A specific antibody to neuropeptide AF1 (KNEFIRFamide) recognizes a small subset of neurons in Ascaris suum: differences from Caenorhabditis elegans

Abstract

A monoclonal antibody, AF1-003, highly specific to the Ascaris suum neuropeptide AF1 (KNEFIRFamide), was generated. This antibody binds strongly to AF1 and extremely weakly to other peptides with C-terminal FIR-Famide: AF5 (SGKPTFIRFamide), AF6 (FIRFamide), and AF7 (AGPRFIRFamide). It does not recognize 35 other AF (A. suum FMRFamide-like) peptides at the highest concentration tested, nor does it recognize FMRFamide. When crude peptide extracts of A. suum are fractionated by two-step HPLC, the only fractions recognized by AF1-003 are those comigrating with synthetic AF1. By immunocytochemistry, antibody AF1-003 recognizes a small subset of the 298 neurons of A. suum: these include the paired URX and RIP neurons, two pairs of lateral ganglion neurons in the head, and the unpaired PQR and PDA or -B tail neurons that send processes to the head along the dorsal and ventral nerve cords, respectively. AF1 immunoreactivity is also seen in three pairs of pharyngeal neurons. Mass spectroscopy (MS) shows the presence of AF1 in the head, pharynx, and dorsal and ventral nerve cords. In A. suum, the neurons that contain AF1 show little overlap with neurons that express green fluorescent protein constructs targeting the flp-8 gene, which encodes AF1 in Caenorhabditis elegans (Kim and Li [2004] J. Comp. Neurol. 475:540– 550); the URX neurons express AF1 in both species, but, in C. elegans, flp-8 expression was not detected in RIP, PQR, and PDA or -B or in the pharynx. Other, less specific monoclonal antibodies recognize AF1, as well as other peptides to differing degrees; these antibodies are useful reagents for determination of neuronal morphology.

Changes in cyclic nucleotides, locomotory behavior, and body length produced by novel endogenous neuropeptides in the parasitic nematode Ascaris suum

“…We investigate both the transmitting cell and the receiving cell that constitute the intercellular signaling dyad mediated by the peptide. To identify the cells that produce and presumably secret these peptides, we use three different approaches: immunocytochemistry with highly specific antibodies against individual peptides [31], in situ hybridization with riboprobes derived from cloned genes encoding the peptides [32], or direct analysis of peptide content by mass spectrometry of single dissected neurons [6]. In almost all cases the three techniques give the same answer.…”

The Neurobiology of Ascaris and Other Parasitic Nematodes

“…87 In other cases there is apparently secretion into the PCF, as mentioned above for the serotonin-containing neurons of the pharynx. In some cases, e.g.…”

Sensory neurons control heritable adaptation to stress through germline reprogramming

“…elegans and A . freiburgensis (within and across generations, respectively) could be due to the expression of particular neurotransmitters in homolog neurons [33, 34]. Future comparative studies with closely related species will reveal how mechanisms of intergenerational inheritance evolved.…”