BACKGROUND
comparative sequence analysis is an approach proven to aid in recognition of genes and defining of their function, especially when comparing genomes of close evolutionary distance.
in addition, when partial genomes are placed in a context of a well-studied and fully sequenced model organism they can greatly facilitate the understanding of the less studied organisms' biology.
hookworms are blood-feeding nematodes that infect one billion people causing iron deficiency anemia and retarded physical and cognitive development in children  <cit> .
the two major species infecting humans are necator americanus and ancylostoma duodenale.
the closely related hookworm species of canids, ancylostoma caninum, and canines and felines, a. ceylanicum, are minor parasites of humans, but are important as laboratory models for hookworm infection and disease.
other hookworms infect raccoons, sheep, seals and a variety of other mammals  <cit>
adult  hookworms inhabit the small intestine and produce eggs that pass in the feces and hatch in the soil.
the first stage larva  feeds on bacteria and molts twice to form the non-feeding, infective third stage .
il <dig> enters the host by penetrating the skin, or orally in the case of ancylostoma species, molts twice, and matures to ad in the small intestine.
a. duodenale and a. caninum l3s can also infect a host, temporarily abort maturation and enter an arrested state  within the host's somatic tissues  <cit> , reactivating in response to host physiological changes such as pregnancy  <cit> .
current hookworm control strategies are limited to de-worming of infected people using anthelmintic drugs.
however, rapid re-infection in endemic areas and the lack of sterile immunity necessitates repeated treatments and can in turn result in resistance.
additionally, tissue-arrested stages are relatively resilient to the effects of anthelmintics  <cit> .
the human hookworm vaccine initiative is beginning clinical trials of a larval hookworm antigen, asp- <dig>  from n. americanus, as a vaccine antigen  <cit> .
there is a critical need for further research to identify new vaccine and drug targets as well as to better understand hookworm biology.
lack of sequence information has been a major hindrance to hookworm molecular studies.
high throughput sequencing of expressed sequence tags  has proven a cost-effective tool for discovering genes  <cit> .
because the hookworm superfamily  falls within nematode clade v  <cit> , which also contains the well-studied model nematode caenorhabditis elegans  <cit> , predictions may be made and tested based on their close relatedness.
previous genome-based characterization of hookworms has been limited to sampling of few hundred ests  <cit>  and molecular studies of individual genes of interest .
est approaches have also been initiated for other strongylid parasites including haemonchus contortus  <cit>  and nippostrongylus brasiliensis  <cit> .
in this report we describe the comparative analyses of almost  <dig>  ests from  <dig> different cdna libraries representing pre-parasitic and parasitic larval through adult stages of the hookworms a. caninum and a. ceylanicum.
the dataset defined nearly  <dig>  hookworm genes, including a number of putative developmentally expressed genes and candidates for further study as drug target or vaccine components.
RESULTS
nearly  <dig>  ancylostoma derived ests were submitted to genbank between  <dig> and  <dig> .
for simplicity, the results and analysis described are presented in the same order beginning with a. caninum and followed by a. ceylanicum, except where specified.
est acquisition and nemagene organization
ests originated from  <dig> cdna libraries, representing three and two life-cycle stages respectively .
clustering, implemented to reduce data redundancy and improve sequence quality and length, grouped ests into contigs which were further organized into clusters , providing a non-redundant catalog of represented genes.
ests within a contig derive from nearly identical transcripts while contigs within a cluster may arise from splice isoforms, alleles, or closely related paralogs  <cit> .
fifty-one potentially chimeric ests were discarded.
clusters ranged in size from a single est to  <dig> and  <dig> for a. caninum and a. ceylanicum respectively .
most clusters for each species  have ten or fewer ests.
gc content for coding sequences was similar in the two species  and consistent with other clade v nematodes like c. elegans and c. briggsae  <cit> .
the number of clusters may overestimate gene discovery, as one gene may be represented by multiple non-overlapping clusters .
by using c. elegans as a reference genome  and discounting for fragmentation calculated as  <dig> % and  <dig>  % respectively  <cit> , the estimated gene numbers were reduced to  <dig>  for a. caninum and  <dig>  for a. ceylanicum giving a gene discovery rate of 41%  and 30% .
these numbers also indicate 20% and 16% representation of all genes for each species respectively.
the number of genes in common for more stage-specific ancylostoma libraries analysed was as low as 9% and 11% .
this may reflect the est sample size or stage-specific expression, as will be discussed.
in either case, the results clearly show the advantage gained for gene discovery in ancylostoma by including diverse life stages in the analysis.
functional classification based on gene ontology and kegg assignments
thirty-four percent of a. caninum and 54% of a. ceylanicum clusters align to interpro domains and 21% and 36% map to gene ontology  respectively.
following this same pattern, a. caninum also had fewer blast matches .
seven of the ten most abundantly represented interpro domains were common to both species .
go representation is shown by biological process, cellular component, and molecular function .
among the most common go categories are protein metabolism  and catalytic activity .
within ancylostoma spp.
clusters that had extracellular mappings, 70% and 56% respectively were in the category of allergen v5/tpx- <dig> proteins  related to the secreted venom proteins from hymenopteran insects .
the ancylostoma secreted proteins  belong to this large gene family  <cit> , members of which been shown to play roles in host-parasite interactions for both mammalian  <cit>  and plant parasitic nematodes  <cit> , and to induce protective responses  <cit> .
asp- <dig> is one of the major proteins secreted by serum-stimulated a. caninum il <dig>  <cit> .
in addition, four a. ceylanicum clusters were classified in extracellular matrix  as tissue inhibitor of metalloprotease  domain proteins.
a timp homolog is reported as the most abundant protein in adult hookworm excretory/secretory products and may inhibit host metalloproteases  <cit> .
