Molecular detection of a novel Ancylostoma sp. by whole mtDNA sequence from pangolin Manis javanica.

BACKGROUND: Ancylostoma species are hematophagous parasites that cause chronic hemorrhage in various animals and humans. Pangolins, also known as scaly anteaters, are mammals that live in soil environments where they are readily exposed to soil-borne parasitic nematodes. However, only a limited number of helminth species have been identified in this animal host so far.
METHODS: Ancylostoma sp. was isolated from a wild pangolin, and the complete mitochondrial (mt) genome of Ancylostoma sp. was obtained by Illumina sequencing of total genomic DNA.
RESULTS: The circular complete mt genome that was assembled had a total length of 13,757 bp and comprised 12 protein-coding genes (PCGs), 22 transfer ribosomal RNAs, two ribosomal RNAs (rRNAs), two non-coding regions and one AT-rich region, but lacked the gene coding for ATPase subunit 8 (atp8). The overall AT content of the mt genome of Ancylostoma sp. was 76%, which is similar to that of other nematodes. The PCGs used two start codons (ATT and TTG) and three stop codons (TAA, TAG, and T). The nucleotide identity of the 12 PCGs ranged from 83.1% to 89.7% and had the highest sequence identity with Ancylostoma caninum among species in the Ancylostomatidae family. Also, the pangolin-derived Ancylostoma sp. lacked repeat sequences in the non-coding regions and in the unique sequence of the short non-coding regions, which differentiated it from other Ancylostoma species. In addition, phylogenetic analyses of 18S rRNA and mtDNA sequences revealed that the Ancylostoma sp. was positioned in a separate branch in the subfamily Ancylostomatinae along with other Ancylostoma species.
CONCLUSIONS: The Ancylostoma sp. isolated from a pangolin in this study was identified as a possible new Ancylostoma species. The identification of this Ancylostoma sp. from pangolin enriches our knowledge of the species in the Ancylostomatidae family and provides information that will lead to a better understanding of the taxonomy, diagnostics, and biology of hookworms.

Background

Hookworms are hematophagous nematodes of mammals, and adult parasites reside in their host’s gastrointestinal tract, causing anemia, stunted growth, tissue damage, inflammation in dogs and cats, and significant neonatal mortality through transmission to unborn pups [1, 2]. Ancylostoma species are members of the family Ancylostomatidae and infect animals and humans by penetrating the skin or being ingested as third-stage larvae, via paratenic hosts, or by transplacental passage [3]. Although some Ancylostoma species have been identified in wild bears, hyenas, red foxes, raccoons and pandas by morphological investigation [4-10], newly emerging Ancylostoma species have not been well identified in other wild animals.

Morphological and morphometric methods have been used to classify nematodes based on the shape of their mouth, tail and sexual organ, the size of the worm body, eggs and larvae [11, 12]. However, these traditional methods for nematode identification have been challenged for a number of reasons. Firstly, some species share similar morphological characteristics; for example, eggs of Necator americanus, Ancylostoma species and Strongylids have similar shapes, and it is not easy to discriminate between closely related species [13]. Traditional identification methods also face some challenges in identifying cryptic species of parasitic nematodes due to their identical morphological features [6]. In addition, nematode collection is also complicated by seasonal fluctuations in the prevalence and intensity of specific species; consequently long-term monitoring is required to collect all nematodes of particular hosts [14]. Another complication is obtaining intact nematodes for morphological identification. Therefore, molecular approaches have been used to discriminate nematodes via nuclear genetic markers and mitochondrial genomes. The mitochondrial (mt) genome has important unique features of maternal inheritance and rapid evolution, but an absence of recombination [15, 16]. Hence, mt genomes provide genetic markers for molecular identification, epidemiological and genetic studies, as well as for phylogenetic and population studies [17-20].

Pangolins, also known as the scaly anteater, are endangered and rare animals that require special protection [21]. These small mammals live in soil environments and can be easily exposed to soil parasitic nematodes. However, only a few helminth parasites have been identified, using egg and adult morphological characteristics, after being isolated from the pangolin gastrointestinal tract; many others are still unknown. A total of 13 parasitic helminths have been reported from pangolins to date. Of these, eight helminth parasites were isolated from the gastrointestinal tract in egg, larvae and adult morphological investigations, including Cylicospirura sp., Leipernema leiperi, Manistrongylus meyeri, Necator americanus, Strongyloides sp., Trichochenia meyeri, Ancylostoma sp. and Gendrespirura sp. [22-27]. Until recently, the identification of Ancylostoma species in pangolin was limited to the genus level. In the family Ancylostomatidae, only N. americanus has been identified in pangolin to the species level [28]. However, there is a paucity of molecular data for identifying Ancylostoma species in pangolins. The aims of this study were to obtain a molecular characterization of a novel Ancylostoma sp. originated from a wild pangolin through the sequencing of total DNA using the Illumina sequencing platform (Illumina, Inc., San Diego, CA, USA).

