  <dig> to  <dig> kb  <cit> .
the metazoan mt genome usually contains a complement of genes encoding 12– <dig> protein subunits of the enzymes involved in oxidative phosphorylation,  <dig> transfer rnas, and two ribosomal rnas.
there are no introns within genes, and no to limted spacer regions between genes  <cit> .
studying mt genomes has important implications for various fundamental areas, including mt biochemistry, physiology and molecular biology.
in addition, mt genome sequences have provided useful markers for investigating population genetic structures, systematics and phylogenetics of organisms due to their maternal inheritance, higher mutation rates than nuclear genes and relatively conserved genome structures  <cit> .
although nematoda is the second largest animal phylum, to date only over  <dig> complete mitochondrial dna sequences of nematode species have been deposited in genbank™  <cit> .
in the order ascaridada, the mt genomes of only two species have been sequenced  <cit> .
the lack of knowledge of mt genomics for parasitic nematodes in this order is a major limitation for population genetic and phylogenetic studies of the pathogens in the order ascaridada including the species in the genus toxocara.
toxocara canis, toxocara cati and toxocara malaysiensis are the common ascaridoid nematodes of dogs and cats, causing significant health problems.
infection of dogs with t. canis is quite common, with the prevalence ranging from  <dig> % to  <dig> %  <cit> .
infection of cats with t. cati is also found worldwide, with infection rates of up to  <dig> % to  <dig> %  <cit> .
more importantly, t. canis and t. cati are of public health significance due to their larvae of being able to invade humans and cause diseases such as ocular larva migrans , visceral larva migrans , eosinophilic meningoencephalitis  and/or covert toxocariasis   <cit> .
although they are of importance in human and animal health, there is still no information on the mt genomes available for any of toxocara species.
the objectives of the present study were to fill some of these knowledge gaps by determining the structure, organization and sequence of the complete mt genomes of t. canis, t. cati and t. malaysiensis of human and animal significance, and to provide mtdna evidence for the recently described species t. malaysiensis  <cit> .
also, the features of mt genomes of the three ascaridoid nematodes, such as the gene arrangements, structures, compositions, as well as translation and initiation codons and codon usage patterns were compared with those of other nematodes in the same order ascaridata, namely ascaris suum and anisakis simplex.
the phylogenetic relationships among these ascaridoid nematodes were also investigated using the protein-coding amino acid sequences.
RESULTS
general features of the mt genome of three toxocara species
the complete mt genomes of t. canis, t. cati and t. malaysiensis are  <dig>  bp,  <dig>  bp and  <dig> bp in length, respectively.
these complete mt genome sequences have been deposited in the genbank™ under the accession numbers am <dig> , am <dig>  and am <dig> .
all three mt genomes contain  <dig> protein-coding genes ,  <dig> transfer rna genes and two ribosomal rna genes, but lack an atp <dig> gene .
these circular genomes are typical of other nematode mitochondrial genomes except for trichinella spiralis in which the atp <dig> gene is encoded.
all genes are transcribed in the same direction as found in other members of the secernentean nematodes sequenced to date, but in contrast to t. spiralis and xiphinema americanus  <cit> .
tcan: toxocara canis, tcat: toxocara cati, tmal: toxocara malaysiensis
the mt genome arrangement of t. canis, t. cati and t. malaysiensis are the same as those of a. suum and a. simplex and almost identical to the genome structures of strongylida nematodes ancylostoma duodenale, necator americanus, cooperia oncophora and rhabditid nematode caenorhabditis elegans, with the exception of the relative position of an at-rich region and the number of non-coding regions .
in the mt genomes of the three toxocara species, the at-rich region is located between trns <dig> and trnn, whereas it is positioned between trna and trnp in a. duodenale, n. americanus, co. oncophora and c. elegans.
the only ncr found in toxocara spp.
was between nad <dig> and cox <dig>  as has been identified in the aforementioned species.
the second ncr located between nad <dig> and nad <dig> in the hookworms a. duodenale and n. americanus was not found in toxocara spp.
