Mitochondrial genome characterization of the stomach nematode Mastophorus muris (Nematoda: Spiruroidea) and phylogenetic relationships with nematodes

Abstract

Mastophorus muris is a prevalent nematode parasite of rodents that can cause pathogenic manifestations or mortality. As the only 1 species in the genus Mastophorus and the sole member of the family Spirocercidae, its biology, molecular identification, and population genetic structure remain poorly studied. In this study, the complete mitochondrial genome of M. muris was fully assembled and annotated through high-fidelity next-generation sequencing for the first time, to resolve its molecular architecture with nucleotide-level precision. The 13,668 bp mitogenome encodes 36 genes, including 12 protein-coding genes (PCGs), 22 transfer RNAs, and 2 ribosomal RNAs (s-rRNA and l-rRNA). The 10,384 bp PCGs account for 76.0% of the mitogenome, with AT (71.7%) and GC (28.3%) contents, AT skew (-0.494) and GC skew (0.477), indicating pronounced nucleotide bias. Analysis of 12 PCGs revealed that TTG, ATT, ATA, or GTG were the common start codons. TAA was the predominant termination codon, while some PCGs (cob, nad1, and nad3) were deduced to end with an incomplete codon T. T-rich codons such as TTT-Phe (16.3%), TTG-Leu (8.5%), GTT-Val (6.7%), ATT-Ile (6.3%), TAT-Tyr (5.6%), TTA-Leu (4.6%), and TCT-Ser (4.3%) were used more frequently. Phylogenetic analysis using the concatenated nucleotide sequences of 12 PCGs, and the NJ tree analysis results showed that M. muris was more closely related to the genus Gongylonema, which indicated that the family Spirocercidae was more closely associated with Gongylonematidae. This study provides valuable mitogenomic data for nematode phylogeny, diagnostics, population genetics, and comparative mitogenomics.

Introduction

The stomach nematode Mastophorus muris (Gmelin, 1790) (Nematoda: Spiruroidea) is a cosmopolitan and important parasitic pathogen of wild and urban rodents (voles and rats), and even other less frequent hosts (marsupials and carnivores) [1]. The genus M. muris (Gmelin, 1790) was identified as nematode based on morphological similarities. The M. muris body is elongated, medium in size, tail is coiled with a lateral lip on the head, with a keratinized mouth capsule and unequal copulatory spicule [2]. While the life-cycles of all species in the family Spirocercidae involve 2 hosts, namely definitive and intermediate hosts. The life cycle of Mastophorus spp. is indirect, with various insects (e.g., cockroaches, flour beetles, locusts, dipterans, and earwigs) acting as intermediate hosts. The infective larvae after being ingested by definitive hosts (e.g., rodents, rats, and voles), where it will develop into gravid adults in the stomach in 28 days [3]. The breeding adult nematodes will shed their eggs inside the definitive hosts’ stomach, and these will be released into the open environment along with the definitive hosts’s faeces. The intermediate hosts (insects) will ingest the eggs, and then the larvae are developed, the definitive hosts will consume the intermediate hosts, thus continuing the life cycle of the Mastophorus spp. nematode. In the current classification, the family Spirocercidae includes 6 genera, such as Ascarops (1 species), Cylicospirura (3 species), Mastophorus (1 species and 7 unclassified species), Physocephalus (2 species and 1 unclassified species), Spirobakerus (1 species), Streptopharagus (1 species and 1 unclassified species). The number of genera is so large and with a rich variety of species, but there are few studies investigating the phylogeny of the Spirocercidae including Mastophorus.

Molecular biology tools have been used to study the phylogenetic relationships among groups of Spiruroidea nematodes [4]. However, the limited availability of sequence data (amounts and length of sequences) has greatly hampered studies of the phylogenetic relationships among families in the order Spirocercidae. Compared to some genes (18S rRNA, 12S rRNA, and cox1 gene), the complete mitochondrial DNA sequences are more appropriate and informative at the generic level [5]. Complete mitochondrial DNA sequences can provide information not only about individual and combined mitochondrial genes, but also about the order of the genes [6]. Mitochondrial gene rearrangements are generally rare events that occur over long evolutionary periods, although mitochondrial sequences evolve rapidly in metazoans [7]. Relative gene rearrangements found in a group of species (insects and crustaceans) are considered evidence of a closer relationship among them [8]. In fact, the use of gene rearrangement comparisons as a phylogenetic tool has been well discussed [9], and has been used to analyse several evolutionary relationships in insects (Aleurodicus rugioperculatus [10] and Pachyneuron aphidis [11]), primate species [12], birds [13]. However, only 1 complete nematode mitochondrial genome sequences (G. pulchrum NC_026687.1) are available for species of the order Spiruroidea in the National Center for Biotechnology Information (NCBI) database and no complete mitochondrial genome was available in the family Spirocercidae until now.