ten % and 15% unique clusters for a. caninum and a. ceylanicum respectively, mapped to  <dig> metabolic pathways grouped in  <dig> categories .
complete listings and graphical representations of the kegg mappings are available at .
pathways well represented by both species include glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation and fatty acid biosynthesis and metabolism.
kegg analysis  also suggests specific biochemical differences among ancylostoma stages.
for example, while serum stimulated l3-specific clusters make up to 27% of all ac clusters, they account for 40% of all kegg pathway mappings.
in contrast, il3-specific clusters that account for 55% of all ac clusters make-up only 38% of kegg pathway mappings.
it is unclear whether the predominance of enzyme mappings from the ssl <dig> stage versus il <dig> stage is indicative of greater metabolic activity, greater metabolic complexity, differences in library construction methods, or other differences.
aa.
caninum –  <dig> multiple and  <dig> unique mappings; a. ceylanicum –  <dig> multiple and  <dig> unique mappings.
b cluster, c enzymes
homologs in other organisms, orthologs within ancylostoma spp.
and estimates of selective pressure
within a. ceylanicum clusters, 83% had homology to proteins from other organisms as compared to only 66% for a. caninum .
to investigate why contigs from closely related species would show a difference in identified homologies, we compared sequence lengths and the open reading frame  lengths of contigs with and without homologies in both species.
est lengths and contig lengths, respectively, were shorter for a. caninum  than for a. ceylanicum .
the differences were even more striking for orfs .
hence, a. caninum contigs very likely identify fewer homologs because these sequences are shorter, contain smaller orfs, and probably include more 3' utr versus the superior quality dataset from a. ceylanicum.
most likely, differences in library construction and sampling rather than intrinsic differences between the species explain this discrepancy.
accounting for such differences is important as it keeps analysis focused upon interesting features of the dataset related to the organisms' biology rather than artifactual differences arising from data collection.
the distribution of identified homologs  was consistent with earlier observations  <cit> .
besides c. elegans, one of the more informative nematode datasets for this study is a collection of  <dig>  ests from the human hookworm necator americanus to which homologies were commonly found .
within ancylostoma itself homologies were common with 34% of total a. caninum clusters matching the a. ceylanicum dataset and 44% of total a. ceylanicum clusters matching a. caninum.
searching for putative orthologs between all a. caninum and a. ceylanicum contigs resulted in  <dig>  reciprocal best tblastx hits.
the ortholog pair members were very similar in gc composition  and the average length of alignment was  <dig> bp.
all ortholog pairs  were under purifying selection  and the average ds was  <dig>  ±  <dig>  and dn was  <dig>  ±  <dig> .
the average dn/ds ratio  is higher than that reported for c. briggsae/c.
elegans , and closer to that for mouse/human , indicating that the levels of purifying selection are somewhat different.
in addition, to examine if this purifying selection is more frequently detected in genes with essential function, we cross-referenced the c. elegans genes matched by ancylostoma orthologs with a list of c. elegans genes with available rna interference  information .
of the 67% of the orthologous genes matching c. elegans genes with available rnai data, 45% had an observable phenotype after transcript knock-down.
a vast majority of the observed phenotypes were severe .
ancylostoma orthologs matching c. elegans genes that had phenotypes showed a somewhat lower dn/ds ratio than those matching genes that remained wild type after rnai treatment, though the difference was not statistically significant at p <  <dig>  .
in a 4-way comparison of orthologs in c. elegans, c. briggsae, a. caninum, and a. ceylanicum, the phylogenetic distance between a. caninum and a. ceylanicum is almost identical to that between c. elegans and c. briggsae and the distance between the two genera is just over four times the within genera distance .
average branch lengths for the set of  <dig> orthologous proteins did not show a significant difference in relative rate of molecular change.
maximum likelihood trees  <cit>  were constructed for each 4-way ortholog and relative branch lengths compared between ancylostoma spp.
for both the protein and nucleotide sequences.
for protein sequences,  <dig> trees had equal branch lengths for the hookworm species while  <dig> trees had longer a. caninum branches and  <dig> had longer a. ceylanicum branches.
to look for differences between genes in the ancylostoma species, we constructed a distribution of branch length differences between each of the sister species pairs in our tree.
because some genes may be rapidly evolving in all nematode lineages we evaluated a subset of the trees where the difference in branch lengths between c. briggsae and c. elegans were less than one standard deviation from the mean but which had significantly different branch lengths in the two ancylostoma species.
this final dataset had  <dig> genes with significantly longer branch lengths in a. ceylanicum and  <dig> in a. caninum .
however the set did not show any significant bias towards either species .
this suggests there is no significant rate difference in protein evolution between the two hookworms, although some of these genes are relatively rapidly evolving.
repeating the analysis for nucleotide sequences we find marginally significant differences .
using the c. elegans genome to interpret hookworm sequences
as expected, the c. elegans genome provides the best source of information for interpreting hookworm sequences as a majority of a. caninum and a. ceylanicum clusters with blast homologies outside ancylostoma had c. elegans homologs .
furthermore, c. elegans orthologs of hookworm genes with available rnai or other data provide information that may be relevant to understanding the role of the parasite genes.
of all the ancylostoma clusters with c. elegans homology, 97% and 92% matched c. elegans genes with available rna interference knock-down information , and in turn 33% and 37% of these c. elegans genes produce rnai phenotypes .
phenotype classification  showed that c. elegans genes with expressed ancylostoma homologs were somewhat more likely to have severe phenotypes  <cit> .
hence, certain genes in the ancylostoma datasets encode proteins if disabled may disrupt survival of the parasite.
some examples include abundant clusters .
a group of particular interest is proteins that are required for nematode survival and lack strong homologies outside of the phyla , since these targets could provide for nematode control without toxicities to the host or other non-target organisms.
