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Comparative analysis of mitochondrial genome data for Necator americanus from two endemic regions reveals substantial genetic variation
International Journal for Parasitology 33 (2003) 955–963 www.parasitology-online.com
Comparative analysis of mitochondrial genome data for Necator americanus from two endemic regions reveals substantial genetic variationq Min Hu, Neil B. Chilton*, Youssef G. Abs El-Osta, Robin B. Gasser Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia Received 6 March 2003; received in revised form 6 May 2003; accepted 7 May 2003
Abstract Necator americanus is a blood-sucking, intestinal nematode of major human health importance in many tropical and subtropical regions of the world. The aim of the present study was to compare the complete mitochondrial genome sequence from one N. americanus individual from Togo with another from China, in order to estimate the magnitude of genetic variability for different mitochondrial genes and noncoding regions. For the 12 protein genes, this comparison revealed sequence differences at both the nucleotide (3 – 7%) and amino acid (1 – 7%) levels. The most conserved of these was the nad4L gene, whereas the nad1 gene was least conserved at both the nucleotide and amino acid levels. Nucleotide differences were also detected in 14 of the 22 transfer RNAs (trns) (1 – 13%), the AT-rich region (,8%), non-coding regions (8 – 25%) and in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rrn) (,1%). Comparison of the rrnL sequences among multiple individual worms revealed nine unequivocal nucleotide differences between N. americanus from the two countries. Consistent with previous studies, these findings provide evidence for substantial genetic variation within N. americanus, which may have implications for the transmission and control of hookworm disease. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Hookworm; Necator americanus; Mitochondrial genome; rrnL; Genetic variation
1. Introduction It is estimated that over one billion people are infected with hookworms, of which , 6% are clinically affected (Albonico et al., 1999). Necator americanus is one of the most important hookworm species infecting humans (Albonico et al., 1999). This parasite is endemic in many tropical and subtropical regions of the world, including parts of Africa, India, China, south-east Asia, the south-west Pacific Islands, South and Central America, the Caribbean Islands and southern USA (e.g. Schad and Warren, 1990; Liu et al., 1999; Behnke et al., 2000; Gandhi et al., 2001). The adult worms live in the small intestine where they attach to the mucosa and suck blood, consequently causing anaemia. In children, the anaemia can seriously affect both q The nucleotide sequences reported herein have been deposited in the DDBJ, EMBL and GenBank databases under the Accession numbers AJ556111– AJ556133, AJ556178– AJ556180 and AJ556134. * Corresponding author. Tel.: þ 61-3-97312330; fax: þ 61-3-97312366. E-mail address: [email protected] (N.B. Chilton).
physical and cognitive development, the latter of which can also result in intellectual impairment (Hotez and Pritchard, 1995). Diagnosis of hookworm infections usually relies on the detection of eggs in human faeces and/or the identification of larvae by copro-culture (Polderman et al., 1991; Blotkamp et al., 1993; Polderman and Blotkamp, 1995). Throughout much of its distribution, N. americanus occurs in sympatry with other hookworms, such as Ancylostoma duodenale or Ancylostoma ceylanicum (see Nelson, 1990; Schad and Warren, 1990; Hotez and Pritchard, 1995) or with the nodule worm Oesophagostomum bifurcum in Africa (Polderman et al., 1991, 1999; Polderman and Blotkamp, 1995). However, these nematode species differ significantly in their life cycle, pathogenesis and epidemiology (e.g. Schad and Warren, 1990; Polderman et al., 1999). Since the eggs from these different species (shed in human faeces) cannot be distinguished unequivocally using morphological characters, species-specific diagnosis by coproscopy is compromised. Hence, an effective PCR-based approach
0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0020-7519(03)00129-2
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M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
has been established for the specific amplification of N. americanus DNA from human faeces in regions of Africa where this hookworm occurs in sympatry with O. bifurcum (see Verweij et al., 2001). Previous studies of N. americanus have detected sequence variation in the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA (rDNA) among individuals from Togo (Africa) and from China or Malaysia (e.g. Gasser et al., 1998; Romstad et al., 1998). Four nucleotide differences (i.e. purine substitutions) in part of the mitochondrial (mt) cytochrome c oxidase subunit 1 gene (cox1) were also detected between N. americanus individuals from Togo and those from China (Hu et al., 2002c). This finding raised the possibility that N. americanus from different geographical regions may represent more than one species (Romstad et al., 1998). However, it has been argued that the phylogenetic species concept (i.e. considering only derived character states in analyses) should be used for the delineation of species (e.g. Nixon and Wheeler, 1990; Adams, 1998; Wheeler, 1999; Nadler et al., 2000). A re-examination of the cox1 sequence data from Hu et al. (2002c) suggests four derived character states for the N. americanus population from Togo but none for the Chinese population, using A. duodenale as the outgroup. Thus, a greater number of genes need to be sequenced, in order to define derived character states to distinguish N. americanus from different geographical origins. Mitochondrial genes are considered to be well-suited for investigating the population genetics of nematodes and for differentiating sibling species, because they have higher substitution rates than nuclear genes (Anderson et al., 1998; Blouin, 2002). Recently, a long PCR-based approach was used to obtain the complete mt genome sequence (comprising 36 genes) from an individual N. americanus from China (Hu et al., 2002a,b). In the present study, this mt genome sequence was compared with that obtained from an individual of N. americanus from Togo, in order to estimate the levels of nucleotide and/or predicted amino acid sequence differences (for individual genes). Extending from this, the genetic variation between N. americanus from these two geographical regions was assessed by comparing nucleotide sequence data (from multiple individuals) for the longest of the two most conserved mt genes.
2. Materials and methods 2.1. Parasites and DNA isolation Adults of N. americanus from the village of Naki-Est, Region des Savannes, Togo, Africa ( ¼ Na-Togo) and from Yiwu county, Zhejiang province, China ( ¼ Na-China), and a morphologically distinct species of hookworm, A. duodenale, from the same county in China (Table 1) were collected from faeces following the treatment of humans with pyrantel pamoate (see Polderman et al., 1991). Worms were washed extensively in physiological saline, identified morphologically, fixed in 70% ethanol and then frozen at 2 20 8C until use. Total genomic DNA was extracted from individual worms using sodium dodecylsulphate/proteinase K treatment (Gasser et al., 1993), followed by spin-column purification (Wizarde Clean-Up, Promega).
2.2. Mitochondrial genome sequencing and analyses A long-PCR approach (Hu et al., 2002a) was employed to determine the complete mt genome sequence from an individual worm of Na-Togo (sample T17), the morphological identification of which had been verified by comparison of its ITS-2 sequences with those reported previously for N. americanus from Togo (Romstad et al., 1998). In brief, the entire mt genome was amplified in two overlapping fragments (, 10 and , 5 kb) by PCR (Expande 20 kb plus kit, Roche) using primer sets 5F-40R and 39F-42R, respectively (Hu et al., 2002b). The cycling conditions used were: 92 8C, 2 min (initial denaturation); then 92 8C, 10 s (denaturation); 50 8C, 30 s (annealing); 68 or 60 8C, 10 min (extension) for 10 cycles, followed by 92 8C for 10 s, 50 8C for 30 s and 68 or 60 8C for 10 min for 20 cycles, with a cycle elongation of 10 s of each cycle, and a final extension at 68 or 60 8C for 7 min. The extension temperatures of 68 and 60 8C were used specifically for primer sets MH5F-MH40R and MH39F-MH42R, respectively. Amplicons were detected upon ultraviolet transillumination of 1% (w/v) agarose gels after electrophoresis in TBE (65 mM Tris – HCl, 27 mM boric acid, 1 mM EDTA, pH 9) buffer and ethidium bromide staining. They were then
Table 1 Individuals of Necator americanus from Togo (Na-Togo) and China (Na-China) and of Ancylostoma duodenale, and accession numbers for their mt rrnL sequences Parasite
No. of individuals
Sample codes
Accession numbers
Na-Togo Na-China A. duodenale
14 11 4
Nal–Na5, T1, T2, T4– T6, T16, T17a, T18, TN1 NaQ6, NaQ8, NaQ9, NaQ11–NaQ15, NaQ18, NaQ19, NaQ22b AdQ22, AdQ23, AdQ34, AdQ21b
AJ556111–AJ556123, AJ556134a AJ556124–AJ556133, AJ417719b AJ556178–AJ556180, AJ417718b
a b
The complete mitochondrial genome sequence for this individual was determined during the present study. Complete mitochondrial genome sequences for these individuals were published previously in Hu et al. (2002a).