Overall, the present study aimed to characterize the complete mitochondrial genome of M. muris, the first representative of the family Spirocercidae; to compare its mitochondrial gene content, arrangement with those of other nematodes; and to infer the phylogenetic position of M. muris in relation to other nematodes based on the concatenated mitochondrial amino acid sequences.

Methods

Ethics statement

In the present study, no prior permission was required for the collection as the species (parasite host) is neither endangered nor protected in IUCN Red List or Law of the People’s Republic of China on the Protection of Wildlife. Furthermore, we followed all the applicable international, national, and/or institutional guidelines for the care and use of animals during experiment.

Sample collection, and DNA extraction

The individual parasitic nematode specimen of M. muris was collected in Qinghai vole (Neodon fuscus) from Maqin County, Qinghai Province, located in the Three Rivers Source Region of the Qinghai-Tibetan Plateau, China in August 2022. The morphological traits of the parasite specimen were promptly recognized by the Academy of Animal and Veterinary Sciences, Qinghai University, and then they were immediately preserved in 100% ethanol during collection and stored at -80 ℃ until DNA extraction. Total genomic DNA was extracted from the parasite body using the TlANamp Genomic DNA Kit (Tiangen Biotech) according to the manufacturer’s instructions. The quality of extracted DNA was detected by electrophoresis and ultraviolet spectrophotometer—specifically the NanoDrop 2000 (Thermo Fisher Scientific), and the genomic DNA was stored at -20℃ until PCR amplification.

PCR amplification identification and sequencing

In order to identify the suspected parasitic nematode, a fragment from the cytochrome c oxidase subunit 1 (cox1) was amplified with primers cox1F (5'-CTCGTGTTAATGCTTTGTCTTTTT-3') and cox1R (5'-AGCAGTACTCAAAATAATACCTCTT-3'), and another from the 18S rRNA gene was amplified with primers 18S F1 (5'-TGGATAACTGTGGTAATTCTAGAGC-3') and 18S R1 (5'-TTAATGATCCTTCCGCAGGT-3'). The PCR was performed on Nexus GX2 Thermal Cycler (Eppendorf) in 50.0 μl reaction volumes containing 2.0 μl of template DNA, 2.0 μl of each primer (10 μmol/L), 25.0 μl TaKaRa PrimeSTAR Max Premix (2×), and 19.0 μl sterilized distilled water. Condition for PCR amplification was as follows: an initial denaturation for 5 min at 98°C followed by 35 cycles of denaturation for 20 sec at 98°C, annealing for 30 sec at 54°C and extending for 2 min at 72°C, finally reactions were held at 72°C for 10 min. The PCR products were assessed through electrophoresis in 1.5% agarose gel, stained with GelStain, and photographed under ultraviolet light, and then the PCR products were sequenced and aligned using BLAST in NCBI database to identify the parasite species.

Mitochondrial genome sequencing and assembly

After precise identification of the parasite species, the mitogenomes of M. muris were sequenced by next-generation sequencing on the illumina HiseqXten/Novaseq/MGI2000 System (Genewiz Biotechnology). Raw sequences data were performed with quality control and then the clean data were de novo assembled by the NOVOPlasty software [14]. The MITOS2 gene-finding software had been used for finding coding genes [15]. tRNAs were detected in the genome using the program tRNAscan-SE with default parameter settings [16]. Furthermore, rRNA and tRNAs were identified by using RNAmmer [17]. We also compared the assembled genome with the previous 2 confirmed sequences by PCR and Sanger sequencing methods to evaluate the single-base accuracy of the assembled mitogenome.

Sequence analysis and gene annotation

The genes sequences were annotated with NCBI nr database by Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and by comparison with G. pulchrum (NC_026687.1) and Spirocerca lupi (NC_021135.1) to ensure that the sequences were correct. The locations of the protein-coding genes (PCGs) were identified using ORF Finder via NCBI and MITOS WebServer (http://mitos2.bioinf.uni-leipzig.de/index.py) comparing with the selection of invertebrate mitochondrial genetic code by the default settings. The ClustalX program was used to observe and identify the start/stop codon of PCGs, especially, the abnormal start/stop codons were identified and manually corrected once again on the basis of comparison with other nematode species [18]. The MEGA X was used to align the PCGs with other nematode species, and then the relative synonymous codon usage (RSCU) was also obtained, noteworthily, the incomplete codons were removed when the RSCU was calculated using PCGs [19]. In addition, the boundaries of rRNA genes were determined based on the locations of adjacent tRNA genes and by alignment with homologous genes of other related nematodes using DNAMAN version 8.0 (Lynnon Biosoft). The composition skew values were calculated according to the following formulae: AT skew=(A-T)/(A+T) and GC skew=(G-C)/(G+C) [20], where A, T, G, and C denoted the percentages of the 4 bases [21]. Functional annotations were performed using protein sequence similarity BLAST searches with the typical cut-off E-value of 10^-5 against several publicly available protein databases: the NCBI non-redundant protein database, Swiss-Prot, Clusters of Orthologous Groups, and Gene Ontology terms [22]. The secondary cloverleaf structures of tRNAs were predicted using the MITOS WebServer (http://mitos2.bioinf.uni-leipzig.de/index.py). The tandem repeats in A+T-rich regions were predicted using the Tandem Repeats Finder program(http://tandem.bu.edu/trf/trf.basic.submit.html). The graphical map of the complete mitochondrial genome was drawn using the online mitochondrial visualization tool Organellar Genome DRAW [23]. The overlapping and intergenic spacer regions were examined manually. The assembled mitogenome with gene features was submitted to GenBank: Bankit (NCBI) to get the accession number (https://www.ncbi.nlm.nih.gov/WebSub/).

