Reconstruction of the insulin-like signalling pathway of Haemonchus contortus
© Mohandas et al. 2016
Received: 12 January 2016
Accepted: 26 January 2016
Published: 3 February 2016
In the present study, we reconstructed the insulin/insulin-like growth factor 1 signalling (IIS) pathway for Haemonchus contortus, which is one of the most important eukaryotic pathogens of livestock worldwide and is related to the free-living nematode Caenorhabditis elegans.
We curated full-length open-reading frames from assembled transcripts, defined the complement of genes that encode proteins involved in this pathway and then investigated the transcription profiles of these genes for all key developmental stages of H. contortus.
The core components of the IIS pathway are similar to their respective homologs in C. elegans. However, there is considerable variation in the numbers of isoforms between H. contortus and C. elegans and an absence of AKT-2 and DDL-2 homologs from H. contortus. Interestingly, DAF-16 has a single isoform in H. contortus compared with 12 in C. elegans, suggesting novel functional roles in the parasitic nematode. Some IIS proteins, such as DAF-18 and SGK-1, vary in their functional domains, indicating distinct roles from their homologs in C. elegans.
This study paves the way for the further characterization of key signalling pathways in other socioeconomically important parasites and should help understand the complex mechanisms involved in developmental processes.
Roundworms (nematodes) are one of the most diverse groups of organisms on the planet. Some are free-living, and many are parasitic, causing substantial disease and socioeconomic problems globally. For example, Haemonchus contortus (the barber’s pole worm; order Strongylida) is one of the most destructive parasitic nematodes of livestock animals (small ruminants, including sheep and goats) due to its high pathogenicity and widespread occurrence around the world . This nematode feeds on blood from capillaries in the stomach (abomasum) wall, and causes haemorrhagic gastritis, anaemia, oedema and associated complications, often leading to the death of severely affected animals. H. contortus is transmitted orally from contaminated pasture to the host through a direct life cycle involving three free-living larval stages, of which the infective third larval stage (iL3) is ingested . After a histotropic phase in the host animal, the larvae develop to the fourth stage (L4) and then to adults, which both feed on blood and cause pathogenic effects in the host animal.
The recent characterization of transcriptomes and draft genomes of H. contortus [3, 4] provides a solid basis for future studies of its developmental and reproductive biology using genetic, genomic, proteomic and metabolomic tools. However, a lack of tractable functional genomic tools for H. contortus and related parasitic nematodes, and an inability to maintain their complete life cycles in vitro, hampers functional investigations of genes and gene products in these nematodes (cf. [5–12]). This contrasts the situation for the free-living nematode, Caenorhabditis elegans, the best characterized metazoan organism, which can be readily maintained, and used to investigate fundamental processes and mechanisms, such as dauer formation .
Caenorhabditis elegans, which belongs to “clade V” , is relatively closely related to H. contortus. Published information [7, 8, 15–17] indicates similarity in entry into and exit from the “dauer state” between C. elegans and strongylid nematodes . This arrested state occurs in C. elegans when the nematode encounters harsh environmental conditions, such as starvation, crowding and/or a high temperature [19, 20]. The dauer form can survive for several months and then resume development to reproductive adults when conditions improve . Consistent with C. elegans, H. contortus and related nematodes have a similar third larval stage (L3), which is relatively resistant to unfavourable conditions and does not feed because it is encased by a cuticular sheath . The “dauer hypothesis”  contends that the resumption of iL3 development in parasitic nematodes is functionally and developmentally analogous to the exit from dauer in C. elegans, and is regulated by similar mechanisms [13, 15, 16, 21].
Dauer development is governed by multiple signalling pathways, including the insulin/insulin-like growth factor 1 (IGF1)-like signalling (IIS) pathway , which, in C. elegans, comprises proteins such as DAF-2 (insulin-like receptor kinase [22–24]), AGE-1 (phosphoinositide-3 (PI3) kinase [25–27]) and DAF-16 (FOXO-class transcription factor [28–30]). In C. elegans, signalling via DAF-2 activates AKT-1/2 by phosphorylation which, in turn, negatively regulates DAF-16, which functions as a central mediator of multiple biological processes, such as growth, development, reproduction, longevity, age and stress resistance .
While much is known about the IIS pathway in C. elegans (reviewed in ), only a few studies have explored the functions of selected parts of this pathway in H. contortus [32–34], and no study has yet investigated its full composition in this parasitic nematode. Therefore, in the present study, we (i) curated the full-length open reading frames (ORFs) and defined the complement of genes that encode peptides/proteins involved in IIS, (ii) studied the interactions of these genes and (iii) examined their transcription profiles in all key developmental stages of H. contortus.
We employed data relating to a published draft genome as well as transcriptomes of all key developmental stages (egg, first- to fourth-stage larvae (L1, L2, L3 and L4) and adult) and both sexes (L4 and adult) of H. contortus (NCBI BioProject accession no. PRJNA205202; [4, 35]). This draft genome is ~ 320 Mb in size and has been predicted to encode 23,610 proteins .
