The invasive species Achatina fulica (Giant African Snail) is an infamous culprit for triggering a tremendous number of ecological issues and lethal diseases over 50 countries and regions, especially in our city, Shenzhen, China. Its invasion has triggered considerable damage to agricultural harvest and ecological balance. There are also growing concerns about its primary nematode accomplice, Angiostrongylus cantonensis, parasitizing in the snails' secretions that result in human severe meningitis, facial paralysis, dementia and even death. This year, GreatBay-SCIE proposed a synthetic biology project named GAStroPurifier, a comprehensive solution aiming at minimizing the harmful influences caused by Achatina fulica, with the six-step solution called ALERTS that can responsively meet multiple needs in different communities: First, we will synthesize papaya and banana odors to ATTRACT Giant African Snails, LOCKing the snails in our hardware, and then we will ELIMINATE A.cantonensis within the snails with the toxic proteins we produce; after that, the collected snails' mucus can be extracted for detecting the A.cantonensis biomarkers to REASSURE the effects of our eliminating process manually, and if it's successful, snails can be TRANSFERed out of our hardware, with a kill SWITCH functioned on the genetically engineered yeasts to ensure the biosafety of our design. Through these attempts, GreatBay-SCIE will not only provide our local community with a templated approach for integrated control of invasive species, but also significantly contribute to worldwide agriculture and healthcare from the roots of parasitic infections.
We, GreatBay-SCIE, as a group of high school students in Shenzhen, China, have consistently witnessed dozens of large and alluring snails creep around the tarmac, cement and walls after some moments of heavy rain, leaving random destruction along their trace (Fig 1.). For a certain range of Shenzhen citizens, it is rather annoying to get in touch with the snails unintentionally; but from time to time, some children and adults could not help getting closer to these creatures, touching them and catching them. Despite ceaseless news reports conveying the tragedies of people being parasitically infected, we are still able to see with our naked eyes that these snails are mostly the same as those snails being petted, appreciated, and even cooked in Chinese households and restaurants. These snails are Giant African Snails.
Fig 1: Huge groups of Giant African Snails in the streets of Shenzhen at night
What is happening? Can we do something to help our citizens, community and environment?
Achatina fulica (Giant African Snails, or GAS) are originated from eastern Africa, but for their potential value in cooking and petting, they have invaded numerous countries and regions through agricultural transport, international trades, smuggling and many other accidental or purposeful approaches [1] (Fig 2.) , being listed among the very first alien species that have invaded China since 1931. GAS has a broad diet of over 500 plants and can consume materials up to half of its original body mass [2], resulting in up to 56.82% income loss in indigenous agriculture and economy [3], further threatening the sustainability of food supply and public welfare.
Fig 2: Distribution of Giant African Snails Source: ScienceDirect
To add insult to injury, each adult Giant African Snail reproduces their offsprings at a staggering rate with up to 1,200 eggs laid per year [4], explaining the reasons why the invasion has remarkably destroyed the natural food chains and the balance of local ecological systems by occupying the living spaces and consuming resources of indigenous species. Due to the relentless invasion of Giant African Snails that is far more rapid than human beings can even react, it significantly draws the attention, in terms of sustainable development and biodiversity conservation, from a variety of eco-communities on a global scale.
Another heart-stricken concern about the GAS, however, is its capability of carrying numerous pathogens, mostly Angiostrongylus cantonensis. As adult A. cantonensis, living in the pulmonary arteries of the rodent definitive hosts, lays eggs and hatches them in the lungs, L1 larvae migrate to the alimentary canal of the definitive hosts, and then swallowed and passed in the faeces of the definitive host. When the L1 larvae penetrate or are ingested by the intermediate hosts, L3 larvae will be produced after two molts, which are either ingested by the definitive hosts to restart the parasitic cycle, or ingested by mammalian hosts without continuous growth, leading to the vital human infection, named Angiostrongyliasis (Fig 3.).
Fig 3: Living cycle of A. cantonesis Source: Centers for Disease Control and Prevention
Angiostrongyliasis makes the global patients painfully suffer from varying degrees of somatic symptoms (e.g. headache, fever, malaise), ocular angiostrongyliasis (e.g. uveitis, blurred vision, and loss of visual acuity), neurological dysfunction, and most seriously, death [5] (Fig 4.). The most notable manifestation of A. cantonensis infection is eosinophilic meningitis caused by the presence of larvae in the brain and resultant local host reactions. This disease was first discovered in 1935 in Canton (Guangzhou), China. Between 1965 and 2021, 11 nematode species were recorded in association with Achatina fulica in 21 countries [6] (Fig 5.), of which A. cantonesis triggers the greatest attention with an infection rate as high as 31.0% locally in Shenzhen, China [7], and a total record of over 3,000 human infected cases of Angiostrongyliasis reported worldwide [8].
