Quantitative study of camouflage behaviour in cuttlefish (Sepia officinalis)

Coleoid cephalopods (cuttlefish, octopus, squid) experts in camouflage. They are unique in the animal kingdom for their ability to rapidly change body colour. While adaptive colour change can be found across different classes of vertebrates and invertebrates, Coleoid cephalopods alone can alter their appearance in sub-second speed, affording them the unmatched capability to camouflage dynamically and adaptively. It is unsurprising therefore that the cephalopod colour changing behaviour had piqued the interests of humans since the ancient times. However, scientific studies in this fascinating behaviour had not started until the early 20th century. In the last century, one of the critical discoveries was that colour change in cephalopods is facilitated through an array of pigment cells (chromatophores) under neuromuscular control. The chromatophore radial muscles, which dictate the size of each chromatophore on the body surface, are directly innervated and hence controlled by motoneurons whose cell bodies are found in the central brain. This not only allows for rapid colour change, but also the diversity and precision in pattern control. It is this behavioural flexibility that allows cephalopods to disguise themselves reliably in a diverse underwater environment. The flexibility of the chromatophore system in the adaptive camouflage behaviour is particularly interesting to neuroscientists. How is the appropriate behaviour response computed based on the stimulus? How are the input and output abstracted and represented in the brain? How is the sensorimotor programme organised? How does the memory and/or its substrates persist through a lifetime and over generations? And more specifically for this particular sensorimotor system, what might the selection of their camouflage strategy reflect about the visual processing in vertebrates, including ours? Indeed, what makes these questions more tractable in the camouflage system compared to many other sensorimotor systems is that both its input and output operate in the visual modality. Consequently, this sensorimotor transformation is primed for quantitative interrogation in a naturalistic setting, more than ever, thanks to the rapid advancement in imaging hardware and image analysis techniques in recent years. This dissertation set out to develop the necessary tools to study cephalopod camouflage with modern methods, using the European common cuttlefish (Sepia officinalis) as a model. By choosing this species, we were able to leverage the wealth of qualitative and semiquantitative behavioural studies from the past few decades. Apart from that, Sepia officinalis also offers other practical advantages over other species, including having a relatively rigid body for imaging and being relatively easy to transport and maintain in the laboratory environment. This effort enabled us to begin to address fundamental questions about this unique behaviour. The project was divided into two main phases, concerning the tracking of single chromatophore activity and camouflage pattern change, respectively. Phase 1 focused on developing the techniques in high-resolution analysis of the chromatophore motor system, as a proof of principle. Phase 2 built upon the first and brought the camouflage behaviour to the forefront. Using a combination of electrophysiological and behavioural methods, as well as two computational analysis pipelines developed specifically for our data, we addressed fundamental questions in cuttlefish camouflage, its dimensionality, adaptability, and dynamics. Taken together, this dissertation established the camouflage behaviour unique to Coleoid cephalopods as a study system for neuroscience using Sepia officinalis as a model. The experimental and analysis methodologies thus developed, as well as biological findings, will inform future investigations of this intriguing behaviour that has captivated humans for millennia. Furthermore, our study showed that new technology, once again, not only opens doors to new ventures, but also provides immense opportunities for a deeper understanding of neural functions.