Monoamine synthesis and transport pathways

Proteins encoded by identified nematode genes are indicated in black, bold, capital letters (abbreviations: TPH – tryptophan hydroxylase, AAADC – aromatic amino acid decarboxylase, VMAT – vesicular monoamine transporter, SERT – serotonin transporter, TH – tyrosine hydroxylase, DAT – dopamine transporter, TDC – tyrosine decarboxylase, TBH – tyramine beta-hydroxylase); nematode gene names (originally from C. elegans) are italicized below the protein name abbreviations. Function of the cat-1 gene, encoding VMAT, is required in all monoaminergic neurons for packaging monoamines into synaptic vesicles to allow subsequent synaptic release. Other genes pictured are required for monoamine biosynthesis or transport across the plasma membrane (‘reuptake’) to stop the action of the neurotransmitter following vesicular release at synapses (or extrasynaptically).

Monoaminergic neurons in C. elegans hermaphrodite, canonical and other likely or possible cells. Pluralized names represent bilaterally symmetric pairs, singular names are unpaired of ventral nerve cord cells.

[Canonical: Serotonin / 5-HT: NSMs, ADFs, AIMs*, RIH, HSNs; Dopamine: CEPVs, CEPDs, ADEs, PDEs; Tyramine: RICs, uv1s(4); Octopamine: RIMs] *AIMs uptake 5HT via MOD-5/SERT but do not express CAT-1/VMAT; therefore, they likely do not function as serotonergic neurons. Neurons expressing the cat-1/VMAT gene in C. elegans hermaphrodite but lacking expression of a typical or proper complement of neurotransmitter synthesis genes required for either serotonin, dopamine, tyramine or octopamine synthesis; these cells may use an unknown / novel monoamine (Wang et al., 2024): Cells expressing cat-1/VMAT alone; these cells may use betaine as a neurotransmitter (Wang et al., 2024).

Nematode strains – P. pacificus and other species

Primer sequences

P. pacificus serotonin immunoreactive and tph-1 transgene expressing neurons.

Anterior is to the left in all panels. (A) Anti-serotonin staining in adult hermaphrodite head, approximately lateral view, MaxIP of confocal image. Four pairs of neurons and one unpaired neuron are apparent, as identified. Three pairs of neurites extend anteriorly to the tip of the pharynx or head, consistent with the known structures of I1, RIP and ADF. In this preparation, the pharynx is considerably kinked (as apparent by the bending of the posterior NSM neurites), making it appear that the ventral unpaired soma (RIH?) is anterior to the nerve ring when it is actually posterior to the nerve ring. (See supplemental figure S1 for a stained head showing this soma posterior to the nerve ring.) (B) Expression of tph-1p::rfp reporter in adult hermaphrodite head showing strong expression in a pair of pharyngeal neurons (NSMs) and anterior ganglion neurons in front of the nerve ring (RIPs). [Construct previously described in (Okumura et al., 2017).] Scale bars in B, D - 50 µm. (C) Same head as B, with DIC. (D) MaxIP with Apotome of tph-1p::rfp reporter showing neuronal morphologies, ventral view. Asterisks mark RIP endings in the nerve ring, arrow heads mark dendrites. (E) Left lateral view; otherwise, same as D. Bracket indicates NSM endings at pharyngeal-intestinal junction. (F-I) 3D rendering of neurons from serial section EM reconstruction (Cook et al., 2025); all left side neurons are rendered in magenta; right side neurons in blue. (F) NSML and NSMR inside a mostly transparent pharynx (gray-green), dorsal view. (G) NSMs, left lateral view. Bracket as in E. (H). RIP neurons, ventral view; pharynx (green). As in D, asterisks mark RIP endings in the nerve ring. (I) RIP neurons, left lateral view, as in H.

Expression pattern of a cat-1p::gfp reporter in P. pacificus.

The ∼2 kb promoter fusion reporter cat-1p::gfp shows expression in known and other likely monoaminergic neurons. (A) GFP expression in the serotonergic amphid neuron ADF(AM9) and likely ring interneurons RIM and RIC. (B) Same with DIC. (C) GFP expression in pharyngeal neuron NSM and anterior ganglion RIP in the same worm. (D) Same with DIC. (E) Expression in mid-posterior lateral body dopaminergic PDE neuron with anteriorly extending dendrite ending near the dorsal midline. (F) Same with DIC. (G) MaxIP showing expression in likely CEPD and CEPV (dopaminergic) neurons, and NSM, RIP, ADF, RIM, and RIC neurons. (H) Expression in a dauer larva showing cat-1p::gfp that is similar to that seen at other developmental stages. (I) Expression in all four VC neurons (VC1-4) in the ventral nerve cord surrounding the vulva, young adult hermaphrodite. (J) A representative adult male shows expression in one likely ventral nerve cord CP motor neuron and a sensory ray neuron in the tail. Scale bar in (G) represents 50 µm in all panels. Because the reporter transgene is propagated as a complex extrachromosomal array resulting in mosaic animals from somatic loss of the array, we examined the expression pattern in many individual hermaphrodites for specific neuronal types across all post-larval developmental stages (n>100).

