Meeting with the Head of the Water Biology Section from the Swiss canton Vaud
To be able to understand the dimension of the issues with quagga mussels, we contacted Nathalie Menetrey Perrottet, the Head of the Water Biology Section in Vaud in Switzerland. She is also a member of the General Department of Environment and Water Protection from Vaud.
Nathalie Menetrey Perrottet gave us information about the characteristics of quagga mussels and why the quagga mussels are invasive. She also showed us a video made in collaboration with Fabwildpix (Une Écrevisse En PLS, Rien N’arrête Ces Envahisseurs!, n.d.), a channel from a Youtube influencer talking about nature-related topics, and the National Institute of Agronomic Research (INRAE), to raise awareness about this plague. This video highlights the damage of this invasion. For example, a crayfish is shown to be completely covered by mussels. We learned from this fact that quagga mussels were much more worrying than the related species, the zebra mussels, because of its ability to colonize any type of support. The video also emphasizes the impact on biodiversity and on an economic level.
By sharing various official statements that have been established by their service, she confirmed the damage that is happening on an economic level. This being the consequence of mussels blocking water pipes and the pollution they lead to by secreting ammonium. All this increasing the maintenance and purification costs by millions of dollars.
She also mentioned the ecological impact to us, specifically caused by mussels filtering a great amount of suspended particles and phytoplankton, which causes a decline of the latter and an increase in water transparency. Water transparency results in an over-proliferation of algae during the day which in turns leads to a drastic decrease in oxygen concentration in freshwater and negatively impacts biodiversity. This leads to an alteration of the food chain by reducing food resources and a modification of the communities of freshwater species.
She finally emphasized the major work that the fishery guardians and the water police do in order to warn and sensitize the people to the good practices in order to avoid the additional propagation of this invasive species, for example, cleaning the boat regularly.
Meeting with researchers from the Eawag Institute at Lake Constance
Broadening our knowledge about quagga mussels and how people deal with this invasive species led us to contact experts in another of the six affected lakes in Switzerland: Lake Constance, which is 11,500 km2 big and located at the borders of Germany, Austria and Switzerland.
There, we met with a team composed of researchers from Eawag Institute (“Swiss Federal Institute of Aquatic Science and Technology”) consisting of P. Spaak, A. Anh-Thu Weber, L. Haltiner, S. Rossbacher to discuss their point of view of the issues with quagga mussels and their strategies to eliminate them.
L. Haltiner is doing research on quagga mussels. Using population genetics, the aim of her study is to understand the invasion pathway and dispersal route of the quagga mussels in Switzerland and to measure the effects of their presence on the ecosystem (Eawag - Swiss Federal Institute of Aquatic Science and Technology - Eawag, n.d.).
By that time, we had trouble keeping quagga mussels alive when feeding them with chlorella for our project, so we asked them for advice. They also experienced that mussels can get killed if feeding too much chlorella. They only used a tiny amount of chlorella. They said, depending on the container, the amount of chlorella needs to be determined. To keep them in the Eawag Institute, they use a 30 L aquarium containing water at a temperature of at 20°C and a bubblestone (stone with air) that keeps the water in motion. We took their advice and came up with a solution to be able to keep quagga mussels alive (see contribution. Additionally, the researchers from the institute gave us more information about how quagga mussels live and why they like to colonize the pipes. quagga mussels can reproduce and live at low temperatures around 4-6 °C similar to the temperatures you find in the pipes. About the colonization of the pipes, they mentioned that because of the water flow, more food gets brought to them than without it.
Another point that we found very interesting in the discussions with the researchers is the negative impact of quagga mussels on the ecosystem. As they are invasive species and no effective solutions have been found nowadays, their populations are still growing. This species is an important filter, which leads to a drastic decrease in nutrients and phytoplankton found in the lake. Weakening the base of the food chain, populations of aquatic species already vulnerable (such as the Arctic char and local crayfishes) are more threatened since the quagga mussels settled in our lakes. If the actual situation doesn’t change at all, aquatic species would either coexist with these mussels, or become extinct.
Conclusion
All in all, the meetings with the researchers of the Eawag institute and the Head of the Water Biology Section were very informative and we could understand better than before how quagga mussels can be kept alive outside of the water. Those meetings also allowed us to obtain information about quagga mussels and their impact on a the ecosystem as well as the economy. We realized how important it is to act against the quagga mussels. With this new knowledge, we are more determined than ever to find a solution to kill the mussels.
