Team:TUDelft/Human Practices

Team

Team Members Collaborations Team Bonding

Project

Project Description Contribution Design Engineering Success Partnership Sustainability

Lab

Experimental Details Notebook Results

Models

Overview Phage infection and toxin production 2D biofilm simulation

Parts

Overview Basic parts Composite parts Part collection

Human Practices

Human Practices Integrated Human Practices Education & Engagement Inclusion Proposed Implementation Safety

Entrepreneurship

Overview Customer Product Company Business Plan

Acknowledgements

Attributions Crowdfunding Sponsors

Achievements

Judging Form Achievements Awards and Nominations

Human Practices

In the Human Practices part of our project, we take a step beyond the lab and make sure that PHOCUS would do no harm. Also look at how we integrated Human Practices into our project. At the start of our project, we identified stakeholders relevant to our project and investigated their values, norms and needs, on which we based design requirements for PHOCUS.

To assess whether PHOCUS can really benefit the people and the environment, we have put a lot of effort into engaging many relevant stakeholders in the progress of our project. Our contact with stakeholders was handled with care. We tried to do this as ethically as possible according to the principles of ethical research by Vanclay et al. [1], which will be explained in more detail below. This contact was crucial for placing PHOCUS in the local context of parts of Africa and Asia. These environments are hardly familiar to our team and therefore, we put in extra efforts to identify their needs. For instance, we conducted a questionnaire among Kenyan and Indian farmers that were affected by locust swarms, we interviewed authorities (the FAO and a national ministry) on how they operate locust control, and had a discussion with locust control experts, governments and non-governmental organizations at the Virtual Practitioners Conference on Desert Locust Control Management 2020, where we presented a keynote lecture. Over the entire course of our project, the design requirements for PHOCUS changed by the input of many stakeholders, who gave us multiple rounds of feedback on the design of our biopesticide.

Not only the local social and economic context where we aimed to innovate was considered, also ethical, legal, biosafety and biosecurity factors were thoroughly investigated by talking to experts and other stakeholders. An elaborate overview of the biosafety design measures we deduced from these insights and literature can be found in Safety. Most importantly, our solution was designed to be safe to humans and the environment.

Finally, we considered any unintended effects of PHOCUS. We learned that, from a safety risk perspective, our project is a very interesting one to study. Our project inspired Andrea van Langen to write her Bachelor Thesis at the TU Delft Biotechnology & Society group about responsibility and safe-by-design considerations of PHOCUS. Also, PHOCUS is proposed as a case study that eventually could be used by partner organizations, of which i.a. the Dutch Ministry of Infrastructure and Water Management. Within the team, we also discussed our responsibilities and risks involving our project with the guidance of the iGEMers Guide to the Future. From this discussion, we learned that we as a team may hold different perspectives of the notion of responsibility, but we should always do our best to mitigate risks and prevent unintended usage.

Responsible innovation

Important to us, and the iGEM competition in general, is that the technology and ideas we develop aid in the improvement of society, by solving a large societal problem. It is paramount to consider how a project can impact society with a broader view than just solving the identified problem. By participating in Responsible Research and Innovation, the goal of innovation shifts from "science in society" to "science for society" [2]. It is vital to identify different groups of people, who will be affected by or have influence on your project, these groups are called "stakeholders". Stakeholders have different values, norms and needs that could clash and need to be taken into account during the design of our project. Overcoming these differences is challenging, but could lead to a more complete solution to the problem that is beneficial to society. On top of that, it is key to recognize that our own values are embedded in the technologies we develop, which opens up our project to prejudice among other things [3].

The transfer of our project from our personal perspectives to that of the stakeholders was done through ensuring that their values and norms are incorporated into a Value-Sensitive Design. First, the key stakeholders were identified. These stakeholders were positioned on a power-interest grid, which is a visual tool that aids in understanding the implications of the power and influence of stakeholders on our project [4]. From this, we identified appropriate communication strategies and prioritized the values and needs of powerful stakeholders with a lot of interest in our project. Second, interviews with the key stakeholders were conducted to obtain an understanding of their values and norms. Specifically, the purpose of these interviews was to gain insights in the stakeholders’ perceptions of the problem and the current and desired solution. The different values and their associated norms of the stakeholders not only gave us insight into what each individual finds important, but also provided an overview of the collective. This helped to identify potential clashes between values and indicated which values should form the core of our technology. These values were, in turn, translated into design and operation requirements. Due to the size of our analysis, we only show an example here. Figure 1 depicts how the value “food safety” has been translated to a norm and design requirements. A norm depicts how we interpret the value as a sort of rule, e.g. for something to be safe, food needs to have no or little hazardous compounds on or in it. From thereon, specific design requirements can be derived as to how to uphold the norm. There are multiple ways as to achieve this, hence multiple design requirements can be derived.

Figure 1. Value pyramid of the value “food safety”. This pyramid describes how the value of food safety is translated to a norm and how from this norm design requirements can be derived.

After identifying the values and norms of stakeholders and deducing design requirements, clashes between these were determined. As an illustration, one of the main clashes that was identified is the clash between the values “environmental safety” and “food security” in choosing between a lytic or lysogenic bacteriophage. A lysogenic phage does not spread as fast as a lytic phage and therefore, may be considered to be more environmentally safe. On the other hand, the fast spread of such a phage in the gut microbiome may result in the production of higher toxin concentrations, which benefits food security. However, a lysogenic phage integrates into the bacterial genome, which allows for horizontal or vertical gene transfer, which may have unintended side-effects. Thus, a lysogenic phage may be less environmentally safe than a lytic phage. Ultimately, we decided on a lytic phage.

