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

Since the original isolation of the neuropeptide FMRFa-mide from the clam Macrocallista nimbosa (Price and Greenberg, 1977), many variants of FMRFamide (FMRFa-midelike peptides or FLPs) have been identified by immunocytochemistry, by direct isolation, or by cloning the genes encoding the peptides. FLPs are ubiquitous in the animal kingdom and have been found from coelenterates to mammals; most species express multiple FLPs (for review see Li et al., 1999a). Nematodes in particular are known to contain a great variety of FLPs. In one species, Caenorhabditis elegans, the availability of the complete sequence of the genome has allowed an extensive search for genes that encode FLP precursor proteins; 31 such genes (flp genes), encoding 71 putative FLPs, have been identified, and new members of this family are still being discovered (Li et al., 1999b; McVeigh et al., 2005; Husson et al., 2005, 2007; Li and Kim, 2008). The existence of several of these putative peptides has been confirmed by direct chemical isolation (Marks et al., 1995, 1997, 1999; Davis and Stretton, 1996; Maule et al., 1996; Brownlee and Fairweather, 1999; Husson et al., 2005, 2007). Other C. elegans genes encoding putative peptides with different C-terminal sequences are also being discovered (Li et al., 1999b; Nathoo et al., 2001; Husson et al., 2005, 2007; Li and Kim, 2008; McVeigh et al., 2008; L.A. Messinger, unpublished); it is now clear that there is a rich and complex set of peptides in C. elegans. In the parasitic nematode Ascaris suum, there is also evidence for many families of peptides (Sithigorngul et al., 1990; Brownlee et al., 1993; McVeigh et al., 2008). The FLPs are prominent, being present in about 70% of the neurons (Cowden et al., 1993), and there is much variety in their structure. In this laboratory, we have directly sequenced 40 FLPs from A. suum: AF1–AF11 and AF13–AF41 (Cowden et al., 1989; Cowden and Stretton, 1993, 1995; Davis and Stretton, 1996, 2001; Yew et al., 2003, 2005, 2007; Jarecki et al., 2010; Jarecki, Andersen, and Stretton, unpublished). In many cases, the afp transcripts that encode these peptides have also been characterized, either by cloning or by sequence mining from EST libraries (Nanda, 2004; McVeigh et al., 2005; Yew et al., 2007; Nanda and Stretton, 2010; Jarecki et al., 2010). AF1 (KNEFIRFamide) occurs in other nematodes. It has been isolated from C. elegans (Sithigorngul; reported in Davis and Stretton, 1996). The generation of large EST libraries from 30 species of parasitic nematodes has allowed the discovery of transcripts that encode putative FLPs in parasitic nematodes (McVeigh et al., 2005). Several nematode ESTs include AF1-encoding sequences. In all cases, the predicted precursor proteins include multiple copies of AF1, flanked by classical dibasic amino acid cleavage sites: Necator americanus (4 × AF1), Ancylostoma ceylanicum (4 × AF1), Onchocerca volvulus (2 × AF1; McVeigh et al., 2005), and A. suum (5 × AF1; McVeigh et al., 2005; revised by Nanda, 2004); the free-living nematodes C. elegans and C. briggsae each encode four copies of AF1 (Li et al., 1999b). Considering that nematodes are estimated to have diverged about 550 million years ago (Vanfleteren et al., 1994), the sequence conservation of AF1 is remarkable. A major task is now to determine the role that these peptides play in the overall biology of A. suum. Because RFa-mide immunoreactivity is located in the nervous system of A. suum, we conclude that the FLPs are neuropeptides (Cowden et al., 1993). To understand their function in the nervous system, it is necessary to identify both the cells that contain and presumably release the peptide, and the cells that respond physiologically to the peptide. We have previously addressed the sites and mode of action of AF1, showing that it has dramatic effects on the inhibitory motorneurons in the nerve cords by decreasing their input resistance, thus disrupting their ability to conduct electrical signals (Cowden et al., 1989). This study addresses the cellular distribution of AF1 by generating an antibody with an exceptionally high specificity to AF1. This reagent allowed us 1) to trace the processes of the AF1-containing neurons and thereby identify them definitively with homologous neurons in C. elegans and 2) to demonstrate that the cellular pattern of expression of AF1 differs in C. elegans and A. suum. In addition, two other anti-AF1 antibodies with broader cross-reactivity enabled us to trace the processes of additional neurons and to identify them as morphological homologs of C. elegans neurons.