Identification of genomic scaffolds containing genes encoding IIS pathway components
From the complete, assembled transcriptome representing all eight stages or sexes of H. contortus , we identified and extracted assembled transcripts based on their homology matches (E-value cut-off: 10−5) to all genes encoding insulin/insulin-like growth factor 1 signalling (IIS) proteins in C. elegans . Then, we identified genomic scaffolds containing regions of homology to known IIS genes by mapping (E-value cut-off: 10−5) all assembled transcripts using BLAT . We also used IIS genes from the H. contortus draft genome predicted previously using MAKER2 [4, 37]. Open reading frames (ORFs) of individual assembled transcripts were inferred using the program GeneMark-ES [38, 39]. Using the Integrative Genomics Viewer (IGV) [40, 41], we then visually integrated all of these data to obtain a consensus sequence for individual coding regions.
Identification of protein domains, families and subfamilies
Identifying IIS protein genes encoded in the draft genome allowed us to then define the complete set of full-length transcripts. ORFs were verified and corresponding coding regions inferred from these full-length transcripts using ORF-finder . Each predicted protein was characterized by its primary amino acid sequence and structural and/or functional domains, inferred using all databases (i.e., PROSITE, HAMAP, Pfam, PRINTS, ProDom, SMART, TIGRFAMs, PIRSF, SUPERFAMILY, CATH-Gene3D and PANTHER) within InterProScan v.5.14.53 [43, 44]. Individual predicted proteins were classified according to family and/or subfamily using information in the PANTHER database v.9.0 [45, 46]. Following a comparison of those inferred from transcripts and genomic exons encoding IIS genes with the corresponding C. elegans homologs, we were able to infer the full complement of full-length transcripts and protein sequences.
Analysis of differential transcription
Each set of quality-filtered, paired-end RNA-seq reads for each individual developmental stage or sex of H. contortus was mapped to the final complement of full-length IIS transcripts using Burrows-Wheeler Aligner (BWA) software . For each stage/sex, the numbers of reads that mapped to individual transcripts were established using the SAM tools algorithm . The resultant read counts per transcript per developmental stage were used as input data for DESeq2 and edgeR [49, 50]. Differential transcription was calculated by pairwise comparison of all free-living (egg, L1, L2 and L3) and parasitic (L4 and adult) stages. Genes were recorded as differentially transcribed, using edgeR-calculated common and gene-wise dispersion factors, if the log2 fold change (log2 FC) between free-living and parasitic stages compared with the normalised read count data was ≥ 2, with a false discovery rate (FDR) of ≤ 0.05. A heat map (representing mapped reads) was produced using the heatmap.2 function in the gplots package in R .
Identification and characterization of IIS signalling protein genes
From the complete, assembled transcriptome of H. contortus, we identified and extracted 3792 assembled transcripts based on their homology matches (E-value cut-off: 10−5) to the 45 IIS protein genes, and then located the regions in genomic scaffolds to which these assembled transcripts mapped. The manual curation of the matching genomic and transcriptomic data for H. contortus identified 27 of 41 IIS gene homologs, and 4 of 40 insulin-like peptide (ILP) gene homologs using C. elegans genes as references.
Pairwise comparisons of sequence identity (%) of proteins representing the insulin/insulin-like growth factor 1(IGF1)-like signalling pathway (IIS) between Haemonchus contortus and Caenorhabditis elegans
Transcripts representing the IIS genes
Homologs in C. elegans (Gene code)
Pairwise sequence identity (%)
Y55D5A.5a, b, c*, d, e, f, g
Locus_5794_Transcript_4/8_Confidence_0.650_Length_4263 + Locus_5794_Transcript_6/8_Confidence_0.500_Length_3032
C12D8.10a*, b, c
C12D8.10a, b*, c
C13G3.3a*, b, c*, d*
Locus_4342_Transcript_5/6_Confidence_0.667_Length_1898 + Locus_4342_Transcript_3/6_Confidence_0.667_Length_1669
R13H8.1a, b, c, d, e, f, g, h, i, k, l, m*
T19E7.2a, b, c, d*
Locus_4675_Transcript_1/2_Confidence_0.750_Length_767 + Locus_8707_Transcript_2/3_Confidence_0.667_Length_738
F22E12.4a, b, c, d*, e
F22E12.4a, b, c, d*, e
F22E12.4a, b, c*, d, e
F22E12.4a, b, c*, d, e
InterProScan analysis allowed the classification of predicted IIS proteins, based on domains and protein signatures (Additional file 1: Figure S1). The proteins predicted for H. contortus (i.e., Hc-PDK-1, −PPTR-1, −AKT-1, −DAF-16, −HSB-1, −SKN-1 and -EGL-9) had the domains and signatures that were consistent with their respective C. elegans homologs. Hc-DAF-2 consisted of a protein kinase ATP-binding region signature (PS00107), an iron-sulfur binding domain (PS51379) and a furin-like cysteine-rich region (SM00261), which have been found in a number of eukaryotic proteins, such as epidermal growth factor receptor, endoprotease-4 and receptor tyrosine-protein kinase (LET-23), known to be involved in signal transduction by receptor tyrosine kinases [52–55]. Hc-DAF-18 lacked the protein tyrosine phosphatase catalytic domain (PTPc) motif and, instead, contained a dual-specificity phosphatase catalytic domain (PF00782). The pleckstrin homology (PH) domain (SM00233) was not present in Hc-PDK-1. Hc-PPTR-2 (protein phosphatase 2A) consisted of a unique leucine-rich repeat variant (G3DSA: 184.108.40.206) that