Complete mitogenome sequence of Caryophyllaeus brachycollis (Cestoda: Caryophyllidae) from China: Characterization and phylogenetic analyses of Caryophyllidea

Ethics statement

The experimental protocol was established according to the ethical guidelines of the Institutional Animal Care and Use Committee and approved by the Ethics Committee of Hunan Agricultural University (No. 202282).

Sample collection and DNA extraction

Samples were collected from the intestines of Cyprinus carpio from Beijing Zoo, China. They were washed in physiological saline, and those morphologically identified as caryophyllidean were fixed in 70% (v/v) ethanol and stored at −40°C until use.

Total genomic DNA was extracted using sodium dodecyl sulfate/proteinase K treatment, followed by spin-column purification (Wizard SV Genomic DNA Purification System; Promega, Madison, WI, USA). The purified DNA was used as the template in PCR assays with the primer pair JB3/JB4.5 targeting a fragment of cox1 for amplification [22].

Sequencing, assembling, and verification

A genomic DNA library (with 350 bp inserts) was prepared and sequenced by Novogene (Tianjin, China) using a HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA) to obtain 250 bp paired-end reads. Geneious v11.1.5 software was used to filter and clean the raw data and assemble the complete CBCB mitogenome using the amplified cox1 sequences as initial references. The parameters were set as follows: minimum overlap identity, 99.0%; minimum overlap, 150 bp; and maximal gap size, 5 bp. The assembled mitogenome was further verified by long PCR experiments (Supplementary Fig. S1) using the primers listed in Supplementary Table S1.

Annotation, visualization, and sequence analysis

The boundaries of protein-coding genes (PCGs) were identified using the NCBI tool ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). The initiation/termination codons for each PCG were identified following alignment with the CBCW mitogenome sequence using MAFFT v7.122 software. All PCG nucleotide sequences were translated to amino acid sequences using MEGA v11.0. Twenty-two transfer RNA (tRNA) genes were detected using ARWEN [23] and tRNAscan-SE [24]. Nucleotide composition and codon usage frequencies of the PCGs were analyzed using MEGA v11.0. The annotated CBCB mitogenome was visualized using the online server Proksee (https://proksee.ca/). The CBCW sequence was used as the reference genome for comparisons with CBCB.

Phylogenetic analyses

Phylogenetic analyses were performed using 6 mitogenome sequences of tapeworms within the order Caryophyllidea (Table 1) and Paruterina candelabraria (GenBank accession No. NC_039533) as the outgroup. Amino acid sequences were aligned using MAFFT v7.122 and concatenated into a single dataset. Ambiguous bases or gaps were excluded using Gblocks 0.91b software with the default parameters [25]. Phylogenetic analyses were performed using Bayesian inference (BI) and maximum likelihood (ML) methods. BI analysis was run in MrBayes 3.1.1 using an approach similar to that of Ronquist and Huelsenbeck [26]. ML analysis was performed using PhyML 3.1 software. “JTT+I+G+F” was selected as the best suitable model by ProtTest v3.4.2 based on the Akaike information criterion [27] and was used for the BI and ML analyses. Phylograms were drawn using FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree).

Table 1

Complete mitogenome sequences of the order Caryophyllidea used for phylogenetic analysis

cox1 sequence analysis

To determine the systematic relationship of the families in Caryophyllidea, BI analysis was performed using 20 cox1 sequences of the order Caryophyllidea and Gigantolina magna (GenBank accession No. JQ268545) as the outgroup.

Genome composition and organization

Sequencing of the CBCB mitogenome generated 4.08 Gb of raw data, from which approximately 7,577,818×2 clean reads were obtained following filtering and used for further assembly. The complete circular CBCB mitogenome (GenBank accession No. OP359069) was 14,273 bp and comprised 36 genes, including 12 PCGs (excluding atp8), 22 tRNA genes, 2 ribosomal RNA genes (rrnS and rrnL), and 2 noncoding regions (NCRs; control or AT-rich regions) (Fig. 1; Table 2). All 36 genes were transcribed in the forward direction. In C. brachycollis mitochondrial DNA, the AT-skews were all negative (−0.481 to −0.125), and the GC-skews were all positive (0.167 to 0.465) (Table 3), suggesting a bias toward T and G bases. The lengths of the 2 NCRs in CBCB were 845 and 423 bp, respectively, while those in CBCW were 641 and 871 bp, respectively (Table 2). The large NCR (LNCR) was located between tRNA-Leu2 and cox2, and the small NCR (SNCR) was located between tRNA-Gly and cox3 (Fig. 1). Alignment using BLAST revealed 100.0% nucleotide similarity with CBCW (GenBank accession No. NC_035430).