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
purified over spin columns (Wizarde PCR-Prep, Promega) and used as templates for subsequent automated sequencing (ABI BigDye chemistry v.1; in a PE Applied Biosystems 377 sequencer), employing a primer walking strategy. Primers used previously for the sequencing of the mt genome of NaChina (sample code NaQ22; see Fig. 1 in Hu et al., 2002b) were also used herein, although five additional primers (details available from authors) were designed to sequence the ATrich region. Contiguous sequences were aligned with the mt genome sequence of Na-China (accession number: AJ417719) using the Clustal X program v.1.53b (Thompson et al., 1997). The nucleotide sequences of the 12 protein genes, two rrn genes and two non-coding regions were compared between the two Necator mt genomes. The amino acid sequences were conceptually translated from individual mt protein genes of Na-Togo using the MacVector v.4.1.4 program (Kodak), and their identities compared with those from Na-China. 2.3. Sequencing of the rrnL gene and phylogenetic analysis Two primers, 65F and 80R (see Fig. 1 of Hu et al., 2002b), designed to regions within the cox2 and nad5 genes, respectively, were employed for long-PCR amplification and subsequent sequencing of the large subunit of mt ribosomal RNA (rrnL) from individual N. americanus (Table 1). The rrnL sequence was also obtained from three individuals of A. duodenale (Table 1). These sequences and a previously published rrnL sequence of A. duodenale (accession number AJ417718, sample code AdQ21; Hu et al., 2002a) were used as the outgroup for phylogenetic analyses, because this morphologically distinct species represents a sister taxon to N. americanus (see Lichtenfels, 1974). All rrnL sequences were aligned using Clustal X, and the alignment was modified manually, based on the predicted secondary structure of the rrnL for hookworms (Hu et al., 2002a). Phylogenetic trees were constructed using a maximum parsimony (MP) analysis, employing PAUP* v.4b10 (Swofford, 1999). Characters were weighted equally and treated as unordered. Alignment gaps were treated as a fifth character state. An heuristic search with TBR-branch swapping was used to infer the shortest trees. The length, consistency index excluding uninformative characters (C.I.) and retention index (R.I.) of the most parsimonious trees were recorded. A bootstrap analysis (using 1,000 replicates) was conducted using heuristic searches and TBR branch swapping with the MulTrees option, in order to determine the relative support for clades of the consensus tree.
3. Results 3.1. Comparative analysis of the mt genome sequence of Na-Togo with that of Na-China The full mt genome sequence of Na-Togo (sample code
957
T17) was 13,606 bp in length, 1 bp longer than that reported for Na-China (accession number AJ417719). The identity, number and arrangement of the mt genes (i.e. 12 protein genes, two rrn genes and 22 transfer RNA [trn ] genes) and non-coding regions were the same as that of Na-China (Hu et al., 2002a). A comparison of the nucleotide sequences of each mt gene and non-coding region, as well as the amino acid sequences, conceptually translated from all protein genes of the two N. americanus, are given in Table 2. The sequence lengths of each gene and non-coding region were the same for Na-Togo and Na-China, except for variation of one nucleotide in the rrnS gene and of one to three nucleotides for each of three trn genes (Table 2). The magnitude of sequence variation in each gene and non-coding region between Na-Togo and Na-China ranged from 1.1 to 24.5% (except for the 8 trns for which no variation was detected). The greatest variation between Na-Togo and Na-China was in the non-coding regions, whereas the least difference (, 2%) was detected in the two rrn subunits (Table 2). Comparison of transition/transversion ðTs=TvÞ ratios for individual genes and regions revealed 2.3– 11 times more transitions than transversions in coding genes compared with non-coding regions ðTs=Tv , 1Þ. Amino acid sequences inferred from individual mt protein genes of Na-Togo were compared with those of Na-China. The amino acid sequence differences ranged from 1.3 to 7.2%, with NAD4L being the most conserved protein and NAD1 the least conserved. There was a total of 120 amino acid substitutions (for an alignment length of 3,418 positions) in the 12 proteins (Table 3), the majority of which were in the proteins COB ðn ¼ 24Þ, NAD1 ðn ¼ 21Þ and NAD5 ðn ¼ 13Þ. The number of substitutions at different codon positions was also examined, revealing the following pattern: third codon position ðn ¼ 8 – 59Þ . first codon position ðn ¼ 1 – 14Þ . second codon position ðn ¼ 0 – 10Þ. Of the 120 amino acid substitutions, 87 (73%) represented potentially informative characters (for a cladistic analysis using A. duodenale as the outgroup), the greatest number of these being in the proteins COB ðn ¼ 17Þ, NAD1 ðn ¼ 16Þ and COX1 ðn ¼ 10Þ (Table 3). 3.2. Analysis of the expanded rrnL sequence data set Comparison of the mt genomes of Na-Togo and Na-China showed that the rrnS and rrnL were the two most conserved genes, the latter of which was , 258 bp longer (Table 2). Therefore, the sequence variation in the rrnL gene among multiple individuals of N. americanus from the two geographical origins was assessed (see Table 4). The complete rrnL sequence determined for 12 of the 14 Na-Togo individuals was 958 bp. Individual Na5 had one insertion, while individual T2 had two insertions. Nucleotide variation among Na-Togo samples was detected at nine other sites (i.e. alignment positions 160, 229, 259, 270, 295, 493, 558, 577 and 618; Table 4). Sequences for eight of the 11 Na-China individuals were of the same
958
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
Table 2 Nucleotide and/or predicted amino acid sequence differences for each mt gene and non-coding region for Necator americanus from Togo, compared with N. americanus from China (accession number AJ417719) Gene/region
atp6 cob cox1 cox2 cox3 nad1 nad2 nad3 nad4 nad4L nad5 nad6 All 22 trns rrnS rrnL AT-rich region 1st Non-codingf 2nd Non-codingh Otheri
Nucleotide sequence length
Nucleotide difference (%)
Ts/Tva ratio
1st
2nd
3rd
Amino acid sequence length
No. of substitutions at codon position
Amino acid difference (%)
598 1,113 1,575 696 766 873 845 336 1,230 234 1,582 435
5.2 6.1 5.1 6.0 3.5 7.4 5.7 6.5 4.9 3.4 4.5 5.3
9.3:1 2.8:1 4.3:1 9.5:1 5.8:1 4.5:1 8.6:1 10.0:1 11.0:1 9.0:0 5.5:1 2.3:1
1 14 6 3 5 14 8 2 8 1 4 3
0 10 4 0 2 8 2 3 2 0 8 2
30 44 70 39 20 44 38 17 50 8 59 18
199 370 524 231 255 290 281 111 409 77 527 144
1.5 6.5 2.1 1.7 2.7 7.2 3.9 4.5 2.7 1.3 2.5 5.6
1,223b
2.5c
3.1:1c
–d
–
–
–
–
700e 958
1.1 1.4
2.5:1 10.0:1
– –
– –
– –
– –
– –
173 68g 67 134
8.1 8.2 20.9 24.5
0.7:1 1.0:0 0.6:1 1.1:1
– – – –
– – – –
– – – –
– – – –
– – – –
a
Ts, transition and Tv, transversion. Nucleotide lengths of Na-Togo trns were 52 (S[UCN]), 53 (S[AGN]), 54 (R, N, G, P and T), 55 (A, C, Q, L[CUN], L[UUR], F, Y and V), 56 (D, H and W), 59 (E and I), 60 (M) or 62 (K). The trn sequence for Na-China was one nucleotide shorter for M, three shorter for E and one longer for F. Standard abbreviations are used for each trn. c Represents mean value; number of nucleotide differences between Na-Togo and Na-China were either 1 (A, N, C, Q, I, K, P and V), 2 (L[UUR]), 3 (D, M, F and S[UCN]) or 8 (E). No differences were detected in other trns. d Not applicable. e The sequence of Na-China was one nucleotide shorter. f Located between the genes nad4 and cox1. g The sequence of Na-China was five nucleotides longer. h Located between the genes nad3 and nad5. i Representing intergenic regions of currently unknown function. The sequences of Na-China were two nucleotides shorter (over the total length). b
length (958 bp), whereas three (NaQ12, NaQ18 and NaQ19) were one base longer. Also, nucleotide variation occurred at nine other sites (i.e. alignment positions 7, 258, 262, 292, 370, 482, 553, 618 and 634; Table 4), only one of which was variable among all Na-Togo individuals examined. The length of the rrnL sequences for the four A. duodenale individuals was 957 or 958 bp. Alignment of the rrnL sequences revealed that all individuals of Na-Togo differed at nine nucleotide positions when compared with Na-China (Table 4). These differences represented two indels, six purine transitions (A $ G) and one transversion (A $ T). A MP analysis yielded two equally most parsimonious trees with a length of 199, a C.I. of 0.95 and a R.I. of 0.98. The strict consensus tree is shown in Fig. 1. There was moderate support (bootstrap values of 74 –78%) for the separation of Na-Togo from Na-China individuals into two distinct clades. Six of the nine nucleotide differences were considered as derived (i.e. autapo-
morphic) characters, with A. duodenale as the outgroup in a cladistic analysis, three for Na-Togo individuals (at positions 25 and 344, and between positions 260 and 261, with respect to the published sequence of Na-China sample NaQ22; see Table 4) and three for Na-China individuals (at positions 278, 371 and 790; Table 4).