of the ancylostoma nematode-specific clusters ,  <dig> and  <dig> respectively had c. elegans matches with rnai phenotypes.
among these, ac <dig> cl and ae <dig> cl matched hypothetical protein f42a <dig>  , a gene without a mammalian homolog yet likely involved in multiple developmental processes based on observed mutant phenotypes  <cit> .
homologs are found in at least  <dig> nematode species to date including free-living species , and parasites of mammals  and plants .
further analysis will identify additional genes which warrant detailed investigation.
a sw/tr is swiss-prot and trembl proteinknowledgebase .
b c. elegans homolog has higher probability match than the best genbank descriptor.
a sw/tr is swiss-prot and trembl proteinknowledgebase .
b c. elegans homolog has higher probability match than the best genbank descriptor.
repeating the analysis in stein et al.
<cit>  indicates that 6–7% of c. elegans and c. briggsae proteins are candidate "orphans", lacking homologs outside of the species.
we examined whether these genes are truly orphans that have arisen in a caenorhabditis sub-lineage or are instead genes present in an ancestral nematode that have been lost or evolved beyond recognition in one species.
of candidate orphan proteins, ten from c. elegans  and  <dig> from c. briggsae  match a. caninum and/or a. ceylanicum clusters, with three and eight, respectively, having matches in both species.
most of the c. elegans orphans are hypothetical proteins of unknown function though some had functional information from interpro domains  or mutant phenotypes .
therefore, at least a portion of the genes identified in either c. elegans or c. briggsae as "orphans" are actually ancestral nematode genes with homologs found in other clade v species and further clade v sequencing will likely reveal more such cases.
a of  <dig>  proteins  <dig>  were candidate orphans
b ac, ancylostoma caninum; ae, a. ceylanicum
abundant transcripts expressed in ancylostoma species
the  <dig> most abundantly represented clusters account for 14% and 19% of ests for a. caninum and a. ceylanicum respectively.
the representation of the abundant transcripts varied from shared to stage-specific .
hookworm developmental stages differ in habitat, morphology and behavior, hence highly represented gene transcripts may identify functions that are important to the parasites at various stages.
differences in gene expression between a. ceylanicum stages have been demonstrated previously for several genes  <cit> .
our comparison of il <dig> and adult suggests additional examples .
in fact, only 9% of the a. ceylanicum clusters are shared between il <dig> and adult  and of the  <dig> largest clusters,  <dig> were biased toward one of the developmental stages .
while representation in est collections generally correlates with source expression level  <cit> , artifacts can occur  <cit> .
differences in expression are most likely to be accurate when comparing the most abundant transcripts in each stage.
therefore, while follow-up work is needed to confirm expression levels, examination of ests provides a list of candidates for various expression profiles.
discussion
overview
we have taken a genomics approach to the study of hookworm species, key parasites of humans and domestic animals that are related to the model nematode caenorhabditis elegans.
nearly  <dig>  ests from ancylostoma caninum and a. ceylanicum identified approximately  <dig>  genes including over  <dig>  likely orthologs represented in both species.
close to  <dig> genes encode putative enzymes involved in  <dig> metabolic pathways.
over  <dig>  genes contain recognizable protein domains many of which have been categorized in the gene ontology hierarchy.
<dig>  genes have homologs in c. elegans including numerous nematode-specific genes and hundreds with observable rnai phenotypes.
ests originated from libraries representing infective l <dig> larva, stimulated l <dig>  tissue arrested l <dig>  and adults resulting in an improved rate of gene discovery and allowing the identification of transcripts abundant in various stages.
gene expression in il <dig> and dauers
infective l <dig>  are developmentally-arrested, non-feeding pre-parasitic stages, which when triggered by the infection process and host-specific signals reactivate, molt and complete development.
a similar stage in c. elegans is called the dauer larva.
in ancylostoma species host factors such as dog serum stimulate feeding and an activation response in serum stimulated l <dig>   <cit>  that approximates the transition to parasitism in the host  <cit> .
a. caninum tissue-arrested l <dig>  recovered from infected mice are a distinct population that potentially share properties with the arrested il <dig>  developmentally arrested, non-feeding larvae would be expected to be dependent on stored energy reserves and lipid metabolic pathways; accordingly, the kegg biochemical pathway mappings show a substantive number of clusters for fatty acid metabolism especially with the a. ceylanicum il <dig> clusters .
c. elegans microarray experiments identified  <dig> dauer-enriched genes along with genes involved in dauer-recovery  <cit> .
c. elegans sage experiments identified  <dig> candidate dauer-specific genes  <cit> .
genes shown to be abundantly expressed in c. elegans dauers include a variety of genes that may play roles in extended survival including heat shock protein encoding genes like hsp- <dig>  and daf- <dig> , ctl- <dig> , sod- <dig> , and hil- <dig> and hil- <dig> .
a number of genes identified both in ancylostoma l3s and c. elegans dauers are discussed below.
heat-shock proteins
hsp- <dig> , a member of the hsp- <dig> family, was one of the most highly represented clusters in a. caninum il <dig> and tal <dig> as well as a. ceylanicum il <dig> .
among a strongyloides stercoralis est collection, the gene is also found in il <dig> but not l <dig>  <cit> .
c. elegans hsp- <dig>  is upregulated in dauer and starved l1s  <cit>  and is a transcriptional target of the foxo transcription factor daf- <dig>  <cit> .
unlike other hsps, c. elegans hsp- <dig>  is not stress-induced and does not prevent aggregation of unfolded proteins, suggesting a novel role.
ae <dig> cl, found exclusively in adult ests, encodes an ortholog of the nippostrongylus brasiliensis hsp- <dig> protein.
