Research Paper
Chung et al., 2022, Current Biology 32, 97–110 January 10, 2022 Crown Copyright 2021 Published by Elsevier Inc. https://doi.org/10.1016/j.cub.2021.10.070
Funding
Funding for this research was acquired and supervised by Emeritus Professor N. Justin Marshall of the University of Queensland’s Queensland Brain Institute. Ethics approval and acknowledgements suggest that funding was supplied by the Queensland Government, the Australian Research Council, the Office of Naval Research (US), and the United States (US) Air Force. The funding provided by Australian Research Council grant DP200101930 was $710,048
Ethics Approval
Octopus collection was conducted under a Great Barrier Reef Marine Park Permit (G17/38160.1) and Queensland General Fisheries Permit (180731) and approved by animal ethics permit (QBI/236/13/ARC/US AIRFORCE & QBI/304/16) indicating a collaborative effort by the Queensland Brain institute (QBI) at the University of Queensland, the Australian Research Council (ARC), and the United States (US) Air Force.
Terminology
Species
Abdopus capricornicus: Day arm-drop octopus
Hapalochlaena fasciata: Blue-lined octopus
Octopus cyanea: Day reef octopus
Octopus vulgaris: European common octopus
Vampyroteuthis infernalis: Deep-sea vampire squid
Phrases
Foliation: The surface of a developing (gestational) brain.
Gyrification: Changes to the brain surface folding.
MRI: Magnetic resonance imaging uses a magnetic field and radio waves to take pictures inside the body.
Nociception: The neural detection of painful stimuli, of which pain is a subjective experience
Octopod: Cephalopod mollusks (such as an octopus) that have eight arms bearing sessile suckers
Octopodiformes: A superorder of Coleoidea, comprising of octopuses and vampire squid
PFA: Paraformaldehyde
PBS: Phosphate-buffered saline
Background
In this project, researchers were attempting to learn about how environmental and behavioural differences contribute to the central nervous system of four species of octopus, and how they might differ from that of the often-studied European common octopus, octopus vulgaris.
The project proposal intended to include mantis shrimp in the study to “investigate vision, neural coding, and behavioural innovations” with suggested benefits including “GPS-free navigation in marine engineering and rapid exposure of research results to millions of people through existing communication programs.”
The Experiment
Twenty-one individual coastal octopuses were wild-caught and used during 2018-2020, consisting of:
- Two (both used for MRI) mature male Blue-lined octopuses collected using a hand-net from Moreton Bay Research Station, Stradbroke Island, Queensland;
- Ten (two used for MRI) juvenile Day reef octopuses collected from collected using a hand-net from the reef flat of Lizard Island, Queensland;
- Nine (three used for MRI) mature male Day arm-drop octopuses were collected using a hand-net from the reef flat of Lizard Island, Queensland;
Additionally, three (one used for MRI) Deep-sea vampire squid were collected using a trawl net from the Indian Ocean on deep-sea expedition (SO258-L1).
Four further octopus species were selected from existing published research and included in the comparison.
The coastal octopuses were anesthetised in 15°C seawater combined with 2% magnesium chloride (MgCl2) then killed with “an overdose” of magnesium chloride (MgCl2). The deep-sea vampire squid were anesthetised in 4°C seawater combined with 2% magnesium chloride (MgCl2) then killed with “an overdose” of magnesium chloride (MgCl2). For the deep-sea vampire squid, “First, each eyeball was injected with 1-2 mL 4% PFA-PBS fixative to avoid eyeball collapse.”
The isolated brain and eyes were imaged using a digital camera, MRI scans were taken of the brains and eyeballs, and the optic nerves were also imaged. The brain lobes (identifiable sections of the brain) and the number of vertical lobe gyri (ridges on the outer layer of the brain) were compared, taking into account the environment and behaviours of each species.
