The mitochondrial genomes of the human hookworms, Ancylostoma duodenale and Necator americanus (Nematoda: Secernentea)

Abstract

The complete mitochondrial genome sequences were determined for two species of human hookworms, Ancylostoma duodenale (13,721 bp) and Necator americanus (13,604 bp). The circular hookworm genomes are amongst the smallest reported to date for any metazoan organism. Their relatively small size relates mainly to a reduced length in the AT-rich region. Both hookworm genomes encode 12 protein, two ribosomal RNA and 22 transfer RNA genes, but lack the ATP synthetase subunit 8 gene, which is consistent with three other species of Secernentea studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T, but low in G and C. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. For both hookworm species, genes were arranged in the same order as for Caenorhabditis elegans, except for the presence of a non-coding region between genes nad3 and nad5. In A. duodenale, this non-coding region is predicted to form a stem-and-loop structure which is not present in N. americanus. The mitochondrial genome structure for both hookworms differs from Ascaris suum only in the location of the AT-rich region, whereas there are substantial differences when compared with Onchocerca volvulus, including four gene or gene-block translocations and the positions of some transfer RNA genes and the AT-rich region. Based on genome organisation and amino acid sequence identity, A. duodenale and N. americanus were more closely related to C. elegans than to A. suum or O. volvulus (all secernentean nematodes), consistent with a previous phylogenetic study using ribosomal DNA sequence data. Determination of the complete mitochondrial genome sequences for two human hookworms (the first members of the order Strongylida ever sequenced) provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance.

Introduction

Most metazoa, with the exception of some cnidarians (Bridge et al., 1992, Boore, 1999), possess compact, circular mitochondrial (mt) genomes which vary in size from 14 to 19 kb (Saccone et al., 1999). The metazoan mt genome usually contains a complement of genes encoding 12–13 protein subunits of the enzymes involved in oxidative phosphorylation (cytochrome c oxidase subunits I–III, cytochrome b, the ATPase complex subunits 6 and/or 8 and the NADH dehydrogenase subunits 1–6 and 4L), 22 transfer RNA (trn) genes, and the small and large ribosomal RNA (rrnS and rrnL) genes. In addition to these genes, there is a non-coding region (D-loop, AT-rich or control region), which is known to regulate the initiation and control of replication and transcription in vertebrates (Clayton, 1991, Clayton, 1992, Shadel and Clayton, 1996) and invertebrates (Clary and Wolstenholme, 1985, Boyce et al., 1989). Given the eubacterial ancestry of the mitochondrion (Saccone et al., 2000), genetic code, codon usage, trn structures and some other features of the metazoan mt genome are distinct from those of the nuclear genome (Osawa et al., 1992, Wolstenholme, 1992). For example, in addition to ATG (the ‘universal start codon’ for methionine in the nuclear genome), the codons ATT, GTT and ATA can be used as translation initiation codons, and some genes may end in T or TA rather than in a full stop codon, TAA or TAG (Wolstenholme, 1992).

To date, over 100 complete mt genome sequences have been published for metazoan organisms representing 11 different phyla, of which more than 70 represent chordates (Boore, 1999, Saccone et al., 1999). In spite of advances in mt genomics, there are serious gaps in our knowledge for many groups of metazoan parasites, although significant progress has been made recently in characterising the mt genomes of a range of flatworms (Platyhelminthes), such as species of Schistosoma, Paragonimus, Echinococcus and Taenia (see Le et al., 2000a, Le et al., 2000b, Le et al., 2000c, von Nickisch-Rosenegk et al., 2001). In contrast, only three full-length mtDNA sequences have been published for parasitic nematodes of socio-economic importance, namely Ascaris suum (Ascaridida), Onchocerca volvulus (Spirurida) and Trichinella spiralis (Enoplida) (see Okimoto et al., 1992, Keddie et al., 1998, Lavrov and Brown, 2001), and that of the free-living nematode Caenorhabditis elegans (Rhabditida) (Okimoto et al., 1992), while partial genome sequences exist for the rootknot nematode, Meloidogyne javanica (Tylenchida) and the mosquito parasite, Romanomermis culicivorax (Stichosomida) (Okimoto et al., 1991, Hyman and Beck Azevedo, 1996). This lack of knowledge of mt genomics for the large phylum of the Nematoda constitutes a major limitation for population genetic and phylogenetic studies, and for investigating fundamental aspects, such as cell metabolism, apoptosis and ageing (Boore, 1999, Le et al., 2000a). The aim of this study was to commence filling some of these knowledge gaps by determining the structure and organisation of the complete mt genome (sequence) for two species of human hookworm, Ancylostoma duodenale and Necator americanus (order Strongylida), which are blood-sucking intestinal parasites of major socio-economic importance, clinically affecting ∼78 million of the ∼1.3 billion humans infected globally (Albonico et al., 1999). Features of both hookworm genomes, such as the gene arrangements, structures, compositions, as well as translation and initiation codons and codon usage patterns were compared with those of the other nematodes (C. elegans, A. suum and O. volvulus) of the class Secernentea for which complete mtDNA sequences were available.