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. 2021 Apr 7;6(15):10288–10305. doi: 10.1021/acsomega.1c00650

Peptidomics of Haemonchus contortus

Armelle Buzy †,*, Camille Allain †, John Harrington ‡, Dominique Lesuisse †, Vincent Mikol †, David F Bruhn ‡, Aaron G Maule §, Jean-Claude Guillemot †
PMCID: PMC8153747  PMID: 34056183

Abstract

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The nematode Haemonchus contortus (the barber’s pole worm) is an endoparasite infecting wild and domesticated ruminants worldwide. Widespread anthelmintic resistance of H. contortus requires alternative strategies to control this parasite. Neuropeptide signaling represents a promising target for anthelmintic drugs. Identification and relative quantification of nematode neuropeptides are, therefore, required for the development of such therapeutic targets. In this work, we undertook the profiling of the whole H. contortus larvae at different stages for the direct sequencing of the neuropeptides expressed at low levels in these tissues. We set out a peptide extraction protocol and a peptidomic workflow to biochemically characterize bioactive peptides from both first-stage (L1) and third-stage larvae (L3) of H. contortus. This work led to the identification and quantification at the peptidomic level of more than 180 mature neuropeptides, including amidated and nonamidated peptides, arising from 55 precursors of H. contortus. The differential peptidomic approach provided evidence that both life stages express most FMRFamide-like peptides (FLPs) and neuropeptide-like proteins (NLPs). The H. contortus peptidome resource, established in this work, could add the discovery of neuropeptide system-targeting drugs for ruminants.

Introduction

Haemonchus contortus is a very common endoparasite and one of the most pathogenic nematodes infecting wild and domesticated ruminants worldwide.1 It lives in the abomasum, causing hemorrhagic gastritis, anemia, edema, and even death of the infected animals.2

H. contortus exhibits a monoxenous life cycle that consists of a free-living phase in the external environment and a parasitic phase in the infested host animal.3 The life cycle begins with the laying of eggs by H. contortus females. The first-stage larvae (L1s) develops inside the eggs after their excretion in the host feces and molts to the second stage (L2s) and then into the third-stage larvae (L3s) within approximately 1 week. The infective L3s are then ingested by the host animal, undergo an exsheathment process to develop to become fourth-stage larvae (L4s) and then to dioecious adults within 3 weeks in the stomach. The last two stages feed on blood from capillaries of the abomasal wall.

Haemonchosis is mainly controlled by anthelmintics, which act by compromising the nematode motor function.4−6 However, the increase in resistance to existing chemotherapeutics warrants the identification of new parasiticides with novel modes of action.7−9 As neuropeptides and their receptors regulate many vital biological processes (such as development, behavior, movement, metabolism, and reproduction5,10), the nematode neuropeptide signaling system has been proposed as a promising target for novel drugs against helminths.11 Neuropeptides consist of short peptides that are derived from larger precursor proteins by the action of processing enzymes and are commonly subjected to post-translational modifications.12 Three large neuropeptide groupings occur in nematodes, two of which are defined by conserved structural features (the FMRFamide-like peptides (FLPs)12−15 and insulin-like peptides (INSs)).16 The FMRFamide-like peptides (FLPs) are a group of neuropeptides that are similar to the tetrapeptide FMRF-amide (H-Phe-Met-Arg-Phe-NH2), a cardioexcitatory peptide first isolated from the mollusk Macrocallista nimbosa.17 Neuropeptides sharing a C-terminal RFamide motif have been further identified from other organisms and were defined as FLPs. The third group comprises all other neuropeptides and includes diverse family groupings (the neuropeptide-like proteins NLPs.) Unlike FLPs and INSs that each comprise single families, the NLPs were originally defined as encompassing 11 distinct peptide families.18,19 The most studied neuropeptide family in nematodes is the FLP gene family: all contain a variation in the tetrapeptide motif X/Y-X-RF-amide where X is a nonpolar hydrophobic (L, I, M or V) residue and Y is aromatic.12,14,15,20

Detailed knowledge on neuropeptide sequences in parasitic nematodes and their post-translational modifications is required to help building an understanding of their in vivo biology and physiological role. The availability of genomic and transcriptomic data sets and the development of in silico mining tools have enabled the identification of neuropeptide genes and further prediction of neuropeptide sequences.21−23 However, these in silico discovery approaches suffer major drawbacks in that the end products are bioactive peptides that can be modified, nonclassically cleaved, or even mispredicted.24 The development and application of sensitive mass spectrometry-based peptidomic technologies25 have enabled the biochemical identification of many nematode neuropeptides.22,26,27 Of particular interest is the recent work performed on Caenorhabditis elegans in which a peptidomic analysis was performed to identify unprecedented 203 mature neuropeptides from C. elegans.28,29 Previous high-throughput peptidomic approaches on parasitic nematodes have been confined to the large gastrointestinal parasite of pigs, Ascaris suum.22,30 No such studies have been reported on other nematode parasites and only two FLP neuropeptides have been characterized biochemically from H. contortus.31,32 In this paper, we report on a comprehensive peptidomic study to biochemically monitor, identify, and quantify endogenous peptides from two larval stages (the first-stage larvae (L1s) and the third-stage larvae (L3s)) of H. contortus through a peptidomic workflow using the recent release of two genome assemblies for H. contortus.33,34

This study aimed at characterizing the whole-worm peptidome of L1 and L3 larval stages of H. contortus using a label-free peptidomic approach. Peptidome exploration of essential developmental stages of H. contortus will provide a valuable repository for a better understanding of this nematode at the biochemical level.

Results

Genomic and Transcriptomic Data Set Interrogation

The direct identification of bioactive peptides using a mass spectrometry (MS)-based strategy relies on mapping the peptide masses identified to a reference data set (predicted from genome and/or transcriptome). There are two genomic and transcriptomic data sets publicly available for H. contortus.33,34 The two versions of the genome and transcriptome presented in both publications are available on the WormBase site (https://wormbase.org) (BioProject PRJNA205202 and BioProject PRJEB506). The protein FASTA files for both BioProjects can be downloaded from the WormBase site. It is noteworthy that for the BioProject PRJEB506, the genome reported for H. contortus has been updated in the WormBase version 11.0, whereas all of the PRJNA205202 versions have remained unchanged.

Before submitting MS/MS data for database searching, we analyzed the two H. contortus draft genomes and transcriptomes, PRJNA205202 version 14 (WBPS14) and PRJEB506 version 14 (WBPS14) and version 10 (WBPS10), for the presence of potential FLPs using the H. contortus C-terminal FLP motifs and FLP-gene sequelogues identified by McCoy et al. in a pan-phylum bioinformatics study14 (Table 1).

Table 1. Presence of FMRFamide-like Peptide Encoding Genes (FLPs) in Genomic and Transcriptomic Data Sets of H. contortus.

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a

From McCoy et al.14 gray shading indicates the presence of a gene. The number of copies of a predicted peptide is indicated as (no.×). Complete sequence of flp-32 was found in Atkinson et al.35 X0 denotes a hydrophobic amino acid. One peptide among the four amidated peptides predicted from the flp-11 precursor is not an flp peptide. This peptide is indicated in italics.

b

flp-19 sequence was present in previous PRJEB506 release (versions to 10) but not in versions 11–14.