4. Discussion In the present study, substantial nucleotide differences were detected in the complete mt genome between an individual of N. americanus from Togo and another from China. The variation detected in the 12 protein-coding genes (3 –7%) and in non-coding regions (8 – 25%) was consistent with previous findings of variation in part of the mt cox1 gene among N. americanus individuals (Hawdon et al., 2001; Hu et al., 2002c) and in the nucleotide sequences of
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
959
Table 3 Amino acid substitutions at particular sites in all 12 conceptually translated mt proteins of Necator americanus (Na) from Togo or China and of Ancylostoma duodenale (Ad). Position numbers in bold-type indicate potentially phylogenetic informative sites for N. americanus, using A. duodenale as the outgroup Gene
Position
Na
Ad
Togo
China
atp6
33 53 63
K L L
N V F
K L L
cob
1 17 63 72 145 155 156 157 193 229 236 241 242 262 284 286 295 302 316 320 358 360 362 370
V I V Y N P A I H V G M Y D V S G M V N V L Y V
I T G C S S T F D M V C C V I G V L F I L F F I
I T V Y S P S I H V I F Y V V G V F M G M L F I
3 65 255 302 428 436 456 461 462 467 491
F V S D G H M A P V S
L A L V V Y V S S I N
L I L V V H V S S I S
cox1
Gene
Position
Na
Ad
Togo
China
cox2
37 40 153 173
V I V V
I V I I
V V I V
cox3
23 38 47 67 176 247 250
G I A H K I G
S V V D S V V
G L V H S V V
49 55 61 137 154 157 160 170 177 207 211 212 230 243 257 263 265 271 277 283 284
T I K A T V K V I A K G V S T D L R I S H
A V E V A L S I V V S V I M S Y M W V V Y
A V E A A I S L L V S V V M S Y M W I M F
16 22 28 51 97 125 133 191 203 262 278
S I V N Y M I V M I V
G V I I F V V I V V I
S I V N F I F V M F I
nad1
nad2
the nuclear ITS rDNA (Gasser et al., 1998; Romstad et al., 1998). However, for N. americanus, as for other species of nematode (e.g. Blouin, 2002), there is greater within-species variation in mt protein-genes than in the ITS. For example, the magnitude of the nucleotide sequence variation in the 12 mt protein genes (. 3%) was greater than the 15 (2%) variable positions in the ITS (over 852 bp) detected among
Gene
Position
Na
Ad
Togo
China
nad3
3 13 63 65 110
N M S T V
S L F I I
N L F I V
nad4
5 16 24 86 87 94 205 218 347 349 387
F I L G A D V H I I Y
L V F V S Y L Y V V C
L L M V S Y A Y I V F
6
V
I
I
nad5
27 246 284 322 326 347 354 359 363 412 427 438 522
F I I I L S S L S A N S I
L V S M P L F M L V S L F
L V S M L A F I F V S L L
nad6
13 19 31 34 72 99 100 101
D K F I S L I T
A N L M K V V A
G N L L K V L Y
nad4L
multiple N. americanus individuals (e.g. Romstad et al., 1998; Chilton et al., unpublished data). Comparison between the N. americanus from Togo and China also revealed variation at 120 amino acid positions in the 12 predicted mt protein sequences (Table 2). This magnitude of amino acid variation seems high, given that mt proteins are considered to be highly conserved within a
960
Table 4 Nucleotide differences in the mt rrnL between Necator americanus from Togo (Na-Togo) and from China (Na-China), and from the consensus sequence for 4 Ancylostoma duodenale (Ad) individuals Samples Positions 7
13/14 15 25 160 193 229 258 259 260/261 262 270 271/272 278 292 295 344 370 371 374 482 493 553 558 577 618 634 675/676 790 953/954
A – A – A – A – A T A – A T A – A – A – A – A – A – A –
A A A A A A A A A A A A A A
G G G G G G G G G G G G G G
G G G G G T G G G G G G G G
A A A A A A A A A A A A A A
G G G G A G G G G G G G G G
G G G G G G G G G G G G G G
T T T T T T T C T T T T T T
T T T T T T T T T T T T T T
A A A A A A A A A A A A A A
A T T T T T T T T T T T T T
– – – – – – – – – – – – – –
A A A A A A A A A A A A A A
T T T T T T T T T T T T T T
G G G G G G G G G A G G G G
– – – – – – – – – – – – – –
A A A A A A A A A A A A A A
G G G G G G G G G G G G G G
A A A A A A A A A A A A A A
A A A A A A A A A A A A A A
T T T T T T T T T T T A T T
G G G G G G G G G G G G G G
A A A A A A A A A A A A G A
A A A G A A A A A A A A G A
G G G A G G G G G G G G G G
G G G G G G G G G G G G G G
– – – – – – G – – – – – – –
A A A A A A A A A A A A A A
– – – – – – – – – – – – – –
NaQ6 NaQ8 NaQ9 NaQ11 NaQ12 NaQ13 NaQ14 NaQ15 NaQ18 NaQ19 NaQ22
G G G A G A G A G G G
– – – – – – – – – – –
G G G G G G G G G G G
A A A A A A A A A A A
G G G G G G G G G G G
G G G G G G G G G G G
G G G G G G G G G G G
A A A A A G A A A A A
T T T T T T T T T T T
– – – – – – – – – – –
A A A G A A A G A A G
T T T T T T T T T T T
– – – – T – – – – T –
G G G G G G G G G G G
T T T T T C T T T T T
G G G G G G G G G G G
T T T T T T T T T T T
A G G A A A G A G A A
A A A A A A A A A A A
T T T T T T T T T T T
A A A A G A A A A G G
T T T T T T T T T T T
G G G G G G G A G G G
A A A A A A A A A A A
A A A A A A A A A A A
A A G A A G A G A A A
G G A G G G G G G G G
– – – – – – – – – – –
G G G G G G G G G G G
– – – – – – – – T – –
Ad
T
–
T
A
T
T
T
R
T
–
T
T
T
A
T
T
T
A
G
G
A
T
T
T
G
A
A
–
A
–
Position numbers correspond to the published mt genome sequence of N. americanus (accession number AJ417719; Hu et al., 2002a) ( –, deletion; nucleotides and deletions in bold-type represent fixed differences between Na-Togo and Na-China).
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
Na1 Na2 Na3 Na4 Na5 T1 T2 T4 T5 T6 T16 T18 TN1 T17
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
Fig. 1. Strict consensus tree, constructed using MP and depicting the relationships of Necator americanus from China (Na-China), and from Togo (Na-Togo) and Ancylostoma duodenale (outgroup) based on sequence data for the mt rrnL. Numerals above the branches represent bootstrap values (%). Two sets of three derived character states separating Na-Togo (positions 25, 344 and between positions 260/261) from Na-China (positions 278, 371 and 790) are shown on each branch, respectively. Position numbers correspond to those in Hu et al. (2002a).
species due to structural and functional constraints (see Simon et al., 1994). Also, previous studies of hookworms have detected little to no within-species variation in protein sequences. For instance, amino acid substitutions were recorded at only two of 196 positions (based on a comparison of conceptually translated sequences originating from GenBanke accession numbers AF303135–AF303159) in partial COX1 among 151 N. americanus samples from four locations in China (Hawdon et al., 2001). Similarly, Hu et al. (2002c) detected no within-species variation in a different COX1 region of 131 amino acids for N. americanus and for related hookworms, including A. duodenale and Ancylostoma caninum. The greatest numbers of amino acid differences recorded herein were in the proteins COB ðn ¼ 24Þ and NAD1 ðn ¼ 21Þ. The significance of the amino acid sequence variation between N. americanus from different geographical origins needs to be assessed further, because for invertebrates generally, there are presently limited data on the magnitude of within-species variation in mt protein sequences.