nb-hsp- <dig> is more similar to the hsp- <dig> group of the hsp- <dig> family of small hsps in c. elegans, is also expressed in the adult  <cit> , and is not stress regulated, suggesting that it may function as an adult version of hsp- <dig> .
candidate stress-response proteins
a. caninum il <dig> showed abundant clusters encoding homologs of the mitochondrial cytochrome oxidase subunits i, ii, iii and a stress associated endoplasmic reticulum protein not seen in ssl <dig> .
one a. ceylanicum il <dig> abundant cluster encoded a ribosome-associated membrane  <dig> protein  involved in er protein translocation  <cit>  which is over-expressed in hypoxia and suppresses degradation of er membrane proteins  <cit> .
a homolog of c. elegans spp- <dig> was also expressed at high levels in a. ceylanicum il <dig>  spp- <dig> encodes an amoebapore, a member of the saposin-like protein superfamily that kill bacteria by forming membrane ion channels  <cit> .
amoebapore proteins are one of a number of putative stress response proteins regulated by daf- <dig> in c. elegans  <cit> .
these proteins, also including lysozyme and thaumatin, may provide a defense against worm pathogens and contribute to dauer longevity  <cit> .
hookworm free-living stages are also soil dwelling microbiverous organisms exposed to soil pathogens, so it is possible that spp- <dig> plays an antibacterial role in a. ceylanicum.
gene expression in ssl <dig>  adults, and multiple stages
in contrast to il <dig>  a. caninum ssl <dig> showed a cmp-sialic acid transporter, cathepsin d-like aspartic protease, calponin-like protein, and ham- <dig> zinc finger protein among the abundant transcripts.
while the significance of these molecules is unknown, upregulation of an aspartic protease during the transition to parasitism and tissue penetration/migration is consistent with its role in degradation of serum proteins and collagens  <cit> .
abundant adult-specific clusters are likely to be involved in reproduction.
for example, a. ceylanicum  encodes an ortholog of the c. elegans vit- <dig> protein, a lipid binding protein and major yolk component  <cit> .
vit- <dig> is expressed exclusively in the c. elegans adult hermaphrodite intestine, secreted, and taken up by oocytes.
two clusters encode genes involved in metabolism of chitin, an important constituent of the nematode eggshell  <cit> .
one encodes a protein similar to c. elegans protein c34d <dig> , and shows some similarity to a beet chitinase; the other encodes an ortholog of c. elegans b <dig> , a protein required for early embryonic development  <cit> .
b <dig>  mrna is expressed specifically in the adult hermaphrodite germ line and is a target of gld- <dig>  an rna binding protein required for oocyte meiotic cell cycle progression  <cit> .
asp's
while there are differences in the cluster profiles among ancylostoma stages, there are shared transcripts as well.
for example, the ancylostoma secreted protein asp- <dig> and asp-like cdnas are present in abundance in both a. caninum il <dig> and ssl <dig>  the secretion of asp- <dig> protein by ssl3s was noted as a marker of the transition to parasitism  <cit> .
these results support conclusions made by wang and kim  <cit>  that arrested larvae are transcriptionally prepared for dauer exit and upon receipt of appropriate stimulatory signals, exit from the arrested state is accompanied by a burst of translational activity in addition to further transcriptional activity.
in contrast to asp- <dig>  ests for asp- <dig> were exclusively detected in a. caninum il <dig>
far proteins
two of the most abundant a. ceylanicum transcripts encode fatty acid/retinol binding  proteins .
far proteins are novel fatty acid and retinol binding proteins described in nematodes including a. caninum, other strongylida, filarial, and plant parasitic species  <cit> .
in c. elegans  <dig> far members are divided into  <dig> groups.
all the parasitic nematodes fars described to date are most similar to the c. elegans a group containing ce-far- <dig>  - <dig>  and - <dig>  seven a. ceylanicum clusters encode far proteins.
four of which  were found in the adult cdna library; clusters ae <dig> cl and ae <dig> cl were nearly identical to ac-far- <dig> and ac-far- <dig>  whereas cluster ae <dig> cl showed the highest similarity to ce-far- <dig> and ae <dig> cl to ac-far- <dig>  the il <dig> specific clusters ae <dig> cl  and ae <dig> cl  were both most closely related to a far protein from ostertagia ostertagi  <cit> , and more distantly to ac-far- <dig> and - <dig>  therefore, as seen in other parasitic nematodes, most a. ceylanicum far proteins are related to c. elegans group a far proteins.
however, the a. ceylanicum cluster ae <dig> was most similar to group c far protein ce-far- <dig>  group c proteins differ from the other fars in important ways including lacking an n-terminal signal peptide , containing several cysteines, and failing to bind dauda  <cit> .
ae <dig> cl represents the first report of a far- <dig> like protein from a parasitic nematode.
the function of far proteins is unknown but may represent a lipid acquisition system in which released fars bind to lipids followed by uptake of the complex by a specific receptor mediated process.
retinoids are required for nematode growth and development, but are not synthesized by the worms.
in parasitic nematodes, release of far proteins may also modify local inflammatory and immunological responses by delivering or sequestering biochemically important lipids  <cit> .
CONCLUSIONS
the application of genomic approaches to hookworms has resulted in more than a 100-fold increase in available sequence data from ancylostoma species thereby allowing an initial bioinformatic analysis of transcripts from these important parasites and establishing a foundation for the eventual completion of a hookworm genome.
semi-automated informatic approaches that are now being applied to all nematode sequences  <cit>  allow uniform comparisons across many genomes and provide databases for further exploration.
transcripts in a. caninum and a. ceylanicum include clear candidates for stage specific expression representing the very different biological processes underway in different points of the lifecycle.
the availability of the c. elegans and c. briggsae genomes has allowed highly informative comparisons to the two hookworm species showing extensive overlap in gene complements, including genes demonstrated to be essential in c. elegans and numerous genes specific to nematodes.
as the most closely related major human pathogen to c. elegans, hookworms provide an attractive near-term application for using a model organism to better understand and eventually control a key disease-causing species.
beyond categorization of hookworm genes, clear research avenues are available to apply this information to improved methods for hookworm control including anthelmintic and vaccine development, diagnostics, population studies, as well as better understanding of fundamental aspects of hookworm biology, such as host immune system modulation.