represents a super-helical structure predicted to aid the binding of large substrates . In comparison to the 14-3-3 proteins FTT-2 and PAR-5 of C. elegans, which have two protein signatures in PROSITE, the predicted H. contortus proteins have only protein signature 1 (PS00796). PANTHER family and subfamily classifications could not be assigned to some predicted IIS proteins of H. contortus, such as Hc-INS-1, Hc-DDL-1 and Hc-PAR-5. The regulatory subunit, Hc-IST-1, which contains the PH domain (SM00233; PS50003) belonging to the PH domain-like superfamily (SSF50729) is not present in C. elegans. The other adaptor protein, Hc-AAP-1, also contains the SH2 (Src homology 2) domain (PR00401), which is a 5-element fingerprint, and corresponds to the core structural element of the protein. The predicted Hc-SGK-1 protein consists of the PX (phox) domain (PS50195; SSF64268), which is an important phosphoinositide-binding module with variable lipid-binding specificity [57, 58].
The IIS signalling pathway of H. contortus
Of a total of 81 genes involved in the IIS pathway of C. elegans , 31 homologs were identified in H. contortus. Specifically, in H. contortus, four insulin-like peptide (ILP) homologs of 40 in C. elegans as well as 9 of 11 downstream components of the IIS pathway were identified. However, C. elegans akt-2 and ddl-2 homologs were not found in H. contortus, which was confirmed by searching independent genomic and transcriptomic data sets . Therefore, overall, the number of core IIS genes (Hc-daf-2, −ist-1, −aap-1, −age-1, −daf-18, −pdk-1, −sgk-1, −akt-1, −pptr-1, −ftt-2, −par-5 and -daf-16) was similar, with variation mainly in the numbers of isoforms between H. contortus and C. elegans.
Using a bioinformatic approach, we reconstructed the IIS pathway of Haemonchus contortus from transcriptomic and genomic data sets for this nematode, and explored the transcription of individual genes as well as their interactions. The composition of the core IIS pathway in H. contortus was relatively consistent with that of C. elegans, although there were considerable differences between these nematodes in the upstream and downstream targets of this pathway. In the present study, four insulin-like peptides (ILPs) were identified in H. contortus, as opposed to 40 ILPs found in C. elegans [61–64]. A similar, marked reduction in the repertoire of ILP-encoding transcripts has been observed in the parasitic nematode Strongyloides stercoralis (clade IV), where only seven of the 40 ILPs of C. elegans are represented . The four ILPs predicted for H. contortus all have representative functional domains and protein signatures, including an insulin-like domain representing the insulin/IGF/relaxin family, a disulphide-rich alpha-helical domain and an insulin family signature (cf. Additional file 1: Figure S1). In C. elegans, several ILPs (encoded by ins-1, ins-6, ins-7) have been shown to regulate dauer formation, longevity and development . Although the functions of all 40 ILPs are not yet known, some (e.g., encoded by ins-1, ins-6, ins-7 and daf-28) have been extensively studied [63, 64, 66–68]. An interesting feature of these peptides is that they can either act as agonists or antagonists of DAF-2, the only insulin-like receptor kinase in C. elegans [63, 66, 67, 69]. While the basis of the differences in function is presently unknown, it is hypothesized that neural inputs from chemosensory neurons in the amphids and transduced by G protein-coupled receptors (GPCRs) therein trigger specific profiles of ILP expression, possibly including optimum levels of agonists and antagonists in one or a few developmental stages, which combine to precisely regulate the insulin signalling pathway response (i.e., downstream gene expression) to the complex set of environmental cues experienced by this free-living nematode . It is likely that ILP profiles are similarly regulated in parasitic nematodes. Indeed, the regulation of ILP expression by upstream cyclic GMP signalling has been observed in S. stercoralis, where the administration of 8-bromo-cGMP to cultured larvae elicits naturally occurring profiles of ILP expression, in contrast to the baseline levels of expression observed in untreated controls . The marked reduction in numbers of ILPs in parasitic nematodes examined to date might reflect a rather more specific interaction with their environments relative to more opportunistic free-living organisms, such as C. elegans. The homologs of the C. elegans ILPs identified in H. contortus were encoded by Hc-ins-1a, −ins-1b, −ins-17 and -ins-18, which all represent antagonists, implying that the regulation of the IIS pathway is dependent on their expression patterns in all life stages of the parasite. High transcription in the L1, L2 and L3 stages of H. contortus and considerably lower transcription in L4 and adult stages indicate that the ILPs of H. contortus are transcriptionally regulated during the transition from free-living to parasitic stages. By contrast to H. contortus, the seven ILPs of S. stercoralis constitute a set of peptides whose structures and patterns of expression in free-living and parasitic stages suggest members that are either agonists or antagonists of Ss-DAF-2. This apparent diversity of ILP function in S. stercoralis could explain the capability of the parasite to undertake either direct development to infective L3s or development to a generation of free-living male and female worms with many biological attributes in common with non-parasitic nematodes.