Fig. 1

Mitogenome organization of Caryophyllaeus brachycollis isolate from China. Gene scaling is approximate. CD, coding region; NCR1, long noncoding region; NCR2, short noncoding region; tRNA, transfer RNA; rRNA, ribosomal RNA.

Table 2

Comparative organization of the complete mitogenome of Caryophyllaeus brachycollis isolates from Beijing and Wuhan, China

Table 3

Nucleotide composition and skews of the Caryophyllaeus brachycollis mitogenome

Composition of PCGs, ribosomal RNA genes, and tRNA genes

ATN was extensively used as the initiation codon, especially ATG. GTG is an uncommon start codon in tapeworms, but it was observed in cytb, nad1, and nad4L in this study (Table 2). Furthermore, TAA and TAG were frequently used as termination codons (TAG: cytb, atp6, nad3, nad4, and nad4L; TAA: nad6) (Table 2). Incomplete stop codons T and TA were also prevalent in termination, with cox1, cox3, and nad2 using T and cox2, nad1, and nad5 using TA (Table 2). The AT-skews of the 12 protein genes were all negative (−0.481 (nad3) to −0.181 (cox2)), and the GC-skews were all positive (0.167 (cox2) to 0.465 (nad4L)), suggesting a large T base bias in nad3, a G base tendency in nad4L, and a minor tendency of T+G bases in cox2.

The 22 tRNA genes ranged from 54 to 68 bp (Table 2). rrnL and rrnS were 951 and 712 bp, respectively, with an A+T content of 62.2% and 62.4%, respectively (Table 3).

Comparative analyses of CBCB and CBCW

Comparisons between the CBCB and CBCW mitogenome sequences revealed that CBCB was approximately 231 bp shorter than CBCW. In both CBCB (Fig. 1) and CBCW, the SNCR and LNCR were located in “tRNA-Gly and cox3” and “tRNA-Leu2 and cox2,” respectively. The size (cox2 and nad5) and the initiation/termination (cox2) codons of PCGs in CBCB and CBCW differed (Table 2). cox2 was 539 bp and 551 bp in CBCW and CBCB, respectively, while nad5 was 1,547 and 1,550 bp, respectively. The initiation/termination codons of cox2 were ATG/TAG and ATG/TA in CBCW and CBCB, respectively.

Comparison of the nucleotide sequences of PCGs between CBCB and CBCW revealed differences of 0% (nad4L) to 2.2% (cox3) (Table 4). Differences in the amino acid sequences of CBCB and CBCW ranged from 0% (nad4L) to 2.7% (cox2) (Table 4). nad4L typically shows interspecies variability. However, our study revealed that it was highly conserved in C. brachycollis. These findings suggested that cox2 might be a better molecular marker for identifying cryptic species within C. brachycollis.

Table 4

Nucleotide and amino acid sequence differences between the isolates from Beijing, China and Wuhan, China mitogenomes

Phylogenetic analyses

The phylogeny of Caryophyllidea is currently unclear due to a lack of complete mitogenome sequences. To better clarify the phylogenetic relationships among the members of this order, 6 mitogenome sequences were phylogenetically analyzed using BI and ML methods. The BI and ML tree topologies were identical (Fig. 2). There were 2 main clades: 1 clade contained Caryophyllaeidae and Lytocestidae, and the other clade contained Capingentidae, Caryophyllaeidae, and Lytocestidae, indicating that families Caryophyllaeidae and Lytocestidae were paraphyletic. The mitochondrial DNA sequences of CBCB and CBCW grouped with short topological distance (BPP =1, BF=100) (Fig. 2), indicating that they were closely related. Furthermore, phylogenetic analyses of cox1 also showed that Caryophyllaeidae and Lytocestidae were paraphyletic and revealed a close evolutionary distance between CBCB and CBCW (Supplementary Fig. S2).

Fig. 2

Inferred phylogenetic relationships among species in the order Caryophyllidea. The concatenated amino acid sequences of 12 mitochondrial protein-coding genes were analyzed utilizing maximum likelihood (ML) and Bayesian analysis (BI) methods, with Paruterina candelabraria as the outgroup. The Caryophyllaeus brachycollis form was isolated from carp tapeworm in Beijing in this study.