Co-expression of tph-1 and cat-1 transcripts in the head of cat-1::GFP strain

Anterior is to the left in all images; each is a single focal plane. Panels include (as noted in upper left corners) HCR for tph-1 transcripts (orange) and cat-1 transcripts (red), GFP (green), DAPI (cyan). (A-C) Ventro-lateral view (same focal plane) showing co-expression of tph-1 and cat-1 in serotonergic neurons I1, NSM, RIP, and lack of expression in other cat-1-positive lateral neurons (RIM, RIC). Outline of the pharynx is clear via the DIC, showing the locations of the cells (and DAPI-stained nuclei) in the ventral pharynx (I1, NSM) and anterior ganglion (RIP). (D – F) More dorsal view (single focal plane) showing co-expression of tph-1 and cat-1 in dorso-lateral serotonergic neuron ADF, and adjacent other cat-1-positive dorsal neurons (CEPD, + other unidentified nearby cell). Note that the strong signal from a ventral NSM is still visible in this focal plane. (G-J) Another ventro-lateral view (single focal plane) showing serotonergic neurons on the other side of the head, including (G) tph-1 HCR signal alone (with DIC). (I, J) Match of cat-1 HCR signal (I) with mosaic transgene cat-1::GFP (J) in a subset of cat-1 transcript-positive cells. [In this preparation, HCR for the gfp gene was included, using an amplifier having the same excitation/emission properties as GFP. This is apparent by the particulate signal in the cells.] In the ventral region of the anterior ganglion, there are additional cat-1 transcript-positive cells (+) adjacent to the CEPVs.

Co-expression of cat-1 and cat-2 transcripts in head dopaminergic neurons

Anterior is to the left in all images, all images of the same head. (A) DIC view of head, single focal plane to show outline of pharynx. (B) Single focal plane with cat-1 (magenta), cat-2 (green), DIC, showing colocalization in CEPVL. (C) MaxIP of several planes in dorsal and right side lateral; colocalization in CEPDs, ADER. (D) MaxIP of several planes in ventral and left side lateral; colocalization in CEPVs, ADEL. (E) MaxIP with all planes shown in C, D.

Expression of tdc-1 and tbh-1 transcripts in the head

Anterior is to the left and ventral down, all images of the same head; tdc-1 transcripts (magenta), tbh-1 transcripts (green). (A-E) Same single focal plane, right side. (A) DIC view, to show outline of pharynx. (B) tdc-1 and DIC, showing two cells in the right lateral ganglion, presumptive RIMR and RICR. (C, D). Same focal plane as B, tdc-1, tbh-1, with (C) and without DIC (D) showing colocalization in the posterior cell (RICR). (F-J) Same single focal plane, left side. (F) tdc-1, showing two cells in the left lateral ganglion, presumptive RIML and RICL. (G). tdc-1, tbh-1 showing colocalization in the posterior cell (RICL). (H) tbh-1 alone, showing signal only in the posterior cell (RICL). (I) tdc-1, DAPI (blue, due to cyan + magenta background). (J) DAPI (cyan) alone, showing adjacent nuclei associated with tdc-1 signal (I).

Expression of tdc-1 and tbh-1 transcripts in the non-neuronal gonadal & vulval cells

Anterior is up and to the left, ventral up as seen by the location of vulva and ventral nerve cord (VNC). (A-C) Same focal plane. (A) Expression of tbh-1 transcripts (green) in the gonad, likely in gonadal sheath cells, with DIC. (B) tbh-1 (green) with DAPI staining of nuclei (cyan). (D-F) Same focal plane. (D) Expression of tdc-1 transcripts (magenta) in vulval cells, with DIC. (E) tdc-1 transcripts alone (magenta). (F) tdc-1 (magenta) with DAPI staining of nuclei (cyan/blue). See also Supplemental Figures for closeups of gonadal and vulval expression.

cat-1- and tdc-1-expressing cells near the vulval opening are not vulval muscles

(A-D) same focal plane, more superficial. On the left side, adjacent tdc-1 (orange) and cat-1 (red) expressing cells, presumptive vulval epidermal cells, are particularly apparent, and distinct from vulval muscles showing strong myosin immunoreactivity (green) centered and close to the vulva. (The tdc-1 and cat-1 expressing cells can also be seen on the right side but are less clear.) (E, F) deeper focal plane at level of the VNC, showing VC neurons that flank the vulval pore. (G,H) slightly deeper focal plane showing continuation of tdc-1 transcript-positive cells anterior and posterior to the vulva.

