Unraveling flp-11/flp-32 dichotomy in nematodes

Abstract

FMRFamide-like peptide (FLP) signalling systems are core to nematode neuromuscular function. Novel drug discovery efforts associated with nematode FLP/FLP receptor biology are advanced through the accumulation of basic biological data that can reveal subtle complexities within the neuropeptidergic system. This study reports the characterisation of FMRFamide-like peptide encoding gene-11 (flp-11) and FMRFamide-like peptide encoding gene-32 (flp-32), two distinct flp genes which encode the analogous peptide, AMRN(A/S)LVRFamide, in multiple nematode species - the only known example of this phenomenon within the FLPergic system of nematodes. Using bioinformatics, in situ hybridisation, immunocytochemistry and behavioural assays we show that: (i) flp-11 and -32 are distinct flp genes expressed individually or in tandem across multiple nematode species, where they encode a highly similar peptide; (ii) flp-11 does not appear to be the most widely expressed flp in Caenorhabditis elegans; (iii) in species expressing both flp-11 and flp-32, flp-11 displays a conserved, restricted expression pattern across nematode clades and lifestyles; (iv) in species expressing both flp-11 and flp-32, flp-32 expression is more widespread and less conserved than flp-11; (v) in species expressing only flp-11, the flp-11 expression profile is more similar to the flp-32 profile observed in species expressing both; and (vi) FLP-11 peptides inhibit motor function in multiple nematode species. The biological significance and evolutionary origin of flp-11 and -32 peptide duplication remains unclear despite attempts to identify a common ancestor; this may become clearer as the availability of genomic data improves. This work provides insight into the complexity of the neuropeptidergic system in nematodes, and begins to examine how nematodes may compensate for structural neuronal simplicity. From a parasite control standpoint, this work underscores the importance of basic biological data, and has wider implications for the utility of C. elegans as a model for parasite neurobiology.

Keywords: FLP-11; FLP-32; FMRFamide-like peptide; Nematode; Neuropeptide; Neuropeptide signalling; flp-11; flp-32.

Graphical abstract

Fig. 1

FMRFamide-like peptides (FLPs) are abundant in the nervous system of nematodes. Confocal microscopy images of FLP immunoreactivity (IR; green) in (A, B) Caenorhabditis elegans, (C, F) Panagrellus redivivus, (D, G) Globodera pallida, (E, H) Teladorsagia circumcincta and (I) Haemonchus contortus. Body wall muscle is counterstained in red. (A–E and I) Dense FLP-immunoreactivity (IR) in the circumpharyngeal nerve ring (CNR) of all nematode species examined. The CNR is positioned posterior to the metacorpus (MC) and anterior to the terminal bulb (TB) in C. elegans (A, B), anterior to the basal bulb (BB) in P. redivivus (C), and anterior to the pharyngeal bulb (PB) in G. pallida (D). Immunoreactive cell bodies in the major head ganglia (anterior ganglia (AG), lateral ganglia (LG), ventral ganglia (VG) and retrovesicular ganglion (RVG)) are shown (A–C), while FLP IR is also evident in the ventral (VC) and dorsal (DC) nerve cords which emanate from the CNR and run parallel to the body wall muscle (C–H). In addition, three FLP IR cell bodies (∗) can been seen in the VC of P. redivivus (C). FLP positive cell bodies are also shown in the region of the preanal (PAG) and lumbar (LBG) ganglia in the tail of G. pallida (G) and T. circumcincta (H). Scale bars: A = 7 μm, B = 6 μm, C = 10 μm, D = 40 μm, E = 20 μm, F = 8 μm, G = 35 μm, H = 36 μm, I = 40 μm.