Design
During the design of our Quagg’out project, by dint of information, many ideas came to our mind. Among them, for example, is a bacteria biofilm that would cover the pipe and actively secret your toxin and the anti-attachment molecule. Unfortunately, biofilm or Engineered Living Material did not seem like a feasible solution for preventing and removing mussels attachment on water treatment pipes. This is why we decided to go for a more traditional approach. In the end, we decided to study the FitD toxin in order to eradicate the mussels. We designed plasmids, constructs and experiments that would allow us to study the efficacy of enhanced FitD toxin expression compared to wild-type FitD from P. protegens that is currently used as a commercial product, Zequanox, for combating mussels in the US.
Build
With our design in the pocket, it's time to go into practice and create our plasmids. To do this, we created our plasmids in silico. As we described previously (cf. design), we inserted our genes of interest into the plasmids. Thus, the fitD gene, as well as the fitG gene, an activator for fitD expression, were inserted into plasmids allowing their expression after transformation into bacteria (cf. design). Subsequently, in the laboratory, the steps of our design were put into practice and modified according to our results when necessary. We then had to think of the form our product would take. After some deliberation, we decided to present it in powdered form, mimicking the commercialized product Zequanox, with higher mussel killing efficacy compared to the wild-type strain used for Zequanox (cf. proof of concept).
Stakeholder feedback
When carrying out a project, a very important step is to (1) do our research and (2) talk directly to professionals. Therefore, we contacted many scientists. Brigitte Schmidt, working at the water service, informed us about the legislation concerning GMOs. For example, she explained to us the danger of releasing living cells into nature. In addition, we decided, after discussion with the water service, to create a sub-project consisting of the expression of zosteric acid with anti-adhesive properties (Jendresen & Nielsen, 2019). Brigitte Schmidt and Nathalie Menetrey also gave us good indications regarding the survival of mussels in the laboratory. Other changes have taken place in the design of our project. Initially, we thought we could apply a coating for the water pipes, which was not feasible according to the experts which have led to some redesign of our project. Our current idea is to use a powder. However, the professionals at the Maison de la Rivière advised us against using this solution because of its instability and the fact that it must be suspended in a liquid solution so that it can be released easily into the pipes. We considered this input, and looked into ways of potentially commercializing a liquid product. The powdered product is, however, commercially used at the moment and remains for now the most stable and the most straightforward way of implementing our product. Thus, for FitD we would like to try solutions, if possible, or a powder. For ZA, we might put a high concentration of the product, or try to chemically link the molecule to the pipe surface to produce a protective coating.
Learn and re-design
Stakeholders gave us their opinion on the design of our project. Fruitful discussions with professionals and people affected by the quagga mussel invasion allowed us to question our project outline and redefine it to meet their expectations. Here are the crucial adjustments we have decided to make:
- For the FitD sub-project,we decided to lyse transformed cells rather than using unlysed cells, in compliance with Swiss legislation in connection with GMOs.
- Our stakeholders made clear that a solution to prevent the mussels invasion would be extremely valuable. This led us to create a “Zosteric acid” sub-project, to prevent the attachment of the mussels.
- Finally, we have changed our design to try and use solutions rather than powders ast our end products to meet the needs of the consumers. Thus, with these modifications we allow our project to be as close as possible to reality and that the final product is crowned with success.
Interview with the Water Department
Quagga mussels colonize a diverse range of surfaces. We find them blocking installations such as water pumps, and invading lake bottoms, boat hulls, fishing nets, and any facility or surface that are located in waters where mussels are present. This generates impressive maintenance costs, not to mention the major and worrying environmental damages (see description for ecological impact).
To understand the existing regulations of water treatment and the water system in the city we live in, we contacted the Water Department of Lausanne for this reason, B. Schmidt and E. Villalpando, engineers of the "study and construction division of the Water Department of Lausanne", agreed to meet with us. Actions of this Institution include “the collection, treatment, storage and pumping, distribution, disposal and purification, protection, and analysis of water (webmaster@lausanne.ch, 2018)”.
During our first meeting, they summarized the observation report of the current damages on their installations for us.
They are currently in charge of two sites close to Lausanne: St-Sulpice and Lutry which are respectively 50m and 60m deep. In the end, the strainer meshes are invaded as well as the uprights by quagga mussels.
This is a considerable threat for drinking water due to the fact that mussels may be present in several steps of the drinking water treatment plant that can release ammonium to the water. In addition, mussel larvae are very difficult to control and also abundantly present (it can spike up to 800 per m3). Therefore, some organizations use ultrafiltration to minimize the mussels’ passage while others (e.g. Lake Zurich) use chlorine shocks to eradicate them. In order to have maximum control of the situation, inspections are often carried out to evaluate where quagga mussels have spread and to reduce their number and their spread as much as possible. All of these details are what B. Schmidt and E. Villalpando try to counteract by proposing sustainable solutions to this invasive species.
