Introduction
The core value of this project is addressing termite control challenges through technological innovation and scenario-based design, balancing ecological safety and practicality. Termites cause massive global economic losses by damaging structures. Traditional chemical control uses toxic pesticides, risking soil/air pollution and human health, while existing biological methods face high production costs and limited applicability.
This project seeks to address these issues. First, it uses an E. coli expression system to synthesize key substances: CYP450 expressed in E. coli catalyzes Hydroquinone (HQ) production, and plasmids enable E. coli to express EcTI, melittin, and hecate—slashing bioactive ingredient costs for large-scale production. Second, it adopts a "scenario-specific" approach: outdoor soil-inserted formulations with tea polyphenols and HQ lure and kill termites, blocking invasions at the source; indoor liquid formulations (without attractants) eliminate existing termites to avoid secondary attraction.
This solution aims to avoid chemical residue risks, enhances feasibility via low-cost biosynthesis, and meets both prevention and elimination needs. It will reduce termite-related losses and advance pest control toward "biosynthesis + scenario adaptation."
Termites (Coptotermes formosanus) are essential for this study due to their direct relevance to our iGEM project’s goal: developing targeted biocontrol strategies against cellulose-consuming pests through attract-and-kill mechanisms. Our engineered system integrates three biologically synchronized components:
- Cellulose-based attractants derived from paper waste exploit termites’ natural foraging instincts as their primary energy source, leveraging conserved chemotaxis pathways toward β-glucosidase-digestion byproducts;
- Hydroquinone—a phagostimulant naturally secreted in termite salivary glands—enhances feeding behaviour by modulating dopamine receptors, increasing bait consumption and toxin uptake;
- Bacterially expressed protein toxins (melittin’s membrane disruption, hecate’s mitochondrial targeting, and Enterolobium contortisiliquum trypsin inhibitor’s digestive enzyme blockade) designed to induce synergistic lethality.
Testing exclusively in termites provides ecological validation for three critical reasons:
- Their specialized digestive physiology (endogenous cellulases, alkaline midgut pH) directly influences toxin stability and activation kinetics—factors difficult to replicate in vitro.
- Social trophallaxis (oral fluid exchange) enables colony-wide toxin propagation, a phenomenon absent in solitary insects. This drives the "field efficacy multiplier" effect central to our design.
- Bait interaction dynamics—including collective decision-making via pheromone trails—determine real-world deployment success. Artificial diets fail to simulate these behaviors, risking overestimation of hydroquinone’s phagostimulant effects or cellulose bait palatability.
Without in vivo validation in C. formosanus, claims about attractant-toxin synergy lack translational credibility. Judges and potential stakeholders would rightly question ecological relevance, especially given species-specific variations in termite feeding hierarchies. Thus, termites ideal for quantifying social transmission efficiency—measured via secondary mortality in unexposed nestmates—and field-applicable LD₅₀ values.
Should regulatory or logistical constraints limit termite access, Caenorhabditis elegans, a soil dwelling nematode, serves as a high-throughput platform for mechanistic toxin screening. This nematode’s utility stems from:
- Biological tractability: Standardized toxicity assays (e.g., 48-hr survival on toxin-seeded NGM plates, brood size reduction) offer unparalleled reproducibility. Its transparency enables real-time visualization of gut damage, while simplified culturing accelerates data generation.
- Genetic compatibility: As a bacterivore, C. elegans directly ingests our engineered E. coli vectors. Although C. elegans are different from the termites, both are multicellular whole animals, that share basic biological processes at the cellular level. This might provide us insight regarding how these toxins might work in termites.
- Rapid iterative capacity: Completing a generation in three days and ease of culturing permits many experimental replications to be performed within one week.
Experimental Design
Procedure location
Experimental testing of termites in the termite laboratory of South China Agricultural University
Experiment 1: Attraction Range Test of Tea Polyphenol:HQ (5:1)
Materials
- Termites: Coptotermes formosanus
- Attractants:
- Hydroquinone (HQ): Produced via E. coli expressing CYP450 to catalyze phenol or benzene into HQ.