Fig. 2

Alignment of partial sequence data from nematode species which express FMRFamide-like peptide encoding gene-11 (flp-11) and FMRFamide-like peptide encoding gene-32 (flp-32). (A) Analysis of partial sequence data from nematode species which express flp-11, showing conservation of FMRFamide like peptide (FLP)-11-like peptides across 26 nematode species, demonstrating that up to three conserved peptides (highlighted in yellow, pink and blue) can be encoded. FLP-11-like peptide encoding transcripts display a highly conserved pattern of di- and mono-basic cleavage site residues (highlighted in green; with the exception of Nippostrongylus brasiliensis and Heterodera glycines), which characteristically incorporate a di-basic KR site preceding (A/T)(M/I)RNAL(V/I)RFG (highlighted in yellow), a mono-basic R residue prior to (A/S)(G/R/A)(G/S/T)(M/I)R(N/S)A(L/M)VRFG (highlighted in pink), and a di-basic KR and mono-basic R residue flanking (N/Q/T/Y)(G/S/A)AP(Q/E)PFVRFG. Clade 4 nematodes (Laxus oneistus) are boxed in brown; clade 6 nematodes (Plectus murrayi) are boxed in grey; clade 8 nematodes (Brugia malayi, Wuchereria bancrofti, Dirofilaria imitis, Loa loa, Onchocerca ochengi, Onchocerca volvulus, Ascaris suum) are boxed in blue; clade 9 nematodes (Caenorhabditis elegans, Oscheius tipulae, Pristionchus pacificus, Pristionchus expectatus, Dictyocaulus viviparous, Ostertagia ostertagi, Haemonchus contortus, Teladorsagia circumcincta, Cooperia oncophora, N. brasiliensis, Heligmosomoides polygyrus, Oesophagostomum dentatum, Necator americanus, Ancylostoma caninum, Ancylostoma cantonensis, Ancylostoma ceylanicum, Heterorhabditis bacteriophora) are boxed in purple; clade 10 nematodes (Steinernema monticolum, Bursaphelenchus xylophilus, Aphelenchus avenae, Panagrellus redivivus, Strongyloides stercoralis, Strongyloides ratti), are boxed in red; clade 12 nematodes (Meloidogyne floridensis, Meloidogyne incognita, Meloidogyne hapla, Meloidogyne graminicola, Pratylenchus penetrans, H. glycines, Globodera rostochiensis, Globodera pallida) are boxed in green (Holterman et al., 2006). Species name suffix (a), (b) or (c) represents alternatively spliced gene isoforms. (B) Analysis of partial sequence data from nematode species which express flp-32, showing conservation of FLP-32-like peptides across 17 nematode species, demonstrating that only one conserved peptide (highlighted in yellow) is encoded. In contrast to cleavage site arrangement displayed by FLP-11-like peptide encoding transcripts, FLP-32-like peptide encoding transcripts characteristically encode two di-basic cleavage site residues (KK/R and KR; highlighted in green) flanking the single encoded peptide AMRN(A/S)L(V/I)RFG (highlighted in yellow). Clade 9 nematodes are boxed in purple; clade 10 nematodes are boxed in red; clade 12 nematodes (including Meloidogyne paranaensis and Radopholus similis) are boxed in green.

Fig. 3

FMRFamide-like peptide encoding gene-11 (flp-11)/FMRFamide-like peptide (FLP)-11 expression is restricted to a single RIS-like (RIS) cell in nematodes. Light microscopy in situ hybridisation images show flp-11 gene expression in a single RIS cell body positioned in the right side of the ventral ganglion in (A, B) Caenorhabditis elegans, (D, E) Panagrellus redivivus, (G, H) Globodera pallida, (J, K) Teladorsagia circumcincta and (M, N) Haemonchus contortus. RIS is positioned between the metacorpus (MC) and terminal bulb (TB) in C. elegans (A–C), posterior to the basal bulb in P. redivivus (D–F), and posterior to the pharyngeal bulb (PB) and stylet (S) in G. pallida (G, H). In T. circumcincta (J, K) and H. contortus (M, N) RIS is shown in the anterior third of the nematode in proximity to the head (HD). (C, F, I, L and O) Confocal microscopy of FLP-11 (anti-NGAPQFPVRFamide) immunoreactivity (green) in C. elegans (C), P. redivivus (F), G. pallida (I), T. circumcincta (L) and H. contortus (O) in a single RIS-like cell (RIS). An anterior neuronal projection running into the circumpharyngeal nerve ring (CNR) is also evident in C. elegans (C), P. redivivus (F) and H. contortus (O). Body wall muscle is counterstained red. Scale bars: A = 20 μm, B = 10 μm, C = 20 μm, D = 25 μm, E = 6 μm, F = 25 μm, G = 15 μm, H = 7 μm, I = 15 μm, J = 20 μm, K = 8 μm, L = 15 μm, M = 20 μm, N = 10 μm, O = 10 μm.

