Description

1. Inspiration

Why and how we choose the AHPND theme ?

1.1 Background

    Shrimp, a tasty seafood with a plenty of protein, is now the dominant player in crustacean aquaculture production owing to its rapidly expanding farming and market size (1).
    The Eastern coast of China is where the domestic Vannamei shrimp farms are mainly situated (Fig. 1). Fujian, the absolute prominent province where Xiamen University is located, in which 443.127 billion of Vannamei shrimps are cultivated per year, accounts for 29.4% of the total national production (2).
Fig. 1 a typical shrimp farm we visited in Fujian province.
    Consequently, shrimp farming is a lucrative industry that not only provides a significant food source consumed on a global scale but also benefits the local economy by fostering job growth and producing massive profits for companies.

1.2 The problem

    We visited Fujian Fisheries Technology Extension Center in order to fully comprehend the status of the farmed shrimp business before settling the content of this season.
    By speaking with the staff, we learned that bacterial sickness is a continuous hindrance to numerous aquaculture companies, resulting in huge shrimp mortality and substantial economic losses, as well as a threat to food safety.
    Acute hepatopancreatic necrosis disease (AHPND), originally known as early mortality syndrome (EMS), is one of the major bacterial challenges which has a devastating impact on the shrimp aquaculture industry (Fig. 2).
    Vibrio parahaemolyticus (Vp), the primary pathogen of AHPND, possesses a distinct plasmid, pVA1. The plasmid encodes the binary toxins, PirA and PirB, that attack the digestive gland of shrimp and then cause subsequent death (3).
Fig. 2 Illustration of pathogenesis and economic effects of Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp Aquaculture.

1.3 The current solution

    The conventional approaches for preventing and treating AHPND are either ineffective or unsustainable. Antibiotics, which could be added in the diet or the rearing water of shrimps, are used as potent medications to combat bacterial infections in shrimp farming. However, the repercussion of antibiotics abuse may pose a potential microbial concern to public health and the environment (4). What’s more, the current diagnosis of AHPND are clinical examination and molecular identification based on PCR amplification, which are laborious and time-consuming, hence the urgent need for effective methods, which could be used on-site and in-time (5).

2. Project

    Motivated by the previous field investigation and literature research, we then focus on tripartite tasks, developing effective diagnosis, treatment, and prevention methods against AHPND, and the synthetic biology methods need to be arranged elaborately.

2.1 Detection

Fig. 3 Our detection method for AHPND.
    The Ribozyme-Enabled Detection of RNA (RENDR) and cell-free sensing system are employed to establish a rapid, highly sensitive, economical and environment-friendly detecting method. The sensor would be activated and generate a visible fluorescent or colorimetric signal when the target RNA of pVA1 plasmid shows up (6).

2.2 OMVs

    Dr. Lina Wu, the professor of Xiamen University, was interviewed and inspires us much. The preliminary framework employing the OMVs (outer membrane vesicles) and surface display system is adopted, as suggested by Prof. Wu.
    The OMVs, produced by E. coli, are modified to enclose the recombinant plasmids and/or just nail the surface-displayed proteins. When the OMVs dock on the external surface of Vp, the membrane of both of them would fuse together, which makes the recombinant plasmids get inside the cytoplasm of Vp for the subsequent actions. What’s more, the displayed proteins help to enhance the binding efficiency towards Vp. If there is no plasmid be involved, the displayed proteins could also work to fight.
Fig. 4 Our treatment and prevention method for AHPND.

2.2.1 Treatment

    The OMVs are used to deliver the recombinant plasmid which would express endolysin directly in the cytoplasm of the pathogenic Vp. The endolysin has a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain that would contribute to the hydrolysis of peptidoglycan of Vp which could specifically kill the Vp and heal the sick shrimps (7).
    To increase the targeting specifity of OMVs, the tail tubular protein A (TTPA) and tail tubular protein B (TTPB), which are from a phage of Vp and can target the receptor Vp0980 on Vp, come into use as the surface-displayed proteins in this situation (8). With TTPA/TTPB present on the membrane surface, OMVs can be recruited and then concentrated around Vp, enhancing the effect of plasmid delivery.

2.2.2 Prevention

    Aminopeptidase N1 of Litopenaeus vannamei (LvAPN1) and Fetuin-B of mouse (FET), serving as the receptors of toxic PirAvp and PirBvp, are displayed on the surface of OMVs and released by the colonized E. coli in the shrimps’ intestine to bind with PirAvp and PirBvp, isolating the pathogen and attenuating the toxicity (9,10). Additionally, a designed CRISPR/Cas system is used to selectively knock out the pirA/pirB gene.

3. Future work

    With the booming of the global economy, the value of agriculture and breeding industry is highlighted more. We expect that our efforts could provide methodology of treatment and prevention for other types of bacterial diseases. Taking care of the earth by tiny bacteria, we here promise a better future of food and health.

4. Reference

      1. M. L. Situmorang et al., Supplementation of ex situ produced bioflocs improves immune response against AHPND in Pacific whiteleg shrimp (Litopenaeus vannamei) postlarvae. Appl. Microbiol. Biotechnol. 106, 3751-3764 (2022).
      2. Lily, "Short history of the Chinese Vannamei shrimp industry and trends."(roda International, 2020), [unpblished or access by title]
      3. S. J. Lin, K. C. Hsu, H. C. Wang, Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirAvp and PirBvp Toxins. Mar. Drugs. 15, (2017).
      4. F. Asche et al., The economics of shrimp disease. J. Invertebr. Pathol. 186, (2021).
      5. H. M. Santos et al., Diagnosis and potential treatments for acute hepatopancreatic necrosis disease (AHPND): a review. Aquacult. Int. 28, 169-185 (2020).
      6. L. Gambill et al., https://www.biorxiv.org/content/10.1101/2022.01.12.476080v1 (2022).
      7. W. Y. Wang, M. Z. Li, H. Lin, J. X. Wang, X. Z. Mao, The Vibrio parahaemolyticus-infecting bacteriophage qdvp001: genome sequence and endolysin with a modular structure. Arch. Virol. 161, 2645-2652 (2016).
      8. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerging Microbes Infect. 9, 855-867 (2020).
      9. W. Luangtrakul et al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS Pathog. 17, e1009463 (2021).
      10. M. V. De Los Santos et al., The Vibrio parahaemolyticus subunit toxin PirBvp recognizes glycoproteins on the epithelium of the Penaeus vannamei hepatopancreas. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 257, 110673 (2022).