Over the past century, economic globalization and global warming have hastened the pace of planetary change and biological invasions. The economic damage caused by invasive alien ants is steadily increasing.
Using the InvaCost database, Taheriet and colleagues analyzed the cost records associated with invasive ants . It was found that since 1930, invasive ants alone have caused direct economic losses exceeding $10.95 billion globally.
Moreover, when factoring potential costs (which incorporate both temporary and spatial dimensions, such as the cost of overseeing the period in which long-term management actions are planned or executed, and the cost of projecting areas that have no actual expenses), the projected expenses for the period between 1980 and 2084 surpassed $40 billion .(See Fig. 1)(Taheriet al.,2022)
Figure 1 economic costs invasive ants worldwide
[Data source: Elena Angulo et al. (2022). “Economic costs of_invasive alien ants worldwide”Biol Invasions (2022) 24:2041–2060. doi:10.1007/s10530-022-02791-w]
What is surprising is that, over 60% of the economic costs resulting from invasive ants were attributed to Solenopsis spp., with Solenopsis invicta (S. invicta) causing the most significant economic losses, as depicted in Fig. 2. It is estimated that the annual cost for the S. invicta in the US was initially $1 billion . (David Pimentel,2005).
Figure 2 Solenopsis invicta（S. invicta） accounted for most of the cost entries
[Data source: Elena Angulo et al. Biol Invasions, 2022 April Nobile / AntWeb.org / CC-BY-SA-3.0]
Global issue
S.invicta invasion from South America has occupied over 100 million acres in 17 American states since 1930s. These ants also invaded states like California and New Mexico and other countries like New Zealand and Australia in 2001 (Haotao, Chen,2010). With global warming, S.invicta has continued to spread to higher latitudes in recent years(Haoran Wanget al.,2022)
The following diagram provides an assessment of the potential global invasion risk posed by S.invicta. (Fig. 3)(Shuai Chenet al.,2020)
Figure 3
[Data source: Chen, et al. (2019). "Global Distribution of Red Imported Fire Ants." Sustainability, 12, 10182. doi:10.3390/su122310182.]
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
Recognized as a top 100 invasive species worldwide, S.invicta presence in invaded regions seriously threatens agriculture, livestock, public infrastructure, ecological communities and human health.(Fig. 4)
Figure 4 S. invicta Inflict Significant Damage in Invaded Regions [Data sourse: California Agriculture, 2002; Journal of Environmental Entomology, 2022; Journal of Environmental Entomology, 2022]
Specifically manifested as:
1. Extremely aggressive, hazardous to human health.
2. Damaging agricultural and livestock production, destroying public facilities, and cumulatively causing huge economic losses.
3. Destroy the ecological balance and reduce biodiversity.
(For details on S.invicta hazard, see Human Practice: Overview of the Problem).
Through this project, we hope to provide eco-friendly, safe, user-friendly, low-cost, more effective, more targeted, and water-resistant biological control agents for S. invicta control worldwide. That is why our team decided to develop Queen Ant's Assassin (QAA).
QAA is an engineered intestinal bacterium that allows us to efficiently eliminate S. Invicta Queen.(See Fig. 9)
Figure 9
1. Why were engineered live bacterial products chosen for S. Invicta control?
During this year's IGEM program, in order to move our project forward, we consulted with a number of wet lab experts in related fields to listen to and incorporate their advice into our project.
As a result of our consultations, we learned about the following current biological control methods and characteristics of S. Invicta:
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Introducing Natural Enemies
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Insect fungus control
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Engineered Live Bacteria Drug
Click on the images to learn more about the limitations of the control method
lt is easy to bring new ecological problems into the world.
The social defensive behavior of S. Invicta (especially the "corpse abandonment'behavior) interrupts the transmission chain of the fungi, which is an important reasonwhy the effectiveness of insect-borne fungal control is not widely used Still now, thereis no breakthrough solution.
The bacteria must be able to adapt to the invader's qut environment and not bepoisoned by the invader's toxins
Research by Prof. Cheng Dai-Feng, an expert in the field of S. Invicta gut symbiotic bacteria, has shown that S. Invicta gut symbiotic bacteria is a possible new biological control method because it's not easy to induce S. Invicta social defense behaviors, especially the "carcass dumping" behavior.
Based on this, we decided to modify the engineered live bacteria to achieve S. invicta extermination.
2. Chassis Selection --- Why E. coli (Escherichia coli)?
When choosing the chassis of the bacteria, we need to focus on whether it can adapt to the environment of the S. Invicta gut and whether it can be poisoned by the active ingredients of the S. Invicta venom, because this directly determines whether our engineered bacteria can reproduce, express the drug, and ultimately produce the venom in a better way in the environment of the S. Invicta gut.
