Reefing After Dark Part 2: Bioluminescence Observations & Research

Collage of bioluminescent displays that I have captured on camera.

Initial Observations
I started watching the 150-gallon refugium tank as that is where I first sighted bioluminescence in my aquarium system. The brittle starfish and isopods that I observed producing bioluminescent light were showing different displays such as constantly glowing or producing a reactive burst of light when disturbed. I also observed some random bioluminescent displays in the 150-gallon refugium tank, that were coming from organisms that could not be identified. The main reason for not being able to identify these organisms is that I never got to see them. I am not sure if they were simply moving through the water column or if they had retreated into a hiding place before I could turn on a light on to see what kind of organism was present. It was also observed in the sump tanks that vibrations from knocking on the side of the sump would set off some bioluminescent responses, but I was unable to identify these organisms. These initial observations made me want to learn more about this amazing phenomenon.



Pictured Top: The reactive burst of light from a brittle starfish. Pictured Bottom: A single Isopod in a constant glow surrounded by other Isopods backlit with a red light both photos taken in November 2017

Challenges
At the start of 2018, I had shifted my focus on the aquarium system towards the stocking of fish and corals. I was also going through an algae bloom that sometimes comes with a new system which lasted for several months. Once the system started to balance out the algae was mostly under control, and I finally dedicated some time towards a bioluminescence observation session. When I sat down for my first observation session in several months, I realized there were several new challenges to overcome in the form of light pollution. In early 2018 I added 4 x XR30 Radion G4 Pro LEDs along with an 80-watt HO UV sterilizer to the system. This equipment was putting out a lot of light in the fish room area which made it impossible to achieve near total darkness. Initially, when I had a light pollution issue, I would break out the black electrical tape to cover the light sources, but with these pieces of equipment, it was easier to shut off the UV sterilizer and unplug the Radion’s. Normally I would not recommend shutting down equipment for a bioluminescence observation session, but in this case the aquarium life support systems would not be significantly impacted if I forgot to turn the UV sterilizer back on or plugin the Radion’s. I learned early on that getting near total darkness allowed me to see more bioluminescent light which tended to come from small organisms such as the isopods or even smaller unidentified organisms. With the light pollution eliminated I finally could start my first observation session in several months.



Light pollution challenges of the equipment - Pictured Top: UV sterilizer, skimmer pump LEDs, & Apex LED indicators. Pictured Bottom: Radion indicator LEDs and circulation pump LEDs. (Note the blue LEDs from the pumps are already covered up with black electrical tape)

What a difference a few months make
When the 1600-gallon system was started, it was seeded with ~60lbs of live rock added to the 150-gallon refugium and ~60lbs of live sand that was added to the 480-gallon reef aquarium. The intention was to establish the micro and macro organisms that are present in the rock and sand that originated in the ocean. When I started to observe for bioluminescent activity, I started by observing the 150-gallon refugium tank due to the concentration of live rock. I did observe some bioluminescence in the display tanks and sump tanks, but the activity was sparse. I did expect to see more bioluminescent activity in the 480-gallon reef tank as the live sand was added to that display, but initially, that was not the case. In the months that had passed, there were significant changes in the life in the 150-gallon refugium tank. The types of algae growing had changed but what was more visible was the lack of the isopods that sometimes showed a bioluminescent display. The 150-gallon refugium was taken over by amphipods and mysid shrimp. On a rare occasion, I see an isopod, but the large groups of them that once dominated that tank were gone. I did see the bioluminescent brittle starfish were still happily living in the rocks, but the lack of isopods was a bit disappointing. I decided that I would start to split my bioluminescent observation sessions between the different tanks in the system.



150gallon-refugium pictured Top November 2017 & pictured Bottom September 2018

150-Gallon Refugium Observations
While the isopods that I enjoyed viewing were no longer present in the 150-gallon refugium, I decided to start looking for other bioluminescent organisms. The level of bioluminescent activity in the refugium has shifted quite a bit since I started my viewing sessions. I no longer observe the constant glow of isopods on the glass or in the rocks, but I still see random flashes of light along with the light generated by the brittle starfish when they are disturbed. The unidentified bright green light source I discussed in my first article (pictured below) has not been seen again, but with that being such a random event its difficult know if I will get to see that brilliant display in the future. I am finding though that many of the bioluminescent flashes of light that I am observing now are coming from organisms that I have yet to identify. For the most part, these flashes of light are just tiny points of light. On occasion, a brighter flash of light will occur. I do suspect though that some of the organisms that I observe during the day or through a microscope are candidates for continued research.