Among the 32 FLP-encoding genes identified in 17 nematode parasites, 26 have been reported for H. contortus, highlighted in gray in Table 1.14,36 Each flp gene encodes one or several FLPs, up to 8, for a total of 62 different predicted FLPs. Eleven FLP sequences (flp-1, flp-5, flp-6, flp-14, flp-15, flp-17, flp-18, flp-21, flp-25, flp-33, and flp-34) were identified within both the H. contortus databases reported in PRJEB506 and PRJNA205202 (WormBase), with some discrepancies in sequences for flp-5, flp-14, and flp-18 (Table 1). Eleven flp transcripts (flp-2, flp-7, flp-8, flp-9, flp-11, flp-12, flp-13, flp-16, flp-19, flp-22, and flp-24) were found only in PRJEB506 with some discrepancies in sequence for flp-7 and flp-16. Flp-28 was only identified in PRJNA205202. Surprisingly, flp-19 was present in previous PRJEB506 releases (versions 1–10) but not in versions 11–14. Finally, three flp sequences (flp-20, flp-23, and flp-32) were not identified in either database (Figure 2); flp-32 was reported by Atkinson et al.35

Figure 2.

Figure 2

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MS/MS spectra of two unmodified peptides of the H. contortusflp-6. The fragmentation schemes enabled the identification of the peptide SEALDEDPMDVE in the WormBase, PRJEB506.WBPS10 database analysis (A), and of the peptide SSEVEDSPDAIDME upon the analysis of both PRJEB506.WBPS14 and PRJNA205202.WBPS14 WormBase (B).

To maximize peptide identification using our approaches, we used a combination of the two transcriptome databases (PRJEB506 and PRJNA205202) with a homemade database that we constructed from the sequelogue sequences described14 (Supplementary Data 1).

Identification of Neuropeptides

To biochemically identify endogenous FLP peptides and other bioactive peptides of H. contortus, a peptide acidic/methanol extraction method was used (see the Methods section). Peptides extracted from both first-stage (L1) and third-stage larvae (L3) of H. contortus were analyzed by LC/MS/MS, and the results were processed in the MaxQuant environment implemented with the three FASTA databases described in the above paragraph. This way, we sequenced 181 endogenous peptides belonging to 55 different peptide precursor proteins.

FMRFamide-like Peptides

Among the 26 FLP genes described for H. contortus, there are 62 predicted FMRFamide-like peptides (Table 1). In addition, two peptides not strictly belonging to the FLPs can be found, the flp-11 peptide (YLATDDDYATAAAQG) described as a neuropeptide and the RYamide flp-34 peptide (SDLSDFASAINSAGRLRYG).

In this work, we isolated and identified 54 of the 62 predicted peptides (Table 2) across the two larval stages. Only two of these peptides were previously biochemically characterized, KHEYLRF.NH2 (flp-14) and KSAYMRF.NH2 (flp-6), by Keating et al.31 and Marks et al.32

Table 2. FLP-Amidated Peptides Identified in the First-Stage (L1) and Third-Stage Larvae (L3) of H. contortus.
gene precursor namea database sequence modifications mass start position end position
flp-1 HCON_00103480; maker-scaffold1982-snap-gene-0.20-mRNA-1 PRJEB506; PRJNA205202 KPNFMRFG Gly-loss+Amide 937.4956 70 77
flp-1 HCON_00103480; maker-scaffold1982-snap-gene-0.20-mRNA-1 PRJEB506; PRJNA205202 GSDPNFLRFG Gly-loss+Amide 1050.525 87 96
flp-1 HCON_00103480; maker-scaffold1982-snap-gene-0.20-mRNA-1 PRJEB506; PRJNA205202 NQPNFLRFG Gly-loss+Amide 1033.546 98 106
flp-1 HCON_00103480; maker-scaffold1982-snap-gene-0.20-mRNA-1 PRJEB506; PRJNA205202 AAGDPNFLRFG Gly-loss+Amide 1105.567 118 128
flp-1 HCON_00103480; maker-scaffold1982-snap-gene-0.20-mRNA-1 PRJEB506; PRJNA205202 GAGDPNFLRFG Gly-loss+Amide 1091.551 130 140
flp-1 HCON_00103480; maker-scaffold1982-snap-gene-0.20-mRNA-1 PRJEB506; PRJNA205202 GVDPNFLRFG Gly-loss+Amide 1062.561 143 152
flp-1 HCON_00103480; maker-scaffold1982-snap-gene-0.20-mRNA-1 PRJEB506; PRJNA205202 KPNFLRFG Gly-loss+Amide 919.5392 154 161
flp-2 HCON_00188000 PRJEB506 FRGEPIRFG Gly-loss+Amide 1019.567 39 47
flp-2 HCON_00188000 PRJEB506 VPREPIRFG Gly-loss+Amide 1011.598 50 58
flp-5 HCON_00164350; maker-scaffold856-augustus-gene-0.7-mRNA-1 PRJEB506; PRJNA205202 APKFIRFG Gly-loss+Amide 876.5334 37 44
flp-5 HCON_00164350; maker-scaffold856-augustus-gene-0.7-mRNA-1 PRJEB506; PRJNA205202 GGGAKFIRFG Gly-loss+Amide 950.545 46 55
flp-5 HCON_00164350; maker-scaffold856-augustus-gene-0.7-mRNA-1 PRJEB506 AAKFIRFG Gly-loss+Amide 850.5177 80 87
flp-6 HCON_00155670; augustus-scaffold18780-abinit-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 KSAYMRFG Gly-loss+Amide 900.464 32 39
flp-7 HCON_00164220 PRJEB506 TPMVRSSMVRFG Gly-loss+Amide 1308.68 42 53
flp-7 HCON_00164220 PRJEB506 APMDRSAMVRFG Gly-loss+Amide 1278.633 56 67
flp-7 HCON_00164220 PRJEB506 APMDRSSMVRFG Gly-loss+Amide 1294.627 97 108
flp-8 HCON_00180390 PRJEB506 KNEFIRFG Gly-loss+Amide 951.529 3 10
flp-9 HCON_00131250 PRJEB506 KPSFVRFG Gly-loss+Amide 878.5127 69 76
flp-11 HCON_00176100 PRJEB506 AMRNALVRFG Gly-loss+Amide 1075.607 31 40
flp-11 HCON_00176100 PRJEB506 AGGSMRNALVRFG Gly-loss+Amide 1276.682 42 54
flp-11 HCON_00176100 PRJEB506 YLATDDDYATAAAQG Gly-loss+Amide 1486.658 57 71
flp-11 HCON_00176100 PRJEB506 NGAPQPFVRFG Gly-loss+Amide 1130.599 74 84
flp-12 HCON_00164300 PRJEB506 NKFEFIRFG Gly-loss+Amide 1098.597 74 82
flp-13 HCON_00095850 PRJEB506 SFEENASPLIRFG Gly-loss+Amide 1407.715 44 56
flp-13 HCON_00095850 PRJEB506 DLSGAPLIRFG Gly-loss+Amide 1086.619 59 69
flp-13 HCON_00095850 PRJEB506 APEAHPLIRFG Gly-loss+Amide 1148.646 71 81
flp-13 HCON_00095850 PRJEB506 APDSAPLIRFG Gly-loss+Amide 1084.603 84 94
flp-13 HCON_00095850 PRJEB506 DPEASPLIRFG Gly-loss+Amide 1142.608 96 106
flp-13 HCON_00095850 PRJEB506 SPAAPLIRFG Gly-loss+Amide 969.576 109 118
flp-13 HCON_00095850 PRJEB506 SPNASPLIRFG Gly-loss+Amide 1099.614 120 130
flp-14 HCON_00084500; maker-scaffold19612-snap-gene-0.18-mRNA-1 PRJEB506; PRJNA205202 KHEYLRFG Gly-loss+Amide 990.5399 93 100
flp-15 HCON_00084140 PRJEB506; PRJNA205202 AGPQGPLRFG Gly-loss+Amide 940.5243 41 49
flp-15 HCON_00084140 PRJEB506; PRJNA205202 GPSGPLRFG Gly-loss+Amide 828.4606 53 61
flp-16 HCON_00035475 PRJEB506 AQTFVRFG Gly-loss+Amide 866.4763 70 77
flp-17 HCON_00123460; maker-C469629-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 KSAFVRFG Gly-loss+Amide 852.497 72 79
flp-17 HCON_00123460; maker-C469629-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 KSQYIRFG Gly-loss+Amide 939.529 112 119
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 DLDGGMPGVLRFG Gly-loss+Amide 1274.644 54 66
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 EVPGVLRFG Gly-loss+Amide 914.5338 76 84
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 SMPGVLRFG Gly-loss+Amide 904.4953 91 99
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 SVPGVLRFG Gly-loss+Amide 872.5232 102 110
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 EMPGVLRFG Gly-loss+Amide 946.5059 113 121
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 AMPGVLRFG Gly-loss+Amide 888.5004 124 132
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 TEIPGMMRFG Gly-loss+Amide 1079.526 135 144
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 NVPGVLRFG Gly-loss+Amide 899.5341 161 169
flp-19 HCOI00587500b PRJEB506.WBPS10 WANQVRFG Gly-loss+Amide 918.4824 50 57
flp-19 HCOI00587500a PRJEB506.WBPS10 ASSWASSIRFG Gly-loss+Amide 1109.562 60 70
flp-22 HCON_00012150 PRJEB506 TPSAKWMRFG Gly-loss+Amide 1121.58 37 46
flp-22 HCON_00012150 PRJEB506 SPNAKWMRFG Gly-loss+Amide 1134.576 49 58
flp-22 HCON_00012150 PRJEB506 TPDAKWMRFG Gly-loss+Amide 1149.575 61 70
flp-24 HCON_00094680 PRJEB506 VPSAGDMMVRFG Gly-loss+Amide 1207.584 53 64
flp-25 HCON_00078750; maker-scaffold165-snap-gene-0.6-mRNA-1 PRJEB506; PRJNA205202 HYDFVRFG Gly-loss+Amide 981.4821 47 54
flp-25 HCON_00078750; maker-scaffold165-snap-gene-0.6-mRNA-1 PRJEB506; PRJNA205202 ASYDYIRFG Gly-loss+Amide 1032.503 63 71
flp-33 HCON_00009870; maker-scaffold18501-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 SIDEIQKPRFG Gly-loss+Amide 1230.672 66 76
flp-34 HCON_00140260; maker-scaffold19714-augustus-gene-0.9-mRNA-1 PRJNA205202 SDLSDFASAINSAGRLRYG Gly-loss+Amide 1940.97 51 69
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a

