Characterization of the Complete Mitochondrial Genome Sequence of Contracaecum sp. (Nematoda: Ascarididae) from China

doi:10.21203/rs.3.rs-1513649/v1

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Characterization of the Complete Mitochondrial Genome Sequence of Contracaecum sp. (Nematoda: Ascarididae) from China

https://doi.org/10.21203/rs.3.rs-1513649/v1

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Contracaecum spp. is a zooparasitic anisakid nematode and occurs in gastrointestinal tracts of vertebrate/invertebrate animals, humans included, causing gastrointestinal pain, diarrhea, and increasingly severe vomiting. To date, thousands of sequences have been published in GenBank, while complete mitochondrial genomes (mitogenome) of Contracaecum spp. are still limited. Although the complete mitogenome of Contracaecum species isolated from night herons in Beijing has been reported, the detailed information about it is still puzzling. In the present study, we describe the detailed characteristics within the complete mt DNA Contracaecum sp.. The AT-content in the complete mitogenome of Contracaecum sp. was 72.2%. The nucleotide diversity (Pi) among genus Contracaecum ranged from 0.124 (cox1) to 0.181 (nad4). Based on the computational algorithms Maximum Likelihood (ML) and Bayesian Inference (BI) within Ascaridoidea and Heterakoidea, the results supported Contracacum sp. Beijing isolate was a new species in the genus Contracaecum, and supported the family Ascaridiidae was paraphyletic.

Contracaecum sp.

Contracaecum

mitochondrial genome

Contracaecum species were known as globally parasitizing nematodes causing severe pathogenic influences on vertebrate and invertebrate animals, including humans (Shamsi 2019). It has an indirect life cycle and piscivorous birds are its definitive hosts, leading to hemorrhages, necrosis, and severe ulcerative eosinophilic granulomas in intestinal tracts in birds (Zhang et al. 2021). In Australia, Contracaecum spp. was the first human anisakidosis detected in the human body with symptoms of gastrointestinal pain, diarrhea, and increasingly severe vomiting, though it was not identified at the species level (Shamsi and Butcher 2011; Shamsi et al. 2019).

In the past decades, over 100 species from the genus Contracaecum have been described the morphologies and partial mitochondrial (mt) sequences (Shamsi et al. 2019; Zhang et al. 2021). Some of them could be identified and distinguished by morphological features, while it is difficult to judge if there are cryptic species complexes within Contracaecum spp., such as C. rudolphii, C. ogmorhini, and C. osculatum. Studies show that even in cryptic species from the same species, considerable differences within hosts and biographylogy still exist (Shamsi et al. 2009; Timi et al. 2014; Mattiucci et al. 2014; Liu et al. 2016). Herein, it is better to understand more molecular information about Contracaecum spp. to proport a new insight to identify potential cryptic species complex, mitogenomes including. Recently, although the complete mitogenome of Contracaecum sp., which was collected from black night herons from Beijing, China, has been published (GenBank no. MN892395), the uploaded sequence was not annotated. Yet, the detailed information of the complete mt sequence was still in a puzzle that was inconvenient to cite the mt sequence of Contracaecum sp. for further studying.

Therefore, in the present study, we aim to (i) reassemble and annotate the complete mitogenome of Contracaecum sp., which was isolated from black herons in Beijing, China, and describe detailed information of the complete mt sequence of Contracaecum sp.; (ii) based on uploaded annotated sequences of Ascaridoidea spp. and Heterakoidea spp., conducting phylogenetic analyses to support Zhang et al. (2021) hypothesis; and (iii) provide more detailed molecular features within genus Contracaecum for successive studies.

Parasites and molecular identification

Specimens’ helminths were obtained from the digestive tracts of hosts grey and night herons in Beijing Zoo, China. The species were washed with ultrapure water and physiological saline solution, then fixed and collected into 75% ethanol (v/v) until further studying. The species were preliminarily identified as Contracaecum roundworms based on hosts and primary characteristic morphology (Zhang et al. 2021). For additional examination of molecules, the total genomic DNA of several species was extracted using QIAamp® DNA Micro Kit as the instruction recommended. The identity of these roundworms was confirmed based on polymerase chain reaction (PCR) amplification of partial cox1 (with primers JB3-JB4.5) (Bowles et al. 1992; Bowles and McManus 1994) and ITS (with primers NC5-NC2) (Newton et al. 1998; Chilton et al. 2001). The obtained ITS sequence totally matched with that of Contracaecum sp. (GenBank no. MW538933 ~ 36). And partial cox1 sequence showed 99.7% identity with Porrocaecum reticulatum (GenBank no. MF113244).