Top: Bioluminescent display of an unidentified organism that has not been seen since taking this photo in November 2017. Bottom: Bioluminescent display of an unidentified organism photo taken in September 2018

New Observations
The reasons for a bioluminescent display can vary between organisms which is why passive observations are not the only method I utilize. One of the most incredible sights in the 150-gallon refugium took place when I decided to disturb the masses of algae growing on the rocks at night with a feeding stick. Hundreds of bioluminescent flashes were set off by this action. These dim flashes of light were coming from small unidentified organisms. At the time of this observation, I did not have the tools needed to make an identification. I also did not have the proper camera equipment needed to try and capture this beautiful bioluminescent display. In September 2018 I attempted to try this same experiment of disturbing the algae with a feeding stick with some unexpected results. I managed to get a reaction out of the algae however the bioluminescent display was very dim but similar to my first experience. I attempted to capture this display on camera the next night, but I did not see any activity when I disturbed the algae with a feeding stick. I did make a discovery though when I bumped into the in-tank algae scrubber PVC cage. When I disturbed the algae scrubber PVC cage, I set off a brilliant display that I could see from above the tank. I also decided to try and pick up and move around part of the floating algae mat and discovered that in most cases when I picked up the algae and moved it around between my fingers, it would trigger a bioluminescent display. I again decided to try and capture this on camera, but I needed to do some tank cleaning first. I was leaving the sides of the glass by the algae scrubber covered in coralline algae, but with this discovery, I got the scraper out and cleaned that part of the glass so I could try to take photos the next night. I was all setup with the camera the next night and attempted to take some photos, but the camera was not able to pick up most of the bioluminescent display that I was able to trigger as it was just too dim to be picked up by my camera. I plan to continue observing this tank for bioluminescence, but I will admit that it is difficult to keep my attention on the refugium tank when the there are two giant display tanks only a few feet away.



150-Gallon Refugium: in-tank algae scrubber and algae mat that has a large concentration of bioluminescent activity when disturbed.

480-Gallon Reef Observations
The 480-gallon reef tank was running for about nine months before I seriously started to watch for bioluminescence. I was shocked at the level of bioluminescent activity observed the first night. I expected to see some activity, but the bioluminescence seemed like it was all over the place especially in the sand. Now when I sit down in front this tank in the dark, I feel as though I am preparing to watch a movie for the first time. I have come to expect that each night of observation is its own unique show. It is worth mentioning that the bioluminescent displays discussed are visible to the naked eye when adjusted to the dark and can be captured with my current camera setup which will be discussed later in this article.


480-Gallon Reef tank September 2018

The Sand
The sand is full of bioluminescent activity, and it has been the primary focus of my observations in this system. I have observed several different patterns of bioluminescent light generated in the sand throughout the last few months. I am uncertain if these bioluminescent displays are all related to defensive mechanisms or if they could be related to general communication, attracting a mate, or attracting food. Many of the bioluminescent displays in the sand seem to be triggered as a defensive mechanism or burglar alarm when a bioluminescent organism encounters another organism via touch. The cleanup crew that goes to work after the lights go out seem to be the trigger of many of these bioluminescent displays as they can be observed moving over or near the area where the flashes of light originate. I have also been able to duplicate the defensive bioluminescent behavior by dragging a feeding stick through the sand. A great example of the defensive behavior is pictured below. The two conchs were moving along the sand and triggered a defensive bioluminescent display when they disturbed an unidentified bioluminescent organism.



Conchs disturbing a bioluminescent organism which triggered a brilliant flash of blue light

Light Patterns
The patterns of bioluminescent light in the sand come in a few varieties that I have classified simply as dashes, lines, and dots which are pictured below. The dashes seem to be generated by organisms that when disturbed swim out into the water just above the sand while emitting a bioluminescent trail into the water. The dashes are very fast displays that are random and normally do not repeat very often. The lines and dots I believe could be the same type of organism in some cases. Because of the camera angle to the bioluminescent flash of light and where the organism is located either at the surface or just below the surface of the sand can provide different appearances to still images. The lines and dots often flash multiple times when disturbed, and at times they will pulsate light for a second or two. The similar behavior between the lines and dots in the sand led me to believe that in some cases these might be the same type of organism. There is at least one image of dots in the sand though that appears to be a different type of organism as the color of the bioluminescent display differs from the other photos of dots.