Phylogenetic and gene arrangement analysis

To reconstruct phylogenetic trees for the estimation of M. muris taxonomic status, 32 available nematode species mitogenomes were retrieved and downloaded from the GenBank, and used in the present study along with newly generated M. muris sequence. In addition, the mitogenome of Schistosoma mansoni (NC_002545.1, Trematoda) were used as the outgroup and included in each analysis. Phylogenetic analyses were performed using the concatenated nucleotide sequences of all the 12 PCGs and 2 rRNAs. Subsequently, the nucleotide sequences were concatenated into a single alignment and converted into phylip and nexus format files for phylogenetic analyses, which were performed using Neighbor-Joining tree method. Comparative analyses were carried out using the MEGA software (version 5.0), by comparing the distribution of the arrangement of individual genes in this order, in particular, the arrangement of tRNA genes among the PCGs.

Results

Morphological characteristics observation

The parasitic nematode was collected from the stomach of Qinghai voles and examined under a stereomicroscope. The worms were elongated and medium sized, reaching up to 80.0 mm in length. The tail was coiled, the head bore lateral lips, and a keratinized buccal capsule and unequal copulatory spicules were present. Females were considerably larger than males, lacked a copulatory bursa, and possessed prominent caudal alae. The right spicule was long and blunt, while the left was shorter and pointed, consistent with previous morphological descriptions [1].

Molecular identification

Genomic DNA was extracted from the nematode and subjected to PCR amplification targeting the cox1 and 18S rRNA genes. Two distinct bands of approximately 797 bp and 1,664 bp were obtained, matching the expected amplicon sizes. Following sequencing, BLAST searches against GenBank revealed high sequence identities with M. muris: 98.3% for cox1 (MG821081.2) and 99.8% for 18S rRNA (MG818763.2). These results confirmed that the nematode isolate was M. muris.

Structure of the complete mitochondrial genome

The complete mitochondrial genome of M. muris was a circular double-stranded DNA molecule13,668 bp in length (GenBank accession No. NC_085209.1). It encoded 36 genes: 12 PCGs (cox1–3, nad1–6, cob, nad4L, and atp6), 2 ribosomal RNA genes (s-rRNA and l-rRNA), 22 transfer RNA genes (tRNAs), and non coding control regions (Fig. 1; Table 1). Similar to other nematode and flatworm mitogenomes, the atp8 gene was absent [20]. All genes were transcribed unidirectionally from the plus strand. The nucleotide composition was 21.1% A, 52.0% T, 7.1% C, and 19.8% G, with an overall AT content of 73.1% and GC content of 26.9%. The mitogenome exhibited negative AT skew (-0.422) and positive GC skew (0.470).

Fig. 1.

The mitochondrial genome of Mastophorus muris. Genome organization of the complete mitochondrial genome of M. muris is a circular DNA molecule containing 36 genes. All genes are coded by the same DNA strand and are transcribed in the same direction. Protein-coding and rRNA genes are shown with the standard nomenclature. Gene scaling is approximate.

Table 1.

