Quagga mussel description
As we are the very first iGEM team working with quagga mussels, we had to establish from scratch protocols to carry out our experiments. Here, you can find explanations on the experiments we have done and some troubleshootings that we sincerely hope will help and benefit the iGEM community to better understand how to work with quagga mussels. At the end of this page, you can read the summary of our most important observations.
Before describing our troubleshootings and experiments, we provide the iGEM community with some information about quagga mussels and their development.
Invasion of quagga mussels in Switzerland
As surprising as it may seem, quagga mussels are not the first freshwater mussels to colonize Swiss freshwaters. The first invaders were the zebra mussels (Dreissena polymorpha), a related species that was first introduced around 1960. However, this species have not been as aggressive as the quagga mussels (Dreissena rostriformis), which have rapidly expanded since their first discovery in 2015. These two related species are originally from the Ponto-Caspian region and made their way up the waterways from the Black Sea to Europe and North America.
Reproduction of Quagga mussels
One of the reasons why it is so difficult to stop the spread of quagga mussels is their external and fast reproduction cycle. In fact, within 28 days, the larvae can evolve into juvenile mussels. Their spawning is characterized by expelling male and female gametes in fresh and brackish water. Larval reproduction occurs in open waters, which is said to be external. According to the water department's figures, adult females can release up to 40,000 eggs per spawning cycle and go through 20 spawning cycles each year. Thus, one female can release up to 1 million eggs, and a male up to 200,000,000 sperm per year. Subsequently, the formed embryos will become veliger larvae, which are the larval stage of mollusks from the trochophore larva composed of cilia that allow them to move. This is known as the planktonic veliger stage, where the larvae will reach an average size of 100-150 micrometers. This is considered the most problematic stage because, being only microscopically visible, it is a phase that is difficult to control.
The next stage of mussel life is called the “planktonik postveliger”, which is characterized by the metamorphosis of the larvae into juvenile mussels. In this stage, the mussels will grow from approximately 150 micrometers to 8 mm during one month and develop their shell and organs. Here, they develop their byssus, a fixation apparatus composed of extracellular filaments and a foot, allowing them to move to find a surface on which to fix themselves in order to finish their development. Juveniles have an adult bivalve shell but are not yet able to close it effectively at first and remain sensitive to dessiccation and various chemicals at this stage. They reach sexual maturity at 7 mm and live sedentarily for up to 5 years. Their density of occupation remains impressive and can go up to 100 000 individuals per m2.
Differences between quagga and zebra mussels
As stated earlier, quagga and zebra mussels are very similar and sometimes difficult to distinguish. However, there are a few ways to differentiate them. First, visually, they can be distinguished by their stripes. Zebra mussels are darker with neat stripes, while quagga mussels are often lighter with less neat stripes or even white, depending on the water depths at which they are found. In addition their shape also differs: quagga mussels have a ventral face with rounded valves that make them tilt when they are on their ventral face, while the zebra ones are triangular and hold on to the surface better without tilting compared to quagga mussels.
As for their mode of reproduction, there is also a clear difference. Zebra mussels are characterized by only reproducing during six months of the year, depending on the conditions, and only in waters at temperatures of around 10-12°C. In contrast, quagga mussels can reproduce all year round in waters at temperatures as low as 5-6°C, which is even more problematic.
Finally, zebra and quagga mussels colonize different depths. Once zebra mussels reach 35m they stop spreading. This information has led some entities to build deeper installations to avoid zebra mussels colonization in the pipes. However, this turned out to be a futile effort after the appearance of quagga mussels, which can colonize up to 200 m deep, tenaciously, even in low nutrient environments.
Working with quagga mussels – Experiments and troubleshooting
To test the effectiveness of our products, we decided to apply them directly to the mussels. However, we had to find the best way to keep them alive and perform our experiments. To do so, we tested several conditions to find the optimal environment.
Collection
First, we collected quagga mussels from Lake Geneva at Vidy’s port (Lausanne). This invasive species is abundantly present, therefore, it is easy to find a spot where the rocks are covered with hundreds of them. We took some rocks covered with mussels from inside the lake, and we carefully detached them by hand, placing them directly in small buckets filled with lake water. The quagga mussels were well attached to the rocks; some were not easy to remove, mainly because their shells were very fragile.
Experiment 1
We transported the collected mussels to the laboratory and tested various conditions to determine which one allowed the mussels to live long enough for the experiments to take place.
We first looked at the requirements that could be used for maintaining standard aquatic animals. We first chose to mimic their natural habitat placing some rocks in a tank filled with 6 L of lake water, and installed a pump that would create a current. Unfortunately, despite good signs of acclimatization in the first two days (exit of the filtering apparatus, retraction during movements, and mussels moving by using their foot), we quickly realized we were not on the right path for good maintenance of the mussels, as they died within four days. Thus, some challenges had to be overcome.