 comparative sequence analysis is an approach proven to aid in recognition of genes and defining of their function, especially when comparing genomes of close evolutionary distance. in addition, when partial genomes are placed in a context of a well-studied and fully sequenced model organism they can greatly facilitate the understanding of the less studied organisms' biology. hookworms are blood-feeding nematodes that infect one billion people causing iron deficiency anemia and retarded physical and cognitive development in children. the two major species infecting humans are necator americanus and ancylostoma duodenale. the closely related hookworm species of canids, ancylostoma caninum, and canines and felines, a. ceylanicum, are minor parasites of humans, but are important as laboratory models for hookworm infection and disease. other hookworms infect raccoons, sheep, seals and a variety of other mammals adult hookworms inhabit the small intestine and produce eggs that pass in the feces and hatch in the soil. the first stage larva feeds on bacteria and molts twice to form the non-feeding, infective third stage. il enters the host by penetrating the skin, or orally in the case of ancylostoma species, molts twice, and matures to ad in the small intestine. a. duodenale and a. caninum l3s can also infect a host, temporarily abort maturation and enter an arrested state within the host's somatic tissues, reactivating in response to host physiological changes such as pregnancy. current hookworm control strategies are limited to de-worming of infected people using anthelmintic drugs. however, rapid re-infection in endemic areas and the lack of sterile immunity necessitates repeated treatments and can in turn result in resistance. additionally, tissue-arrested stages are relatively resilient to the effects of anthelmintics. the human hookworm vaccine initiative is beginning clinical trials of a larval hookworm antigen, asp- from n. americanus, as a vaccine antigen. there is a critical need for further research to identify new vaccine and drug targets as well as to better understand hookworm biology. lack of sequence information has been a major hindrance to hookworm molecular studies. high throughput sequencing of expressed sequence tags has proven a cost-effective tool for discovering genes. because the hookworm superfamily falls within nematode clade v, which also contains the well-studied model nematode caenorhabditis elegans, predictions may be made and tested based on their close relatedness. previous genome-based characterization of hookworms has been limited to sampling of few hundred ests and molecular studies of individual genes of interest. est approaches have also been initiated for other strongylid parasites including haemonchus contortus and nippostrongylus brasiliensis. in this report we describe the comparative analyses of almost ests from different cdna libraries representing pre-parasitic and parasitic larval through adult stages of the hookworms a. caninum and a. ceylanicum. the dataset defined nearly hookworm genes, including a number of putative developmentally expressed genes and candidates for further study as drug target or vaccine components.  nearly ancylostoma derived ests were submitted to genbank between and. for simplicity, the results and analysis described are presented in the same order beginning with a. caninum and followed by a. ceylanicum, except where specified. est acquisition and nemagene organization ests originated from cdna libraries, representing three and two life-cycle stages respectively. clustering, implemented to reduce data redundancy and improve sequence quality and length, grouped ests into contigs which were further organized into clusters, providing a non-redundant catalog of represented genes. ests within a contig derive from nearly identical transcripts while contigs within a cluster may arise from splice isoforms, alleles, or closely related paralogs. fifty-one potentially chimeric ests were discarded. clusters ranged in size from a single est to and for a. caninum and a. ceylanicum respectively. most clusters for each species have ten or fewer ests. gc content for coding sequences was similar in the two species and consistent with other clade v nematodes like c. elegans and c. briggsae. the number of clusters may overestimate gene discovery, as one gene may be represented by multiple non-overlapping clusters. by using c. elegans as a reference genome and discounting for fragmentation calculated as % and % respectively, the estimated gene numbers were reduced to for a. caninum and for a. ceylanicum giving a gene discovery rate of 41% and 30%. these numbers also indicate 20% and 16% representation of all genes for each species respectively. the number of genes in common for more stage-specific ancylostoma libraries analysed was as low as 9% and 11%. this may reflect the est sample size or stage-specific expression, as will be discussed. in either case, the results clearly show the advantage gained for gene discovery in ancylostoma by including diverse life stages in the analysis. functional classification based on gene ontology and kegg assignments thirty-four percent of a. caninum and 54% of a. ceylanicum clusters align to interpro domains and 21% and 36% map to gene ontology respectively. following this same pattern, a. caninum also had fewer blast matches. seven of the ten most abundantly represented interpro domains were common to both species. go representation is shown by biological process, cellular component, and molecular function. among the most common go categories are protein metabolism and catalytic activity. within ancylostoma spp. clusters that had extracellular mappings, 70% and 56% respectively were in the category of allergen v5/tpx- proteins related to the secreted venom proteins from hymenopteran insects. the ancylostoma secreted proteins belong to this large gene family, members of which been shown to play roles in host-parasite interactions for both mammalian and plant parasitic nematodes, and to induce protective responses. asp- is one of the major proteins secreted by serum-stimulated a. caninum il. in addition, four a. ceylanicum clusters were classified in extracellular matrix as tissue inhibitor of metalloprotease domain proteins. a timp homolog is reported as the most abundant protein in adult hookworm excretory/secretory products and may inhibit host metalloproteases. ten % and 15% unique clusters for a. caninum and a. ceylanicum respectively, mapped to metabolic pathways grouped in categories. complete listings and graphical representations of the kegg mappings are available at. pathways well represented by both species include glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation and fatty acid biosynthesis and metabolism. kegg analysis also suggests specific biochemical differences among ancylostoma stages. for example, while serum stimulated l3-specific clusters make up to 27% of all ac clusters, they account for 40% of all