India Viola et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Ascaris Suum Is Not Just A Giant Version Of C. Elegans"

India Viola, Jennifer Cho-Nanda, Ivan Chevere, Joanne Yew, Antony Stretton

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The morphology of the neurons of A. suum and C. elegans is remarkably similar: neurons in A. suum can be named for their homologs in C. elegans. This similarity extends to the cellular expression pattern of the classical transmitters acetylcholine, GABA, dopamine and serotonin, although a few differences have been found. The world of neuropeptides in both of these organisms is very rich. Many neuropeptides are identical or closely sequence-related. It is therefore astounding to find that the cellular expression patterns of identical or near-identical peptides are almost completely different. In A. suum we have determined the expression patterns of 13 peptides by 3 techniques. Mass spectrometry has been carried out on dissected ganglia from single worms, and does not yet have single cell resolution. Immunocytochemistry and in situ hybridization identify individual cells expressing each peptide. Some examples of differences between expression patterns are a) In the dorsal ganglion, AF8 is expressed in the ALA neuron, and AF3, 4, 10, 13, 14 and 20 (encoded by afp-1) are expressed in RID. GFP constructs in C. elegans show no expression of flp-6 (encodes AF8) or flp-18 (encodes 6 afp-1 related peptides) in these cells (Nelson et al., 1998). b) In A. suum the RIS neuron expresses AF 21, 22, and 23 (encoded by afp-6); in C. elegans the related gene flp-11 is not expressed in RIS. c) Inhibitory motor neurons express AF9 and AF24 in A. suum, but GFP constructs of the flp-21 and flp-12 genes (encode AF9 and AF24) are not expressed in these neurons. AF1 is the only case in which we have seen identical peptide expression in the two organisms, but it applies to only one neuron, URX; all other neurons in which AF1 is expressed are different. One possible explanation for these differences is that the GFP constructs are not faithfully reporting the endogenous expression of the normal gene; however, staining of C. elegans with an AF1-specific antibody completely confirms the GFP construct data (Nelson et al., 1998) for the flp-8 gene (encodes AF1). We believe that the dissimilarities of peptide expression in A. suum versus C. elegans leads to a more accurate and interesting story of divergent evolution and hints at the true level of complexity of neuron-peptide interactions.

John McKenna et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Biological Effects of Neuropeptide DYRPLQFamide in Ascaris suum."

Dianne Witek, Antony Stretton, John McKenna

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The nlp-12 transcript of C. elegans (Nathoo et al, 2001) encodes 3 or 4 predicted peptides. There is strong conservation of related sequences in other nematodes, including A. suum, and the predicted peptides DGYRPLQFamide, SYRPLQFamide, pQDRDYRPLQFamide and DYRPLQFamide have been detected in A. suum by mass spectrometry (Yew et al, 2005). An antibody specific to the QFamide family stains 3 head neurons and 2 ventral cord interneurons. Each of these peptides produces a ventral coil when injected into intact A. suum (see Reinitz et al, 2000), so the tension of the ventral muscles must exceed that of the dorsal muscles. This response might be due to differences in the relative sensitivity of dorsal and of ventral muscles to these neuropeptides. Alternatively it might be due to specific effects on selected neurons of the motor nervous system. In experiments on dissected muscle strips, prior to the application of the neuropeptide, acetylcholine was applied to the muscle strip as a control to ensure the muscle strip was functioning within normal parameters. DYRPLQFa was then applied to individual ventral and dorsal muscle strips. Upon completion of the neuropeptide tests, ACh was applied again to determine whether the contractile response to ACh had been affected by the peptide. Both the ventral and dorsal muscle strips contract upon application of DYRPLQFa, and there was no statistical difference between them. These results did not explain the ventral coil produced by this peptide in the intact worm. Similar results were obtained by McVeigh et. al. (2006). In these experiments the dorsoventral commissures were cut in order to isolate each muscle strip; it is possible that the dorsoventral signaling mediated by the commissures may play a role in the overall activity of this neuropeptide in A. suum. Therefore further experiments are being conducted using muscle strips which contain both the dorsal and ventral muscle strips but leaving selected commissures intact.

Stretton, Antony et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "A Role for Neuropeptides in Nematode Speciation?"

Stretton, Antony, Konop, Christopher, Knickelbine, Jennifer

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Nematodes are extremely numerous and occupy an enormous range of ecological niches, yet their body plans are highly conserved. This morphological conservation includes the nervous system. For example, the large pig parasite Ascaris suum and the minute free-living Caenorhabditis elegans have very similar numbers of neurons (298 in A. suum females and 302 in C. elegans). In addition, the neurons in each are virtually identical in morphology and connectivity: essentially they just differ in size. The motor nervous system in both species comprises the same subtypes of motorneurons, and homologous neurons use the same small neurotransmitters, acetylcholine in excitors and GABA in inhibitors. How do nematodes with essentially the same wiring diagrams produce the different behaviors that allow them to speciate into different niches? We suggest that at least one mechanism is the role of the numerous neuropeptides that modulate the physiological properties of the neurons.From mass spectrometric (MS) analysis and from EST libraries, we estimate that there are at least 250 neuropeptides in A. suum. We have isolated and sequenced 102 peptides so far (Yew et al., 2005; Jarecki et al., 2013), the majority of which we have tested physiologically: almost all affect motor neurons and/or muscles. Recent improvements in dissection techniques have enabled us to isolate single neurons, including the motorneurons. MS is sensitive enough not only to detect the neuropeptide complement of each dissected cell, but also to sequence each neuropeptide by tandem MS. We have confirmed the localization of peptides by in situ hybridization, and, where we have generated sufficiently specific antibodies, by immunocytochemistry. In C. elegans and many other nematodes, EST libraries include transcripts that encode peptides identical to those isolated from A. suum (McVeigh et al., 2005, 2008), implying strong positive selection pressure. However, localization studies in C. elegans using GFP constructs (Kim and Li, 2004) show in almost all cases a completely different neuronal expression pattern in the two species. We suggest that differences in the cellular expression patterns of neuropeptides is an important evolutionary mechanism by which nervous systems with essentially identical structure and synthesizing the same neuropeptides can generate the different behaviors that enable them to occupy different ecological niches.Supported by NSF 1145721, NIH R21-AI103790, and the UW-Madison Graduate School