Investigating acantharians with imaging and sequencing

Acantharians are small, single-celled organisms that can be found in ocean water all across the planet. Despite being small, or maybe because they are small, they are incredibly beautiful—acantharians are perfectly symmetrical and at first glance they remind many people of tiny crystal stars or snowflakes. Their exquisite shapes have attracted scientists since the 1800s, when Ernst Haeckel, a famous biologist and science illustrator, completed several studies on their diversity and structure (he was part of the famous Challenger expedition from 1872-1876 and one of the best sources for looking up acantharians is still his “Report on the Radiolaria Collected by H.M.S. Challenger”). In addition to their eye-catching morphology, acantharians can play large roles in important elemental cycles occurring in the oceans, such as the carbon cycle. This is, in part, thanks to the microalgae that some acantharians can keep within their cytoplasm. Many acantharians host algal endosymbionts from the Haptophyte genera Phaeocystis and Chrysochromulina. These tiny servants actively photosynthesize within acantharian hosts and share some of their sun-fueled organic carbon with the acantharians in return for nutrients, like nitrogen, that they need to keep their photosynthetic machinery in good working condition. The fancy acantharian skeletons are very heavy, though, and can cause acantharians to sink very rapidly. Sinking acantharians effectively transport the carbon that was photosynthetically fixed by algal symbionts down into the deep ocean. This carbon may eventually make its way back to the atmosphere, but the transport of photosynthetically fixed carbon to the deep ocean is an important process for removing CO2 from the atmosphere and regulating climate.  

Example of acantharians drawn and classified by Ernst Haeckel (image credit: Wikimedia Commons). The beautifully symmetric skeletons are made of Strontium Sulfate (celestite) crystals secreted by the acantharians.

Despite catching the eyes of many, acantharians are still rather mysterious in many ways. This is primarily because they are not easily collected using traditional plankton sampling methods (e.g. nets or sediment traps) and because they will not stay alive in the lab. The delicate acantharian skeleton often breaks in plankton nets and also dissolves in the chemicals used to preserve plankton samples. As a result, it is difficult to get a full understanding of acantharian abundance and distribution in varied locations. Since acantharians do not survive in laboratory conditions, it has been impossible to observe many aspects of their biology, such as how they eat or how they reproduce. In the first chapter of my PhD thesis (now published as an open-access article in Limnology & Oceanography: https://aslopubs.onlinelibrary.wiley.com/doi/full/10.1002/lno.11567), I brought together two more modern technologies to try to answer questions about acantharian abundance, distribution, and biology. DNA metabarcoding involves extracting DNA from all the cells in a water sample and then copying and sequencing a small segment of DNA that is present but variable in all organisms. DNA metabarcoding is an amazing tool for assessing what organisms are present in a sample, but it can only tell you what proportion of your sample each organism makes up and can never provide information about how many actual cells of each type were in the sample. High throughput in-situ imaging takes pictures of cells in their natural environment and allows the abundance of different cell types to be quantified. But, imaging cannot differentiate between organisms that look very similarly but are genetically different. In my study, I hoped to assess how well the results from DNA metabarcoding and in-situ imaging lined up for acantharians and learn more about how abundant acantharians are at different depths in the western North Pacific (where Okinawa is located). I hoped the results would inform hypotheses about important aspects of acantharian biology. 

Acantharians imaged with high throughput in situ imaging during this research project.

To collect the samples and images that I needed for this project, I was lucky to join a research cruise led by the Japan Agency for Marine Earth Science and Technology (JAMSTEC) on their research vessel the R/V Mirai. The Mirai, at 128.5 m, is longer than a football field and, without a doubt, is the largest research vessel I have been on—maybe the largest ship I’ve ever been on. For comparison, the two NOAA ships on which I joined cruises during my master’s degree—the R/V Oregon 2 and the R/V Delaware 2—were only 52 and 47 m, respectively. In addition to the massive size, there were several other notable differences between cruising on the Mirai and the previous cruises I had joined. The biggest was probably the working hours. On all other cruises I joined, scientists and crew worked around the clock, taking alternating 12 or 8 hour shifts. On the Mirai, we all worked from approximately 9 am to 5 pm. This gave me time to enjoy the amenities on the Mirai, such as the large fitness room and baths. Other differences included the food (fish for breakfast everyday!) and alcohol consumption (especially at the karaoke party on the last night). In contrast, there is a very strict no-alcohol policy on NOAA vessels.