We then introduced our project in order to bring a new and eco-friendly approach to their service. It is important to note that at this stage of the project, we had designed our project around a main method: the FitD toxin in order to have a molluscicide that we wanted to implement as a powder product.
FitD toxin is produced by living bacteria (Pseudomonas protegens) that we have genetically modified to enhance the toxin production (see design). The precious advice from these experts in this field pushed us to re-evaluate the real-life implementation of our idea. Why? First of all, products that contain or are produced by genetically modified organisms (GMOs) are subject to a specific set of regulations in Switzerland. They require authorization before being placed on the market. That means that it is not recommendable to use genetically modified P. protegens overexpressing FitD toxin. To ensure that our product does not contain any living GMOs, we have decided to lyse our strains and tested the cell lysate to kill the mussels (see measurement for further information).
We also chose to reconsider our product formulation as powder for several reasons. First, there is an American product on the market that is in the form of a powder: Zequanox®. It is derived from Pseudomonas fluorescens. After ingestion, it causes mortality by degrading the stomach and digestive glands of the mussels. However, it has not been widely used in Switzerland due to its composition and its powder form which requires resuspension step into a solution. This extra step shortens the shelf life of their product. Besides, putting a powder in solution requires an additional step that is time consuming. For this reason, we decided to implement our product as a solution (for FitD, we would use the cell lysate) (see implementation for further information). This is also in line with the feedback we received from the Maison de la Rivière.
Afterwards, we had a final meeting with E. Villalpando in order to review our project and get her opinion on it. Our project would be a solution to the problem of mussel invasion as long as it respects certain criteria that we are still working on. In this sense, the water department is in charge of water potabilization and is therefore very careful about all substances that might come into contact with it. E. Villalpando made a major point of saying that "the water composition must not be modified". Moreover, having very long pipes (1 km for St-Sulpice, 460 m for Lutry), it remains a prerequisite to know the full composition of our product, which must explicitly demonstrate that it does not represent any risk for other species. The reason behind this requirement is that it is difficult to control the flow of any substance through pipes that stretch for miles with very little access to them. Another aspect that is not often highlighted is the clogging of certain networks caused by a massive accumulation of dead mussels, presenting not only a risk for the pipes, that can get blocked by the accumulation, but also for the water quality due to substances such as ammonium released by the mussels.
Understanding the importance of avoiding any risk of altering the quality of drinking water and not relying solely on one approach to eliminate mussels, we have highlighted the other effects of wanting to kill mussels alone. For a definitive and permanent eradication of this invasive species, it is necessary to combine several actions: prevention, control, eradication and disposal of the potential waste generated by the latter. After intensive research, we have developed a second approach based on zosteric acid to prevent mussels attachment. The main goal is to prevent any contamination of the water without releasing GMOs (see Implementation).
In conclusion, all of these meetings were of utmost importance because they allowed us to better understand the current situation with the quagga mussels residing in the pipes, and to hear about the requirements for potential products against quagga mussels. This gave us the opportunity to adapt the formulations of our FitD toxin products and to develop an additional approach with zosteric acid so that we can tackle the Quagga mussel invasion both curatively and preventively.
Collaboration with La Maison de la Rivière
La Maison de la Rivière (literally : River’s House) is a foundation located in Tolochenaz, next to the Geneva lake and the Boiron river. This foundation aims to protect Swiss aquatic species and perform the research for their conservation, but also facilitates a place where academics, citizens and children can meet and exchange information and opinions about the aquatic biodiversity. You can learn more on the webpage : https://www.maisondelariviere.ch/.
As quagga mussels cause many damages to the aquatic environment, we contacted La Maison de la Rivière to present our project and discuss the subject with environmental professionals and scientists. On the 26th of July, we met La Maison de la Rivière co-director, Damien Robert-Charrue. He showed us the museum, the aquariums and explained to us the various studies that were taking place on quagga mussels. Mr. Robert-Charrue felt very concerned about this invasive species and gave us an example why. He told us that in 2018 a new canalisation that pumps the lake water for some of their infrastructures had been installed. The strainer arrived up to 25m in depth, which was enough to avoid zebra mussels invasion. Unfortunately, quagga mussels started to be more and more numerous in Lake Geneva. As a consequence, this canalisation, typically operational for 50 years, lost functionality within 2 years. In 2020, the foundation sent divers to clean the strainer but only a week later it was completely blocked by quagga shells again. Since sending divers on a weekly basis is not a viable solution, they gave up and the pump is currently on standby.