<cit> .
the genome structures of these toxocara species differ significantly from those of onchocerca volvulus, strongyloides stercoralis, dirofiliria immitis, x. americanus and t. spiralis in the location of the at-rich region, and some trna and protein-coding genes.
comparison of the gene arrangements of these three toxocara species with those of the other nine representatives of the secernentean nematodes suggests that t. canis, t. cati and t. malaysiensis are more closely related to a. suum and a. simplex than to c. elegans, a. duodenale, n. americanus, co. oncophora, o. volvulus,d.
immitis and s. stercoralis.
the nucleotide compositions of the entire mtdna sequences for t. canis, t. cati and t. malaysiensis are biased toward a and t, with t being the most favored nucleotide and c the least favored, in accordance with mt genomes of other nematodes.
the content of a+t is  <dig> % for t. canis , which is the lowest of the nematodes studied to date .
the content of a+t is  <dig> % for t. cati  and  <dig> % for t. malaysiensis , respectively .
tcan: toxocara canis, tcat: toxocara cati, tmal: toxocara malaysiensis, asu: ascaris suum, asi: anisakis simplex, ad: ancylostoma duodenale, ov: onchocerca volvulus, ss: strongyloides stercoralis, emtg: entire mitochondrial genome.
protein-coding genes and codon usage patterns
the boundaries between protein-coding genes of mt genomes of t. canis, t. cati and t. malaysiensis were determined by aligning their sequences and by identifying translation initiation and termination codons with those of a. suum.
for each of the three toxocara species, the lengths of protein-coding genes cox <dig>  nad <dig>  nad <dig>  nad <dig> and nad <dig> are the same as those of a. suum, whereas the lengths of genes atp <dig>  nad4l,nad <dig> and nad <dig> are reduced, and the lengths of cox <dig> and cytb are increased  when compared to those of a. suum.
the length of cox <dig> in t. canis and t. cati is the same as that of a. suum, but the length of cox <dig> in t. malaysiensis is increased .
tcan: toxocara canis, tcat: toxocara cati, tmal: toxocara malaysiensis, asu: ascaris suum, asi: anisakis simplex, emtg: entire mitochondrial genome.
the inferred nucleotide and amino acid sequences for each of the  <dig> mt proteins of t. canis, t. cati and t. malaysiensis were compared with those of a. suum and a. simplex.
the identity of the nucleotide and amino acid sequences is 71~90% and  <dig> ~ <dig> %, respectively .
based on identity, cox <dig> is the most conserved protein-coding gene, while the cytb is the least conserved.
for all  <dig> proteins, the amino acid sequence identities are higher when compared between each of the three toxocara species than between each of toxocara specis and a. suum or a. simplex.
the identities of the nucleotide and amino acid sequences among five ascaridoid species are higher than those among c. elegans, a. duodenale, n. americanus and co. oncophora .
these findings reinforce the conclusion that the three toxocara species are genetically more closely related to a. suum and a. simplex than to c. elegans, a. duodenale, n. americanus and co. oncophora.
the predicted initiation and termination codons for the protein-coding genes of the three toxocara species were compared with those of ascaridoid species  and with selected species representing different nematode orders including the human hookworm a. duodenale, filarioid worm o. volvulus, and rhabditid species s. stercoralis.
the most common start codon for three toxocara species is ttg , followed by att , ata , and atg, gtg, gtt and gta are used as initiation codons.
gtg, which is used in the cytb of t. canis and t. malaysiensis, and cox <dig> of t. malaysiensis, is not used as a start codon in the other nematodes compared.
gta used in the cox <dig> of t. malaysiensis is also used as a start codon for nad4l in d. immitis .
seven of the  <dig> protein-coding genes were predicted to have a tag or taa translation termination codon.
