Human Practices | SCAU-China

01 Understand the problem and empathize with it

Background

Invasion status of S. invicta

Global issue

Solenopsis invicta invasion from South America has occupied over 100 million acres in 17 American states since 1930s. These ants also invaded other countries like New Zealand and Australia in 2001 (Chen, 2010). With global warming, S. invicta has continued to spread to higher latitudes in recent years(Wang et al., 2022)

The following diagram provides an assessment of the potential global invasion risk posed by S. invicta. (Fig. 1)(Chen et al., 2020)

Fig 1.Global infestation risk level of S. invicta

Local issue

In China,S. invicta was first reported in 2003 in Taiwan. As of October 2021, the epidemic encompassed 129 cities across all 21 counties in Guangdong Province, covering a total area of 555,171.192 acres, which constitutes half of the impacted area of China.

The statistical data from June 2022 showed that another 131 cities were reported the distribution of S. invicta, thereby signifying that the prevention and control situation remains critical.

S. invicta cause damage to invaded areas.

Recognized as the top 100 invasive species worldwide, S. invicta cause serious harm to agriculture, animal husbandry, public infrastructure, ecological communities, and human health in invasive areas, as follows(Fig. 4):

1. Extremely aggressive, hazardous to human health.

When a person is stung by S. invicta, the skin becomes red, swollen, blistered and accompanied by a burning pain. Research has shown that 0.5% of the stings produce an immune response that can lead to anaphylactic shock in the patient(Travis et al.,1998).In severe cases, the patient may be in shock or even death from the sting.

According to incomplete statistics, more than 40 million people in the U.S. live in the S. invicta's range, 14 million people were stung each year, medical costs are approximately $7.9 million, of which more than 200,000 people require hospitalization, and cause several deaths case from allergic reactions.(Klotz,2002)

2. Damaging agricultural and livestock production, destroying public facilities, and cumulatively causing huge economic losses.

2.1 Crops and public facilities: S. invicta can damage or kill some plants by destroying roots and stems. Since S. invicta prefer food with high concentration of sugar, certain orchards planted with cash crops are also major disaster areas for S. invicta epidemic, such as lychee, longan, citrus orchards. During harvest, S. invictacan damage equipment like irrigation systems and power stations. In addition, S. invicta protects some pest species that produce "honeydew", which leads to a reduction in the quality of the product and contributes to the spread of some pest diseases.(Dussutouret al., 2009)

2.2 Livestock: S. invicta have been known to attack young animals and livestock, stinging their eyes, mouths and noses, causing blindness and even suffocation. Grazing livestock are discouraged from feeding in areas where S. invicta are present for fear of their stings.

2.3 Economic Losses: Since its invasion from Argentina to the United States in the 1930s, S. invicta has caused economic losses of approximately 5 to 6 billion USD per year in the U.S. Gutrich discussed the potential impacts of S. invicta invasion in Hawaii and speculated that the economic losses caused by the S. invicta invasion after 20 years would amount to 2.5 billion USD(FU et al.,2022). Aoyama speculated that the loss would reach 43 billion yen （291 million USD） if S. invicta invaded Okinawa, Japan. Based on Aoyama ‘s model, the annual loss in China due to S. invicta invasion would be more than $25 billion.(FU et al., 2022)(David Pimentel, 2005)

3. Destroy the ecological balance and reduce biodiversity.: S. invicta is highly aggressive and interspecific competitive, and in agroecosystems it is a greedy arthropod predator that indiscriminately attacks pests and beneficial insects. Studies have shown that the invasion of S. invicta has resulted in a 64.7% decline in arthropod species in cornfields. In addition, S. invicta also prey on other small animals, including reptiles, mammals, birds, mollusks, etc. For example, in the southern states of the United States, S. invicta have had a severe impact on the activities of invertebrates, small rodents, birds, turtles, etc., especially individuals at the egg and hatchling stages.(Li et al., 2022)

Contact with affected people

Government

Farmer

Medical Facilities

Biotech company

Scientific researchers

For more information, click on the images

Government

The government plays a pivotal role in the control and management of S. invicta, overseeing the local control efforts. We consider government agencies to be important stakeholders in the prevention and control of S. invicta.

Investigation at Baitu Town, Qujiang District, Shaoguan County

Shaoguan County is one of the heavily affected areas by S. invicta in Guangdong Province, China, with the ants having spread throughout the region. It serves as a representative case for S. invicta prevention and control. On one hand, Shaoguan County is an important agricultural production base and an ecological barrier in Guangdong Province, making it more susceptible to the harm and spread of S. invicta due to its unique agricultural characteristics. On the other hand, Shaoguan County is located in the northern mountainous area of Guangdong Province, which is relatively remote, lacks convenient transportation, and faces resource limitations, posing challenges for monitoring and controlling S. invicta. Therefore, Shaoguan County faces significant challenges and pressure in addressing the issue of S. invicta prevention and control.

Shaoguan County places great importance on S. invicta prevention and control. It has innovatively established a new model of community-based S. invicta prevention and control. Leveraging intelligent monitoring technology for S. invicta data collection, the city encourages residents to actively participate in prevention and control efforts through a "destroy ant nests with subsidies" paid service approach. Currently, all 18 towns (or subdistricts) in Nangxiong city have implemented community-based S. invicta prevention and control, and each natural village has established professional prevention and control teams comprising 2 to 5 individuals. The total number of ant nests controlled by the community has exceeded 140,000.

Fig 3.Investigation at Baitu Town

Investigation at Agriculture and Rural Affairs Bureau of Jieyang City Jiexi County

Jiexi, located in the eastern part of Guangdong Province, China, is one of the heavily affected areas by S. invicta in China. Jiexi faces certain geographical and agricultural challenges in addressing the issue of S. invicta prevention and control. On one hand, Jiexi is situated in the coastal region of eastern Guangdong Province with a warm and humid climate, fertile soil, and abundant water resources, making it conducive to the survival and reproduction of S. invictas. On the other hand, Jiexi's economy is primarily based on agriculture, with main crops including peanuts, sweet potatoes, soybeans, which are all preferred food sources for S. invicta. Therefore, Jiexi faces significant challenges and pressure in addressing the issue of S. invicta prevention and control.

