Intramantle Inking: A Stress Behavior in Octopus bimaculoides (Mollusca: Cephalopoda)

Abstract

Several Pacific 2-spot octopuses (Octopus bimaculoides) shipped from California and held in a recirculating seawater system at Illinois College exhibited an unusual postshipping stress behavior not previously documented in the literature. Ink, normally ejected into the surrounding seawater, was uncharacteristically retained in the mantle cavity. We describe the resulting behaviors, discuss successful resuscitation efforts, and briefly consider the possible role(s) that ink may have played in the death of one octopus.

Cephalopods such as squid, cuttlefish, and octopuses are held in laboratory facilities for use in a variety of biomedical studies² and are known to exhibit an array of stress behaviors when kept in captivity. Autophagy³ and agitated swimming behaviors resulting in repeated contact of the head with the sides of the aquaria in which they are held¹⁴ can lead to the formation of bacterial lesions and ultimately death. In addition, stress-induced normal inking behavior has been reported after shipping of animals.^10,11,15

Although octopuses have successfully been shipped long distances, little attention has been paid to the stress that shipping and initial introduction into the laboratory environment places on the animal. We here report a case of postshipping stress behavior, intramantle inking, first observed in several animals being used in our laboratory investigations but not yet documented in the literature. We also report a subsequent incidence of intramantle inking in a postsurgical animal.

Animal care and husbandry.

The mesoscale (700 L) recirculating seawater system used is similar to those described previously^2,16 (Figure 1) is capable of housing up to 12 live octopodids at a time, each in its own 5-gal aquarium. Animals have been successfully maintained in this system over the past 4 y. Four wild-caught Octopus bimaculoides were received by one of us (HB) on 26 January 2010. These animals were caught by Aquatic Research Consultants (San Pedro, CA) and shipped overnight by air on a commercial carrier. According to our usual animal handling practice, the plastic bags containing the octopuses were removed gently from their foam insulated shipping container, and the animals, still within the plastic bags, were placed into individual aquaria for 1 h to allow for temperature equilibrium. Before the octopuses were released into the aquaria, the salinity of both the shipping water and the water in the recirculating system were measured with a refractometer and found to be identical. Octopuses were released into the aquaria without visible incident.

Figure 1.

The recirculating seawater system in use on the Illinois College campus.

Case Report

Stress behavior.

The stress behavior reported here did not begin until the octopuses had been free of their bags and in their individual aquaria for approximately 4 h. Three of the 4 animals exhibited stress behavior, which occurred repeatedly for as long as 6 h after it began (as long as 10 h after release). At the onset of the behavior, the octopuses became very dark in coloration and began to writhe and curl the tips of all of their arms. Next, sucker activity and respiration ceased simultaneously and instantly. Each of the animals then stiffened, as if in rigor mortis, and floated (head down) at the surface of the water, appearing dead (Figure 2). Gross observation of the animals immediately after removal from the aquarium revealed that the siphon and mantle were closed tightly, causing the mantle cavity to be effectively sealed from the aquarium water. Gentle probing of the funnel and mantle apertures revealed a substantial quantity of ink inside the mantle cavity (Figure 3).

Figure 2.

The animal is in ‘rigor’ and floating upside-down in its tank. Scale bar, 1 cm.

Figure 3.

Ink in the mantle cavity.

Recovery efforts.

Because octopus ink has been reported to be self-toxic to the animals themselves if they are in contact with it for a long period of time^10,15 and can harm the animals’ ctenidia,¹¹ efforts were undertaken promptly to remove the ink from the mantle cavity and to restore respiration. Once the abnormal behavior had begun, the octopus was removed from the recirculating seawater system and placed into an aquarium containing clean, aerated seawater.

The mantle musculature was massaged and compressed gently and rhythmically in an effort to stimulate respiration. Once the mantle was open, a piece of flexible tubing was used as a siphon to suction the ink from the mantle cavity (Figure 4). After all of the ink was removed, clean, fresh seawater was used to flush the mantle cavity until spontaneous respiration commenced. This procedure was suggested previously³ to assist octopuses recovering from the after-effects of narcotization and seems to have been efficacious as used here.

