Pivotal octopus, ocean acidity study attracts international media

Collaboration is key

They both earned master’s degrees in biology from Walla Walla University, Trueblood in 2002, six years ahead of Onthank who was undergraduate at the time. The two never crossed paths, however, until Trueblood was three or four years in his doctoral studies at the University of Rhode Island, around 2006. “I was having a hard time doing some data management, and I called my professor at Walla Walla for ideas and he’s like, ‘I don't know how to do that but there’s this kid here [who] probably does. And so I got on the horn with Kirt and he helped me develop this pivot table thing to do some data management stuff,” said Trueblood.

Later on, as Onthank was pursing doctoral research on eye lenses for various cephalopods as a means of tracking their life events, he contacted Trueblood in search of eyeball specimens to analyze from the cephalopod species Trueblood had been studying. Trueblood made arrangements for the requested material to be mailed to Onthank. “Since then we just kind of kept track of each other,” said Trueblood.

Onthank earned a doctorate in 2013 from Washington State University and Trueblood received his from the University of Rhode Island in 2010. Onthank had worked with octopuses during undergraduate and graduate training at Walla Walla and currently studies marine invertebrate physiology, particularly cephalopods such as octopus and squid in the Onthank Physiology Lab. He viewed the study of the impacts of ocean acidification “as kind of the big pressing question about what’s going to happen to octopuses going forward,” he said.

Similarly, Trueblood had worked with a research group during his doctoral program that had pursued “ground floor” studies on the physiological effects of ocean acidification on squid, a problematic effort due to the squid’s inability to handle being inside of a tank for long periods. “And so we were both kind of starting out our labs and Kirt at one point was like, ‘hey man, I'm interested in doing this acidification stuff on octopus, what are your thoughts,’ and we just kind of started talking from there and comparing notes and ideas,” Trueblood said. He was intrigued by the opportunity of studying octopus in part because of the creature’s amenability to life in a lab tank.

The scientists initiated the five-week study of Octopus rubescens in the summer of 2014. They harvested the ruby octopuses from Washington’s Puget Sound where a naturally low pH level results in continuously high water acidity that is on par with predictions for ocean acidic levels this century. “We basically have a future test ground,” said Trueblood.Puget Sound, a shallow inlet of the Pacific Ocean along the northwestern coast of Washington State, incurs an upwelling of deeper ocean water washing in at its mouth that is higher in acidity than typical ocean water. Additionally, through a phenomenon called the biological curve, carbon dioxide is deposited into deeper water when phytoplankton, which normally live in light-infused water near the surface, die and transport CO2 to the bottom of the deep ocean where the phytoplankton are consumed by bacteria and other things which likewise respire out CO2. Because of the sound’s comparative shallowness and lack of direct exposure to the open sea, the deep water and its CO2 deposits mixes with the surface water leading to an accumulation and mixing of carbon dioxide deposits throughout the water levels of Puget Sound.

Mother of invention

Substantive, long-term scientific study of the effects of ocean acidity can be cost-prohibitive. Scientists who are short on budget and big on desire to test hypotheses must invent ways to achieve their goals. To conduct a longer-range study of the ruby octopus, the scientists needed a way to control pH and CO2 levels and temperatures in specially formulated saltwater tanks, one tank per octopus. Using inexpensive Arduino computer electronics boards and grant funding, Onthank designed systems for multiple tanks that would precisely control functions for the five-week analysis.

Meanwhile, Trueblood used equipment he had previously built for research on another project to conduct the short-term study of the ruby octopus. He tossed a variety of technical gear, optodes, electrodes, waterbaths, respirmeters and other equipment, into his car and drove north to Washington. “So that way we were able to get a short-term-long-term data set because we were pooling our resources …and we could end up with this big study,” said Trueblood.

The long-term study was particularly important as prior ocean acidification effect studies had all focused on shorter periods of exposure, Onthank said. “That doesn’t really replicate what’s going to happen down the road. [It’s] another way to be showing do these short-term experiments really reflect what we would actually see for a long-term chronic problem that's actually going to be occurring in nature or are you seeing more of a stress response in these short-term studies,” he said.

“We're still trying to figure out the mechanism, right, we see this big spike in routine metabolic rate, and then it comes right back down,” added Trueblood. “And, you know, there’s a couple different areas that were suspect of that spike, and this is in part why my students are trying to do blood samples now. …it’s a stress response but we are still searching for the mechanisms that drive it.”

To avoid investing in costly equipment for analyzing blood samples, Trueblood is again turning to invention by using a 3D printer to fabricate a small instrument that holds a very small sample of blood. The blood samples are placed in a machine called a spectrophotometer that shines a light through the blood. The level of oxygen in the blood is determined based on the color. “Octopus blood goes from blue to clear, so measuring how blue it is telling us how much oxygen is there,” he said.