A stakeholder value matrix was used to establish to what extent our design iterations were in accordance with the stakeholders’ values and was used as a tool to monitor our progress towards meeting as much of the values as possible. Whether design iterations complied with the values of the stakeholders was repeatedly tested through feedback loops of stakeholder interaction.

Stakeholder approach

A potential pitfall for scientists and engineers is to be too focused on their invention being the only solution to a problem, they do not take into account potential environmental influences. Moreover, they may not consider that their potential adopters may have different perceptions of the problem and the desired solution. By improper communication about the perspectives of stakeholders, scientists and engineers may not take into account the context they are inventing for. This could mean that the implementation of this solution will not have the impact its inventors aspired it to have.

We acknowledge that, to make a societal impact with our innovation, we have to be aware of who our stakeholders are, what they value, and keep them involved in our project. Therefore, from the start of our project, we placed great emphasis on approaching, engaging and analysing stakeholders in the appropriate way. Here, we describe our process of identifying and managing our stakeholders.

Identification of stakeholders

By identifying groups who have an interest in solving the locust crisis or in our project itself, we have identified and engaged stakeholders who may play a role in developing and implementing PHOCUS in the real world (Figure 2).

Figure 2. Interactive stakeholder web, containing most stakeholders with whom we had contact throughout the development of PHOCUS. Clicking on a specific person will display a specific quote each person gave, that influenced the development of PHOCUS. A more detailed information on what we learned from these people can be found in the timeline.

• Farmers in affected areas are the ones that would benefit the most from the implementation of Phocus, as these people suffer from crop losses and are exposed to harmful chemical pesticides. Hence, the integration of their values and perceptions into our project have high priority.
• The end-users of Phocus are the people and organizations that are currently responsible for locust control management:
  • The Food and Agriculture Organisation (FAO) of the United Nations is responsible for global locust control. It aids national governments financially and operationally in their locust control efforts.
  • National governments of affected areas are responsible for national locust control efforts. In practice, they are highly dependent on the FAO.
• The end-users are also regulators, such as national governments, and therefore, play a key role in acquiring admission for Phocus to be applied.
• The public at large has to be generally accepting of using genetically modified bacteriophages in their country. This includes civil society organisations, such as environmental organisations.
• Companies in the agrochemical industry may support our project through providing us information or funding, or, in case they feel threatened by upcoming competition, form hurdles against us through lobbying.
• It is important to engage the scientific community in our project to obtain technical feedback on our project.

Reaching out to stakeholders and establishing feedback loops

We approached our stakeholders through email. In this email, we introduced ourselves, our project and why we are interested in approaching that particular stakeholder. Also, we asked whether they would be interested in sharing their insights in an interview. Unfortunately, due to the COVID-19 pandemic, these interviews had to be held online. However, meeting online lowered the threshold to meeting with people worldwide, it allowed us to reach out to stakeholders not only in the Netherlands, but also, for example, in Africa and North-America.

In general, we tried to adhere to principles of ethical research by Vanclay et al. [1], for instance, informing interviewees about our intentions, not giving them hope that PHOCUS will actually be implemented anytime soon, ensuring their voluntary participation, communicating the results and allowing them to change these, and asking for permission to share any of the results on the Wiki.

Before the interview, we prepared questions and sent these to the interviewee in advance. At the start of the interview, we introduced ourselves, our role in the team, and our project to the interviewee through a presentation. In addition, we asked for informed consent to share our insights from this interview, including the name and photograph of the interviewee, on our Wiki page and whether it was okay if we would make a transcript. After the presentation, we performed a semi-structured interview using the questions that we sent. Once the interview was finished, we summarized the key points from the interview and sent it to the interviewee for additional permission to publish the exact words. In some cases, we introduced our project idea in a later stage of the interview as not to influence their description of the problem.

Every two months, we sent the stakeholders that we talked to up to that point an email, in which we communicated the progression of our project and the way in which their interview impacted our project, to keep them engaged. Sometimes, this yielded interesting replies that also resulted in an adjustment of our project.

Managing stakeholders

The following chapter will explain with whom and how we have had contact throughout the project.

• End-users: We had continuous contact with two locust control experts, Dr. Mamoon AlSarai Alalawi and Prof. Alexandre Latchininsky, from the FAO throughout the project, both via video calls and emails. They taught us locust control is only performed by professionals. In addition, we have had online meetings with two locust control experts who used to work for the FAO for many years: Em. Prof. Arnold van Huis and Harold van der Valk. We presented our initial plan to them, which was to stop the swarming behavior of the gregarious locusts. When both of them advised us not to do this because this strategy may do more harm than good, and to kill the locusts instead, we changed our entire project. We also had continuous contact through video calls and emails with Susan Nguku, Agriculture Extension Officer of the Kenyan Ministry of Agriculture. Susan is a representative of the national government of Kenya, which is one of the countries that is most affected by locust swarms. She told us about how scared people got when locust swarms arrived.