Fig 4. Symptoms of Angiostrongyliasis, original picture
Fig 5: Global Distribution of A.cantonesis Source: BMC (Biomedicalcentral)
There are three main control measures to Giant African Snails have been attempted all over the world: firstly, biological controls with the snail's natural enemies [9], which automatically disturbs the natural food chains and interactions among different species; secondly, chemical controls with highly toxic compounds acted as killing agents of the snails, which is only likely to be adapted in rural areas but not urban regions; and thirdly, mechanical controls by crushing and drowning the snails, which requires enormous amount of labour to collect and dispose the snails, followed by increasing probability of accidental injuries to other indigenous species[10]. These treatments, however, are so far from being integrated and efficient, indicated by the negligible number of countries and regions achieving the eradication of GAS, compared to the considerable number of those influenced by the invasion of Giant African Snails.
There is no specific treatment for A. cantonensis infection, either. Although it is proven that certain supportive treatments may reduce the severity of headache and the duration of symptoms [11], for example, the drug Albendazole is used in most cases with the combination of anti-inflammatory drugs [12], a vast majority of diagnostic treatments for Angiostrongyliasis are currently limited to the infected hosts, including human and other animals [13][14]. These approaches are neither sufficient enough compared to the severeness of the diseases, nor putting us, the entire human race, in a proactive position in terms of parasitic disease treatment.
What if ...?
What if GreatBay-SCIE can work on developing a systematic approach for the integrated controls of Giant African Snails by minimizing their harmful effects with the unique insight on targeting the parasites in their bodies?
In order to tackle the issues above, we proposed our solution, "GAStroPurifer", that not only controls the outbreak of Achatina fulica but also safely eliminate the A. cantonensis larvae.
This is based on the brand-new ALERTS (Attract - Lock - Eliminate - Reassure - Transfer - Switch) procedure (Fig 6.).
Fig 6. Visual demonstration of "ALERTS" procedure
Instead of enticing Giant African Snails manually or chemically, the "ATTRACT" stage in "ALERTS" solution is where GAStroPurifier starts to strive for an ecological balance with the delicate integration of the advantages of synthetic biology and the natural habitats of the snails.
During our exploration of appropriate attracting agents, some of our team members recalled their memories on their way home in the late afternoon: fresh doughs with fruits from bakery stores were surrounded by several Giant African Snails, predictably due to sweet fruit odours and smells that active dry yeasts originally have, supported by the research from Akan et al. suggesting that Giant African Snails favor the smells of yeasts, more specifically, the alcoholic fragrances of S.cerevisiae[15]. To investigate the functionality of yeasts and sweet fruit smells in attracting Giant African Snails, we conducted the verification experiment in one member's garden: home-made doughs with active dry yeasts, active dry yeasts + 10% fruit papaya, and active dry yeasts + 10% fruit banana are prepared respectively, placing distantly in the garden for hourly recording the numbers of snails being attracted by each dough (Fig 7.). This success significantly inspired us to utilize the potential functionality of yeasts in both odour synthesis and snail attraction, motivating our continuous attempts to optimize substance synthesized and amounts produced to achieve the best possible solution for the ATTRACT process.
Fig 7. Giant African Snails were attracted by doughs, original picture
𝛼-pinene, being identified as one of the crucial compounds favours unique aroma in papaya oil [16], is the first substance we would like to increase its production in S.cerevisiae in a controllable manner (Fig 8.). Wei's and Ma's studies shifted our attention to the mevalonate pathway (MVA pathway) [17][18]. By the overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMG1) and isopentenyldiphosphate delta-isomerase (IDi) on the relatively weak upstream pathway, the transcription level of MVA pathway can be enhanced. In order to manipulate the downstream metabolic pathway to flow towards geranyl pyrophosphate (GPP), the precursor of 𝛼-pinene, and away from farnesyl diphosphate (FPP), the by-product, we not only overexpressed the endogenous gene farnesyl pyrophosphate synthetase (ERG20ww), mutated from ERG20, but also used flexible linker GGGGSGGGGS to fuse it with pinene synthase (PS), originated from Pinus taeda, with the first 48 amino acids truncated forming t48PS. The enhancement in the upstream MVA pathway and the adjustment in the downstream pathway constructively contributed to a rise in the production yield and efficiency in 𝛼-pinene synthesis.