With these suggestion in mind, we chose E. coli as our chassis organism after a thorough literature review. Here is why:
Video: Project Demo
Drug selection
We hope to achieve the goal of eco-friendly, safe and more effective by slecting suitable protein -based bioactive materials with insecticidal function.
For this reason, we chose to make QAA secrete Bt toxin Cry3A-like protein and cowpea trypsin inhibitor (CPTI) under specific time and space, so as to control S. Invicta, especially the queen ants.
Simultaneously, we exploited the different insect resistance mechanisms of the two proteins to establish a multi-insect resistance mechanism (Xuhong-lin et al.,2008).
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Cry3A-like protein
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Cowpea trypsin inhibitor
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Mechanism
For more information, click on the images
Bacillus thuringiensis UTD-001, as well as the protoxin and toxin of this isolate, maybe used to control pests such as fire ants, carpenter ants, Argentine ants, and Pharaoh's ants, including S. Invicta. Cry3A-like toxins isolated from UTD-001 have been shown to be toxic to S. Invicta.(Lee A. Bullaet al.,2003)(See Fig. 10)
A. How does it work?
It has been shown that after treatment with papain in vitro, the Cry3A-like toxin prototoxin (73KD) forms an active toxin (67KD) that is toxic to S. Invicta.
When the active toxin binds to the aminopeptidase nitrogen and calreticulin analog receptors on the midgut rim membrane of S. Invicta, or penetrates the cell membrane and forms a perforation, the ionic balance between the periplasm of the cell membrane and the midmembrane lumen is disrupted, resulting in cell swelling and even lysis, ultimately leading to paralysis or death of the insect.(Lee A. Bullaet al.,2003)(See Fig. 11)
B. What we have done?
The Cry3A-like gene was designed to directly produce an active, soluble 67 kD form of the toxin, and the secretory expression of the toxin was successfully achieved, while the biological activity of the designed protein was preliminarily verified by molecular simulations, etc. (See Proof of Concept for more details)
C. Is it eco-friendly and safe?
Cry3A-like toxins are a type of Bt protein. Bt protein is recognized as a safe and environmentally benign insecticide that is harmless to human health, environmentally friendly, and non-toxic to mammals and most beneficial insects.(For details, see Safety page)
CPTI is a plant-derived anthelmintic protein commonly used in genetic engineering for insect resistance due to its broad spectrum of resistance and low susceptibility to insect tolerance.(See Fig. 12)
A. How does it work?
It was found that in order to achieve successful delivery of the modified cryA-like protein from the larval gut to the queen through the cross-feeding behavior of S. Invicta (please set a redirect link)(See Fig. 13), the problem to be solved is to reduce the degradation rate of the cryA-like protein during the delivery process.
In addition, a review of the literature revealed that S. Invicta have amide-solubilizing activity only in the larval intestine, while adult ants have little or no amide-solubilizing activity.(Travis, Jameset al.,1998) This means that Inhibiting the activity of intestinal proteases (mainly serine proteases) in fourth instar larvae would effectively reduce the rate (DAVID P. BOWN,1997; Travis, Jameset al.,1998), at which cryA-like protein is degraded by intestinal proteases during intestinal delivery, thereby improving the efficiency of toxin delivery.(See Fig. 14)
CPTI's mechanism of action is to interact with and inhibit the insect gut serine protease active site. Simultaneously, CPTI inhibits insect feeding by stimulating the production of insect feedback signals.(ＸＵＨｏｎｇ-ｌｉｎet al.,2008). In this case, CPTI can lead to reduced egg production or death of the queen due to lack of essential nutrients. (SORENSEN,1986)
B. What we have done?
Based on this, we introduced CPTI and successfully secreted and expressed the active protein. (See Proof of Concept for details)
Is it eco-friendly and safe?
A large number of medical studies have shown that this family of proteins is not harmful to human health. Pest spectrum testing has shown that CPTI inhibits nearly all major agricultural pests tested.(Xuhong-Lin et al.,2008)(For details, see Safety page)
It has been shown that pests are susceptible to developing tolerance to a single gene. An important measure that is now more feasible is the creation of multiple insect resistance mechanisms. Using Cry3A-like toxin and CPTI, two genes with completely different insect resistance mechanisms, together would greatly reduce the chances of an insect developing tolerance. Theoretically, the probability of an insect developing tolerance is the product of the probability of two separate genes developing tolerance.(Xuhong-lin et al.,2008)
Design of drug modulation circuits
As mentioned above, to enrich the targeting drug to the queen, we introduced CPTI to inhibit the activity of intestinal digestive enzymes in fourth instar larvae.