Fig 4.Investigation at Jiexi County

Jiexi places high importance on S. invicta prevention and control. They have established a city-level command center and township working groups, formulated prevention and control plans and responsibilities, and allocated funds and resources for prevention and control efforts. Jiexi has adopted scientific prevention and control measures, tailored strategies, and community-based control methods. They conduct two concentrated "eradication" campaigns in spring and autumn, have established an intelligent monitoring and control management system, and set up two demonstration areas for prevention and control. Additionally, they have conducted technical training and educational outreach 4,352 times. According to statistics, during the concentrated "eradication" campaign in spring 2021, approximately 18,000 kilograms of bait were distributed, covering an area of approximately 108,000 mu (about 18,000 acres). In the autumn campaign, around 24,000 kilograms of bait were distributed, covering an area of approximately 144,000 mu (about 24,000 acres).

(The Agricultural Bureau is responsible for overseeing agricultural and rural affairs throughout the city, including monitoring and controlling major crop pests and diseases, guiding the development of the plant quarantine and inspection system, organizing and supervising plant quarantine and inspection work, conducting surveys of major plant quarantine pests, and managing invasive species in accordance with the law.)

Q: Why is the efficiency of S. invicta prevention and control not high?

Baitu Town: "In the prevention and control of S. invicta, local villagers here have a low willingness to actively participate and apply pesticides. Most local villagers are senior citizens, and the penetration and usage rates of smartphones are low, posing challenges for S. invicta community monitoring and digital control. Furthermore, the shortage of pesticides and their insufficient effectiveness often led to frequent S. invicta resurgence and incomplete eradication, seriously impacting the effectiveness of S. invicta prevention and control."

Jiexi City: "Local farmers here lack enthusiasm in addressing S. invicta issues and often rely on government-provided free pesticides rather than taking the initiative to purchase pesticides. The main participants in S. invicta prevention and control/dispersal of pesticides are middle-aged and elderly individuals, who have limited awareness of the proper use of control pesticides, often resulting in overuse or incorrect application, leading to poor effectiveness in S. invicta prevention and control."

our reflection: We have observed that the success of S. invicta prevention and control efforts is highly dependent on favorable weather conditions. The critical period for S. invicta control in Guangdong coincides with heavy rainfall, and the contact of S. invicta bait with water during rainy days can render the pesticides ineffective. Considering this situation, we have placed a significant emphasis on the water resistance of pesticides in our drug design approach.

Q: What measures have been taken locally for S. invicta prevention and control awareness?

Baitu Town: "We conduct regular training for villagers on how to prevent and control S. invicta and how to use pesticides. Additionally, we actively organize volunteers to engage in prevention and control activities."

Jiexi City: "We establish communication groups with farmers to address issues that arise during the prevention and control process promptly. We distribute a large number of informational pamphlets and organize regular S. invicta prevention and control training sessions. We have also established S. invicta prevention and control teams to disseminate knowledge on how to use pesticides and how to use monitoring platforms."

our reflection: Currently, S. invicta prevention and control awareness efforts in the region are progressing well. Through our visits, we have found that there is a relatively high level of public awareness regarding S. invicta. There is room for further development in the team's efforts in propaganda and public education, utilizing innovative approaches to disseminate knowledge about S. invicta to the public.

Q: What are the challenges encountered in practical S. invicta prevention and control?

Baitu Town: " S. invicta resurgence is common in the local area, with nests often re-establishing themselves within two to three months after unified control efforts. Insufficient availability of control pesticides and frequent S. invicta resurgence negatively impact the actual effectiveness of prevention and control."

Jiexi City: "The primary participants in S. invicta prevention and control/dispersal of pesticides are middle-aged and elderly locals, who have a weak awareness of the proper use of control pesticides and tend to overuse them, leading to the wastage of bait. Additionally, non-scientific control methods, such as using regular insecticides or burning, accelerate the spread and dissemination of S. invicta. These methods not only fail to eradicate the nests but also cause the queen ants to relocate, further expanding the distribution of ant colonies."

our reflection: During the interviews, we recognized the importance of considering user-friendly pesticide application methods that can be used correctly even without specialized knowledge, in order to achieve effective S. invicta eradication.

Q: What factors does the government consider in the process of selecting pesticides?

Baitu Town: "Due to the limited budget and resources available for S. invicta prevention and control at the township government level, we tend to prioritize the cost of pesticides when selecting them."

Jiexi City: "In our pesticide selection process, we primarily consider the effectiveness of the pesticides, their cost, and ease of use."

Through these interviews, our project is placing increased emphasis on the "financial feasibility" of pesticides.

Farmer

S. invicta, poses a threat to local agricultural development and human health and safety. It directly leads to reduced crop yields, damage to arable land, and threats to public health and safety. Farmers are the direct stakeholders affected by this hazard. Additionally, in the current efforts to control S. invicta, farmers often serve as the primary implementers of control measures (with the government distributing chemicals to farmers, who apply them individually). Therefore, we believe that conducting interviews with farmers can help us gain a detailed understanding of their perception of the dangers posed by S. invicta and their opinions on government control measures.

During interviews with local farmers in Baitu Town, we learned that S. invicta bites are relatively common among the farming community, but in most cases, the bites are not severe enough to warrant a visit to the hospital. One farmer told us that he has been bitten by S. invicta many times while working in the fields, but he didn't experience severe symptoms, so he didn't seek medical treatment. Currently, the area on his legs that is frequently bitten by the ants has become less sensitive to the bites.

Fig 5.Communication with farmers

Regarding the government-distributed medication for S. invicta, they expressed dissatisfaction with its effectiveness, claiming that it does not effectively control the ants and, as a result, the ant problem persists. This directly leads to a decrease in farmers' motivation to engage in ant control efforts. Specifically, they mentioned that after applying the medication, there is no noticeable reduction in the spread of S. invicta, and the process of applying the medication is time-consuming and labor-intensive, making them reluctant to invest their efforts into what they perceive as ineffective measures. During the interviews, we also learned that S. invicta nests are often located near water bodies, and farmers are concerned that the medication might pose a threat to other aquatic organisms.