Expression of cat-1 transcripts in the head of cat-1::GFP strain

Anterior is to the left in all images; each is a single focal plane. (A – D) HCR for cat-1 transcripts (magenta) in 3 different focal planes – ventral / ventro-lateral (A), lateral (B), dorsal / dorso-lateral (C, D). Both cat-1 HCR fluorescence and DIC (A-C); cat-1 alone (D), same focal plane as C. Panels E – H are the same focal plane as panel A. (C, D) Dorso-lateral unidentified cat-1- transcript positive cells anterior and posterior to the nerve ring – possible OLQDs in anterior ganglion, and unidentified cell adjacent to CEPD and ADF; candidates include URX, AWA neurons (see also Supplemental Figure X). (E-F) Nuclear staining with DAPI shows stereotyped ‘diamond’ arrangement of ventral pharyngeal anterior bulb nuclei as labeled. cat-1 HCR fluorescence and DAPI (E); DAPI alone (F). NR = nerve ring location. (G, H) Ventro-lateral GFP+ cells also show cat-1 HCR fluorescence. Transgene expression cat-1::GFP alone (G); cat-1::GFP and cat-1 HCR fluorescence colocalization.

Serotonin staining in 13 different nematode species

Serotonin immunoreactivity in the heads of representative species examined or re-examined. Likely identifications of cells based on location are indicated. Cells in the pharynx directly anterior to NSMs and sending a neurite anteriorly to the tip of the pharynx were identified as I1s. Single pairs of neurons located dorsolaterally in the anterior ganglion were identified as likely RIPs. In the case of Acrobeloides and Plectus species, the presence of multiple bilaterally-paired neurons in the anterior ganglion suggested that the cells might be members of a 4- or 6-fold symmetric class of neurons, possibly not including RIPs; the numbers are listed under ‘other’ in Figure 14, which includes the two possible RIPs.

Cladogram of species with 5HT cells; serotonin-IR in I1 and RIP neurons is ancestral.

Cladogram of species examined or re-examined for serotonin-IR head neurons. (Branch lengths do not indicate phylogenetic distance.) Symbols: ‘ +’ = bilateral pair of cell(s) present; ‘ -’ = cell(s) absent. ‘ -?’ = indicates that a possible matching cell was seen in only a few of the heads examined. For these purposes, if a single pair of cells was observed in the correct location (e.g., a likely RIP in the lateral portion of the anterior ganglion), then the cell is marked as present. Other columns: v-mid = ventral midline cells posterior to the nerve ring – either 1 unpaired cell or 2 cells (that might be a bilateral pair or 2 unpaired cells); in C. elegans, this cell is RIH. Other ant = additional unidentified cells anterior to the nerve ring (i.e., not RIP). Other post = additional unidentified cells posterior to the nerve ring (i.e., not ADF). These are all cells not matching an identified serotonin-IR cell known from C. elegans or P. pacificus. For numbers of ‘other’ cells, a ‘2’ indicates a single bilaterally symmetric pair. For ‘other ant’ cells in clades 6 and 11 (H), these appear to be 6-fold symmetric cells, and may be IL2 neurons. For ‘other post’ cells, in the genus Caenorhabditis, these are AIM neurons, seen only in this genus, with the possible exception of O. myriophila and P. strongyloides. For ‘other post’ cells in clades 6, 10, and 11 (H), these are bilaterally symmetric pairs of cells adjacent to ADF in the dorsolateral ganglion. Species in red are presented for the first time in this study. Species marked with an asterisk (*) have been given new genus names (i.e., since Loer & Rivard, 2007 and Rivard et al., 2010); the matching species can be identified by the strain number. Clade designations (left side of cladogram) are from either (H) Holterman et al., 2006 or (B) Blaxter et al., 1998; Plectus is not within a B-designated clade number.

Egg laying is defective in cat-1 mutants but is increased by exogenous serotonin.

