The ability to recognize a reflection in a mirror as an image of self is considered a hallmark of cognition in animals. And the classic way of testing this self-awareness involves passing the ‘mark’ test. This test, also known as the mirror self-recognition (MSR) test, involves marking an individual animal in a place on the body that is only visible when the individual sees it in a mirror reflection. If the animal then investigates or touches the mark, it is considered to have demonstrated that it recognizes the image in the mirror as belonging to itself.
Very few species have passed the mark test. These include chimpanzees and other great apes, a single Asian elephant, some rays, dolphins, orcas and Eurasian magpies. A 2019 research paper that claimed cleaner wrasses (Labroides dimidiatus) also passed the mark test was criticized by some members of the scientific community, including the test’s inventor, American psychologist Gordon G. Gallup. In response, the authors of the 2019 paper have undertaken new and more extensive experiments with cleaner wrasses to determine the validity of the criticisms.
The new research, published today in the journal PLOS Biology, was led by Professor Masanori Kohda from the Graduate School of Science, Osaka City University. It tackled the methodological criticisms levelled at the initial experiments and has provided further evidence to suggest the fish do have the capacity for MSR.
One of the criticisms related to the small sample of cleaner wrasses used in the mark test. “Previously, using a brown marking on the throat area of L. dimidiatus, we had shown three out of four cleaner fish to scrape their throats several times after swimming in front of a mirror, a number on par with similar studies done on other animals like elephants, dolphins, and magpies,” explained Professor Kohda.
However, in response to the issue of small sample size, the researchers managed to increase the number of fish in the study to 18. They teamed up with scientists from the Max Planck Institute of Animal Behavior in Germany, and the University of Neuchâtel in Switzerland, and observed that 17 of the 18 cleaner fish (94 percent) demonstrated the same scraping behavior that was recorded for fish that had been marked in the 2019 study.
The critics of the initial study had also questioned the researchers’ use of a brown mark on the throat of the cleaner wrasses, when the classic mark test employs a bright red spot or sticker.
“After looking at similar studies done on monkeys, pigs, dogs, cats, etc. that clearly tested negative, we wondered if the reason these animals didn’t attend to the mark was because it didn’t represent something in their natural environment that they would be concerned about,” said Professor Kohda. “In our previous study, we used a brown mark as it may look like a small parasite that is a main food source for L. dimidiatus.”
At the time, critics had asserted that the physical sensation of the mark, together with seeing the brown mark in the mirror, may possibly have triggered behavior that didn’t conclusively suggest MSR. To address this, the team now tested how the fish would respond to a physical stimulus in their throat by injecting the brown mark 3 mm deep (as opposed to 1mm). At such a depth the mark was hardly visible, yet they found that fish with the deeper insertion scraped their throat at similar rates, whether a mirror was absent or present.
To further support their choice of a brown mark that was ecologically relevant to the cleaner fish, the team tested fish with green or blue marks and found that no fish injected with green or blue marks demonstrated the scraping behavior.
A further criticism of the initial research was that the experiments had not proven that L. dimidiatus recognized the mirror image as itself and not another fish. When training a fish to look at the image of itself in a mirror, three steps are involved. Initially, when faced with a mirror image, a fish will express aggressive behavior, probably because it perceives the image as another fish. Then, after some time, fish presented with the image in the mirror show some unnatural behavior but are not aggressive as they realize that there is no other fish associated with the mirror. And lastly the fish repeatedly looks at itself in the mirror, with no aggression. At this stage, MSR is possible as the fish can look intently at the image and can potentially see the mark and then respond by going to scrape it off.
“Our previous study demonstrated MSR in L. dimidiatus,” said Professor Kohda, “however studies with other animals have shown that simply moving a mirror reignites aggressive behavior, suggesting the animal has only learned a spatial contingency, not MSR.” To address this, the team transferred mirror-trained cleaner fish to a tank with a mirror on one side of the tank and then, three days later, to a different tank with a mirror on the other side. They found that the fish showed no aggression towards their mirror image in either of the tanks.
To further support the conclusion that the L. dimidiatus are actually capable of recognizing themselves during the mark test, the researchers placed mirror-trained fish in adjacent tanks that were separated by transparent glass. After two to three days, when fish largely reduced their aggressive behavior towards each other, they were all marked with a brown mark the following night. When they were exposed to the fish in the adjacent tank for 120 minutes the following morning, none of them scraped their throat.
“This result suggests that a visual ecologically relevant stimulus on another fish is not enough to induce throat scraping on marked subjects,” said Professor Kohda. The evidence arising from these new experiments supports the conclusion of the 2019 study that found cleaner wrasses show behavioral responses that can be interpreted as passing the mark (or mirror) test and could therefore be attributed with a sense of self-awareness.
“We still have much work to be done, especially quantitatively, to show that fish, as well as other animals have the capacity for MSR, however as a result of this study we reiterate the conclusion of our previous study that either self-awareness in animals or the validity of the mirror test needs to be revised,” concluded Professor Kohda.