While these different bioluminescent displays are far more interesting to observe in person I have managed to capture photos of these displays. Below are some still photos of the different bioluminescent displays that have been observed in the sand of the 480-gallon reef tank. The accompanying video with this article contains additional footage of bioluminescence in the sand of the 480-gallon reef tank.






Unidentified bioluminescent organisms that I have classified as creating dashes of light



Unidentified bioluminescent organisms that I have classified as creating lines of light


Unidentified bioluminescent organisms that I have classified as creating dots of light (notice the last picture in this series shows a different color display compared to the other pictures)

The Rocks and Water Column
The sand is not the only place where there is bioluminescent activity in the 480-gallon reef tank. I have started to pull some of my attention away from the sand recently, and I have started to notice that there are other organisms in the rocks and water column which are also generating bioluminescent displays. In the rocks, there have been a few different types of bioluminescent patterns being picked up which vary in color when compared to most of the activity that takes place in the sand. Most noticeable is the flashes of green bioluminescent light coming from the Christmas tree rock where a brittle starfish resides and can be seen sometimes during the daylight hours. There have also been several other bright green flashes of light along with some dim flashes of green light in the rocks which are unidentified organisms. The rocks also have also been the source of several small dots of bioluminescent light that appear as small white dots on the photos. It is possible though that these tiny dots of bioluminescent light that appear as white dots are also related to the activity seen in the water column. The organisms in the water column put on a bioluminescent display which is only lasts for a fraction of a second. These organisms in the water column are suspected to be very small and fast as they seem to jet off in a single direction leaving a bioluminescent trail of dots or a streak if light. During observation sessions, I have seen flashes appear in the water column, but they happen so fast that I cannot see the trail of light the camera is picking up. I honestly felt that these bright white dots and streaks appearing in the photos might be noise from the camera or another anomaly in the image when I first saw them. However, these little white dots and streaks of light appear randomly in different parts of photos and in different patterns that do not match each other. I decided to test out the theory of these white dots on the photos being noise the camera is picking up due to the long exposure times. I placed the camera so that half of the frame would be pointed at the aquarium while the other half of the frame was pointed at a wall. After taking several hundred photos and reviewing them, I have concluded that some of the white dots that are the size of a single pixel, square-shaped, and mostly filter out with a contrast adjustment are likely camera noise. The other white dots and streaks of white light with dots that cover multiple pixels do not appear to be camera noise as they only occurred within the side of the camera frame pointed towards the aquarium. Since I see these small random flashes of bioluminescent light with my own eyes, I feel comfortable in classifying these images captured by the camera as a bioluminescent display, but I will continue test that theory as my research continues. I am sure that as I continue to make observations and review photos that additional discoveries of bioluminescent light displays will be found in the rocks and water column of the 480-gallon reef tank as it matures.


Christmas Tree worm rock with a bioluminescent brittle starfish taking up residence. Pictured Top: The red arrow points to the bioluminescent display of the brittle starfish in this early morning picture taken after the refugium lights turned on in the fish room. Pictured Middle: Brittle starfish bioluminescent display during the night. Pictured Bottom: Brittle starfish arms hanging out of the Christmas Tree worm rock.


Bioluminescent displays from unidentified organisms in the rocks. (Note: In the last photo of this series the small white dots of light appear in the same image as a bright green flash of light in top of the photo)


Bioluminescent displays of white dots and streaks from unidentified organisms or possible camera noise or maybe both.

720-Gallon Reef Observations
The 720-gallon reef tank has been a bit of a surprise with its bioluminescent activity since there was no live rock or live sand added to this tank when the system was started. In a way, this tank has been a little experiment that has shown how fast life can spread between the interconnected tanks of a large aquarium system. There is only a small tray of sand in this tank for the wrasses that like to sleep in the sand at night, and the rest of the tank is bare bottom. Out of all the tanks in my aquarium system, the 720-gallon reef receives the least amount of attention and time for bioluminescent observation along with camera time. There is still plenty of bioluminescent activity to be seen in this tank though, that covers the same spectrum of bioluminescent displays seen in the 480-gallon reef tank. The one difference is that the bioluminescent displays that mostly appear in the sand of the 480-gallon reef tank occur on the bottom and rocks of the 720-gallon reef tank. I plan to spend some additional time observing this display in the future, and I might even drag a feeding stick around a few times to see if any defensive bioluminescent displays are triggered. Since there is a minimum level of cleanup crew in this tank due to the aggressive nature of the fish being kept, there have been fewer observations of the cleanup crew triggering defensive bioluminescent displays. Below is an overall tank photo and some photos of the bioluminescent displays that have been caught during photo sessions of the 720-gallon reef tank.


