Spider mites (Acariformes: Tetranychidae) are ubiquitous pests that had vigorously evoked several massive product loss, including 330,000 acres of wheat land affected in Jiangsu China, 2024. Also, scattered product loss escalates as their population exponentially grows on leaves. [1] However, current arrays of chemical acaricides targeting spider mites are shown to be inefficacious. For instance, the most popular acaricides, including bizenafate and etoxazole, are already experiencing regular resistance development. Specifically, spider mite resistivity to etoxazole could escalate by ten times in merely 5 generations [2] with a generation capable of being completed within a week [3]. Additionally, there were reported cases of Tetranychus urticae, a prominent member of the spider mites family, being more numerous on sprayed plots than unsprayed plots [Fig.1].

Cross-resistance is also pervasive. Spider mite strains reported to be resistive against one acaricide often have resistance against many more [4]. For instance, a common cross-resistance would be between etoxazole and hexythiaz [5]. Their resistances are genetically linked to each other, suggesting that the use of only one of these acaricides would cause resistance to both.

Multiple incidents reveal that their population occasionally hits zero but always rises again after a seemingly extermination. For instance, there was an outbreak of Tetranychus urticae in apple orchards in Akita Japan caused by broad-spectrum usage of insecticides [6]. Therefore, the urgency for such a problem to be resolved is elucidated.


But to the ultimate question, what are spider mites?

Spider mites (acariforme: Tetranychidae) are a family of ubiquitous pests that arbitrarily embroiled itself in uncountable major horticultural loss. Whether it is large-scale agriculture, household greenery, or public forestry [Fig.2]. As long as there is cultivation involved, there is a possibility of being parasitized by spider mites. What's trepidatious is that each spider mite would lay an approximate of 80 eggs in their voracious life time, resulting in significant distribution of them on affected plants . During a recent spider mite infection in Hainan China, there appeared to be averagely 57.4 red spider mites per leaf [Fig.3].

After settling themselves on their "habitat", they begin their plundering of plant nutrition. This process damages plant leaves in a way that causes noticeable changes to their appearance and function. When infested by spider mites, leaves typically develop small, light yellow or white spots due to the mites sucking out the cell sap. As the damage intensifies, these spots may enlarge and merge, causing the overall leaf color to turn yellow or even a bronzed hue. However, the result is not only a decline in aesthetic value, but also a significant reduction in the plant's productivity and growth rate [Fig.4]. The spider mites often produce thin webbings on the underside of leaves to protect themselves from predators, which reduces the efficiency of photosynthesis, impacting the overall health and growth of the plant. comparing to the non infested plant, the infested plant show a relatively low productivity [25].

The current array of strategies against spider mites are usually either biological or chemical. However, none of them are proven effective enough to prevent nor reduce damage.

Biological control involves introduction of various natural predators of Tetranychidae mites, however, the most widely used predator (due to its selectivity on the spider mites), Phytoseiulus persimilis , depends heavily on the environmental conditions, such as humidity and temperature. Various other predators, including N. californicus, F. acarisuga, and N. Cucumers are either predators for an array of various insects, and thus may impact local ecosystem, or ineffectual due to low birth rate comparing to the exponential growth of spider mites. In addition, biological methods could not be utilized along with chemical pesticides, resulting in lack of efficiency for personal cultivators.  

Chemical approaches involve a range of acaricides that are either potentially dangerous to mammals or are already resulting in signs of resistance development in the spider mites.

Unlimited to the most widely-used bizenafate and etoxazole, a number of other acaricides have been assessed and proven inefficacious to some extent against spider mites, including fenbutatin oxide, fenpyroximate, amitraz, et cetera [7]. Furthermore, cross resistance against other acaricides is also as significant. That said, various spider mite strains reported around the world that is found to be resistant to one type of acaricide is highly likely to display resistance against multiple other types of acaricide. Thus, it will ultimately result in a global development of extended resistance in spider mites.

In addition, chemical pesticides have the ability to incur adverse severe health effects on humans, including an increased risk of Alzeheimer's disease, congenital abnormalities, and neurodegeneration [8]. 

There are also traditional treatments of painting tree trunks with lime water or submerging the plant in water, but such practice had proved to be ineffectual or unfitting for various scenarios.

While there are on-going selective breeding programs that are aiming to produce mite-resistant and economically viable crop species from the crop’s wild relatives that had already developed spider mite resistance, it is impossible to do so for all affected plant species, and such programs all take at least years to complete.