(A) P. pacificus wild type adult hermaphrodites typically retain 2 eggs; cat-1 mutants accumulate many eggs (in both worms, white arrows indicate internal eggs). The cat-1(csu116) mutant shown has 13 internal eggs. (B) Percentage of adult hermaphrodites that are Egl (with ≥8 eggs) in wild type and two cat-1 alleles, in young adults (5 days after individual isolated as a newly laid egg), and 1 day later (6 days post egg isolation). Mean ± SEM, 3 tests each condition (n = 50-100 worms per test). (C) Many cat-1 mutant adults have a protruding vulva (Pvl, black arrowhead), indicating abnormal vulval development, which may contribute to the Egl phenotype. (D) Number of eggs laid by individual P. pacificus adult hermaphrodites in liquid media, counted over two hours. Control is M9 buffer (yellow) vs. 10 mM serotonin in M9 (blue), mean ± SEM shown (n = 68-72 worms for each condition). Statistics: Multiple unpaired t-test; * indicates False-Discovery Rate for significant difference between control versus serotonin treated worms (P<0.001). “nd” denotes no discovery and “NP” indicates that the serotonin treatment for Ppa-cat-2 was not performed due to the lack of an egg laying defect.

Multiple monoamines are required for nictation in P. pacificus.

(A) The loss of cat-1/VMAT or genes for biosynthetic enzymes for serotonin (tph-1), tyramine (tdc-1), or octopamine (tdc-1, tbh-1), reduced the percentage of nictating dauer larvae. (B) Genetic ablation of the RIP and NSM neurons in tph-1::cas-3 transgenic dauer larvae phenocopied the tph-1(tu628) loss of function nictation defect. Between 30-60 animals were used in each nictation assay and at least 6 assays were performed for each genotype. *P<0.05, ***P<0.001, ****P<0.0001 Dunnett’s multiple comparisons test show significant difference to wildtype (WT).

Anti-5HT staining of wildtype P. pacificus, larval head showing all serotonin-IR head neurons as in Figure 1 adult, Max IP, anterior to the left, ventral view.

In the pharynx: I1s, NSMs; anterior ganglion in front of the nerve ring: RIPs; posterior to the nerve ring: ADFs and unpaired possible RIH. Because the pharynx is not kinked as it Figure 5, the unpaired neuron is shown clearly to be posterior to the nerve ring.

Additional reporter expression in aminergic neurons in P. pacificus.

(A) cat-1p::gfp expression in the amphid neuron homolog ADF(AM9) with double ciliated ending (arrowhead). Anterior is to the left. (B) 3D-rendering of the ADF neuron pair in lateral and dorsal orientations. (C-D) F1 hermaphrodite adult showing overlap of cat-1p::gfp and tph-1p::rfp reporter expression in the NSM neuron.

Expression of cat-1 transcripts via HCR in head in cat-1::GFP strain showing RIP neuron in anterior ganglion with neurite to dorsal nerve ring.

Anterior to the left, ventro-lateral view of head. All images are the same focal plane, except (E), slightly different to show pharynx outline better. (A) Mosaic expression of cat-1::GFP reporter in NSM and RIP on one side of the head. Note the characteristic morphology of RIP neurites. (B) Colocalization of cat-1 transcripts with cat-1::GFP in NSM and RIP. (C) cat-1 transcripts (magenta). (D) cat-1 transcripts and DAPI showing RIP in anterior ganglion anterior to nerve ring (nucleus-free region, marked in H). (E) DIC alone (F) DIC with cat-1::GFP. (G) cat-1::GFP with DAPI showing RIP ending in dorsal nerve ring. (H) DAPI showing nuclei and location of nerve ring (NR).

Coexpression of tph-1 and cat-1 transcripts in vulval region in cat-1::GFP strain.

Anterior to the left, ventral down, all images of the same focal plane. (A) tph-1 transcripts expressed in VC1-4 in the VNC (B) cat-1 transcripts expressed in VC1-4 and in vulval cells in ventrolateral body wall. (C) tph-1 and cat-1 transcripts colocalized in VC1-4. (D) cat-1 transcripts colocalized with cat-1::GFP reporter fluorescence of a single labeled VC neuron, VC2. (E) DAPI staining of vulval region, showing compact nuclei of VNC. (F) cat-1 transcripts colocalized with DAPI-stained VNC nuclei. (G) Including DIC shows the location of vulval pore between VC2 & VC3 (marked with ‘V’). (H) cat-1::GFP alone, showing VC2 neuron. Interestingly, the neurite crosses to the other side of the vulval pore and grows back anteriorly with varicosities. Inset: schematic outline of VC2 major branches. (Note that fine branches may not be seen.)

Cells expressing the tph-1p::rfp reporter in the mid-body of P. pacificus

(A, B) VC neurons in the VNC express the tph-1p::rfp reporter. (A) Arrowheads indicate somas in ventral nerve cord near the vulva; neurites are also apparent. (B) VC3 neuron soma just posterior to the vulva, including DIC.

