Conservation of a Tritonia Pedal peptides network in gastropods

Adults of the nudibranch mollusc Tritonia diomedea crawl using mucociliary locomotion. Crawling is controlled in part by the large Pedal 5 (Pd5) and Pedal 6 (Pd6) neurons that produce Tritonia Pedal peptides (TPeps). TPeps elicit an increase in ciliary beat frequency, thereby increasing crawling speed. In adults of T. diomedea, an extensive network of TPep-containing neurites adjacent to the basement membrane of the pedal epithelium delivers TPeps to the ciliated cells. In this study, we show that diverse nudibranchs all have a pattern of TPep-like immunoreactivity similar to that of T. diomedea, with thin tracts of TPep-like immunoreactive (TPep-LIR) neurites projecting to the epithelial layer. We also show that members of two non-nudibranch gastropod species have a pattern of TPep-innervation similar to that of the nudibranchs. In addition, we characterized two pairs of motor neurons in adults of the nudibranch Armina californica that are possible homologues of the Pd5 and Pd6 cells in T. diomedea. Activity in one of these pairs, the Pedal Peptidergic Dorsal 1 (PPD1) cells, was correlated with mucociliary locomotion. The second pair, the Pedal Peptidergic Ventral 1 cells, shared synchronous synaptic input with the PPD1 cells, a pattern consistent with the shared synaptic input of the T. diomedea Pd5 and Pd6 cells. These findings suggest that the roles of the Pd5 and Pd6 cells as mucociliary motor neurons in nudibranchs are conserved evolutionarily. Additionally, the extensive network of TPep-LIR neurites seen in the foot of T. diomedea appears likely to be a common feature among gastropods. Additional key words: Armina, TPeps, mucociliary, locomotion, crawling Members of many species of nudibranch molluscs, or sea slugs, crawl primarily using mucociliary propulsion (Agersborg 1922; Audesirk 1978a; Crow & Tian 2003). Mucociliary crawling in members of Tritonia diomedea BERGH 1894 is mediated in part by the bilaterally symmetrical Pedal 5 (Pd5) and Pedal 6 (Pd6) neurons, which are located on the dorsal surface of the pedal ganglia (Wang et al. 2003; Cain et al. 2006). Pd5 and Pd6 are large neurons (300–500 lm in diameter) which produce Tritonia Pedal peptides (TPeps). These cells project to the foot of the animal, where an extensive network of TPep-like immunoreactive (TPep-LIR) neurites adjacent to the basement membrane of the epithelium delivers TPeps to the ciliated cells (Willows et al. 1997; Cain et al. 2006). Similar TPep-LIR cells have been identified in the pedal ganglia of diverse nudibranchs, but the function of these cells has not been established (Croll et al. 2001; Newcomb & Katz 2007; Baltzley & Lohmann 2008). Although putative Pd5 and Pd6 homologues have been identified in members of other species of nudibranchs based on TPep-LIR, size, and location, these cells do not necessarily control mucociliary locomotion. For example, the Pedal 7 (Pd7) cells in individuals of T. diomedea are large, TPepproducing neurons located on the dorsal surface of Author for correspondence. E-mail: [email protected] Invertebrate Biology 130(4): 313–324. © 2011, The American Microscopical Society, Inc. DOI: 10.1111/j.1744-7410.2011.00242.x the pedal ganglia adjacent to Pd5 and Pd6. However, unlike the Pd5 and Pd6 cells, the Pd7 cells innervate the anterior body wall, oral veil, and mouth, rather than the foot, and are therefore unlikely to control crawling (Wang et al. 2004). Similarly, there are a number of TPep-LIR cells in the cerebral, pleural, and buccal ganglia of T. diomedea that, based on location and morphology, are unlikely to be involved in locomotion (Beck et al. 2000). TPep-like peptides have also been localized in the central nervous system and adjacent to the foot epithelium in adults of the pulmonate gastropod Cornu aspersum (MÜLLER 1774) (formerly known as Helix aspersa) (Pavlova & Willows 2005). While members of C. aspersum have an extensive network of TPep-LIR neurites adjacent to the foot epithelium, the animals crawl using muscular contractions of the foot rather than mucociliary propulsion (Pavlova & Willows 2005). Because TPep-LIR has been observed at a number of different ciliated epithelia in both T. diomedea and C. aspersum (Gaston 1998; Pavlova & Willows 2005), it is possible that the presence of TPep-like peptides at ciliated epithelia is highly conserved across the gastropods. Thus, the Pd5 and Pd6 neurons may have evolved a specialized role in mucociliary locomotion in T. diomedea, whereas the homologues of Pd5 and Pd6 may have a different role in other gastropods. In this study, we examined the large TPep-LIR neurons of the nudibranch Armina californica COOPER 1863, which are likely homologous to the Pd5 and Pd6 cells in members of T. diomedea (Baltzley & Lohmann 2008). We also examined the pedal epithelium of members of nine other species of nudibranchs, representing all four nudibranch suborders, as well as two non-nudibranch gastropods, Phyllaplysia taylori DALL 1900 and Calliostoma ligatum GOULD 1849, to determine if the network of TPep-LIR neurites is conserved across the nudibranchs and possibly more widely in the gastropods. We found that the presence of TPep-LIR neurites adjacent to the pedal epithelium is a widespread trait in nudibranchs. We also found TPep-LIR neurites adjacent to the pedal epithelium of P. taylori and C. ligatum, indicating that this TPep network may be a general feature in all gastropods. Finally, in adults of the nudibranch A. californica, we characterized the activity of the PPD1 and PPV1 neurons. The results are consistent with the hypotheses that the PPD1 and PPV1 cells are mucociliary motor neurons and are homologous to the Pd5 and Pd6 cells in T. diomedea. Methods