Ascaris suum Informs Extrasynaptic Volume Transmission in Nematodes

Abstract

Neural circuit synaptic connectivities (the connectome) provide the anatomical foundation for our understanding of nematode nervous system function. However, other nonsynaptic routes of communication are known in invertebrates including extrasynaptic volume transmission (EVT), which enables short- and/or long-range communication in the absence of synaptic connections. Although EVT has been highlighted as a facet of Caenorhabditis elegans neurosignaling, no experimental evidence identifies body cavity fluid (pseudocoelomic fluid; PCF) as a vehicle for either neuropeptide or biogenic amine transmission. In the parasitic nematode Ascaris suum, FMRFamide-like peptides encoded on flp-18 potently stimulate female reproductive organs but are expressed in cells that are anatomically distant from the reproductive organ, with no known synaptic connections to this tissue. Here we investigate nonsynaptic neuropeptide signaling in nematodes mediated by the body cavity fluid. Our data show that (i) A. suum PCF (As-PCF) contains a catalog of neuropeptides including FMRFamide-like peptides and neuropeptide-like proteins, (ii) the A. suum FMRFamide-like peptide As-FLP-18A dominates the As-PCF peptidome, (iii) As-PCF potently modulates nematode reproductive muscle function ex vivo, mirroring the effects of synthetic FLP-18 peptides, (iv) As-PCF activates the C. elegans FLP-18 receptors NPR-4 and -5, (v) As-PCF alters C. elegans behavior, and (vi) FLP-18 and FLP-18 receptors display pan-phylum distribution in nematodes. This study provides the first direct experimental evidence to support an extrasynaptic volume route for neuropeptide transmission in nematodes. These data indicate nonsynaptic signaling within the nematode functional connectome and are particularly pertinent to receptor deorphanization approaches underpinning drug discovery programs for nematode pathogens.

Keywords: Ascaris; Extrasynaptic volume transmission; nematode; neuronal signaling; neuropeptide; parasite; pseudocoelomic fluid.

Figure 1.

Ascaris suum pseudocoelomic fluid (As-PCF) contains a rich neuropeptide library. (A) 41 neuropeptides, including 6 FMRFamide-like peptides (FLPs) and 35 neuropeptide-like proteins (NLPs), and 35 antimicrobial peptides (AMPs) are present in As-PCF (n = 24 LC–MS/MS runs representing 67 individual worms; combined data from single worm, pooled female and male samples). This represents 9.09%, 14.89%, and 97.20% of FLP, NLP, and AMP peptides (respectively) predicted from the in silico libraries used to query the As-PCF LC–MS/MS data. (B) Pooled As-PCF (P As-PCF) derived from female nematodes has a richer neuropeptide complement than single worm As-PCF samples (SW As-PCF) [54 peptides detected in pooled As-PCF (n = 17) vs an average of 17.75 ± 1.2 peptides across single worm samples (n = 20)]. (C) The frequency of detection of individual peptides is variable in As-PCF. Dotted line represents detection rate in 50% of samples. Nine peptides are detected consistently in >50% of As-PCF samples [As-Cecropin P1, As-Cecropin P2, As-FLP-18A, As-Cecropin P3, As-Defensin 2, As-Defensin 1, As-Nemapore 1, As-GRSP-16, As-Cecropin 6]. See File S2 for all peptide sequences detected. (D) Mass spectra of (i) FLP-18A detected in As-PCF (GFGDEMSMPGVLR) and (ii) As-FLP-18A isotopic standard (GFGDEMSMPGVLRF-NH₂). Amino acid alignments indicate similar fragmentation patterns for As-FLP-18A detected in As-PCF and As-FLP-18A isotopic standard but that As-PCF-derived As-FLP-18A is truncated at the C-terminus. All data are represented as the mean ± SEM.

Figure 2.