When discussing the "cloud-based monitoring" system that has been implemented for S. invicta, one farmer expressed skepticism. They believed that the system relies heavily on the use of smartphones, which are not widely adopted in rural areas with lower smartphone penetration rates. Therefore, they considered it challenging to promote the use of such a system.

In agricultural production, farmers are crucial stakeholders in the fight against S. invicta. Our approach needs to gain significant acceptance among the farming community.

Medical Facilities

Health centers and hospitals are the most direct stakeholders when it comes to cases of S. invicta bites, as they are responsible for treating patients who have been bitten by these ants. Through interviews with members of medical facilities, we can gain a detailed understanding of the current number and frequency of S. invicta bite cases, the facilities and resources available for post-bite treatment, and other relevant information. This will help us assess the medical burden caused by S. invicta bites, common characteristics of such bites, and preventive and treatment measures. Ultimately, this information will assist us in further improving our project.

Health Care Station of Baitu Town

The staff at the Health Care Center of Baitu Town informed us that currently, in the rural areas of Shaoguan County, medical facility coverage is high. There is typically one health care station equipped with basic treatment medications for S. invicta bites in each village, which can meet the general treatment needs for such bites. However, these health centers do not have emergency medical equipment. In the case of severe allergic reactions, patients need to be transferred to a health care center or a larger hospital for emergency treatment.

Fig 6.Investigation at Health Care Station of Baitu Town

People's Hospital of Jiexi City

Dr. Zhendong Huang, the head of the Emergency Department at People's Hospital of Jiexi City, participated in our interview. Dr. Huang graduated from Jinan University Medical College with a the major of clinical medicine and has nearly twenty years of clinical experience, making him highly experienced in handling S. invicta bite cases.

Dr. Huang introduced the treatment plan for S. invicta bites. The hospital recommends rinsing the affected area with clean water, preferably alkaline water, and if possible, squeezing the wound to release any fluid, followed by the application of medication to the wound.

Dr. Huang expressed strong support for our project's environmental friendliness. From a medical perspective, he emphasized the significant impact of S. invicta on human health and offered great encouragement and hope for our project. He hopes that our efforts can further reduce public health risks primarily associated with S. invicta.

Fig 7.Investigation at People's Hospital of Jiexi City

Biotech company

Biotechnology companies play a vital role in the control and detection of S. invicta, as they are at the forefront of developing technologies and testing products. They can provide us with information about commercially available insecticides and devices for eradicating S. invicta. These companies can also offer product manufacturing standards and assist in testing our new products.

Guangdong Red Fire Ant Technology Development Co.,LTD.: The company was established in early 2020, specializing in S. invicta control services. Our technology is based on the "Natural Pesticides and Chemical Biology" Key Laboratory of the Ministry of Education under the supervision of Professor Hanhong Xu at South China Agricultural University. The work related to S. invicta within the company is conducted under the guidance of Professor Xu Hanhong, Dr. Lihua Lu, Dr. Zhixiang Zhang, and Dr. Dongmei Cheng. The company has independently developed the S. invicta Cloud Detection" mobile application and the highly effective medication called "Wei An," achieving remarkable results.

We visited the Guangdong Red Fire Ant Technology Development Co.,LTD. and conducted an interview with the CEO, Mr. Weizhuan Wang. Our team's approach received strong support from them in terms of the design of medications and devices. The specific questions and outcomes of the interview are as follows:

Q: How do you view our bait-based poisoning plan? Is it feasible?

A: "I believe that your idea is correct. Accumulating toxicity within the ant queen and eventually killing her is practical. The key to eradicating S. invicta lies in targeting the queen, given her long lifespan and strong reproductive capabilities. It's possible to slowly eliminate her with a chronic bait without disturbing her."

our reflection: Based on literature and Mr. Wang Weizhuan's assessment, our design approach has received preliminary affirmation. Targeting and eliminating the ant queen during behaviors such as fourth instar larvae trophallaxis and multiple queens within a nest are the right directions, and they hold significant feasibility.

Q: We have initially considered the use of drones for bait deployment in our project. Do you think this approach is feasible?

A: "The use of drones for bait deployment is not very common in the market. It is often limited to large-scale experimental sites, where the environmental conditions are controlled, resulting in favorable outcomes. However, in practical applications, it can be easily affected by the complexity of the terrain and may not be practical. Manual deployment is often more precise and efficient."

our reflection: During the process of innovatively designing the bait deployment method for the project, Mr. Wang offered insights based on his past experiences and pointed out the challenges associated with using drones. We considered the complexity of environmental conditions and the need for precise targeting of ant nests, ultimately deciding against drone usage. Therefore, our goal is to innovate and design a suitable device for deployment.

Q: We are considering a device designed to attract S. invicta for feeding. What are your thoughts on this design?

A: "A device resembling a small box might have limited effectiveness, and such designs are currently scarce in the market. S. invicta is quite 'smart' and sensitive. Even minor changes in the environment within the device can make them reluctant to enter. Additionally, we need to consider factors like temperature, humidity, and lighting."

our reflection: The initial idea behind the small box design was to provide a container for the project's medication while minimizing the impact of external environmental factors, such as rain causing the medication to become damp. However, the development of the small box design is facing several challenges, including the sensitivity of S. invicta to the device, internal ventilation, humidity, temperature, and a lack of precedent in the design. Perhaps we can boldly innovate to address these challenges or reevaluate the purpose and design a new device based on the specific requirements of the project.

Q: We place great importance on biosafety issues such as the accidental ingestion of bait by other organisms and its environmental impact. How do you suggest we adjust our plan to address safety concerns?

A: "The biosafety concerns you're considering touch upon the core safety issues. We have had very few cases related to safety concerns during our usage because the bait has a low toxicity, and it kills through accumulation, resulting in minimal environmental impact. Furthermore, due to the aggressiveness of S. invicta, other nearby organisms such as other ant species and insects cannot survive, so the risk of accidental ingestion is almost non-existent."