Serotonin-IR is unchanged in mod-5 mutants

Anti-serotonin staining in mod-5(tu587) mutant. Anterior to the left. The same result was seen with the other allele, mod-5(tu586). (A) Adult hermaphrodite head, MaxIP of several focal planes, approximately dorsal-ventral view. All serotonin-IR cells (I1s, NSMs, RIPs, and ADFs) are seen, as in wildtype. Staining of the unpaired ventral serotonin-IR neuron, which is weak and unreliable, was not seen in wildtype worms in these experiments, so this cell might still be uptake-dependent. (B) Adult midbody vulval region, ventro-lateral view. VC neurons stain as in wildtype. We observed the same results for worms treated with SERT blockers fluoxetine or imipramine at various concentrations; no cells were affected except at the highest concentration used, which eliminated or greatly reduced serotonin staining uniformly in all cells, a likely non-specific effect since these drugs can bind other targets beside SERT.

Serotonergic neurons in the head express mod-5/SERT transcripts

Anterior to the left, ventrolateral views with ventral approximately down. MaxIPs of a few focal planes on left (A,C,E) and right sides (B, D, F) of head. (A) Expression of mod-5 transcripts (green) in identified pharyngeal (I1, NSM) and anterior ganglion (RIP) serotonin neurons, left side, with DAPI (cyan). (B) Same on the right side. (C) DAPI alone, same as A, with other adjacent DAPI-stained pharyngeal neurons identified. (D) Same on the right side. (E) Including DIC to show outlines of pharyngeal bulbs. (F) Same on the right side.

Head serotonergic and other neurons in the head sometimes express mod-5/SERT

Anterior to the left, ventral down, all images. MaxIPs of the same few focal planes (A, C, E), or a nearby set of focal planes (B, D). (A) Expression of mod-5 transcripts (green) in identified pharyngeal (I1, NSM) and anterior ganglion (RIP) serotonin neurons, plus IL2 neurons (D, V, lateral), with DAPI (cyan). (B) Nearby focal planes showing mod-5 transcripts in serotonin neurons posterior to the nerve ring, ADF and possibly RIH. (C, D) Including DIC to show outlines of pharyngeal bulbs. (E) Closeup of anterior ganglion, lateral neurons (green box). Several adjacent DAPI-stained neuronal nuclei are identified by position. (F) Map of identified anterior ganglion neuronal nuclei, left side lateral view (boxed as in E), 3D rendering from EM reconstruction (Cook et al., 2025).

Vulval-proximal VC neurons express mod-5/SERT

Closeup of vulval region; anterior to the left, ventral down. Images are MaxIPs of several focal planes. (A) mod-5 transcripts expressed in VC2 and VC3 (proximal VCs) in the VNC, but not obviously in distal VCs (VC1, VC4). Asterisk indicates intestine, which displays high background and autofluorescence in this preparation. (B) mod-5 transcripts are associated with compact VNC nuclei shown with DAPI staining. (C) DIC reveals location of vulval opening. Four embryos in the uterus are seen above the VNC. (D) cat-1 transcripts are expressed in VC1-4, but colocalized with mod-5 only in proximal VCs. (E) DAPI staining of vulval region, showing compact nuclei of VNC. (F) cat-1 transcripts colocalized with DAPI-stained VNC nuclei.

Expression of cat-2 transcripts in identified dopaminergic neurons

Whole adult (anterior to the left) and larval head (anterior to right) expression of cat-2 transcripts (magenta), MaxIP. Head neurons CEPs and ADEs are seen both in the adult head (top) and larval head (below). The posterior body shows PDE neurons on left and right sides.

Colocalization of cat-1 transcripts and cat-1::GFP reporter in posterior body dopaminergic neurons (PDEs).

Anterior is to the left in all panels. (A-E) Single focal plane. (A) cat-1 HCR fluorescence (magenta) and DIC. (B) As in A, with DAPI. (C) DIC and DAPI, which shows a small, compact nucleus associated with the cat-1 HCR signal. (D) cat-1::GFP (green) and cat-1 transcripts. (E) cat-1::GFP alone (F) MaxIP of several focal planes to show colocalization in PDE neurons on both sides of the body wall.

Co-expression of cat-2 and cat-1 transcripts in PDE neurons.

Anterior is to the left in all panels. (A-E) Right side of posterior body, MaxIP of same set of focal planes. (A) cat-2 HCR fluorescence (green) and DIC. (B) cat-1 HCR fluorescence (magenta) and DIC. (C) Colocalization of cat-2 and cat-1 transcripts in PDE neuron. (D) DAPI and DIC (E) cat-2 and cat-1 transcripts are associated with compact PDER nucleus associated with the cat-1 HCR signal. (F) MaxIP including left side focal planes to show colocalization in PDE neurons on both sides of the body.