the remaining protein-coding genes were inferred to end with an abbreviated stop codon, such as ta or t. for the three toxocara species, the 3'-end of most of these genes is immediately adjacent to a downstream trna gene , which is consistent with the arrangement in the mt genomes of a. suum and a. simplex  <cit> , but in contrast to that of c. elegans where both the nad <dig> and nad <dig> genes terminate in t or ta, and are followed by the putative att translation initiation codon of their downstream protein-coding genes.
the protein-coding gene nad <dig> ended with an abbreviated stop codon ta is followed by the putative att translation initiation codon of its downstream protein coding gene nad4l, which is similar to c. elegans.
in general, the nucleotides of metazoan mt genomes are not randomly distributed, and such nucleotide bias is often associated with unequal usage of synonymous codons.
the mt genome nucleotide composition of nematodes is biased toward a and t. the a+t content of protein-coding genes ranged from  <dig> % to  <dig> % for all three toxocara species .
this bias in nucleotide composition toward at  affects both the codon usage pattern and amino acid composition of proteins.
in these three species examined, all  <dig> possible codons were used, and the most frequently used codon was ttt  while the least used codon was cgc .
the preferred nucleotide usage at the third codon position of toxocara mt protein-coding genes reflects the overall nucleotide composition of the mtdna.
at this position, t is the most frequently used, and c the least frequently used.
the codons ending in g have higher frequencies than the codons ending in a, which is similar to a. suum, but opposite to c. elegans, a. duodenale and n. americanus.
the protein-coding genes of the three toxocara genomes are biased toward using amino acids encoded by t-, a- and g-rich codons .
the at-rich codons represent amino acids phe, ile, met, tyr, asn or lys, and the gc-rich codons represent pro, ala, arg or gly.
t-rich codons  comprise phe , leu , ile , val , tyr , ser , and cys , and account for approximately half  of the total amino acid composition.
a- and g-rich codons  represent  <dig> % and  <dig> % of the total amino acid composition, respectively .
in contrast, the proportion of c-rich codons  is much lower .
this codon bias against c is even more evident when only the third codon positions are considered in both four fold and two fold degenerate codon families.
when the frequencies of synonymous codons within the at-rich group were compared, the frequency was always decreased if the third position was substituted with a c. for instance, the relative frequencies of codons for phe are  <dig> % ttt and  <dig> % ttc, respectively.
this result suggests that the third codon positions mostly reflect mutational bias against c. a greater translational efficiency has also been considered to be a potential cause underlying observed codon usage bias  <cit> .
transfer rna genes
twenty-two trna sequences  were identified in the mt genomes of the three toxocara species.
their putative secondary structures  are similar to those of other nematode mtdnas  <cit> , with the exception of t. spiralis  <cit> , and differ from the conventional cloverleaf-like structures found in other metazoan mtdna molecules.
common features of the predicted secondary structures  of  <dig> trna genes in t. canis, t. cati and t. malaysiensis mtdna include a  <dig> bp amino-acyl stem, a  <dig> bp dhu stem with a 4– <dig> bp loop, a  <dig> bp anticodon stem with a loop of  <dig> bp , and a tv replacement loop of 6– <dig> bp with some exceptions, in accordance with other nematodes.
the exception is trns <dig>  in which the dhu-arm is lacking.
ribosomal rna genes
the rrns and rrnl genes of the three roundworm species were identified by sequence comparison with those of a. suum.
the rrns is located between trne and trns , and rrnl is located between trnh and nad <dig>  the two genes are separated from one another by the protein-coding genes nad <dig>  nad <dig>  nad <dig> and nad4l.
the size of the rrns gene is  <dig> bp for t. canis,  <dig> bp for t. cati and  <dig> bp for t. malaysiensis.
the size of the rrnl gene is  <dig> bp for t. canis,  <dig> bp for t. cati and  <dig> bp for t. malaysiensis.
the sizes of the two rrna genes for the three toxocara species are similar to those of other nematodes .
the a+t contents of the rrns for t. canis, t. cati and t. malaysiensis are  <dig> %,  <dig> % and  <dig> %, respectively, whereas those of the rrnl are higher , and a+t contents of the two genes are the lowest among the nematodes studied to date .