Our colleague, Dr. Daifeng Cheng (Second PI), believes that "our biosafety concerns should mainly focus on genetically modified technology. We should assess the engineering bacteria used in the project and determine the potential harm if products produced by genetically modified organisms are consumed. This is what we need to evaluate and prevent."

our reflection: In response to on-site issues, we can engage in more discussions with users (farmers) and implement measures to avoid potential biosafety concerns like accidental ingestion. Additionally, the application of genetically modified technology is the most critical safety concern we need to address

Fig 8.Investigation at Guangdong Red Fire Ant Technology Development Co.,LTD.

Q: In the design of the bait, besides the main component responsible for poisoning, we also plan to add substances that can efficiently lure S. invicta. Is this feasible?

A: "The use of attractants in bait is highly significant as it speeds up the time for S. invicta to consume the bait, improving the effectiveness of the pesticide application. It also helps prevent other organisms from ingesting the bait, reducing environmental residues. One of the major strengths of our company is attractants, so this aspect is confidential."

our reflection: There is still a lot of room for exploration in the use of attractants in biopesticides. Our idea has received support, and although the company cannot disclose further details, we can continue our efforts to find effective attractants that are suitable for our project.

Fig 9.Photo in Guangdong Red Fire Ant Technology Development Co.,LTD.

Scientific researchers

Scientists are the driving force behind the study of behavior and characteristics of S. invicta, as well as the development of eradication methods. The preliminary information for our project was gathered through extensive literature review, and we have received significant support from them in designing and refining our experiments to ensure the feasibility and rigor of the project.

Dr. Lei Wang: Dr. Wang specializes in insect population ecology, pest control theory, and technology research. He focuses on studying important pests in the Southern China, such as S. invicta, with research areas including population biology ecology, damage patterns and mechanisms, and environmentally friendly eradication techniques.

In the early stages of our project, we proposed three directions: detection, prevention, and treatment. Dr. Wang Lei recommended that we abandon the detection and treatment directions. On one hand, the primary application of S. invicta detection is at customs entry points, import goods, and container storage areas to monitor and prevent the invasion of foreign S. invicta. However, the technology in this direction is already mature, with detection products available on the market that can complete the testing in a short time frame. Moreover, detection is not the most pressing issue. On the other hand, treatment measures for S. invicta are also relatively mature, with many general insect bite remedies available on the market that are suitable for S. invicta bite cases.

Dr. Wang affirmed our approach in prevention and control, suggesting that there is significant potential in developing biopesticides using synthetic biology and genetic engineering. However, he emphasized the need for further investigation into the current gaps in the market's prevention and control measures. We should identify the problems and make improvements to overcome challenges such as limited options, non-user-friendly approaches for non-specialists, and complex application environments.

Fig 10.Communication with Dr. Wang Lei

Fig 11.A tour of Dr. Wang Lei's laboratory

Dr. Daifeng Cheng (Second PI): Dr. Daifeng Cheng applies research methods from fields such as insect behavior, chemical ecology, and microbiology. He systematically studies different ways and mechanisms through which microorganisms influence the population expansion of S. invicta, yielding a series of original results that offer new insights and methods for managing the expansion and harm caused by S. invicta. Dr. Cheng also serves as our second PI.

Q: Typically, what is the collaboration model behind the development and launch of S. invicta control measures or a specific pesticide for S. invictas in a particular region?

A: "Usually, government departments initiate the demand, and companies with research and development capabilities undertake the project. During this process, companies may collaborate with efficient teams for technical cooperation. Another model involves research teams being responsible for technical research and development, while companies focus solely on large-scale production of products. Ultimately, the product needs to go to market for use in the required fields."

our reflection: We have learned that the launch of pesticides primarily involves collaboration among government, companies, and universities. As we innovate in the development of S. invicta pesticides, we can learn from their collaboration models and engage in communication and cooperation with government and industry stakeholders. The development of pesticides emphasizes practicality as the primary factor, while also considering cost, ease of operation, and factors related to environmental safety and sustainability. To bring a pesticide to market, three certifications are usually required: pesticide registration certificate, pesticide production permit, and pesticide standards. It is also important to understand the existing products in the market, select outstanding indicators, and continuously improve the solution through practical testing.

Fig 12.Communication with Dr. Daifeng Cheng

Q: In the mechanism of the pesticide, is it necessary to target the elimination of ant queens?

A: "Targeting the elimination of S. invicta queens is actually correct. The ant queen has a strong reproductive capacity and can produce between 800 to 5,000 eggs per day. Whether the pesticide can effectively eliminate the queens hidden within the ant nests is an important consideration for its effectiveness."

our reflection: In China, invasive S. invicta exhibit a "multiple queens in one nest" behavior. The ant queens have strong reproductive abilities and long lifespans, while other worker ants are sterile and have shorter lifespans. Effective control requires the elimination of the ant queens to halt population growth. ultimate concept behind the pesticide we are designing is to accumulate and exert its toxic effects within the ant queens.

We also inquired with Dr. Cheng about the current situation of S. invicta control measures in the market. Professor Cheng Daifeng pointed out:

"Currently, most of the control measures available in the market are chemical pesticides. Biological control measures are rare, and there are none domestically available. This is primarily due to the novelty of the approach, limited research, and the relative difficulty in achieving results compared to chemical pesticides."

our reflection: Biological control measures for S. invicta are scarce, and there are no precedents to reference. When aiming to introduce a pesticide to the market, our team needs to consider product standards, review criteria, legal regulations, safety aspects, and more. Therefore, our goal is to gather input from stakeholders extensively and design a comprehensive plan for bringing a pesticide to market.

Fig 13.Photo with Dr. Daifeng Cheng

02 Define a good solution

The rapid spread and severe impact of S. invicta have garnered significant attention from relevant authorities since their discovery in China. Domestic efforts to control S. invicta are challenging. However, conventional control measures can only temporarily contain the harm and spread of S. invicta. To achieve long-term and fundamental control of these ants in China, it is imperative to conduct in-depth research on their native occurrence patterns and basic biological characteristics. This is because, as a highly adaptable invasive species, they have the potential to undergo significant biological changes when introduced to new regions.