The precursor name corresponds either to the one reported in project PRJEB506 or to the one described in project PRJNA205202 on the WormBase site. When sequences are predicted by both databases, the peptide sequence start and end positions refer to the database indicated first.

b

Denotes identified sequences from the PRJEB506 release (versions previous version 10).

All predicted FLP precursors were found except flp-20, flp-21, flp-23, flp-28, and flp-32. It is noteworthy that flp-20, flp-23, and flp-32 were not identified in either of the WormBase data sets, although the complete flp-32 sequence was previously reported.35 In the study performed on C. elegans by Van Bael et al.,28,29 the bioactive peptides issued from these precursors were either not detected or could not be confirmed using MS/MS. Peptides predicted to be encoded by flp-1, flp-2, flp-6, flp-8, flp-9, flp-11, flp-12, flp-13, flp-15, flp-17, flp-19, flp-24, flp-25, flp-33, and flp-34 transcripts, which had identical sequences between those reported in MacCoy et al.14 and both WormBase databases, were all unambiguously identified in this study, except for one peptide (APITSKLIQSLNEAERLRFG) arising from flp-34 (Table 1), not detected in this study. Peptides arising from flp-19 (WANQVRFG and ASSWASSIRFG) whose sequences were removed from the updated versions of PRJEB506 were unambiguously identified in this study.

For peptides showing sequence discrepancies across those reported14 and the WormBase data sets (Figure 1; Table 1), the use of a combination of all three data sets enabled sequence confirmation. For flp-5, flp-7, flp-16, and flp-18, the correct predicted sequences are those reported in the WormBase data sets. The peptide KHEYLRFSRG arising from flp-14 and predicted only by the sequelogue database was not detected in this study, whereas the other flp-14-predicted peptide (KHEYLRFG) was unambiguously identified. This raises the question of the occurrence of the undetected peptide.

Figure 1.

Figure 1

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Presence of flp-gene sequences in the H. contortus databases reported in PRJEB506 (pink circle) and PRJNA205202 (blue circle) on WormBase (https://wormbase.org). Flp-20, flp-23, and flp-32 were described by McCoy et al.14 and flp-32 by Atkinson et al.35 * indicates discrepancies between sequences reported in McCoy et al.14 and databases from Wormbase. ** Flp-19 sequence was present in the previous PRJEB506 release (versions to 10) but not in versions 11–14.

In addition to the confirmation that peptides were all amidated at the C-terminus, we also searched for additional processed peptides as described for C. elegans.29 The authors looked for potential peptides derived from predicted neuropeptides precursors, which are flanked by (di)basic residues and which were not identified as mature peptides. Applying the same strategy to our H. contortus peptidomic data set, we could identify 14 additional peptides arising from 10 FLP precursors (Table 3). Here again, the interrogation of both worm databases led to the identification of the correct sequences for these additional peptides derived from flp-5, flp-6, flp-15, and flp-17.

Table 3. flp-Gene-Encoded Non-FLP Peptides Identified in the First-Stage (L1) and Third-Stage Larvae (L3) of H. contortus.
gene precursor namea database sequence modifications mass start positionb end positionb
flp-2 HCON_00188000 PRJEB506 GPMFEPYFDY unmodified 1264.511 61 70
flp-5 HCON_00164350 PRJEB506 SGTNTWDDDSSDITSYAHQDD unmodified 2328.889 57 77
flp-6 HCON_00155670; augustus-scaffold18780-abinit-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 SDPELADQMMME unmodified 1395.536 41 52
flp-6 HCON_00155670 PRJEB506 SEALDEDPMDVE unmodified 1348.534 65 76
flp-6 HCON_00155670; augustus-scaffold18780-abinit-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 SSEVEDSPDAIDME unmodified 1522.598 99 112
flp-11 HCON_00176100 PRJEB506 SGHLDHIHDILSTLQKLQLANYH unmodified 2652.377 86 108
flp-13 HCON_00095850 PRJEB506 VDTLSRES unmodified 905.4454 35 42
flp-15 U6PJU1 CBN-FLP-15protein GN=HCOI 01474400 PRJEB506 EIEDITDDSK unmodified 1163.519 22 31
flp-15 U6PJU1 CBN-FLP-15protein GN=HCOI 01474400 PRJEB506 STFDYPTVFDQQPYYYFV unmodified 2279.01 64 81
flp-17 HCON_00123460; maker-C469629-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 AAEESAEIE unmodified 947.4084 82 90
flp-17 maker-C469629-augustus-gene-0.17-mRNA-1 PRJNA205202 SAAEFDMPE unmodified 995.3906 102 110
flp-18 HCON_00164730; augustus-scaffold2866-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 STYDTIPLELLD unmodified 1378.687 147 148
flp-25 HCON_00078750; maker-scaffold165-snap-gene-0.6-mRNA-1 PRJEB506; PRJNA205202 SQIDSDDLNARFSPYQFL unmodified 2114.991 74 91
flp-33 HCON_00009870; maker-scaffold18501-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 SPLEGFEDISSMM unmodified 1441.611 52 64
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a

The precursor name corresponds either to the one reported in project PRJEB506 or to the one described in project PRJNA205202 on the WormBase site.

b

When sequences are predicted by both databases, the peptide sequence start and end positions refer to the database indicated first.

