Sequencing And Analysis Of The Complete Mitochondrial Genomes Of Four Baylisascaris Spp. Within Ascarididae Family

The mitochondrial genome (mtDNA) has been regarded as a good mode for studying the genome structure function and species evolution as well as an ideal molecular marker for investigating molecular classification, systematics, population genetics and variation, due to its small gene numbers, high polymorphism, more rapid mutation rates than nuclear genes, presumed lack of recombination and maternal inheritance. Roundworms of the genus Baylisascaris are the most common parasitic nematodes of the intestinal tracts of wild mammals, with high infection rate and extreme threats among these animals. Of them, Baylisascaris schroederi, a specific-parasitic nematode for the giant panda (Ailuropoda melanoleuca), is the leading cause of death from primary and secondary infection in wild and captive population. Baylisascaris ailuri is an important cause of death of the wild red panda (Ailurus fulgens). Baylisascaris transfuga and Baylisascaris procyonis, the common parasites in Ursidae animals and raccoons (Procyon lotor), respectively, are emerging as an important helminthic zoonosis because of serious or fatal larval migrans, such as ocular larva migrans (OLM), visceral larval migrans (VLM) and neural larva migrans (NLM) in incidental hosts including humans. Despite their significant veterinary and public health impact, the epidemiology, molecular ecology and population genetics of these parasites remain largely unexplored. Mitochondrial (mt) genomes can provide a foundation for investigations in these areas and assist in the precaution and control of baylisascariasis. Herein, the complete mt genome sequences of B. schroederi, B. ailuri, B. transfuga and B. procyonis were determined using a long-polymerase chain reaction (Long-PCR)-based primer-walking strategy. According to the information of Ascaris suum and Toxocara canis mtDNAs, four mt genomes were assembled and annotated, and were then subjected to comprehensive comparative analysis with other related species available in GenBank, including genome structure, base content, codon usage patterns, as well as the secondary structures of tRNAs, rRNAs and non-coding region (NCR). Finally, combined with other published Ascaridida species mtDNAs, phylogenetic relationships within the order Ascaridida were reconstructed. The main results of research are summarized as following:1. Study of complete mitochondrial genomes of B. schroederi, B. ailuri and B. transfuga1.1The complete mt genomes of B. schroederi, B. ailuri and B. transfuga were determined to be14,778bp,14,657bp and14,898bp in size, respectively. Each genome contains36genes, consisting of12protein-coding genes,22transfer RNA (tRNA) genes,2ribosomal RNA (rRNA) genes and2non-coding regions (AT-rich region and NCR). The gene arrangements (including NCR) for the three Baylisascaris species are the same as those of the Ascaridata species (e.g. Ascaris suum and Toxocara canis), but radically different from those of the Spirurida species (Dirofilaria immitis and Onchocerca volvulus).1.2The overall base compositions (coding strand) for the mt genome sequences of B. schroederi, B. ailuri and B. transfuga are biased toward T and A, with total AT content68.62%,69.47%and69.45%, respectively. Compared with other Ascaridida species, these AT contents are the lowest of the nematodes reported so far. The initiation codons used in12protein-coding genes within the three Baylisascaris species are TTG, ATT, GTG and ATG, while TAG, TAA, T and TA are predicted as the termination codons. Moreover, among all codons, the codon TTT (Phe) is the most frequently used, whereas codon CTC (Leu) is the least used.1.3Twenty-two tRNAs and two rRNAs in B. schroederi, B. ailuri and B. transfuga mtDNAs were similar to those of other Ascaridida nematodes characterized to date in length, but the AT-rich regions seem to represent the longest studied thus far among most other secernentean nematodes, with1406bp for B. schroederi,1282bp for B. ailuri and1516bp for B. transfuga in size. The secondary structures of tRNA, rRNA and NCR (including AT-rich and non-coding regions) inferred from the three Baylisascaris species were similar with those exhibited A. suum as well as other nematodes described in Ascaridida to date. 1.4Phylogenetic analysis (NJ, MP and ML) based on mtDNAs (concatenated amino acid sequences of12protein-coding genes of9taxas (B. schroederi, B. ailuri, B. transfuga, T. canis, Toxocara cati, Toxocara malaysiensis, A. suum, Anisakis simplex and O. volvulus)) indicated that B. ailuri belongs to the genus Baylisascaris within the order Ascaridida, and that a closer relationship between B. αiluri and B. transfuga than between B. αiluri and B. schroederi among the genus Baylisascaris. Additionally, These three Baylisascaris species were determined to be more closely related to A. suum (Ascarididae) than to the Toxocara species (T. canis, T. cati and T. malaysiensis)(Toxocaridae) and A. simplex (Anisakidae) in our phylogenetic analyses, which was consistent with results of previous morphological and molecular studies.2. Study of the complete mitochondrial genome of B. procyonis2.1The complete mt genome of B. procyonis was14781bp in size and encoded36genes, including12protein-coding genes,22transfer RNA (tRNA) and the small and large subunit ribosomal RNAs (rrnS and rrnL). All genes were distributed on the