720-Gallon Reef tank September 2018


Examples of the bioluminescent displays seen in the 720-gallon reef tank by unidentified organisms.

Sump Tank Observations
Beyond the display tanks, the sump tanks have also been observed to have bioluminescent activity. I have spent some time observing the sump tanks and just by lightly tapping on the sides or the bottom of the sump tanks there are multiple bioluminescent displays triggered. I did attempt to take some photos of the sump tank pictured below using a viewing box but when I was setup to try and trigger the bioluminescent displays it did not occur thus I did not manage to get any useful photos of these bioluminescent displays. It is evident though that some of the bioluminescent displays in the sump tanks are coming from little tunnels that have been constructed using detritus. I know that some bioluminescent organisms are calling these detritus tunnels home.


Detritus tunnels on the bottom of the sump tanks

What is making this light?
Identifying the organisms generating bioluminescent light in my aquarium system has not been an easy task. When I started observing bioluminescent activity, I managed to positively identify isopods and brittle starfish with relative ease as these organisms were in plain sight and big enough to be identified without additional research tools. However, several other organisms were eluding identification as I was unable to identify them visually after a bioluminescent display occurred. In some instances, the organisms that were producing light were just never seen. I could see a flash of light take place, but when I would flip on the flashlight to see what was there, I would not see anything. Having failed to spot a potential bioluminescent organisms hundreds of times with the flashlight technique, I decided it was time to change my research strategy. I started to believe that these bioluminescent organisms were too small to see, they left the area, or they went into hiding after setting off their light show. None of these possibilities would make it easy to identify bioluminescent organisms. It’s this challenge which has been driving my continued research of these organisms. I had to ask myself a few questions though before I could settle on a research strategy that would help me identify these bioluminescent organisms.

• Where do I focus my research?
• What tools are needed to continue this research?
• How can I be certain that I have identified a bioluminescent organism?

New Research Tools
After asking myself these questions, I took some time to think about them until I had answers. I had no idea where I should focus my research, but I knew that with the right tools I could answer that question. I needed to know where the bioluminescent activity was occurring the most in the aquarium system then I could focus my research. I decided that continued observation sessions were required to identify the bioluminescent activity hot spots. The only issue was that I could not stay up all night every night for observation. I have a family and career that must come first. However, I did figure out a way to complete observations of bioluminescent activity almost every night while I was sleeping. I already had a DSLR camera, and I had captured photos of bioluminescence for my first article using this camera. I attempted to utilize this same camera for time-lapse photography but, my initial attempts failed as the camera was just not sensitive enough to pick-up most of the bioluminescent light. After doing some research, I determined that I needed to try a different camera lens that had a larger aperture and was capable of gathering additional light when a photo was taken. After talking with some co-workers, one offered to lend me a lens to try out. I want to thank my co-worker Brad as the lens that he let me borrow was just what I needed to capture the bright flashes of bioluminescent light on camera. After a few nights of time-lapse photography, the bioluminescent activity in the sand of the 480-gallon reef tank is where I decided to focus my research. Based on all the bioluminescent displays I saw during observations sessions, and with photos, I determined that I would need a microscope to assist with the identification of bioluminescent organisms. I decided that a stereo microscope would offer me the most flexibility with magnification and lighting options. I did not want to break the bank on a microscope, so I set out to find a reasonably priced model. I ended up purchasing an AmScope Cordless LED Stereo Microscope 20x-40x-80x Model: SE306R-PZ-LED, some petri dishes, and a Gosky Universal Cell Phone Adapter Mount. Now I had the tools needed to view, image, and identify small organisms that might be bioluminescent. All I needed to do was isolate a bioluminescent organism and observe that organism generating a bioluminescent display while viewing through the microscope. Much easier said than done.


Microscope setup and ready to take pictures with some sand samples ready for viewing.