sequence identity in the rrns and rrnl genes is  <dig> % and  <dig> % between t. canis and t. cati,  <dig> % and  <dig> % between t. canis and t. malaysiensis, and  <dig> % and  <dig> % between t. cati and t. malaysiensis, respectively.
non-coding regions
like a. suum and a. simplex, the longest non-coding region  in the three toxocara mt genomes is located between the trns <dig> and trnn.
their sizes are  <dig> bp ,  <dig> bp  and  <dig> bp , and a+t contents are  <dig> % ,  <dig> %  and  <dig> % , respectively, which are significantly lower than the comparable ncrs of nematodes studied to date .
repeated sequence motifs  present in the c. elegans at-rich region  <cit>  are not found in toxocara spp.
however, there are a lot of at dinucleotide repeats in the at-region of toxocara mt genomes of which the longest consists of repeat units .
similar at dinucleotide repeats has been found in a. suum  <cit> .
the function or role of these at repeats is currently unknown  <cit> .
although nothing is yet known about the replication process in the mtdna of parasitic nematodes, the high a+t content and the predicted structure of the at-rich non-coding region suggests an involvement in the initiation of replication  <cit> .
for the three roundworm species, the second longest non-coding region is located between genes cox <dig> and nad <dig>  as in the mt genomes of a. suum  <cit> .
its length is  <dig> bp ,  <dig> bp  and  <dig> bp , with an a+t content of  <dig> %,  <dig> % and  <dig> %, respectively, and is shorter than that of a. suum .
the non-coding region for the three toxocara species could form a hairpin loop structure .
phylogenetic analyses
the final alignment of the amino acid sequences of  <dig> proteins for the six taxa  yield  <dig> characters .
in all three phylogenetic analyses, three toxocara species were clustered together .
t. malaysiensis, the recently described toxocara species from cat  <cit> , was inferred to be the sister species of t. cati with high bootstrap values.
this result was consistent with that of a previous study  <cit>  which used sequences of internal transcribed spacers of nuclear ribosomal dna, thus providing mt dna evidence for the validity of t. malaysiensis as an ascaridoid of cats.
t. malaysiensis is more closely related to t. cati, the common ascaridoid of cats, than to t. canis, the common ascaridoid of canids.
toxocara species was resolved being more closely related to a. suum than to a. simplex with moderate support in the phylogenetic analyses, which was consistent with results of previous morphological and molecular studies  <cit> .
but relationship between a. suum and a. simplex was poorly inferred in the mp and ml analyses .
CONCLUSIONS
toxocara species are the important socio-economic parasites because they have significant impact on human health.
the determined mt genomes of the three roundworms, t. canis, t. cati and t. malaysiensis, add the mtdna data to the order ascaridida, which includes a broad range of parasites of major socio-economic importance.
determination of the complete mt genome sequences for three toxocara species of human and animal health significance provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance.