• Farmers in affected areas: Affected farmers were very difficult to reach out to. We tried to establish contact with multiple farmer organisations in parts of Africa, but with no success. Ideally, we would have gone to one of the affected regions, but unfortunately, travel restrictions due to COVID-19 did not allow us to go there. Luckily, Susan Nguku, from the Kenyan Ministry of Agriculture, works very closely with farmers that have been affected by locust swarms and was able to articulate their experiences to us. In addition, she helped us in reaching out to farmers through conducting our questionnaire with Kenyan farmers. We also got in contact with farmers from India through our contact person, Shanu. Although both countries suffer from the same problem, we were wondering if there were any differences in how they perceive the problem and solution.


• Regulators: The use and release of genetically modified material is under strict regulation. We reached out to Cécile van der Vlugt, a senior risk assessor from the Dutch National Institute for Public Health and the Environment. She is specialized in risks associated with GMOs. We discussed and assessed different types of risks associated with PHOCUS. Our lack of additional biocontainment strategy should not pose a problem if we can prove our claim that it will not propagate in nature.


• Public at large: We reached out to many environmental organizations, such as Greenpeace, Friends of the Earth, ETC group and Milieudefensie. Unfortunately, none of them wanted to discuss the impact of chemical pesticides on the environment or their perception of PHOCUS with us.


• Companies in the agrochemical industry: Companies in the agrochemical industry: We reached out to many agrochemical companies, both small and large, to raise awareness of the biopesticide we were developing, to obtain feedback on PHOCUS and to see whether we could help each other in any other way. We had a meeting at our faculty in Delft with Remco and Gerard Bod from Simonis BV, which is a Dutch insecticide and pesticide exporter and one of the official suppliers of the FAO. They elaborated on the approval process of new pesticides. They were enthusiastic about our project and even showed great interest in making an investment. After our meeting, we kept email contact.


• Other people from the industry we have had contact with include dr. Frank van Steenbergen from MetaMeta, a company involved in bottom-up locust control, and dr. Steven Hagens from MICREOS, a company involved in improving food safety and quality using bacteriophages. After talking to dr. Frank van Steenbergen, we even got invited to present a keynote lecture about our biopesticide at the Virtual Practitioners’ Conference on Locust Management 2020. This conference was also attended by representatives of a.o. FAO, Oxfam Novib UK, International Centre for Insect Physiology and Ecology, Pakistan Ministry of National Food Security & Research, Kenyan Ministry of Agriculture, University of Bath, Penn State University, University of Graz, Arizona University. Speaking at this conference provided us with the great opportunity to talk to locust control experts and scientists from all over the globe. This would have never happened if we did not repeatedly keep dr. Frank van Steenbergen involved in our project. From this, we learned about the state of locust control by the FAO and the national governments of India, Pakistan and Kenya, and about the newest scientific accomplishments in this field. Current trends in locust control include a.o. community-based surveillance and satellite-based monitoring of developing locust swarms. From a talk by Mohammed Khurshid from the Pakistan Ministry of National Food Security Research, we learned that locusts are a very nutritious source of food. This inspired us to investigate the idea of using dead locusts as a sustainable food source. This idea is elaborated upon further in the Integrated Human Practices section.


• During the course of our project, multiple researchers from the scientific community were of great help for us regarding the technical details, and safety, security and ethical considerations. For instance, based on advice from Em. Prof. Arnold van Huis and Harold van der Valk, we stopped our initial plan of stopping the swarming behavior of locusts and took on the strategy of killing the locusts. From Harold van der Valk, we were taught that Bacillus thuringiensis (Bt) toxins generally do not pose safety concerns to humans or the environment, which made the use of the locust-specific Bt toxin Cry7Ca1 a potential candidate for killing locusts. From Prof. Jozef Vanden Broeck we learned that desert locusts have a strong response to RNA interference (RNAi). This can be used as an effective way of halting essential gene functions, thereby killing the locusts. Therefore, we pursued this strategy. From Omer Lavy, we learned that the composition of the locust gut microbiome is not stable over time. Therefore, we decided to use a cocktail of engineered phages to infect the bacteria that are most prevalent in the desert locust gut according to his metagenomic data.
  
  The field of research in which the scientists we talked to work include:
  • Bacteriophage biology: Dr. Franklin Nobrega and Dr. Steven Hagens
  • Plant pathology: Prof. Jeffrey B. Jones
  • (Tropical) Entomology: Prof. Jozef Vanden Broeck and Em. prof. Arnold van Huis
  • Insect physiology and ecology: Prof. Baldwyn Torto
  • Locust population dynamics: Dr. Cyril Piou
  • Toxicology: Harold van der Valk
  • Microbiology (locust gut microbiome): Omer Lavy
  • Biosafety and biosecurity: Dr. Piers Millet
  • Bioethics: Dr. Zoë Robaey

Responsibility and risks

When designing and constructing a novel technology, it is important to be conscious about the risks and responsibilities that this brings about. Our project revealed to be a very interesting case for studying responsibility and safe-by-design. It inspired Andrea van Langen to use PHOCUS as a case study for her entire Bachelor Thesis at the TU Delft Biotechnology & Society group, titled “Bacteriophages as agents against locust swarms: A Safe-by-Design study on the application of gene and microbiome therapies” [5]. In addition, the PHOCUS project is proposed to be used as a case study for a workshop which is currently being developed by a PhD researcher at TU Delft. The goal of this workshop is for participants to gain insight in what values are associated with potential risks, and how to identify ‘new’ risks. Eventually, this workshop could be used by, amongst others, the Dutch Ministry of Infrastructure and Water Management (IenW).