Sequencing, assembling, and annotation

The genomic DNA sample was fragmented to a size of 350 bp. The DNA libraries were sequenced for high throughput sequencing (HTS) on Illumina Hiseq 6000 platform (Novogene Co. Ltd. Tianjin, China) and 250 bp paired-end reads were generated. The raw data then were obtained and recorded in FASTQ format. Then, the reads with low-quality bases (Phred quality < 5) or uncertain reads with repetitive “N” bases were discarded to acquire clear data. The partial cox1 sequence was used as the initial reference to assemble complete sequences of Contracaecum sp. using Geneious Prime 2022.0.1 (Kearse et al. 2012). The assembly was operated with parameters as follows: (i) minimum overlap within the ranges of 150–200 bp; (ii) minimum overlap identity among 98% − 100%; and (iii) maximum gap of 5 bp. The completely circular mt genome of roundworm was then got verification by long PCR with primers as listed (Table S1, Fig. 1).

All 12 protein-coding regions were preliminarily identified the start and stop codons by ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) in NCBI. Later, two ribosomal RNAs (rRNAs, rrnL and rrnS) were framed using the Tandem repeats finder (Benson 1999). The 12 genes were then further confirmed by identifying similar sequences with former published Ascarididae (Ascaris lumbricoides, GenBank no. NC_016198). The tRNAscan-SE 2.0 (Chan et al. 2021) with cutoff score of 1.0, and MITOs (Bernt et al. 2013) were applied to search 22 potential transfer RNAs (tRNAs).

Nucleotide variation in mtDNA genomes among Contracaecum spp.

Based on available mitogenome sequences of genus Contracaecum in NCBI, pairwise alignment of 12 protein genes was conducted in Clustal X1.83 to compute nucleotide diversity. The complete protein alignments from the genus Contracaecum, C. osculatum, C. rudolphii, C. ogmorhini, and Contracaecum sp. in the present study, were analyzed by DnaSP v5 using sliding windows (Librado and Rozas 2009). The parameters of the sliding window were followed with 300 bp window length and a default 25 bp step site to calculate the nucleotide diversity (Pi or π). Each boundary of protein genes was identified due to mid-point position, and we then graphed nucleotide diversity for 12 protein genes from Contracaecum.

Phylogenetic analysis

Total of 41 mt sequences of nematodes, from families Ascaridoidea and Heterakoidea, were applied to analyze phylogeny with outgroups Enterobius vermicularis (GenBank accession no. EU281143) and Wellcomia siamensis (GenBank accession no. NC_016129) (Table S2). Each amino acid sequence was aligned using MAFFT computational algorithm (Katoh et al. 2019). The aligned sequences were then concatenated to obtain a single alignment dataset. The ambiguous gaps in the alignment were excluded by Gblocks 0.91b with default parameters “less stringent” (Dereeper et al. 2008). Computational algorithm Maximum likelihood (ML) (Guindon et al. 2010) was conducted to perform a phylogenetic tree with the best model “JTT + I + G + F” screened by ProtTest 3.4.2 (Darriba et al. 2011) and 1,000 replicates. Bayesian analysis was operated with MrBayes 3.2 (Ronquist et al. 2012) and “GTR + F + G” was selected as the best suitable model by ModelFinder in IQTree v.2.1.3 (Kalyaanamoorthy et al. 2017). Four Markov chains were progressed with 1,000,000 MCMC generations, with sampling analysis tree every 100 generations. The residual trees were calculated with Bayesian posterior probabilities (BPP), burning first 250 trees.