961
Genetic variation within N. americanus was also detected herein for the two mt ribosomal RNA gene subunits (rrnS and rrnL). These subunits are considered to be more conserved in sequence than the protein genes (see review of Simon et al., 1994), which was supported by the data for N. americanus. Comparison of the complete genome data set between the two individuals (samples T17 and NaQ22) demonstrated less sequence variation in the rrnS and rrnL genes (, 1%) compared with the protein genes (. 3%) and the non-coding regions (8 –21%). The rrnL gene was the larger of these two most conserved genes in N. americanus, thus being more likely to provide a greater number of variable sites to examine the magnitude of genetic variation in the parasite between different geographical origins. A comparison of the complete rrnL sequences (958 bp) for 25 N. americanus individuals revealed 30 (3%) variable positions. The nucleotide sequences of all 12 mt protein genes, the rrnS and rrnL genes and the non-coding regions were used to assess the genetic variation among N. americanus from different geographical origins. For instance, the rrnL sequences of all Chinese N. americanus individuals differed from those from Togo at nine unequivocal nucleotide positions. This result is consistent with previous findings of genetic differences among geographically isolated populations of N. americanus (i.e. from Togo, China and/or Malaysia) based on the sequences and/or single strand conformation polymorphism (SSCP) analyses of nuclear ITS-1 and/or ITS-2 regions (Gasser et al., 1998; Romstad et al., 1998) and the mt cox1 gene (Hu et al., 2002c). For example, Chinese N. americanus differed from those from Togo at four unequivocal nucleotide positions in a 395 bp region of cox1 (Hu et al., 2002c). Taken together, this molecular evidence (Gasser et al., 1998; Romstad et al., 1998; Hu et al., 2002c) indicates that the N. americanus population from Togo and from that China each represents a distinct set of genotypes, as a consequence of geographical isolation over a long period of time. Whether each set of genotypes represents a genetically distinct but morphologically similar (i.e. cryptic) species remains to be determined. Previously, the significance of the genetic differences between N. americanus populations has been interpreted phenetically (e.g. Romstad et al., 1998) rather than in an evolutionary context (i.e. based on the phylogenetic species concept; e.g. Nixon and Wheeler, 1990; Wheeler, 1999). The latter approach (i.e. based on shared, derived characterstate data) is considered to be more objective and is proposed to eliminate some of the predictive errors associated with phenetic analyses (Adams, 1998). For example, Nadler et al. (2000) employed a cladistic approach in analyses of sequence data (nuclear ITS-1 and 28S rDNA), in combination with morphometric data, to infer that hookworms (Uncinaria spp.) from the Californian sea lion (Zalophus californianus) and from the northern fur seal (Callorhinus ursinus) collected in sympatry each represent a separate species. A similar approach, using sequence data for several
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mitochondrial and nuclear DNA genes, could be adopted to test the specific status of N. americanus. For instance, of the nine rrnL nucleotide positions differing between the Chinese and African N. americanus, six represented autapomorphic (i.e. derived) characters (three for African individuals and three for Chinese individuals), using A. duodenale as the outgroup. There are also other informative characters in the ITS rDNA (Romstad et al., 1998) and in mt cox1 (Hu et al., 2002c). Even though these results indicate isolation of the Chinese and African N. americanus for a long period of time, some caution is warranted with an interpretation supporting the ‘cryptic species hypothesis’ for the following reasons. Firstly, although A. duodenale is a sister taxon to N. americanus (see Lichtenfels, 1974), there may be other hookworm species which represent more suitable outgroups to test the specific status of N. americanus populations. For example, Necator suillus from pigs in the Caribbean or Necator congolensis from the chimpanzee in Africa (Ackert and Payne, 1923) could be used in cladistic analyses, provided that they indeed represent distinct morphospecies. Secondly, more samples of N. americanus need to be examined from several locations in China and in Africa, and preferably from other continents (e.g. South and Central America), in order to assess the biological and evolutionary significance of the sequence data obtained. For instance, sampling from Central America would be informative, because preliminary SSCP-based analysis of the ITS-1 and ITS-2 for three N. americanus individuals from Guatemala suggests that some have ‘mixed genotypes’ compared with the parasite from Africa and Asia (cf. Gasser et al., 1998). The significance of this finding requires investigation using the rrnL and mt protein sequence data, together with morphological study of specimens. In conclusion, analysis of mt nucleotide and/or amino acid sequence data sets supports the proposal that the gene pools of N. americanus from Togo and China have been isolated for a substantial period of time. Whether each gene pool represents a distinct species is still unclear. Given that N. americanus is widely distributed across the world (in tropical and subtropical regions) and is a significant human pathogen, answering this question may provide insights into the population biology and epidemiology of the parasite, and may assist in the implementation of control measures.
Acknowledgements Funding support was from various sources, including the Australian Research Council. Min Hu has been the recipient of a postgraduate scholarship from The University of Melbourne and a Travel Award from the Australian Society for Parasitology. Samples used in this study were originally provided by Anton Polderman and Qian Bao-Zhen. The two anonymous reviewers are thanked for their constructive comments on the manuscript.
References Ackert, J.E., Payne, F.K., 1923. Investigations on the control of hookworm disease. XII. Studies on the occurrence, distribution and morphology of Necator suillus, including descriptions of the other species of Necator. Am. J. Hyg. 3, 1–25. Adams, B.J., 1998. Species concepts and the evolutionary paradigm in modern nematology. J. Nematol. 30, 1–21. Albonico, M., Crompton, D.W.T., Savioli, L., 1999. Control strategies for human intestinal nematode infections. Adv. Parasitol. 42, 277–341. Anderson, T.J.C., Blouin, M.S., Beech, R.N., 1998. Population biology of parasitic nematodes: applications of genetic markers. Adv. Parasitol. 41, 219–283. Behnke, J.M., De Clercq, D., Sacko, M., Gilbert, F.S., Quattara, D.B., Vercruysse, J., 2000. The epidemiology of human hookworm infections in the southern region of Mali. Trop. Med. Int. Health 5, 343 –354. Blotkamp, J., Krepel, H.P., Kumar, V., Baeta, S., van’t Noorende, J.M., Polderman, A.M., 1993. Observations on the morphology of adults and larval stages of Oesophagostomum sp. isolated from man in northern Togo and Ghana. J. Helminthol. 67, 49–61. Blouin, M.S., 2002. Molecular prospecting for cryptic species of nematodes: mitochondrial DNA versus internal transcribed spacer. Int. J. Parasitol. 32, 527–531. Gandhi, N.S., Chen, J., Khoshnood, K., Xing, F., Li, S., Liu, Y., Zhan, B., Xue, H., Tong, C., Wang, Y., Wang, W., He, D., Chen, C., Xiao, S., Hawdon, J.M., Hotez, P.J., 2001. Epidemiology of Necator americanus hookworm infections in Xiulongkan village, Hainan province, China: high prevalence and intensity among middle-aged and elderly residents. J. Parasitol. 84, 739 –743. Gasser, R.B., Chilton, N.B., Hoste, H., Beveridge, I., 1993. Rapid sequencing of rDNA from single worms and eggs of parasitic helminths. Nucleic Acids Res. 21, 2525–2526. Gasser, R.B., Monti, J.R., Qian, B.-Z., Polderman, A.M., Nansen, P., Chilton, N.B., 1998. A mutation scanning approach for the identification of hookworm species and analysis of population variation. Mol. Biochem. Parasitol. 92, 303 –312. Hawdon, J.M., Li, T., Zhan, B., Blouin, M.S., 2001. Genetic structure of populations of the human hookworm, Necator americanus, in China. Mol. Ecol. 10, 1433–1437. Hotez, P.J., Pritchard, D.J., 1995. Hookworm infection. Sci. Am. 272, 42 –48. Hu, M., Chilton, N.B., Gasser, R.B., 2002a. The mitochondrial genomes of the human hookworms, Ancylostoma duodenale and Necator americanus (Nematoda: Secernentea). Int. J. Parasitol. 32, 145–158. Hu, M., Chilton, N.B., Gasser, R.B., 2002b. Long PCR-based amplification of the entire mitochondrial genome from single parasitic nematodes. Mol. Cell. Probes 16, 261 –267. Hu, M., Chilton, N.B., Zhu, X.Q., Gasser, R.B., 2002c. Single-strand conformation polymorphism-based analysis of mitochondrial cytochrome c oxidase subunit 1 reveals significant substructuring in hookworm populations. Electrophoresis 23, 27–34. Lichtenfels, J.R., 1974. Keys to genera of the superfamilies Ancylostomatoidea and Diaphanocephaloidea No.8. In: Anderson, R.C., Chabaud, A.G., Willwott, S. (Eds.), CIH Keys to the Nematode Parasites of Vertebrates, Commonwealth Agricultural Bureaux, Farnham Royal. Liu, C., Zhang, X., Qiu, D., Xiao, S., Hotez, P.J., Zhen, D., Zhen, H., Li, M., Ren, H., Zhan, B., Xue, H., Hawdon, J., Feng, Z., 1999. Epidemiology of human hookworm infections among adult villages in Hejiang and Santai counties, Sichuan province, China. Acta Trop. 73, 243 –249. Nadler, S.A., Adams, B.J., Lyons, E.T., Delong, R.L., Melin, S.R., 2000. Molecular and morphometric evidence for separate species of Uncinaria (Nematoda: Ancylostomatidae) in California sea lions and northern fur seals: hypothesis testing supplants verification. J. Parasitol. 86, 1099–1106. Nelson, G.S., 1990. Hookworms in perspective. In: Schad, G., Warren, K.S.
