Peptides nature

When we talk about biopesticides, it refers to substances that come from a living organism such as fungus, bacteria, plants, etc. Spidicide-CX is composed by two peptides (ω-HXTX-Hv2a and U1-theraphotoxin-Sp1a) from two venom spiders: Selenotypus plumipes and Hadronyche versuta.

These peptides have been reported with toxic functions to neuronal receptors and voltage-gated sodium (NaV) and calcium (CaV) ions channels in insects (Shen et al., 2018). It is worth noting that their functions are highly specific for some insect orders, mainly the Coleoptera order, the order that belongs to the pests we intend to control.

Also, these peptides are not toxic for human being according to literature, and they have a high degradation rate wich avoid the bioaccumulation. So, its use does not represent any risk to the environment either (Hardy et al., 2013).

For more security, we search for related allergies to these peptides and similar peptides to be sure that its constant use does not affect the farmer's health. Fortunately, there are no allergies reported. To see if the Spidicide-CX peptides cause allergies, we used the I-TASSER server. This server helped us to find similar peptides according to our peptides sequence. This is because there is no recorded data that our peptides are harmful to humans. Several peptides were related to the ones of Spidicide-CX. Nevertheless, after in-deep research, we found out that there are no related allergies to similar peptides. However, in future, we could plan to prove if they cause any allergy in long-term exposure.

Proposed application of Spidicide-CX

The growth of the world population causes a proportional increase in the demand for food, so the agricultural sector is one of the main sectors that are affected and under pressure. Due to this, the use of fertilizers, pesticides, herbicides, and other agrochemical compounds to maintain this demand has also increased, causing unwanted effects on crops and farmer’s health (Kah & Hofmann, 2013).

Therefore, it is essential to search for new tools that, with the advancement of science, are potentially applicable to combat these problems and, at the same time, reduce costs, especially for stakeholders, increase productivity, improve efficiency and reduce environmental impact.

The iGEM teams from Tec Chihuahua and UAM are solving similar local problems since both are developing biopesticides. Tec Chihuahua's active ingredients are two antimicrobial peptides and interfering RNA to counteract the losses in chili production caused by the oomycete Phytophthora capsici, which is one of the most destructive pathogens in chili crops. On the other hand, UAM is developing a biopesticide based on two spider venom peptides from Australian spiders to counteract the pests that cause the most damage to cactus, avocado, and agave crops. These pests are insects that belong to the Coleoptera order.

Therefore, the two teams collaborated through in-depth research to determine a delivery method that could work for both biopesticides. Finally, we concluded that the nanotechnology field could be a great solution.

The emerging application of nanotechnology-based pesticides is known as nanopesticides. These methods have made it possible to improve the efficacy of pesticides, reducing doses and improving the stability of the active ingredients, which reduces environmental impact. It is expected that these nanopesticides could contribute to traditional pest control strategies through the use of nanoparticles to more easily penetrate the target organism, be resistant to environmental conditions, and at the same time not affect the surrounding crops and wildlife (Smith et al., 2008).

Comparative table of this research

In this collaboration, both teams are from Mexico. In our country, we face several problems related to the lack of regulations in the synthetic biology field and the fact that our stakeholders have many fears related to biotechnological products. Because of this and after thorough research, both teams concluded that the chitosan nanoparticles could be a suitable delivery method because this presents many advantages. So, we have to bring the information about this technology to our stakeholders in a friendly way.

Chitosan is the second largest available biopolymer in nature and is a polysaccharide copolymer. It is produced after the deacetylation of chitin harvested from crustacean shells. The applications of chitosan have been explored in many industrial sectors such as the food industry, packaging, pharmaceutical, and agriculture, among others (Ahmed Wani et al., 2022). There is increasing interest in using nanoencapsulation as a tool for delivery systems of many components, such as antimicrobial peptides, iRNA, and active ingredients (Patiño, 2017).

General characteristics

The chitosan nanoparticles have high biodegradability, biocompatibility, and low toxicity. These characteristics generate more interest in the application as carriers of compounds such as pesticides (Sharifi-Rad et al., 2021).

These nanocapsules have components hydrophobic and hydrophilic, converting the product to amphiphilic. Therefore, this property promotes the formation of micellar structures and provides a stabilizing interface between the nucleus of the nanoparticle and the aqueous environment.

Biodegradation

In the nanotechnology field, synthetic nanoparticles have issues due to materials used as nanocarriers being toxic and having poor degradability. However, the use of different kinds of materials as nanocarriers has emerged, such as chitosan-based nanoparticles, which counteract these effects caused by synthetic nanoparticles. The chitosan-based nanosystems can be used as advanced delivery systems due to their capacity to alter protein loading and adjust the value of each parameter during preparation. They also have high stability, high protein packing efficiency, and are easy to store and transport (Sharifi‑Rad et al., 2021).

