We show that Enteroctopus dofleini experiences behavioral hypersensitivity and significant deterioration of the peripheral neural and epithelial tissues during the physiological period of senescence. The behavioral changes revealed through touch tests indicate a period of hypersensitivity that begins around the time an individual is entering the reproductive phase, persists through to late senescence and then declines rapidly until the animal is almost completely unresponsive in the peri-mortem period. Likewise, we report a clear decline in the health of nervous and sucker edge tissue in the periphery through senescence, primarily indicated by progressive reduction in cell density both in neural and non-neuronal tissue.
The behavioral responses we measured were in response to two qualitatively different sensory experiences; the light von Frey filament delivered a non-noxious, possibly even sub-detection threshold stimulus to the skin, while the 26g filament was likely noxious and aversive (Bazarini & Crook, 2020; R. J. Crook et al., 2011). Although behavioral responses to the two filaments varied among animals tested in their pre-reproductive phase, in early and mid-senescence we found evidence for pronounced aversive reactions to the light filament that were similar behaviorally to responses to the heavy filament, suggesting significant reduction in activation thresholds of nociceptive neural pathways. The functional consequences of this shift toward hypersensitivity in the early reproductive phase are not clear; it is possible that these changes in sensory function serve to heighten reproductive receptivity or to enhance protective behaviors that are associated with egg care in females. The more applied implication of this finding is that even very mild dermal stimulation in animals in early senescence - when their outward appearance is quite healthy - may be perceived as aversive and thus this is a significant concern for welfare (Fig. 1).
Behavioral changes in other cephalopod species during senescence (Anderson et al., 2002; Bellanger et al., 1997; Holst & Miller-Morgan, 2020), along with declines in cognitive performance (M. P. Chichery & Chichery, 1992; R. Chichery & Chichery, 1992; Halm et al., 2000), suggest that changes to the nervous system are a result of degeneration, and may not be adaptive or functional, however, previous studies have focused on the later stages of senescence when brooding behavior is well advanced. A recent study (Z. Y. Wang & Ragsdale, 2018) showed changes in expression levels of neurotransmitter and other neural-function associated proteins in brooding and senescent females, indicating a prolonged and progressive suite of hormonally driven changes the nervous system occur throughout senescence. In other invertebrate species, age-associated changes to nociception and mechanosensation have been attributed to changes within sensory neurons (Ghimire & Kim, 2015), while in mammals, where the most extensive study of age-related changes to pain perception have been conducted, there is evidence for changes in both peripheral and central compartments (Devor, 1991; Lautenbacher et al., 2005; Taguchi et al., 2010).
Interestingly, we find that E. dofleini exhibits hypersensitivity at the very early stages of senescence. There is limited evidence for an onset of hypersensitivity during early senescence in other animals, but several studies suggest that decline in inhibitory neurotransmitter is associated with chronic pain (Yang & Chang, 2019) or neurodegenerative diseases, which often increase with age (Hou et al., 2019). Gamma-aminobutyric acid (GABA) is a conserved inhibitory neurotransmitter that modulates transmission of nociceptive signals across synapses of the central nervous system, and loss of inhibitory neurotransmitters is at least partly responsible for some aspects of chronic pain in mammals (Yang and Chang, 2019). Thus, we hypothesize the onset of hypersensitivity exhibited by E. dofleini at the early stages of senescence may be caused by disproportionate loss of inhibitory interneurons in the arms, resulting in hypersensitive responses to previously non-nociceptive stimulus. This could also explain observations of excessive arm-spinning during grooming, and increased movement of the arms and body in early and late senescence. Identification of neural sub-types in cephalopods is challenging, but further studies will investigate the hypothesis that loss of inhibitory control is associated with the onset of hypersensitivity in early senescence.
After early senescence, there is a clear downward trend of behavioral response from early stage to perimortem senescence. In some cases, behavioral responses completely ceased in the final few days before euthanasia. This sudden increase in response threshold after a period of hypersensitivity (abnormally low response threshold) is likely to have multiple causes, including loss of mechanoreceptor function, loss of afferent pathway integrity, or loss of motor control over withdrawal reflexes. In this study we examined tissue health only in the arms, and we also did not evaluate changes in the central nervous system, (in part due to the challenges of central brain dissections for local caretakers, compared with the relative ease of taking arm sections from euthanized animals). How the central brain declines, and how this contributes to changes in behavior, is not currently known.
It is clear that total cell density in the arms, both for neuronal and non-neuronal cell types, shows a clear downward trend as animals approach end-of-life. We had hypothesized that an increase in the rate of apoptosis and necrosis was responsible for loss of arm sensitivity at end-of-life, but unexpectedly we found no such pattern; proportions of TUNEL positive cells were the same for healthy control animals. Instead, we found greatly reduced cellular density in two of the three regions we examined (and a clear trend in the third). It is possible that the observed physiological and behavioral shift that occurs in E. dofleini is a result of reductions in the rate of normal cell replacement (López-Otín et al., 2013), rather than an increase in the rate of cell death.
All animals in the study were tracked by their local caretakers for outward signs of senescence, and the evaluation tool (Holst & Miller-Morgan, 2020) was used to aid euthanasia decisions. All animals in the study were euthanized; none died naturally. Thus, the physiological state at euthanasia was reflective of care-takers’ decision making, and animals were euthanized in various conditions. In an effort to determine how closely external signs of decline correlated with the physiological measures of tissue health, we correlated cell density with the number of concerning, “Level 4” welfare observations reported for that animal prior to death (correlation was not possible for touch-test data since not all tissue samples came from animals with peri-mortem touch-tests). We found significant association between outward condition and cellular health in the arms, suggesting that outward measures of welfare correlate reliably with physiological tissue health.
Management of end-of-life care is of paramount importance for animal caretakers in zoos and aquariums, as well as in research labs. There has been relatively little research on welfare and euthanasia in invertebrates, but concern is growing for cephalopods in particular (Jacquet, Franks, & Godfrey-Smith, 2019; Jacquet, Franks, Godfrey-Smith, et al., 2019; Mather, 2022). The sudden drop in response thresholds in early senescence that we observed in this study suggest that proactive management and welfare assessment of senescent E. dofleini should begin at the onset of the reproductive phase, rather than in the terminal period. Hypersensitivity in the early senescent phase may imply that routine maintenance and handling (either in research laboratories or in public aquaria) may be perceived as aversive or painful and may have strong influence on research findings if the onset of reproductive maturity is not accounted for.
Educational facilities often tend to manage end-of-life in terminal animals by providing care that would extend the life of an animal as long as possible. However, our study indicates that cellular decline and possible loss of cellular function may lead to increased, rather than decreased, sensitivity to external stimuli that only declines as the animal enters the last days of life. Thus, efforts that focus on prolonging life until animals are extremely compromised may not be in the best interest of the animal. This study provides new evidence of a link between predictable behavioral changes during senescence in octopuses, and degeneration of peripheral tissues, and raises important new questions about sensory function, perception, and welfare of cephalopods over the course of senescence and death.