Therefore, through this project, we aim to provide a green, cost-effective, and highly queen-targeted biological control method for S. invicta, contributing to global efforts in combating this invasive species.

Project Objectives

1.Green and safe, easy to operate

During our interview with Guangdong Red Fire Ant Technology Development Co., Ltd., we learned that traditional chemical baits primarily achieve green control through the selection of chemicals, application rates, and application methods. However, this method appears to be more suitable for professionals. Based on our initial research, we found that the primary group actively involved in nationwide S. invicta control is farmers, most of whom are middle-aged to senior citizens with limited expertise and relatively weak awareness of pest control. In this situation, overly complex pesticide application procedures may discourage their participation in control efforts.

At the same time, when selecting control methods, the impact on environmental safety and personal safety is one of the government and farmers' major concerns. During the interviews, one farmer specifically mentioned concerns about whether the use of chemicals would harm domestic animals and nearby water bodies.

Therefore, our team hopes to leverage synthetic biology methods to develop an engineered live bacterial pesticide as a replacement for chemical pesticides in traditional baits. This live bacterial pesticide will be regulated by genetic switches to minimize environmental and biological safety risks to the greatest extent. Additionally, farmers will only need to perform simple pesticide application with this product to achieve the goal of S. invicta control.

In the future, we hope that the engineered live bacterial pesticide we develop can be safely applied in water source protection areas, ecologically sensitive areas, organic farms, and other contexts.

2.Efficient and higher targeting of queen ants

According to our research, the traditional baiting method relies on the enrichment of low-toxicity chemical pesticides in the queen to achieve the goal of eradicating ant colonies. However, this method is less effective when dealing with populations with multiple queens. While the control objective can be achieved by increasing bait application, it raises the cost of control and chemical pesticide residues. Additionally, in our discussions with local farmers, we learned that the long control cycle and the lack of significant results in single-dose treatments are major reasons for the low willingness of farmers to use pesticides.

Although there are ongoing research efforts to develop targeted drugs for S. invicta, there have been no breakthroughs in the development of drugs specifically targeting the queen. In contrast, professional S. invicta control companies focus on developing more attractive baits to accelerate the feeding rate of S. invicta to promote control effectiveness. Our project aims to improve the queen-targeting ability of the drug by introducing genetic circuits to regulate the timing of drug release. This will achieve faster and more efficient S. invicta control with the same or even fewer bait applications.

3. Low cost

Based on our previous interview with the government, we have learned that high costs lead to limited purchases of S. invicta control drugs. As a result, farmers cannot access a sufficient quantity of these drugs, which is one of the significant barriers to the effective implementation of nationwide S. invicta control efforts.

We aim to produce our final drug at a cost that is at most equivalent to traditional chemical baits, and ideally, even lower

Additionally, we are also attempting to reduce costs at the township level by designing suitable hardware devices. This would help lower the expenses associated with organizing specialized teams for S. invicta control, thereby reducing overall control expenditures. This approach will enable more townships to adopt this cost-effective control measure.

4. High waterproofing

According to research conducted by the Agriculture and Rural Affairs Bureau of Jixi City, we have learned that a drawback of S. invicta ant baits is that they can only be used on sunny days and should not come into contact with water sources. In the Guangdong region, the critical period for S. invicta control is during the autumn and winter seasons, which coincides with heavy rainfall. This limited number of suitable days for control hinders the progress of control efforts.

Therefore, we aim to enhance the waterproof properties of our project's outcomes or develop an additional set of devices or baits specifically designed for use in situations where water resistance is crucial for effective control.

03 Conceive and design our solutions

In the conception and design of our project proposal, we have consistently sought input from experts in both wet laboratory experiments and dry laboratory experiments. This approach ensures that our final solution is not only technically feasible but also aligns with the values and perspectives of relevant stakeholders.

Concept and design

In this year's IGEM project, we aim to leverage synthetic biology to address the prominent issues in the current S. invicta control methods, and provide a green, cost-effective, and more efficient biological control solution for S. invicta worldwide. To facilitate the development of our project, we have consulted a series of experts in wet laboratory experiments, incorporating their advice into our project.

Our choice of the S. invicta as the target species stems from a team member's personal experience during a hiking trip where he suffered painful bites from these ants in his hometown. After initial research into the related information about S. invicta, our team recognized them as highly invasive species, and their rapid spread in our country is a cause for concern. Therefore, we decided to propose a synthetic biology solution to address the problem of S. invicta.

Initially, we brainstormed three project directions: eradication, detection, and post-bite treatment, all of which we considered important aspects of S. invicta control. However, we were unsure which direction to pursue. Dr. Wang, who specializes in insect population ecology, control theory, and technology, suggested that we abandon the detection and treatment directions. This decision was based on various factors, as detailed in the section on interactions with affected individuals in the earlier discussion.

Therefore, we focused our goal on eradicating S. invicta. In terms of eradication methods, Dr. Wang pointed out that there have been studies using entomopathogenic fungi as a basis for biological control, but the presence of ant social immunity, particularly the corpse removal behavior, significantly hampers the spread of entomopathogenic fungi within ant colonies, resulting in low efficacy. Hence, we have chosen to explore disrupting the corpse removal behavior of S. invicta to enhance the effectiveness of entomopathogenic fungi as our research direction.

Therefore, we decided to focus on disrupting the corpse removal behavior and found that some research suggests it is regulated by specific genes in S. invicta. We contemplated whether we could use RNAi technology to interfere with the specific genes associated with corpse removal behavior. However, Dr. Cheng, who specializes in studying symbiotic bacteria that regulate the fire ant's corpse removal behavior, pointed out that this behavior is regulated by multiple genes, and the specific mechanism is not well understood. RNAi interference with multiple genes simultaneously is challenging, and the presence of nucleases in the ant's gut can affect RNAi efficiency. Thus, the overall approach is difficult and not recommended.