Therefore, GreatBay 2024 presents Dienamite⸺A powerful pesticide that addresses all aspects of pest infection, moreover, specializes in eliminating spider mites with a safer, more efficient and complete method. Whether you are a horticulturist or a farmer, Dienamite will be a desirable weapon against the attack of spider mites.

During the pre-infection stage, the first component of our product, 7epiZ, 9HZ and 9H10epoZ establish repelling and progeny-reducing effects towards adult mites, preventing further infestation. During the mid-infection stage, the second component of our product, venom peptides derived from predatory mites and various spider species exterminate adult mites by targeting their central nervous system. These neurotoxins can bind to various ion channels including Na_V, K_V, and Ca_V, making it difficult for the mites to develop resistance. During the post-infection stage, the third component of our product, harpin protein can be used to boost plant immunity and promote plant growth with a recovery impact [Fig.5].

Being a complete product that not only serves the purpose of pesticides, but also takes plant recovery and pest prevention into consideration, we proudly claim that Dienamite is the absolute solution for the spectrum of demands from stakeholders: safety, odorless, hard to develop resistance, et cetera.

Dienamite is an insectidal spray containing zingiberene, spider venom peptides, mite venom peptide and harpin protein. We produce 7epiZ through the use of E. coli, utilizing double plasmids. Then, 9HZ and 9H10epoZ is produced through oxidation of 7epiZ, using the co-culture of Saccharomyces cerevisiae and E. coli to produce the oxidase and reductase. Synthesis/production of spider venom peptides and mite venom peptides involve both E. coli and Pichia pastoris. Lastly, Harpin protein is expressed successfully in E. coli .


In order to make our pesticide both powerful and safe for usages, we came up with the idea of incorporating venom peptides from predatory mites, natural predators of spider mites. During our initial research, we discovered Dr.Chen et al.’s study that described two venom peptides from the polyphagous predatory mite Neoseiulus barkeri. The peptide, named NbSP2, contains an inhibitor cystein knot (ICK) enabling it to have strong stability and, as its name suggests, inhibits its molecular targets. It is found to be highly toxic against T. cinnabaribus, and mortality occurred mainly within 24 hours.[9] According to the research, injection of NbSP2 caused a mortality of 54.7% in 24 h and 58.5% in 48 h.[9]

However, incorporation of mite venom peptides (MVP) with even higher toxicity into our project is desired. Thus, with further research, we believe Phytoseiulus persimilis, a specialized predatory mite that is widely used as a biological control agent against spider mites, will be a satisfactory candidate as the source of MVPs. We therefore conducted genome mining to uncover the P. persimilis venoms.

Using coding sequences of the two venom peptides from Dr. Chen et al.'s research as the query against the P. persimilis genome[10], we located the orthologous loci of the persimilis venoms. We then filtered out the exons and obtained the two venoms' protein sequences. Looking into molecular structure of MVPs, we discovered spider venom peptides, a class of well-characterized, cysteine-rich venom peptides that originates from spiders shows a high degree of homology towards the MVPs. We then realized that MVPs can be truncated to allow better heterologous expression and toxicity.

Looking into molecular mechanisms of the SVPs, we realized they paralyze and eliminate their targets by binding to ion channels that lead to blocking of relay of neural impulses. And, wishing to identify potential molecular targets of our MVPs, we constructed a phylogenic tree of the MVPs along with SVPs with characterized targeted ion channels. We thus identified all of the MVPs to be likely targeting voltage-gated calcium channels. That said, mutations in the Ca_V channel will likely lead to resistance against all MVPs. Knowing the spider mites to be highly adaptive and develops resistance extremely rapidly—one of the very reasons why our project was established—we decided to incorporate several spider venom peptides that target several other ion channels. MVPs can therefore be used along with the SVPs to target a more diverse range of molecular targets and therefore prevents resistance from developing.

In nature, the venom peptides are employed by predator spiders and mites to subdue preys and deter predators through injection. However, for a sprayed acaricide, it is necessary for the venom peptides to display oral and contact toxicity. Thus, Galanthus nivalis agglutinin (GNA), a plant derived mannose-specific lectin that provides resistance to enzyme-meditated proteolytic activity in insect gastrointestinal tract is introduced and fused with the venom peptides. GNA is able to transport attached peptides across the insect gut, allowing delivery to the circulatory system [11]. Through fusion with GNA, the fusion protein HxTx-Hv1h-GNA was observed to possess both enhanced oral and contact efficacy [12]. In addition, the lectin is toxic to both chewing and sucking pests, thus further enhancing the toxicity of the fusion protein [13].