Methods

Parasite collection

Guangzhou customs confiscated two pangolins from poachers and placed them in the Guangzhou Zoo, Guangdong Province, China. No information on the origin and species of the pangolins was available. One pangolin suffered severe trauma and a purulent infection of the forelimb and ultimately died due to complicated infections. During the post-mortem examination, a total of 15 adult parasites were collected from the duodenum of the naturally infected pangolin. The parasites were washed completely in phosphate- buffered saline, preserved in 70% ethanol and frozen for further identification. Prior to examination under a microscrope, the worms were cleaned with lactophenol and mounted in glycerine. We examined several frozen worms to obtain a complete description of their morphological features under dissecting microscopes (magnifications: 10–40×) and light microscopes (magnifications: 40–100×), but it was difficult to obtain precise morphological features.

DNA extraction and whole-genome amplification

Total genomic DNA was extracted from a single adult worm using the Wizard® SV Genomic DNA Purification System (Promega, Guangzhou, China) according to the manufacturer's instructions and then stored at − 20 °C until use. Complete genomic DNA was amplified using a whole genome amplification kit (REPLI-g® Midi Kit; Qiagen, Hilden, Germany). All procedures were performed according to the manufacturer’s instructions. The amplified DNA was sequenced with an Illumina Novaseq 6000 sequencing platform using a 150-bp paired-end technique (Illumina, Inc.). Approximately 12 Gb of sequence data had a quality score (Q-score) ≥ 20.

PCR amplification and DNA sequencing

The 18S ribosomal ribonucleic acid (rRNA) gene was amplified from the total extracted DNA of the observed worm using DreamTaq DNA Polymerase with the primers NC18S (AAAGATTAAGCCATGCA) and NC5B (GCAGGTTCACCTACAGAT) [29]. The amplification procedure was: 95 °C for 5 min; followed by 35 cycles of 95 °C for 30 s, 54 °C for 30 s, 71 °C for 75 min and 72 °C for 5 min. The amplified fragments were visualized and verified by electrophoresis in a 1.5% agar gel (Sangon Biotech Co., Ltd. Shanghai, China) with staining (0.2 mg/ml ethidium bromide). The PCR fragments were sequenced by Sanger sequencing (Sangon Biotech Co.).

Assembly of the complete mt genome of pangolin and worm

The raw data was mapped to the pangolin genome and then filtered using Samtools (v1. 7) to remove the host gene sequences [30]. The filtered data were assembled into contigs and scaffolds using SPAdes (v3.14.1) [31]. Contigs were aligned into the nucleotide (nt) database using BLAST+ (v2.11.0) [32]. We extracted contigs that contained worm mt genomes with a sequencing depth > 100 and a length > 150 bp. Finally, eight contigs were randomly chosen as a seed sequence, and each seed sequence was assembled using Novoplasty (v.4.2) to reconstruct the complete mt genome of the worm [33]. To determine host identity, the filtered host data were also assembled into contigs and scaffolds using SPAdes, and all the mitochondrial contigs were aligned to the nt database using BLAST+ (v2.11.0). We identified the pangolin mtDNA by comparing it with the known mtDNA of pangolin species available in GenBank.

Gene annotation and sequence analysis

Gene annotation of the assembled mt genome was conducted using MITOS and Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) [34]. The Mitos webserver was employed to predict protein-coding genes (PCGs) and non-coding regions (NCRs) of parasitic nematodes using the genetic code of invertebrate mtDNA (http://mitos.bioinf.uni-leipzig.de) [35]. Initiation and termination codons were identified using the Expasy translation tool (https://web.expasy.org/translate/) [36]. The secondary structures of transfer RNA (tRNA) were predicted and shown by MiTFi and the webserver FoRNA on Mitos [37]. Both rRNA genes (small and large ribosomal subunits [rrnS and rrnL, respectively]) were identified by MiTFi. The codon usage of amino acids for PCGs was determined by the sequence manipulation suite [38]. The complete mt genome was visualized by the MTviz (http://pacosy.informatik.uni-leipzig.de/mtviz/). A comparison of the nucleotide identity (%) of the observed worm mt genome with 13 closely related species of the Ancylostomatidae family was conducted using Clustal Omega [39].

Phylogenetic analysis of 18S rRNA and PCGs of mt genome of worm

We obtained 18S rRNA sequences of 14 nematodes from the NCBI database and used these and the amplified 18S rRNA of the worm to construct a phylogenetic tree (Additional file 1: Table S1). The maximum likelihood (ML) method was performed to evaluate the phylogenetic tree, and the ML tree was made with the TPM3 + G4 model using RAxML-ng (v. 1.0.2) [40]. ML bootstrap > 70% was considered to be strong support [41].