However, he also suggested that we could use gut microbes residing in the fourth-instar larval gut as a chassis and engineer them to produce toxic substances harmful to S. invicta, achieving an eradication effect. Furthermore, Dr. Cheng emphasized that targeting the eradication of the queen ant (the reproductive ant) is crucial for achieving a significant impact on the colony. If a treatment only kills worker ants and not the queen, the colony's robust reproductive capacity could lead to a rapid resurgence. Traditional baiting techniques achieve this by targeting the queen through a process of accumulation.

Based on all the advice we have received so far and the literature we have consulted, we have proposed a specific project idea: to modify the dominant gut microbes present in the fourth-instar larva of S. invicta. We aim to make them express toxic proteins or chemical substances and find a way to engineer these microbes to accumulate in the queen ant's body, thereby achieving targeted eradication by expressing the toxin or chemical inside the queen ant.

Research

Regarding the choice of the eradication agent, Dr. Cheng recommended that we prioritize exploring the direction of toxic proteins. We should select several toxic proteins that have been studied and shown to be effective against S. invicta. He emphasized that validating their eradication efficacy after expression is crucial for the project's success. Dr. Cheng believes that researching toxic proteins can temporarily bypass the issue of resistance that can develop against traditional chemical agents. However, he also advised that if the research on toxic proteins encounters difficulties, we could consider expressing existing chemical agents, although constructing the expression pathways for chemical agents is quite complex and should only serve as an alternative.

Furthermore, Dr. Cheng provided suggestions for selecting our chassis organism. He mentioned that Enterococcus faecalis is a prominent symbiotic bacterium found in the gut of S. invicta and could be considered as one of our chassis options. Professor Xu also advised us to focus on the adaptability of the chosen chassis organism to the S. invicta gut environment and whether it would be susceptible to the toxic components of the S. invicta venom. These factors directly influence the ability of our engineered bacteria to reproduce and express the drug effectively within the S. invicta gut. After considering these suggestions and reviewing various literature, we ultimately chose Escherichia coli (E. coli) as our chassis organism.

Gut microbes take some time to colonize the gut after entering, increasing the concentration of the bacterial population to secure more resources for survival before massive expression occurs. Therefore, we considered finding a signal that controls the behavior of engineered bacteria to release drugs only after reaching a certain concentration. At this point, Dr. Zhe Hu (Primer PI) introduced us to the Quorum Quenching strategy used in Xanthomonas oryzae pv. oryzae (Xoo), an organism causing diseases in Oryza sativa L. He explained the concept of quorum sensing and suggested that we could incorporate it into our gene circuit to achieve the goal of controlling the behavior of the bacteria based on their population density. Quorum sensing also helps coordinate the collective behavior of the bacterial population, ensuring uniform drug release and reducing the amount of drug susceptible to degradation by endogenous proteases, thereby increasing effective expression.

However, Dr. Cheng pointed out a problem in our design. Gut symbiotic bacteria can only colonize specific structures inside the larval gut, and they cannot stay in the queen ant's gut for an extended period; they are excreted within a day or two after ingestion. This realization led us to understand that if we use quorum sensing to coordinate drug release and rely on gut symbiotic bacteria's colonization in larvae, quorum sensing would activate prematurely in the larval gut due to their distinct colonization characteristics. This would not achieve the intended goal of engineering bacteria to accumulate in the queen ant before expressing the drug. Therefore, we shifted our project direction to explore the possibility of first expressing the drug within the larvae and then enriching the drug in the queen ant for targeted eradication.

In this new direction, the key challenge was to ensure that the drug expressed within the larvae would not be excessive and kill the larvae. We needed to find a method to control the drug's expression level within the larvae, keeping it below the lethal dose. This way, the drug could still be transmitted back to the queen ant after expression. Therefore, we introduced a binary oscillation system. By designing a well-structured oscillation circuit, we ensured that the amount of expressed drug would oscillate below a specific predetermined value.

Fig 14.The Schematic of binary oscillation circuits

Fig 15.The previous project schematic

The construction of binary oscillation circuits is not a straightforward task. We posed questions to experts regarding the selection and construction of binary oscillations. Mr. Boxiang Wang from Lingzhu Co.,LTD. commended our innovative use of binary oscillations as a promising concept but acknowledged the significant challenges it presents. The construction of binary oscillations is indeed complex. He pointed out some principles that we may inadvertently violate when designing artificial dynamic circuits and recommended reading materials by Christopher A. Voigt, as well as the gene circuit design software Cello Cad, to facilitate a deeper understanding of our project's binary oscillation circuit. This helped us identify potential issues in the design of dynamic gene circuits. These challenges prompted us to reevaluate the difficulties in achieving binary oscillation and its practical implications.

During the CCIC conference, we also consulted Dr. Dong from ATANTARE, who has expertise in oscillator-related research. Our discussions revolved around the specific design details of the experiment program intended to validate the binary oscillator. Through these exchanges, we came to realize the instability associated with the project's binary oscillator system. Consequently, we made the decision to abandon the oscillator circuit and opt for the construction of a digital circuit instead.

We have proposed a new plan that omits the oscillation route (with a page link to the experimental plan) to regulate the quorum sensing threshold and control the rate of toxic protein production. This approach allows us to maintain a balance between production and output, effectively controlling the dosage of drugs for the larvae in the intestine. Some of the details of this plan were inspired by our team's communication with the Tsinghua University team during the CCIC conference, focusing on the following two aspects: 1. The Tsinghua University team's use of the OmpA signaling peptide was found to meet our project's requirements for secreting two proteins, which we later confirmed through our experiments. 2. The Tsinghua University team's biosafety design inspired us to develop a nutrient-deficient safety switch based on the loss of single gene expression, allowing engineered bacteria to survive only in bait with added diaminobenzoic acid (DAP) or in the anaerobic environment of the S. invicta gut. This prevents the engineered bacteria from surviving in the environment and causing harm.

Dr. Hu has expressed support for this new plan, as it is expected to be more stable than the application of a binary oscillation system."