kegg pathway mappings. in contrast, il3-specific clusters that account for 55% of all ac clusters make-up only 38% of kegg pathway mappings. it is unclear whether the predominance of enzyme mappings from the ssl stage versus il stage is indicative of greater metabolic activity, greater metabolic complexity, differences in library construction methods, or other differences. aa. caninum – multiple and unique mappings; a. ceylanicum – multiple and unique mappings. b cluster, c enzymes homologs in other organisms, orthologs within ancylostoma spp. and estimates of selective pressure within a. ceylanicum clusters, 83% had homology to proteins from other organisms as compared to only 66% for a. caninum. to investigate why contigs from closely related species would show a difference in identified homologies, we compared sequence lengths and the open reading frame lengths of contigs with and without homologies in both species. est lengths and contig lengths, respectively, were shorter for a. caninum than for a. ceylanicum. the differences were even more striking for orfs. hence, a. caninum contigs very likely identify fewer homologs because these sequences are shorter, contain smaller orfs, and probably include more 3' utr versus the superior quality dataset from a. ceylanicum. most likely, differences in library construction and sampling rather than intrinsic differences between the species explain this discrepancy. accounting for such differences is important as it keeps analysis focused upon interesting features of the dataset related to the organisms' biology rather than artifactual differences arising from data collection. the distribution of identified homologs was consistent with earlier observations. besides c. elegans, one of the more informative nematode datasets for this study is a collection of ests from the human hookworm necator americanus to which homologies were commonly found. within ancylostoma itself homologies were common with 34% of total a. caninum clusters matching the a. ceylanicum dataset and 44% of total a. ceylanicum clusters matching a. caninum. searching for putative orthologs between all a. caninum and a. ceylanicum contigs resulted in reciprocal best tblastx hits. the ortholog pair members were very similar in gc composition and the average length of alignment was bp. all ortholog pairs were under purifying selection and the average ds was ± and dn was ±. the average dn/ds ratio is higher than that reported for c. briggsae/c. elegans, and closer to that for mouse/human, indicating that the levels of purifying selection are somewhat different. in addition, to examine if this purifying selection is more frequently detected in genes with essential function, we cross-referenced the c. elegans genes matched by ancylostoma orthologs with a list of c. elegans genes with available rna interference information. of the 67% of the orthologous genes matching c. elegans genes with available rnai data, 45% had an observable phenotype after transcript knock-down. a vast majority of the observed phenotypes were severe. ancylostoma orthologs matching c. elegans genes that had phenotypes showed a somewhat lower dn/ds ratio than those matching genes that remained wild type after rnai treatment, though the difference was not statistically significant at p <. in a 4-way comparison of orthologs in c. elegans, c. briggsae, a. caninum, and a. ceylanicum, the phylogenetic distance between a. caninum and a. ceylanicum is almost identical to that between c. elegans and c. briggsae and the distance between the two genera is just over four times the within genera distance. average branch lengths for the set of orthologous proteins did not show a significant difference in relative rate of molecular change. maximum likelihood trees were constructed for each 4-way ortholog and relative branch lengths compared between ancylostoma spp. for both the protein and nucleotide sequences. for protein sequences, trees had equal branch lengths for the hookworm species while trees had longer a. caninum branches and had longer a. ceylanicum branches. to look for differences between genes in the ancylostoma species, we constructed a distribution of branch length differences between each of the sister species pairs in our tree. because some genes may be rapidly evolving in all nematode lineages we evaluated a subset of the trees where the difference in branch lengths between c. briggsae and c. elegans were less than one standard deviation from the mean but which had significantly different branch lengths in the two ancylostoma species. this final dataset had genes with significantly longer branch lengths in a. ceylanicum and in a. caninum. however the set did not show any significant bias towards either species. this suggests there is no significant rate difference in protein evolution between the two hookworms, although some of these genes are relatively rapidly evolving. repeating the analysis for nucleotide sequences we find marginally significant differences. using the c. elegans genome to interpret hookworm sequences as expected, the c. elegans genome provides the best source of information for interpreting hookworm sequences as a majority of a. caninum and a. ceylanicum clusters with blast homologies outside ancylostoma had c. elegans homologs. furthermore, c. elegans orthologs of hookworm genes with available rnai or other data provide information that may be relevant to understanding the role of the parasite genes. of all the ancylostoma clusters with c. elegans homology, 97% and 92% matched c. elegans genes with available rna interference knock-down information, and in turn 33% and 37% of these c. elegans genes produce rnai phenotypes. phenotype classification showed that c. elegans genes with expressed ancylostoma homologs were somewhat more likely to have severe phenotypes. hence, certain genes in the ancylostoma datasets encode proteins if disabled may disrupt survival of the parasite. some examples include abundant clusters. a group of particular interest is proteins that are required for nematode survival and lack strong homologies outside of the phyla, since these targets could provide for nematode control without toxicities to the host or other non-target organisms. of the ancylostoma nematode-specific clusters, and respectively had c. elegans matches with rnai phenotypes. among these, ac cl and ae cl matched hypothetical protein f42a, a gene without a mammalian homolog yet likely involved in multiple developmental processes based on observed mutant phenotypes. homologs are found in at least nematode species to date including free-living species, and parasites of mammals and plants. further analysis will identify additional genes which warrant detailed investigation. a sw/tr is swiss-prot and trembl proteinknowledgebase. b c. elegans homolog has higher probability match than the best genbank descriptor. a sw/tr is swiss-prot and trembl proteinknowledgebase. b c. elegans homolog has higher probability match than the best