We obtained nucleotide sequences of 12 PCGs from the mt genome of the worm isolated from the pangolin. We also downloaded the complete mt genome sequences of 13 species in the Ancylostomatidae family and 4 species in the Chabertiidae family (outgroup) from NCBI GenBank and aligned these for sequence comparison (Additional file 1: Table S2). A phylogenic tree was reconstructed with RAxML-ng (v. 1.0.2) and a ML method was used with the GTR+G+I model.

Results

Identification of pangolin species

To identify the pangolin species implicated in this case, we obtained the mt genome of the animal, with a total length of 16,574 bp, from Illumina sequencing data. This mtDNA showed the highest sequence identity (99.50%) and coverage (99.0%) with Manis javanica (Malayan pangolin) available from GenBank (accession number: MG196302.1).

Observation on the worm

The worms were isolated from the wild pangolin's duodenum and frozen immediately in 75% ethanol for further identification. The worms were round and tapered at both ends. However, it was challenging to observe precise morphological features due to frozen state of the worms. Therefore, we performed molecular characterization using total genomic DNA by Illumina sequencing.

Primary identification of worm by molecular markers

The amplified 18S rDNA sequence of the worm was 1681 bp and was deposited in GenBank databases under accession number: MZ681936.1. It showed 99.88% sequence identity with the 18S rDNA sequence of A. caninum from GenBank (accession number: AJ920347.2). Phylogenetic analysis of 18S rRNA sequences showed that the amplified 18S rDNA sequence of the worm clustered with Ancylostoma duodenale, A. caninum and N. americanus in the family of Ancylostomatidae (Fig. 1). This worm was relatively closer to Ancylostoma species than N. americanus. Thus, we proposed that this worm might be closely related to Ancylostoma species in the Ancylostomatidae family.

Fig. 1

Phylogenetic tree of 18S rDNA sequences from Ancylostoma sp. and species of the orders Strongylida and Ascaridida. The phylogenetic relationship of this tree is inferred using the maximum likelihood (ML) method and order Ascaridida as outgroup (Toxocara cati and Toxocara canis). Bootstrap values are shown in the nodes. Scale bar represents the number of nucleotide substitutions per site

Features, gene organization and composition of the mt genome

For further identification of this worm, we obtained 12 Gb of raw data with 80,271,718 reads from the complete genomic DNA of the worm using Illumina sequencing. The assembled sequence showed that the complete mt genome of the worm was 13,757 bp; this sequence was deposited in GenBank with accession number MZ665481.1. The mt genome of this worm was a circular DNA molecule and contained 36 genes, comprising 12 PCGs, 22 tRNA genes (2 coding for leucine and 2 coding for serine), two rRNA genes, two NCRs (a long non-coding region [LNCR] and a short non-coding region [SNCR]) and an AT-rich region. Interestingly, the ATPase subunit 8 gene (atp8) was missing from the mt genome (Fig. 2). Twelve PCGs of this worm were transcribed in the same direction. In general, the overall base composition of the mt genome of this worm was: A = 27%, T = 49%, C = 7% and G = 17%, with an entire AT content of 76%, which was greatly inclined towards A and T bases. The AT- and GC-skews of the worm’s mt genome were determined to be: AT-skew (A−T)/(A+T) = − 0.26; GC-skew (G−C)/(G+C) = 0.41; Additional file 1: Table S3).

Fig. 2

Mitochondrial genome organization of Ancylostoma sp. The map shows 12 PCGs, 22 tRNAs (shown as abbreviations with the initial letter of their amino acids) and 2 rRNAs. Each of the 2 leucines (L1 and L2) is identified for the codon families CUN and UUR, respectively, and each of the 2 serines (S1 and S2) is identified for the codon families AGR and UGN, respectively. Inner circle indicates GC content of the mt genome. Abbreviations: CDS, coding DNA sequence; LNCR, long non-coding region; SNCR, short non-coding region

PCGs and codon usage

The total length of the 12 PCGs was 10,283 bp, which accounts for 74.7% of the entire mt genome of the worm. These PCGs ranged in size from 234 bp of NADH dehydrogenase subunit 4L (nad4L) to 1578 bp of cytochrome c oxidase subunit I (cox1). The overall base composition of the PCGs in the worm mt genome was: A = 25%, T = 50%, C = 7% and G = 18%, with AT skew = − 0.32 and GC skew = 0.42, which was largely biased towards the A and T bases. The most favored nucleotide was the T base, but the C base was the least favored in PCGs of the worm. The nad4L gene had the highest AT content (81%) among the 12 PCGs, while cox1 had the lowest AT content (68%) (Additional file 1: Table S3). All of the AT-skew values of the 12 PCGs were negative, and all of the GC-skew values were positive.