Fig 16.The project schematic

04 Implementation and evaluation

Drug carriers and hardware devices

During the project design phase, we are exploring strategies to transform our genetically modified bacteria into practical biological pesticides. Concurrently, we are focused on enhancing overall effectiveness and implementing safety precautions through the prototyping of hardware devices.

Fig 17. Improving the moisture resistance of baits

Selection of drug carriers

On the basis of ensuring the attraction rate of S. invicta and the activity of protecting engineering bacteria, we focus on improving the moisture resistance of bait. Through literature research, we have learned about the S. invicta bait capsule developed by Xie Feng, which aligns with our project's requirements. Therefore, we have decided to build upon his research findings to further design our drug carrier (Dong et al., 2021).

According to the author's research, the optimal formula of the rubber shell is as followed: Gelatin 20%, Glycerin 3%, Triethyl citrate 1%, Corn meal 2%, Fish meal 7%, The bait capsule shells formulated as described above showed the highest attraction to S. invictas, along with high water resistance, good mold resistance, and overall effectiveness in pest control.

In the future, our plan is to build upon the author's formulation by using gelatin microspheres (GMS) as the carrier for the live bacterial drug. We will introduce varying concentrations of diaminopimelic acid (DAP) (for more details, see the Safety page link). Additionally, we will conduct single-factor attraction tests with bait formulations containing different concentrations of DAP in gelatin microspheres to determine the optimal DAP concentration for the bait formulation.

Our experimental method of orthogonal test method will refer to the scheme provided by Xie Feng (2019), and the single factor attractant test is detailed in (set as hidden column).

Experimental method:

Several GMS (0.5 g for each) with varying concentrations of DAP were prepared for single-factor attraction tests against S. invicta. A total of 100 S. invicta worker ants, selected for uniform size and good activity, were subjected to 24 hours of fasting.

Fig 18.diagram of experimental device

The experimental setup consisted of two plastic cups connected by a flexible tube, as illustrated in Figure 2.2. The plastic cups had dimensions of 9 cm (bottom length) × 14 cm (top length) × 7.5 cm (height), and the semi-transparent rubber tube measured 10 cm (length) × 0.6 cm (inner diameter) × 0.8 cm (outer diameter).

The S. invicta were placed in one side of each plastic cup, with the other side containing the bait (the connecting tube's outer diameter bottom height was parallel to and closely adhered to the bottom of the plastic cup). The number of S. S. invicta in each test tube was recorded every 10 minutes for a total duration of 320 minutes. The experiment was conducted in five parallel sets.

The tests were carried out indoors at a temperature of (26±0.5)°C and a humidity level of (60±5)%. In the control group (blank test), only S. invicta were placed in the cup, with nothing added on the other side. Each group consisted of 5 sets of parallel experiments.

Note: Due to biosafety issues, this experimental method is only theoretical, and no related zoological experiments have been carried out. In the future, we will conduct further experimental verification if experimental conditions permit.

The choice of hardware devices

We focus on reducing costs and improving prevention and control measures when designing our hardware. During our research, we learned that Shenzhen City, China, had employed the strategy of hiring professional teams to conduct regular control measures against S. invicta. This approach addressed several challenges associated with relying primarily on farmers for control efforts, including issues related to professionalism, efficiency, and low willingness among farmers to participate in control activities. Additionally, it facilitated supervision, data collection, and improved the overall efficiency of S. invicta.

However, the success of such control measures depends on adequate financial support. While well-funded municipalities like Shenzhen City may not face significant challenges in this regard, smaller township governments with limited financial resources often prioritize cost-effectiveness when considering S. invicta control measures. Therefore, we aim to design appropriate devices to reduce the costs associated with organizing specialized control teams at the township level while enhancing the safety of control operations. This approach can assist financially constrained township governments in establishing control teams and ultimately improve the efficiency of S. invicta control efforts.

Check out our hardware related content here

The object of consideration for the project

During our discussions with stakeholders, we recognized the importance of identifying the target audience for our project as a significant issue. As a result, we placed a strong emphasis on determining which individuals or groups would benefit the most from our project, how to enhance their user experience, and how to attract and address the concerns of potential buyers. This approach aimed to improve the overall effectiveness of our project.

As a result of these considerations, our team members engaged in focused discussions, taking into account the needs and concerns of various target user groups. We brainstormed and proposed effective suggestions for advancing the project and developed preliminary plans based on this collaborative effort.

Ease of operation

During our research, we discovered that cities like Shenzhen and Jiexi in China have employed dedicated teams to organize regular and targeted control measures for S. invicta. These regions have become exemplary areas for S. invicta control, demonstrating effective eradication of the ants. The scheduled pesticide application work is carried out by these specialized S. invicta control teams, whose members have received professional scientific training and are familiar with the proper use of S. invicta control pesticides. However, routine pesticide application is often performed by local villagers in areas with complex terrain and a wide scope. Despite training provided to local farmers on pesticide application, their awareness of scientific S. invicta control methods is often limited, resulting in suboptimal control outcomes due to improper use or incorrect dosage of S. invicta control pesticides.On the left, the staff of Jiexi County Bureau of Agriculture and Rural Affairs demonstrates spraying medicine

We also encountered similar issues in Baitu Town. Taking this phenomenon into consideration, we focused on the target user group with limited scientific knowledge and skills. In this context, Hardware development should prioritize simplicity of operation. While professionally trained control teams can use control pesticides correctly, there is a need for hardware devices that enable user-friendly operation and deliver optimal results for those with limited scientific awareness.

Therefore, our Hardware development primarily focuses on ease of operation and quantitative usage. We initially designed a prototype that caters to both simplicity and accurate dosing. However, through field experiments, we found that the preliminary device was prone to disturbing ant nests and lacked sufficient quantification. Consequently, we plan to continually refine the device's ease of operation through ongoing field testing and innovation.