Isolating Organisms
Isolating a bioluminescent organism for identification can be a challenge. As I was trying not to reinvent the wheel, I started off by utilizing one of the best tools that I have available as an aquarist which is a poking stick. During observation sessions, I learned that in many cases I could trigger a bioluminescent response by using a feeding stick repurposed as a poking stick. With that knowledge, I needed to reduce the territory of my search for these bioluminescent organisms. As I focused my research on the sand of the 480-gallon reef tank, I knew that I needed to limit the search area since there are ~460lbs of sand covering an area of ~32 square feet in this tank. I decided that I needed to take some sand samples for more detailed observation. I repurposed my top down viewing box for searching sand samples. I kept the process simple. I would take a cup or two of sand with water and place it in the top down viewing box where it was kept submerged and spread into a layer about 1/4” deep. In the dark, I would take a poking stick and run it through the sand from the top to the bottom of the tray slowly in a grid pattern to try and trigger a bioluminescent response. If the poking stick triggered a bioluminescent display, I would stop moving the poking stick and turn on my red flashlight to see the area. I would move the sand away from that area slowly with the flashlight on to try and isolate a small portion of sand which is moved to a petri dish for further analysis through the microscope. I have found that this method has been successful in isolating several different organisms in the sand one of which I have confirmed to be bioluminescent. While I have found many different organisms living in the sand that could be bioluminescent it not always easy to trigger multiple bioluminescent displays from the same organism repeatedly. When a bioluminescent display is triggered, the light is the result of an internal chemical reaction in the organism. As stated by Haddock, Moline, Case “Bioluminescence is typically produced by the oxidation of a light-emitting molecule—generically called the luciferin—in conjunction with a catalyzing enzyme—either a luciferase or photoprotein.” (2010, pg. 446). The organisms that produce these chemical compounds to create light can also exhaust their supplies of these chemicals which requires them to in a sense recharge. During bioluminescence research expeditions many organisms exhaust their bioluminescent ability when they are captured using nets from the depths of the ocean (TEDEducation, Widder, 2013). I find that the in some cases these bioluminescent organisms have exhausted their abilities when I am searching for them in the top-down viewing box or a petri dish. As this is the case, I normally go through a sand sample several times at 15-20-minute intervals before I return the sand to the 480-gallon reef tank. I have had several samples of sand where more than a dozen bioluminescent displays are triggered by the poking stick which is why it’s a good idea to go through each sand sample multiple times. Recently I have utilized a similar method of isolating organisms living in the algae mat of the 150-gallon refugium tank. With the algae, I have been picking up the mat, and if a bioluminescent reaction is triggered, I tear off a small section of algae where I believe the bioluminescent organism is located.


Sand sample tray, a poking stick, and petri dishes. I just need to turn out the lights, let my eyes adjust, and then it’s time to find some bioluminescent organisms.

Confirmed Bioluminescent Organisms
After many months of observation and research, I have identified two more organisms living in my aquarium system that are bioluminescent. Utilizing the method described above for isolating bioluminescent organisms I managed to not only isolate these organisms, but I was also able to trigger them to produce a bioluminescent display while observing through the microscope. I found two different species of spaghetti or medusa worms pictured below that are bioluminescent. As there are countless numbers of worm species that fit the general description of the worms identified I did not try to determine the exact species. The first worm was found living in the sand of the 480-gallon reef tank inside of a detritus burrow. This worm was brown and is only a few millimeters in length. The bioluminescence was triggered by touching the tentacles of the worm using a poking stick. The bioluminescent display was blue to the eye and extended along the tentacles that were in contact with the poking stick. The light was emitted as a flicker, and through the microscope, it appeared to look like a filament from an incandescent light bulb. I attempted to take a video of the bioluminescent display over several nights of observation through the microscope, but the camera on my phone is not sensitive enough to record the bioluminescent light.


Unidentified species of spaghetti worm or medusa worm that is bioluminescent. This worm was found living in the sand of the 480-gallon reef tank.

The Second Worm
The second worm was located in the algae mat of the 150-gallon refugium tank. The worm is white and at most is 1-2mm in length. The bioluminescent display was blue and appeared to be emitted from the entire worm. Since this worm was living in an algae mat, it went slightly out of focus when touched with the poking stick. I was surprised how bright the display of this worm was since I spotted it from over a foot away when I was looking for bioluminescent flashes in the algae mat. The burst of light emitted was flickering but was a bit muted through the microscope. This worm had flashed light on several occasions, and the final burst of light came when viewing through the microscope. Again, I attempted to take a video, but I was unable to trigger any further bioluminescent displays from this worm so it is not clear if the camera would be capable of recording this light. Additional worms will need to be located before attempting to film their bioluminescent displays.


