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Squid Hatchlings React to Changes in Light Levels

Hi everyone! The summer is finally coming to a close and I am excited to share all that I have learned from my time as a Backyard Brains fellow with you. If you’ve been here since the beginning, thank you so much for following along with me and my squiddos for the past ten weeks and if not, feel free to check out my previous two blog posts to see how we got to this point.

If you missed them… my first post Behavioral Study of Baby Squids and my second post Recording the Behavior of Squid Hatchlings

Honestly what even happened to me this summer

At the end of my last blog, I had finally gotten a recording box setup that worked well for me, and software that successfully tracked the squid over time. I was pondering what I wanted my final experiment with the squid to be and what I would test them for as they developed. I think that the most interesting part of the project was to be able to relate my experiments to actual conditions that the squid would experience in the ocean so that we could reveal something about the early lives of longfin inshore squid.

With that in mind, I first asked a very simple question. Do the squid prefer to be at the surface of the water or further down? In order to test this, I first put LEDs above the squid in the experimental tank and set them to produce very low light, around 100 lux. I found that the squid stayed right up at the top of the tank, and appeared very attracted to the light. Next, I ordered a really intense flashlight off of Amazon which according to the product description can be used for ‘calamity search and rescue.’ Super hardcore. I proceeded to blind all my intern friends and abuse the strobe function on the flashlight before getting down to experimenting.

Sunlight on a very bright day is around 120,000 lux, which is around the intensity of my flashlight. By placing it on top of the squid tank, I mimicked the experience that the squid would have should they swim right at the surface of the water. What I saw was pretty impressive; the squid dropped dramatically in the water and hung around the bottom until the light was turned off. This was especially interesting to me, because as you might have read in my previous post, squid are strongly negatively geotaxic. This means that they prefer not to be low in the column of water if they can help it, so seeing this reaction must mean that the squid have a very strong instinct to stay away from bright sunlight.


I saw this same behavior across different ages of squid and I believe that it is a pretty general response. While this was an interesting and informative experiment, it seems a little broad. We’ve learned that squid of all ages don’t like SUPER SUPER bright light and they do like SUPER SUPER dim light, but what about the kinds of light in between?

This is where my idea to film the squid moving right to left came into play. I became very obsessive about checking my eggs every hour to see if they had hatched, so I could be sure to test the squid under different conditions in their very first hours of life. I tested each group with 100 lux, 600 lux and 1,200 lux light shining from the right. I first tested the groups within 24 hours of hatching, and tested them again when they were 48 hours old.

The first result was similar to what I learned from the surface-light experiment which was that the squid are attracted to 100 lux light. In both age groups, there was a strong movement towards the LED as soon as it turned on, and clustering around the light for the entire duration of the video.

The result of the next experiment was totally different. Under 600 lux light, the newly hatched squid had a totally random response and were not attracted to the light source at all. After two days, the same group of squid would respond very strongly to the light and move right towards it.


Under 1,200 lux light, the young squid also did not respond and the older squid were somewhat attracted to the light, but less than they were to the 600 lux light.

So what does all of this mean??? We can only guess. It is apparent that in all age groups, squid are attracted to conditions that are dimly lit, probably because this is a safe location for avoiding predators. The differentiation of response is more interesting, however. In order to understand why younger squid are more repelled by bright light, we can consider the early migration patterns of the squid. While they are born at the coastal shelf of the ocean, the squid quickly move out towards the open ocean within their first few days of life.

As you can see in the diagram, there is a significant difference between how the light penetrates water on the coastal shelf and how it penetrates water in the open ocean. When the squid are born in the murky waters off the coast, even moderately strong light probably signals to them that they are dangerously close to the surface of the water. A few days later when they move to the open ocean. light penetrates more deeply, so moderately strong light probably still represents a safe distance from the surface.

Although these hypotheses could be totally wrong, the behavioral development that is apparent here is an extremely interesting model of infant reflexes and their changes over time. In human babies, we see strong behaviors such as the rooting reflex that allow infants to survive, which go away with time. This same general concept is being seen in the squid hatchlings, and we could perhaps work to study the mechanisms for how these instincts develop genetically in an organism. NEW PROJECT DIRECTION!??