The PCGs of the worm contained a total of 3417 amino acids. Two different types of codons (ATT and TTG) were used as start codons, while three different codons (TAA, TAG and T) were used as stop codons (Table 1). ATT was used as a start codon in 10 genes, namely cox1, cox2, nad3, nad5, nad6, nad4L, nad1, atp6, cob and cox3, while TTG was used as a start codon in the nad2 and nad4 genes. TAA was used as a stop codon in seven genes: cox1, cox2, nad6, nad4L, nad1, cytochrome b (cob) and nad4. TAG was used as a stop codon in three genes, including nad3, atp6 and nad2; moreover, an incomplete codon (T) was used in the genes nad5 and cox3 for transcription termination. Thus, in 12 PCGs, ATT and TAA were the most frequently used start and stop codons, respectively. Phenylalanine (TTT: 13.0%) was the most repeatedly employed amino acid in the mt genome of the worm, followed by leucine (TTA: 8.6%) and isoleucine (ATT: 7.0%). However, some transcription codons were absent, such as CGC and CGG coding for arginine and CTC coding for leucine (Table 2).

Table 1

Mitochrondrial genome organization, showing start and stop codons of PCGs and as anticodons of tRNA of Ancylostoma sp.

Gene/region	Position	Length		Codon		Anticodon
	Start to end	Number of nucleotides	Number of amino acids	Start	Stop
cox1	1–1578	1578	525	ATT	TAA
trnC	1577–1630	54				GCA
trnM	1637–1695	59				CAT
trnD	1714–1772	59				GTC
trnG	1773–1829	57				TCC
cox2	1830–2525	696	231	ATT	TAA
trnH	2526–2581	56				GTG
rrnL	2582–3548	967
nad3	3549–3884	336	111	ATT	TAG
SNCR	3885–3984	100
nad5	3985–5560	1576	525	ATT	T
trnA	5567–5622	56				TGC
AT-rich	5623–5883	261
trnP	5884–5938	55				TGG
trnV	5957–6011	55				TAC
nad6	6012–6446	435	144	ATT	TAA
nad4L	6450–6683	234	77	ATT	TAA
trnW	6686–6742	57				TCA
trnE	6751–6809	59				TTC
rrnS	6810–7507	698
trnS2(UGN)	7508–7561	54				TGA
trnN	7562–7617	56				GTT
trnY	7618–7673	56				GTA
nad1	7674–8546	873	290	ATT	TAA
atp6	8553–9152	600	199	ATT	TAG
trnK	9152–9214	63				TTT
trnL2(UUR)	9231–9285	55				TAA
trnS1(AGR)	9286–9338	53				TCT
nad2	9339–10,184	846	281	TTG	TAG
trnI	10,189–10,247	59				GAT
trnR	10,251–10,304	54				ACG
trnQ	10,306–10,360	55				TTG
trnF	10,376–10,432	57				GAA
cob	10,433–11,545	1113	370	ATT	TAA
trnL1(CUN)	11,546–11,600	55				TAG
cox3	11,601–12,366	766	255	ATT	T
trnT	12,367–12,421	55				TGT
nad4	12,422–13,651	1230	409	TTG	TAA
LNCR	13,652–13,757	106

LNCR, Long non-coding region; SNCR, short non-coding region; see Abbreviations for other abbreviations and gene names

Table 2

Amino acid codons and percentage of codon usage for PCGs in the Ancylostoma sp. mt genome

Amino acid	Codon	Number	Frequency (%)	Amino acid	Codon	Number	Frequency (%)
Ala	GCA	24	0.70	Leu	TTA	297	8.67
Ala	GCC	7	0.20	Leu	TTG	209	6.10
Ala	GCG	6	0.18	Lys	AAA	50	1.46
Ala	GCT	61	1.78	Lys	AAG	57	1.66
Arg	CGA	2	0.06	Met	ATA	124	3.62
Arg	CGC	0	0.00	Met	ATG	110	3.21
Arg	CGG	0	0.00	Phe	TTC	18	0.53
Arg	CGT	29	0.85	Phe	TTT	446	13.01
Asn	AAC	9	0.26	Pro	CCA	7	0.20
Asn	AAT	132	3.85	Pro	CCC	5	0.15
Asp	GAC	9	0.26	Pro	CCG	11	0.32
Asp	GAT	56	1.63	Pro	CCT	57	1.66
Cys	TGC	2	0.06	Ser	AGA	57	1.66
Cys	TGT	40	1.17	Ser	AGC	7	0.20
End	TAA	7	0.20	Ser	AGG	43	1.25
End	TAG	3	0.09	Ser	AGT	119	3.47
Gln	CAA	21	0.61	Ser	TCA	47	1.37
Gln	CAG	21	0.61	Ser	TCC	4	0.12
Glu	GAA	35	1.02	Ser	TCG	9	0.26
Glu	GAG	42	1.23	Ser	TCT	92	2.68
Gly	GGA	12	0.35	Thr	ACA	30	0.88
Gly	GGC	6	0.18	Thr	ACC	1	0.03
Gly	GGG	33	0.96	Thr	ACG	17	0.50
Gly	GGT	139	4.06	Thr	ACT	62	1.81
His	CAC	12	0.35	Trp	TGA	48	1.40
His	CAT	42	1.23	Trp	TGG	20	0.58
Ile	ATC	10	0.29	Tyr	TAC	11	0.32
Ile	ATT	240	7.00	Tyr	TAT	171	4.99
Leu	CTA	6	0.18	Val	GTA	68	1.98
Leu	CTC	0	0.00	Val	GTC	10	0.29
Leu	CTG	9	0.26	Val	GTG	55	1.60
Leu	CTT	8	0.23	Val	GTT	142	4.14