To prevent unintended consequences of PHOCUS, we tried to think of as many bad effects as possible. The most relevant bad effects from this list were discussed within our team and with biosecurity expert Dr. Piers Millet. For the discussion within our team, we made use of instructions of the SynBio conversation tool from the iGEMers Guide to the Future. On posters we wrote down five different provocative statements or scenarios involving risks of our project and the notion of responsibility. On the top, our team had to write down the possible causes using post-its and at the bottom, the possible consequences. Afterwards, the findings were discussed in the group, from which we drew conclusions. The statements or scenarios, including their possible causes and consequences and the conclusions we drew from the discussion, are stated below.

"Malicious use of PHOCUS is our responsibility"

We feel like the benevolent use of PHOCUS is our responsibility as we are, to a certain extent, able to anticipate potential uses. Also, as engineers we hold the responsibility to limit the extent to which PHOCUS is prone to malicious use. However it is difficult to foresee everything, but it is our responsibility to anticipate most. As a consequence of our responsibility, we make it as safe as possible, which is an upside of this sense. However, it also hampers technological advancement when there is a fear of dual use potential, which can be both good and bad. This is because together with responsibility may come accountability. In conclusion, we should do our best at making PHOCUS as inherently safe as possible, but we are also aware that not everything can be reasonably prevented and safety can never be guaranteed and therefore we cannot be responsible for everything PHOCUS is used for.

"PHOCUS has become an uncontrollable and mutated virus"

PHOCUS may become uncontrollable when it would spread everywhere and persist in nature. Would we have the tools and resources to monitor it or to stop it? We discussed that no kill-switches have ever been designed for phages, meaning we cannot just kill the phage in a particular condition to prevent it from spreading. We came to the conclusion that due to competition with wild-type phages for host infection, together with reduced fitness, PHOCUS will not persist in nature. Also, PHOCUS kills its hosts, which decreases its own chances of replication, a form of self-limitation. This means that PHOCUS becoming uncontrollable is improbable, but still should remain at the back of our minds throughout the development.

We also discussed what could happen if PHOCUS becomes mutated. A chance exists that a random mutation in the phage genome occurs. However, for this mutation to form a problem, it must be one that leads to pathogenicity of the phage or the production of a compound that is harmful for anything other than the desert locust. Furthermore, for this mutation to persist in nature and take over the population, it would be one that has a competitive advantage. Thus, we feel this is improbable, but still not impossible and should not be forgotten.

"Overzealous use of PHOCUS has eradicated the desert locust"

The overzealous spraying of PHOCUS could be caused by a lack of communication between countries and insufficient training of the people who would apply PHOCUS. The eradication of the desert locust could lead to an imbalance in the ecosystem, since it is part of a food chain. The exact consequence is difficult to predict. However, we seriously doubt whether this could ever lead to the distinction of the desert locust. Still, we turned to locust population dynamics expert Dr. Cyril Piou for advice.

"PHOCUS is harmful for (human) health and environment"

We already thought extensively about whether the phage, the Cry7Ca1 toxin or shRNA for RNA interference could be bad for (human) health and the environment. Using this statement, we wanted to investigate whether any unforeseen risks exist.

PHOCUS could be harmful for the environment if the phages that are sprayed on the plant kill all of the bacteria, and if this would affect the wellbeing of the plant or animals or hamper the role bacteria play in nutrient cycling. However, the chances that this happens are small, because we aim to place a physical barrier between the phage and bacteria when the phage is not in the locust gut.

Another way in which PHOCUS can be bad for people or animals is when the phage mutates in a way that the toxins become toxic for other beings than locusts. We decided to talk about this issue with phage expert Dr. Steven Hagens.

Another issue we have considered is what would happen if, for instance, a bee would ingest PHOCUS. Would this kill the bacteria in its gut? And if the bee has a symbiotic relationship with its bacteria, would this affect its wellbeing? We came to the conclusion that, similar to humans, these symbiotic bacteria probably maintain in the gut through a sort of mucus layer formation, meaning that the chance that all of it will get killed by phage infection is small. Still, we should be careful in determining a target bacterium and should do appropriate safety testing before release.

The consequences of this scenario occurring could be detrimental: it may lead to serious injury, loss of biodiversity, trigger phage resistance, and reduce crop variability. Moreover, people would blame us and biopesticide may get a bad reputation and therefore, may not be used anymore.

"PHOCUS can be used as a bioweapon"

It was discussed that theoretically, a phage can be engineered to make a bacterium produce anything. As bacteria are everywhere, this would allow you to produce anything anywhere. We decided to reach out to biosecurity expert Dr. Piers Millet to help us to assess this risk.

From the first discussion point, we obtained awareness of the fact that we all have different perceptions of responsibility. Still, we all find it important to do our best at ensuring that risks involved with PHOCUS are minimized.

Even though we think that PHOCUS becoming a mutated virus is not probable, we decided to ask phage expert Dr. Steven Hagens what the chances are to be sure. Dr. Hagens agreed with our thoughts on the chances of PHOCUS becoming mutated. In addition, he told us that the phage genome has evolved for millions of years to get to the optimal state where it is at, and therefore, generally any mutation will reduce the phage’s fitness. This means that this will not lead to a competitive advantage and will not sustain in nature. Based on this statement, it could be said that the chances of PHOCUS becoming mutated in a way that would be harmful for humans and other animals is negligible.