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963 (Eds.), Hookworm Disease: Current Status and New Directions, Taylor and Francis, London, pp. 417–430. Nixon, K.C., Wheeler, Q.D., 1990. An amplification of the phylogenetic species concept. Cladistics 6, 211–224. Polderman, A.M., Blotkamp, J., 1995. Oesophagostomum infections in humans. Parasitol. Today 11, 451–456. Polderman, A.M., Krepel, H.P., Baeta, S., Blotkamp, J., Gigase, P., 1991. Oesophagostomiasis, a common infection of man in northern Togo and Ghana. Am. J. Trop. Med. Hyg. 44, 336 –344. Polderman, A.M., Anemana, S.D., Asigri, V., 1999. Human oesophagostomiasis: a regional public health problem in Africa. Parasitol. Today 15, 129 –130. Romstad, A., Gasser, R.B., Nansen, P., Polderman, A.M., Chilton, N.B., 1998. Necator americanus (Nematoda: Ancylostomatidae) from Africa and Malaysia have different ITS-2 rDNA sequences. Int. J. Parasitol. 28, 611–615. Schad, G., Warren, K.S., 1990. Hookworm Disease: Current Status and New Directions, Taylor and Francis, London.
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Simon, C., Frai, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651 – 701. Swofford, D.L., 1999. PAUP* 4.0b10, Sinauer Associates, Sunderland, MA. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Verweij, J.J., Pit, D.S.S., van Lieshout, L., Baeta, S.M., Dery, G.D., Gasser, R.B., Polderman, A.M., 2001. Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples. Trop. Med. Int. Health 6, 726 –731. Wheeler, Q.D., 1999. Why the phylogenetic species concept?—elementary. J. Nematol. 31, 134–141.
Comparative analysis of mitochondrial genome data for Necator americanus from two endemic regions reveals substantial genetic variationq Min Hu, Neil B. Chilton*, Youssef G. Abs El-Osta, Robin B. Gasser Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia Received 6 March 2003; received in revised form 6 May 2003; accepted 7 May 2003
Abstract Necator americanus is a blood-sucking, intestinal nematode of major human health importance in many tropical and subtropical regions of the world. The aim of the present study was to compare the complete mitochondrial genome sequence from one N. americanus individual from Togo with another from China, in order to estimate the magnitude of genetic variability for different mitochondrial genes and noncoding regions. For the 12 protein genes, this comparison revealed sequence differences at both the nucleotide (3 – 7%) and amino acid (1 – 7%) levels. The most conserved of these was the nad4L gene, whereas the nad1 gene was least conserved at both the nucleotide and amino acid levels. Nucleotide differences were also detected in 14 of the 22 transfer RNAs (trns) (1 – 13%), the AT-rich region (,8%), non-coding regions (8 – 25%) and in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rrn) (,1%). Comparison of the rrnL sequences among multiple individual worms revealed nine unequivocal nucleotide differences between N. americanus from the two countries. Consistent with previous studies, these findings provide evidence for substantial genetic variation within N. americanus, which may have implications for the transmission and control of hookworm disease. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Hookworm; Necator americanus; Mitochondrial genome; rrnL; Genetic variation
1. Introduction It is estimated that over one billion people are infected with hookworms, of which , 6% are clinically affected (Albonico et al., 1999). Necator americanus is one of the most important hookworm species infecting humans (Albonico et al., 1999). This parasite is endemic in many tropical and subtropical regions of the world, including parts of Africa, India, China, south-east Asia, the south-west Pacific Islands, South and Central America, the Caribbean Islands and southern USA (e.g. Schad and Warren, 1990; Liu et al., 1999; Behnke et al., 2000; Gandhi et al., 2001). The adult worms live in the small intestine where they attach to the mucosa and suck blood, consequently causing anaemia. In children, the anaemia can seriously affect both q The nucleotide sequences reported herein have been deposited in the DDBJ, EMBL and GenBank databases under the Accession numbers AJ556111– AJ556133, AJ556178– AJ556180 and AJ556134. * Corresponding author. Tel.: þ 61-3-97312330; fax: þ 61-3-97312366. E-mail address: [email protected] (N.B. Chilton).
physical and cognitive development, the latter of which can also result in intellectual impairment (Hotez and Pritchard, 1995). Diagnosis of hookworm infections usually relies on the detection of eggs in human faeces and/or the identification of larvae by copro-culture (Polderman et al., 1991; Blotkamp et al., 1993; Polderman and Blotkamp, 1995). Throughout much of its distribution, N. americanus occurs in sympatry with other hookworms, such as Ancylostoma duodenale or Ancylostoma ceylanicum (see Nelson, 1990; Schad and Warren, 1990; Hotez and Pritchard, 1995) or with the nodule worm Oesophagostomum bifurcum in Africa (Polderman et al., 1991, 1999; Polderman and Blotkamp, 1995). However, these nematode species differ significantly in their life cycle, pathogenesis and epidemiology (e.g. Schad and Warren, 1990; Polderman et al., 1999). Since the eggs from these different species (shed in human faeces) cannot be distinguished unequivocally using morphological characters, species-specific diagnosis by coproscopy is compromised. Hence, an effective PCR-based approach
0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0020-7519(03)00129-2
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has been established for the specific amplification of N. americanus DNA from human faeces in regions of Africa where this hookworm occurs in sympatry with O. bifurcum (see Verweij et al., 2001). Previous studies of N. americanus have detected sequence variation in the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA (rDNA) among individuals from Togo (Africa) and from China or Malaysia (e.g. Gasser et al., 1998; Romstad et al., 1998). Four nucleotide differences (i.e. purine substitutions) in part of the mitochondrial (mt) cytochrome c oxidase subunit 1 gene (cox1) were also detected between N. americanus individuals from Togo and those from China (Hu et al., 2002c). This finding raised the possibility that N. americanus from different geographical regions may represent more than one species (Romstad et al., 1998). However, it has been argued that the phylogenetic species concept (i.e. considering only derived character states in analyses) should be used for the delineation of species (e.g. Nixon and Wheeler, 1990; Adams, 1998; Wheeler, 1999; Nadler et al., 2000). A re-examination of the cox1 sequence data from Hu et al. (2002c) suggests four derived character states for the N. americanus population from Togo but none for the Chinese population, using A. duodenale as the outgroup. Thus, a greater number of genes need to be sequenced, in order to define derived character states to distinguish N. americanus from different geographical origins. Mitochondrial genes are considered to be well-suited for investigating the population genetics of nematodes and for differentiating sibling species, because they have higher substitution rates than nuclear genes (Anderson et al., 1998; Blouin, 2002). Recently, a long PCR-based approach was used to obtain the complete mt genome sequence (comprising 36 genes) from an individual N. americanus from China (Hu et al., 2002a,b). In the present study, this mt genome sequence was compared with that obtained from an individual of N. americanus from Togo, in order to estimate the levels of nucleotide and/or predicted amino acid sequence differences (for individual genes). Extending from this, the genetic variation between N. americanus from these two geographical regions was assessed by comparing nucleotide sequence data (from multiple individuals) for the longest of the two most conserved mt genes.