Some of the intracellular protein components of the IIS pathway showed variation in the functional domains present and in the number of isoforms predicted. For instance, Hc-DAF-18 is a homolog of the mammalian PTEN protein, which is a well-recognised phosphatase and a tumour suppressor . In C. elegans, DAF-18, a lipid phosphatase, acts by inhibiting the activation of PDK-1 by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2) by removing the phosphate in the D3 position of the inositol ring . Ce-DAF-18 is also suggested to have tyrosine phosphatase activity, based on the presence of its functional catalytic domain. However, in H. contortus, while Hc-DAF-18 might have the same lipid phosphatase function as its C. elegans homolog, the presence of a dual-specificity phosphatase catalytic domain indicates its ability to dephosphorylate both tyrosine- and serine-/threonine-phosphorylated proteins. This suggested, additional function of Hc-DAF-18 might hint to a functional role of the signalling pathway in recovery from developmental arrest.
The C. elegans insulin-signalling pathway activates two Akt family members, AKT-1 and AKT-2, as well as a serum and glucocorticoid-inducible kinase, SGK-1 downstream of the phosphoinositide-dependent kinase, PDK-1. All of these serine-threonine kinases are activated by AGE-1/PI3K. Previous studies [74–76] have shown that null-mutants of Ce-akt-1 and Ce-akt-2 result in non-conditional dauer arrest and an extension of lifespan. A knockdown of sgk-1 by RNAi also induces an extended lifespan, indicating that it functions in a similar manner to AKT-1 and AKT-2 . Interestingly, in H. contortus, only two kinases were predicted downstream of Hc-PDK-1, namely Hc-AKT-1 and Hc-SGK-1. The absence of AKT-2 might indicate a distinct regulation of IIS pathway in this nematode during the switch to the parasitic stage and also during reproduction. The transcription of Hc-akt-1 was higher in females than in males. The assessment of the functional domains of Hc-SGK-1 revealed the presence of a PX (phox) domain, which was not present in its respective C. elegans homolog. PX, a phospholipid-binding domain, primarily interacts with PIP3 lipids . The presence of this unique domain is likely to compensate for the deficiency of AKT-2 by allowing an activation of Hc-SGK-1 by both Hc-PDK-1 as well as PIP3, thereby effecting the expression of downstream Hc-DAF-16 target genes.
DAF-16 is a member of the FoxO family of forkhead transcription factors, which are regulators of growth, metabolism, stress response, cell cycle control and longevity in many organisms . The nuclear translocation of DAF-16 from the cytoplasm is inhibited by the phosphorylation at its RxRxxS/T motifs [80, 81], which are conserved among C. elegans DAF-16, mammalian FoxOs and predicted H. contortus daf-16. The C. elegans genome encodes twelve DAF-16 transcripts. Although the functions of these isoforms are not known, it is hypothesized that they have distinct tissue distributions in hypodermis, muscle, neurons, and intestine [29, 80–83]. Studies of Ce-DAF-16 indicate that biological functions of the isoforms vary according to their tissue distribution [80, 83]. In contrast, in H. contortus, a single homolog of DAF-16 was predicted. This finding implies that the array of target genes predicted to be transcriptionally regulated by Hc-DAF-16 is controlled by a single homolog, possibly resulting in a more complex regulation of downstream gene targets and distinct phenotypes. Given that the input signals of IIS pathway are also lesser in number compared with C. elegans, this information suggests a novel regulatory mechanism that differs between free-living and parasitic nematodes. This hypothesis is bolstered by a similar reduction in transcripts from the daf-16 ortholog in S. stercoralis. Here, there are only two transcripts, designated Ss-daf-16a and Ss-daf-16b, each expressed under the control of a different promoter [65, 84].
The ‘dauer hypothesis’ suggests that a similar mechanism of action takes place in major signalling pathways including the IIS, cyclic GMP and TGF-β pathways, which control the entry into and exit from arrested development in C. elegans, as in parasitic nematodes . The present investigation of genomic and transcriptomic data sets from H. contortus suggests that the major intracellular signalling components of IIS, such as daf-2, age-1, pdk-1 and akt-1, likely have similar functions to C. elegans homologs. This hypothesis has been supported for age-1 orthologs in other parasitic nematodes by the fact that the PI3K inhibitor LY294002 suppresses developmental activation of iL3 under host-like culture conditions [85–87]. However, the roles of key genes encoding ILPs, DAF-18, SGK-1 and DAF-16 in H. contortus appear to be distinct.