Summary data for the annotated mitochondrial genome of Mastophorus muris

Gene	Position/length	Initiation/termination codon	Anticodon	No. of amino acids	No. of intergenic nucleotides
cox1	1-1656/1656	ATG/TAA	-	551	+1
trnW (tRNA-Trp)	1662-1719/58	-	TCA	-	+5
Non-coding region (NC1)	1720-1757/38	-	-	-	0
nad6	1758-2219/462	ATT/TAA	-	142	0
trnR (tRNA-Arg)	2218-2272/55	-	ACG	-	-1
trnQ (tRNA-Trp)	2274-2327/54	-	TTG	-	+1
cob	2327-3414/1088	TTG/TAA	-	362	-1
trnL1 (tRNA-Leu)	3414-3468/55	-	TAG	-	-1
cox3	3468-4247/780	ATG/TAA	-	259	-1
Non-coding region (NC2)	4248-4562/315	-	-	-	0
trnA (tRNA-Ala)	4563-4614/52	-	TGC	-	0
trnL2 (tRNA-Leu)	4618-4671/54	-	TAA	-	+3
trnN (tRNA-Asn)	4673-4729/57	-	GTT	-	+1
trnM (tRNA-Met)	4730-4785/56	-	CAT	-	0
trnK (tRNA-Lys)	4785-4841/57	-	CTT	-	-1
nad4l	4849-5091/243	TTG/TAA	-	80	+7
s-rRNA	5092-5752/661	-	-	-	0
trnY (tRNA-Tyr)	5759-5813/55	-	GTA	-	+6
nad1	5811-6687/877	TTG/TAA	-	291	-3
trnF (tRNA-Phe)	6688-6742/55	-	GAA	-	0
atp6	6750-7328/579	ATC/TAG	-	192	+7
trnI (tRNA-Ile)	7329-7387/59	-	GAT	-	0
trnG (tRNA-Gly)	7390-7446/57	-	TCC	-	+2
cox2	7447-8145/699	ATA/TAA	-	232	0
trnH (tRNA-His)	8146-8200/55	-	GTG	-	0
l-rRNA	8201-9147/947	-	-	-	0
nad3	9148-9496/349	ATG/T	-	115	0
trnC (tRNA-Cys)	9497-9552/56	-	GCA	-	-2
trnS2 (tRNA-Ser)	9553-9604/52	-	TGA	-	0
trnP (tRNA-Pro)	9604-9672/69	-	AGG	-	-1
Non-coding region (NC3)	9673-9722/50	-	-	-	0
trnD (tRNA-Asp)	9723-9779/57	-	GTC	-	0
trnV (tRNA-Val)	9781-9834/54	-	TAC	-	+1
nad5	9839-11428/1590	TTG/TAG	-	529	+4
trnE (tRNA-Glu)	11428-11485/58	-	TTC	-	-1
trnS1 (tRNA-Ser)	11485-11537/53	-	TCT	-	-1
nad2	11546-12382/837	GTG/TAG	-	278	+8
trnT (tRNA-Thr)	12384-12438/55	-	TGT	-	+1
nad4	12444-13667/1224	ATG/TAA	-	402	+5

Protein coding genes, initiation and termination codons

The 12 PCGs spanned 10,384 bp, accounting for 76.0% of the total mitogenome (Table 2). The AT and GC contents were 71.7% and 28.3%, respectively (Table 3). The AT skew was -0.494 and the GC skew was 0.477, indicating strong nucleotide compositional bias (Table 4).

Table 2.

Properties of protein-coding genes, length of the mt genomes and rRNA genes and AT content of nematode mt genomes

Gene	Species
	B. m.	W. b.	M. p.	C. m.	C. l.	S. d.	G. p.	H. l.	S. l.	M. m.
No. of aa
cox1	548	548	550	548	517	548	550	530	551	551
nad6	158	150	142	145	157	149	143	145	152	153
cob	362	362	362	360	365	360	354	362	351	362
cox3	259	259	259	258	253	259	257	258	259	259
nad4l	71	80	77	88	77	80	75	77	75	80
nad1	292	292	292	290	286	299	290	289	292	291
atp6	193	193	194	191	222	193	188	192	194	192
cox2	232	233	233	231	226	232	230	231	229	232
nad3	94	112	81	112	111	113	113	108	111	107
nad5	512	531	516	531	526	531	527	529	530	529
nad2	273	281	278	275	272	284	276	282	280	278
nad4	409	409	413	405	407	409	402	408	401	407
Length of rRNA gene (bp)
s-rRNA	672	672	665	571	709	672	681	682	688	661
l-rRNA	1034	972	1016	765	937	971	965	973	987	947
Deduced initiation codon
cox1	ATT	ATT	ATG	AAT	TTG	ATT	ATG	ATT	ATG	ATG
nad6	TTG	---	GTG	ATT	ATT	TTG	ATT	ATA	TTG	ATT
cob	ATT	ATT	ATG	GTG	ATT	ATT	ATT	ATT	ATG	TTG
cox3	ATT	ATT	ATT	TTG	ATT	ATA	TTG	ATT	ATT	ATG
nad4l	TTG	GTA	TTG	TTG	TTG	TTG	TTG	ATA	ATG	TTG
nad1	TTG	TTG	TTG	ATT	TTG	TTG	TTG	TTG	TTG	TTG
atp6	ATT	ATA	ATT	TTG	TTG	TTT	TTG	ATT	ATT	ATC
cox2	ATT	ATT	ATT	TTG	TTG	ATT	ATG	ATT	ATG	ATA
nad3	ATA	CTT	ATG	ATT	TTG	TTT	GTG	ATA	TTG	TTG
nad5	TTG	TTT	ATA	TTT	TTG	TTT	TTG	ATT	TTG	TTG
nad2	ATG	TTA	TTG	ATT	GTG	TTT	ATT	TTG	TTG	GTG
nad4	TTG	TTG	TTG	ATT	ATG	ATG	ATT	ATA	TTG	TTG
Deduced termination codon
cox1	TAG	TAA	TAA	TAA	TAG	TAG	TAA	TAA	TAA	TAA
nad6	TAA	TAA	TAG	TAA	TAA	TAA	TAA	TAA	TAA	TAA
cob	GTT	TAA	TAA	TAA	TAA	TAA	TAA	TAA	TAA	T
cox3	TAA	TAA	TAA	TAA	TAA	TAA	TAA	TAA	TAA	TAA
nad4l	TAA	TAA	TAG	TAG	TAA	TAA	TAA	TAA	TAG	TAA
nad1	TAA	TAA	TAA	TAA	TAG	TAA	TAA	TAA	TAA	T
atp6	TAG	TAG	TAA	TAA	TAA	TAG	TAA	TAG	TAA	TAG
cox2	TAA	TAA	TAG	TAA	TAA	TAG	TAA	TAA	TAA	TAA
nad3	TAG	TAA	TAA	TAA	TAA	TAA	TAA	TAG	TAA	T
nad5	TAG	TAG	TAA	TAA	TAA	TAG	TAA	TAG	TAA	TAG
nad2	TTT	TAA	TAA	TAA	TAA	TAG	TAG	TAA	TAG	TAG
nad4	TAA	TAA	TAG	TAA	TAG	TAA	TAA	TAA	TAG	TAA
Length of mt genome (bp)	13658	13635	13619	13604	18935	13839	13798	13610	13804	13668
AT content of complete mt genome (%)	75.5	74.6	74.1	75.0	75.9	75.1	76.0	79.1	74.4	73.1