 mitochondria are sub-cellular organelles involved in oxidative phosphorylation, offering energy to organisms. they play important roles in cellular metabolism, living and apoptosis. within these organelles, most metazoan species possess a compact, circular mitochondrial genome, which varies in size from to kb. the metazoan mt genome usually contains a complement of genes encoding 12– protein subunits of the enzymes involved in oxidative phosphorylation, transfer rnas, and two ribosomal rnas. there are no introns within genes, and no to limted spacer regions between genes. studying mt genomes has important implications for various fundamental areas, including mt biochemistry, physiology and molecular biology. in addition, mt genome sequences have provided useful markers for investigating population genetic structures, systematics and phylogenetics of organisms due to their maternal inheritance, higher mutation rates than nuclear genes and relatively conserved genome structures. although nematoda is the second largest animal phylum, to date only over complete mitochondrial dna sequences of nematode species have been deposited in genbank™. in the order ascaridada, the mt genomes of only two species have been sequenced. the lack of knowledge of mt genomics for parasitic nematodes in this order is a major limitation for population genetic and phylogenetic studies of the pathogens in the order ascaridada including the species in the genus toxocara. toxocara canis, toxocara cati and toxocara malaysiensis are the common ascaridoid nematodes of dogs and cats, causing significant health problems. infection of dogs with t. canis is quite common, with the prevalence ranging from % to %. infection of cats with t. cati is also found worldwide, with infection rates of up to % to %. more importantly, t. canis and t. cati are of public health significance due to their larvae of being able to invade humans and cause diseases such as ocular larva migrans, visceral larva migrans, eosinophilic meningoencephalitis and/or covert toxocariasis. although they are of importance in human and animal health, there is still no information on the mt genomes available for any of toxocara species. the objectives of the present study were to fill some of these knowledge gaps by determining the structure, organization and sequence of the complete mt genomes of t. canis, t. cati and t. malaysiensis of human and animal significance, and to provide mtdna evidence for the recently described species t. malaysiensis. also, the features of mt genomes of the three ascaridoid nematodes, such as the gene arrangements, structures, compositions, as well as translation and initiation codons and codon usage patterns were compared with those of other nematodes in the same order ascaridata, namely ascaris suum and anisakis simplex. the phylogenetic relationships among these ascaridoid nematodes were also investigated using the protein-coding amino acid sequences.  general features of the mt genome of three toxocara species the complete mt genomes of t. canis, t. cati and t. malaysiensis are bp, bp and bp in length, respectively. these complete mt genome sequences have been deposited in the genbank™ under the accession numbers am, am and am. all three mt genomes contain protein-coding genes, transfer rna genes and two ribosomal rna genes, but lack an atp gene. these circular genomes are typical of other nematode mitochondrial genomes except for trichinella spiralis in which the atp gene is encoded. all genes are transcribed in the same direction as found in other members of the secernentean nematodes sequenced to date, but in contrast to t. spiralis and xiphinema americanus. tcan: toxocara canis, tcat: toxocara cati, tmal: toxocara malaysiensis the mt genome arrangement of t. canis, t. cati and t. malaysiensis are the same as those of a. suum and a. simplex and almost identical to the genome structures of strongylida nematodes ancylostoma duodenale, necator americanus, cooperia oncophora and rhabditid nematode caenorhabditis elegans, with the exception of the relative position of an at-rich region and the number of non-coding regions. in the mt genomes of the three toxocara species, the at-rich region is located between trns and trnn, whereas it is positioned between trna and trnp in a. duodenale, n. americanus, co. oncophora and c. elegans. the only ncr found in toxocara spp. was between nad and cox as has been identified in the aforementioned species. the second ncr located between nad and nad in the hookworms a. duodenale and n. americanus was not found in toxocara spp.. the genome structures of these toxocara species differ significantly from those of onchocerca volvulus, strongyloides stercoralis, dirofiliria immitis, x. americanus and t. spiralis in the location of the at-rich region, and some trna and protein-coding genes. comparison of the gene arrangements of these three toxocara species with those of the other nine representatives of the secernentean nematodes suggests that t. canis, t. cati and t. malaysiensis are more closely related to a. suum and a. simplex than to c. elegans, a. duodenale, n. americanus, co. oncophora, o. volvulus,d. immitis and s. stercoralis. the nucleotide compositions of the entire mtdna