And, if you missed it, I made and presented a poster! Check it out here if you’re interested in a more formal presentation of my results:

Anyway, this is almost the end of my last blog as a BYB fellow and if you want to peace out before things get sappy/ grateful/nostalgic, now would be the time 😉

 

Are we happy to be done filming or are we still arguing about the merits of subplot(‘Position’) vs subsubplot??

BUT ANYWAY I am so grateful to have had the opportunity to work at Backyard Brains this summer and for all the amazing experiences I’ve had here. Infinite thanks to Greg for taking the time to work with and teach us so much this summer and for his dedication to broadening the availability of neuroscience education. I will miss your excellent renditions of the entire soundtrack from The Book of Mormon and being your (unconfirmed) favorite intern.

Thank you also to the entire staff at Backyard Brains (looking at you Stan, Will and Zach) for being there every time I got stuck or frustrated this summer and providing helpful suggestions and thumbs ups. Same to my fellow interns. You’re all like siblings to me and I am so grateful to have gotten to know such a quality group of people; I know you’ll all do amazing things in life. The open-apartment Friday night policy applies forever please come visit me.

TO CONCLUDE this summer has been incredible. It has really deepened my passion for neuroscience and experimentation, and introduced me to the satisfaction of DIY science. I can only hope that the rest of my career involves so much gratifying research and such a wonderful community of scientists and makers. TO THE NEUROREVOLUTION!!

 


Recording the Behavior of Squid Hatchlings

Introduction to the project

Hi, everyone! Last week marked the halfway point of my time as a fellow here at Backyard Brains!  Recently, I’ve succeeded in building a rig and recording video footage of my Squid Hatchlings! I’m excited because it means I can start gathering quantifiable data! The squiddos have kept me pretty busy during these weeks, and they have been so fascinating to study (and photograph obsessively, like the proud parent that I am).

The main goal of my summer project is to document and begin to understand the behavior of squid hatchlings. Mostly I have been investigating how they complete the complex task of navigating away from where they’re born to seek prey and space to grow up. From previous studies, we suspect that they use their vision to help them navigate because they tend to swim towards sources of light, especially when they are very young. Since we already know a lot about what kind of light exists at the different depths, being able to specifically quantify the strength of their preference for different colors and intensities of light would help us understand where they like to be in the ocean at different ages.

In order to study how their light preferences change over time, I first had to figure out how to separate the eggs into individual containers that could still be aerated from a single pump. The containers needed to be able to let in oxygenated water, but keep hatchlings inside once they were born. The solution was a series of seven water bottle covered in tiny pinholes which rest in a bucket of water. As long as I check them regularly, I always know the age of a hatchling in any given bottle.

Home sweet home.

Next, I needed to write the computer software that could track the squid when I filmed them reacting to different environmental conditions. Since my camera and the background of the tank are stationary when I film, the solution was pretty simple. I break down the video into frames and find the average image over time to figure out what the background looks like. Then, I remove the background from each frame, apply some filters and am left with an image of just the squid! This method is called foreground detection and it is a commonly used method in image processing and computer vision. Once I have this simplified image, I can calculate the population of squid that are in each segment of their tank and see how they move over time. (Click the Gif’s to see the sweet action!)

 

Next, I needed to build an experimental setup in order to actually test the squid with variable conditions. After several failed attempts, I ended up with an acrylic box covered in black paper that has a hole for the iPhone to take a video through. This way, the only light that the squid are exposed to while I record them is the light that I produce and control. At first, I wrongly assumed that I should be testing squid with light from the top (since the sun is above them in their natural environment), and was getting very… boring results. Besides their sense of light, the squid also have a sense of gravity and are negatively geotactic, meaning that they like to stay at the surface of the water. So the squid in the dark would be at the surface of the water and I’d turn on a light and they’d just… stay at the surface. I wasn’t sure how to quantify this and I knew that these weren’t the results I wanted, since there was no differentiation of their reactions. I felt exactly like this:

My exact face after four hours of attempting to get results… I brought them outside the lab to see if the fresh air would help them? I just got soaked in the rain and still saw nothing interesting…

Finally, I had the idea to put the LEDs on the sides of the tank to test their reactions. I wouldn’t be forcing them to swim away from the surface, but I could hopefully still see them track the light. It worked! I could finally see the squid making an active effort to follow the light and can quantify their reactions to it.