Results obtained from GenBank accessions.

B. m., Brugia malayi; W. b., Wuchereria bancrofti; M. p., Mansonella perstans; C. m., Crassicauda magna; C. l., Camallanus lacustris; S. d., Setaria digitata; G. p., Gongylonema pulchrum; H. l., Heliconema longissimum; S. l., Spirocerca lupi; M. m., Mastophorus muris.

Table 3.

Comparison of A+T content (%) of gene and region of the mt genomes of Spiruromorpha nematode species, including Mastophorus muris

Gene	Species
	B. m.	W. b.	M. p.	C. m.	C. l.	S. d.	G. p.	H. l.	S. l.	M. m.
cox1	67.9 (1119)	67.7 (1115)	67.2 (1110)	67.2 (1106)	65.3 (1014)	69.1 (1138)	68.6 (1134)	71.7 (1142)	67.4 (1116)	67.6 (1120)
nad6	81.6 (389)	80.0 (361)	77.4 (332)	82.7 (362)	73.4 (348)	82.4 (371)	81.0 (350)	81.7 (358)	77.3 (355)	77.3 (357)
cob	74.2 (806)	72.7 (791)	73.2 (796)	72.6 (785)	70.2 (769)	72.3 (782)	74.7 (796)	79.3 (862)	72.8 (767)	72.6 (790)
cox3	72.8 (568)	74.3 (579)	74.4 (580)	73.1 (568)	69.6 (529)	72.6 (566)	73.0 (565)	75.9 (590)	70.6 (551)	71.3 (556)
nad4l	81.0 (175)	80.7 (196)	75.6 (177)	76.4 (204)	75.6 (177)	76.8 (185)	77.6 (177)	82.1 (192)	78.5 (179)	76.1 (185)
nad1	73.7 (646)	72.5 (636)	73.2 (642)	72.9 (636)	68.6 (591)	72.8 (655)	73.0 (636)	75.7 (657)	75.0 (659)	70.2 (616)
atp6	75.3 (438)	76.6 (446)	73.7 (431)	75.6 (434)	70.6 (471)	74.2 (432)	78.6 (444)	77.9 (451)	75.2 (440)	72.9 (422)
cox2	69.2 (484)	70.6 (494)	68.2 (479)	68.8 (479)	65.8 (447)	69.4 (485)	71.6 (495)	74.7 (520)	69.0 (475)	67.2 (470)
nad3	77.9 (222)	76.9 (259)	75.4 (184)	76.9 (259)	72.2 (241)	77.1 (262)	82.9 (282)	83.2 (272)	81.0 (272)	77.4 (270)
nad5	74.1 (1141)	74.7 (1192)	73.1 (1134)	74.8 (1193)	73.2 (1157)	74.8 (1194)	75.0 (1186)	78.9 (1255)	73.1 (1163)	71.6 (1138)
nad2	77.1 (632)	75.7 (639)	75.7 (632)	79.5 (658)	73.3 (599)	76.5 (654)	77.9 (647)	82.9 (704)	74.9 (631)	74.6 (624)
nad4	76.7 (943)	73.9 (908)	75.9 (942)	76.9 (936)	71.9 (880)	76.9 (946)	76.2 (933)	80.4 (986)	74.5 (898)	73.6 (901)
PCGs	73.9 (7563)	73.4 (7616)	72.8 (7439)	73.8 (7620)	70.3 (7223)	73.7 (7670)	74.5 (7645)	77.8 (7989)	72.8 (7506)	71.7 (7449)
Complete mt genome	75.5 (10314)	74.6 (10170)	74.1 (10090)	75.0 (10198)	75.9 (14371)	75.1 (10398)	76.0 (10479)	79.1 (10767)	74.4 (10268)	73.1 (9991)

Values are presented as A+T content (%) and length (bp). Results obtained from GenBank accessions.