Monoaminergic cells in the central and posterior body of P. pacificus

Anterior is to the left. Serotonergic and presumptive dopaminergic cells and in the central and posterior body of adult hermaphrodite P. pacificus revealed by 5-HTP-induced serotonin immunoreactivity; animal incubated with 5-HTP and subsequently stained with anti-serotonin. Dopaminergic neurons take up 5-HTP and convert it to serotonin. In the ventral nerve cord, VC motor neurons stain for serotonin (and are also seen without 5-HTP treatment). On either side of the vulva in the lateral body wall are teardrop-shaped cells that are likely homologs of C. elegans uterine vulva uv1 cells. In the mid-posterior lateral body wall is the dopaminergic PDE mechanosensory neuron, which extends a long dendrite anteriorly and laterally (with a slight turn dorsally at the end, in contrast to that seen in other free-living nematodes in which a much shorter dendrite extends dorsally from the soma (Rivard et al., 2010). The PDE dendrite is seen more clearly in Fig. 6E-F, showing cat-1p::gfp expression.

5HTP-induced serotonin immunoreactivity requires bas-1 / AAADC function

Anterior is to the left, each panel shows anti-serotonin staining in adult hermaphrodite central and posterior body in worms exposed to 5mM 5-HTP in NGM plates for 24 hr. Such treatment typically results in high background staining in nearly all tissues. (A) Wild type (PS312) worms show both serotonin-IR uv1s and PDEs. Ventral view (uv1s and PDEs seen on both sides). (B) bas-1(tu629) mutant worms show no stained uv1s or PDEs, showing the requirement for AAADC function to decarboxylate 5HTP to 5HT. Interestingly, bas-1 mutants show weak to moderately-stained VC neurons and sometimes NSM neurons in the head, independent of 5HTP exposure (not shown). Lateral view. (C) tdc-1(tu1007) worms show staining in uv1s and PDEs. Note that in some worms (unrelated to 5HTP exposure), as shown here, only a single uv1 cell is found on a given side. Lateral view. (D) tbh-1(cbh32) worms show staining in uv1s and PDEs. Ventral view (uv1s and PDEs seen on both sides).

Ppa bas-1 locus is a tandem duplication – bas-1 mutants may have residual function

(A) Ppa-bas-1 locus has a tandem duplication of BAS-1/AADC coding sequence (tracks from Wormbase genome browser, WS297). Gene model showing coding exons (pink boxes, upper track) and RNASeq coverage (lower track); existing bas-1 mutations (causing premature stops) are in the second section of coding sequence (C-terminal duplicate) indicated by red triangle. Differences in RNASeq coverage suggest that the regions are expressed independently. (B) Dot plot of 1022 AA predicted protein (made with https://www.bioinformatics.nl/cgi-bin/emboss/dotmatcher) showing duplicated region starting around 500 AAs. (C) Multiple sequence alignment of human AADC, C. elegans BAS-1, Ppa N-terminal BAS-1 and C-terminal BAS-1 proteins. Examples of known critical function AAs are highlighted (red letters with yellow background); all but one are conserved in the N-terminal BAS-1 which appears likely to be functional, either by itself or as a truncated internally duplicated protein.

P. pacificus RIM and RIC neuron 3D renderings

Putative tyraminergic (RIM) and octopaminergic (RIC) neurons, lateral views. Anterior to the left. Pharynx in light green, left neuron in magenta, right side blue (Cook et al., 2025).

Colocalization of cat-1 transcripts with tdc-1 and tbh-1 transcripts in RIM and RIC neurons.

Anterior left, ventral down, ventro-lateral view. All images of the same adult hermaphrodite head, MaxIP of a few focal planes showing one side. (A) tbh-1 transcripts (green) expressed in RIC. (B) tdc-1 transcripts (orange), showing two cells in lateral ganglion, RIM and RIC. (C) cat-1 transcripts (magenta) with DIC to show location of the pharynx as a landmark. Some cat-1-positive cells are identified. (D) tdc-1 with DIC, expression in RIM and RIC. (E) Colocalization of cat-1 with tbh-1 transcripts in RIC. (F) Colocalization of cat-1 with tdc-1 transcripts in RIM and RIC.

Expression of tbh-1 transcripts in the gonad.

Anterior to the left, ventral down, showing posterior body. (A-H) distal (and dorsal) gonad staining; (A-E) same single focal plane; (F-H) different focal plane. (I, J) same single focal plane, showing proximal (ventral) gonad. ‘Proximal’ indicates closer to the vulva, distal is further away within the gonad. Regions identified based on (Rudel et al. 2005). (A, F) HCR of tbh-1 transcripts (green) shows expression in likely gonadal sheath cells. (B, G) Both tbh-1 and DAPI signals. (C, H) DAPI staining with nuclei strongly tbh-1-expressing indicated. Some adjacent nuclei (in front and behind) appear to express tbh-1 at a lower level. (D) DAPI and DIC. (E) DIC showing that the region of tbh-1 expression is distal to the gonad bend where the gonad shifts from dorsal to ventral. (I) tbh-1 transcripts are expressed at a lower level in a second region after the gonad bend (proximal to the vulva). (J) DAPI staining with some tbh-1-expressing nuclei indicated.