For example, we found two additional non-FLP peptides encoded on flp-17, with one (AAEESAEIE) correctly predicted by both databases and the other (SAAEFDMPE) arising from the PRJEB506 database only. Flp-6 gives rise to two additional non-FLP peptides (SDPELADQMMME and SSEVEDSPDAIDME) having the same predicted sequences on both databases: PRJEB506.WBPS14 and PRJNA205202.WBPS14. We also identified the peptide (SEALDEDPMDVE) predicted only by the database PRJEB506.WBPS10 (version before the update). The PRJEB506.WBPS14 database predicted the sequence (SEALEEDPMDVE), which was not identified in this study. Interestingly, the peptide (SSEVEDSPDAIDME) was wrongly predicted by the previous version, PRJEB506.WBPS10. Figure 2 shows the MS/MS spectra of the peptides SSEVEDSPDAIDME and SEALDEDPMDVE. Figure S1 illustrates different predicted flp-6 sequence alignments.

All FLP precursor sequences identified in this study were aligned with the corresponding C. elegans FLP gene precursors (Figure S1). These alignments emphasize the strong homologies of bioactive peptides and their mono- and di-basic cleavage sites across nematode species, as highlighted.14 This is illustrated in Figure 3 with the alignment of flp-11 precursors for both H. contortus and C. elegans. The four amidated peptides observed for C. elegansflp-11 were also detected for H. contortus, with one of them being amidated on a glutamine residue in both species. Compared to the studies of Van Bael et al.,28,29 we were able to sequence the C-terminal peptide (SGHLDHIHDILSTLQKLQLANYH).

Figure 3.

Figure 3

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Sequence alignments of H. contortusflp-11 (sequence in the BioProject PRJEB506 protein fasta file, Wormbase source) with C. elegansflp-11 (UniProtKB source). Sequences were aligned using the Clustal Omega program (http://www.clustal.org). Predicted signal peptide is indicated in italics. Putative mono- and di-basic cleavage sites are shown in red. The potential C-terminal glycine residues for amidation are indicated in brown. C. elegans peptide data shown in the study of Van Bael et al.29 are highlighted in blue. H. contortus FLP peptide sequences identified in this study are shown in bold green with the sequence of the unmodified peptides being underlined. The amidated glutamine residues identified in both species are indicated in purple.

NLP Peptides

The identification of NLP peptides was carried out in association with the two PRJEB506 (version 10 and version 14) and PRJNA205202 WormBase data sets (there were no additional resources for NLP precursors as there was for FLP precursors). In addition, we performed a BLAST analysis of the 82 NLP precursors of C. elegans reported,28,29 against the PRJEB506 and PRJNA205202 data sets. In total, 42 nlpH. contortus genes could be found using this BLAST approach (Table S1). Among these 42 nlp genes, 20 were predicted by both PRJNA205202 and PRJEB506 BioProjects and 22 were found only in the PRJEB506 data set. As noticed for FLP precursors, some NLP precursors (those from nlp-1, nlp-19, nlp-35, and nlp-69) were only predicted by the PRJEB506 versions before the update (versions 1–10), whereas nlp-10, nlp-12, and nlp58 were found in the updated PRJEB506 versions (versions 11–14).

In this study, we clearly detected and identified 110 putative bioactive neuropeptides encoded by 33 of these 42 predicted NLP precursors (Table 4). We also identified three additional peptides arising from the precursor HCON_00135420 (PRJEB506 nomenclature), which could not be assigned by BLAST searches to any C. elegans precursor.