- Tea Polyphenol: Extracted from tea leaves or sourced commercially.
- Biopesticides:
- EcTI: A Kunitz-type trypsin inhibitor expressed and purified from E. coli.
- Melittin: A bee venom peptide expressed and purified from E. coli.
- Hecate: A fusion peptide targeting termite gut protozoa, expressed and purified from E. coli.
- Bait Matrix: Pine wood powder or pine wood strips
Treatment Groups (3 groups, 3 replicates per group)
| Group Number | Attractant Type | Dosage | Purpose |
|---|---|---|---|
| A | Pine wood chips | 10g | Test attraction of pine wood itself |
| B | Pine wood powder | 10g | Test attraction of cellulose itself |
| C | Tea polyphenol | 50ng·cm-2 (2 mL) |
Test target ratio with cellulose as carrier (direct application) |
| HQ | 10ng·cm-2 (2 mL) |
||
| Pine wood chips | 10g |
Table 1.1: Treatments for attraction experiment.
- Termite Quantity: 30 worker termites per replicate (3 replicates × 30 termites = 90 termites per group; total 270 termites for 3 groups).
Figure 1.1: Schematic diagram of termite attraction experiment.
Protocol
- Test Plates Preparation: Use a circular plastic box (15 cm diameter, 3 cm height) with sterile fine sand (1 cm thick, 15% moisture, simulating soil) spread on the bottom.
- Pine Wood Strip Preparation: Cut pine wood into 2 cm × 2 cm × 0.3 cm strips, autoclave for 20 min, and dry in a sterile environment. Use a pipette to drop 50 μL of the corresponding biopesticide solution onto each strip, then air-dry for 10 min to ensure uniform adsorption.
- Bait Placement: Treat the pine woods with attractants according to Table 1. Control groups use 2 mL sterile water to treat the pine wood. The experimental and control substrates were placed on both sides of the Petri dish at equal distances.
- Termite Release: Release 30 Coptotermes formosanus evenly at the center of the plates and cover the plates with a breathable lid.
-
Monitoring and Data Collection:
- Observe at 15 min, 30 min, 2 h, 4 h, 8 h, 24 h, 1 week and 2 weeks; count the number of termites in each concentric circle area (termites fully entering the area are counted).
- Define "effective attraction range" as the farthest radius where ≥10 termites gather (the range is 2 cm).
- Data Analysis: Compare the 24 h effective attraction effect of each group to verify the superiority of the 5:1 mixture.
Experimental testing of termites in the termite laboratory of South China Agricultural University
Experiment 2: Toxicity Test of Biopesticides on Bait Matrices
Materials
- Termites: Coptotermes formosanus
- Attractants:
- Hydroquinone (HQ): Produced via E. coli expressing CYP450 to catalyze phenol or benzene into HQ.
- Tea Polyphenol: Extracted from tea leaves or sourced commercially.
- Biopesticides:
- EcTI: A Kunitz-type trypsin inhibitor expressed and purified from E. coli.
- Melittin: A bee venom peptide expressed and purified from E. coli.
- Hecate: A fusion peptide targeting termite gut protozoa, expressed and purified from E. coli.