B. m., Brugia malayi (MT149211.1); W. b., Wuchereria bancrofti (JN367461.1); M. p., Mansonella perstans (MT361687.1); C. m., Crassicauda magna (OQ834322.1); C. l., Camallanus lacustris (NC_070137.1); S. d., Setaria digitata (NC_014282.1); G. p., Gongylonema pulchrum (NC_026687.1); H. l., Heliconema longissimum; S. l., Spirocerca lupi (MK922357.1); M. m., Mastophorus muris (NC_085209.1); PCGs, protein-coding genes.

Table 4.

Nucleotide content and skewness (AT-skew/GC-skew) of Spiruromorpha mitochondrial PCGs and rRNAs, including Mastophorus muris

Gene	Species (AT-skew/GC-skew)
	B. m.	W. b.	M. p.	C. m.	C. l.	S. d.	G. p.	H. l.	S. l.	M. m.
cox1	-0.382/0.258	-0.433/0.301	-0.387/0.289	-0.356/0/295	-0.361/0.315	-0.422/0.253	-0.365/0.260	-0.312/0.251	-0.403/0.341	-0.382/0.295
nad6	-0.532/0.636	-0.551/0.644	-0.506/0.485	-0.580/0.684	-0.575/0.603	-0.553/0.671	-0.554/0.634	-0.469/0.375	-0.572/0.635	-0.557/0.733
cob	-0.395/0.288	-0.482/0.360	-0.445/0.333	-0.406/0.399	-0.506/0.541	-0.563/0.465	-0.442/0.390	-0.364/0.289	-0.395/0.373	-0.443/0.369
cox3	-0.370/0.368	-0.396/0.480	-0.393/0.390	-0.384/0.464	-0.478/0.463	-0.449/0.533	-0.345/0.407	-0.319/0.444	-0.401/0.467	-0.370/0.384
nad4l	-0.577/0.610	-0.602/0.574	-0.638/0.649	-0.539/0.587	-0.492/0.684	-0.654/0.786	-0.627/0.804	-0.490/0.619	-0.620/0.714	-0.622/0.793
nad1	-0.529/0.299	-0.541/0.427	-0.551/0.447	-0.575/0.494	-0.479/0.407	-0.612/0.494	-0.557/0.481	-0.440/0.327	-0.530/0.427	-0.604/0.548
atp6	-0.498/0.389	-0.498/0.426	-0.503/0.545	-0.479/0.429	-0.376/0.388	-0.620/0.507	-0.455/0.438	-0.459/0.281	-0.559/0.503	-0.507/0.490
cox2	-0.360/0.330	-0.356/0.369	-0.332/0.462	-0.420/0.401	-0.351/0.302	-0.427/0.458	-0.293/0.347	-0.308/0.352	-0.309/0.362	-0.340/0.406
nad3	-0.541/0.714	-0.598/0.590	-0.598/0.667	-0.552/0.462	-0.560/0.742	-0.565/0.641	-0.418/0.586	-0.456/0.455	-0.390/0.656	-0.437/0.544
nad5	-0.558/0.422	-0.609/0.500	-0.556/0.482	-0.475/0.451	-0.526/0.590	-0.620/0.478	-0.530/0.525	-0.390/0.373	-0.560/0.523	-0.576/0.562
nad2	-0.538/0.574	-0.590/0.600	-0.541/0.606	-0.429/0.365	-0.556/0.550	-0.590/0.542	-0.549/0.663	-0.403/0.269	-0.572/0.623	-0.577/0.671
nad4	-0.521/0.415	-0.597/0.495	-0.546/0.460	-0.513/0.447	-0.593/0.488	-0.575/0.465	-0.516/0.505	-0.446/0.378	-0.548/0.526	-0.558/0.529
PCGs	-0.474/0.378	-0.519/0.441	-0.486/0.435	-0.459/0.418	-0.486/0.468	-0.547/0.460	-0.464/0.445	-0.390/0.334	-0.485/0.466	-0.494/0.477
complete mt genome	-0.429/0.375	-0.460/0.433	-0.433/0.431	-0.399/0.420	-0.322/0.438	-0.483/0.459	-0.413/0.448	-0.337/0.354	-0.419/0.471	-0.422/0.470

Results obtained from GenBank accessions.

