Human-Practices

Overview

    Xiamen is a coastal city where fisheries and aquaculture play important roles in providing food and generating income. To make a living by using the local resources is common wise for the mass. So, seafood has brought up the local people, among which shrimp is a kind of economic marine product particularly rich in protein, calcium, and astaxanthin. After the interview of fisherfolks and further discussion with workers in relevant enterprise and government, we know that acute hepatopancreatic necrosis disease (AHPND) triggers the quick death of shrimp and economic losses to the farmer every year, which is also a universal phenomenon in shrimp aquaculture in coastal cities all around the world. Thus, this year, our project focuses on solving the problem from AHPND that threatens shrimp aquaculture using synthetic biology tools.
    After collecting information from fisherfolks, technicists, and references (1,2,3), we can conclude that AHPND possesses the characteristics of a rapid pathogenetic rate, hard to treat, high fatality ratio and low treatment efficiency, which is still a significant challenge to fisherfolks, technicists, and scientists. Thus, our project was designed as three parts to solve this intractable problems, including detection, prevention, and treatment. After communication with the professors and technicists, a feasible approach was established based on synthetic biology tools, including outer membrane vesicles and a cell-free system. In the experimental process, the advice from scientists and technicists revises the experimental protocol constantly, which makes it more complete and more systematic. For example, a technicist suggested us that fresh water was poured into the breeding ponds to replace the wastewater every day in the later period. So, they wished us to take full consideration of the method in the treatment and prevention part, which can be disturbed by the dilution and replacement effect. Thus, their advice was constructive and effective for us to obtain a more perfect project.
    At last, we also introduced our experimental results to the fisherfolks and relevant enterprises, which brought them out with knowledge, experience, and effective method based on synthetic biology to deal with AHPND. We also carried out offline community science popularization education to adolescents in the science and technology museum and Qun Xian high school. We never forget the power of synthetic biology can be employed to solve practical problems and promote social development. We also believe that the success of our project will not only provide a paradigm in the detection, prevention, and treatment of AHPND but also prevent economic losses for farmers and aquaculture.

Background

    The team of XMU-China comes from Xiamen University, which is located in the seagirt islands in the southeast coastal areas of China. Similar to that in many coastal cities all over the world, the fishery industry is an important source of food, while aquaculture acts as an important complement to the resource of seafood in the fishing off-season. Shrimp is a kind of economic marine product that receives attention all over the world, which is also particularly rich in protein, calcium, and astaxanthin. However, some news and reports tell us that many diseases caused by bacteria will trigger not only the death of shrimp but also the huge economic loss of farmers. Among these diseases, acute hepatopancreatic necrosis disease (AHPND) is a really nasty contagion, which will cause rapid death on a large scale in the shrimp farming process. So, we further collect a lot of information about AHPND from the reference. Thus, we can conclude that the AHPND is mainly caused by the infection from Vibrio parahaemolyticus which carries the virulence genes of pirA and pirB. Using drugs and antibiotics is the common approach to deal with this Vibrio currently. However, abuse of antibiotics and drugs neither specifically targeted this Vibrio nor prevented the shrimp from death quickly. It will also destroy the ecological microenvironment and increase the risk of antibiotic resistance. 

Survey

    So far, AHPND is still not only a significant threat to farmers and aquaculture but also a great challenge to scientists and technicists. Therefore, we tried to develop a comprehensive method that combined the detection, prevention, and treatment strategies to deal with the AHPND based on synthetic biology. We could also obtain viewpoints and professional advice from interviews and discussions which could provide a theoretical foundation for experimental route design.

1 Interview with the ecological company

Mata Ecology Company
Xiamen Mata Ecology Company (hereinafter referred to as "Mata Ecology") has rich experience in aquaculture which also provides commodity and technical supports for the farmers of shrimp aquaculture.

Interview:

    In the communication offline, Mata Ecology introduced the general situation, the breeding process, and the key points of shrimp aquaculture. We learned that Penaeus vannamei is the most popular species in shrimp aquaculture, which has become an important pillar of aquaculture. Mata Ecology has developed many protection products for shrimp aquaculture, which has brought a lot of benefits for growth and immunity.
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    After that, we carried out a deep discussion on the disease of AHPND which bring substantial losses to shrimp aquaculture. The technicist, Mr. Chen Minghua, gave us a scientific lecture entitled “the background and current situation of shrimp aquaculture”, including the characteristics of shrimp disease AHPND. He told us that AHPND possesses the characteristics of a rapid pathogenetic rate, hard to treat, high fatality ratio and low treatment efficiency, which make it a great challenge for detection, prevention, and treatment. The farmer could only deal with AHPND based on their experience. Mr. Chen also emphasized that there was still no effective solution for such a serious disease, while the outbreak of AHPND resulted in the widespread death of shrimp and great economic losses.

2 Interview with shrimp farmer

Shrimp Farmer
A shrimp farmer from the shrimp aquaculture center.

Interview:

    After the communication with the technicist, we further carried out a field investigation in the shrimp aquaculture center. The fisherfolk told us AHPND was a devastating disease that would cause the completely annihilated death of shrimps in the pond in a very short time. The method to deal with this AHPND at present could be summarized as follows. First, purchase the shrimp fry with high disease resistance; second, monitor the quality (Vibrio parahaemolyticus) of the water in the pond; last, pour the probiotics into the pond to provide a better living environment. However, these strategies were still unable to cope with the threats and destruction from AHPND. The warming climate promoted the proliferation of Vibrio parahaemolyticus, resulting in shrimp death and economic losses. They could only do their best in shrimp aquaculture and search for technical support to reduce economic losses.
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    Through this interview and field investigation, we knew that AHPND can cause massive shrimp mortality and severe economic losses to the shrimp farming industry, posing a threat to food safety. The farmer still wanted effective detection, prevention, and treatment strategies. 