Table 4. NLP Peptides Identified in the First-Stage (L1) and Third-Stage Larvae (L3) of H. contortus.
NLP precursor namea database sequence modifications mass start positionb end positionb
nlp-1c HCOI00158100 PRJEB506.WBPS10 AVMFPRTFGALFG Gly-loss+Amide 1354.722 31 43
nlp-1c HCOI00158100 PRJEB506.WBPS10 MDMKHYFVGLG Gly-loss+Amide 1238.594 82 92
nlp-3 HCON_00188680 PRJEB506 AINPFLDSMG Gly-loss+Amide 1005.495 28 37
nlp-3 HCON_00188680 PRJEB506 AVNPFLDSFG Gly-loss+Amide 1007.508 40 49
nlp-3 HCON_00188680 PRJEB506 SSRYQPYYHLD unmodified 1427.647 52 62
nlp-3 HCON_00188680 PRJEB506 YFDSLAGQALG Gly-loss+Amide 1082.54 65 75
nlp-5 HCON_00046600; maker-C469189-snap-gene-0.14-mRNA-1 PRJEB506; PRJNA205202 ALSSFDTLGGIGLG Gly-loss+Amide 1248.671 42 55
nlp-5 HCON_00046600 PRJEB506 TQLSSIDSLGGLGLG Gly-loss+Amide 1358.741 46 60
nlp-5 HCON_00046600; maker-C469189-snap-gene-0.14-mRNA-1 PRJEB506; PRJNA205202 SEDTAKKALSSFDTLGGIGLG Gly-loss+Amide 2008.048 123 143
nlp-5 HCON_00046600; maker-C469189-snap-gene-0.14-mRNA-1 PRJEB506; PRJNA205202 DDMLAGEKKSVSSFDTLAGIGLG Gly-loss+Amide 2252.136 146 168
nlp-5 maker-C469189-snap-gene-0.14-mRNA-1 PRJNA205202 SRLFSTYYYLPYRDSLEDMDQNAQE unmodified 3103.387 171 195
nlp-5 HCON_00046600 PRJEB506 SRLFSTYYYLPYRDSLEDMDQNVQE unmodified 3131.418 127 151
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 LVIPSYLSSHYD unmodified 1392.693 32 43
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 TLDFDDPRLFSTAFG Gly-loss+Amide 1642.799 46 60
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 NGLVTTTLNRPRFI unmodified 1600.905 63 76
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 SPFLGTNVGLAYI unmodified 1350.718 79 91
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 ADMDPRFISNSFG Gly-loss+Amide 1397.64 94 106
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 STLYDFDDPRFASLSFG Gly-loss+Amide 1878.879 109 125
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 SGFDFDDPRFSSMSFG Gly-loss+Amide 1739.725 128 143
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 SGFDLEDPRFASMSFG Gly-loss+Amide 1703.761 146 161
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 SGFN FEDPRFASLSFG Gly-loss+Amide 1718.805 164 179
nlp-7 HCON_00165470; maker-C452207-snap-gene-0.3-mRNA-1 PRJEB506; PRJNA205202 SGFDLDDPRFASMSFG Gly-loss+Amide 1689.746 182 197
nlp-7 HCON_00165470 PRJEB506 SGSDLEDPRYWSMSFG Gly-loss+Amide 1774.762 200 215
nlp-8 HCON_00010710; maker-scaffold14524-snap-gene-0.4-mRNA-1 PRJEB506; PRJNA205202 AFDRIESN DFGLF unmodified 1529.715 49 61
nlp-8 HCON_00010710; maker-scaffold14524-snap-gene-0.4-mRNA-1 PRJEB506; PRJNA205202 AFDRIEMADFGF unmodified 1417.634 69 80
nlp-8 HCON_00010710; maker-scaffold14524-snap-gene-0.4-mRNA-1 PRJEB506; PRJNA205202 AFDRVGRTEFGFEGVL unmodified 1798.9 85 100
nlp-8 HCON_00010710; maker-scaffold14524-snap-gene-0.4-mRNA-1 PRJEB506; PRJNA205202 TEFGFEGVL unmodified 997.4757 92 100
nlp-8 HCON_00010710; maker-scaffold14524-snap-gene-0.4-mRNA-1 PRJEB506; PRJNA205202 AADRLADIGFRN unmodified 1317.679 104 115
nlp-9 HCON_00136940; maker-scaffold10126-snap-gene-0.11-mRNA-1 PRJEB506; PRJNA205202 GGARAFHGYFNMPSS unmodified 1597.71 48 62
nlp-9 HCON_00136940; maker-scaffold10126-snap-gene-0.11-mRNA-1 PRJEB506; PRJNA205202 LSGEYPYYLYE unmodified 1395.623 65 75
nlp-9 maker-scaffold10126-snap-gene-0.11-mRNA-1 PRJNA205202 GGGRAFFGGWQPYESLGARMD unmodified 2258.033 78 98
nlp-9 HCON_00136940; maker-scaffold10126-snap-gene-0.11-mRNA-1 PRJEB506; PRJNA205202 SSSLWEFLEDRNAL unmodified 1665.8 127 140
nlp-10 HCON_00073270; maker-scaffold13486-augustus-gene-0.18-mRNA- PRJEB506; PRJNA205202 AVMPFSGGLYG Gly-loss+Amide 1039.516 64 74
nlp-10 HCON_00073270; maker-scaffold13486-augustus-gene-0.18-mRNA- PRJEB506; PRJNA205202 SEMPDDMYIERPVLPLSAGWQE unmodified 2562.177 90 111
nlp-10 HCON_00073270; maker-scaffold13486-augustus-gene-0.18-mRNA- PRJEB506; PRJNA205202 AVMPFSGGLYGKRAVMPFSGGLYG Gly-loss+Amide 2403.223 114 137
nlp-10 HCON_00073270; maker-scaffold13486-augustus-gene-0.18-mRNA- PRJEB506; PRJNA205202 AAMPFSGGLYG Gly-loss+Amide 1011.485 140 150
nlp-10 HCON_00073270; maker-scaffold13486-augustus-gene-0.18-mRNA- PRJEB506; PRJNA205202 ADRYIRSPMPISGGIFG Gly-loss+Amide 1777.93 153 169
nlp-10 HCON_00073270; maker-scaffold13486-augustus-gene-0.18-mRNA- PRJEB506; PRJNA205202 SPMPISGGIFG Gly-loss+Amide 1003.516 159 169
nlp-11 HCON_00062360; augustus-C472037-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 LETELHPLVMGMYGFGPENNAY unmodified 2481.135 36 57
nlp-11 HCON_00062360; augustus-C472037-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 HISPSFDVEEDVGNMRTLMDIG Gly-loss+Amide 2403.12 67 88
nlp-11 HCON_00062360; augustus-C472037-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 QLSVADDVGRQMQMYHRLFEAG Gly-loss+Amide 2492.205 91 112
nlp-11 HCON_00062360; augustus-C472037-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 AALSPSQDLQSAVELSNYLERAG Gly-loss+Amide 2360.197 116 138
nlp-12 HCON_00021180; maker-scaffold5805-snap-gene-0.2-mRNA-1 PRJEB506; PRJNA205202 DYRPLQFG Gly-loss+Amide 936.4818 31 38
nlp-12 HCON_00021180; maker-scaffold5805-snap-gene-0.2-mRNA-1 PRJEB506; PRJNA205202 DGYRPLQFG Gly-loss+Amide 993.5032 41 49
nlp-12 HCON_00021180; maker-scaffold5805-snap-gene-0.2-mRNA-1 PRJEB506; PRJNA205202 SPLASAFLVPAL unmodified 1184.681 62 73
nlp-13 HCON_00136950; maker-C471727-snap-gene-0.19-mRNA-1 PRJEB506; PRJNA205202 NDFSRDIMHFG Gly-loss+Amide 1279.577 33 43
nlp-13 HCON_00136950; maker-C471727-snap-gene-0.19-mRNA-1 PRJEB506; PRJNA205202 AYGNGRLVAYGGPAFERDMMAFG Gly-loss+Amide 2391.125 46 68
nlp-13 HCON_00136950; maker-C471727-snap-gene-0.19-mRNA-1 PRJEB506; PRJNA205202 SGGFEREMMSFG Gly-loss+Amide 1275.538 71 82
nlp-13 HCON_00136950 PRJEB506 SPFEREFLSFG Gly-loss+Amide 1256.61E 85 95
nlp-13 HCON_00136950; maker-C471727-snap-gene-0.19-mRNA-1 PRJEB506; PRJNA205202 GSEFDREMLSFG Gly-loss+Amide 1315.587 98 109
nlp-13 HCON_00136950; maker-C471727-snap-gene-0.19-mRNA-1 PRJEB506; PRJNA205202 DEFERSMMAFG Gly-loss+Amide 1260.527 112 122
nlp-14 HCON_00190000; maker-scaffold4554-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 ALDSLEGDGFGGLF unmodified 1396.651 52 65
nlp-14 HCON_00190000; maker-scaffold4554-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 SLDSLEGDGFGFD unmodified 1357.567 68 80
nlp-14 HCON_00190000; maker-scaffold4554-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 ALNALDGTGFGFD unmodified 1296.599 83 95
nlp-14 HCON_00190000; maker-scaffold4554-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 ALNSLEGTGFGFD unmodified 1326.609 98 110
nlp-14 HCON_00190000; maker-scaffold4554-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 SLNSIEGTGFGFD unmodified 1342.604 113 125
nlp-14 HCON_00190000; maker-scaffold4554-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 SLDSTEGTGFGYRRG Gly-loss+Amide 1543.738 160 174
nlp-14 HCON_00190000; maker-scaffold4554-snap-gene-0.8-mRNA-1 PRJEB506; PRJNA205202 TLPQITGTHPYLRLY unmodified 1771.962 177 191
nlp-15 HCON_00024600; maker-C472057-snap-gene-0.5-mRNA-1 PRJEB506; PRJNA205202 AFDSLSGSGLTPFN unmodified 1411.662 47 60
nlp-15 HCON_00024600; maker-C472057-snap-gene-0.5-mRNA-1 PRJEB506; PRJNA205202 AFDSLAGSGFTGFD unmodified 1390.604 79 92
nlp-15 HCON_00024600 PRJEB506 SPIALNGGYYQPLREDALLL unmodified 2202.169 95 114
nlp-17 HCON_00114940 PRJEB506 NSLSN MMRLG Gly-loss+Amide 1063.527 44 49
nlp-17 HCON_00114940 PRJEB506 VDALKEQEPCVDCSLGNLMRLG 2 Cys-Cys; Gly-loss+ 2329.123 52 73
nlp-18 HCON_00035340; augustus-scaffold7830-abinit-gene-0.1-mRNA-1 PRJEB506; PRJNA205202 SDEVVEDDGELE unmodified 1334.536 52 63
nlp-18 HCON_00035340; augustus-scaffold7830-abinit-gene-0.1-mRNA-1 PRJEB506; PRJNA205202 SPYRQFAFA unmodified 1085.529 74 82
nlp-18 HCON_00035340 PRJEB506 GSPYGFAFA unmodified 915.4127 85 93
nlp-19c HCOI00354200 PRJEB506.WBPS10 IGMRLPNIIYM unmodified 1319.709 52 62
nlp-20 HCON_00131138 PRJEB506 DLDNSKKFSFA unmodified 1270.619 64 74
nlp-20 HCON_00131138 PRJEB506 FADRDDRLIR unmodified 1275.668 105 114
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGGRAFVPIEE unmodified 1130.572 27 37
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFFGDE unmodified 1025.457 39 48
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFVVDT unmodified 991.5087 51 60
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGGRAFLPVEE unmodified 1130.572 73 83
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFFKDD unmodified 1082.515 85 94
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFVPVKEEDEGQSFTDLE unmodified 2380.118 97 118
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFFVP unmodified 920.4868 121 129
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFFVPK unmodified 1048.582 121 130
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGGRAFLPIEE unmodified 1144.588 132 142
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFFQEP unmodified 1078.52 144 153
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGARAFP unmodified 674.35 156 162
nlp-21 HCON_00067330; maker-C471673-augustus-gene-0.17-mRNA-1 PRJEB506; PRJNA205202 GGGRAFVPQ unmodified 887.4614 165 173
nlp-24 HCON_00126360 PRJEB506 VNPLQGAMMGAMMGAMRG Gly-loss+Amide 1763.813 66 83
nlp-31 HCON_00105250 PRJEB506 PVVVERTIIYPG Gly-loss+Amide 1283.76 68 79
nlp-35c HCOI00356900; snap-scaffold16547-abinit-gene-0.6-mRNA-1 PRJEB506.WBPS10; PRJNA205202 DQLAFSGQNAYLRQLLQN LKPK unmodified 2544.381 46 67
nlp-37 HCON_00163850 PRJEB506 NNAEVVNHILKNFGTLDRLGDVG Gly-loss+Amide 2436.287 60 83
nlp-40 HCON_00017810.1; maker-C461709-augustus-gene-0.1-mRNA-1 PRJEB506; PRJNA205202 QLTAPTTMEKEEA Gln->pyro-Glu 1430.66 41 53
nlp-40 HCON_00017810.1; maker-C461709-augustus-gene-0.1-mRNA-1 PRJEB506; PRJNA205202 MVAWQPM unmodified 861.3877 67 73
nlp-42 HCON_00142640 PRJEB506 SAVGELSYPRRFL unmodified 1493.799 36 48
nlp-42c HCOI01162100 PRJEB506.WBPS10 SVDWHSLGWAWG Gly-loss+Amide 1341.626 57 68
nlp-44 HCON_00163905 PRJEB506 STLPLSSLLVPYPRVG Gly-loss+Amide 1639.966 34 49
nlp-44 HCON_00163905 PRJEB506 SFFTGDRNVYPPTSI unmodified 1699.821 52 66
nlp-44 HCON_00163905 PRJEB506 RYLYTARVG Gly-loss+Amide 1039.593 79 87
nlp-54 HCON_00096380 PRJEB506 GNM WGTPSKSYGYTN LA E unmodified 1974.878 43 60
nlp-58 HCON_00137190 PRJEB506 SLYGVDDGFTFKGFRGL unmodified 1877.931 41 57
nlp-58 HCON_00163905 PRJEB506 MPYMNLKGLRG Gly-loss+Amide 1220.652 62 72
nlp-59 HCON_00096380; maker-scaffold14296-augustus-gene-0.22-mRNA-1 PRJEB506; PRJNA205202 FEGLADYVALEDPNA unmodified 1622.746 63 77
nlp-59 HCON_00096380; maker-scaffold14296-augustus-gene-0.22-mRNA-1 PRJEB506; PRJNA205202 LAILSARGFG Gly-loss+Amide 945.576 85 94
nlp-67 HCON_00186320; maker-scaffold993-snap-gene-0.2-mRNA- PRJEB506; PRJNA205202 AVPVEVEQRE unmodified 1154.593 54 63
nlp-67 HCON_00186320; maker-scaffold993-snap-gene-0.2-mRNA- PRJEB506; PRJNA205202 SYPRNCYFSPIQCLFT 2 Cys-Cys 1935.865 71 86
nlp-68 HCON_00070810; augustus-scaffold17454-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 LVREPPLI unmodified 935.5804 50 57
nlp-68 HCON_00070810; augustus-scaffold17454-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 GIDPFSIPTLIKEPPL unmodified 1735.976 60 75
nlp-68 HCON_00070810; augustus-scaffold17454-abinit-gene-0.0-mRNA-1 PRJEB506; PRJNA205202 DSYVYPSTNSQLSDRIPLREPPL unmodified 2646.329 82 104
nlp-69c HCOI01768700 PRJEB506.WBPS10 FYRTGGTILLG Gly-loss+Amide 1138.65 58 68
nlp-71 HCON_00122860 PRJEB506 ALNQFKNCYFSPIQCVLME 2 Cys-Cys 2245.037 62 80
nlp-74 HCON_00082180; maker-scaffold4879-augustus-gene-0.4-mRNA-1 PRJEB506; PRJNA20222 APQMYDDVQFV unmodified 1311.581 28 38
nlp-74 HCON_00082180; maker-scaffold4879-augustus-gene-0.4-mRNA-1 PRJEB506; PRJNA205202 SNAELINGLIGMDLGKLSAVG Gly-loss+Amide 2013.093 41 61
nlp-74 HCON_00082180; maker-scaffold4879-augustus-gene-0.4-mRNA-1 PRJEB506; PRJNA205202 SNAELINGLLGMNLNKLSSAG Gly-loss+Amide 2057.094 64 84
nlp-81 HCON_00107730 PRJEB506 FTNGDFFVP unmodified 1101.524 48 56
nlp-81 HCON_00107730 PRJEB506 WIPSGGAGLVSGRG unmodified 1312.689 60 73
nlp-81 HCON_00107730 PRJEB506 DWRSAIAEPNF unmodified 1304.615 83 93
HCON_00135420 HCON_00135420; maker-scaffold6512-augustus-gene-0.7-mRNA-1 PRJEB506; PRJNA205202 ARNPYSWMVVDQS unmodified 1551.714 45 57
HCON_00135420 HCON_00135420; maker-scaffold6512-augustus-gene-0.7-mRNA-1 PRJEB506 SRNPYSWMVHSKG Gly-loss+Amide 1489.725 60 72
HCON 00135420 HCON_00135420; maker-scaffold6512-augustus-gene-0.7-mRNA-1 PRJEB506; PRJNA205202 ARNPYSWMNE unmodified 1266.545 101 110
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a