Based on our discussion of the third scenario, in which overzealous use of PHOCUS has eradicated the desert locust, we asked locust dynamics expert Dr. Cyril Piou whether this is possible. He assured us that there will always be some solitary locusts somewhere minding their own business, hence, our method does not risk eradicating the locust species.

The discussion of PHOCUS being potentially used as a bioweapon motivated us to talk to biosecurity expert Dr. Piers Millet. From our discussion with Dr. Millet, we learned that biosecurity issues arise from platform technologies. Therefore, we decided not to advocate nor explore the potential of PHOCUS being used as a platform technology, but instead, maintain focus on its niche application, which is killing locusts.

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Make sure to read how interaction with stakeholders has shaped our project in Integrated Human Practices!

References

1. Esteves, A. M., Franks, D., & Vanclay, F. (2012). Social impact assessment: the state of the art. Impact Assessment and Project Appraisal, 30(1), 34–42.
2. Owen, R. Magnaghten, P., Stilgoe, J. 2012. Responsible research and innovation: From science in society to science for society, with society. Science and Public Policy. 38 751-760
3. Winner, L. (1980). Do artifacts have politics? Daedalus, 109 (1), 121–136.
4. Ackermann, F., Eden, C. 2011. Strategic Management of Stakeholders: Theory and Practice. Long Range Planning 44 179-196.
5. van Langen, A. (2020) Bacteriophages as agents against locust swarms: A Safe-by-Design study on the application of gene and microbiome therapies. Retrieved on 20-8-2020 from https://repository.tudelft.nl/islandora/object/uuid%3A813d761c-8a6f-481c-8e51-ee6a594327a1?collection=education

Dr. Franklin Luzia de Nobrega

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Who?

Dr. Franklin Nobrega is a head of the Microbial Interactions Lab in the School of Biological Sciences at the University of Southampton.

Why?

The goal of this meeting was to discuss our idea of genetic engineering a bacteriophage to use it to turn the gut microbiome of locusts into a cell factory that produces toxins and kills the locust from inside out.

What?

• As proposed by Franklin, for the genetic engineering of our bacteriophages, we used the bacteriophage recombineering with electroporated DNA (BRED) protocol.
• The lytic property of the bacteriophages is part of our biosafety plan. When the bacteriophages kill all their hosts, they will die as well, because quorum sensing ensures that the amount of host bacteria always maintains a level that is sufficient for bacteriophage survival, we decided that it is safer to identify the genes responsible for quorum sensing in bacteriophages and remove them.

Susan Nguku

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Who?

Susan Nguku is an Agriculture Extension Officer at the Ministry of Agriculture in Kenya.

Why?

To learn how the locust swarms affect the lives of people in Kenya and how the Kenyan government approaches this crisis.

What?

• Some people in Kenya have never seen locusts before. When these arrived, they were in shock. There was a nationwide warning by the FAO that locusts would come. However, in the local communities, there were rumors, but no real communication. Still, even if the message could reach these locals properly, they would not have the tools to do anything about the locusts.
• Locals revert to anything at hand to scare away the locusts from their land. Such as banging pieces of metal to black magic.
• Barriers for acceptance by locals include usage of a virus, as the term virus is associated with disease. Even though it might be beneficial that mindset is hard to get out of people's minds.
• Even with all the fears, some people would be interested if the pesticide is fully approved for safety by a governing body (such as ICIPE, KEBS and FAO) and if there is some lobbying for the product.

Shanu

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Who?

Shanu is a resident of Jaipur, India where the locust swarms raided the city earlier this year.

Why?

To consider the cultural and socio-demographic differences in the perception of the locust problem by interviewing someone from another affected country.

What?

• Indian citizens trust the government when it comes to the safety of approved pesticides. Even more so if the government is responsible for the application.
• Since this was the first time the locusts had been to the area in decades, the city dwellers were only startled by the locusts but those who made a livelihood of agriculture were severely affected.

Value Sensitive Design

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As described within the Human Practice (silver) page, we have conducted a Value-Sensitive Design (VSD) assessment. The main conclusion from this where:

• An overview was created of our key stakeholders, indicating those whom we needed to reach out to throughout the progression of the project. Our Power-Interest grid indicated the appropriate communication strategy, as described in our stakeholder approach.
• We identified the values and norms of our stakeholders and translated these to design requirements for our project. Especially the value of safety came up extensively.

Omer Lavy

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Who?

Omer Lavy is a PhD student at the Tel Aviv University of Israel at the Department of Zoology. His major research focus is on behavior and neural mechanisms of behavior in invertebrates, and specifically insect models.

Why?

Omer Lavy investigated the Effect of Density-Dependent Phase on the Locust Gut Bacterial Composition. In his publication, he characterized the microbiome of the desert locust.

What?

• As we could not confidently identify a certain bacterial species in the locust gut from literature, we decided to use the conclusion of Omer Lavy that the Enterobacter genus was widely present in most Locusts. Therefore, we decided to tackle the locust problem by creating a phage cocktail targeting the Enterobacter genus.
• In the dry lab, a key parameter is the amount of (target) bacteria that are present in the locust gut as this is directly correlated to the amount of toxin that can be produced. He, therefore, sent us some papers that quantified the number of bacteria in the locust gut, giving us a good estimate to use in our models.

