APHiGO — A Next-Generation RNA Pesticide Platform for Citrus Aphid Control

China is the country with the earliest global citrus cultivation history, having a history of over 4,000 years to date ^[1]. In China, citrus is far more than just a post-meal fruit or baking ingredient; it is also a pillar industry deeply rooted and closely related to people's livelihood.

As the world's largest citrus producer, China's citrus planting area has remained stable at 43 million mu for many years ^[2]. According to a report released by the US Department of Agriculture, in 2022, China's net citrus output reached 60.0389 million tons ^[3], achieving an agricultural output value of $27.87 billion. According to 'Citrus World Statistics' released by The World Citrus Organisation (WCO), China's citrus output accounts for 28% of the global total ^[4]. This means that for every four citrus fruits harvested worldwide, one comes from China.

Fig 1. Annual Output Value of Chinese Citrus and Distribution of Main Production Areas of Chinese Citrus

Within this context, South China — where our team is based — contributes over 38% of the country's total citrus output, leveraging its exceptional conditions of sunlight, temperature, soil, and water. This not only meets domestic and international demand but also plays a crucial role in securing the livelihoods of millions of farming households.

However, this fertile land that harbors the hope of a bountiful harvest is now facing a severe and irreversible invasion. The invader is an insect less than 2 millimeters in length—the brown citrus aphid (Toxoptera citricida) ^[5].

This pest usually hides on the undersides of leaves to feed and reproduce, making early detection difficult in the early stages. Meanwhile, its high reproductive capacity and rapid dispersal ability lead to a rapid increase in its population. Aphids affect citrus health in three primary mechanisms ^[6]:

Fig 2. Aphids damage citrus by direct feeding, inducing sooty mold, and transmitting viruses. Click on the image to learn more.

The economic losses inflicted by this crisis are staggering. According to monitoring and forecasts from agricultural departments in China's major production regions, citrus aphid infestations occur annually across the country, affecting vast areas. Each year, over 25 million mu (approximately 1.67 million hectares)^[7] of citrus crops are impacted, with control costs exceeding 67 million USD. Furthermore, the sooty mold they trigger and the Citrus Tristeza Virus they spread inflict long-term damage on trees, further exacerbating economic losses.

Fig 3. The physiological reproduction cycle of aphids typically involves parthenogenesis, where offspring are produced without fertilization.

Yet in facing this invisible crisis, traditional chemical pesticide control has reached an impasse ^[8]. Aphids have developed significant resistance to common insecticides, compelling growers to increase dosage and application frequency even as effectiveness continues to decline. This overreliance not only drives up production costs but also poses serious food safety and environmental risks. The indiscriminate harm pesticides cause natural pest predators like ladybugs and parasitic wasps to weaken the ecosystem's inherent pest control capacity, creating a vicious cycle.

Compounding the crisis, pesticides pose even greater health risks to farmers through direct exposure. Research indicates that chemical components in pesticides can enter the human body via inhalation or dermal absorption ^[9]. A study on occupationally exposed populations found a significant association between pesticide exposure and the risk of developing cancers, including non-Hodgkin lymphoma .Furthermore, the chemical pesticide DDT has been linked to an increased risk of breast cancer.

A Sustainable Approach to Pest Management——APHiGO

APHiGO is an integrated green pest management system that incorporates synthetic biology to achieve "reconnaissance-strike integration." Its core technology is RNA interference (RNAi) — which enables precise molecular-level targeting of key survival genes in pests, delivering highly specific control with minimal environmental risk^[10]. The system facilitates early warning and precise intervention, fundamentally regulating field ecological balance and providing farmers with a sustainable pest control solution.

The concealment of citrus aphids is the main challenge in their prevention and control. They usually hide and reproduce on the underside of leaves, and may have already passed the optimal intervention period before becoming visible to the naked eye. To address this issue, this system shifts the monitoring target from the aphids themselves to the honeydew they secrete, enabling early and discreet pest warning.

We employed Bacillus subtilis as a biological reconnaissance platform and genetically engineered it to "sense honeydew and release signals"^[11]. When the engineered bacteria detect sucrose—the main component of honeydew—their internal sucrose-inducible promoter is activated, initiating the downstream biosynthetic pathway. Through the introduced pchBA gene cluster and BSMT gene expression, the volatile signaling molecule methyl salicylate (MeSA) is ultimately produced ^[12].