2. Materials and methods 2.1. Parasites and DNA isolation Adults of N. americanus from the village of Naki-Est, Region des Savannes, Togo, Africa ( ¼ Na-Togo) and from Yiwu county, Zhejiang province, China ( ¼ Na-China), and a morphologically distinct species of hookworm, A. duodenale, from the same county in China (Table 1) were collected from faeces following the treatment of humans with pyrantel pamoate (see Polderman et al., 1991). Worms were washed extensively in physiological saline, identified morphologically, fixed in 70% ethanol and then frozen at 2 20 8C until use. Total genomic DNA was extracted from individual worms using sodium dodecylsulphate/proteinase K treatment (Gasser et al., 1993), followed by spin-column purification (Wizarde Clean-Up, Promega).
2.2. Mitochondrial genome sequencing and analyses A long-PCR approach (Hu et al., 2002a) was employed to determine the complete mt genome sequence from an individual worm of Na-Togo (sample T17), the morphological identification of which had been verified by comparison of its ITS-2 sequences with those reported previously for N. americanus from Togo (Romstad et al., 1998). In brief, the entire mt genome was amplified in two overlapping fragments (, 10 and , 5 kb) by PCR (Expande 20 kb plus kit, Roche) using primer sets 5F-40R and 39F-42R, respectively (Hu et al., 2002b). The cycling conditions used were: 92 8C, 2 min (initial denaturation); then 92 8C, 10 s (denaturation); 50 8C, 30 s (annealing); 68 or 60 8C, 10 min (extension) for 10 cycles, followed by 92 8C for 10 s, 50 8C for 30 s and 68 or 60 8C for 10 min for 20 cycles, with a cycle elongation of 10 s of each cycle, and a final extension at 68 or 60 8C for 7 min. The extension temperatures of 68 and 60 8C were used specifically for primer sets MH5F-MH40R and MH39F-MH42R, respectively. Amplicons were detected upon ultraviolet transillumination of 1% (w/v) agarose gels after electrophoresis in TBE (65 mM Tris – HCl, 27 mM boric acid, 1 mM EDTA, pH 9) buffer and ethidium bromide staining. They were then
Table 1 Individuals of Necator americanus from Togo (Na-Togo) and China (Na-China) and of Ancylostoma duodenale, and accession numbers for their mt rrnL sequences Parasite
No. of individuals
Sample codes
Accession numbers
Na-Togo Na-China A. duodenale
14 11 4
Nal–Na5, T1, T2, T4– T6, T16, T17a, T18, TN1 NaQ6, NaQ8, NaQ9, NaQ11–NaQ15, NaQ18, NaQ19, NaQ22b AdQ22, AdQ23, AdQ34, AdQ21b
AJ556111–AJ556123, AJ556134a AJ556124–AJ556133, AJ417719b AJ556178–AJ556180, AJ417718b
a b
The complete mitochondrial genome sequence for this individual was determined during the present study. Complete mitochondrial genome sequences for these individuals were published previously in Hu et al. (2002a).
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
purified over spin columns (Wizarde PCR-Prep, Promega) and used as templates for subsequent automated sequencing (ABI BigDye chemistry v.1; in a PE Applied Biosystems 377 sequencer), employing a primer walking strategy. Primers used previously for the sequencing of the mt genome of NaChina (sample code NaQ22; see Fig. 1 in Hu et al., 2002b) were also used herein, although five additional primers (details available from authors) were designed to sequence the ATrich region. Contiguous sequences were aligned with the mt genome sequence of Na-China (accession number: AJ417719) using the Clustal X program v.1.53b (Thompson et al., 1997). The nucleotide sequences of the 12 protein genes, two rrn genes and two non-coding regions were compared between the two Necator mt genomes. The amino acid sequences were conceptually translated from individual mt protein genes of Na-Togo using the MacVector v.4.1.4 program (Kodak), and their identities compared with those from Na-China. 2.3. Sequencing of the rrnL gene and phylogenetic analysis Two primers, 65F and 80R (see Fig. 1 of Hu et al., 2002b), designed to regions within the cox2 and nad5 genes, respectively, were employed for long-PCR amplification and subsequent sequencing of the large subunit of mt ribosomal RNA (rrnL) from individual N. americanus (Table 1). The rrnL sequence was also obtained from three individuals of A. duodenale (Table 1). These sequences and a previously published rrnL sequence of A. duodenale (accession number AJ417718, sample code AdQ21; Hu et al., 2002a) were used as the outgroup for phylogenetic analyses, because this morphologically distinct species represents a sister taxon to N. americanus (see Lichtenfels, 1974). All rrnL sequences were aligned using Clustal X, and the alignment was modified manually, based on the predicted secondary structure of the rrnL for hookworms (Hu et al., 2002a). Phylogenetic trees were constructed using a maximum parsimony (MP) analysis, employing PAUP* v.4b10 (Swofford, 1999). Characters were weighted equally and treated as unordered. Alignment gaps were treated as a fifth character state. An heuristic search with TBR-branch swapping was used to infer the shortest trees. The length, consistency index excluding uninformative characters (C.I.) and retention index (R.I.) of the most parsimonious trees were recorded. A bootstrap analysis (using 1,000 replicates) was conducted using heuristic searches and TBR branch swapping with the MulTrees option, in order to determine the relative support for clades of the consensus tree.
3. Results 3.1. Comparative analysis of the mt genome sequence of Na-Togo with that of Na-China The full mt genome sequence of Na-Togo (sample code
957
T17) was 13,606 bp in length, 1 bp longer than that reported for Na-China (accession number AJ417719). The identity, number and arrangement of the mt genes (i.e. 12 protein genes, two rrn genes and 22 transfer RNA [trn ] genes) and non-coding regions were the same as that of Na-China (Hu et al., 2002a). A comparison of the nucleotide sequences of each mt gene and non-coding region, as well as the amino acid sequences, conceptually translated from all protein genes of the two N. americanus, are given in Table 2. The sequence lengths of each gene and non-coding region were the same for Na-Togo and Na-China, except for variation of one nucleotide in the rrnS gene and of one to three nucleotides for each of three trn genes (Table 2). The magnitude of sequence variation in each gene and non-coding region between Na-Togo and Na-China ranged from 1.1 to 24.5% (except for the 8 trns for which no variation was detected). The greatest variation between Na-Togo and Na-China was in the non-coding regions, whereas the least difference (, 2%) was detected in the two rrn subunits (Table 2). Comparison of transition/transversion ðTs=TvÞ ratios for individual genes and regions revealed 2.3– 11 times more transitions than transversions in coding genes compared with non-coding regions ðTs=Tv , 1Þ. Amino acid sequences inferred from individual mt protein genes of Na-Togo were compared with those of Na-China. The amino acid sequence differences ranged from 1.3 to 7.2%, with NAD4L being the most conserved protein and NAD1 the least conserved. There was a total of 120 amino acid substitutions (for an alignment length of 3,418 positions) in the 12 proteins (Table 3), the majority of which were in the proteins COB ðn ¼ 24Þ, NAD1 ðn ¼ 21Þ and NAD5 ðn ¼ 13Þ. The number of substitutions at different codon positions was also examined, revealing the following pattern: third codon position ðn ¼ 8 – 59Þ . first codon position ðn ¼ 1 – 14Þ . second codon position ðn ¼ 0 – 10Þ. Of the 120 amino acid substitutions, 87 (73%) represented potentially informative characters (for a cladistic analysis using A. duodenale as the outgroup), the greatest number of these being in the proteins COB ðn ¼ 17Þ, NAD1 ðn ¼ 16Þ and COX1 ðn ¼ 10Þ (Table 3). 3.2. Analysis of the expanded rrnL sequence data set Comparison of the mt genomes of Na-Togo and Na-China showed that the rrnS and rrnL were the two most conserved genes, the latter of which was , 258 bp longer (Table 2). Therefore, the sequence variation in the rrnL gene among multiple individuals of N. americanus from the two geographical origins was assessed (see Table 4). The complete rrnL sequence determined for 12 of the 14 Na-Togo individuals was 958 bp. Individual Na5 had one insertion, while individual T2 had two insertions. Nucleotide variation among Na-Togo samples was detected at nine other sites (i.e. alignment positions 160, 229, 259, 270, 295, 493, 558, 577 and 618; Table 4). Sequences for eight of the 11 Na-China individuals were of the same