Mitogenome organization and composition

The filtered dataset of the sequenced mt genome of Contracaecum sp. is nearly 2Gb with a total of 8,677,194 x 2 clean reads for further assembling. The circular mt genome of Contracaecum sp. (GenBank accession: WM056322) assembled was 14,082 bp in size, shorter than Zhang et al. (2021) published, with 12 PCGs, 22 tRNAs, 2 rRNAs, and two non-coding regions (NCRs) (Table 1, Fig. 2). Total of 36 genes were transcribed in the forward direction and gene arrangement was recognized into the typical GA3 pattern which is mostly observed in roundworms (Liu et al. 2013a). Consistent with previous reports, there was a tendency of T base (48.7%) companying high A-T bases biased (71.2%). Total ten intergenic regions among the complete mt genome of Contracaecum sp. ranging from 1 bp to 16 bp (Table 1). One short (122 bp) was located between nad4 and cox1, and one long non-coding region (691 bp) was placed in tRNA-Ser₂ and tRNA-Asn. The values of AT-skew were negative from − 0.475 (nad6) to -0.111 (NCRs), inversely, the values of GC- skew were positive with scope 0.226 (nad4) – 0.674 (nad3), suggesting Ts and Gs were more frequently used in the genome.

Table 1

The organization of the complete mt genome of *Contracaecum* sp. from Beijing, China.
Gene/Region	Strand	Positions	Size (bp)	Number of aa^a	Ini/Ter codons	Anticodons	In
tRNA-Asn (N)	H	1-60	60			GTT	0
tRNA-Tyr (Y)	H	61-116	56			GTA	0
nad1	H	117-989	873	290	TTG/TAG		0
atp6	H	993-1591	599	199	ATT/TA		+3
tRNA-Lys (K)	H	1592-1653	62			TTT	0
tRNA-Leu2 (L₂)	H	1654-1708	55			TAA	0
tRNA-Ser1 (S₁)	H	1709-1759	51			TCT	0
nad2	H	1760-2605	846	281	TTG/TAA		0
tRNA-Ile (I)	H	2619-2678	60			GAT	+13
tRNA-Arg (R)	H	2679-2732	54			GCG	0
tRNA-Gln (Q)	H	2733-2787	55			TTG	0
tRNA-Phe (F)	H	2788-2846	59			GAA	0
cytb	H	2847-3953	1107	368	TTG/TAA		0
tRNA-Leu1 (L₁)	H	3961-4017	57			TAG	+7
cox3	H	4018-4782	766	255	TTG/T		0
tRNA-Thr (T)	H	4783-4843	60			TGT	0
nad4	H	4844-6073	1230	409	TTG/TAA		0
Intergenic region	H	6074-6195	122				0
cox1	H	6196-7771	1576	525	TTG/T		0
tRNA-Cys (C)	H	7772-7829	58			GCA	0
tRNA-Met (M)	H	7831-7890	60			CAT	+1
tRNA-Asp (D)	H	7907-7963	57			GTC	+16
tRNA-Gly (G)	H	7965-8021	57			TCC	+1
cox2	H	8022-8713	692	230	TTG/TA		0
tRNA-His (H)	H	8714-8778	65			GTG	0
rrnL	H	8779-9737	959				0
nad3	H	9738-10073	336	111	TTG/TAG		0
nad5	H	10077-11659	1583	527	ATT/TA		+3
tRNA-Ala (A)	H	11660-11716	57			TGC	0
tRNA-Pro (P)	H	11724-11780	57			TGG	+7
tRNA-Val (V)	H	11781-11837	57			TAC	0
nad6	H	11838-12272	435	144	TTG/TAA		0
nad4L	H	12275-12505	231	76	ATT/TAA		+2
tRNA-Trp (W)	H	12506-12563	58			TCA	0
tRNA-Glu (E)	H	12565-12624	60			TTC	+1
rrnS	H	12625-13335	711				0
tRNA-Ser2 (S₂)	H	13336-13391	56			TGA	0
Non-coding region	H	13392-14082	691				0
In: Intergenic nucleotides.
^aThe inferred length of amino acid (aa) sequence of 13 protein-coding genes; Ini/Ter codons: initiation and termination codons.