Fig 8. The metabolic pathway of synthesizing 𝛼-pinene, original picture
Fruit banana has also demonstrated impressive potential in trapping Giant African Snails [19], of which isoamyl acetate is present as one of the major ingredients of banana fragrance (Fig 9.). To achieve the largest possible production of this compound in S.cerevisiae, ketoacid decarboxylase (ARO10) and alcohol dehydrogenase (ADH7) in the Ehrlich degradation pathway and alcohol acetyl transferase (ATF1) were overexpressed to directly boost the production of isoamyl acetate [20]. In response to the translocation of 2-ketoisocaproate from mitochondrion to cytosol, the overexpressed enzymes in the relatively long metabolic pathway, isopropyl malate isomerase (LEU1), 3-isopropylmalate dehydrogenase (LEU2) and 2-isopropylmalate synthase (LEU4, leucine-insensitive mutant with amino acid S547 deleted employed) have experienced mitochondrial compartmentalization [21][22] driven by signal peptides CDC9, COX6 and COX4 [23][24], aiming at breaking through the bottleneck of the transport of intracellular metabolite, increasing the bioactivity of enzymes and the concentrations of involved enzymes and intermediates, and enhancing isoamyl acetate's yield specificity.
Fig 9. The metabolic pathway of synthesizing isoamyl acetate, original picture
The inspiration of the LOCK process, namely locking the snails enticed temporarily, is sourced from numerous feedback we gained by interacting with our projects' stakeholders and manifested mainly by our hardware design (See Hardware). In this process, we not only took the possibility of biorelease of genetically modified organisms (GMOs) into serious considerations, but also provided multiple choices of applications for the different needs of various groups of future users: the trapped snails can either undergo the subsequent ELIMINATE and REASSURE processes automatically, or be collected for other purposes decided by the people in charge.
Prioritizing concerns on ecological balance and public healthcare concluded from massive research and mutual communications with the stakeholders, toxic protein Cry1518-35, originated from Bacillus thuringiensis, was chosen as the killing agent of A.cantonensis, for its significant specificity to the target nematodes and negligible impacts on human beings and other creatures [25]. Given that most nematodes infesting in the guts of Giant African Snails are L3 larva A.cantonensis that will instantaneously trigger pains if a person is infected, letting the snails ingest killing agents of nematodes is considered a desirable proposal. As the snails ingest food with Cry1518-35, the toxins function by binding with the cadherin receptor in the nematode's midgut and activating the magnesium dependent signal transduction pathway that possibly results in the death of living cells of the nematodes [26] (Fig 10.).
Fig 10. The mechanisms of Cry1518-35 killing A.cantonensis Source: Journal of Entomology and Zoology Studies
Considering the continuity of our project approaches, S.cerevisiae is innovatively chosen as the ultimate chassis for expressing Cry proteins. To ensure the direct functionality of the toxic proteins on the target nematodes, expressing synthetic Cry proteins in the supernatant of S.cerevisiae was accomplished by the introduction of signal peptides. Besides the native signal peptide of S.cerevisiae (ScMα), the optimized version of ScMα (OPT) with A9D and A20T mutations [27], signal peptide originated from the inulinase gene in K. maxianus (JF) and its optimized version with P10L mutation (JFm) were separately employed [28][29], of which each secretion effect on heterologous proteins are proven in our experiments.
While developing a more sustainable expression with a higher secretion level in S.cerevisiae, we conducted further attempts in the expression of Cry1518-35 with alterations of vectors and optimization of the coding sequence in E.coli for a more efficient verification of the toxin's characteristics and bioactivity. Either engineered E.coli or bacterial whole-cell liquid expressing Cry proteins and E.coli OP50 were co-cultured to test the corresponding survival rates of C.elegans as a close reference of the protein's toxicity on A.cantonensis, together unveiling a great potential of Cry1518-35 being synthesized in different chassis to satisfy different demands of nematode ELIMINATION in different circumstances (Fig 11.).