Unidentified species of spaghetti worm or medusa worm that is bioluminescent. This worm was found living in the algae mat of the 150-gallon refugium.

Original Organisms Identified
It has been almost a year since I started my research on bioluminescence and I don’t think I will ever forget the first time I spotted that faint light in my aquarium system. I still see the light show of the brittle starfish at night from time to time in the 150-gallon refugium. Below are my most recent pictures of the bioluminescent display of the brittle starfish. The isopods as I have said earlier are no longer seen in the 150-gallon refugium, but recently my wife started a small 25-gallon reef tank. I used sand from the 480-gallon reef tank to seed her tank with life, and a few weeks later I started so see isopods on the glass. I plan to complete some observation sessions soon with her small tank to see if that species of isopods shows the same beautiful display of bioluminescence.


Bioluminescent brittle starfish living in the 150-gallon refugium tank.


Bioluminescent Isopods that that would sometimes glow at night.

Why Continue the Search?
While I have confirmed four different organisms to be bioluminescent in my aquarium system, I suspect there are other organisms that I have yet to find. There are many reasons why I suspect some organisms to be bioluminescent. One of the primary reasons is that many of the organisms which inhabit our tanks are taxonomically identified to have species within their class which are known to be bioluminescent. Figure 1 shows a taxonomic break down of many different classes of life that are known to be bioluminescent. While not all species in these taxonomic classifications are bioluminescent, it is a good place to start looking for potential suspects that are living in my aquarium system. Another reason is that the purpose of a bioluminescent display can differ between organisms. Three out of the four organisms that I have identified have a defensive bioluminescent response which means that there might be other bioluminescent activity which does not occur as often or requires to be in the right place at the right time to observe. Finally, in several instances, I have isolated an organism that I believe to be bioluminescent under the microscope, but I am unable to trigger another bioluminescent response. It is these reasons that drive me to continue my search for other organisms in my aquarium system that are bioluminescent.


Figure 1. Luminous genera (Haddock, Moline, Case, 2010, para. 6).

Suspected Bioluminescent Organisms
Several suspected organisms have been observed in my aquarium system that have the possibility of being bioluminescent. The most interesting organisms that I have found thus far have a good chance of being bioluminescent since I have isolated these organisms on multiple occasions while checking sand samples for bioluminescence. I also have no idea what these organisms are as I have been unable to classify them. These organisms are only a few millimeters long living in burrows which are built using a mixture of rock, sand, and detritus which are all glued together into a flexible structure of tubes which are pictured in Figure 2. I have noticed in some cases that when I take sand samples even just a tablespoon or less of sand that I find several of these organisms living in pieces of burrows. I am not sure if they live as individuals or as a colony since my disruption of the sand to take samples inevitably disturbs their homes. When separated from their burrow these organisms can be incredibly fast, and on several occasions, they have broken the surface tension of the water in the petri dish when they are swimming around. I have also noticed what appears to be filaments being pulled behind these organisms as they walk around in the petri dish which reminds me of a spider laying silk. I took some additional photos in Figure 2 of one of these organisms and will continue to try and identify what they are.


Figure 2. Unknown species living in my sand that might be bioluminescent.

Ostracods
The next organisms living in my aquarium system that I feel has a high probability of being bioluminescent are ostracods. I have found several different species of ostracods living in my aquarium system pictured in Figure 3 which are ~1 millimeter in length. I am not sure if any of these species of ostracods are known to be bioluminescent as I have yet to try and identify the species I have found. There are several thousand species of ostracods that live in both freshwater and marine environments. Many species of marine ostracods are known to be bioluminescent, but since I have not tried to identify these species, I am not sure if any of them are known to be bioluminescent. I plan to continue my research and observation into the ostracods living in my aquarium system to see if any of them turn out to be bioluminescent.