Protein-coding genes

TTG was the most common initial codon in this study, followed by ATT. TTG was used as the start codon for nine genes (cox1-3, cytb, nad1-4, and nad6), excluding genes atp6, nad4L, and nad5 (Table 1). The rest three PCGs used ATT as the initial codon. Generally, TAG and TAA were shared as common stop codons in metazoan (Hu et al. 2004). In this study, TAA was the most frequent termination among nad6, nad4L, nad4, cytb, and nad2. the genes nad1 and nad3 used TAG as the stop codon. The rest genes respectively used incomplete stop codons T (cox1 and cox3) or TA (atp6, cox2, and nad5).

A sum of 3422 amino acids was translated by 12 PCGs. TTT (480) was the most common codon used in encoding with Phe. Followed by codons GTT (Val), TTG (Leu), and ATT (Ile), the amounts were 219, 216, and 214 (Table 2), respectively. Leu (519) and Phe (499) were the most frequently coded amino acids, while Arg (34) was the rarest. There was a tendency of Gs and Ts in the same amino acid by comparing the relative synonymous codon usage (RSCU) (Table 2). The AT content of 12 protein genes ranged from 66.7% (cox1) to 78.9% (nad6) (Table 3). There was an obvious use of Ts and Gs, all rates of AT-skews were negative ranging from − 0.475 to -0.111, and whole values for GC-skew were positive from 0.226 to 0.674. Ts were the most used bases in nad6 (-0.475), followed by nad3 (-0.464) and nad2 (-0.451). Similarly, Gs were the most used bases in nad3 (0.674), nad4L (0.569) and atp6 (0.526).

Table 2

Amino acids frequency of *Contracaecum* sp. mitochondrial protein-coding genes.
Amino acid	Codon	Number	RSCU (%)	Amino acid	Codon	Number	RSCU (%)
Phe	TTT	480	1.92	Tyr	TAT	154	1.84
Phe	TTC	19	0.08	Tyr	TAC	13	0.16
Leu	TTA	199	2.3	Stop	TAA	5	1.43
Leu	TTG	216	2.5	Stop	TAG	2	0.57
Leu	CTT	76	0.88	His	CAT	54	1.86
Leu	CTC	2	0.02	His	CAC	4	0.14
Leu	CTA	10	0.12	Gln	CAA	20	0.98
Leu	CTG	16	0.18	Gln	CAG	21	1.02
Ile	ATT	214	1.92	Asn	AAT	100	1.79
Ile	ATC	9	0.08	Asn	AAC	12	0.21
Met	ATA	76	0.86	Lys	AAA	35	0.71
Met	ATG	101	1.14	Lys	AAG	63	1.29
Val	GTT	219	2.61	Asp	GAT	62	1.65
Val	GTC	13	0.16	Asp	GAC	13	0.35
Val	GTA	49	0.59	Glu	GAA	32	0.84
Val	GTG	54	0.64	Glu	GAG	44	1.16
Ser	TCT	139	3.08	Cys	TGT	53	1.96
Ser	TCC	6	0.13	Cys	TGC	1	0.04
Ser	TCA	14	0.31	Trp	TGA	21	0.57
Ser	TCG	5	0.11	Trp	TGG	53	1.43
Pro	CCT	66	3.11	Arg	CGT	33	3.88
Pro	CCC	7	0.33	Arg	CGC	1	0.12
Pro	CCA	9	0.42	Arg	CGA	0	0
Pro	CCG	3	0.14	Arg	CGG	0	0
Thr	ACT	89	3.24	Ser	AGT	121	2.68
Thr	ACC	6	0.22	Ser	AGC	2	0.04
Thr	ACA	9	0.33	Ser	AGA	36	0.8
Thr	ACG	6	0.22	Ser	AGG	38	0.84
Ala	GCT	72	2.5	Gly	GGT	112	2.22
Ala	GCC	24	0.83	Gly	GGC	21	0.42
Ala	GCA	11	0.38	Gly	GGA	23	0.46
Ala	GCG	8	0.28	Gly	GGG	46	0.91
Excluding abbreviated stop codons (TA and T).
Stop = Stop codon.