BeNeLux Meetup

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From the 22nd till the 25th of July iGEM Groningen organised the digital Belgium, Netherlands, Luxembourg (BeNeLux) iGEM meetup. During these days we had the opportunity to present our project not only to our peer teams, but also to iGEM judges. From these judges we received all kinds of feedback. Most important was the potential parallel between our project and the Insect Allies project from the Defense Advanced Research Projects Agency (DARPA) from The United States Department of Defense. A project that had gotten a lot of attention for its dual-use concerns. Hereafter we reached out to iGEM HQ, to address these concerns, and had an internal discussion on our responsibility as scientists.

Dr. Cyril Piou

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Who?

Dr. Cyril Piou is an ecologist specialized in locust population dynamics, affiliated with CIRAD.

Why?

Experienced with locust control in Western Africa and has experience with the FAO.

What?

• Taking preventative measures against locust swarms is very important because these are hard to control once there is an outbreak. Since locusts tend to breed in remote locations, good forecasting methods have to be in place to understand where a locust can breed. Drones can be used to monitor and control the locust population in these remote areas.
• The relationship between media attention and locusts is cyclic. When there's a locust upsurge, the media pays attention to it. Media attention is important to obtain financial donations from other countries, such as France, to help set up locust control operations. The affected countries themselves are often not sufficiently responsive. The development of the logistics and practice of these operations continue for a few years until the locusts are gone and people don't find locusts a problem anymore. At that point, preventative measures are not being taken anymore and the upsurge returns.
• In the case of Green Muscle: Many people didn't know how to use it. Education of farmers needs to be encouraged so they understand how to use the biopesticide, the limited side effects, why it's much safer than chemical pesticides and why it's worthwhile waiting for 10 days for the locusts to die.

Em. Prof. Arnold van Huis

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Who?

Emeritus Professor at Wageningen University. Currently spending all his time on the journal, Insects as food and feed. Worked on Integrated Pest Management projects from the FAO.

Why?

Worked on locust control in both the African region and Australia, including being the dutch representative at meetings concerning the desert locust.

What?

• People will only use locust as food if they are caught alive. The risk of potential health hazards is too big when they are plucked off the ground dead. Still there exists a health hazard if sub-lethal amounts of pesticide are sprayed on the locust, as these can be eaten during an upsurge. Making PHOCUS safe for human consumption would relieve this health hazard.
• Controlling locusts by returning them to their solitary state is infeasible as a control option, as now you have to deal with many individual and dispersed locusts.
• Usage of pesticides is a political problem as well. People want an answer to their problems that is quick. Efficiency and effectiveness of pesticide spraying in general is under some debate, as sometimes spraying does very little to a swarm, while changes in weather conditions often have huge impacts on swarms.

FAO CRC

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Who?

Dr. Mamoon Alsarai AlAlawi, Executive Secretary for the FAO Commission for Controlling the desert locust in the Central Region (CRC). Cairo, Egypt.
Prof. Alexandre Latchininsky, Agriculture Officer at the Locusts and Transboundary Plant Pests Team (AGPMM) from the Plant Production and Protection Division (AGP)of the FAO. Rome, Italy.

Why?

FAO is the global authority responsible for locust control.

What?

• The FAO policy is that locust management should stay in the hands of educated professionals who are specifically trained to deal with locusts. Next to experts in locust control, the FAO works closely with governments. Governments lead the control of desert locusts because it is costly. Farmers cannot afford it. The Member States of the FAO trust that this organization chooses the right technology for control operations, pesticides and sprayers.
• We still rely on conventional pesticides and this is not feasible from an ecological and human health standpoint. There is a need to produce a pesticide that is fast, easy to use and environmentally safe. Metarhizium works but it is not the best solution. The main benefit is its specificity, but the drawback is that it takes 1-2 weeks to actually kill the locust.
• It is important to construct a biopesticide that is able to withstand high temperatures and UV radiation. Behavioral fever has been a problem when using biopesticides. When locusts are treated with the fungus Beauveria bassianaBeauveria bassiana, the locusts sense that they are infected. As a result, they position their body in a particular angle towards the sun, which increases the temperature and the UV radiation on the fungus. As a result, the efficacy was only 20-30% on the field, whereas it was high in the lab.

Harold van der Valk

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Who?

Harold is an environmental toxicologist for over 20 years. Previously he worked with international organisations like the FAO, UNEP and WHO.

Why?

Early on, he worked with the FAO on pesticides and locust control. Currently he has his own consultancy bureau called Falconsult, once in a while he is still involved with locusts when it concerns health and environmental impact.

What?

• In an ideal situation locust control is done by small organisations. Small groups of people travel into the desert for multiple weeks to search for locusts, somewhat resembling guerrilla warfare. Although they have coolers, often these reach temperatures of 20 degrees celsius and we should keep this in mind during the design of our biopesticide.

"the people tackling locusts need to be like locusts, who move quickly"

• As locusts move a lot a specific area might be only sprayed once or twice. Leaving the effect of the chemical pesticide on the environment to a minimal level. This is an acceptable level concerning human health if this is done by professionals, who are properly trained and protected. If lay people use these techniques there are health concerns.
• There are concerns with the part of our project aiming to "de-gragarize" locusts. Harold is concerned we will effectively create "super grasshoppers". Solitary locusts are too spread out to target with pesticides, making control of them impossible. It is actually a virtue that locusts form dense swarms, as now there is concentrated target to spray. By degragerizing the locusts, the large number of individuals will spread out and reproduce, only spreading out the problem and possibly making it harder to control.
• There are many BT insecticides/pesticides being used around the world, producing these with PHOCUS should not pose a problem.

Prof. Jozef Vanden Broeck

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Who?