Figure 3. Different Ostracods species I have found living in my aquarium system. Many species of Ostracods are bioluminescent so it’s possible

Copepods, Mysid Shrimp, & Amphipods
Other potential organisms that I regularly observe in my aquarium system that have the potential to be bioluminescent are copepods - Figure 4, mysid shrimp - Figure 5, and amphipods - Figure 6. As shown in Figure 1 copepods, mysid shrimp, and amphipods all fall into taxonomic classifications with known bioluminescent species. Due to their high population in the aquarium system, I think that they deserve closer observation as the frequency in which I see very faint flashes of bioluminescent light could be attributed to these organisms. I have not been able to trigger a bioluminescent response out of these organisms yet but if their bioluminescent displays are not intended to be used for defense then continued observation in conjunction with correct timing might also play a role in determining if any of these organisms are bioluminescent. From a big picture standpoint, I spent very little time observing these organisms as I find other organisms can be triggered to create a bioluminescent display which makes identification a little less tricky. As my bioluminescence research progresses, I plan to spend more time observing copepods, mysid shrimp, and amphipods with the hope that some of the species living in my aquarium system can be confirmed to be bioluminescent.


Figure 4. Some species of copepods are known to be bioluminescent.


Figure 5. Mysid Shrimp in some cases are bioluminescent.


Figure 6. Amphipods are also known to have some species that are bioluminescent.

Photographing Bioluminescence
Capturing images of bioluminescence was not an easy task when I first started my research. In the past few months though I have managed to take some great pictures of bioluminescence which has created a much better viewing experience for still photos. My current camera setup is a Cannon Rebel T5i with a Cannon EF 50mm F1.8 lens (80mm equivalent with my APS-C camera). I was also using a borrowed Canon EF 50mm 1.4 lens (Thanks Brad) for many of the pictures captured in this article. Both the F1.4 and F1.8 lens can capture bioluminescent displays with still images. There are some limitations to what can be photographed with this camera though. Dim bioluminescent displays are not picked up by the camera as it is not sensitive enough to work in such dark conditions. Image noise in the pictures is another issue which is due to the high ISO needed to capture the bioluminescent light. This camera is also not capable of recording video of bioluminescent displays even with the F1.8 lens. While I could not record videos of bioluminescence activity, I did utilize my time-lapse photos along with a video editor to create videos of bioluminescent activity. I merely imported all the photos taken from each night generally around 400-600 photos into the video editor (Power Director). I would adjust the duration each photo is shown to between .15-.05 seconds which will create a video of all the pictures stitched together. The only other adjustment I made to photos for editing is turning up the contrast to the highest level which eliminates most of the noise and brings out the light of the bioluminescent activity in the photos. Creating videos using this method allowed me to locate hotspots of bioluminescent activity while also providing a way to review the hundreds of photos taken every night quickly. Despite having a few drawbacks my camera as done an excellent job for my research and eventually I will upgrade my camera to a model that is better suited for taking photos and video in low light conditions. Below are the camera settings that I have been using to capture photos of bioluminescent activity.

Camera settings:

• Manual Mode
• File Type: Highest resolution .jpeg
• Noise reduction: highest setting
• ISO: 3200-6400
• Auto Focus: Off
• Aperture: F1.8 with my current lens
• Exposure time: 10-30 seconds
• Camera modifications: Magic Lantern installed to utilize the intervalometer settings (WARNING MAGIC LANTERN IS A THIRD-PARTY SOFTWARE THAT CAN DAMAGE OR RENDER A CAMERA UNUSABLE – USE AT YOUR OWN RISK)
  • Intervalometer set to take a new exposure every 1 second after the previous picture is taken. This comes out to about 1 photo taken every 50 seconds due to exposure time and processing to save the image.

Conclusions: Lessons learned and Future Research
All the research that I have conducted thus far has made me want to learn more about bioluminescence. I have some new ideas about how I want to continue my research, but I also think that it is important to reflect on some lessons learned from observing bioluminescence. How I observe bioluminescence has changed over the last few months as my original methods of trying to achieve near total darkness have changed with observations in the main display tanks. I have found that the indicator LED lights on the main lighting fixtures for both display aquariums can generate just enough light to make out the aquascape which, create a much better lighting environment for photography and visual observation. If a light source is not in the direct or reflected line of sight with the camera or the naked eye, then those light sources in many cases will not adversely impact the observation or photography of bioluminescence. I have also learned that I need to concentrate on observation of specific areas of the aquariums for extended periods rather than following the flashes of light to the parts of the displays where they occur. It is not easy observing the 480-gallon or 720-gallon reef tanks for bioluminescence as these tanks are eight feet long and four feet front to back. My eyes constantly wonder, and when my peripheral vision picks up a flash of light, I tend to shift my observation to that area of the tank to see if more bioluminescent flashes occur. I can sit back from the tank a few feet, and I will catch the bright bioluminescent flashes, but the tiny/dim flashes of light cannot be observed unless my nose is practically on the glass. It has not been easy, but I have been learning to resist the urge to change my observation area due to what is being picked up in my peripheral vision and concentrate on a single area which normally yields some great light shows that would have been missed if I continued to change observation areas. The lessons learned come down trying new methods, along with time, and patience.