Table 3

Nucleotide composition and skews of *Contracaecum* sp. mitochondrial genome.
Gene	Nucleotide frequency				A + T (%)	AT-skew	GC-skew
Gene	A (%) G (%) T (%) C (%)				A + T (%)	AT-skew	GC-skew
atp6	22.0	22.0	49.1	6.9	71.1	-0.380	0.526
cox1	19.5	21.8	47.2	11.5	66.7	-0.416	0.307
cox2	21.2	22.1	46.5	10.1	67.7	-0.373	0.372
cox3	18.9	20.9	49.8	10.4	68.7	-0.449	0.333
cytb	19.7	22.0	47.6	10.7	67.3	-0.415	0.343
nad1	19.5	20.5	50.5	9.5	70.0	-0.444	0.364
nad2	20.7	18.2	54.6	6.5	75.3	-0.451	0.474
nad3	20.0	21.4	54.4	4.2	74.4	-0.464	0.674
nad4	21.4	17.0	50.9	10.7	72.3	-0.408	0.226
nad4L	22.9	17.3	55.0	4.8	77.9	-0.411	0.569
nad5	21.2	18.8	51.9	8.1	73.1	-0.420	0.398
nad6	20.7	13.3	58.2	7.8	78.9	-0.475	0.261
rrnS	30.2	19.7	40.4	9.7	70.6	-0.143	0.340
rrnL 22 tRNA	27.3 31.5	17.5 18.7	48.3 40.8	6.9 9.0	75.6 72.3	-0.277 -0.129	0.436 0.352
NCR	37.4	10.3	46.7	5.6	84.1	-0.111	0.290
Total	23.5	19.0	48.7	8.9	72.2	-0.350	0.364

Transfer RNA genes, ribosomal RNA genes and non-coding region

The length of 22 tRNAs ranged from 51 bp (tRNA-Ser₁) to 65 bp (tRNA-His). As one of the most conserved and amplest RNA, the secondary structure of a typical cloverleaf consisted of one acceptor stem, a dihydrouridine loop (D-loop), an anticodon loop, a TΨC loop, and related arms fixing with them (Su et al. 2020). However, in nematodes, most tRNAs were different from other metazoan animals. In our study, 16 of 20 tRNAs (excluding tRNA-Ser₁ and tRNA-Ser₂) lacked a TΨC loop, replaced by several nucleotide residues which compromised the TV-replacement loop (Hu et al. 2004). The tRNAs tRNA-His, tRNA-Ile, and tRNA-Met were observed in a relatively standard cloverleaf structure with a TΨC loop, though the latter two (tRNA-Ile and tRNA-Met) lacked DHU-stem. The tRNA-Ser1 and tRNA-Ser2 were similar to previous reports with one TΨC-loop but lacked D-loop (Su et al. 2020), while tRNA-Lys had a TΨC-arm with a short of TΨC-loop.

Ribosomal RNAs of Contracaecum sp. were fixed as GA3 pattern. The rrnL was located between tRNA-His and nad3 with a size of 959 bp, the rrnS gene was located between tRNA-Glu and tRNA-Ser₂ with a size of 711 bp (Table 1). The content of A + T for rrnL and rrnS were 75.6% and 70.6%, respectively. There were two NCRs among the mt genome of Contracaecum sp.. One short region was placed in nad4 and cox1 with a length of 122 bp, and the long region was situated between tRNA-Ser₂ and tRNA-Asn with a length of 691 bp.

Nucleotide variation of genus Contracaecum

Based on aligned nucleotide sequences among species C. osculatum, C. rudolphii, C. ogmorhini, and Contracaecum sp., nucleotide diversities (Pi) were calculated based on the sliding window. The values of Pi were ranged from 0.124 to 0.181 by analyzing window 300 bp and default step 25 bp (Fig. 3). The most variable genes were cytb (0.178), nad2 (0.181), nad4 (0.179) and nad6 (0.172), and the most conserved genes were cox1 (0.124) and cox2 (0.130) in Contracaecum (Fig. 3). Protein genes cox1 and cox2 seemed to be the most stable genes in Contracaecum nematodes with the least variation, which could be used as molecular markers to identify species from Contracaecum. Results also supported that nad2 and nad4 could act as alternative markers among nematodes isolated from different environments.