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Table 2 Nucleotide and/or predicted amino acid sequence differences for each mt gene and non-coding region for Necator americanus from Togo, compared with N. americanus from China (accession number AJ417719) Gene/region
atp6 cob cox1 cox2 cox3 nad1 nad2 nad3 nad4 nad4L nad5 nad6 All 22 trns rrnS rrnL AT-rich region 1st Non-codingf 2nd Non-codingh Otheri
Nucleotide sequence length
Nucleotide difference (%)
Ts/Tva ratio
1st
2nd
3rd
Amino acid sequence length
No. of substitutions at codon position
Amino acid difference (%)
598 1,113 1,575 696 766 873 845 336 1,230 234 1,582 435
5.2 6.1 5.1 6.0 3.5 7.4 5.7 6.5 4.9 3.4 4.5 5.3
9.3:1 2.8:1 4.3:1 9.5:1 5.8:1 4.5:1 8.6:1 10.0:1 11.0:1 9.0:0 5.5:1 2.3:1
1 14 6 3 5 14 8 2 8 1 4 3
0 10 4 0 2 8 2 3 2 0 8 2
30 44 70 39 20 44 38 17 50 8 59 18
199 370 524 231 255 290 281 111 409 77 527 144
1.5 6.5 2.1 1.7 2.7 7.2 3.9 4.5 2.7 1.3 2.5 5.6
1,223b
2.5c
3.1:1c
–d
–
–
–
–
700e 958
1.1 1.4
2.5:1 10.0:1
– –
– –
– –
– –
– –
173 68g 67 134
8.1 8.2 20.9 24.5
0.7:1 1.0:0 0.6:1 1.1:1
– – – –
– – – –
– – – –
– – – –
– – – –
a
Ts, transition and Tv, transversion. Nucleotide lengths of Na-Togo trns were 52 (S[UCN]), 53 (S[AGN]), 54 (R, N, G, P and T), 55 (A, C, Q, L[CUN], L[UUR], F, Y and V), 56 (D, H and W), 59 (E and I), 60 (M) or 62 (K). The trn sequence for Na-China was one nucleotide shorter for M, three shorter for E and one longer for F. Standard abbreviations are used for each trn. c Represents mean value; number of nucleotide differences between Na-Togo and Na-China were either 1 (A, N, C, Q, I, K, P and V), 2 (L[UUR]), 3 (D, M, F and S[UCN]) or 8 (E). No differences were detected in other trns. d Not applicable. e The sequence of Na-China was one nucleotide shorter. f Located between the genes nad4 and cox1. g The sequence of Na-China was five nucleotides longer. h Located between the genes nad3 and nad5. i Representing intergenic regions of currently unknown function. The sequences of Na-China were two nucleotides shorter (over the total length). b
length (958 bp), whereas three (NaQ12, NaQ18 and NaQ19) were one base longer. Also, nucleotide variation occurred at nine other sites (i.e. alignment positions 7, 258, 262, 292, 370, 482, 553, 618 and 634; Table 4), only one of which was variable among all Na-Togo individuals examined. The length of the rrnL sequences for the four A. duodenale individuals was 957 or 958 bp. Alignment of the rrnL sequences revealed that all individuals of Na-Togo differed at nine nucleotide positions when compared with Na-China (Table 4). These differences represented two indels, six purine transitions (A $ G) and one transversion (A $ T). A MP analysis yielded two equally most parsimonious trees with a length of 199, a C.I. of 0.95 and a R.I. of 0.98. The strict consensus tree is shown in Fig. 1. There was moderate support (bootstrap values of 74 –78%) for the separation of Na-Togo from Na-China individuals into two distinct clades. Six of the nine nucleotide differences were considered as derived (i.e. autapo-
morphic) characters, with A. duodenale as the outgroup in a cladistic analysis, three for Na-Togo individuals (at positions 25 and 344, and between positions 260 and 261, with respect to the published sequence of Na-China sample NaQ22; see Table 4) and three for Na-China individuals (at positions 278, 371 and 790; Table 4).
4. Discussion In the present study, substantial nucleotide differences were detected in the complete mt genome between an individual of N. americanus from Togo and another from China. The variation detected in the 12 protein-coding genes (3 –7%) and in non-coding regions (8 – 25%) was consistent with previous findings of variation in part of the mt cox1 gene among N. americanus individuals (Hawdon et al., 2001; Hu et al., 2002c) and in the nucleotide sequences of
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
959
Table 3 Amino acid substitutions at particular sites in all 12 conceptually translated mt proteins of Necator americanus (Na) from Togo or China and of Ancylostoma duodenale (Ad). Position numbers in bold-type indicate potentially phylogenetic informative sites for N. americanus, using A. duodenale as the outgroup Gene
Position
Na
Ad
Togo
China
atp6
33 53 63
K L L
N V F
K L L
cob
1 17 63 72 145 155 156 157 193 229 236 241 242 262 284 286 295 302 316 320 358 360 362 370
V I V Y N P A I H V G M Y D V S G M V N V L Y V
I T G C S S T F D M V C C V I G V L F I L F F I
I T V Y S P S I H V I F Y V V G V F M G M L F I
3 65 255 302 428 436 456 461 462 467 491
F V S D G H M A P V S
L A L V V Y V S S I N
L I L V V H V S S I S
cox1
Gene
Position
Na
Ad
Togo
China
cox2
37 40 153 173
V I V V
I V I I
V V I V
cox3
23 38 47 67 176 247 250
G I A H K I G
S V V D S V V
G L V H S V V
49 55 61 137 154 157 160 170 177 207 211 212 230 243 257 263 265 271 277 283 284
T I K A T V K V I A K G V S T D L R I S H
A V E V A L S I V V S V I M S Y M W V V Y
A V E A A I S L L V S V V M S Y M W I M F
16 22 28 51 97 125 133 191 203 262 278
S I V N Y M I V M I V
G V I I F V V I V V I
S I V N F I F V M F I
nad1
nad2
the nuclear ITS rDNA (Gasser et al., 1998; Romstad et al., 1998). However, for N. americanus, as for other species of nematode (e.g. Blouin, 2002), there is greater within-species variation in mt protein-genes than in the ITS. For example, the magnitude of the nucleotide sequence variation in the 12 mt protein genes (. 3%) was greater than the 15 (2%) variable positions in the ITS (over 852 bp) detected among
Gene
Position
Na
Ad
Togo
China
nad3
3 13 63 65 110
N M S T V
S L F I I
N L F I V
nad4
5 16 24 86 87 94 205 218 347 349 387
F I L G A D V H I I Y
L V F V S Y L Y V V C
L L M V S Y A Y I V F
6
V
I
I
nad5
27 246 284 322 326 347 354 359 363 412 427 438 522
F I I I L S S L S A N S I
L V S M P L F M L V S L F
L V S M L A F I F V S L L
nad6
13 19 31 34 72 99 100 101
D K F I S L I T
A N L M K V V A
G N L L K V L Y
nad4L
multiple N. americanus individuals (e.g. Romstad et al., 1998; Chilton et al., unpublished data). Comparison between the N. americanus from Togo and China also revealed variation at 120 amino acid positions in the 12 predicted mt protein sequences (Table 2). This magnitude of amino acid variation seems high, given that mt proteins are considered to be highly conserved within a
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Table 4 Nucleotide differences in the mt rrnL between Necator americanus from Togo (Na-Togo) and from China (Na-China), and from the consensus sequence for 4 Ancylostoma duodenale (Ad) individuals Samples Positions 7
13/14 15 25 160 193 229 258 259 260/261 262 270 271/272 278 292 295 344 370 371 374 482 493 553 558 577 618 634 675/676 790 953/954
A – A – A – A – A T A – A T A – A – A – A – A – A – A –
A A A A A A A A A A A A A A
G G G G G G G G G G G G G G
G G G G G T G G G G G G G G
A A A A A A A A A A A A A A
G G G G A G G G G G G G G G
G G G G G G G G G G G G G G
T T T T T T T C T T T T T T
T T T T T T T T T T T T T T
A A A A A A A A A A A A A A
A T T T T T T T T T T T T T
– – – – – – – – – – – – – –
A A A A A A A A A A A A A A
T T T T T T T T T T T T T T
G G G G G G G G G A G G G G
– – – – – – – – – – – – – –
A A A A A A A A A A A A A A
G G G G G G G G G G G G G G
A A A A A A A A A A A A A A
A A A A A A A A A A A A A A
T T T T T T T T T T T A T T
G G G G G G G G G G G G G G
A A A A A A A A A A A A G A
A A A G A A A A A A A A G A
G G G A G G G G G G G G G G
G G G G G G G G G G G G G G
– – – – – – G – – – – – – –
A A A A A A A A A A A A A A
– – – – – – – – – – – – – –
NaQ6 NaQ8 NaQ9 NaQ11 NaQ12 NaQ13 NaQ14 NaQ15 NaQ18 NaQ19 NaQ22
G G G A G A G A G G G
– – – – – – – – – – –
G G G G G G G G G G G
A A A A A A A A A A A
G G G G G G G G G G G
G G G G G G G G G G G
G G G G G G G G G G G
A A A A A G A A A A A
T T T T T T T T T T T
– – – – – – – – – – –
A A A G A A A G A A G
T T T T T T T T T T T
– – – – T – – – – T –
G G G G G G G G G G G
T T T T T C T T T T T
G G G G G G G G G G G
T T T T T T T T T T T
A G G A A A G A G A A
A A A A A A A A A A A
T T T T T T T T T T T
A A A A G A A A A G G
T T T T T T T T T T T
G G G G G G G A G G G
A A A A A A A A A A A
A A A A A A A A A A A
A A G A A G A G A A A
G G A G G G G G G G G
– – – – – – – – – – –
G G G G G G G G G G G
– – – – – – – – T – –
Ad
T
–
T
A
T
T
T
R
T
–
T
T
T
A
T
T
T
A
G
G
A
T
T
T
G
A
A
–
A
–
Position numbers correspond to the published mt genome sequence of N. americanus (accession number AJ417719; Hu et al., 2002a) ( –, deletion; nucleotides and deletions in bold-type represent fixed differences between Na-Togo and Na-China).