Toxicity

When chitosan is degraded by enzymes, amino sugars and other non-toxic compounds are released. Therefore, chitosan is considered a biocompatible compound with low toxicity. Only one data has reported that chitosan presents minimal LD50 toxicity of 16 g/kg, which is similar to the toxicity of salt and sugar, so it is considered safe for plants and animals (National Toxicology Program, 2017).

Methodology

Chitosan nanoparticle production

Chitosan nanoparticle production is easy. Currently, there are a lot of methods by which it can be achieved. The protocol that both teams have chose reduces costs and doesn’t require sophisticated equipment or reactants in the lab. This process is known as ionic gelation.

1. To prepare a chitosan solution of 1.5% w/v, dissolved chitosan in glacial acetic acid solution at 1% (v/v) or hydrochloric acid (HCI) at 0.1% v/v concentration.
2. A pH adjustment to the solution must be performed at 5 by adding Sodium hydroxide (NaOH) 1.0 M. Stirring the solution for around 40 minutes.
3. Simultaneously, the preparation of the cross-linking agent can be done using sodium tripolyphosphate (TPP). It is prepared as 0.1% wt solution in deionized water. Adjust the pH with the additions of HCl 0.1 M.
4. Dissolve the chitosan solution in the polyanionic solution (TPP) to obtain the cation of chitosan. A stabilizing agent, such as Poloxamer can also be added but is optional.
5. Stir the chitosan/TTP solution constantly for 10 minutes at room temperature; the precipitation forms the nanoparticles.
6. The suspension is centrifuged to separate the nanoparticles from unreacted chitosan and TPP. The pellet of nanoparticles could be resuspended in water (ChitoLytic, 2022).

Figure 1. Diagram of chitosan nanoparticles production through ionic gelation

The specifications of quantity and concentrations of each reagent are variable because those parameters can change depending on the characteristics of the nanoparticles that could be needed. A variability in concentration or times could change their size or porosity. This is one of the reasons that makes chitosan nanoparticles able to encapsulate many kinds of molecules.

Nanoencapsulation

To encapsulate the active ingredients of both pesticides, we based on a protocol made by Mudo et al. (2022). They have analyzed the best parameters for dsRNA encapsulation without compromising its integrity or stabilization.

1. Prepare a solution with the chitosan particles at a concentration of 3.0 µg µL−1.
2. Add dropwise a 1000 µL solution composed of 700 µL of TPP (1.2 µg µL−1) and 300 µL of dsRNA (550 µg mL−1) to the chitosan solution under magnetic stirring. The nanoparticles are separated through centrifugation (de Britto et al., 2014).

Figure 2. Encapsulation of protein or active ingredients in chitosan nanoparticles

Proof of Concept

To verify the efficacy of Spidicide-CX over commercial pesticides, we designed this proof of concept. It consisted of feeding five groups of larvae and adult insects with different treatments (control, buffer, malathion, OAIP-1 with lectin, AcTx-Hv2 with lectin, and AcTx-Hv2 with lectin + OAIP-1 with lectin) every 12 hours. And during that, we will observe the mortality of the insects and do an ANOVA and Tukey tests to compare the data recollected.

Proof of concept results

Due to the lack of insect samples because it is no longer cactus weevil season, we modified the initial protocol. These new changes consisted in reducing the treatments, as shown below. Another inconvenience in the realization of this protocol was the lack of time. So, we collect the first hours of data without finishing the minimum data to run statistical tests.

We collected the larvae and adult cactus weevils since we got permission from the Safety and Security Committee. In the first place, we submitted the Animal Use form, but we were notified that this form wasn’t necessary. Instead, we must submit just a check-in form. We received the answer by October 5th. So, we tried to hurry to collect the insect samples on October 8th.

These are the results we were able to obtain:

Table 3. Alive adult insects

Table 4. Alive larvae insects

These preliminary data have shown positive results, in which the peptides of Spidicide-CX have a similar effect to the chemical pesticide (Malathion). It is worth mentioning that the concentration of the Malathion solution was high (84% w/v). So, this could explain the rate of dead insects fed with this commercial pesticide.

In addition, we have to comment that larvae are more sensitive to external conditions. An example of this is that they are photosensitive. Therefore, it is probably correlated with the death rate in Table 2 because most of them died with such a high concentration of Malathion.

Figure 3. Proof of concept carried out on larvae and adults of the enemy: Cactus weevil

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