Expression of tbh-1 transcripts in the spermatheca (proximal gonad).

Adult hermaphrodite with embryos, vulval region, dorsal to the upper left, ventral is lower right. Same focal plane for each panel. (A) DIC showing locations as indicated. A single embryo is present in the uterus between the vulval opening and more distal spermatheca. (B, D, F) Expression of tbh-1 transcripts by HCR. (B) tbh-1 transcripts (green) + DIC. (C) DIC and DAPI (cyan) to show nuclei. (D) tbh-1 alone. (E) DAPI with sperm nuclei within spermatheca indicated. Just distally is gonad constriction with tbh-1-expressing nuclei indicated. (F) tbh-1 transcripts + DAPI staining.

Expression of tdc-1 and cat-1 transcripts is in adjacent vulval region cells.

Adult hermaphrodite vulval region, anterior to the right and ventral down, MaxIP of 21 z-planes on the left side to approximately the midline. (A) tdc-1 transcripts (orange) associated with two nuclei (DAPI, cyan) per quandrant. (B) Co-labeling shows that cat-1 transcripts (magenta) are associated with two nuclei closer to the vulval pore in each quadrant. Although this image suggests possible overlap in expression in some cells (left / anterior), most preparations show clear separation of expression in adjacent cells, like that seen here in the posterior cells. (C) cat-1 transcripts expressed close to the vulval pore, lateral to the ventral nerve cord. Some smaller VC neurons expressing cat-1 are seen at the bottom of the image.

Expression of cat-1 transcripts in the midbody VC neurons and vulval cells.

Ventral view; anterior is to the left in all images. Each panel is the same single focal plane (panels B-D, however, a partly montages that include a small region from a nearby focal plane to better show the ventral nerve cord). (A-C) HCR for cat-1 transcripts (magenta) with DIC and/or DAPI (cyan). (D) DAPI alone to better show vulval region and VNC.

Other cat-1-expressing cells in the head.

Expression of cat-1 transcripts via HCR in dorsal head in cat-1::GFP strain. Anterior to the left. (A, C) same dorsal focal plane. (A) cat-1 HCR fluorescence and DAPI, particularly showing likely identification of OLQDs as dorsal anterior ganglion nuclei just anterior to the nucleus-free region of the nerve ring (NR). Readily identifiable DAPI stained nuclei in the dorsal anterior pharyngeal bulb are indicated. (B) Dorsal view map of dorsal neuronal nuclei around NR, including dorsal pharyngeal neurons (green) identified in (A) from EM reconstruction (Cook et al., 2025); pharynx, partly transparent, in darker green. Other head nuclei (non-pharyngeal) colored by apparent function: sensory (red), motor (blue), interneuron (yellow). (C) cat-1::GFP transgene expression and cat-1 HCR fluorescence colocalization in one putative OLDQ neuron. Same focal plane as (A).

Acrobeloides nanus ES501 head serotonin-IR neurons.

Anterior left, ventral down, lateral view; confocal MaxIP of a few focal planes on left side (top); likely cell and neurite identifications as indicated. Brighter cells and neurites are over-exposed to show less brightly stained somas. nr – location of nerve ring. Schematic showing outlines of somas matching image (below).

Plectus sambesii ES601 head serotonin-IR neurons.

Anterior left, ventral down, lateral view; montage of confocal focal planes on left side (top); likely cell and neurite identifications as indicated. nr – location of nerve ring. Schematic showing outlines of somas and neurites matching image (below).

Plectus sp. WWL501 head serotonin-IR neurons.

Anterior left, ventral down, lateral views; confocal MaxIPs of a few focal planes on left (top) and right (bottom) sides. Likely cell identifications as indicated.

P. pacificus CAT-1 mutant alignments

P. pacificus DAT-1 mutants (N-terminal deletions)

Anti-5HT staining in P. pacificus cat-1 mutants is variably reduced.

