Comparative analysis of mitochondrial genome data for Necator americanus from two endemic regions reveals substantial genetic variation

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 non-coding 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.

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 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, Polderman et al., 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 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, Hu et al., 2002b). 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.