As cysteine-rich neurotoxins, our venom peptides could not guarantee correct folding in reductive environments such as the cytoplasm[14], therefore resulting in inclusion bodies. To tackle inconveniences during synthesis of venom peptides due to their intracellular expression, we attempted to utilize the annex of G1M5-SUMO tag before MVP and SVP [Fig. 8]. G1M5 is a signal peptide that is originally found in prokaryotes and can transport attached protein directly into the less reductive environment in the extracellular milieu[15]. SUMO fusion leads to enhanced expression and solubility.[16]

According to an interview with Professor Fitches, Pichia pastoris was recommended to be used as the chassis instead of E. coli for extracellular expression, increasing production level and reducing production and purification costs. Thus, we incorporated P. pastoris as our chassis for efficient secretion of the venom peptides.[11]


We discovered a type of sesquiterpene—7epiZ—originally produced and utilized in glandular trichomes of accessions of wild tomato species Solanum habrochaites, which is proven to have repellent, fecundity-reducing and fatal properties. During our further research, we soon discovered the presence of two oxidation products of 7epiZ, 9HZ and 9H10epoZ, which are considerably more effective in repelling spider mites as indicated by their apparent reduction in EC₅₀ values [17]. Hence, we decided to produce the three terpenes in a 2-step pathway involving sequential oxidations to enhance the insecticidal effects of the insecticide [Fig. 9].

To synthesize 7epiZ, 9HZ and 9H10epoZ, we are first going to utilize the Mevalonate pathway, also known as the MVA pathway, to synthesize isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) from glucose as the precursors to sesquiterpenes. Meanwhile, we employ the enzyme SltNPPS to transform IPP and DMAPP into neryl diphosphate(NPP). NPP is then used to produce Z,Z-farnesyl diphosphate (Z,Z-FPP) by Mvan_4662, an enzyme derived from M. vanbaalenii, which is manifested to be the most productive Z,Z-FPP synthase compared to others [18]. At the downstream of the pathway, the zingiberene synthase LA2167-ZIS is introduced to transform Z,Z-FPP into our first desired product, 7epiZ [19]. Furthermore, with the use of ShZPO [Fig.9], a cytochrome P450 oxidase found in Solanum habrochaites and SlCPR2/AtCPR1, cytochrome P450 reductases found in Solanum lycopersicum and Arabidopsis thaliana respectively, 9HZ and 9H10epoZ are produced from further oxidation of 7epiZ [20]. To imitate anchorage to ER of the reductase and oxidase in plants, that brought about the two enzymes to proximity to allows efficient electron transfer, we opted to use SpyTag-SpyCatcher system[Fig.10], acting as a flexible bridge linking the oxidase and the reductase [21]. This ensures efficient electron transfer and thus efficient biosynthesis.

According to Dr. Su from Earlham Institute, using yeast instead of E. coli as our chassis can increase the efficiency of production due to the eukaryotic nature of the involved oxidases and reductases. For efficient production of 9HZ and 9H10epoZ, we explored the co-culture of E. coli with yeast Saccharomyces cerevisiae, with the former producing 7epiZ and the latter oxidizing it for synthesis of 9HZ and 9H10epoZ.

To prevent immunodeficiency and even death of plants even after the elimination of the spider mites via insecticides that is reported by many horticulturists we have interviewed; harpin, a glycine-rich and thermal-stable protein found in prokaryotic secretions, is introduced to stimulate immune response and promote growth in plants [Fig.12] that had been devastated by spider mites' invasion [22].

Synthesis of harpin is achieved via engineered E. coli. For more efficient expression, synonymous codon optimization is carried out in the translation initiation region (TIR) of the harpin protein. As demonstrated by our BCA assay, this leads to a significant increase in the final protein yield [23].

Through the design and implementation of our project Dienamite, we devised our core beliefs of consistently reflecting and incessantly improving. By executing the core beliefs at all parts of the project, we came up with our comprehensive project framework that integrates Wet Lab, Human Practices, Entrepreneurship, Education, Inclusivity, and Sustainable Development Goals concurrently [Fig.13].

In the early stages, by conducting Wet Lab, Human Practices, and Entrepreneurship activities, we focused on developing our products, embracing others’ opinions, and refining our project designs.