The precursor name corresponds either to the one reported in project PRJEB506 or to the one reported in project PRJNA205202 on the WormBase site.

b

When sequences are predicted by both databases, the peptide sequence start and end positions refer to the database indicated first.

c

Denotes identified sequences from the PRJEB506 release (versions to 10).

As seen for FLP peptides, the use of the two WormBase data sets (PRJEB506 versions 10 and 14, and PRJNA205202) yielded an increase in the number of identified NLP peptides and allowed us to inform the predicted sequences where discrepancies existed between databases, as shown for nlp-5, nlp-7, nlp-15, nlp-9, nlp-13, and nlp-18 (Table 4; Figure S2). Detection of the peptide GGGRAFFGGWQPYESLGARMD encoded on nlp-9 shows that the correct sequence was the one predicted in PRJNA205202, whereas for nlp-7, nlp-13, nlp-15, nlp-18, and one peptide of nlp-5, the correct sequences were predicted in PRJEB506. More surprising was the fact that we clearly identified both of the WormBase predicted sequences for the C-terminal peptide of nlp-5 (SRLFSTYYYLPYRDSLEDMDQNAQE, predicted in PRJNA205202, and SRLFSTYYYLPYRDSLEDMDQNVQE, predicted in PRJEB506); the corresponding MS/MS spectra are shown in Figure 4.

Figure 4.

Figure 4

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MS/MS spectra of two peptides of the H. contortus neuropeptide precursor encoded by nlp-5. The fragmentation schemes allowed the identification of the peptide SRLFSTYYYLPYRDSLEDMDQNVQE from the WormBase PRJEB506 data set analysis (A) and of the peptide SRLFSTYYYLPYRDSLEDMDQNAQE from analysis of the WormBase PRJNA205202 data set (B).