Here, to further improve drug delivery efficiency, we introduced the Tra* quorum sensing system(QS system-1) to achieve a cascading delayed release of Cry-3A like toxins and CPTI.
As the cell density increases, the concentration of toxic proteins would accumulate to the point of premature larval death, which would be detrimental to the enrichment of our drug into the queen via back-feeding. Therefore, we introduced the Las quorum sensing system(QS system-2) , which was modified to be complete orthogonal to the QS system-1.(Jianget al.,2020).
QAA will initiate a T4 Lysis Device in response to activation of the QS System-2, thereby preventing the continued overaccumulation of toxic proteins. (More design details can be found on the Design page.)(see Fig. 17)
Figure 17
Biological Safety Device (BSD)
Inspired by our interaction with Tsinghua University at the Conference of China iGEMer Community (CCiC), we successfully designed our biosafety device based on constructing a nutrient-deficient strain (see Human Practice for details).
The modified bacteria will have the following characteristics: They can only survive in baits with exogenously added added Diaminopimelic acid(DAP) or in the anaerobic environment of the S. Invicta gut, and they will die due to nutritional deficiencies when released into an aerobic environment without DAP(see Fig. 18).
Figure 18
Oleic acid is the attractive high fatty food to S. Invicta, on the other hand, it also improves the waterproof performance of the drug to a certain extent. (For more design details, please refer to: design page)
Figure 19
(Activated at a cell density of 1(ρ-1); Activated at a cell density of 2(ρ-2). For more design details, please refer to: design page)
At the same time as completing the schematic design of our project, our team also consider how to implement our engineered bacteria as an operational biopesticide, and how to achieve efficiency and safety through the design of our hardware equipment. On this basis, we also conducted research and consultations with various stakeholders. (See more details in Human Practice: Implement)
We focused on improving the moisture resistance of the bait, while ensuring the bait attracts S. Invicta and protecting the activity of the engineered bacteria. Through literature research, we learned that the S. Invicta bait encapsulation developed by Feng Xie met the needs of our project, so we decided to build on his research to further develop our drug carrier(see Fig. 20).
Figure 20
(For more design details, please refer to: Intergrated Human Practice page)
We hope that by designing a suitable device, we can reduce the cost of S. Invicta control and improve the safety and efficiency of control. Based on this, we confirm that the design criteria are:
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simple operation
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high dosing efficiency
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controllable single dose
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low production cost
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low energy consumption or manual
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durable and easy to maintain device
For more information, click on the images
(For details on the research process, see Human Practice: Engaging with Affected People ).
After iterative design, we ended up producing and designing a series of devices based on the original dispersal device that were expected to achieve our desired goals (See Hardware for details).
From a synthetic biology standpoint, our efforts in controllingS.invicta have been closely linked to promoting public awareness of synthetic biology. We've engaged in knowledge-sharing with experts in red ant research and pesticide specialists. By collaborating with local government officials, we've developed a tailored communication approach for our synthetic biology project, facilitating better understanding of synthetic biology applications among the public, communities, and stakeholders."(See more detail in Education)
Our choice of S.Invicta as a target initially stemmed from a backcountry trail experience of one of our members who was suffering from bites caused by the S. Invicta that were proliferating in his hometown. After doing some initial research on red fire ants, our team realized that this is a very aggressive invasive species and that the rapid spread of red fire ants in our country was a concern. So we decided to develop a synthetic biology solution to the S. Invicta problem.
A good solution for S.invicta control
Contribute to the popularization of S.invicta control knowledge, outreach, and education
Expanding Synthetic Biology Public Awareness.
Engaging Stakeholders for Community-Centric Projects
Our project introduces a novel strategy for global biological control of invasive ants, focusing on eco-friendliness, lost cost and efficiency. (Fig. )
Our approach comprises three key elements:
1. Intestinal Flora as Carrier Bacteria:
Modifying ants' gut bacteria to produce insecticidal proteins, targeting invasive ants during feeding.
2. Cross-Feeding Behavior:
Use of ants for cross-feeding behavioral enrichment with the goal of eradicating ant queens.
3. Three-Tier Cascade Control Circuit:
A precise delivery system minimizes collateral damage when applying substances.
Over a year and a half of HP activities, we iteratively improved our how-to. (For a brief overview, see the Engineering Success)