sequences for t. canis, t. cati and t. malaysiensis are biased toward a and t, with t being the most favored nucleotide and c the least favored, in accordance with mt genomes of other nematodes. the content of a+t is % for t. canis, which is the lowest of the nematodes studied to date. the content of a+t is % for t. cati and % for t. malaysiensis, respectively. tcan: toxocara canis, tcat: toxocara cati, tmal: toxocara malaysiensis, asu: ascaris suum, asi: anisakis simplex, ad: ancylostoma duodenale, ov: onchocerca volvulus, ss: strongyloides stercoralis, emtg: entire mitochondrial genome. protein-coding genes and codon usage patterns the boundaries between protein-coding genes of mt genomes of t. canis, t. cati and t. malaysiensis were determined by aligning their sequences and by identifying translation initiation and termination codons with those of a. suum. for each of the three toxocara species, the lengths of protein-coding genes cox nad nad nad and nad are the same as those of a. suum, whereas the lengths of genes atp nad4l,nad and nad are reduced, and the lengths of cox and cytb are increased when compared to those of a. suum. the length of cox in t. canis and t. cati is the same as that of a. suum, but the length of cox in t. malaysiensis is increased. tcan: toxocara canis, tcat: toxocara cati, tmal: toxocara malaysiensis, asu: ascaris suum, asi: anisakis simplex, emtg: entire mitochondrial genome. the inferred nucleotide and amino acid sequences for each of the mt proteins of t. canis, t. cati and t. malaysiensis were compared with those of a. suum and a. simplex. the identity of the nucleotide and amino acid sequences is 71~90% and ~ %, respectively. based on identity, cox is the most conserved protein-coding gene, while the cytb is the least conserved. for all proteins, the amino acid sequence identities are higher when compared between each of the three toxocara species than between each of toxocara specis and a. suum or a. simplex. the identities of the nucleotide and amino acid sequences among five ascaridoid species are higher than those among c. elegans, a. duodenale, n. americanus and co. oncophora. these findings reinforce the conclusion that the three toxocara species are genetically more closely related to a. suum and a. simplex than to c. elegans, a. duodenale, n. americanus and co. oncophora. the predicted initiation and termination codons for the protein-coding genes of the three toxocara species were compared with those of ascaridoid species and with selected species representing different nematode orders including the human hookworm a. duodenale, filarioid worm o. volvulus, and rhabditid species s. stercoralis. the most common start codon for three toxocara species is ttg, followed by att, ata, and atg, gtg, gtt and gta are used as initiation codons. gtg, which is used in the cytb of t. canis and t. malaysiensis, and cox of t. malaysiensis, is not used as a start codon in the other nematodes compared. gta used in the cox of t. malaysiensis is also used as a start codon for nad4l in d. immitis. seven of the protein-coding genes were predicted to have a tag or taa translation termination codon. the remaining protein-coding genes were inferred to end with an abbreviated stop codon, such as ta or t. for the three toxocara species, the 3'-end of most of these genes is immediately adjacent to a downstream trna gene, which is consistent with the arrangement in the mt genomes of a. suum and a. simplex, but in contrast to that of c. elegans where both the nad and nad genes terminate in t or ta, and are followed by the putative att translation initiation codon of their downstream protein-coding genes. the protein-coding gene nad ended with an abbreviated stop codon ta is followed by the putative att translation initiation codon of its downstream protein coding gene nad4l, which is similar to c. elegans. in general, the nucleotides of metazoan mt genomes are not randomly distributed, and such nucleotide bias is often associated with unequal usage of synonymous codons. the mt genome nucleotide composition of nematodes is biased toward a and t. the a+t content of protein-coding genes ranged from % to % for all three toxocara species. this bias in nucleotide composition toward at affects both the codon usage pattern and amino acid composition of proteins. in these three species examined, all possible codons were used, and the most frequently used codon was ttt while the least used codon was cgc. the preferred nucleotide usage at the third codon position of toxocara mt protein-coding genes reflects the overall nucleotide composition of the mtdna. at this position, t is the most frequently used, and c the least frequently used. the codons ending in g have higher frequencies than the codons ending in a, which is similar to a. suum, but opposite to c. elegans, a. duodenale and n. americanus. the protein-coding genes of the three toxocara genomes are biased toward using amino acids encoded by t-, a- and g-rich codons. the at-rich codons represent amino acids phe, ile, met, tyr, asn or lys, and the gc-rich codons represent pro, ala, arg or gly. t-rich codons comprise phe, leu, ile, val, tyr, ser, and cys, and account for approximately half of the total amino acid composition. a- and