same strand and transcribed in the same direction (5’to3’), typical for other nematodes reported to date (except for Trichinella spiral is and Xiphinema americanum). Gene order for B. procyonis mtDNA was similar with described previously for members of the orders Ascaridida and Strongylida as well as for Caenorhabditis elegans. The total AT content of this mt genome is70.5%, comprising68.6%of theprotein-coding genes,72.0%of the rRNAs,69.6%of the tRN As and82.8%of the AT-rich region.2.2B. procyonis mtDNA contained17intergenic spacers ranging in length from1to118bp (accounting for1.42%in total) including the NCR region, which was the highest number of intergenic spacers sequenced in a nematode mt genome thus far. In addition, there were only two overlaps found between the genes, with one (1bp) between coxl and trnC and another (3bp) between trnF and cytb.2.3Within the entire B. procyonis mtDNA, the AT and GC skews for the coding strand were20.376and0.448, respectively, which were significant compared with those of other nematodes characterized to date, indicating that this genome is biased toward T and G. The nucleotide frequency of12protein-coding genes was observed to be in the order T> G> A> C, which excessively favored T (50.8%) and was skewed against C (8.31%).2.4As for the choices of initiation and termination codons for12protein-coding genes in B. procyonis, the most frequently used start codon was TTG (6genes; cox1-2, nadl, nad3-4and nad6) followed by GTG (three genes; nad2, cytb and cox3), and ATT(nad4L and nad5) and ATA (atp6) were also used as initiation codons. Ten of the12protein-coding genes were deduced to use TAG (eight genes; atp6, cytb, coxl-3, nadl, nad4and nad6) or TAA (nad3and nad4L) as the termination codons, while the remaining genes (nad2and nad5) were predicted to end with an incomplete codon T.2.5Twenty-two tRNAs were predicted in B. procyonis mtDNA, ranging from51to62bp in size, and their corresponding locations and anti-codon sequences were the same as in B. schroederi, B. ailuri, B. trans fuga, T. canis, T. cati and T. malaysiensis. All but the two serine tRNAs (AGN and UCN) had a predicted secondary structure containing a DHU loop and a TV-replacement loop, while the two serine tRNAs each contained the TΨC loop but lacked the DHU arm and loop. Interestingly, similar secondary structures were observed in those of other secernentean nematodes including Ascaridida species. Moreover, the lengths and secondary structures of rrnS and rrnL found in B. procyonis appeared to be similar with those of B. schroederi, B. ailuri, B. transfuga, implying to a certain extent that they may have something to do with the relative conservation in structure and function.2.6The length of the AT-rich region was1375bp in the B. procyonis mtDNA. This region along with its counterpart in B. schroederi (1406bp), B. ailuri (1282bp) and B. transfuga (1516bp) represented the longest studied thus far among the Ascaridida nematodes. Additionally, B. procyonis AT-rich region contained31regions with varying numbers of the dinucleotide (TA) repeat (n=3to21) within a total of344bp. Similar multiple TA repeats have been described in the AT-rich region of the mt genomes for almost other secernentean nematodes. Other repetitive elements, such as CR1-CR6identified in the C. elegans AT-rich region were not found in B. procyonis.2.7Based on the results from pairwise comparisons and sliding window analysis of mt genes among the available Ascaridida mtDNAs, coxl and cox2were the most conserved protein-coding genes, whereas cytb and nad4were the least conserved ones. So genes rrnL and cytb were selected as potentially new mt fragments for PCR specific identification within the Ascaridida species. Subsequently, PCR primer pairs were designed targeting a200bp fragment in rrnL and a548bp fragment in cytb and the corresponding primer pairs were:A (rrnL) forward5’-GAGAACTGGCGGGG-3’ reverse5’-CTCACACTGACTTACACACC-3’; B (cytb) forward5’-TCCTTAGTAATGAGTATTGCGT-3’, reverse5’-TATAACGACATTTGAAAAACACC-39.2.8Phylogenies (NJ, MP and ML) in this study inferred from the concatenated amino acid sequence dataset derived from12protein-coding mitochondrial genes for the11taxas (B. procyonis, B. schroederi, B. ailuri, B. transfuga, A. suum, T. canis, T. cati, T. malaysiensis, A. simplex, C. rudolphii B and O. volvulus) showed that among the genus Baylisascaris, there was a closer relationship between B. procyonis and B. schroederi than between B. procyonis and B. αiluri or B. transfuga, and that in the order Ascaridida, the genus Baylisascaris was determined to be more closely related to Ascaris than to Toxocara, Anisakis and Contracaecum. These results were congruent with results of previous morphological and molecular studies.The mt genomes of B. schroederi, B. ailuri, B. transfuga and B. procyonis determined herein represent the first complete mt genome sequences for any member of the genus Baylisascaris. These mitogenome sequenced not only augment the mtDNA dataset of the order Ascaridida which contains a broad range of parasites of major socio-economic importance, but also provide a rich resource of bioinformation (e.g. molecular markers) for systematic, population genetic, epidemiological and ecological studies of parasitic nematodes of importance in wildlife, which together are expected to render implications for further prevention and control of these four Baylisascaris species and other related ascaridoids.