Continued research into bioluminescence can take many different paths depending on my observations. I do have some preliminary plans that focus on organism identification, obtaining new species of bioluminescence organisms, looking beyond defensive bioluminescent displays, and working to improve my ability to capture bioluminescent displays on camera. Organism identification has been a difficult task to complete due to the lack of time needed for these observations. I plan to use some of my available time towards isolating organisms and attempting to observe bioluminescent displays directly. Given the amount of bioluminescent activity seen in my aquarium system, I wonder what other bioluminescent organisms are obtainable while still being able to thrive in a reef aquarium. I am not sure how the search for additional bioluminescent organisms will progress, but I look forward to the opportunity to expand my research with new organisms. Part of the reason that I want to increase my biodiversity of bioluminescent organisms is to expand research into other forms of bioluminescence beyond defensive responses. Seeing bioluminescent displays related to breeding, general communication, or gathering food would be fascinating to research. I am also considering the use of creating artificial bioluminescent displays to see if they can be utilized to trigger bioluminescent responses as I have seen scientists utilize this technique in the ocean with some rather interesting results. While I am looking forward to my journey with continued research, I would like to be able to share better my experiences of bioluminescence which is why I will be looking to obtain a new camera which is better suited for the photography and videography of bioluminescent light. I am just beginning to research new cameras that could improve the quality of my bioluminescence photography and videography, but until a new camera is obtained, I will continue to produce the best imagery I can with my current camera setup. I hope everyone can take something away from this article on bioluminescence. It’s hard to describe the sight of what I see during an observation session which is why I have included a video with this article. I highly encourage anyone reading this article to watch the accompanying video which gives a small preview into the amazing world of bioluminescence that I get to observe in my aquarium system.

Bioluminescent Reef Aquarium


Aquarium System Specs:

• Total System Volume: ~1600 Gallons
  • 2 Plywood Displays:
    • 720 Gallons 96”L x 48”W x 36”H
    • 480 Gallons 96”L x 48”W x 24”H
  • 2 HDPE Sump Tanks: 265 Gallons & 120 Gallons
  • 1 Glass Refugium Tank: 150 Gallons
• Average water chemistry
  • Salinity: 1.026
  • Temp: 77-79F
  • pH: 7.8-8.02
  • Alk: 8.0-8.3 DKH
  • Cal: 500ppm
  • Mg: 1440ppm
  • PO4: .03ppm
  • NO3: 5ppm
• Equipment
  • Main Return: Reeflo Hammerhead
  • Gravity fed DIY Protein Skimmer
    • Main body constructed from a 60 gallon HDPE cone bottom induction tank
    • Air injection 2 x Jebao 15000 pumps with DIY needle wheels
  • Emperor Aquatics 80 watt HO UV Sterilizer
  • Geo CR1218 Commercial Calcium Reactor & Secondary Chamber SMC618
  • Heating: DIY hot water heater driven radiant heating system
  • Lighting
    • 480 Gallon Display:8x39watt T5s & 4x XR30 Radion G4 Pro LEDs
    • 720 Gallon Display:3xAI Vega Color LEDs
    • Refugium: AI SOL, LED Grow light, outdoor LED floodlight
  • Apex Aquarium Controller
  • 200 Gallons RO/DI water storage
• Humidity controls
  • Dehumidifier
  • Exhaust Fan
  • Slightly negative pressure fish room with moisture barriers to contain and minimize humidity
• Water Flow
  • 480 Gallon Display
    • 2xGyre 250s
  • 720 Gallon Display
    • 3xRossmont MX4100 Mover Pumps

References
Haddock, S., Moline, M., Case, J. (2010). Bioluminescence in the Sea. Annual Review of Marine Science 2010 2:1, 443-493, Retrieved from: http://www.annualreviews.org/doi/full/10.1146/annurev-marine-120308-081028

TEDEducation, Widder, E. (29 Mar. 2013) “The Weird, Wonderful World of Bioluminescence - Edith Widder.” YouTube, www.youtube.com/watch?v=lKeDBpkrDUA