g-rich codons represent % and % of the total amino acid composition, respectively. in contrast, the proportion of c-rich codons is much lower. this codon bias against c is even more evident when only the third codon positions are considered in both four fold and two fold degenerate codon families. when the frequencies of synonymous codons within the at-rich group were compared, the frequency was always decreased if the third position was substituted with a c. for instance, the relative frequencies of codons for phe are % ttt and % ttc, respectively. this result suggests that the third codon positions mostly reflect mutational bias against c. a greater translational efficiency has also been considered to be a potential cause underlying observed codon usage bias. transfer rna genes twenty-two trna sequences were identified in the mt genomes of the three toxocara species. their putative secondary structures are similar to those of other nematode mtdnas, with the exception of t. spiralis, and differ from the conventional cloverleaf-like structures found in other metazoan mtdna molecules. common features of the predicted secondary structures of trna genes in t. canis, t. cati and t. malaysiensis mtdna include a bp amino-acyl stem, a bp dhu stem with a 4– bp loop, a bp anticodon stem with a loop of bp, and a tv replacement loop of 6– bp with some exceptions, in accordance with other nematodes. the exception is trns in which the dhu-arm is lacking. ribosomal rna genes the rrns and rrnl genes of the three roundworm species were identified by sequence comparison with those of a. suum. the rrns is located between trne and trns, and rrnl is located between trnh and nad the two genes are separated from one another by the protein-coding genes nad nad nad and nad4l. the size of the rrns gene is bp for t. canis, bp for t. cati and bp for t. malaysiensis. the size of the rrnl gene is bp for t. canis, bp for t. cati and bp for t. malaysiensis. the sizes of the two rrna genes for the three toxocara species are similar to those of other nematodes. the a+t contents of the rrns for t. canis, t. cati and t. malaysiensis are %, % and %, respectively, whereas those of the rrnl are higher, and a+t contents of the two genes are the lowest among the nematodes studied to date. sequence identity in the rrns and rrnl genes is % and % between t. canis and t. cati, % and % between t. canis and t. malaysiensis, and % and % between t. cati and t. malaysiensis, respectively. non-coding regions like a. suum and a. simplex, the longest non-coding region in the three toxocara mt genomes is located between the trns and trnn. their sizes are bp, bp and bp, and a+t contents are %, % and %, respectively, which are significantly lower than the comparable ncrs of nematodes studied to date. repeated sequence motifs present in the c. elegans at-rich region are not found in toxocara spp. however, there are a lot of at dinucleotide repeats in the at-region of toxocara mt genomes of which the longest consists of repeat units. similar at dinucleotide repeats has been found in a. suum. the function or role of these at repeats is currently unknown. although nothing is yet known about the replication process in the mtdna of parasitic nematodes, the high a+t content and the predicted structure of the at-rich non-coding region suggests an involvement in the initiation of replication. for the three roundworm species, the second longest non-coding region is located between genes cox and nad as in the mt genomes of a. suum. its length is bp, bp and bp, with an a+t content of %, % and %, respectively, and is shorter than that of a. suum. the non-coding region for the three toxocara species could form a hairpin loop structure. phylogenetic analyses the final alignment of the amino acid sequences of proteins for the six taxa yield characters. in all three phylogenetic analyses, three toxocara species were clustered together. t. malaysiensis, the recently described toxocara species from cat, was inferred to be the sister species of t. cati with high bootstrap values. this result was consistent with that of a previous study which used sequences of internal transcribed spacers of nuclear ribosomal dna, thus providing mt dna evidence for the validity of t. malaysiensis as an ascaridoid of cats. t. malaysiensis is more closely related to t. cati, the common ascaridoid of cats, than to t. canis, the common ascaridoid of canids. toxocara species was resolved being more closely related to a. suum than to a. simplex with moderate support in the phylogenetic analyses, which was consistent with results of previous morphological and molecular studies. but relationship between a. suum and a. simplex was poorly inferred in the mp and ml analyses.  toxocara species are the important socio-economic parasites because they have significant impact on human health. the determined mt genomes of the three roundworms, t. canis, t. cati and t. malaysiensis, add the mtdna data to the order ascaridida, which includes a broad range of parasites of major socio-economic importance. determination of the complete mt genome sequences for three toxocara species of human and animal health significance provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance. 