B. m., Brugia malayi (MT149211.1); W. b., Wuchereria bancrofti (JN367461.1); M. p., Mansonella perstans (MT361687.1); C. m., Crassicauda magna (OQ834322.1); C. l., Camallanus lacustris (NC_070137.1); S. d., Setaria digitata (NC_014282.1); G. p., Gongylonema pulchrum (NC_026687.1); H. l., Heliconema longissimum; S. l., Spirocerca lupi (MK922357.1); M. m., Mastophorus muris (NC_085209.1); PCGs, protein-coding genes.

Six PCGs (cob, nad4L, nad1, nad3, nad4, and nad5) used TTG as the initiation codon, 2 (cox1 and cox3) used ATG, 1 (nad6) used ATT, 1 (atp6) used ATC, 1 (cox2) used ATA, and 1 (nad2) used GTG. Nine PCGs terminated with complete stop codons (TAA or TAG), whereas cob, nad1, and nad3 ended with the incomplete stop codon T. The lengths of the deduced proteins were comparable to those of other nematodes (Table 2).

Several overlaps were detected: 1-bp overlaps between trnM-trnK, trnS2-trnP, trnE-trnS1, cox3-trnL1, and nad5-trnE; 2-bp overlaps between nad6-trnR and nad3-trnC; and a 3-bp overlap between trnY-nad1.

Ribosomal and transfer RNA genes

The large ribosomal RNA (l-rRNA) was located between trnH and nad3, and the s-rRNA gene between nad4L and trnY (Table 1). Their lengths were 947 bp and 641 bp, respectively. A total of 22 tRNA genes were identified, ranging from 52 to 69 bp, with a cumulative length of 1,230 bp (excluding overlaps), representing ~9.0% of the mitogenome. The AT content was 76.9%, with negative AT skew (-0.184) and positive GC skew (0.488). Gene order was identical to that of S. lupi and Setaria digitata. Twenty tRNAs displayed the typical TV replacement loop structure, while trnS1 and trnS2 lacked the DHU loop but retained the TΨC loop.

Relative synonymous codon usage

RSCU was analyzed for the 12 PCGs (Table 5; Supplementary Table S1). Among the 64 possible codons, 62 were used; CAC (His) and CGC (Arg) were absent. T-rich codons, including TTT (Phe), TTG (Leu), GTT (Val), ATT (Ile), TAT (Tyr), TTA (Leu), and TCT (Ser), were strongly preferred. Codons with high C content were markedly underrepresented. This pattern reflected a strong codon usage bias toward T in the M. muris mitogenome.

Table 5.

Nucleotide codon usage for 12 protein-coding genes of the mitochondrial genome of Mastophorus muris

AmAcid	Codon	Number	%	AmAcid	Codon	Number	%	AmAcid	Codon	Number	%
Ala	GCG	9	0.3	Lys	AAG	70	2.0	Ser	AGG	22	0.6
Ala	GCA	5	0.1	Leu	TTG	295	8.5	Ser	AGA	29	0.8
Ala	GCT	78	2.3	Leu	TTA	159	4.6	Ser	AGT	106	3.1
Ala	GCC	2	0.1	Leu	CTG	7	0.2	Ser	AGC	5	0.1
Cys	TGT	85	2.5	Leu	CTA	5	0.1	Ser	TCG	6	0.2
Cys	TGC	4	0.1	Leu	CTT	28	0.8	Ser	TCA	8	0.2
Asp	GAT	72	2.1	Leu	CTC	1	0.03	Ser	TCT	150	4.3
Asp	GAC	1	0.03	Met	ATG	92	2.7	Ser	TCC	1	0.03
Glu	GAG	43	1.2	Asn	AAA	13	0.4	Thr	ACG	4	0.1
Glu	GAA	28	0.8	Asn	AAT	90	2.6	Thr	ACA	2	0.1
Phe	TTT	565	16.3	Asn	AAC	6	0.2	Thr	ACT	94	2.7
Phe	TTC	5	0.1	Pro	CCG	4	0.1	Thr	ACC	1	0.03
Gly	GGG	44	1.3	Pro	CCA	2	0.1	Val	GTG	47	1.4
Gly	GGA	28	0.8	Pro	CCT	67	1.9	Val	GTA	28	0.8
Gly	GGT	167	4.8	Pro	CCC	2	0.1	Val	GTT	233	6.7
Gly	GGC	10	0.3	Gln	CAG	38	1.1	Val	GTC	2	0.1
His	CAT	56	1.6	Gln	CAA	15	0.4	Trp	TGG	42	1.2
His	CAC	0	-	Arg	CGG	9	0.3	Trp	TGA	39	1.1
Ile	ATA	56	1.6	Arg	CGA	5	0.1	Tyr	TAA	6	0.2
Ile	ATT	218	6.3	Arg	CGT	41	1.2	Tyr	TAT	195	5.6
Ile	ATC	8	0.2	Arg	CGC	0	-	Tyr	TAC	4	0.1

Total number of codon is 3,457.

