Characterization of the complete mitochondrial genome of Brentisentisyangtzensis Yu & Wu, 1989 (Acanthocephala, Illiosentidae)

Abstract The mitogenome of Brentisentisyangtzensis is 13,864 bp in length and has the circular structure typical of metazoans. It contains 36 genes: 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and 12 protein-encoding genes (PCGs). All genes are transcribed from the same strand. Thirteen overlapping regions were found in the mitochondrial genome. The overall A+T content of B.yangtzensis is 68.3% versus 31.7% of G+C content (A = 27.8%, T = 40.5%, C = 9.0%, G = 22.7%). B.yangtzenensis (Illiosentidae) and Leptorhynchoidesthecatus (Rhadinorhynchidae) form a sister clade, showing the relatively close relationship between the Illiosentidae and the Rhadinorhynchidae. The mitochondrial gene arrangements of acanthocephalan species are relatively conserved, with only a few translocations of tRNAs (trnS1, trnS2, trnV, and trnK) detected. An identical gene order was found both in a sister clade (Centrorhynchusaluconis and Plagiorhynchustransversus) and across different classes (B.yangtzensis (Palaeacanthocephala), Acanthosentischeni (Eoacanthocephala) and Macracanthorhynchushirudinaceus (Archiacanthocephala), Oncicolaluehei and L.thecatus (Palaeacanthocephala)). More studies and more sequences of acanthocephalan species are needed to gain a clear understanding of the phylogenetic relationships.


Introduction
Members of the Acanthocephala are obligate endoparasites which utilize arthropods as intermediate hosts and vertebrates as definitive hosts. This phylum contains approximately 1300 documented species, and is classified into three classes (Archiacanthocephala, Palaeacanthocephala and Eoacanthocephala). The Palaeacanthocephala has the highest species richness with 65% of the total acanthocephalan species, and comprises three orders: Echinorhynchida, 472 species; Polymorphida, 372 species; and Heteramorphida, one species (Amin 1987(Amin , 2013. The classifications proposed by Golvan (1960) and Amin (1987) have been challenged by recent phylogenetic studies, which indicated that the genus Leptorhynchoides (Rhadinorhynchidae Kostylew, 1924) is more closely related to the genera of the Illiosentidae Golvan, 1960 rather than those of the Rhadinorhynchidae in morphological (Monks 2001) and the molecular phylogenies (García-Varela and Nadler 2005; García-Varela and González-Oliver 2008). Intriguingly, Illiosentis Van Cleave & Lincicome, 1939 was first placed in the Rhadinorhynchidae (Van Cleave and Lincicome 1939), but Golvan (1960) decided that a new family was required to accommodate the genus, and so erected the Illiosentidae.
This debate continues, and molecular markers carrying stronger phylogenetic signals are needed to resolve the phylogenetic relationships with a higher resolution. The mitogenome is a good candidate, being approximately ten times larger than commonly used single-gene molecular markers (ITS, 18S, and 28S) , and considered to provide the best interrelationship estimate for the Cestoda (Waeschenbach et al. 2012). Mitochondrial genome sequences are becoming prevalent and are increasingly used in population genetics (Yin et al. 2015), phylogenetics (Gazi et al. 2012(Gazi et al. , 2015(Gazi et al. , 2016Weber et al. 2013;Li et al. 2017;Pan and Nie 2013) and the diagnostics (Huyse et al. 2008;Jia et al. 2010) of metazoans. However, many groups of parasitic organisms are unrepresented, and the resolving power of mitochondrial genomics is still limited by the small number of acanthocephalan mitogenomes (only 13 species available), with many taxonomic categories, e.g. the Illiosentidae, poorly represented or unrepresented.
The complete mitogenome of an illiosentid species has not previously been published. In order to fill this knowledge gap, we have sequenced and annotated the complete mitogenome of Brentisentis yangtzensis Yu & Wu, 1989 (Palaeacanthocephala, Illiosentidae), a parasite from the intestines of many freshwater fish species in the middle reaches of the Yangtze River (Yi and Wu 1989). Previous studies on this parasite have focused on its morphology and population ecology (Fang 1999, Fang andDai 2000;Fang et al. 2004); molecular data have not previously been reported.

Specimen collection and DNA extraction
The acanthocephalans were collected on 24 September 2018 from the intestine of 36 bullhead catfish Tachysurus fulvidraco (Richardson, 1846) from east Dongting Lake in Yueyang (29°22'N, 113°06'E), Hunan Province, China. Brentisentis yangtzensis was identified by morphology (e.g., Yu 1989) using a stereomicroscope and a light microscope. The parasites were preserved in 100% ethanol and stored at 4 °C. The total genomic DNA was extracted from an entire acanthocephalan using a TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China) according to manufacturer's recommended protocol, and stored at -20 °C. Eleven acanthocephalans were collected in total.