Although the ‘dauer hypothesis’ usually considers L3 arrest in parasitic nematodes to be analogous to dauer in C. elegans , H. contortus and related nematodes, such as Ostertagia and Cooperia spp., can undergo hypobiosis at the early L4 stage within the host animal . Given that this latter adaptive state enables transitional parasite survival within the host, and regulates parasite transmission and population size , understanding this phenomenon is of critical importance. Hence, future work should evaluate the involvement of IIS and associated signalling pathways in hypobiosis. It would be of particular interest to reconstruct the TGF-β signalling pathway in H. contortus and other trichostrongylids, and assess transcription profiles throughout development, given the major contrast in transcription at the L3 stage between various parasitic nematodes including H. contortus (up-regulation) and C. elegans (down-regulation) (cf. ) that suggests an altogether unique function of DAF-7 in parasitic worms. This line of investigation will be interesting in light of the fact that C. elegans offers no exact counterpart to the early L4 arrest that occurs in these economically important trichostrongyles. As such, mechanisms of early L4 arrest will likely represent a unique adaptation to parasitism for some clade V nematodes.
In conclusion, the availability of transcriptomic and draft genomic data sets for H. contortus has enabled the first detailed bioinformatic exploration of the IIS pathway in this parasite. We curated the full-length transcripts and defined the complement of genes that encode peptides/proteins involved in this pathway by comparison with C. elegans, reconstructed the pathway with these genes and investigated their transcription profiles in key developmental stages of H. contortus. We hope that reconstructing the IIS pathway for H. contortus will provide a stepping stone for future studies of development, reproduction, ageing, longevity, metabolism and/or behaviour in this important parasitic worm, and a stimulus to explore other signalling pathways in socioeconomically important strongylids.
Funding from the Australian Research Council (ARC) and the National Health and Medical Research Council of Australia (NHMRC) is gratefully acknowledged, as is support from the Victorian Life Sciences Computation Initiative (VLSCI; grant number VR0007) (RBG). Other support was from grants from the National Key Basic Research Program (973 program) of China (no. 2015CB150300), the National Natural Science Foundation of China (NSFC) (no. 31172310) (MH) and from the National Institute of Health (NIH), USA (AI-50688) (JBL). We acknowledge the contributions of staff at WormBase (www.wormbase.org). AJS is a recipient of a Melbourne International Research Scholarship (MIRS) and a Melbourne International Fee Remission Scholarship (MIFRS) from The University of Melbourne.
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- Gasser RB, von Samson-Himmelstjerna G. Haemonchus contortus and Haemonchosis Past, Present and Future Trends. Adv Parasitol. in press.Google Scholar
- Veglia F. The anatomy and life-history of the Haemonchus contortus. Vet Res. 1915;4:347–500.Google Scholar
- Laing R, Kikuchi T, Martinelli A, Tsai IJ, Beech RN, Redman E, et al. The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery. Genome Biol. 2013;14:R88.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwarz EM, Korhonen PK, Campbell BE, Young ND, Jex AR, Jabbar A, et al. The genome and developmental transcriptome of the strongylid nematode Haemonchus contortus. Genome Biol. 2013;14:R89.PubMed CentralView ArticlePubMedGoogle Scholar
- Boyle JP, Yoshino TP. Gene manipulation in parasitic helminths. Int J Parasitol. 2003;33:1259–68.View ArticlePubMedGoogle Scholar
- Aboobaker AA, Blaxter ML. Functional genomics for parasitic nematodes and platyhelminths. Trends Parasitol. 2004;20:178–84.View ArticlePubMedGoogle Scholar
- Gilleard JS. The use of Caenorhabditis elegans in parasitic nematode research. Parasitology. 2004;128 Suppl 1:S49–70.PubMedGoogle Scholar
- Britton C, Murray L. Using Caenorhabditis elegans for functional analysis of genes of parasitic nematodes. Int J Parasitol. 2006;36:651–9.View ArticlePubMedGoogle Scholar
- Kalinna BH, Brindley PJ. Manipulating the manipulators: advances in parasitic helminth transgenesis and RNAi. Trends Parasitol. 2007;23:197–204.View ArticlePubMedGoogle Scholar
- Knox DP, Geldhof P, Visser A, Britton C. RNA interference in parasitic nematodes of animals: a reality check? Trends Parasitol. 2007;23:105–7.View ArticlePubMedGoogle Scholar
- Maule AG, McVeigh P, Dalzell JJ, Atkinson L, Mousley A, Marks NJ. An eye on RNAi in nematode parasites. Trends Parasitol. 2011;27:505–13.View ArticlePubMedGoogle Scholar
- Lok JB. Nucleic acid transfection and transgenesis in parasitic nematodes. Parasitology. 2012;139:574–88.PubMed CentralView ArticlePubMedGoogle Scholar
- Hu PJ. Dauer. WormBook, ed. The C. elegans Research Community, WormBook. 2007. doi/10.1895/wormbook.1.144.1.
- Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, et al. A molecular evolutionary framework for the phylum Nematoda. Nature. 1998;392:71–5.View ArticlePubMedGoogle Scholar
- Hotez P, Hawdon J, Schad GA. Hookworm larval infectivity, arrest and amphiparatenesis: the Caenorhabditis elegans Daf-c paradigm. Parasitol Today. 1993;9:23–6.View ArticlePubMedGoogle Scholar
- Bürglin TR, Lobos E, Blaxter ML. Caenorhabditis elegans as a model for parasitic nematodes. Int J Parasitol. 1998;28:395–411.View ArticlePubMedGoogle Scholar
- Hashmi S, Tawe W, Lustigman S. Caenorhabditis elegans and the study of gene function in parasites. Trends Parasitol. 2001;17:387–93.View ArticlePubMedGoogle Scholar
- Crook M. The dauer hypothesis and the evolution of parasitism: 20 years on and still going strong. Int J Parasitol. 2014;44:1–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Golden JW, Riddle DL. The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature. Dev Biol. 1984;102:368–78.View ArticlePubMedGoogle Scholar
- Riddle DL, Albert PS. Genetic and environmental regulation of dauer larva development. In: Riddle DL, Blumenthal T, Meyer BJ, Priess JR, editors. C. elegans II. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1997. ch. 26.Google Scholar
- Blaxter M. Caenorhabditis elegans is a nematode. Science. 1998;282:2041–6.View ArticlePubMedGoogle Scholar
- Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science. 1997;277:942–6.View ArticlePubMedGoogle Scholar
- Apfeld J, Kenyon C. Cell nonautonomy of C. elegans daf-2 function in the regulation of diapause and life span. Cell. 1998;95:199–210.View ArticlePubMedGoogle Scholar
- Gems D, Sutton AJ, Sundermeyer ML, Albert PS, King KV, Edgley ML, et al. Two pleiotropic classes of daf-2 mutation affect larval arrest, adult behavior, reproduction and longevity in Caenorhabditis elegans. Genetics. 1998;150:129–55.PubMed CentralPubMedGoogle Scholar
- Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci. 1997;22:267–72.View ArticlePubMedGoogle Scholar
- Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006;7:606–19.View ArticlePubMedGoogle Scholar
- Ayyadevara S, Tazearslan C, Bharill P, Alla R, Siegel E, Shmookler Reis RJ. Caenorhabditis elegans PI3K mutants reveal novel genes underlying exceptional stress resistance and lifespan. Aging Cell. 2009;8:706–25.PubMed CentralView ArticlePubMedGoogle Scholar
- Lin K, Dorman JB, Rodan A, Kenyon C. daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science. 1997;278:1319–22.View ArticlePubMedGoogle Scholar
- Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA, et al. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature. 1997;389:994–9.View ArticlePubMedGoogle Scholar
- Ogg S, G R. The C. elegans PTEN homolog, DAF-18, acts in the insulin receptor-like metabolic signaling pathway. Mol Cell. 1998;2:887–93.View ArticlePubMedGoogle Scholar
- Murphy CT, Hu PJ. Insulin/insulin-like growth factor signaling in C. elegans. WormBook, ed. The C. elegans Research Community, WormBook. 2013. doi/10.1895/wormbook.1.164.1.
- Hu M, Lok JB, Ranjit N, Massey Jr HC, Sternberg PW, Gasser RB. Structural and functional characterisation of the fork head transcription factor-encoding gene, Hc-daf-16, from the parasitic nematode Haemonchus contortus (Strongylida). Int J Parasitol. 2010;40:405–15.PubMed CentralView ArticlePubMedGoogle Scholar
- Li F, Lok JB, Gasser RB, Korhonen PK, Sandeman MR, Shi D, et al. Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation. Int J Parasitol. 2014;44:485–96.PubMed CentralView ArticlePubMedGoogle Scholar
- Li FC, Gasser RB, Lok JB, Korhonen PK, Wang YF, Yin FY, et al. Exploring the role of two interacting phosphoinositide 3-kinases of Haemonchus contortus. Parasit Vectors. 2014;7:498.PubMed CentralView ArticlePubMedGoogle Scholar
- https://www.wormbase.org/ (2016). Accessed 6 Jan 2016.
- Kent WJ. BLAT - the BLAST-like alignment tool. Genome Res. 2002;12:656–64.PubMed CentralView ArticlePubMedGoogle Scholar
- Holt C, Yandell M. MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinformatics. 2011;2:491.View ArticleGoogle Scholar
- Lomsadze A, Ter-Hovhannisyan V, Chernoff YO, Borodovsky M. Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res. 2005;33:6494–506.PubMed CentralView ArticlePubMedGoogle Scholar
- Ter-Hovhannisyan V, Lomsadze A, Chernoff YO, Borodovsky M. Gene prediction in novel fungal genomes using an ab initio algorithm with unsupervised training. Genome Res. 2008;18:1979–90.PubMed CentralView ArticlePubMedGoogle Scholar
- Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative Genomics Viewer. Nature Biotechnol. 2011;29:24–6.View ArticleGoogle Scholar
- Thorvaldsdóttir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013;14:178–92.PubMed CentralView ArticlePubMedGoogle Scholar
- http://www.ncbi.nlm.nih.gov/projects/gorf/ (2016). Accessed 6 Jan 2016.