genbank descriptor. repeating the analysis in stein et al. indicates that 6–7% of c. elegans and c. briggsae proteins are candidate "orphans", lacking homologs outside of the species. we examined whether these genes are truly orphans that have arisen in a caenorhabditis sub-lineage or are instead genes present in an ancestral nematode that have been lost or evolved beyond recognition in one species. of candidate orphan proteins, ten from c. elegans and from c. briggsae match a. caninum and/or a. ceylanicum clusters, with three and eight, respectively, having matches in both species. most of the c. elegans orphans are hypothetical proteins of unknown function though some had functional information from interpro domains or mutant phenotypes. therefore, at least a portion of the genes identified in either c. elegans or c. briggsae as "orphans" are actually ancestral nematode genes with homologs found in other clade v species and further clade v sequencing will likely reveal more such cases. a of proteins were candidate orphans b ac, ancylostoma caninum; ae, a. ceylanicum abundant transcripts expressed in ancylostoma species the most abundantly represented clusters account for 14% and 19% of ests for a. caninum and a. ceylanicum respectively. the representation of the abundant transcripts varied from shared to stage-specific. hookworm developmental stages differ in habitat, morphology and behavior, hence highly represented gene transcripts may identify functions that are important to the parasites at various stages. differences in gene expression between a. ceylanicum stages have been demonstrated previously for several genes. our comparison of il and adult suggests additional examples. in fact, only 9% of the a. ceylanicum clusters are shared between il and adult and of the largest clusters, were biased toward one of the developmental stages. while representation in est collections generally correlates with source expression level, artifacts can occur. differences in expression are most likely to be accurate when comparing the most abundant transcripts in each stage. therefore, while follow-up work is needed to confirm expression levels, examination of ests provides a list of candidates for various expression profiles. discussion overview we have taken a genomics approach to the study of hookworm species, key parasites of humans and domestic animals that are related to the model nematode caenorhabditis elegans. nearly ests from ancylostoma caninum and a. ceylanicum identified approximately genes including over likely orthologs represented in both species. close to genes encode putative enzymes involved in metabolic pathways. over genes contain recognizable protein domains many of which have been categorized in the gene ontology hierarchy. genes have homologs in c. elegans including numerous nematode-specific genes and hundreds with observable rnai phenotypes. ests originated from libraries representing infective l larva, stimulated l tissue arrested l and adults resulting in an improved rate of gene discovery and allowing the identification of transcripts abundant in various stages. gene expression in il and dauers infective l are developmentally-arrested, non-feeding pre-parasitic stages, which when triggered by the infection process and host-specific signals reactivate, molt and complete development. a similar stage in c. elegans is called the dauer larva. in ancylostoma species host factors such as dog serum stimulate feeding and an activation response in serum stimulated l that approximates the transition to parasitism in the host. a. caninum tissue-arrested l recovered from infected mice are a distinct population that potentially share properties with the arrested il developmentally arrested, non-feeding larvae would be expected to be dependent on stored energy reserves and lipid metabolic pathways; accordingly, the kegg biochemical pathway mappings show a substantive number of clusters for fatty acid metabolism especially with the a. ceylanicum il clusters. c. elegans microarray experiments identified dauer-enriched genes along with genes involved in dauer-recovery. c. elegans sage experiments identified candidate dauer-specific genes. genes shown to be abundantly expressed in c. elegans dauers include a variety of genes that may play roles in extended survival including heat shock protein encoding genes like hsp- and daf-, ctl-, sod-, and hil- and hil-. a number of genes identified both in ancylostoma l3s and c. elegans dauers are discussed below. heat-shock proteins hsp-, a member of the hsp- family, was one of the most highly represented clusters in a. caninum il and tal as well as a. ceylanicum il. among a strongyloides stercoralis est collection, the gene is also found in il but not l. c. elegans hsp- is upregulated in dauer and starved l1s and is a transcriptional target of the foxo transcription factor daf-. unlike other hsps, c. elegans hsp- is not stress-induced and does not prevent aggregation of unfolded proteins, suggesting a novel role. ae cl, found exclusively in adult ests, encodes an ortholog of the nippostrongylus brasiliensis hsp- protein. nb-hsp- is more similar to the hsp- group of the hsp- family of small hsps in c. elegans, is also expressed in the adult, and is not stress regulated, suggesting that it may function as an adult version of hsp-. candidate stress-response proteins a. caninum il showed abundant clusters encoding homologs of the mitochondrial cytochrome oxidase subunits i, ii, iii and a stress associated endoplasmic reticulum protein not seen in ssl. one a. ceylanicum il abundant cluster encoded a ribosome-associated membrane protein involved in er protein translocation which is over-expressed in hypoxia and suppresses degradation of er membrane proteins. a homolog of c. elegans spp- was also expressed at high levels in a. ceylanicum il spp- encodes an amoebapore, a member of the saposin-like protein superfamily that kill bacteria by forming membrane ion channels. amoebapore proteins are one of a number of putative stress response proteins regulated by daf- in c. elegans. these proteins, also including lysozyme and thaumatin, may provide a defense against worm pathogens and contribute to dauer longevity. hookworm free-living stages are also soil dwelling microbiverous organisms exposed to soil pathogens, so it is possible that spp- plays an antibacterial role in a. ceylanicum. gene expression in ssl adults, and multiple stages in contrast to il a. caninum ssl showed a cmp-sialic acid transporter, cathepsin d-like aspartic protease, calponin-like protein, and ham- zinc finger protein among the abundant transcripts. while the significance of these molecules is unknown, upregulation of an aspartic protease during the transition to parasitism and tissue penetration/migration is consistent with its role in degradation of serum proteins and collagens. abundant adult-specific clusters are likely to be involved in reproduction. for example, a. ceylanicum encodes an ortholog of the