Fig. 4

FMRFamide-like peptide encoding gene-32 (flp-32)/FMRFamide-like peptide (FLP)-32 expression is more extensive and less conserved than flp-11/FLP-11. Light microscopy in situ hybridisation images show flp-32 gene expression in multiple cell bodies (CB) in (A, D) Caenorhabditis elegans, (E, H, I) Panagrellus redivivus and (J) H. contortus. In C. elegans two paired flp-32 positive CBs are visible posterior to the metacorpus (MC). In P. redivivus three flp-32 positive CBs are located anterior to the basal bulb (BB; E), while one CB is visible in the tail of a female nematode (H), and at least three CBs are in close proximity to the spicules in males (I). In H. contortus one CB is positioned in the posterior third of the nematode body (J). (B, C, F, G and K) Confocal microscopy of FLP-32 (anti-AMRNALVRFamide) immunoreactivity (IR; green) in C. elegans (B and C), P. redivivus (F and G) and H. contortus (K) in a single RIS-like cell (RIS) and in multiple additional cell bodies (CBs). Anti-AMRNALVRFamide antiserum is cross-reactive for both FLP-11 and FLP-32 explaining the presence of the RIS-like cell in the confocal microscopy images. In C. elegans (B, C), in addition to RIS, two FLP-32 IR CBs are visible in close association with the circumpharyngeal nerve ring (CNR) positioned between the MC and the terminal bulb (TB). In P. redivivus (F, G) FLP-32 positive CBs are evident in the region of the CNR, anterior to the BB and RIS; in addition the ventral nerve cord (VC) is FLP-32 immunoreactive (G). In H. contortus one FLP-32 IR cell is shown (K). Body wall muscle is counterstained red. Scale bars: A = 15 μm, B = 8 μm, C = 5 μm, D = 6 μm, E = 25 μm, F = 7 μm, G = 30 μm, H = 25 μm, I = 6 μm, J = 20 μm, K = 6 μm.

Fig. 5

FMRFamide-like peptide (FLP)-11 is localised to two neuronal cell bodies associated with reproductive muscle and the ventral nerve cord (VC) in Ascaris suum. Immunocytochemistry images showing FLP-11 localisation in the region of the A. suum reproductive apparatus. (A) Two FLP-11 (anti-NGAPQFPVRFamide) immunoreactive (IR; green) cell bodies (arrows) located at the base of the ovijector in the region of the vagina vera muscle (VV; counterstained red), in close proximity to the FLP-11 positive VC which runs parallel to the VV/body wall junction. (B) Two FLP-11-IR neuronal processes (NP) extending from the cell body (arrow) and innervating the VV muscle. Somatic muscle (SM) is also counterstained red (A, B). Scale bars: A = 30 μm, B = 14 μm.

Fig. 6

FMRFamide-like peptide (FLP)-11-like peptides inhibit locomotion in Panagrellus redivivus and Caenorhabditis elegans. FLP-11 peptides (AMRNALVRFamide, ASGGMNALVRFamide and NGAPQPFVRFamide) significantly inhibit locomotion in P. redivivus and C. elegans. (A) In P. redivivus, AMRNALVRFamide and NGAPQPFVRFamide induced the greatest inhibition of locomotion as assessed by a reduction in body bends/min (AMRNALVRFamide, 11.73 ± 3.55 body bends/min; ASGGMNALVRFamide 20.89 ± 4.15 body bends/min; NGAPQPFVRFamide, 11.17 ± 4.22 body bends/min) compared with control (M9 negative control, 38.06 ± 3.75 body bends/min; P < 0.01). Serotonin was used as a positive control (64.82 ± 4.66 body bends/min). (B) In C. elegans, AMRNALVRFamide induced the greatest inhibition of locomotion as assessed by a reduction in body bends/min (AMRNALVRFamide, 4.93 ± 1.17 body bends/min; ASGGMNALVRFamide 8.67 ± 2.32 body bends/min; NGAPQPFVRFamide, 7.33 ± 2.44 body bends/min) compared with control (M9 negative control, 18.40 ± 1.74 body bends/min; P < 0.01). Serotonin was used as a positive control (1.78 ± 0.88 body bends/min). ^*P ⩽ 0.05; ^**P ⩽ 0.01; ^***P ⩽ 0.001.