- Zdobnov EM, Apweiler R. InterProScan - an integration platform for the signature-recognition methods in InterPro. Bioinformatics. 2001;17:847–8.View ArticlePubMedGoogle Scholar
- Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014;30:1236–40.PubMed CentralView ArticlePubMedGoogle Scholar
- Thomas PD, Campbell MJ, Kejariwal A, Mi H, Karlak B, Daverman R, et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 2003;13:2129–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Mi H, Muruganujan A, Casagrande JT, Thomas PD. Large-scale gene function analysis with the PANTHER classification system. Nat Protoc. 2013;8:1551–66.View ArticlePubMedGoogle Scholar
- Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26:589–95.PubMed CentralView ArticlePubMedGoogle Scholar
- Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139–40.PubMed CentralView ArticlePubMedGoogle Scholar
- Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.PubMed CentralView ArticlePubMedGoogle Scholar
- http://www.R-project.org/ (2016). Accessed 6 Jan 2016.
- Livneh E, Glazer L, Segal D, Schlessinger J, Shilo BZ. The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains. Cell. 1985;40:599–607.View ArticlePubMedGoogle Scholar
- Aroian RV, Koga M, Mendel JE, Ohshima Y, Sternberg PW. The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature. 1990;348:693–9.View ArticlePubMedGoogle Scholar
- Raz E, Schejter ED, Shilo BZ. Interallelic complementation among DER/flb alleles: implications for the mechanism of signal transduction by receptor-tyrosine kinases. Genetics. 1991;129:191–201.PubMed CentralPubMedGoogle Scholar
- Thacker C, Peters K, Srayko M, Rose AM. The bli-4 locus of Caenorhabditis elegans encodes structurally distinct kex2/subtilisin-like endoproteases essential for early development and adult morphology. Genes Dev. 1995;9:956–71.View ArticlePubMedGoogle Scholar
- Andrade MA, Perez-Iratxeta C, Ponting CP. Protein repeats: structures, functions, and evolution. J Struct Biol. 2001;134:117–31.View ArticlePubMedGoogle Scholar
- Kanai F, Liu H, Field SJ, Akbary H, Matsuo T, Brown GE, et al. The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol. 2001;3:675–8.View ArticlePubMedGoogle Scholar
- Song X, Xu W, Zhang A, Huang G, Liang X, Virbasius JV, et al. Phox homology domains specifically bind phosphatidylinositol phosphates. Biochemistry. 2001;40:8940–4.View ArticlePubMedGoogle Scholar
- Wang Y, Oh SW, Deplancke B, Luo J, Walhout AJ, Tissenbaum HA. Caenorhabditis elegans 14-3-3 proteins regulate life span and interact with SIR-2.1 and DAF-16/FOXO. Mech Ageing Dev. 2006;127:741–7.View ArticlePubMedGoogle Scholar
- Li J, Tewari M, Vidal M, Lee SS. The 14-3-3 protein FTT-2 regulates DAF-16 in Caenorhabditis elegans. Dev Biol. 2007;301:82–91.PubMed CentralView ArticlePubMedGoogle Scholar
- Duret L, Guex N, Peitsch MC, Bairoch A. New insulin-like proteins with atypical disulfide bond pattern characterized in Caenorhabditis elegans by comparative sequence analysis and homology modeling. Genome Res. 1998;8:348–53.PubMedGoogle Scholar
- Kawano T, Ito Y, Ishiguro M, Takuwa K, Nakajima T, Kimura Y. Molecular cloning and characterization of a new insulin/IGF-like peptide of the nematode Caenorhabditis elegans. Biochem Biophys Res Commun. 2000;273:431–6.View ArticlePubMedGoogle Scholar
- Pierce SB, Costa M, Wisotzkey R, Devadhar S, Homburger SA, Buchman AR, et al. Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Dev. 2001;15:672–86.PubMed CentralView ArticlePubMedGoogle Scholar
- Li W, Kennedy SG, Ruvkun G. daf-28 encodes a C. elegans insulin superfamily member that is regulated by environmental cues and acts in the DAF-2 signaling pathway. Genes Dev. 2003;17:844–58.PubMed CentralView ArticlePubMedGoogle Scholar
- Stoltzfus JD, Minot S, Berriman M, Nolan TJ, Lok JB. RNAseq analysis of the parasitic nematode Strongyloides stercoralis reveals divergent regulation of canonical dauer pathways. PLoS Negl Trop Dis. 2012;6:e1854.PubMed CentralView ArticlePubMedGoogle Scholar
- Murphy CT, Lee SJ, Kenyon C. Tissue entrainment by feedback regulation of insulin gene expression in the endoderm of Caenorhabditis elegans. Proc Natl Acad Sci USA. 2007;104:19046–50.PubMed CentralView ArticlePubMedGoogle Scholar