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
Na1 Na2 Na3 Na4 Na5 T1 T2 T4 T5 T6 T16 T18 TN1 T17
M. Hu et al. / International Journal for Parasitology 33 (2003) 955–963
Fig. 1. Strict consensus tree, constructed using MP and depicting the relationships of Necator americanus from China (Na-China), and from Togo (Na-Togo) and Ancylostoma duodenale (outgroup) based on sequence data for the mt rrnL. Numerals above the branches represent bootstrap values (%). Two sets of three derived character states separating Na-Togo (positions 25, 344 and between positions 260/261) from Na-China (positions 278, 371 and 790) are shown on each branch, respectively. Position numbers correspond to those in Hu et al. (2002a).
species due to structural and functional constraints (see Simon et al., 1994). Also, previous studies of hookworms have detected little to no within-species variation in protein sequences. For instance, amino acid substitutions were recorded at only two of 196 positions (based on a comparison of conceptually translated sequences originating from GenBanke accession numbers AF303135–AF303159) in partial COX1 among 151 N. americanus samples from four locations in China (Hawdon et al., 2001). Similarly, Hu et al. (2002c) detected no within-species variation in a different COX1 region of 131 amino acids for N. americanus and for related hookworms, including A. duodenale and Ancylostoma caninum. The greatest numbers of amino acid differences recorded herein were in the proteins COB ðn ¼ 24Þ and NAD1 ðn ¼ 21Þ. The significance of the amino acid sequence variation between N. americanus from different geographical origins needs to be assessed further, because for invertebrates generally, there are presently limited data on the magnitude of within-species variation in mt protein sequences.
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Genetic variation within N. americanus was also detected herein for the two mt ribosomal RNA gene subunits (rrnS and rrnL). These subunits are considered to be more conserved in sequence than the protein genes (see review of Simon et al., 1994), which was supported by the data for N. americanus. Comparison of the complete genome data set between the two individuals (samples T17 and NaQ22) demonstrated less sequence variation in the rrnS and rrnL genes (, 1%) compared with the protein genes (. 3%) and the non-coding regions (8 –21%). The rrnL gene was the larger of these two most conserved genes in N. americanus, thus being more likely to provide a greater number of variable sites to examine the magnitude of genetic variation in the parasite between different geographical origins. A comparison of the complete rrnL sequences (958 bp) for 25 N. americanus individuals revealed 30 (3%) variable positions. The nucleotide sequences of all 12 mt protein genes, the rrnS and rrnL genes and the non-coding regions were used to assess the genetic variation among N. americanus from different geographical origins. For instance, the rrnL sequences of all Chinese N. americanus individuals differed from those from Togo at nine unequivocal nucleotide positions. This result is consistent with previous findings of genetic differences among geographically isolated populations of N. americanus (i.e. from Togo, China and/or Malaysia) based on the sequences and/or single strand conformation polymorphism (SSCP) analyses of nuclear ITS-1 and/or ITS-2 regions (Gasser et al., 1998; Romstad et al., 1998) and the mt cox1 gene (Hu et al., 2002c). For example, Chinese N. americanus differed from those from Togo at four unequivocal nucleotide positions in a 395 bp region of cox1 (Hu et al., 2002c). Taken together, this molecular evidence (Gasser et al., 1998; Romstad et al., 1998; Hu et al., 2002c) indicates that the N. americanus population from Togo and from that China each represents a distinct set of genotypes, as a consequence of geographical isolation over a long period of time. Whether each set of genotypes represents a genetically distinct but morphologically similar (i.e. cryptic) species remains to be determined. Previously, the significance of the genetic differences between N. americanus populations has been interpreted phenetically (e.g. Romstad et al., 1998) rather than in an evolutionary context (i.e. based on the phylogenetic species concept; e.g. Nixon and Wheeler, 1990; Wheeler, 1999). The latter approach (i.e. based on shared, derived characterstate data) is considered to be more objective and is proposed to eliminate some of the predictive errors associated with phenetic analyses (Adams, 1998). For example, Nadler et al. (2000) employed a cladistic approach in analyses of sequence data (nuclear ITS-1 and 28S rDNA), in combination with morphometric data, to infer that hookworms (Uncinaria spp.) from the Californian sea lion (Zalophus californianus) and from the northern fur seal (Callorhinus ursinus) collected in sympatry each represent a separate species. A similar approach, using sequence data for several
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mitochondrial and nuclear DNA genes, could be adopted to test the specific status of N. americanus. For instance, of the nine rrnL nucleotide positions differing between the Chinese and African N. americanus, six represented autapomorphic (i.e. derived) characters (three for African individuals and three for Chinese individuals), using A. duodenale as the outgroup. There are also other informative characters in the ITS rDNA (Romstad et al., 1998) and in mt cox1 (Hu et al., 2002c). Even though these results indicate isolation of the Chinese and African N. americanus for a long period of time, some caution is warranted with an interpretation supporting the ‘cryptic species hypothesis’ for the following reasons. Firstly, although A. duodenale is a sister taxon to N. americanus (see Lichtenfels, 1974), there may be other hookworm species which represent more suitable outgroups to test the specific status of N. americanus populations. For example, Necator suillus from pigs in the Caribbean or Necator congolensis from the chimpanzee in Africa (Ackert and Payne, 1923) could be used in cladistic analyses, provided that they indeed represent distinct morphospecies. Secondly, more samples of N. americanus need to be examined from several locations in China and in Africa, and preferably from other continents (e.g. South and Central America), in order to assess the biological and evolutionary significance of the sequence data obtained. For instance, sampling from Central America would be informative, because preliminary SSCP-based analysis of the ITS-1 and ITS-2 for three N. americanus individuals from Guatemala suggests that some have ‘mixed genotypes’ compared with the parasite from Africa and Asia (cf. Gasser et al., 1998). The significance of this finding requires investigation using the rrnL and mt protein sequence data, together with morphological study of specimens. In conclusion, analysis of mt nucleotide and/or amino acid sequence data sets supports the proposal that the gene pools of N. americanus from Togo and China have been isolated for a substantial period of time. Whether each gene pool represents a distinct species is still unclear. Given that N. americanus is widely distributed across the world (in tropical and subtropical regions) and is a significant human pathogen, answering this question may provide insights into the population biology and epidemiology of the parasite, and may assist in the implementation of control measures.
Acknowledgements Funding support was from various sources, including the Australian Research Council. Min Hu has been the recipient of a postgraduate scholarship from The University of Melbourne and a Travel Award from the Australian Society for Parasitology. Samples used in this study were originally provided by Anton Polderman and Qian Bao-Zhen. The two anonymous reviewers are thanked for their constructive comments on the manuscript.
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