We read on websites specialized in mussels and tanks that a dead mussel liberates ammonium, which is toxic for neighboring mussels and can lead to a chain reaction that causes rapid mussels death (The Mussel Masquerading as a Clam - Pilsbryoconcha Exilis - Asian Gold, n.d. / Isadora, n.d.). This may explain why, from day 3 to day 4, most of the quagga mussels in the tank suddenly died. To avoid this, we had to answer two questions: first, how can we check if a mussel is dead or alive? Second, how can we remove a dead one quickly, without causing stress to the others?
For the first question, we learned that they actively close their shell. This means dead quagga mussels stay open, whereas alive ones can open and close their shells. To verify it, we simply used an inoculation loop and gently tapped the ground next to it. By sensing the vibrations, the mussel closed itself quickly.
Note: During our experiments, we observed that a mussel that would die soon was weaker and closed itself slower. Sometimes, vibrations were not enough, and to be sure if we had to count this mussel as dead or alive, we had to touch it directly to see if it was moving its shell or not.
For the second question, we thought of finding a method to separate the quagga mussels individually so that they would not get attached to each other. Thus, it would be easier to remove a dead one without disturbing the others.
As our experiments with FitD progressed, a new challenge pointed out: finding the optimal water volume. We needed enough volume to allow them to live and as little volume as possible to have a good concentration of our final solutions.
Therefore, several parameters such as volume, number of mussels and absence of the pump were changed in the following experiments.
Experiment 2
We tested four different environments: the glass tank with a pump containing 62 quaggas and filled with 5 L, a plastic container filled with 2 L of lake water and ten quaggas, another plastic container with 1 L and ten quaggas, and the last setup tested consisted of 10 quaggas in a 50 mL Falcon tube.
To give them nutrients, we decided to change half of the total water volume every two days and replace this volume with freshwater from the lake.
For the aquarium and the plastic container, we placed the quagga mussels in lines and with space between them, so we could easily count them and see their movements along the days (see Figure 3).
Observations in the aquarium
Within 20 minutes after we placed the quagga mussels, we observed that the individuals located opposite of the pump were active – they began to show their feet, looking for a surface to attach to (some of them moved and broke the lines) and they opened, filtering the water. More precisely, they were not moving by themselves, but they were moved by the current, and we noticed that the current was not equally strong within the box, but it appeared to be stronger on the side opposite to the pump (where it collided with the wall). The mussels closer to the pump began to be active only the next day. The mussels, once active, quickly attached to any surface of the aquarium: most of them attached to other mussels (approximately 35%), some of them attached to the black plastic bottom (30%), and one succeeded in getting attached to the glass wall, 5 cm in height. The other mussels (35%) did not fix themselves to a surface, but all were active.
Despite we arranged them in line, with 1 cm between each one, we noticed that they tend to clump together, which can be problematic for our experiments, as it is essential to remove a dead mussel quickly and without causing any additional stress to the others. Furthermore, it would be difficult to determine their attachment rate to surfaces. Therefore, we had to find a way to keep the mussels separated.
Small containers
Within 24 hours, no mussels attached to the small plastic containers. The mussels opened and were still alive. Here we noticed that the mussels having only 1 L of lake water were less active: the opening was reduced, they did not show their feet, and when we touched them gently, they closed themselves much slower than the mussels in the 2 L container. We had some hypotheses explaining this:
- Lack of nutrients: since the total water volume was half, mussels had to grow with half the amount of nutrients, which might result in generalized weakness.
- Type of plastic: mussels attach more easily to certain types of plastic. Unfortunately, the instructions did not specify the type of plastic for the tank and containers, so we could not check if the attachment surface was identical in the two conditions.
- Absence of current: the presence of a current is crucial to reproduce the mussel's natural environment. We speculate that the absence of a current might discourage the search for an attachment surface.
- They can live with less water but are less active. The lack of current may make them less active, too, so we planned a third experiment in which we would create a small current in a small volume.
- Their attachment may depend on the presence of a current and on the type of plastic too.
This indicated 2 things:
50 mL Falcon Tubes
We tested a setup with ten mussels in a 50 mL Falcon tube filled with lake water. By doing so, we would need less water per animal, so it would be easier to get a high concentration of our products. The mussels attached, but not to the plastic surface. However, removing the dead mussels was inconvenient in this setup. Therefore, we abandoned this approach. We did note that mussels can survive for at least four days in the limited amount of lake water mentioned above.