rRNA and tRNA genes

The worm had two rRNAs, including a large subunit (rrnL) of 967 bp and a small subunit (rrnS) of 698 bp. The rrnL was situated between trnH and nad3, while rrnS was found between trnE and trnS2. The position of rRNA in Ancylostoma sp. was similar to that found in other Ancylostoma species but distinct from that found in Trichinella spiralis (class Adenophorea) [43]. The rrnL of the worm was longer than the rrnL of 13 species in the Ancylostomatidae family, which ranged from 957 bp (Uncinaria sanguinis) to 963 bp (A. caninum) (Table 3). In addition, sequence identity of rrnL and rrnS in the observed worm was higher with species in the subfamily Ancylostomatinae than with species in the subfamily Bunostominae. The highest sequence identity of rrnL of the worm was 89.6% with Ancylostoma tubaeforme compared to other species in the Ancylostomatidae family, and rrnS had the highest sequence identity of 94% with Ancylostoma ceylanicum (Table 3).

Table 3

Comparisons of nucleotide identity of PCGs, rRNA and NCRs of the mt genome of Ancylostoma sp. with the mt genomes of other Ancylostomatidae species

Region	Gene/region	Length of gene regions (bp)/nucleotide identity (%)
		Ancylostoma sp. (MZ665481.1)	Ancylostomatinae						Bunostominae
			Ancylostoma caninum		Ancylostoma ceylanicum		Ancylostoma duodenale	Ancylostoma tubaeforme	Uncinaria sanguinis		Nector americanus	Bunostomum phlebotomum		Bunostomum trigonocephalum
			AP017673.1	FJ483518.1	KY640299.1	AP017674.1	AJ417718.1	KY070315.1	KF924756.1	KF924757.1	AJ417719.2	MW067147.1	FJ483517.1	JQ234674.1	KF255998.1
		Length (bp)
PCGs	cox1	1578	1578/87.6	1578/87.5	1578/87.9	1578/87.7	1577/87.9	1578/87.9	1578/86.1	1578/86.7	1575/86.6	1573/86.6	1573/87.0	1573/84.0	1576/84.3
	cox1	696	696/88.6	696/88.7	696/85.8	696/86.0	696/87.7	696/88.2	696/84.3	696/84.9	696/83.9	696/83.9	696/83.9	696/43.0	696/77.0
	nad3	336	336/85.1	336/85.1	336/84.7	336/84.5	336/86.0	336/88.3	336/82.7	336/80.0	336/83.0	336/80.9	336/81.2	336/51.6	336/63.5
	nad5	1576	1582/85.3	1576/85.4	1579/85.5	1582/85.8	1579/84.6	1576/84.9	1582/81.0	1582/81.6	1582/81.8	1582/80.0	1582/79.7	1582/78.9	1582/78.9
	nad6	435	432/83.1	435/83.6	435/83.3	435/81.8	435/80.0	435/82.9	435/78.1	435/76.5	435/79.0	435/76.7	435/76.7	435/60.9	435/41.9
	nad4L	234	234/89.7	234/91.0	234/92.8	234/91.8	234/91.8	234/90.6	234/86.3	234/86.3	234/88.8	234/88.4	234/88.4	234/78.1	234/47.3
	nad1	873	873/86.9	873/85.4	873/85.5	873/85.4	873/86.0	873/86.8	873/83.6	873/84.4	873/82.3	876/80.3	876/78.5	873/44.0	873/78.5
	atp6	600	600/89.1	600/88.8	599/89.8	602/89.6	599/90.1	600/88.6	600/82.0	600/83.6	598/84.6	600/83.8	600/84.1	600/57.6	600/75.6
	nad2	846	846/83.1	846/83.1	846/83.8	846/83.4	846/83.1	846/82.2	846/78.4	846/77.9	846/79.5	849/76.7	849/76.1	849/70.1	849/62.9
	cob	1113	1113/88.1	1113/88.1	1113/87.0	1113/86.8	1112/88.2	1113/87.9	1113/82.2	1113/81.6	1113/83.1	1113/83.9	1113/83.5	1113/78.2	1113/75.8
	cox3	766	766/89.3	766/89.3	766/87.7	766/87.9	766/88.5	766/87.7	766/86.0	766/84.4	766/86.6	766/85.9	766/86.5	766/81.0	766/81.2
	nad4	1230	1230/86.9	1230/86.2	1230/86.5	1230/86.7	1230/85.7	1230/87.4	1230/83.0	1230/81.8	1230/82.6	1230/80.4	1230/80.6	1230/78	1230/72.0
rRNA	rrnL	967	958/88.3	963/87.8	960/89.0	960/88.4	958/87.3	958/89.6	957/86.2	958/85.4	958/80.1	961/82.7	961/82.6	961/76.8	961/69.7
	rrnS	698	695/92.9	694/93.0	694/94.0	694/93.9	697/91.8	697/93.1	699/87.7	697/88.2	699/86.6	694/86.1	694/86.1	693/84.5	696/64.5
NCR	SNCR	100	88/null	87/42.1	61/null	66/null	79/null	88/57.1	81/41.6	82/null	67/null	24/65.0	21/null	2/null	2/null
	LNCR	106	100/70.7	100/68.6	103/70.5	94/52.3	109/78.2	107/73.0	86/73.2	86/73.2	73/null	108/58.3	106/43.9	100/48.1	106/42.8
AT-rich		261	262/76.4	272/80.8	244/80.3	246/78.3	268/74.5	286/76.8	333/73.2	331/74.9	173/null	235/50.6	234/52.3	219/56.0	218/60.0
CmtG		13,757	13,702	13,717	13,660	13,655	13,721	13,730	13,74	13,75	13,605	13,799	13,790	13,764	13,771
TI (%)			87.3	87.2	86.8	86.9	86.8	87.1	83.5	83.2	83.7	80.9	80.8	82.7	82.7