Phylogenetic analyses

The present phylogenetic tree is based on the 12 PCGs of 41 available mt sequences from the superfamily Ascaridoidea and Heterakoidea (Table S2). Two phylogenetic trees, both BI and ML, had similar topologies, excluding species within the superfamily Heterakoidea. The topologies of ML and BI phylogenetic trees were highly similar to previous studies (Liu et al. 2016; Zhang et al. 2021; Zhao et al. 2021). The present sample formed a branch with Contracaecum nematodes, indicating a closer relationship within the genus Contracaecum with strong support (Fig. 4), but a distinct distance from species that had been reported. According to the structure of phylogenetic trees, results supported previous reports that superfamily Ascaridoidea and Heterakoidea were monophyly, and families (including Ascarididae, Anisakidae, Heterocheiidae, Toxocaridae, and Cucullanidae) within it were all monophyletic (Li et al. 2018; Zhao et al. 2021).

For ML and BI analyses, two trees had identical topologies within the superfamily Ascaridoidea. Among the family Ascarididae, the genera Ascaris, Baylisascaris, Toxascaris, and Parascaris had a closer relationship than Ophidascaris similarly to Zhou et al. (2021) reported. According to morphological descriptions of the genus Ophidascaris, Ophidascaris had been classified as a member genus of the superfamily Ascaridoidea (Pinto et al. 2010), and based on phylogenetic analyses, Ophidascaris more related to the family Ascaridae. While compared with other genera in Ascaridae, there was a relatively long evolutionary distance in Ascaridae. A previous study presented that the family Ascarididae was more related to Toxocaridae (Zhou et al. 2021). In this study, the family Ascarididae was closer taxa to Anisakidae than Toxocaridae (Fig. 4). In addition, all five families and all eleven genera within the superfamily Ascaridoidea were monophyletic with strong support (Bpp = 1, Bf > 70, Fig. 4), verifying the correctness of former studies (Liu et al. 2016; Zhao et al. 2018).

In ML analysis, present findings showed strong statistical support (Bf = 100) that Ascaridia galli and Heterakis species were sister taxa, similar to former studies report (Liu et al. 2016). Similar to Liu et al. (2013b) reported Ascaridia columbae was more related to Ascaridia sp. than A. galli. However, especially in the BI analyzing tree, species A. galli formed a separate genus which created sister relationships with genera Heterakis and Ascaridia with strong support (Bpp = 1). The results proposed the hypothesis that Heterakidae had a closer relationship with Ascaridiidae, and the family Ascaridiidae was paraphyly within Heterakoidea. However, due to limited molecular data among Heterakoidea, it is better to catch more mt sequences to verify the relationship within this superfamily.

Based on analyses of molecular statistics for Contracaecum species, the present study showed detailed mitochondrial characteristics of the complete mitogenome of Contracaecum sp., and nucleotide diversity among the published Contracaecum species. Additionally, based on the nucleotide diversity, results emphasized that conservative cox1 and cox2 seem to be better markers in species identifications. In accordance with previous reports, Contracaecum sp. isolated from Beijing might be one new species of the genus Contracaecum based on the phylogenetic analysis, further verifying the hypotheses Zhang et al. (2021) supported before (Zhang et al. 2021). Additionally, consistent with Liu et al. (2016) supported that the families Heterakidae and Ascaridiidae were related, all genera and families cited in the present study were monophyly, excluding genus Ascaridia and family Ascaridiidae.

Author’s contributions

G-HL and Y-PD conceived and designed the study, and critically revised the manuscript. Y-T provided the sample worms and provided initial identification. Y-PD performed the experiments and analyzed the data. G-HL and Y-PD drafted the manuscript. R-L and H-MW helped in study design, study implementation, and manuscript preparation. All authors read and approved the final manuscript.

Funding

This study was supported by the Training Programme for Excellent Young Innovators of Changsha (Grant No. KQ2106044).

Declaration of Competing Interest

The authors declare that there are no competing interests.

Availability of data and material

The datasets generated and analyzed during the current study are available in GenBank.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

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Characterization of the Complete Mitochondrial Genome Sequence of Contracaecum sp. (Nematoda: Ascarididae) from China

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