Fig 11. C.elegans were killed by E.coli expressing Cry1518-35
The REASSURE process is designed to check for any alive residual parasites A. cantonensis after the killing attempts and before manual TRANSFER of the snails, and the necessity of this stage is expected to be uplifted to a convenient and quick screening before precise assessments in laboratories during disease control actions (See Human Practice). Once the mucus of the snails is collected with our hardware or other methods, the precise technology SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing) can be incorporated with CRISPR/Cas 13a for real-time detection of the nucleic acids extracted from A. cantonensis [30][31]. Our detection procedure will not only create a much safer working condition for manual operation, but also be comprehensively responsible and responsive for the communities and environments that our project is dedicated to.
After serious debate and active interactions on efficiency of the overall design with the target users of our project, we decided to end the systematic operation with a manual TRANSFER process through the portable container of our hardware, together with regular replacement of attracting agents and killing agents inside the hardware. Consequently, not only the flexibility of integrated control on invasive species will be maintained in our actions, but also people in environmental management bureau or other properties will be protected from directly being contact with the Giant African Snails.
Acting as a biosafety guarantee, the SWITCH stage represents a kill switch design, mainly consisting of a toxin-antitoxin system, to inhibit the possible release of genetically engineered yeasts into external environments and avoid accidental harms to human beings or other species that have yet to be measured.
The first assurance of the performance of the kill switch is to select an auxotrophic strain of S.cerevisiae, CEN.PK2-1C as the chassis. The presence of URA selectable marker indicates that the genetically engineered yeasts will not successfully function without the consistent supply of uracil from our closed system.
Inspired by Xu's research [32], Bax protein, whose overexpression in mitochondria of S.cerevisiae will lead to lethal phenotype of cell activities, and Bax-inhibitor (BI-1), an apoptosis inhibitor that functions the regulation associated with Bcl-2 homologs in the genome of S.cerevisiae, were selected to perform our second design of kill switch: toxin-antitoxin system. Theoretically, Bax gene is under constitutive expression while BI-1 is under inducible expression until changes happen in the external environment (Fig 12). Specifically, if our genetically engineered yeasts escape the closed storage systems, the absence of inducers resists the expression of BI-1 gene. As the continuous expression of Bax gene overtakes the amount of remaining BI-1 gene, Bax gene will no longer be inhibited and trigger the released cells to die (Fig 13).
Fig 12. Mechanism of kill switch design with the presence of inducers
Fig 13. Mechanism of kill switch design without the presence of inducers
Excitingly, the employment of various designs of synthetic promoters in S.cerevisiae significantly broadens the range of choices of the switch mechanisms, including xylose promoters [33] and arabinose promoters that are optimized from prokaryotic promoters. Remarkably, arabinose inducible promoters (pAra.1, pAra.2, pAra.3) are applied to transcribe the antitoxin gene BI-1, while NOT gate arabinose promoters (pNOTAra.1, pNOTAra.2) are applied to transcribe the toxin gene Bax [34][35], idealizing the application of manipulating the SWITCH stage by simply inputing inducers xylose or arabinose. (Fig 14) (Fig 15)
Fig 14. Improved mechanism of kill switch design with synthetic arabinose inducible promoters
Fig 15. Verification process of kill switch design
GAStroPurifier is a comprehensive solution to the invasion of Giant African Snails, aiming at providing a ecologically friendly template to the integrated control of invasive species in the local and worldwide communities. Through persistent efforts in exploring synthetic biology, conducting lab experiments, integrating social communications, and engaging broad communities, we have been on the way to our ultimate goals with three merits so far.
Integrative: Being integrative has been set as the primary principle since our project initiated. As the control of invasive species requires awareness and actions from multiple communities, the design and manifestation of "ALERTS" solution is consistently refined and developed with the multidimensional voices from different levels of target users of our project, including the government, scientific and bioethical communities, property managers, sanitation workers and medical workers, etc. Moreover, we also integrated our projects with the Sustainable Development Goals "Live on land" by avoiding unnecessary injuries to non-target species in the control of invasive species, and "Quality education" by involving the youths, the peers, the scientific communities and the general public to realize the facts of invasive species and the charms of synthetic biology with in-depth understanding and engagement.