CmtG, Complete mitochondrial genome; TI, total identity, null, no identity

The length of the 22 tRNAs ranged from 53 bp (trnS1) to 63 bp (trnS2 and trnK). The total length of the 22 tRNAs of the worm was 1239 bp with an A+T content of 80%; consequently, most codons were composed of A+T bases relative to G+C bases. Apart from serine (CUN and UUR) and leucine (AGR and UGN), there was a one-to-one binding between codon and anticodon for all other tRNAs. With the exception of trnS1 and trnS2, all tRNA secondary structures of the mt genome of Ancylostoma sp. had the DHU arm and DHU loop, which were similar to those of most nematodes, including Toxocara canis, Ascaris suum, A. tubaeforme, Onchocerca volvulus and Anisakis simplex [44-48]. Only trnI, trnK, trnS1 and trnS2 had a pseudouridine (TΨC) arm. Other tRNAs lacked a pseudouridine (TΨC) arm and changed into a TV replacement loop. Moreover, an undeveloped form of the TΨC loop was only found in trnK; a typical TΨC loop was detected in trnM but it lacked TΨC arm (Additional file 1: Fig. S1).

NCR and AT-rich regions

The LNCR of the worm was located between nad4 and cox1 with a length of 106 bp, whereas the SNCR was found between nad3 and nad5 with a length of 100. The entire base composition of the NCRs was as follows: A = 41%, G = 10%, C = 4%, T = 45%, AT = 86% and GC = 14%. The NCRs of this worm lacked repeat sequences, unlike other Ancylostoma species, including A. caninum, A. ceylanicum, A. tubaeforme and A. duodenale. LNCR sequence identity of the worm was 52.3–78.2% with related species in the subfamily Ancylostomatinae and 42.8–58.3% with species in the Bunostominae subfamily, but there was no sequence identity with N. americanus. The LNCR of the observed worm had the highest nucleotide identity of 78.2% with A. duodenale from GenBank (accession number: AJ417718.1) [49]. Nonetheless, the SNCR of the worm had low identity with a few species in the family Ancylostomatidae, while there was no sequence identity with many species in the family of Ancylostomatidae (Table 3). Thus, the SNCR was the unique region in the mt genome of the worm based on nucleotide identity (Table 3).

The AT-rich region was situated between trnA and trnP in the mt genome of the worm. The size of AT-rich region of the worm (261 bp) lay within range 173 bp (N. americanus) and 333 bp (A. duodenale and B. phlebotomum) (Table 3). The AT-rich region had 90% A+T content and comprised a poly-A stretch, poly-T stretch and microsatellites (such as an TA or TA repeat). The AT-rich region of the worm had a sequence identity of 73.2–80.8% with that of species in the subfamily Ancylostomatinae, and 50.6–60.0% sequence identify with some species in the subfamily Bunostominae. The AT-rich region of the worm had no sequence identity with that of N. americanus in the subfamily Bunostominae (Table 3). Thus, the sequence of the AT-rich region showed that this worm was more closely related to species in the subfamily Ancylostomatinae than to species in the subfamily Bunostominae.