Figure 4.

Rinsing ink (arrow) out of the mantle cavity. Scale bar, 1 cm.

The entire behavior, from rigor to the resumption of respiration, lasted as briefly as 4 min to as long as 9 min. We successfully revived all 3 octopuses. These animals were observed for several hours afterward and remained quiescent for 2 to 6 h. The behavior was not repeated, and none of the animals that exhibited the behavior seemed to have any lasting effects. They all accepted and ate food and behaved in the same way as all of our previously held octopuses.

Intramantle inking and death.

One of the 3 octopuses was narcotized in a 2% ethanol in seawater solution for a surgical procedure on 23 February 2010. After 2 min of immersion in this solution, the octopus was limp and flaccid, and the second right arm was removed for use in a long-term regeneration study. The octopus was removed from the narcotizing solution after a total of 8 min and placed into clean, oxygenated water until activity resumed. Recovery was unremarkable, and the animal was returned to its tank without incident. It was observed periodically for 8 h after surgery and exhibited normal behavior. The next morning (24 February 2010), it was found dead in its tank. Because the death occurred at night (during the dark cycle of laboratory lighting), no direct observations of any predeath behavior were made. On removal of the carcass from the tank, ink was observed oozing from the mantle. An immediate necropsy revealed that ink was indeed in the mantle and was concentrated around the ctenidia (Figure 5). We cannot be sure how long the ink remained in contact with the ctenidia before the octopus died.

Figure 5.

Necropsy of octopus. Arrow indicates ink concentrated around the ctenidia. Scale bar, 1 cm.

The precise role of the ink in the death of this octopus is unknown. The existing literature is paradoxical with respect to the action of the ink. On release, the ink can assume the form of a diffuse cloud or even as a pseudomorph, taking on the shape of the cephalopod itself. Either form acts as a visual decoy that startles potential predators, allowing the cephalopod to escape.^5,7 Chemicals in ink are hypothesized to be either noxious or toxic to other animals, but little experimental or biochemical evidence addresses this.^5,12

Although ink is known to deter predators, the composition and effects of chemical compounds in the ink are poorly understood. The ink of octopuses and other cephalopods is composed primarily of mucus and melanin and may contain other biochemical compounds, such as carotenoids,⁶ precursors to L-dopa,⁹ high concentrations of tyrosinase,¹³ and high concentrations of uracil and uradine.⁸ These compounds are known to kill several cell lines and have antimicrobial properties.¹³ In social cephalopods such as squid and cuttlefish, dopa and dopamine may serve as alarm signals secreted when conditions are dangerous. The effect of these compounds on octopuses (which are largely solitary animals) is unknown. A careful and thorough biochemical analysis of octopus ink would be an important contribution to the literature.

One author¹⁵ asserts that ink is toxic to the octopus that produces it and that octopuses in captivity should be removed from tanks in which ink is present. Another author¹ is careful to repackage giant Pacific octopuses into clean bags for shipping if ink is released during packing. However, the absence of published reports of empirical studies of ink autotoxicity in the literature makes it difficult to support or refute this claim. One group¹¹ cautions against leaving cephalopods (specifically, cuttlefishes) in an inky tank but refute the notion that ink is toxic to the cuttlefishes that produce it. These authors hypothesize instead that ink can foul the ctenidia, leading to suffocation and death.¹¹ Although our evidence must be considered anecdotal (n = 1), the observation of ink concentrated around the ctenidia of the deceased octopus suggests suffocation¹¹ more than toxicity.¹⁵

Conclusion

Intramantle inking and distributed rigor mortis is reported here as an abnormal activity presumably associated with shipping or other stress. Although this behavior is dramatic and potentially fatal, immediate resuscitation can reverse the symptoms and return the animal to a healthy state. Researchers using shipped octopods are cautioned to observe sfor several hours and to be prepared to respond to this behavior as necessary.