All H. contortus NLP sequences revealed in this study were aligned with their C. elegans homologues as shown in Figure S2. As reported for C. elegans,28,29 we also detected equivalent unrecorded H. contortus neuropeptides for nlp-3, nlp-9, nlp-10, and nlp-12 (these peptides are underlined in Figure S2). As already shown for FLP peptides, these alignments emphasize the similarity in neuropeptide sequences between both C. elegans and H. contortus with the identification of peptides bearing the C-terminal glycine residue for amidation and with peptides not modified but being flanked by di- or mono-basic residues. In this study, we were able to isolate and identify peptides arising from nlp-17, nlp-19, nlp-44, nlp-54, nlp-59, nlp-67, nlp-38, nlp-69, and nlp-71 that were not previously reported in similar studies on C. elegans. Of particular interest are the C-terminal peptides of nlp-17, nlp-67, and nlp-71, which were unambiguously identified with one disulfide bridge, as shown by their MS/MS spectra (Figure 5). The study on C. elegans(28,29) reported the homologous sequences with the prediction of disulfide bridges, but there was no biochemical isolation or characterization.

Figure 5.

Figure 5

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MS/MS spectra of the C-terminal peptides of nlp-17 (A), nlp-67 (B), and nlp-71 (C) of H. contortus. The fragmentation schemes allow the unambiguous identification of the presence of a disulfide bridge between two cysteine residues for the three peptides.

Label-Free Quantification of Neuropeptides

Neuropeptide quantification was performed on four L1 and three L3 biological replicates. Normalized intensities measured for all identified peptides arising from predicted neuropeptide precursors are listed in Table S2.

Relative Abundance of Endogenous Peptides

Some of the detected peptides are shorter versions of the same peptide, likely reflecting the degradation of bioactive peptides during sample processing. We calculated the intensities of truncated forms and compared these to the intensity of the corresponding nontruncated bioactive peptides. On average, we found that these degraded forms represented less than 15% of the entire form, indicative of a low level of peptide degradation. It is noteworthy that for most amidated peptides, we also detected two other minor forms, representing less than 5% of the mature peptide, one corresponding to the nonamidated peptide and the second one corresponding to the substrate of the amidating enzymes, which act sequentially on C-terminal Glycine-extended immature peptides (Table S2).

The relative abundance of all endogenous peptides reported in Tables 2 and 4 is shown in Figure 7, spanning four orders of magnitude both in the L1 and L3 life stages. Among the 181 detected bioactive peptides, the most intense flp and nlp peptides quantified are indicated in Figure 6.

Figure 7.

Figure 7

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Volcano plot showing peptides differentially expressed between L1 larvae and L3 larvae of H. contortus. The x-axis represents the log2 fold-change (FC) of L3 versus L1. The y-axis represents the p-value from a t test applied between four L1 biological replicates and three L3 biological replicates. All data are shown in Table S2. Peptides with FC < −3 and p-value <0.01, statistically more expressed in L1 stage, are highlighted in green and peptides with FC > 3 and p-value <0.01, statistically more expressed in L3 stage, are highlighted in red. For the purpose of clarity, peptides are only annotated by the precursor name.

Figure 6.

Figure 6

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Relative abundance of flp-encoded (highlighted in blue) and nlp-encoded peptides (highlighted in red) in the L1 larvae stage (A) and L3 larvae stage (B) of H. contortus. The most intense peptides identified are indicated.

Differential Neuropeptide Expression between L1 Stage and L3 Stage Larvae of H. contortus

We compared the relative peptide expression in L1 (free-living stage) and L3 (infective) life stages for the 181 identified mature peptides (Table S3). All resulting individual boxplots are depicted in Figure S3. Based on an arbitrary fold-change of 3 and a p-value of 0.01, we found 29 peptides more highly expressed in L3s and 22 more highly expressed in L1s (Figure 7).

Data shown in Table S2 and Figure S3 highlight that peptides arising from the same precursor can either follow the same trend or vary greatly in expression between L1 and L3 worms. For example, all four nlp-3 peptides (AINPFLDSMG, AVNPFLDSFG, SSRYQPYYHLD, and YFDSLAGQALG) are more highly expressed in L3s (Figure 8A), whereas nlp-10 has two peptides (AVMPFSGGLYG and ADRYIRSMPISGGIFG) that show opposite trends in the two life stages, being lower in the L3 stage worms (Figure 8B).

Figure 8.

Figure 8

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Relative label-free quantification of four nlp-3 encoded peptides (A) and of two nlp-10 encoded peptides (B) between the first-stage (L1) and third-stage larvae (L3) of H. contortus. The boxplots were obtained from four L1 biological replicates and from three L3 biological replicates. The zone below the inferior red line (value of 16.6) indicates very low or no expression. The zone between the two red lines corresponds to a low expression zone.

Discussion

The present study is the first whole-parasitic peptidome analysis of H. contortus. Previously, only two FLP neuropeptides have been directly sequenced for this nematode.31,32 These new findings enhance the understanding of nematode neuropeptide biology and enhance the FLP-activated G-protein coupler receptors profiling described in McCoy et al.14

This is also one of the most comprehensive neuropeptidome analyses of a nematode with a total of 181 peptides (68 FLPs and 113 NLPs) being identified biochemically using MS/MS. It can be compared with similar studies performed on C. elegans, which led to the identification of 203 neuropeptides based on mass matching,28,29 131 among them with sequenced levels using LC-MS/MS. In our study, all of the identified peptides were fully sequenced by MS/MS. However, five predicted FLP and nine NLP precursors remain undetected in this study. This can be due to various reasons. At first, it is not possible to anticipate which predicted peptides will be expressed and correctly processed into bioactive peptides. Another possibility is that some neuropeptides may be present in other developmental stages or may be expressed under certain conditions. In addition, we cannot exclude experimental bias like neuropeptide degradation during sample processing, leading to low-molecular-weight peptides not detectable by LC/MS. The stochastic precursor selection of DDA may also lead to inconsistent detection of peptides, and finally, some neuropeptides are certainly below the current detection threshold of MS due to their weak expression.

The results obtained from H. contortus larvae peptidomics profiling reveal and expand on the known complexity of neuropeptide expression in nematodes. These data are consistent with nematode parasites, displaying remarkable neuropeptide complexity despite their apparent nervous system simplicity.37 The data further confirm that peptide-based neuronal signaling in parasitic nematodes is similarly complex to that reported for free-living nematodes, at least in these clade V nematodes. This is not surprising considering that H. contortus has both free-living and parasitic stages and the L3 stage transitions from the free-living to a host-based environment.

In this study, the differentially expressed neuropeptides between two key developmental stages of H. contortus (the first-larval stage, L1, and the infective stage, L3) were investigated by comparative peptidomics. The free-living stage L1 is motile and exits the egg to feed on feces, while the L3 stage waits in water droplets on vegetation and enters a resting stage that relies on reserves, slows its metabolic rate, and stops actively feeding, prior to being ingested by the ruminant host. Despite these dramatic differences in life stage behaviors, which have been reflected previously through transcriptomic studies showing significant differences in protein-coding gene expression,34,38 both life stages appear here to express mostly similar FLP and NLP neuropeptides. Among the 181 quantified peptides in this study, 170 were detected in the L1 stage and 171 in the L3 stage. This is consistent with the fact that many of these neuropeptides, especially the FLPs, regulate diverse behaviors through the modulation of sensory and motor functions.

It appears that genes expressed in both life stages are overall upregulated in the L3 phase, with only peptides encoded on flp-2, flp-9, nlp-1, and nlp-7 being upregulated in the L1 stages. These changes in expression could be associated with the maturation of the nervous motor system observed in L3. It is also possible that the peptides upregulated in the L1s associate with feeding behaviors that differ dramatically between the L1 and L3 life stages. More interesting is that for some genes, individual peptides are differentially expressed in one of the life stages compared to the other, e.g., nlp-10, nlp-21, nlp-40, and nlp-81. These data are intriguing and suggest that nematodes can differentially regulate the levels of individual peptides from the same precursor protein. This could be done through more rapid degradation of some component peptides compared to others, which further demonstrates the complexity inherent in nematode neuropeptide signaling.