c. elegans vit- protein, a lipid binding protein and major yolk component. vit- is expressed exclusively in the c. elegans adult hermaphrodite intestine, secreted, and taken up by oocytes. two clusters encode genes involved in metabolism of chitin, an important constituent of the nematode eggshell. one encodes a protein similar to c. elegans protein c34d, and shows some similarity to a beet chitinase; the other encodes an ortholog of c. elegans b, a protein required for early embryonic development. b mrna is expressed specifically in the adult hermaphrodite germ line and is a target of gld- an rna binding protein required for oocyte meiotic cell cycle progression. asp's while there are differences in the cluster profiles among ancylostoma stages, there are shared transcripts as well. for example, the ancylostoma secreted protein asp- and asp-like cdnas are present in abundance in both a. caninum il and ssl the secretion of asp- protein by ssl3s was noted as a marker of the transition to parasitism. these results support conclusions made by wang and kim that arrested larvae are transcriptionally prepared for dauer exit and upon receipt of appropriate stimulatory signals, exit from the arrested state is accompanied by a burst of translational activity in addition to further transcriptional activity. in contrast to asp- ests for asp- were exclusively detected in a. caninum il far proteins two of the most abundant a. ceylanicum transcripts encode fatty acid/retinol binding proteins. far proteins are novel fatty acid and retinol binding proteins described in nematodes including a. caninum, other strongylida, filarial, and plant parasitic species. in c. elegans far members are divided into groups. all the parasitic nematodes fars described to date are most similar to the c. elegans a group containing ce-far- - and - seven a. ceylanicum clusters encode far proteins. four of which were found in the adult cdna library; clusters ae cl and ae cl were nearly identical to ac-far- and ac-far- whereas cluster ae cl showed the highest similarity to ce-far- and ae cl to ac-far- the il specific clusters ae cl and ae cl were both most closely related to a far protein from ostertagia ostertagi, and more distantly to ac-far- and - therefore, as seen in other parasitic nematodes, most a. ceylanicum far proteins are related to c. elegans group a far proteins. however, the a. ceylanicum cluster ae was most similar to group c far protein ce-far- group c proteins differ from the other fars in important ways including lacking an n-terminal signal peptide, containing several cysteines, and failing to bind dauda. ae cl represents the first report of a far- like protein from a parasitic nematode. the function of far proteins is unknown but may represent a lipid acquisition system in which released fars bind to lipids followed by uptake of the complex by a specific receptor mediated process. retinoids are required for nematode growth and development, but are not synthesized by the worms. in parasitic nematodes, release of far proteins may also modify local inflammatory and immunological responses by delivering or sequestering biochemically important lipids.  the application of genomic approaches to hookworms has resulted in more than a 100-fold increase in available sequence data from ancylostoma species thereby allowing an initial bioinformatic analysis of transcripts from these important parasites and establishing a foundation for the eventual completion of a hookworm genome. semi-automated informatic approaches that are now being applied to all nematode sequences allow uniform comparisons across many genomes and provide databases for further exploration. transcripts in a. caninum and a. ceylanicum include clear candidates for stage specific expression representing the very different biological processes underway in different points of the lifecycle. the availability of the c. elegans and c. briggsae genomes has allowed highly informative comparisons to the two hookworm species showing extensive overlap in gene complements, including genes demonstrated to be essential in c. elegans and numerous genes specific to nematodes. as the most closely related major human pathogen to c. elegans, hookworms provide an attractive near-term application for using a model organism to better understand and eventually control a key disease-causing species. beyond categorization of hookworm genes, clear research avenues are available to apply this information to improved methods for hookworm control including anthelmintic and vaccine development, diagnostics, population studies, as well as better understanding of fundamental aspects of hookworm biology, such as host immune system modulation. 
hookworms, infecting over one billion people, are the mostly closely related major human parasites to the model nematode caenorhabditis elegans. to study the evolution of hookworm genes, a. caninum / a. ceylanicum orthologs were identified, all of which were found to be under purifying selection with distribution ratios of nonsynonymous to synonymous amino acid substitutions similar to that reported for c. elegans / c. briggsae orthologs. the phylogenetic distance between a. caninum and a. ceylanicum is almost identical to that for c. elegans / c. briggsae. most genes are represented in single stages including abundant transcripts like hsp- in infective l and vit- in adults. applying genomics techniques to these species, we analyzed and genes from ancylostoma caninum and a. ceylanicum. over 80% of the genes have homologs in c. elegans, and nearly 30% of these were with observable rna interference phenotypes. homologies were identified to nematode-specific and clade v specific gene families.
 hookworms, infecting over one billion people, are the mostly closely related major human parasites to the model nematode caenorhabditis elegans. applying genomics techniques to these species, we analyzed and genes from ancylostoma caninum and a. ceylanicum.  transcripts originated from libraries representing infective l larva, stimulated l arrested l and adults. most genes are represented in single stages including abundant transcripts like hsp- in infective l and vit- in adults. over 80% of the genes have homologs in c. elegans, and nearly 30% of these were with observable rna interference phenotypes. homologies were identified to nematode-specific and clade v specific gene families. to study the evolution of hookworm genes, a. caninum / a. ceylanicum orthologs were identified, all of which were found to be under purifying selection with distribution ratios of nonsynonymous to synonymous amino acid substitutions similar to that reported for c. elegans / c. briggsae orthologs. the phylogenetic distance between a. caninum and a. ceylanicum is almost identical to that for c. elegans / c. briggsae.  the genes discovered should substantially accelerate research toward better understanding of the parasites' basic biology as well as new therapies including vaccines and novel anthelmintics. 