Octopuses are solitary animals that don’t mate very often. So it was a surprise, initially, when two of them hooked up in a lab on Harvard’s campus.
“This was kind of accidental,” says Pablo Villar del Rio, a postdoctoral fellow in Harvard’s molecular and cellular biology department. “We didn’t plan to study octopus mating. We were studying chemosensation in the octopus in general.”
Villar del Rio works with professor of molecular and cellular biology Nicholas Bellono, whose laboratory focuses on the evolutionary impact of chemical and sensory mechanisms in the natural world, particularly in sea animals. The octopus liaison transpired during a study initially meant to observe how the cephalopods make sense of their environments more broadly, and the ways they rely on chemical signals to hunt food, seek shelter, and identify threats.
But the resulting paper, published in Science in April, sheds light specifically on how octopuses use those signals for sex.
“This was an unexpected observation,” Bellono says, “and it’s led to new understandings of some really important biology concepts.”
The paper details how female hormones—progesterone, in particular—help male octopuses recognize potential mates, even in the absence of other environmental signals.
“Male octopuses use a specialized arm called the hectocotylus to identify females and navigate their internal organs to reach the oviduct and deliver sperm,” the paper explains. Villar del Rio (the paper’s first author) and his collaborators discovered that one of the octopus’s eight arms, used specifically for mating and protected during activities like hunting, has receptors that are activated by female progesterone.
To confirm that, the researchers worked with pairs of wild-born California two-spot octopuses (Octopus bimaculoides), a small, hardy species native to the Pacific Ocean. “They’re pretty robust octopuses,” Bellono says. “It’s hard to have [octopuses] happy enough in a lab setting where they’re not stressed and they actually want to mate, so this species is very useful.”
During the observation, pairs were placed in a dark, shallow tank separated by an opaque barrier with a small hole in the middle. Octopuses prefer to be alone and may get violent with each other under the wrong circumstances, so controlling the environment was critical for their safety. The octopuses, however, didn’t let the separation stop them.
“We saw the male so confident going and mating with the female through a wall,” Villar del Rio says. The Science paper elaborates: “To our surprise, even with minimal visual information, the male extended the hectocotylus through the barrier and carefully maneuvered toward and subsequently inserted the specialized appendage within the mantle. After insertion, the hectocotylus extended deep within the mantle and eventually stopped.”
The pair then went still for an hour or so, during the transfer of sperm into the oviduct. The research team saw repeat results across “numerous male-female pairs,” but not in same-sex pairings, a finding that suggests the cue is female-specific.
From there, the researchers drilled down on the molecular underpinnings of the octopuses’ encounters. The hectocotylus, while similar in physiology to the male octopus’s other arms, is more specialized, with more tightly packed receptors and different metabolic responses to its surroundings.
A bait-and-switch confirmed it: when the female octopuses were replaced by “conical tubes that were coated with [progesterone]” and attached to the holes in the experiment’s original tank barriers, the male octopuses tried to mate with them anyway. Experiments with other chemical signals octopuses encounter in the wild, given off by steroid derivatives, bile acids, terpenes, or bitter molecules, didn’t elicit such behavior.
The findings build on the Bellono lab’s prior research on how octopuses use chemical receptors to move through their environments. “We first discovered and published about this receptor class in 2020, and we didn’t know at first what it did,” Bellono says. “Pablo showed [with this research] that they [have] a range of functions, including, it turns out, driving reproductive behaviors.”
“This was kind of accidental. We didn’t plan to study octopus mating.”
—PABLO VILLAR deL RIO
This insight has broad implications for biodiversity, he adds. In the study, similar but separate species of octopuses displayed parallel mating behaviors, but they would not mate with each other. This suggests the chemosensory reactions also help the octopuses mate with their own species, though how that works is still unclear. “Specifically, we don’t know what type of progesterone allows females of different species to tell apart their mating partners,” Villar del Rio says.
Moving forward, Villar del Rio would like to use the lab’s research on chemosensation as a basis to further investigate octopus reproduction, many aspects of which are still a mystery. Two-spot octopuses live for about two years, and females mate multiple times during that period, carrying sperm packages of different mates. Once fertilization is triggered and the female lays and nurtures eggs, she dies.
“There is one event that triggers the fertilization of eggs and possibly the competition between sperm cells from different males in the female. And that is completely unknown,” Villar del Rio says. “We don’t know how it works.”