Water resistance of plant

There are two main reasons for our focus on the waterproof feature of the device. First, S. invicta are primarily found in the southern regions of China, which experience frequent rainfall during the peak S. invicta season due to climatic conditions. Second, S. invicta nests often grow near water bodies such as ponds, which significantly affect the effectiveness of S. invicta control and the work of control teams. During our discussions with Agriculture and Rural Affairs Bureau of Jiexi City, they mentioned that they often encounter rainy weather during pesticide application, causing the pesticides to be washed away and adversely affecting their control efforts. Experts from the Red Fire Ant Technologies also emphasized the importance of device waterproofing in their research. We learned that many existing pesticide application devices on the market do not perform well in wet conditions, requiring multiple manual applications to mitigate the impact of water.

Considering these factors, we focused on the target user group of S. invicta control personnel and proposed the idea of waterproofing the device. In addition to enhancing the effectiveness of the pesticides, Hardware development aims to reduce inconveniences caused by external factors for control personnel, ultimately improving the efficiency of their work. Hardware design primarily addresses device dimensions and waterproofing. We initially designed two pesticide application devices, but after testing, we found that the size of the device was slightly small and had some impact on the foraging behavior of the ant colony. Therefore, Hardware development is further refining the device to maximize waterproofing while ensuring its functionality.

Financial feasibility: During our discussions with Red Fire Ant Technologies and development Co.,LTD., we gained insights into the entire process of S. invicta drug production, market entry, and sales. From this, we continuously considered the needs of the purchasers of S. invicta eradication drugs. Subsequently, in our discussions with government officials, we received answers to these questions. When the government procures S. invicta eradication drugs, their primary considerations are the effectiveness and ease of use of the drugs. Since the government makes bulk purchases, cost-effectiveness is a significant concern for them. Additionally, we noticed that currently, the main purchasers of S. invicta eradication drugs are government departments. Even though these drugs are available in pharmacies, very few individuals purchase them. This phenomenon underscores the importance of financial feasibility. Therefore, we are focusing on the target user group of drug purchasers and proposing ideas for cost-effectiveness. By reducing costs, we aim to further enhance the overall effectiveness of S. invicta control.

Key implementation elements of S. invicta prevention and control concept

Control requirements influenced by characteristics of S. invicta: In Bai Tu Town, we learned that S. invicta control differs significantly from traditional pest control methods. Currently, there are specific chemical or biological agents available in the market for traditional pests such as aphids, armyworms, and red spiders. These pests can be effectively controlled and eradicated through timely and targeted measures. However, in practical S. invicta control, it has been observed that S. invicta infestations frequently reoccur after a period of control and eradication, leading to suboptimal results. Therefore, we believe that the key factor in S. invicta control is the unique nature of S. invicta itself. It requires multiple rounds of control and non-scheduled treatments based on the actual control situation, rather than fixed and scheduled treatments.

Professional quality of S. invicta control personnel

Through our preliminary research and field visits, we have identified a key focus on raising awareness and publicizing information about S. invicta. According to the Agriculture and Rural Affairs Bureau of Jiexi City, the government currently takes the lead in initiating and managing S. invicta awareness campaigns and control efforts.

However, in many rural areas, some staff members lack sufficient knowledge about S. invicta, leading to suboptimal outcomes in control measures such as selecting and administering pesticides, ultimately resulting in less effective control efforts. In view of this element, we believe that it is necessary to focus on the professional quality of S. invicta prevention and control personnel at all levels, focus on training corresponding staff, and further implement the concept of S. invicta prevention and control.

People's initiative in S. invicta control

In response to this factor, we believe it is crucial to prioritize the professional competence of S. invicta control personnel at all levels and provide targeted training for them to better understand and implement S. invicta control concepts. While government-initiated awareness campaigns have been effective in educating the public about S. invicta and its control, we have observed a lack of initiative from the general population. This passive stance among the public limits their active participation in S. invicta control efforts.

Therefore, we propose that S. invicta control efforts should place a greater emphasis on empowering the public with knowledge and encouraging proactive involvement in S. invicta management.

The standards we reached

The comprehensive human practice section has effectively promoted the iterative optimization of experimental plans.

From the beginning of our project, human practices (HP) and wet team have been in a spiral of complementary development: HP's interviews, research and data collection are based on the progress and problems encountered by wet team. Simultaneously, the design of experimental ideas for wet Team is based on the feedback from the human practices for modification and improvement.

Therefore, these two components complement each other, serving as a crucial collaborative model for the successful progression of our project

Fig 19.Project Plan Iteration Process (Partial)

Summarized problems and proposed solutions.

The work of Integrated Human Practices has extensively and in-depth researched the multi-stakeholders in the S. invicta control process as well as related literature, better summarized the background knowledge of the S. invicta control project and in-depth study of the ecology of the industry, based on which the most critical problems currently faced by the industry have been summarized and attempted to come up with some solutions.

Fig 20.Main issues in the prevention and control of RIFA

Extensive research on the values and suggestions of multiple stakeholders in the prevention and control of S. invictas.

The integrated human practice work has clearly summarized the multiple stakeholders deeply involved in the process of S. invicta prevention and control and summarized them into a chart. At the same time, we have also extensively researched the values of the multiple stakeholders in the S. invicta prevention and control and by introducing our project to them and listening to their suggestions, we have achieved a continuous updating of our project and a better alignment with the needs of the society.

Fig 21.Multi stakeholder relationships

Fully consider the suggestions and criticisms of stakeholders, and update the plan to promote project development.

Our live bacterial drugs are facing the market and we have prepared a relatively complete plan for this. The launch of a drug requires multiple investigations and references, and we are currently preparing a safety research report for the project, which also requires public opinion.

In conversations with farmers, government personnel, biological companies, medical service personnel, etc., we have shaped more comprehensive and progressive plans to ensure the widespread absorption of stakeholder opinions.

Fig 22＆23.Needs and Objectives

Innovative use of models for objective evaluation and improvement of projects.

We innovatively used Maturity of HP work to evaluate our HP work and align with the best HP. At the same time, using this table, we filled in the gaps, sought innovation, and achieved self breakthroughs in the expected basic work.

Fig 24. HP Maturity Model

Reference

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