Comparison of the worm mt genome with that of species in the family Ancylostomatidae

Total sequences of the worm mt genome had higher identities of 86.8–87.3% with those of related species in the subfamily Ancylostomatinae than with those in the subfamily Bunostominae (Table 3). Moreover, the entire mt genome of the worm had the highest sequence identity of 87.3% with A. caninum compared to other Ancylostomatidae species (Table 3). The relatively low sequence identity was noted with the Bunostomum species, Uncinaria sanguinis, and N. americanus, with sequence identity ranging from 80.8% to 83.7%. In PCGs, the most conserved gene across the subfamily Ancylostomatinae was nad4L, with a sequence identity of 89.7–92.8%, whereas nad6 was the least conserved gene with 80.0–83.6% sequence identity (Table 3). The 12 PCGs of the collected worm also had the highest sequence identity (83.1–91.0%) with A. caninum compared with other species from the subfamilies Ancylostomatinae and Bunostominae. These results suggest that the reported worm is an undescribed Ancylostoma sp. and genetically related closer to A. caninum than to other Ancylostoma species.

PCGs of the mt genome based on phylogenetic analysis

The PCG sequences of the collected Ancylostoma sp., 12 species from the Ancylostomatidae family and 4 species from the Chabertiidae family (outgroup) were used to reconstruct the phylogenetic tree (Fig. 3). Accordingly, Ancylostoma sp. was grouped into the family Ancylostomatidae, separate from the species of the Chabertiidae family. In the Ancylostomatinae subfamily, Ancylostoma sp. was grouped with A. ceylanicum, A. caninum, A. tubaeforme and A. duodenale, while N. americanus and two Bunostomum species (Bunostomum phlebotumum and Bunostomum trignocephalum) were grouped in the Bunostominae subfamily (Fig. 3). Thus, the worm had a closer relationship with A. ceylanicum, A. caninum, A. tubaeforme and A. duodenale than to species in the subfamily Bunostominae. Phylogenetic analyses of the PCGs showed that Ancylostoma sp. clustered with other Ancylostoma species in the Ancylostomatinae subfamily. Sequence identity showed that the Ancylostoma sp. from the pangolin was distinct from known species of the genus Ancylostoma. Thus, the Ancylostoma analyzed herein may represent a novel species in the genus Ancylostoma.

Fig. 3

Phylogenetic tree of 12 PCG sequences from the mt genomes in the families of Ancylostomatidae, Chabertiidae and Ancylostoma sp. This tree is reconstructed based on the ML method. The numbers at the nodal points indicate the statistical values of the phylogenic tree. Nodal points indicate bootstrap values. Scale bar represents the number of nucleotide substitutions per site

Discussion

Ancylostoma species are one of the most prevalent soil-transmitted helminths, affecting both domestic and wild animals, as well as humans. In this study, we identified a novel Ancylostoma sp. that originated from a Sunda pangolin (Manis javanica) by analysis of the mt genome using Illumina sequencing of total DNA.

The complete mt genome of Ancylostoma sp. was 13,757 bp, which is longer than that of A. caninum (13,717 bp) [50], A. tubaeforme (13,730 bp) [48], A. ceylanicum (13,660 bp) [51], A. duodenale (13,721 bp), U. sanguinis (13,753 bp) [52], and N. americanus (13,606 bp), respectively [53], but shorter than that of B. phlebotomum (13,790 bp) [50]. This difference in mt genome length is due to the longer NCR and rRNA sequences of A. caninum in comparison to those of other Ancylostomatidae species. Thus, differences in mt genome size may be a useful indicator to increase our understanding of mtDNA mutation, mitochondrial genetics and evolutionary biology. The 12 PCGs of Ancylostoma sp. were transcribed in the same direction as those of class Secernentea nematodes of hookworms (A. duodenale and N. americanus) and other species (Ascaris suum and Onchocerca volvulus) [45, 49]. The direction of transcription in the mtDNA of Secernentea nematodes is conserved. The mt genome organization and gene arrangement of Ancylostoma sp. were similar with those of N. americanus and A. duodenale, with the exception of the position of rrnL and rrnS, which were located between trnH and nad3, and trnE and trnS2, respectively [49]. However, the gene arrangement and organization of Ancylostoma sp. were identical with those of A. tubaeforme, A. caninum and B. phlebotomum [48, 50]. The cox1 gene in Ancylostoma sp. was the longest gene among the 12 PCGs, similar to the situation in A. tubaeforme [48]; conversely, nad5 was the longest gene in A. ceylanicum, A. doudenale and N. americanus [48, 49, 53]. Nad4L was the shortest region of the PCGs in Ancylostoma sp., which is consistent with observations in other hookworms [48, 50]. The overall base composition of PCGs in Ancylostoma sp. was inclined towards AT bases. All PCGs from different nematodes have a higher AT base selection that maintains the stability of gene structure through decreasing gene mutation [54]. Thus, the length of mtDNA and PCGs of Ancylostoma sp. was slightly different from that of known Ancylostoma species. A complete mt genome sequence of Ancylostoma species can be used as a genetic marker for molecular investigation and diagnosis of members of the family Ancylostomatidae. Moreover, the entire mt genome data of Ancylostoma sp. would contribute to a further understanding of the pangolin helminth fauna.