Furthermore, the ability to quantify neuropeptides between different samples allows the comparison of peptide profiles. For example, this quantitative strategy allowed the differential analysis of peptide amidation profiles and represents an efficient approach to the characterization of key neuropeptide processing enzymes of the neuropeptide processing pathway or, in the context of drug discovery, could inform target engagement and the efficacy of inhibitors or modulators of the neuropeptide signaling pathways or their processing enzymes. This repository of biochemically identified and quantified peptide sequences provides a unique resource to enable the discovery of compounds active at different developmental stages of the nematode.

In conclusion, the extensive neuropeptide database provided here is a first step toward the understanding of the fundamental biochemistry of H. contortus and can be exploited in further experimental studies aiming at developing new anthelmintics against H. contortus.

Methods

Parasite Collection

All H. contortus samples (L1 and L3 stages) were obtained from Boehringer Ingelheim Animal Health. Worms were pelleted (12 min, 1800g), resuspended in deionized water, and washed several times with water. At the end of the final wash, worms were resuspended in 30 μL of PBS prior to storage at −80 °C. The L1 samples had a similar biomass to the L3 stage samples, i.e., approximately 45 000 L1 and 20 000 L3 in a volume of 20 μL PBS.

Peptide Extraction and Purification

To extract the peptides, 150 μL of an acidic methanol (methanol/water/acetic acid (90/9/1)) solution was added to the larvae. The mixture was then stirred for 30 min at 4 °C, followed by a 10 s sonication step for L3 larvae. Samples were then centrifuged for 15 min at 10 000g at 4 °C. Supernatants were collected and concentrated under vacuum (Concentrator 5301, Eppendorf (SpeedVac)) to obtain a volume of approximately 10 μL. The peptides were purified and desalted using C18 columns (OMIX C18 pipette tips, Millipore, Molsheim, France) according to manufacturer’s instructions. Finally, peptides were concentrated in vacuum and reconstituted in 0.2% formic acid for injection in LC-MS/MS.

Nano-HPLC/Nano-ESI Orbitrap-MS/MS

LC-MS/MS analyses were performed using a liquid chromatograph (LC) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA) using a Nano-spray Flex NG source. Reversed-phase chromatography was performed with a nano-ACQUITY Ultra-Performance LC system (Waters, Milford, MA) fitted with a trapping column (nano-Acquity Symmetry C18, 100 Å, 5 μm, 180 μm × 20 mm) at a 15 μL/min flow rate and an analytical column (nano-Acquity BEH C18, 130 Å, 1.7 μm, 75 μm × 250 mm) directly coupled to the ion source. The mobile phases for LC separation were 0.2% (v/v) formic acid in LC-MS grade water (solvent A) and 0.2% (v/v) formic acid in acetonitrile (solvent B). Peptides were separated at a 300 nL/min constant flow rate with a linear gradient of 5–85% solvent B for 85 min. A full MS1 survey scan was acquired with the Orbitrap for m/z 325–1200 at a 50 ms maximum filling time and 2 × 105 ions. The resolution was set to 120 000 at m/z 200. For MS/MS experiments, fragmentation was performed in HCD fragmentation cell (collision energy at 26%), with isolation of precursor ions in a quadrupole. Target ions previously selected for fragmentation were dynamically excluded for 50 s with a relative mass window of ±10 ppm. The MS/MS selection threshold was set to 5 × 103 ion counts. The detection was performed in an Ion Trap with an Automatic Gain Control (AGC) of 2 × 104 target value and a 50 ms maximum injection time. Each sample was injected twice (technical replicate).

Data Processing

Identification and quantification of peptides were performed using MaxQuant software (Ver. 1.5.3.8, Max-Planck Institute of Biochemistry, Department of Proteomics and Signal Transduction, Munich). Database searching was performed against the FASTA databases downloaded from the WormBase site (https://wormbase.org) (BioProject PRJNA205202 and BioProject PRJEB506). Interrogation of the databanks was based on the following criteria: precursor mass tolerance of 7 ppm, fragment ions mass tolerance of 0.6 Da, and 2 maximum missed cleavages with semi-trypsin as the enzyme. Search parameters for post-translational modifications were variable modifications of oxidation on methionine residues, N-terminal cyclization of glutamine/glutamic acid to pyroglutamate, disulfide bridge on cysteine residues, and glycine loss in combination with amidation (Gly-loss+Amide (C-term G)). The match between runs was performed with a match time window set to 0.7 min and an alignment time window set to 20 min. A false discovery rate of 1% was required for peptides with a minimum Andromeda score for accepting an MS/MS identification for modified peptides set to 40. All of the other parameters were MaxQuant default parameters. Peptides were retained as putative neuropeptides if they were surrounded by (di)basic residues and were kept only if their Andromeda score was higher than 60 or after manual inspection of their MS/MS spectra.

Differential Statistical Analysis

Peptide intensities were exported from the MaxQuant modificationSpecificPeptides file. Missing values were replaced by the minimum value of each acquisition. Intensities were transformed into their log2 values. Medians were calculated over the technical replicates. Data normalization was performed on the set of all identified and quantified peptides. The normalization coefficients thus obtained were applied to the initial intensities of all of the peptides detected. A two-tailed t test for each peptide was performed on the normalized medians to determine the statistical significance between L1 and L3 sample groups, assuming equal variance.

Acknowledgments

The authors would like to thank Veeranagouda Yaligara, Michel Didier, Jean-Luc Zachayus, and Anne Remaury for technical advice.

Glossary

Abbreviations

FLPs

FMRFamide-like peptides

NLPs

neuropeptide-like proteins

LC-MS/MS

liquid chromatography-tandem mass spectrometry

FDR

false discovery rate

FC

fold-change

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c00650.

  • Sequence alignments of H. contortus FLP precursors predicted from the WormBase PRJNA205202 project and from the WormBase PRJEB506 with its homologs in C. elegans, UniProtKB source (Figure S1); sequence alignments of H. contortus NLP precursors predicted from the PRJNA205202 and from the PRJEB506.WBPS14 WormBase projects with their homologs in C. elegans, UniProtKB source (Figure S2); relative label-free quantification of FLP and NLP peptides between the first-stage (L1) and third-stage larvae (L3) of H. contortus (Figure S3); homemade database Fasta file (Supplementary Data 1) (PDF)

  • Blast analysis of C. elegans NLP precursors reported by Van Bael et al.,29 against H. contortus WormBase PRJEB506 and PRJNA205202 databases (Table S1); expressions of all FLP and NLP peptides in the L1 larvae stage and L3 larvae stage of H. contortus (Table S2); expressions of mature FLP and NLP peptides in the L1 larvae stage and L3 larvae stage of H. contortus (Table S3) (XLSX)

Author Contributions

A.B. wrote the paper with input from all other authors. A.B. and C.A. designed the experiments. J.-C.G. supervised the development of the whole analytical workflow. D.F.B. prepared the parasite samples for peptide extraction. C.A. performed the peptide extraction, the LC-MS/MS analysis, and the Database Searching. A.B. processed the data.

The authors declare no competing financial interest.

Supplementary Material

ao1c00650_si_001.pdf (959.2KB, pdf)
ao1c00650_si_002.xlsx (213.4KB, xlsx)

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Associated Data

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Supplementary Materials

ao1c00650_si_001.pdf (959.2KB, pdf)
ao1c00650_si_002.xlsx (213.4KB, xlsx)

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