PCR and DNA sequencing
Partial sequences of rrnL, cytb, nad1, and rrnS genes were amplified via a polymerase chain reaction (PCR) using four primer pairs. Based on these fragments, we designed specific primers for subsequent PCR amplification (Suppl. material 1). PCR reactions were conducted in a 50 ml reaction mixture, containing 18.5 ml double-distilled water (dd H 2 O), 25 ml 2×PCR buffer (Mg 2+ , dNTP plus, Takara, China), 1.5 ml of each primer, 1 ml rTaq polymerase (250U, Takara, China) and 2.5 ml DNA template. Amplification was performed under the following conditions: initial denaturation at 98 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 48-60 °C for 15 s, 68 °C for 1 min/ kb, and a final extension at 68 °C for 10 min. PCR products were sequenced bidirectionally at Sangon Biotech (Shanghai) Co., Ltd. (China) using the primer walking strategy.

Sequence annotation analyses
The mitogenome of B. yangtzensis were assembled manually in a stepwise manner with the help of the DNAstar v7.1 program (Burland 2000), after quality-proofing of the obtained fragment. The mitogenome was annotated mainly following the procedures described previously Zhang et al. 2017a;Li et al. 2017). In detail, protein-coding genes (PCGs) were inferred with the help of BLAST and ORF Finder tools (both available from the National Center for Biotechnology Information (NCBI)), employing the invertebrate mitochondrial code (Codon Table 5), and checking the nucleotide alignments against the reference genomes in acanthocephalan Leptorhynchoides thecatus (Linton, 1891) Kostylew, 1924 (NC_006892). A majority of the tRNAs were identified using the results of ARWEN (Laslett and Canback 2008) and MITOS web server (Bernt et al. 2013), the rest were found by alignment with other acanthocephalans (Suppl. material 2). Two ribosomal RNA genes (rrnL and rrnS) were found by alignment with other published acanthocephalan mitogenomes, and their ends were assumed to extend to the boundaries of their flanking genes. Codon usage and relative synonymous codon usage (RSCU) for 12 protein-encoding genes (PCGs) of the B. yangtzensis and L. thecatus (NC_006892) were computed and sorted using PhyloSuite , and finally the RSCU figure drawn using ggplot2 plugin (Wickham 2016). The circular map of B. yangtzensis mitogenome was drawn with the mitochondrial visualization tool MTVIZ (http://pacosy.informatik. uni-leipzig.de/mtviz/).

Phylogenetic analyses
Phylogenetic analyses were carried out on the newly sequenced mitogenome of B. yangtzensis and the 12 acanthocephalan mitogenomes available in GenBank (Suppl . Table S2). Two species of the Bdelloidea, Rotaria rotatoria (Pallas, 1766) (NC013568.1) and Philodina citrina Ehrenberg, 1832 (FR856884.1), were used as outgroups. Fasta files with the amino acid sequences for all 12 PCGs were extracted from the GenBank files using Phy-loSuite. All the genes were aligned in batches with MAFFT (Katoh et al. 2002) integrated in PhyloSuite, using normal-alignment mode. PhyloSuite was then used to concatenate these alignments into a single alignment and generate phylip and nexus format files for the phylogenetic analyses, conducted using maximum likelihood (ML) and Bayesian inference (BI) methods. The selection of the best-fit partition strategy and models was car-

Genome organization and base composition
The circular duplex molecule mitogenome of B. yangtzensis is 13,864bp in size (Gen-Bank accession number MK651258) and contains all 36 of the typical metazoan genes: 22 tRNA genes, 2 rRNA genes and 12 protein-encoding genes (PCGs) (lacking atp8) (Fig. 1). All genes are transcribed from the same strand, and 13 overlapping regions were found in the genome (Table 1). The lack of the gene atp8 is common in acanthocephalans (Gazi et al. 2016;Song et al. 2016) with one exception; in L. thecatus two putative atp8 genes have been suggested (Steinauer et al. 2005).

Protein-coding genes and codon usage
The total length of the concatenated 12 protein-coding genes is 10,355 bp, with the average A+T content of 68.0%, ranging from 65.9% (nad3) to 69.5% (atp6 and nad4) (Suppl. material 3). ATG (for 6 PCGs) is the most commonly used start codon, whereas nad6, nad4, cox1 and cox2 used GTG, nad1 and nad3 used TTG and ATA, respectively. The most frequent terminal codons are TAG (for 7 PCGs), followed by T (4 PCGs) (Table 1).

Transfer and ribosomal RNA genes
All 22 commonly found tRNAs are present in the mitogenome of B. yangtzensis, ranging from 50 bp (trnS2) to 70 bp (trnT) in size, with a concatenated length of 1,247 bps (Table 1).

Gene order
The mitochondrial gene arrangements of acanthocephalan species are relatively conserved (Fig. 2). Besides the incomplete mitochondrial genome of E. truttae, the gene arrangement of 12 protein coding genes and two rRNA genes are highly conserved, with only a few translocations of tRNAs (trnS1, trnS2, trnV, and trnK) detected. In mitochondrial genomes, conserved gene arrangement is considered to be a typical characteristic (Boore 2000;Li et al. 2012), but the arrangement of tRNAs shows more variability, and there are examples of extensive gene rearrangement (Hyman et al. 2011;Zhang et al. 2017b;Wang et al. 2017). Transfer RNA genes are more often translocated than other genes, probably because of their small size (Kilpert and Podsiadlowski 2006).