- Cornils A, Gloeck M, Chen Z, Zhang Y, Alcedo J. Specific insulin-like peptides encode sensory information to regulate distinct developmental processes. Development. 2011;138:1183–93.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Z, Hendricks M, Cornils A, Maier W, Alcedo J, Zhang Y. Two insulin-like peptides antagonistically regulate aversive olfactory learning in C. elegans. Neuron. 2013;77:572–85.PubMed CentralView ArticlePubMedGoogle Scholar
- Hristova M, Birse D, Hong Y, Ambros V. The Caenorhabditis elegans heterochronic regulator LIN-14 is a novel transcription factor that controls the developmental timing of transcription from the insulin/insulin-like growth factor gene ins-33 by direct DNA binding. Mol Cell Biol. 2005;25:11059–72.PubMed CentralView ArticlePubMedGoogle Scholar
- Baugh LR, Kurhanewicz N, Sternberg PW. Sensitive and precise quantification of insulin-like mRNA expression in Caenorhabditis elegans. PLoS One. 2011;6:e18086.PubMed CentralView ArticlePubMedGoogle Scholar
- Stoltzfus JD, Bart SM, Lok JB. cGMP and NHR signaling co-regulate expression of insulin-like peptides and developmental activation of infective larvae in Strongyloides stercoralis. PLoS Pathog. 2014;10:e1004235.PubMed CentralView ArticlePubMedGoogle Scholar
- Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 2012;13:283–96.PubMedGoogle Scholar
- Hollander MC, Blumenthal GM, Dennis PA. PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer. 2011;11:289–301.View ArticlePubMedGoogle Scholar
- Oh SW, Mukhopadhyay A, Svrzikapa N, Jiang F, Davis RJ, Tissenbaum HA. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16. Proc Natl Acad Sci USA. 2005;102:4494–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Ayyadevara S, Alla R, Thaden JJ, Shmookler Reis RJ. Remarkable longevity and stress resistance of nematode PI3K-null mutants. Aging Cell. 2008;7:13–22.View ArticlePubMedGoogle Scholar
- Alam H, Williams TW, Dumas KJ, Guo C, Yoshina S, Mitani S, et al. EAK-7 controls development and life span by regulating nuclear DAF-16/FoxO activity. Cell Metab. 2010;12:30–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Hertweck M, Göbel C, Baumeister R. C. elegans SGK-1 is the critical component in the Akt/PKB kinase complex to control stress response and life span. Dev Cell. 2004;6:577–88.View ArticlePubMedGoogle Scholar
- Karathanassis D, Stahelin RV, Bravo J, Perisic O, Pacold CM, Cho W, et al. Binding of the PX domain of p47(phox) to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction. EMBO J. 2002;21:5057–68.PubMed CentralView ArticlePubMedGoogle Scholar
- Accili D, Arden KC. FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell. 2004;117:421–6.View ArticlePubMedGoogle Scholar
- Lee RY, Hench J, Ruvkun G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr Biol. 2001;11:1950–7.View ArticlePubMedGoogle Scholar
- Lin K, Hsin H, Libina N, Kenyon C. Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat Genet. 2001;28:139–45.View ArticlePubMedGoogle Scholar
- Henderson ST, Johnson TE. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr Biol. 2001;11:1975–80.View ArticlePubMedGoogle Scholar
- Kwon ES, Narasimhan SD, Yen K, Tissenbaum HA. A new DAF-16 isoform regulates longevity. Nature. 2010;466:498–502.PubMed CentralView ArticlePubMedGoogle Scholar
- Massey Jr HC, Nishi M, Chaudhary K, Pakpour N, Lok JB. Structure and developmental expression of Strongyloides stercoralis fktf-1, a proposed ortholog of daf-16 in Caenorhabditis elegans. Int J Parasitol. 2003;33:1537–44.PubMed CentralView ArticlePubMedGoogle Scholar
- Brand A, Hawdon JM. Phosphoinositide-3-OH-kinase inhibitor LY294002 prevents activation of Ancylostoma caninum and Ancylostoma ceylanicum third-stage infective larvae. Int J Parasitol. 2004;34:909–14.View ArticlePubMedGoogle Scholar
- Huang SC, Chan DT, Smyth DJ, Ball G, Gounaris K, Selkirk ME. Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum. Int J Parasitol. 2010;40:1619–28.View ArticlePubMedGoogle Scholar
- Stoltzfus JD, Massey Jr HC, Nolan TJ, Griffith SD, Lok JB. Strongyloides stercoralis age-1: a potential regulator of infective larval development in a parasitic nematode. PLoS One. 2012;7:e38587.PubMed CentralView ArticlePubMedGoogle Scholar
- Gibbs HC. Hypobiosis in parasitic nematodes - an update. Adv Parasitol. 1986;25:129–74.View ArticlePubMedGoogle Scholar