Jozef Vanden Broeck, Professor in Animal Physiology and Neurobiology at KU Leuven, studies the physiological mechanisms implicated in the regulation of important post-embryonic developmental processes of insects, such as the phase polyphenism in locusts. In addition, he investigates the mechanisms of systemic RNA interference in insects to explore novel strategies for pest control.

Why?

We met with Jozef Vanden Broeck to obtain information about the gut physiology and microbiome of locust species. Furthermore, we were interested in insights we could provide in the use of toxins and RNA interference as pesticide. Finally, we reached out to him to discuss ethical considerations in using insects as a research object.

What?

• It is difficult to bring a toxin into the gut that would survive the gut conditions of the locust. Its enzymatic capacity is huge and the conditions during digestion are very harsh for having biological molecules to survive in.
• RNA interference works perfectly at very low concentrations in locusts if you can get it into the hemolymph. Locusts have evolved to have a very strong defense system against viruses because they are occurring in high density populations when they are swarming.
• According to Dr. Franklin Luzia de Nobrega (University of Southampton), bacteriophages survive in neutral conditions, somewhere between a pH of 6 and 8. According to Jozef Vanden Broeck, the pH of the locust gut is somewhere between 7 and 8, the gut is in terms of pH generally survivable for bacteriophages. This means that we do not have to take this into consideration anymore when deciding on the design requirements of PHOCUS.
• Real locusts are used in Jozef Vanden Broeck's lab, not cell lines. Regulations regarding conducting experiments using locusts are based on biosafety, not at the ethical level. Locusts are legally not considered in the bureaucracy like for mice. This stems from the fact that a lot of insect science is focused on killing them.

Frank van Steenbergen

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Who?

Frank van Steenbergen is the owner of MetaMeta. MetaMeta is a value-driven company that is primarily trying to introduce solutions for water management. They try to do things that otherwise do not get done, i.e. challenges that are not addressed or overlooked also outside of water management.

Why?

Understanding the context of the affected areas from someone with local experience and contacts.

What?

• Frank feels very little is done regarding the locust upsurge, specifically the communication thereof. Media coverage is often superficial and not taking any initiative to find a solution.
• "The knowledge of the few can empower the many", Frank said to describe the current imbalance of knowledge between experts and the ones affected. He sees the lack of knowledge leads people to use ineffective measures.
• Considering implementation and opposition, Frank advised to get some of our potentially strongest opponents on our side as soon as possible. Integrating some of their needs and values into our project would see a lot of benefits regarding acceptance.

Locust Conference

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We presented our project at the 1st digital practitioners conference on desert locust control management, organized by Frank van Steenbergen (link). A wide array of 13 different speakers told their stories. Ranging from different ways of locust forecasting to stories of how locals experience such a crisis. Often with chilling stories of how their livelihoods are threatened. It was a combination of the causes, effects, current solutions and novel potential solutions. Many different facts and insights were gathered over the day, we answered questions and partook in the networking event afterwards.

RIVM Safe-by Design Webinar

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We were invited by the Dutch National Institute for Public Health and the Environment (RIVM) for a webinar on Safe-by-Design (SbD). During this webinar the what, why and how of SbD were discussed with all Dutch iGEM teams. Giving insight into how to effectively apply SbD into your iGEM project.

Dr. Zoë Robaey

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Who?

Zoë Robaey is Assistant Professor in Ethics of Technology at the Philosophy Group of Wageningen University.

Why?

To discuss our project with her, so that we create our product with knowledge of our moral responsibility.

What?

• People work differently in different areas of the world - when a new technology is integrated in a different country it is important to think of the local context and not take how our countries are organised for granted.
• It is not enough to compare biocontrol to chemical pesticides in terms of problems that chemical pesticides bring. There is a need to study the new problem or solutions brought on by bio-control, for example, the effect on the ecosystem and other unintended side effects.
• A discussion with entomologists and/or ecologists would be interesting to know what ecological systems might be disrupted by these phages or killing large amounts of locusts. Therefore we discussed this with both Arnold and Cyril, the latter stressed to us that this would be impossible to eradicate locusts without the use of something resembling a genedrive. Arnold introduced us to a book describing the extinct North American locusts.

Dr. Piers Millet

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Who?

Dr. Piers D. Millett is Vice President for Safety and Security at iGEM and co-chairs iGEM’s Safety Committee.

Why?

The aim of the meeting was to discuss all biosafety and biosecurity measures we took so far. Specifically, we wanted to validate whether we could say that our biopesticide is safe and does not raise any dual-use concerns.

What?

• The safety of our project relies on whether the Cry7Ca1 toxin is as specific as it is claimed to be. We know from two research papers that the Cry7Ca1 is specific to locusts, but how sure are we really? To ensure the specificity of the Cry7Ca1, it can be assessed by testing the selectivity and by performing field trials. Before we can do any field trial, we have to convince regulators to allow for these trials to happen on their ground. Therefore, first of all, we should consider which steps are between the lab and the real-world to find out which data we need to convince the regulator.
• We can back our argumentation that the genetically engineered phage does not require an additional containment mechanism by validating in the lab that, after some time, the genome sequence of the phage will return to wild type. This can be done by determining the genomic stability of our phage using serial passage experiments. It can also be used to assess whether the engineered system is sufficiently stable to be effective.

Prof. Jeffrey B. Jones

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Who?

