Functional investigation of molecular determinants of nociception in Octopus vulgaris

Abstract

Cephalopods are characterised by an exceptional neuroanatomy, formed of a central brain mass and an extended arm sensory system which contributed to the evolution of complex behaviours that might underly brain states comparable to vertebrates, including pain. This possibility is a major reason behind their inclusion in the UK and EU legislations regulating animal welfare in research. Despite irrefutable evidence of nociception in cephalopods, the occurrence of pain in these animals requires further studies.
This thesis investigated putative genes involved in Octopus vulgaris nociception. To overcome the current limited direct genetic tractability in this species, I designed my work around a model hopping approach and used in vivo and in vitro approaches to characterise gene function.
In silico analysis identified O. vulgaris orthologues of well-characterised vertebrate and invertebrate genes involved in nociception based on the rationale that the strong adaptive value of nociception led to the evolution of conserved molecules across metazoans. I prioritised genes that showed orthologues in Caenorhabditis elegans. This allowed me to probe mutant strains for deficiency in avoidance behaviours triggered by low pH. This verified 19 putative nociceptive-related genes from octopus with a discernible nociception defect in C. elegans.
The validity of this approach was reinforced by complementation experiments using TRPV receptors, a family of highly conserved ion channels directly involved in sensory detection of noxious stimuli. Two TRPV sequences, Ovtrpv1 and Ovtrpv2, were identified in O. vulgaris genome and exhibited a tissue distribution consistent with a selective role in nociception. Heterologous expression in C. elegans and successful rescue of loss of function mutants for the orthologous genes (ocr-2 and osm-9 respectively), as well as complementary work carried out in the Xenopus laevis oocyte expression system, confirmed a polymodal function of Ovtrpv1 and Ovtrpv2.
Finally, I searched for potential candidates of an antinociceptive system in O. vulgaris. As the approach described above found no evidence for an opioid peptide system, I used an evolutionary strategy to define the closest protostome orthologue of the opioid system that evolved to modulate pain, namely allatostatin C. In silico and experimental evidence revealed the presence of two receptors, OvAstCR1 and OvAstCR2. These were successfully de-orphanised by the identified putative endogenous peptide OvAstC when investigated for functional signalling in HEK293G5A recombinant system. The tissue distribution of the allatostatin C system implies broad functional outcomes in O. vulgaris.
Overall, I identified conserved genes that could concur to trigger and modulate O. vulgaris nociceptive responses, paving the way for functional studies aimed at investigating pain-like responses in cephalopods, ultimately increasing the level of welfare attention these molluscs deserve in scientific, cultural and economic contexts.

More information

Identifiers

ORCID for Eleonora Maria Pieroni: orcid.org/0000-0002-2258-2780

ORCID for Vincent O'Connor: orcid.org/0000-0003-3185-5709

ORCID for James Dillon: orcid.org/0000-0003-3244-7483

ORCID for Lindy Holden-Dye: orcid.org/0000-0002-9704-1217

Catalogue record

Export record

Altmetrics