Building blocks

The scientists’ labs and their students have continued to build upon the findings of the five-week study from 2014. Onthank’s lab, which typically involves three graduate student researchers focused on octopus ocean acidification projects, has pursued several studies and made additional findings: a refined study in 2015 indicated that an increase in temperature combined with an increase in carbon dioxide resulted in a worsened metabolic rate for the octopus over five weeks; and a study of the octopus’ immune system after exposure to increased CO2 showed an elevation after three weeks of hemocytes, which are equivalent to human white blood cells. “Their immune system is being activated and turned up with long-term exposure,” Onthank said.

“And now we're looking at another thing octopuses and cephalopods do, this really crazy thing where they edit their RNA,” he said. “So what's happening here is essentially all life [has] DNA. The DNA gets turned into RNA, and then the RNA gets turned into proteins and annotated along the way,” a process akin to making ingredient changes on a recipe card in making cake. “It’s something that essentially all animals can do, but they don’t really use it all. Whereas octopus and squid use it a ton,” affecting upwards of 60% of all their genes, said Onthank. “I'm aggressively right now looking to see does the way that they edit their RNA change with high CO2 and it actually looks like they do, it looks like we found a few 100 genes that the editing on those genes changes depending on if they're [exposed to] high or low CO2.”

Just 10 minutes. That’s how long Trueblood’s student researchers have to extract a drop of blood using a tiny syringe to reach a vessel the size of angel hair pasta hidden inside of a small, slippery octopus. It is a difficult procedure, but critical along with work in Onthank’s lab to furthering the scientists’ groundbreaking research.

La Sierra University undergraduates Shannon Grewal and Danny Bazan are the latest students to make blood extraction attempts, currently working with three ruby octopus specimens that arrived to Trueblood’s lab via FedEx on Jan. 13. The students follow strict European-standard animal welfare protocol which involves first anesthetizing the creatures and minimizing their time out of their specially formulated salt water tanks. Trueblood traveled to WWU’s Rosario Beach Marine Laboratory in Washington State in January to work remotely and begin a research sabbatical in March. He guides his students in their blood-drawing efforts from afar via Zoom video conferencing. Over the past year or so students have been attempting to draw blood from octopuses gathered from various locations along the coasts of California and Washington toward assessing blood oxygen levels. Over the years, only a handful of useable blood samples have been collected with only one successful sample collected by his two students thus far, Trueblood said. “That’s half the challenge. Getting blood [from] intact animals is incredibly difficult.”

The octopuses currently residing in Trueblood’s lab were mailed in a special aquarium box in January and arrived 18 hours after being extracted from the Pacific Ocean along California’s central coast by a permitted marine collector. The small cephalopod species typically weighs between 3.5 and 5.3 ounces and has an arm length of roughly 12 to 16 inches.

"I’m so grateful I had a chance to experience this intertwining of the practical and theoretical and I get to really be a part of something that’s going to affect a lot of people for generations to come." Danny Bazan, junior neuroscience major

The blood-drawing process involves one student holding up the octopus to reveal an opening in its mantle and gently extending a gill with tweezers while another student strives to insert the needle into the gill. If this doesn’t work, the students aim for either a cephalic vein next to the systemic heart (octopuses have three hearts) deep inside the octopus’ mantle which houses its organs, or from an artery in one of the octopus’s arms. They study octopus anatomy beforehand and look at references as a guide while they attempt the extraction. “You have to be very sensitive to the feeling of where everything is,” Bazan said. They check for the correct needle gauge, or hole size in the needle’s end and make certain there is no pressure inside the needle. “So there’s a lot of different variables so that even if everything goes perfect, one of those variables can kind of offset us so we don’t get any blood,” he said. “The hardest part is that you can’t see any of these [veins or arteries].”

He continued, “We have a very limited window of opportunity to draw blood because we only have them out [of their tank] for so long. …It’s extremely difficult and frustrating, but on our first try, on the [January] 25th, we were able to get blood.” About half a milliliter was withdrawn. “A few more of those and we should have enough to do some research,” Bazan said.

Trying to draw blood for research is difficult but fun, said senior biomedical sciences major Grewal. “It’s a puzzle you have to solve. There aren’t a lot of people who extract blood from octopuses, so it’s kind of a surprise every time. It’s quite an adventure.” 

Grewal aims for a career in dentistry and will attend the University of Michigan School of Dentistry this fall. She believes her work in the biology lab has increased her critical thinking and problem-solving skills and fine-tuned her manual dexterity. “This lab has taught me skills that will help me succeed in dental school and my future career,” she said.

Bazan, who will travel to join Trueblood at the Rosario Beach Marine Lab later this year studied in virology and neuroscience labs before being accepted into Trueblood’s marine biology lab. He became interested in his present career path after participating in the SEA-PHAGES virology lab his freshman year. Working with live animals has been an eye-opening experience.

“I feel as though there’s a different type of responsibility,” Bazan said, “So for me as a budding researcher and hopefully as a physician scientist, that directly translates to not only practical skills, but as well the patience and the intentionality of treatment. I think that has really opened my eyes to a different way of doing research. I’m so grateful I had a chance to experience this intertwining of the practical and theoretical and I get to really be a part of something that’s going to affect a lot of people for generations to come.”