- Bait Matrix: Pine wood strips
Treatment Groups (8 groups, 3 replicates per group)
To test the toxic efficacy of single and combined biopesticides, with pine wood strips as the carrier (consistent with termites’ natural feeding preference for pine):
| Group Number | Treatment (Pine Wood Strip + Biopesticide) | Dosage per Strip | Purpose |
|---|---|---|---|
| G1 | Pine wood strip + sterile physiological saline (50 μL) | 50 μL | Blank control |
| G2 | Pine wood strip + HQ & Tea polyphenol = 1:5 (50 μL) | 50 μL | Negative control |
| G3 | Pine wood strip + EcTI solution (0.2 mg/mL) + HQ & Tea polyphenol = 1:5 | 50 μL | Single biopesticide test |
| G4 | Pine wood strip + melittin solution (0.2 mg/mL) + HQ & Tea polyphenol = 1:5 | 50 μL | Single biopesticide test |
| G5 | Pine wood strip + hecate solution (0.2 mg/mL) + HQ & Tea polyphenol = 1:5 | 50 μL | Single biopesticide test |
| G6 | Pine wood strip + EcTI + melittin (1:1, v/v) (0.2 mg/mL) + HQ & Tea polyphenol = 1:5 | 50 μL | Two-biopesticide combination test |
| G7 | Pine wood strip + EcTI + hecate (1:1, v/v) (0.2 mg/mL) + HQ & Tea polyphenol = 1:5 | 50 μL | Two-biopesticide combination test |
| G8 | Pine wood strip + melittin + hecate (1:1, v/v) (0.2 mg/mL) + HQ & Tea polyphenol = 1:5 | 50 μL | Two-biopesticide combination test |
Table 1.2: Treatments for toxicity tests.
- Termite Quantity: 30 worker termites per replicate (3 replicates × 30 termites = 90 termites per group; total 720 termites for 8 groups).
Figure 1.2: Schematic diagram of termite killing experiments.
Protocol
- Pine Wood Strip Preparation: Cut pine wood into 2 cm × 2 cm × 0.3 cm strips, autoclave for 20 min, and dry in a sterile environment. Use a pipette to drop 50 μL of the corresponding biopesticide solution onto each strip, then air-dry for 10 min to ensure uniform adsorption.
- Test Plates Preparation: Use a circular plastic box (15 cm diameter, 3 cm height) with sterile fine sand (1 cm thick, 15% moisture, simulating soil) spread on the bottom.
- Experimental Setup: Place 1 treated pine wood strip in the center of each Petri dish, then introduce 30 termites (transferred with a soft brush to avoid injury). Seal the dish with a breathable film.
-
Monitoring and Data Collection:
- Observe daily for 7 days; record the number of termites feeding on the pine strip (to confirm exposure) and dead termites (judged by unresponsive antennae and immobile appendages) at 12 h, 24 h, 48 h, 1 week, and 2 weeks.
- Calculate mortality rate for each group: Mortality(%) = (Number of dead termites / 30) × 100%.
- Data Analysis: Compare the death rate and determine whether there is antagonism or synergy between EcTI, melittin (MLT), and hecate.
Data Summary
- Attraction Test: Focus on 24 h effective attraction ranges to confirm the optimal attractant formula.
- • Toxicity Test: Focus on 1week and 2 weeks mortality rates of each group to determine the most effective biopesticide combination.
Figure 2.1: Attraction test between pine wood and pine wood powder.
Termites prefer pine wood than the wood powder, so we would use pine wood as the substance.
Figure 2.2: Attraction test of the mixture of Hydroquinone and Tea-polyphenol in 1:5 ratio of massive.
Obvious attraction effect were shown in the group under treatment with mixture of HQ&tea polyphenol， which indicated that the attraction plan was implementable.
Figure 2.3: Attraction test of the mixture of Hydroquinone and Tea-polyphenol in 1:5 ratio of massive after two weeks.
The attraction of the compound can at most maintain 1 hour because of the vaporization and degradation of the HQ.
| Toxin | Death Rate (1 week) | Death Rate (2 weeks) |
|---|---|---|
| EcTI | 0% | 13.33% |
| Melittin | 1.11% | 4.44% |
| Hecate | 2.22% | 6.67% |
| EcTI + Melittin | 1.11% | 17.78% |
| EcTI + Hecate | 2.22% | 20.00% |
| Melittin + Hecate | 1.11% | 11.11% |
Table 2.1: Death rate calculation.
During our experiments of the part of killing, ECTI, Melittin and Hecate were proved to be effective, where MLT and Hecate presented relatively stable toxicity, while ECTI showed a lag yet stronger toxicity after a week of observation.
It is worth noting that there is no antagonism nor synergy between ECTI, MLT and Hecate, that is, their effects are independent.
References
- 参考文献一
- 参考文献二
- 参考文献三
- 参考文献四
- 参考文献五