A distinguished professor of the University of Florida at the institute of food and agricultural sciences. Works primarily on strategies against bacteria that affect tomatoes, peppers and citrus fruits.

Why?

Acquired the first EPA registration for use of bacteriophages as biocontrol in agriculture.

What?

• That one of the primary concerns of biosafety concerning the use of wild-type bacteriophages in the environment is transduction.
• If a relatively large amount of toxin is required to kill the locusts, there might be a problem of selecting for locusts with a resistant gut microbiome. This is of little worry when little amounts of toxin are required.
• Bacteria become resistant to phages by a factor of one in a million or ten million. This is faster than resistance to antibiotics. To determine if this would pose a problem for our project, this question of resistance was modeled in the dry lab.
• If we can show that only the host of the phage is very limited, then the spread into the environment and the chance of propagating becomes smaller. This is also aided by the fact that bacteriophages outside of the locust are unstable. Therefore the targeted bacterium, or another in the host range of the bacteriophage, should not be present within the soil. Otherwise this would mean the bacteriophage is able to propagate, as within the soil the effect of heat and UV is reduced, increasing the stability of the phage while also enabling its propagation.

Dr. Steven Hagens

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Who?

Chief Scientific Officer Food Security at Micreos, of which the mother company is PhageGuard (which uses bacteriophages for food safety).

Why?

He has been working with phages for 23 years, during his master, PhD and during employment at Micreos.

What?

• Bacteriophages do not make us sick or cause allergic reactions. It is true that bacteriophages shape the human microbiome. However, clinical trials showed that, even though participants obtained large doses of phages, it had no impact on their microbiota.
• Bacteriophages that integrate into the genome (lysogenic phages) are known to be able to make bacteria toxic and resistant. An example is the Shiga-toxin carrying phage. In contrast, non-integrating phages (lytic phages) have not been shown to be detrimental to humans in any way.
• The easiest way for a bacterium to obtain resistance is by modifying its receptor molecules. The best way around this is to use 2 to 3 different phages that recognise different receptors of the same bacterium. In case resistance becomes a problem this approach would be developed.
• The risk of bacteriophage legs mutating, whereafter another bacterium could be infected, is not very high, although it will not never happen. However, the bacteriophage legs have evolved for millions of years to be perfectly aligned to their hosts. Even if a bacteriophage manages to mutate and infect another bacterium, it will be competing with the natural phages.

Simonis BV

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Who?

Remco and Gerard Bod are Export Manager and Director, respectively, of the Dutch agrochemical company Simonis B.V.

Why?

This company produces and exports insecticides and pesticides to many regions of the world, predominantly to the Middle East and Africa. It is a preselected pesticide supplier for, among others, locust control of the FAO for many years.

What?

• The FAO has standardised dilutions for all used pesticides, so that the same amount of volume is always sprayed on the same amount of land (1L per 1 hectare). In this way, the spraying practice and sprayer settings remain constant regardless of the active ingredient. Therefore it is important PHOCUS can fit into these standards.
• To be absolutely sure that the pesticide is of high quality, it is best to have it produced in Europe or the USA. Local production of pesticides in regions of Africa or Asia does not give sufficient assurance of the quality. The quality of the pesticide is not only important for its efficacy, but also to prevent any accidental side-effects. If something bad happens, the producer and supplier are responsible. Taking this into mind the idea of decentralized local production was dropped.
• The easiest way for a bacterium to obtain resistance is by modifying its receptor molecules. The best way around this is to use 2 to 3 different phages that recognise different receptors of the same bacterium. In case resistance becomes a problem this approach would be developed.
• The UN monitors the health of those involved in the application of pesticides and local community members. Although people are advised not to eat locusts because these may be treated with chemical pesticides, people will still do it because of a lack of other options to eat.

Dr. Cécile van der vlugt

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Who?

Dr. Cécile van der Vlugt is a Senior Risk Assessor at the Dutch National Institute for Public Health and the Environment (RIVM). In that function, she works on risk assessment and risk management issues concerning genetically modified organisms. As an expert on new biotechnological developments, she participates in (intern)national projects on emerging risks and safety measures of these new developments.

Why?

To gain insight into the view of a risk assessor on our project and to understand how PHOCUS would fit into current legislation.

What?

• PHOCUS would need to comply with three different regulatory aspects; GMO regulations, insecticide regulations and food regulations. The latter surprisingly could pose one of the biggest hurdles, as there is the possibility of an association between the applied GM phages and plant cells, resulting in residual phages present in plants grown for food or feed. Although this is still uncertain and so are the risks, this should be further investigated.
• The need for an additional biocontainment strategy in the design, i.e. a kill-switch, would not be necessary as long as we have data to back up that our bacteriophage would not persist in absence of its target host. There will be more problems stemming from the general acceptance of GMO in or on food and feed. In Europe, only one GMO has been approved by risk assessment and not even policy-wise. This is less a problem in pharmaceuticals, like vaccines, so maybe the improved sustainability of PHOCUS could be the convincing factor for some.
• The use of a bacteriophage cocktail can lead to more regulatory complexity, as assessments and the likes have to be conducted for a biological entity (the phages) instead of a pharmaceutical (non-living). The lack of specificity from the cocktail can pose problems for risk assessment, but could also be used as a case study for these type of mechanisms for future applications.
• There is no gap in GMO legislation, rather a gap in data and knowledge. Cultivation of the phages could be difficult as this needs to be standardized, something that is currently very new and hardly done.

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