Fig 4. Mechanism diagram of the engineered Bacillus subtilis detection module

MeSA nnaturally occurs in plants and can be monitored in real-time and remotely through gas sensors deployed in the field ^[13]. When MeSA concentration increases significantly, the system sends alerts to farmers, enabling early intervention and shifting pest management from passive response to proactive warning ^[14]. For details, please visit our CitrusShield page.

Citrus aphid damage often begins during a latent period: when population levels are low, individuals hide on the undersides of leaves, making them difficult for growers to detect and beyond the reach of sprayed pesticides. By the time the infestation becomes visible, the optimal window for intervention has often passed. To address this, we have developed a trunk-injected RNAi strategy. This approach delivers RNAi complexes directly into the citrus vascular system, enabling the entire tree to establish preemptive systemic protection and form a comprehensive anti-pest barrier, even in the early stages^[13].

Our RNAi molecules specifically target essential survival genes in the brown citrus aphid (Toxoptera citricida):

• CHS (Chitin Synthase): Silencing disrupts chitin synthesis, impeding molting and development ^[14].
• CYP450: Weakens detoxification metabolism, reducing environmental tolerance ^[15].
• CP19 Protein: Directly interferes with cuticle formation and growth ^[16].

To ensure stable delivery of RNAi molecules in complex environments, we employ MS2 virus-like particles (VLPs) as carriers. These VLPs undergo self-assembly through specific interactions with pac-RNA. The robust protein shell of the VLP effectively protects the RNAi molecules, significantly enhancing their stability ^[17]. To further improve the delivery efficiency of the RNAi complex within the aphid, we have fused the VLP with the aphid gut-targeting peptide GBP3.1 ^[18] and the cell-penetrating peptide TAT, enabling more efficient cellular uptake.

Once the RNAi formulation is transported via the sap to the sites where aphids feed, ingestion by the aphids triggers gene silencing, leading to effective pest mortality. This approach allows the citrus tree to develop whole-plant systemic resistance while the pest population is still at a low, latent density, suppressing outbreaks at their source. For detailed implementation protocols, please refer to the Implementation page.

In Module 2, we established a "preemptive systemic protection" in citrus trees through trunk injection of RNAi molecules, providing an early defensive line against aphids. However, when localized aphid outbreaks occur or when they breach this preventive barrier, a potent and rapid-response control measure is required. To meet this need, Module 3 introduces a targeted therapeutic with a dual-strike strategy — a "biological special forces" solution based on engineered Metarhizium.

The engineered fungus attacks aphids through two distinct modes:

External Infection: Spores of Metarhizium, upon contact with the aphid, germinate rapidly, penetrate the cuticle, and release toxins, leading to quick mortality.

Internal Gene Silencing: We have further engineered the fungus to function as a living bio-factory, enabling it to continuously produce RNAi molecules inside the aphid. This delivers a "lethal strike" from within, significantly enhancing the killing effect ^[19].

This combined internal and external assault not only eliminates aphids effectively but also delays the development of pest resistance due to its dual mode of action ^[20]. Crucially, Metarhizium is a natural component of the ecosystem, ensuring safety for non-target organisms and achieving a harmonious balance between control efficacy and environmental sustainability.

The APHiGO project is more than a set of tools; it represents a paradigm shift in agricultural pest management—transitioning from reactive spraying and fighting against the environment to proactive early-warning and working in synergy with ecology. Through the foresight of our early detection system, the systemic prevention of trunk-injected RNAi, and the powered intervention of engineered Metarhizium, we have built an integrated "Monitor-Prevent-Treat" solution. These three modules do not simply operate in sequence; they work in deep synergy, aiming to fundamentally break the vicious cycle of chemical pesticide dependence, with the ultimate goal of ensuring the health and productivity of every citrus tree.

Yet our vision extends far beyond this. Each module of APHiGO is a foundational tool that can be decoupled, refined, and repurposed. For instance, the sensor platform in the early detection system can be adapted to monitor other pests or plant pathogens, while the VLP-based RNAi delivery system can be redeployed for precise molecular therapeutics targeting different pests or plant diseases. We envision these modules functioning like Bio-Bricks, providing future synthetic biology researchers with composable foundational units to accelerate the development of sustainable agricultural solutions worldwide.

We are convinced that the implementation of APHiGO will have profound impacts: it will reduce pesticide residues, safeguarding food safety for consumers; it will protect pollinators and natural predators, restoring the ecological balance of orchards to make the citrus groves themselves more resilient. These efforts will, in turn, deliver more stable economic benefits to citrus growers globally for more details, please visit our iHP page.