Phylogenetic analysis

Phylogenetic trees were constructed using the neighbor joining method based on concatenated nucleotide sequences of 12 PCGs plus 2 rRNA genes from M. muris and 30 other Spiruromorpha species (Figs. S1-S14). The phylogenetic topology showed that M. muris clustered closely with G. pulchrum (Gongylonematidae, Spiruroidea) with high support (Fig. 2). Members of Spirocercidae and Gongylonematidae formed a monophyletic group within Spiruroidea, which was paraphyletic with respect to Tetrameridae (Habronematoidea) and Thelaziidae (Thelazioidea).

Fig. 2.

Phylogenetic relationships among 30 species of selected Spiruromorpha nematodes inferred from neighbor-joining method of nucleotide sequences of 12 mitochondrial protein-coding genes. Schistosoma mansoni was used as the outgroup. Black dots represented Mastophorus muris.

Single gene phylogenetic analyses revealed that cox1, cox3, cob, nad1, nad4, nad4L, nad5, nad6, l-rRNA, and s-rRNA produced relatively stable topologies. Notably, nad1, nad4, nad4L, and s-rRNA recovered a tree topology identical to that based on the full mitogenome dataset.

Discussion

Although morphological characteristics observed in the present study were consistent with previous descriptions, accurate species level identification of parasitic nematodes based solely on gross morphology remains difficult due to high morphological similarity and phenotypic plasticity among related taxa [1]. Molecular identification provides higher accuracy and resolution than traditional morphological approaches, and thus represents a more reliable tool for species delimitation.

In the present study, the nematode isolated from the stomach of Qinghai voles was molecularly identified as M. muris based on high sequence identities of cox1 and 18S rRNA genes. At the time of writing, only one complete mitochondrial genome was available for species of the superfamily Spiruroidea in the NCBI database (G. pulchrum, NC_026687.1), and no mitochondrial genome had been reported for the family Spirocercidae. Therefore, characterization of the M. muris mitogenome fills an important taxonomic gap and enriches the mitochondrial genomic resources for Spirocercidae.

The M. muris mitogenome exhibits several typical features of nematode mitogenomes, including the absence of the atp8 gene, unidirectional transcription of all genes from the same strand, and high AT content [20]. Incomplete stop codons (T) detected in three PCGs are commonly observed in nematode mitochondrial genomes and are believed to be completed to TAA via post transcriptional polyadenylation [24-29]. Gene overlaps between tRNA genes are common in metazoan mitogenomes [30,31]; however, overlaps between protein coding genes and tRNA genes are relatively rare, but have been reported in some flatworms and other helminths [6,32].

The M. muris mitogenome displays a strong codon usage bias toward T-rich codons and low usage of C-rich codons, a pattern widely observed in spirurid nematodes such as Dirofilaria immitis, S. digitata, Onchocerca volvulus, and Brugia malayi [27,33,34]. This conserved bias likely reflects strong mutational pressure and selection associated with the high AT content typical of nematode mitogenomes.

Most tRNA genes of M. muris possess the typical TV replacement loop, whereas trnS1 and trnS2 lack the DHU arm, a common feature in nematodes [24,35]. The gene order and arrangement are highly similar to those of S. lupi and S. digitata [33], supporting conserved mitochondrial architecture within Spiruromorpha.

Phylogenetic analysis based on concatenated mitochondrial sequences showed that M. muris clusters closely with G. pulchrum with high nodal support. Members of Spirocercidae and Gongylonematidae formed a monophyletic group within Spiruroidea, which was paraphyletic with respect to Tetrameridae (Habronematoidea) and Thelaziidae (Thelazioidea). These results are consistent with recent mitochondrial phylogenomic studies of Spiruromorpha and challenge traditional morphology based classification systems.

Single gene phylogenetic analyses revealed that nad1, nad4, nad4L, and s-rRNA produced topologies highly consistent with the full mitogenome dataset. These genes may serve as suitable molecular markers for species identification and phylogenetic analysis of Spiruroidea, although wider validation is needed with increased taxon sampling, particularly for the genus Mastophorus [27,33,35].

In conclusion, this study presents the first complete mitochondrial genome of M. muris, representing the first mitogenome for the family Spirocercidae. Comparative and phylogenetic analyses improve our understanding of the systematic relationships, genome evolution, and codon usage patterns within Spiruromorpha. This mitogenome provides a valuable resource for developing molecular diagnostic tools, studying population genetics, and exploring host–parasite co evolution and adaptive diversification in spirurid nematodes.

Supplementary information

Supplementary material is available with this article at https://doi.org/10.3347/PHD.25064.