Experiment 3
We chose to test new small boxes that contained 12 separate compartments, where one mussel was placed in each compartment. The pump could not be used for such small volumes. Therefore, we tried a different technique to create water displacement. Namely, we placed the mussels on shakers that were set at 50 rpm, thereby creating a slow current. We filled the boxes with 350 mL of water.
After reading additional articles (James et al., 2021) and following the advice from Brigitte Schmidt (an ex-worker in the Lausanne Water Service), we decided to use chlorella as a nutrient for the quagga mussels. This would significantly enhance the reproducibility of our experiments since we would not need to rely on lake water for nutrients. We did a small experiment with chlorella with four boxes of twelve compartments each. In order to determine which amount of chlorella would be optimal for the mussels, we tested various concentrations. Our reference article estimated that we needed 0.2 grams per 350 mL of water (James et al. 2021). We additionally tested half the concentration (0.1 grams per 350 mL of water) and had a control box with just lake water. A fourth box was initially planned to test smaller concentrations of chlorella with 2-day intervals.
At 48 hours, all the quagga mussels that received 0.2 g of chlorella died; the following day, mussels that received 0.1 g of chlorella died. We hypothesized that the chlorella was complicating the mussel filtration capacity, and they died of asphyxia or due to too many nutrients.
Additional tests with smaller concentrations of chlorella gave similar results. After seeing this, we decided to stop the chlorella use and chose to proceed with lake water, despite a small loss in standardization.
Compared to the transparent containers in the second experiment, we observed that the small current created by the shaker did not change the mussel fixation rate or their longevity. It is possible that it was not strong enough to bring the same results as observed in the tank equipped with the pump. We would not use the shakers for the next experiments, as their use did not seem relevant. Moreover, there would be hundreds of quagga mussels, and we will not have enough shakers.
As the last boxes had plastic separations (instead of the tank), we thought the attachment rate would have decreased as the quagga mussels could not feel the presence of others around them. To check this hypothesis, we prepared two boxes, with and without separation. We placed three quagga mussels in each. After three days, none of them attached, so we returned to the first observation that the mussels are more active and attach easier when they are in big water volumes.
Final setup
As we prepared our FitD solution, we realized that recipients with 350 mL lake water and 12 quaggas (~30 mL per individual) would demand a high amount of FitD solution, and we would not be able to have the time required to produce such a quantity. We decided to set up a last “quagga mussel condition” experiment, placing one quagga in a tiny glass Petri dish containing 10 mL of water. We added 5 mL of lake water each morning to avoid dehydration from water evaporation. We did the first test with four quagga mussels. As they were all alive after 72 hours, we widened the experiment to 10 mussels and finally to 45. For this last experiment, we observed that 44 mussels were still alive 85 hours later. As we estimated that our FitD experiments should take around four days, these results for the control group were satisfying, and we continued all our experiments using this final setup. Note: we chose 45 quagga mussels per condition, as Petri dishes require space, and many conditions had to be measured. Forty-five mussels are the bigger group we could manage according to our laboratory size.
Summary
In conclusion, we observed that the mussel activity and their attachment rate require sufficient water volume, enough current, and a specific material. We didn't have time to test different surfaces, but this could be something to explore, i.e. with glass and different types of plastic.
To ensure a good concentration of our products, we needed to grow mussels in small water volumes, which could negatively impact their survival rate. The purpose of chlorella was to give them nutrients and to use tap water to increase our experiments' reproducibility further. As chlorella proved more harmful than beneficial, we decided to use lake water and add a small volume of fresh water each day to ensure that the mussels had enough nutrients. The amount of chlorella needed for small volumes would require further testing.
As well, it is easier to keep them separated, so when one is dead, we can quickly remove it without leading to a stressful environment. As a final setup, we chose small Petri dishes that permit small water volume (30 mL) and separation of the mussels.
References
- The Mussel Masquerading as a Clam - Pilsbryoconcha exilis - Asian Gold. (n.d.). Quality Marine. Retrieved October 9, 2022, from https://www.qualitymarine.com/news/the-mussel-masquerading-as-a-clam-pilsbryoconcha-exilis-asian-gold/
- Isadora. (n.d.). How to Keep and Care for Freshwater Clams in Aquariums. PetHelpful - by Fellow Animal Lovers and Experts.https://pethelpful.com/fish-aquariums/Introduction-Keeping-Clams-In-Aquariums
- James, B.D., Kimmins, K.M., Nguyen, MT. et al. Attachment of zebra and quagga mussel adhesive plaques to diverse substrates. Sci Rep 11, 23998 (2021).https://doi.org/10.1038/s41598-021-03227-6