The authors state that changes to the brain surface folding, or ‘gyrification’, and the formation of anchor points on the surface of a developing (gestational) brain, or ‘foliation’, “are an indicator in brain evolution associated with increases in size, complexity, and information processing capability.”
They found that the deep-sea vampire squid has a smooth brain, that is less ‘gyrification’, as a result of their slow pace of life and limited social interactions amongst low-oxygen and dim-light conditions. On the other hand, day reef octopuses have a greater gyrification which is associated with more social interactions, complex visual tasks, and a well-lit environment.
The researchers write that due to having collected and observed only a few species of “octopodiforms [sic]”, “any such test must wait for more comparative data to be meaningful”, acknowledging the limitations of the design of the study.
They then sign off by concluding simply that, “this study reveals new insights into the complexity and evolutionary neuroscience of octopod brain organization and demonstrates correlations with habitat and behavioral ecology.”
Relevance to Humans
Aside from minor conservation through an appreciation of the evolution of oceanic animals or a basic contribution to our understanding of animal senses, it is not clear how this project may be significantly relevant to humans, animals, or the environment. However, the indication of involvement of the United States Air Force suggests that the intention may have been to do with perceived or anticipated applicability to humans.
“[I]t is my stance that experimentation on invertebrate octopuses is ethically wrong and scientifically unhelpful.”
– Dr. Barbara J. King
Discussion
Concerning questions arise when considering the methods of slaughter for cephalopods. Neurobiologist Joshua Rosenthal of the University of Chicago’s Marine Biological Laboratory has stated that, “…it’s an open question, and a difficult one. We don’t know how they sense nociception.” (The word “nociception” means painful feelings). As octopuses have a decentralised nervous system, it is difficult to kill them in accordance with understood ‘humane’ slaughter methods. The method of slaughter briefly described in the highlighted research does have the “potential to cause adverse effects prior to unconsciousness, such as skin or eye irritation, or asphyxia.” The authors do not declare any adverse reactions to the “overdose” of magnesium chloride.
According to Dr. Barbara J. King from the College of William and Mary, there are two primary moral reasons that experiments on octopuses should not be permitted. Dr. King opposes any experiments which are physically invasive, or which require the capture of wild animals or for them to be subjects of breeding colonies. She proposes only allowing “non-invasive observational experiments” on octopuses already held in captivity, but states that, “it is my stance that experimentation on invertebrate octopuses is ethically wrong and scientifically unhelpful.”
A 2021 paper published in Frontiers in Marine Science described the method of breeding Octopus chierchiae, or the lesser Pacific striped octopus, and the culture program they propose. As this species of octopus is small, lays eggs every ~30-90 days, and has a short time to maturity, they have touted them as “ideally suited to laboratory culture”. The proposal suggests that the species is an “advantageous model organism for developmental, neurobiological, and behavioral study.”
The researchers proposing the use of the lesser Pacific striped octopus are proposing their use in place of existing animals used in laboratories, such as mice and zebrafish, and potentially in place of investing and developing alternatives to developmental and neurobiological models.
AFSA Comment
Currently, only the ACT, Queensland, and Victoria include cephalopods under their legal classification of ‘animal’. However, unfortunately, a classification as an ‘animal’ does not always include protections from pain or death in research.
The Australian code for the care and use of animals for scientific purposes (the Code) states that animals can only be used where there will be “potential benefit for humans, animals or the environment” and that projects must only be approved to “establish significant information relevant to the understanding of humans and/or animals” (emphasis added) or “to the understanding, maintenance or improvement of the natural environment”.
The authors themselves indicate that the research project did not in fact meet the criteria the Code, revealing in their second-to-last discussion section paragraph that they did not use the minimum number of animals required to obtain valid data, as required by the Code, saying, “due to the limitation of a small number of [octopus] species” they are not able to make comparisons between similar octopus species and they “must wait for more comparative data to be meaningful.”
Animal-Free Science Advocacy contacted the researchers and Animal Ethics Committee for comment and further information, however we are yet to receive a response.