• Wet lab:
  our group entered the wet lab in May and continuously delved into it until late September. Throughout this lengthy process, we executed our core beliefs into the design of our biological parts. For the synthesis of zingiberene, efforts such as the removal of N-terminus anchoring regions and the addition of the SpyTag-SpyCatcher system were made to boost the production rate. Meanwhile, during the synthesis of spider venom peptides, the solubility of the peptides is improved first through the use of SUMO tags and signal peptides, then through the use of secretion systems.


• Human Practices:
  We then proceeded into Human Practice activities to seek suggestions from the outside for further enhancements of our project design. Through the interview with the prestigious Professor Fitches, Professor Glenn King, and Mr. Tao (the owner of a Hangzhou tea garden), the advice of using P. pastoris as the chassis to synthesize SVPs and the suggestion of designing various pesticide appliances was raised. This valuable and insightful guidance was later integrated into our project, perfecting the experiment designs and making the final product more applicable and inclusive to the target customers.


• Entrepreneurship:
  After finalizing our designs, we then attempted to deliver our product directly to our target customers through Entrepreneurship. With the help received from Mr. Yuan, an employee at the Pesticide Registration Bureau, we gained insights into the registration process of syn-bio pesticides and therefore created a comprehensive protocol summarizing the procedures. (link) Meanwhile, after interviewing Mr. Glenn King, the founder of a biological pesticide company called Vestaron, essential information about business start-ups was obtained. We therefore revised our development plan, making it more realistic and applicative.
  
  However, when the project developed to a certain extent, we began to realize some of the societal inadequacies and were thus determined to apply our core values to broader scenarios. Therefore, through Education, Inclusivity, and SDG activities, we attempted to convert our priorities and passion into the outlooks of solving global issues and making the world a better place.


• Education:
  After analyzing the data we obtained from our public questionnaire and the responses we collected from the interviews, we discovered that the vast majority of amateur florists use acaricides when the mites are already present. Most of them lack the awareness of preventing mite infestation beforehand, thus resulting in belated pesticide appliances and plant death. Realizing this pervasive problem, we started educating the florists to harness prevention strategies before mite invasion to prolong plant lives.


• Inclusivity:
  Inspired by the communication with Mr. Tao, who told us to consider the diverse environments and terrains at different locations, we designed multiple equipment suitable for various groups of people specifically. For instance, regular sprays were designed for amateur florists who require less volume of pesticides; backpack sprayers were designed for farms located in mountainous regions for easier application; irrigation rigs were also designed particularly for farms located on plain lands. Moreover, user instruction was written in different languages, making our product inclusive and friendly to diverse groups of target customers.


• Sustainable Development Goals:
  after conducting extensive research, we found out that most chemical acaricides have adverse health effects on humans and the environment. Disorders such as cardiovascular disease are often incurred and toxic chemical residues that remain on crops are often washed and accumulated on land and in the ocean. Thus, we decided to contribute our efforts to environmental conservation by synthesizing and promoting our nontoxic pesticide Dienamite, and by advocating sustainable development goals to other people.

From our perspective, Synthetic Biology is not only an innovative field but is indeed a discipline that owns unfathomable potential for environmental preservation. Harnessing the power of novel biological systems, numerous beneficial outputs such as renewable fuels and recyclable plastics can be synthesized, replacing conventional chemical production methods and resolving global challenges. Deeply inspired, our project Dienamite emerged, aiming to use E. coli and yeasts as the chassis to produce a sustainable acaricide. By doing that, we hope to influence the world and contribute to conserving a greener earth. Despite this, we aim to spread our passion for synthetic biology to other people by conducting Education and Inclusivity activities. We believe, through our persistent efforts and perpetual enthusiasm, diverse groups of people with different socioeconomic backgrounds and ages can have access to the inventive world of Synthetic Biology.



Harnessing the power of Synthetic Biology, we will continuously improve our current designs, making them more comprehensive in the future. We will attempt to:

• Utilize transcriptome mining to seek for and eventually synthesize more venom peptides from various predatory mite species, thus further enhancing target specificity and lowering the rate of resistance development.


• Perform more in vivo experiments on spider mites to explore the mechanisms of MVPs specifically and thoroughly


• Utilize different chassis to synthesize our product, aiming to discover the most efficient production pathway


• Cooperate with stakeholders and synthesize the products at an industrial scale, reducing the selling price and making the product more accessible to different groups of target customers


• Promote concepts of sustainable agriculture to the target customers by advertising our product and conducting more educational activities