Anterior to the left, ventro-lateral view, montage. Head and midbody VNC region of cat-1(csu116) adult hermaphrodite. In this individual, NSM neurons in the head are moderately stained; other serotonin-IR neurons are not seen. VC neurons in VNC stain similarly to wildtype (VC1-3 shown; VC4, which also stains moderately, is out of the plane of focus). Although both cat-1 mutations likely cause a complete loss of function, we observed a variable reduction in serotonin-IR, as has been seen previously in C. elegans cat-1 mutants (Desai et al., 1988) (Loer and Kenyon 1993). Remaining serotonin-IR is not surprising since neurons should continue to synthesize serotonin via tph-1/TPH and bas-1/AADC expression (Figure 1), even if the cells are unable to package the neurotransmitter into synaptic vesicles. In some preparations, we saw loss of serotonin-IR in most or all head neurons and midbody VC neurons; in other preparations (like that above), we observed reduced but considerable staining in all known serotonin-IR cells. The behavioral phenotypes we observed in P. pacificus cat-1 mutants are consistent with a loss of VMAT function in spite of remaining neurotransmitter within the neurons.

Cholinergic proteins and nematode genes

Proteins encoded by identified nematode genes are indicated in black, bold, capital letters (abbreviations: Ch - choline; ACh - acetylcholine; ChAT - choline acetyltransferase, required for acetylcholine synthesis; VAChT - vesicular acetylcholine transporter, required for packaging acetylcholine into synaptic vesicles); nematode gene names (originally from C. elegans) are italicized below the protein name abbreviations.

Cholinergic neurons and nerves in the head of P. pacificus

Both images are left side views, anterior to the left. (A) Wildtype adult hermaphrodite head staining with a ‘cholinergic mix’ of monoclonal antibodies to C. elegans cholinergic proteins CHA-1 (ChAT) and UNC-17 (VAChT) shows extensive staining in the nervous system. The nerve ring is very strongly stained; head ganglia surrounding the nerve ring, including the anterior ganglion, are moderately stained. Although it is difficult to identify individual neuronal somas, the somas of IL2D and IL2V neurons are well isolated from all other neurons, being just in front of the pharyngeal anterior bulb, so can be clearly identified. In this view, the IL2DL (left) is seen, and both IL2Vs (L & R). Another soma in the anterior ganglion could be IL2L. (B) 3D rendering of IL2 neurons, left lateral view (and slightly ventral), anterior to the left. IL2Ds and IL2Vs are further anterior; IL2 L & R are within the anterior ganglion near to the nerve ring. Left side IL2s are rendered in magenta; right side IL2s in blue. The pharynx is in green.

CHA-1 C-terminus alignments of mutant alleles and wildtype proteins.

(A) Alignments of wildtype P. pacificus CHA-1 protein showing alterations in mutant alleles. * denotes stop codon; bolded region shows in-frame 2x FLAG epitope tag insertion (DYKDDDDK); underlined regions indicate results of frame-shift from indel mutations. Alignments of C-termini from nematode and mammalian proteins. There is considerable conservation of the C-terminus. Between each sequence shows identical and similar (+) AAs. Cel – C. elegans, Ppa – P. pacificus, Hsa – Homo sapiens. Cel sequence highlight: P584L missense mutation results in 99% loss of ChAT activity (Rand and Russel, 1984). Ppa sequence highlight: Ppa CHA-1 wildtype C-terminus portion shown in Ppa mutant alignments (A). Hsa-ChAT highlight: region of Helix 20 in rat ChAT crystal structure (Govindasamy et al., 2004).

Effect of cholinergic function loss on nictation is inconclusive

Reduction-of-function alleles in the acetylcholine biosynthesis gene cha-1, including the C-terminal 2x-FLAG strain (ot5000), and two C-terminal indel mutants were found in the same CRISPR knock-in screen (ot5001, ot5002) (Suppl Figure 19) have temperature-sensitive phenotypes.† In these nictation assays, the alleles showed a partial nictation phenotype (ot5001), paralysis of locomotion (ot5002), or no effect (ot5000, in frame FLAG insertion). Between 30-60 animals participate in each nictation assay and at least 6 assays were performed for each genotype. *P<0.05, ***P<0.001, ****P<0.0001 Dunnett’s multiple comparisons test show significant difference to wildtype (WT). † All three cha-1 strains were found to be temperature-sensitive; this may reflect a temperature-sensitive process in CHA-1 protein function such has been observed in all C. elegans cha-1 mutants (Duerr et al., 2021). Whereas the mutants appeared to move similarly to wildtype when raised at 20°C, mutants raised continuously at 25°C or higher became extremely uncoordinated or paralyzed, and progressively become sterile, particularly at temperatures above 25°C. Therefore, to test cha-1 dauers, we let cultures starve at the permissive 20°C, then shifted to 25°C after food was depleted but before dauer formation, so that dauers were formed at the higher temperature. Given the pleiotropic phenotype of the cha-1 alleles at 25°C, however, including defects in locomotion, whether the phenotype is truly specific to nictation is inconclusive. Furthermore, the nictation phenotype could also result simply from a slower response to the sand substrate beyond the 30-minute incubation time of the assay.