Adaptive: Given that thousands of countries and regions all over the world are suffering from numerous issues of species invasion, the "ALERTS" solution is not only proposed according to the needs of our local community, but also designed to be flexible enough to be separately utilized under different circumstances, especially for those regions without systematic operation system on the control of invasive species. The flexibility of the "ALERTS" design has already been recognized by different entities during our Human Practice activities. For instance, the "ATTRACT" stage enables the health quarantine department to collectively examine and disinfect the imported species according to their own criteria, the "LOCK" stage helps scientific researchers with their studies in Achatina sp. in a less risky manner, and the "ELIMINATION" stage can be further modified into a large-scale and cost-effective production of environmentally friendly pesticides etc.
Innovative: Instead of traditional chemical production that may be difficult to adjust the smell produced and may potentially influence the environment, different aromas are selected to be synthesized in S.cerevisiae for its high level of sustainable production and great flexibility to express the mixed ingredients of smells to an optimum ratio and amount that have the best performances in attracting Giant African Snails. Furthermore, we shed light on the treatment of parasitic infection to the active control of the parasites in the infective chain, rather than the passive diagnosis of the infected cases, which is not only expected to be a creative contribution to the prevention of diseases and the protection of public health, but also a protective measure for the consumption of non-invasive species that also carry the nematodes.
In the future implementation of our project, synthetic biology will remain a crucial role in engineering our solution to reveal more comprehensive values and functions. Therefore, we will:
Seek more desirable insert sites in the genome of S.cerevisiae to achieve a higher integration efficiency of synthesizing the attracting agents and killing agents;
Conclude the optimum recipe for the ratio and amount of attracting agents mixed in fresh doughs through carrying out field research under the guidance of biosafety and bioethics;
Investigate the functionality of synthetic inducible promoters in yeasts and design a set of efficient promoters in performing the kill switch design;
Prolong the elimination effects of Cry proteins to prevent secondary A.cantonensis infection on the Snails by optimizing the mechanisms of the kill switch design;
Explore the appropriate biomarkers for real-time and effective detection of alive A.cantonensis and the most convenient application of SHERLOCK technology in detecting the nucleic acids of nematodes.
Although the "ALERTS" procedure is rooted from the control of Giant African Snails, the idea that "Attracting the specific species - Locking the species for operations - Eliminating the possible sources of threats to the ecological system - Reassuring the effects of operations - Transfering the species out of the locked system - Switching on/off to avoid unnecessary biorelease" has the latent capacity to be adapted in the treatment of other land-living invasive species. With delicate modifications to the ATTRACT and LOCK procedures, our solutions are further expected to be utilized in controlling invasive species under water; for example, the chief in Wildlife Protection & Control Bureau encouraged us to apply our method to Pomacea canaliculata, another invasive species that also carries an enormous quantity of A.cantonensis [36], which is supposed to be far more difficult than controlling invasive species on land with current approaches.
What we have promoted in our dry lab activities also contribute to the bright future of the use of synthetic biology in the treatment of invasive species. In Human Practice, we issued a mini program on WeChat - which Shenzhen government have shown their appreciation and built the collaboration with us - that establishes a platform for the general public to report any emergence of Giant African Snails in their daily lives to the local government and prosperity managers, aiming at solving the problems we discovered that many regions suffering from the invasion of GAS haven't ever run an operating system for the integrated control of invasive species. In Education, regarding the feedback on the understanding and attitude we gain from the iGEM community and scientific community, we published a bioethics handbook for them that provided detailed guidelines on how to include bioethical concerns during the development of science research related to the invasive species and synthetic biology, purposefully adapting the four bioethics value (Be respectful, harmless, beneficial, and just) to achieve higher level of balance and humanity in scientific development. In Hardware, we documented and built a mobile device that can apply the "ALERTS" design in a systematic approach, either automatically or separately depending on the choice of individual users, and through the user testing process, the property managers and sanitation workers expressed their willingness to utilize our hardware if we can continuously communicate with them and make improvement following their suggestions.
The project "GAStroPurifier" generates long-lasting impacts on much more fields and industries besides the control of species invasion and public healthcare. It greatly benefits the balance of ecological system and the preservation of biodiversity as it prevents Giant African Snails from keeping occupying the living spaces of indigenous species without disturbing the delicate ecological equilibrium formed by the invasion for over a century. It also ensures positive economic growth by reducing the staggering annual loss of incomes generated from agriculture, and acts as a protective intensifier to the food industries related to the consumption of Giant African Snails, responding back to one of the major initial incentives of importing the snails.