The cost of a canalisation is, according to the co-director, around 500 - 1’000 swiss francs per meter (= 500 - 1 000$ per meter). During our interview, he has shared his concern with us and has admitted that engineers probably need to rethink the pumping system because of the quagga presence. Since quagga mussels can live up to 130 meters of depth in the lake, placing the strainer deeper is not a feasible solution. Considering the situation, any ideas, however small they are, could get us a step closer to the final solution that we are looking for. Mr. Robert-Charrue suggested testing a few things:
- New materials for canalisation: nowadays, these infrastructures are more often covered with PVC or PE, which are one of the favourite attachment materials for the quagga mussels.
- New strainer morphologies: a strainer is a piece placed at the beginning of the canalisation and serves to collect the water and to take out the big particles that could block the filter. For example, a rotating strainer would prevent quaggas’ entry and attachment, while a removable strainer would facilitate substitution and cleaning (since it could be cleaned outside the water).
In addition to the inputs from the Water Treatment Department of Canton Vaud, this second idea also prompted us to develop a waterproof paint-like solution containing Zosteric Acid.
As we thought to include our genetically modified Pseudomonas protegens and Escherichia coli in the canalisation, the co-director pointed out that current regulations in Switzerland do not allow the release of GMOs in nature. Also, we realized that releasing transformed bacteria could be dangerous for other species, if we couldn’t exclude the idea of eventual dispersion out of the canalisations. Therefore, in order to apply our product with the purpose to protect both the canalisations and the environment, we have to figure out a solution to release only the components that are active against mussels: FitD toxin and zosteric acid. To do so, we thought to add a step of cellular lysis, to kill all viable cells and release their content. In addition, we reached out to biologists from the Chemical Section of EPFL and Microbiology department of the UNIL, to find the most appropriate way to separate our products from the rest of the cell content, so we will not release unnecessary compounds in the environment. For the FitD toxin application, we would use it with the aim to treat plug canalisations, as the zosteric acid would prevent their attachment to the strainer and their entry in the canalisation (cf. implementation).
La Maison de la Rivière gave us some reports written by bachelor and master students. Those reports and the meeting with Mr. Robert-Charrue, showed us the major opportunity for the use of zosteric acid: not only will it decrease the costs for cleaning the pipes and boats, but it will reduce their reproductive capacity as they need to be attached to grow up and become healthy adults.
In conclusion, all of these meetings were of the utmost importance because they allowed us to better understand the current situation with the quagga mussels residing in the pipes, and to hear about the requirements for potential products against quagga mussels. This gave us the opportunity to adapt the formulations of our FitD toxin products and to develop an additional approach with zosteric acid so that we can tackle the quagga mussel invasion both curatively and preventively.
References
- Colonization, Ecology, and Population Structure of the "quagga" Mussel (Bivalvia: Dreissenidae) in the Lower Great Lakes. Authors: Edward L. Mills, Ron M. Dermott, Edward F. Roseman, Donna Dustin, Eric Mellina, David Bruce Conn, and Adrian P. Spidle. Publication: Canadian Journal of Fisheries and Aquatic Sciences November 1993. https://doi.org/10.1139/f93-255
- Literature review: biology, ecology, and potential impacts of Dreissena rostriformis bugensis (quagga mussel), an invasive species in Lake Geneva. Authors: Beisel Jean-Nicolas, Soulignac Frédéric. Publication : Rapport de la Commission internationale pour la protection des eaux du Léman, Campagne 2020.
- Roe, S. L. & MacIsaac, H. J (1997). Deepwater population structure and reproductive state of quagga mussels (Dreissena bugensis) in Lake Erie. Canadian Journal of Fisheries and Aquatic Sciences 54: 2428-2433.
- Eawag - Swiss Federal Institute of Aquatic Science and Technology - Eawag. (n.d.).ch, Retrieved October 11, 2022, from https://www.eawag.ch/en/department/eco/projects/competition-between-filter-feeders-in-lake-constance
- Une écrevisse en PLS, rien n’arrête ces envahisseurs!(n.d.). from https://www.youtube.com/watch?v=Xt4GNt3z-xs
- webmaster@lausanne.ch, B. de la communication-Web & multimédia –. (2018, January 22). Service de l’eau. Site Officiel de La Ville de Lausanne. https://www.lausanne.ch/officiel/administration/securite-et-economie/eau.html