Now I am in the process of designing the exact experiment that I want to study consistently across all groups of squid as they age. While I have been exploring and contemplating, I’ve seen some pretty cool effects. It seems that young squid (within 24 hours of hatching) react extremely strongly to low light levels (around 100 lux) and much less strongly to very high light levels (around 600 lux).

Two recordings from samples at <12 hours since hatching. The x-axis here represents the region of the tank where section 1 is the far left (in this case, closest to the ligt) and section 7 is the far right. The y-axis shows the average percentage of the population that can be found in the section over the course of a 2-minute video.

In testing some older squid, I’ve also observed that they don’t seem to react to red light. Perhaps they don’t see it at all, at the least they don’t care about it. It’s not conclusive yet, and I’d like to test more groups, but you can see the graph of their reaction below.

I’ve also seen that the older squid seem to react strongly to very strong intensities of light, especially compared to the younger squid. This might provide some interesting insight into the life of the squid and where they live in the ocean at different ages.

I think that that is all for now! After recording some more data I hope to have some awesome graphs to share with you that show how the squid react to low light, high light, and colored light as they age! With that information, hopefully we will be able to piece together the narrative of the early life of the loligo pealeii and improve our understanding of their abilities and behaviors.


Studying the Behavior of Baby Squids!

Hi everyone! My name is Christy and I will be a junior at Rice University this coming Fall. I am so excited and grateful to be part of the Backyard Brains fellowship program for this summer and will be studying longfin inshore squid hatchlings for my research project.

My Favorite People and my Favorite Place

At school, I am majoring in Computer Science and Cognitive Science and minoring in Neuroscience. I am specifically interested in the fields of computational neuroscience and artificial intelligence and I’ve really, really loved the classes I’ve taken in those topics so far. In my free time, I enjoy long walks on campus… as a tour guide!  I also perform with my school dance team and work at the McNair Center where I write classifier algorithms to identify startups and accelerators.

A Scenic Ann Arbor Destination

I have really enjoyed my time in Ann Arbor! It has been great meeting the other interns and exploring the city. So far, I’ve eaten a ton of excellent food (ZINGERMANS!), gotten lost in the Dawn Treader Bookstore, visited Lake Erie, willingly touched a cockroach for the first time in my life, and discovered “The Most Well Stocked Walgreens in the History of Time.”

As soon as I arrived here at Backyard Brains, a shipment of squid eggs had already arrived for me, so I hit the ground running! I mixed and aerated some seawater and placed the eggs in their new home. I’ve never worked with any sort of marine creatures before, so I was definitely worried that they were in the wrong hands! Fortunately, my brilliant advisor Eric Edsinger has been helping me remotely, and the squid babies hatched this Tuesday. I became the proud mother of a ton of squiddos (like kiddos, but squid).

Since then, I’ve been scrambling to capture quality videos of my squid swimming so that I can train computer software to track their movements! Once I have a program up and running, I will be receiving another shipment of squid and tracking them as they react to different environmental conditions like strong light, temperature fluctuations, and water movement. Very little is known about how these hatchlings behave, and I hope that by recording information about them, I can contribute to our understanding of these fascinating creatures.

My squid live in aerated buckets of saltwater, but for recording, I needed to come up with a simple, DIY transparent container. So, for now, the tiny squid are swimming around in Tic-Tac containers! (By the end of the summer, I suspect the other interns and I will be very sick of eating Tic-Tacs.) Unfortunately, the squid like to hide in the beveled edges of the container that the camera can’t capture properly, so today I built some blockers to stop them from entering that region. I also constructed a little lightbox to film them from with random materials I found in our makerspace and started filming! I got some images that look like this:

I think my squiddos are pretty cute, but I’m biased of course #momthings

The next step is turning them into images that look something like this:

Negative Image of Squid for Easier Computer Vision Tracking

Using edge detection and high-pass filters, I can track how the population reacts to different stimuli.

I am enjoying building my experimental setup, and I am excited to get started with my experiments!. I hope to produce meaningful results this summer! By recording and analyzing the squid’s behavior in response to certain stimuli, I can begin to hypothesize why the squid behave in such a way, thus prompting further scientific experiments! I feel so lucky to have the opportunity to spend my summer learning and studying at Backyard Brains and can’t wait to update the blog soon!