ATT is the most common start codon found in hookworms, followed by TTG. Likewise, Ancylostoma sp. used ATT and TTG as start codons, similar to A. ceylanicum and A. duodenale [49, 51]. Nonetheless, A. tubaeforme and A. caninum utilize GTG as additional start codons [48, 50]. This variation in codon usage in the different genes of parasite species arises from various factors, but mainly from compositional constraints and translational selection [55]. It is noteworthy that the start codons of nad5 and nad6 in Ancylostoma species were remarkably different from other those of PCGs [8]. With the exception of A. ceylanicum, the nad5 gene of this Ancylostoma sp. and other Ancylostoma species uses ATT as a start codon. Similarly, the nad6 gene of Ancylostoma sp. utilized ATT as a start codon, consistent with A. ceylanicum but distinct from A. tubaeforme and A. caninum, both of which use GTG codons [48, 50]. Ancylostoma sp. utilized three codons (TAA, TAG and T) as stop codons, but A. caninum, A. tubaeforme and A. doudenale use additional TA codons [48-50]. The translation termination in the cox3 and nad5 genes of Ancylostoma sp. used an incomplete codon of T, which is similar to that of cox3 and nad5 genes in A. ceylanicum and N. americanus [51, 53]. It is believed that post-transcriptional polyadenylation has been shown to complete codons by adding A’s to incomplete stop codons, resulting in TAA [56].

The majority of codons were composed of A and T bases, contributing to the high AT content of the entire mt genome of Ancylostoma sp. Nucleotide bias significantly impacts codon usage and amino acid composition. For example, it has been reported that mutational bias at the nucleotide level can alter codon usage and amino acid content [57, 58]. The length of the rrnL gene of Ancylostoma sp. was 967 bp, which is longer than that of other known species of hookworm by 4 bp (A. caninum, B. phlebotumum), 7 bp (A. ceylanicum), 9 bp (A. tubaeforme and N. americanus) and 11 bp (A. doudenale) [48-50]. The rrnS gene of Ancylostoma sp. (698 bp) was slightly longer than that of other hookworm species, with the exception of N. americanus (699 bp) [49]. Thus, the difference in the entire mt genome size of Ancylostoma sp. from other known hookworm species is also due to longer rRNA sizes.

Ancylostoma sp. had an AT-rich region with a length of 261 bp and maximum A+T content of 90%. The placement of the AT-rich region of Ancylostoma sp. was between trnA and trnP, which is consistent with all hookworms [10, 48, 51]. Although the function of the AT-rich region has not yet been explored, it is believed to be the epicenter for the the initiation of gene replication and transcription [15]. The SNCR in Ancylostoma sp. was larger than that of other hookworms in the families Ancylostomatinae and Bunostominae [10, 49–51]. However, the position of the SNCR in the mt genome was identical to that of other Ancylostoma species [50, 53]. The LNCR in Ancylostoma sp. (106 bp) was larger than that in most hookworm species, with the exception of A. tubaeforme (107 bp) and B. phlebotomum (108 bp) [48, 50]. Previous studies showed that the NCR contained repeat sequences of TTTTA in A. caninum and A. ceylanicum; TATATTTAGT in A. tubaeforme; and TTTG in A. doudenale [48]. However, none of these repeat sequences were found in the NCR of Ancylostoma sp. Thus, the NCR of Ancylostoma sp. is an important region that differentiates this species from other Ancylostoma species.

Phylogenetic analyses of 18S rRNA and the complete mt genome showed that the Ancylostoma sp. clustered with A. ceylanicum, A. caninum, A. tubaeforme and A. duodenale in the subfamily Ancylostomatinae. However, some differences in the size of the mt genome, codon usage in PCGs, NCR sequences and tRNA secondary structures of the Ancylostoma sp. mt genome were helpful to differentiate it from other Ancylostoma species. Based on these results, we believe that this is a novel Ancylostoma species in the family Ancylostomatidae.

Conclusions

We characterized the complete mt genome of an Ancylostoma sp. isolated from the Sunda pangolin (Manis javanica) by Illumina sequencing of total DNA. Amplified 18S rRNA and mt genome data identified this Ancylostoma sp. as a novel species in the Ancylostomatidae family. The identification of this novel mtDNA sequence enriches our knowledge of mt genomes in the Ancylostomatidae family.

Additional file 1: Figure S1. Predicted secondary structure of tRNA genes in the Ancylostoma sp. mt genome, Table S1. Retrieved 18S rRNAs in species of Strongylida and Ascaridida from GenBank, Table S2. The mt genome from species in Strongylida, Table S3. Nucleotide composition (%) of PCGs, entire mt genome, and skew value of Ancylostoma sp.