the mt genome structures for three toxocara species, including genes and non-coding regions, are in the same order as for ascaris suum and anisakis simplex, but differ from ancylostoma duodenale, necator americanus and caenorhabditis elegans only in the location of the at-rich region, whereas there are substantial differences when compared with onchocerca volvulus,dirofiliria immitis and strongyloides stercoralis. phylogenetic analyses based on concatenated amino acid sequences of protein-coding genes revealed that the newly described species t. malaysiensis was more closely related to t. cati than to t. canis, consistent with results of a previous study using sequences of nuclear internal transcribed spacers as genetic markers. toxocara canis, toxocara cati and toxocara malaysiensis cause significant health problems in animals and humans. the mt genomes of the three toxocara species all encode proteins, two ribosomal rnas and transfer rna genes, but lack the atp synthetase subunit gene, which is consistent with all other species of nematode studied to date, with the exception of trichinella spiralis. the sizes of the entire mt genome are bp for t. canis, bp for t. cati and bp for t. malaysiensis, respectively. all genes are transcribed in the same direction and have a nucleotide composition high in a and t, but low in g and c. the contents of a+t of the complete genomes are % for t. canis, % for t. cati and % for t. malaysiensis, among which the a+t for t. canis is the lowest among all nematodes studied to date. in addition, mt genome sequences have provided useful markers for investigating population genetic structures, systematics and phylogenetics of organisms. the present study determined the complete mt genome sequences for three roundworms of human and animal health significance, which provides mtdna evidence for the validity of t. malaysiensis and also provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance. their relatively large sizes relate mainly to an increased length in the at-rich region. these circular genomes are amongst the largest reported to date for all secernentean nematodes. studying mitochondrial genomics has important implications for various fundamental areas, including mt biochemistry, physiology and molecular biology. the at bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. although they are of importance in human and animal health, no information on the mt genomes for any of toxocara species is available.
 studying mitochondrial genomics has important implications for various fundamental areas, including mt biochemistry, physiology and molecular biology. in addition, mt genome sequences have provided useful markers for investigating population genetic structures, systematics and phylogenetics of organisms. toxocara canis, toxocara cati and toxocara malaysiensis cause significant health problems in animals and humans. although they are of importance in human and animal health, no information on the mt genomes for any of toxocara species is available.  the sizes of the entire mt genome are bp for t. canis, bp for t. cati and bp for t. malaysiensis, respectively. these circular genomes are amongst the largest reported to date for all secernentean nematodes. their relatively large sizes relate mainly to an increased length in the at-rich region. the mt genomes of the three toxocara species all encode proteins, two ribosomal rnas and transfer rna genes, but lack the atp synthetase subunit gene, which is consistent with all other species of nematode studied to date, with the exception of trichinella spiralis. all genes are transcribed in the same direction and have a nucleotide composition high in a and t, but low in g and c. the contents of a+t of the complete genomes are % for t. canis, % for t. cati and % for t. malaysiensis, among which the a+t for t. canis is the lowest among all nematodes studied to date. the at bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. the mt genome structures for three toxocara species, including genes and non-coding regions, are in the same order as for ascaris suum and anisakis simplex, but differ from ancylostoma duodenale, necator americanus and caenorhabditis elegans only in the location of the at-rich region, whereas there are substantial differences when compared with onchocerca volvulus,dirofiliria immitis and strongyloides stercoralis. phylogenetic analyses based on concatenated amino acid sequences of protein-coding genes revealed that the newly described species t. malaysiensis was more closely related to t. cati than to t. canis, consistent with results of a previous study using sequences of nuclear internal transcribed spacers as genetic markers.  the present study determined the complete mt genome sequences for three roundworms of human and animal health significance, which provides mtdna evidence for the validity of t. malaysiensis and also provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance. 