To collect water for DNA metabarcoding, a rosette of Niskin bottles was deployed at each sampling station. The bottles remain open until triggered, so that they collect water only from the specific depth at which they closed. In this case, I collected water from near the surface, at the subsurface chlorophyll maximum, from the twilight zone, and from near the seafloor. I then filtered the water through different pore-sized filters aimed at separating adult acantharians from their smaller reproductive cells. I froze the filters with liquid nitrogen (-320°F) to preserve the DNA until I could process the filters later in the lab. To collect the images, we attached a special camera to a large frame called the DEEP TOW that can be lowered through the water column. My camera was far from being the only camera attached to the DEEP TOW, which was fully kitted out with GoPros, 6k video cameras, and other plankton imaging systems, including one that takes holographic 3D images of single cells. While the DEEP TOW was traveling to the seafloor, a few researchers at a time would sit in a shipping container on deck and watch all the live video feeds—being in the dark shipping container made it feel as if you were actually in a submarine and the many video screens were windows out into the deep. Most of the time there was not much to see, but on one very exciting occasion we saw a shark swim right past the DEEP TOW. 

The R/V Mirai docked in Okinawa before setting sail for the research cruise.
The rosette of Niskin bottles going overboard to collect seawater from different depths.
The lab space where we filtered approximately 650 liters of seawater to collect cells for DNA extraction. The filtration unit is on the table on the left. In the back is the room where other researchers processed sediment cores that they took from the seafloor.
The DEEP TOW frame about the be deployed (left) and a close up of the camera I used to take plankton images (right).
Inside the dark shipping container where we watched the live feeds from the different video cameras attached to the DEEP TOW frame. We mostly did not see much, but there were a few exciting moments when we saw jellyfish and sharks.

The cruise eventually finished up in Mutsu (Aomori Prefecture), all the way at the northern tip of the main island of Japan. Mutsu was beautiful, with lots of trees in full summer foliage and clean, cool, humidity-free air. We took a train to Tokyo and then flew home to Okinawa, where I extracted DNA from all the water filters and poured over thousands and thousands of plankton images collected during the cruise. When I finally finished processing all the samples and analyzing the data, I found some interesting patterns in the relative abundance of acantharian DNA sequences and the quantities of acantharian cells captured in the images. When I looked at all the sequences classified as belonging to acantharians, the proportion they contributed to each sample increased with depth. This was puzzling because the image data clearly showed that the number of acantharian cells decreased as we went deeper in the water column. I realized that the acantharians that I counted in the images were only those that “looked” like acantharians—the ones that had star-shaped skeletons. But, there are many acantharians that we have no idea what they actually look like; we only know they exist because a bit of their DNA was sequenced in a mixed environmental sample at some point. Interestingly, if I excluded all the sequences belonging to acantharians with unknown morphology, the remaining proportion of acantharian sequences in the sample correlated with the abundance of imaged cells. This suggested to me that the acantharians without documented morphology may not look how we expect acantharians to look, which could explain why they were not counted in the images. It is also possible that they are much smaller than other acantharians (too small for the camera), or that metabarcoding data just didn’t line up well with the imaging results. I also found trends in the data that provided more clues about where different acantharians reproduce. But perhaps the coolest finding was that I saw many acantharians with a peculiar drop-shaped structure at the end of a long tentacle (technically a pseudopod). The structure immediately reminded me of the fishing lures of deep-sea fish we are all familiar with now thanks to the movie “Finding Nemo”. It is possible that this newly observed structure in acantharians is also a fishing apparatus to catch microscopic prey, but there are other possibilities, too. For example, the structure could also be related to locomotion or reproduction. Ultimately, I was able to learn more about the beautiful and mysterious acantharians by bringing together results from two very different technologies in this study. However, the study also really highlights how little we know about tiny organisms like acantharians. I am hopeful that this work will inspire more research in the future regarding acantharians, but also in thinking about how we can bring different methods together to better understand important processes in the plankton.  

Making landfall in Mutsu, Aomori Prefecture, Northern Japan.
The coastline in Mutsu.
Examples of acantharians with the interesting drop-shape structures on pseudopodial extensions. These structures may be used for feeding, or they could be for locomotion or reproduction.

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