Ascaris suum pseudocoelomic fluid (As-PCF) is bioactive on Ascaris ovijector tissue and mirrors the synthetic As-FLP-18A peptide response. (A) Representative muscle tension recordings showing the effects of As-PCF on A. suum ovijector tissue: (i) As-PCF induces two distinct, concentration dependent, myoactivity profiles (known as response types; RT) on the A. suum ovijector; 40 μL of As-PCF (equivalent to ≥0.15× As-PCF where 1× is representative of the biological sample) induces either a transient muscle contraction followed by muscle paralysis (50% of preparations; RT-2) or immediate muscle relaxation (50% of preparations; RT-1). 4 μL of As-PCF (equivalent to 0.015× As-PCF where 1× is representative of the biological sample) induces an RT-1 response only. (ii) Positive controls [phosphate buffered saline spiked with synthetic As-FLP-18A (GFGDEMSMPGVLRF-NH₂) before C18 peptide purification; SB] induce two distinct, concentration dependent myoactivity profiles on the A. suum ovijector. 40 μL of SB (equivalent to final concentration of 1 μM synthetic As-FLP-18A) induces either a RT-2 (50% of preparations) or RT-1 (50% of preparations) response. 4 μL of SB (equivalent to final concentration of 0.1 μM synthetic As-FLP-18A) induces an RT-2 response only. (iii) Positive controls [phosphate buffered saline spiked with synthetic As-FLP-18A (GFGDEMSMPGVLRF-NH₂) after C18 peptide purification (SA) also induced a concentration dependent myoactivity profile on the A. suum ovijector. 40 μL and 4 μL of SA controls (equivalent to final concentration of 1 μM and 100 nM synthetic As-FLP-18A, respectively) induce an RT-2 response in 100% of preparations. (iv) Negative control (perfused activation solution only resuspended in ddH₂O) does not modulate intrinsic ovijector contractility. Test compounds (As-PCF, SB, SA, negative control) were present during the period indicated by arrows. Vertical scales represent 2 mg, and horizontal scales represent 2 min. (B) Effects of As-PCF and positive controls (SB and SA) on (i) contraction frequency, (ii) contraction amplitude, and (iii) muscle tension of the A. suum ovijector. Test compounds (As-PCF, SB, or SA) were added at 0 min and washed out at 10 min. Data are presented as the mean ± SEM (see Table S1). (C) Representative muscle tension recordings showing the effects of synthetic As-FLP-18A on A. suum ovijector tissue. 1 nM to 10 μM synthetic As-FLP-18A induce concentration dependent myoexcitatory effects. 10 μM synthetic As-FLP-18A induces two distinct myoexcitatory profiles consistent with RT-2 (35.7% of preparations) or RT-1 (64.2% of preparations). 1 μM to 10 nM synthetic As-FLP-18A induce RT-2 responses only. The truncated form of synthetic As-FLP-18A (GFGDEMSMPGVLR; 10 μM) does not modulate ovijector contractility. Negative control (ddH₂O) does not modulate intrinsic ovijector contractility. Peptide was present during the period indicated by the arrows. Vertical scales represent 2 mg and horizontal scales represent 2 min. (D) Concentration dependent effects of 1 nM to 10 μM synthetic As-FLP-18A on (i) contraction frequency, (ii) contraction amplitude, and (iii) muscle tension of the A. suum ovijector. Peptide was added at 0 min and washed out at 10 min. All data are presented as the mean ± SEM (see Table S2).

Figure 3.

Ascaris suum PCF (As-PCF) activates heterologously expressed Caenorhabditis elegans neuropeptide receptors NPR-4 and NPR-5 in mammalian cell lines. (A) C. elegans NPR-4 (Ce-NPR-4) and -5 (Ce-NPR-5) expressed in CHO cells are activated by synthetic As-FLP-18 peptides (10 μM; As-FLP-18A, GFGDEMSMPGVLRF-NH₂; As-FLP-18B, GMPGVLRF-NH₂; As-FLP-18C, AVPGVLRF-NH2; As-FLP-18D, GDVPGVLRF-NH₂; As-FLP-18E, SDMPGVLRF-NH₂; As-FLP-18F, SMPGVLRF-NH₂) compared to controls transfected with an empty vector (pcDNA3.1). (B) Ce-NPR-4 is activated by As-PCF in a concentration dependent manner compared to both peptide-free cell medium (BSA) and empty vector (pcDNA3.1) negative controls. The cognate Ce-NPR-4 peptide Ce-FLP-18A (DFDGAMPGVLRF-NH₂) and As-FLP-18A were used as positive controls. (C) Ce-NPR-5 is also activated by As-PCF in a concentration dependent manner compared to both peptide-free cell medium (BSA) and empty vector (pcDNA3.1) negative controls. The cognate Ce-NPR-5 peptide Ce-FLP-18A and As-FLP-18A were used as positive controls. In all cases data are shown as the ratio of peptide/As-PCF response to total calcium response. Error bars represent the mean ± SEM, n > 3 replicates. Statistical significance of peptide or As-PCF-evoked responses compared with BSA or empty vector controls was determined by two-way ANOVA and Tukey’s multiple comparisons test. P-values are denoted by *** (<0.001) and **** (<0.0001).

Figure 4.

Exogenous application of Ascaris suum PCF (As-PCF) and synthetic As-FLP-18A influences Caenorhabditis elegans growth and motility. (A) C. elegans growth (as measured by changes in body length) is significantly reduced in nematodes exposed to 2% and 5% As-PCF and 100 μM synthetic As-FLP-18A (GFGDEMSMPGVLRF-NH₂) compared to the negative control (nematodes exposed to S-medium only). (B) C. elegans motility (as measured locomotory activity over time) is significantly reduced in nematodes exposed to 5%, 7.5%, 10% As-PCF and 10 μM synthetic As-FLP-18A compared to the negative control (nematodes exposed to M9 only). Statistical significance is determined by one-way ANOVA and Dunnett’s multiple comparisons test (A) or two-way ANOVA and Tukey’s multiple comparisons test (B). P-values are denoted by * (<0.05), ** (<0.01), *** (<0.001), and **** (<0.0001).

Figure 5.

Caenorhabditis elegans flp-18 and the cognate FLP-GPCR encoding gene homologues npr-4 and npr-5 display pan-phylum conservation in nematodes. Pan-phylum HMM analysis of 134 nematode genomes (109 species) demonstrates that C. elegans flp-18, npr-4, and npr-5 encoding gene homologues are highly conserved (99%, 93%, 84%, respectively) in nematodes. Black boxes indicate the presence of a putative gene homologue. Presence/absence identified based on motif conservation and reciprocal BLAST. Nematode species are arranged based on 12 clade designation. Asterisk denotes that multiple genomes were mined per species. Genome quality is indicated by CEGMA and BUSCO scores obtained from Wormbase Parasite version 14.^–,, A list of genomes mined, HMM query sequences returned, and retrieved gene homologues are detailed in File S3.