3 Interview with the worker at Fujian Aquatic Technology Extension Station

Nan Lin
Professor Nan Lin:
The vice director of the Fujian Aquatic Technology Extension Station.
Research field:
Disease prevention and control of aquatic animals.

Interview:

    In order to further understand the feasibility of detection, treatment, and prevention strategies for AHPND, we made an online interview with Mr. Lin Nan, the vice director of the Fujian aquatic technology extension station. In this interview, Mr. Lin told us more helpful information about AHPND. First, a large amount of fresh seawater was needed for shrimp aquaculture, in which Vibrio parahaemolyticus was hidden. However, it is difficult to achieve seawater sterilization because of the high cost. Second, not all of the Vibrio parahaemolyticus possess plasmid which carries the virulence genes of pirA and pirB. Thus, our target should not be Vibrio parahaemolyticus but the plasmid carries the virulence genes. Third, the fisherfolk spread samples of seawater and shrimp gut in the plate with TCBs to detect pathogenic Vibrio parahaemolyticus based on the color changes. Unfortunately, this is a time-consuming process with a high error rate. Last, probiotics and antibiotics also didn't work out too well to cope with AHPND.
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    During the discussion with Mr. Lin, we not only learned a lot about the information on shrimp aquaculture but also obtained guidance and suggestions on our project. Detection and prevention were the key points in dealing with AHPND, although the current approach was not working well. Fast and accurate diagnosis of pirA and pirB virulence genes was the key point to stifle the AHPND in its earliest stages. After that, we determined to establish integrated strategies based on synthetic biology tools which were divided into three parts: detection, prevention, and treatment.

Design

    After investigating the information on AHPND in shrimp aquaculture, we tried to design strategies for the detection, prevention, and treatment of this disease based on synthetic biology. However, we also faced many intractable problems. Thus, we consulted some professors about our project for some advice, which could offer us some design guidance.

1 Interview with professor Huiming Ye and Jumei Liu (detection part)

Jumei Liu
Professor Jumei Liu:
An expert in diagnostics.
Research field:
Biosensors and clinical molecular diagnostics.
Huiming Ye
Professor Huiming Ye:
An expert in immunology.
Research field:
Clinical immunology and clinical molecular diagnostics.

Interview:

    In the process of project design, we were uncertain about what is the best way to achieve fast detection. So, we paid a visit to Professor Huiming Ye and Professor Jumei Liu. They told us that precision, specificity, and detection speed was the critical factors based on the characteristics of AHPND. However, the low abundance of targeted genes of pirA and pirB made it difficult to realize the effective detection in water and shrimp tissue samples through conventional methods, such as electrochemical sensors and fluorescent sensors. Thus, we knew that nucleic acid enrichment was a necessary step for targeted gene detection. This discussion enlightened us a lot of the design of detection part.
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2 Interview Professor Lina Wu (prevention part)

Lina Wu
Professor Lina Wu:
An expert in biochemistry.
Research field:
Sensitive and specific detection technology of pathogenic bacteria, development, and characterization of extracellular vesicles (EVs).

Interview:

    For the prevention part, we tried to employ outer membrane vesicles (OMVs) to deliver protein, which could be targeted to the Vibrio parahaemolyticus or the toxin. So, we carried out an interview with professor Wu, who had a rich experience in the development and characterization of extracellular vesicles. She told us that OMVs was the excellent carrier that could transfer material from cell to cell efficiently and safely. She also introduced the backgrounds, characterization, expression, separation, and purification of OMVs which strengthened our understanding and broadened the scope of our knowledge. For more details see the page prevention part in Design.
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3 Interview Professor Shaowei Li (treatment part)

Shaowei Li
Professor Shaowei Li:
An expert in genetic engineering vaccines.
Research field:
The structure and function research of important infectious disease virus immune epitopes, molecular simulation, and molecular design.

Interview:

    For the prevention part, we also tried to employ outer membrane vesicles (OMVs) to deliver toxin protein or CRISPR/Cas9 system, which could cause targeted damage to the Vibrio parahaemolyticus or its genome. While we were wavering, we consulted Professor Li who studied infectious disease virus immune, for the choice between two systems. He believed that targeted delivery through OMVs was a good choice to cause considerable damage to pathogens, while toxin protein was easier to implement compared with CRISPR/Cas9 system. He also proposed some constructive suggestions that we should take more consideration on the efficiency of OMVs and lethality of pathogenic bacteria. Based on his advice, further improvements were added to the experimental scheme. For example, to increase the efficiency and specificity of OMVs, the tail tubular protein A (TTPA) and tail tubular protein (TTPB) from a phage of Vibrio parahaemolyticus are displayed on the surface of OMVs, which can target the receptor Vp0980 on Vibrio parahaemolyticus. For more details see the page prevention part in Design.
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Improvement

    Following the advice from the professors, the preliminary experimental scheme was determined. However, as the experiments went on, many kinds of problems and difficulties were coming. Thus, relevant discussions with experts were carried out to solve these problems.

1 Interview with Professor Yun Tian

Yun Tian
Professor Yun Tian:
An expert in microbial molecular ecology.
Research field:
Microbial molecular ecology, physiology, and genetics.

Interview:

    You can't eat your cake and have it too. We wanted to search for a balance between cost and precision, but we failed. So, we had a discussion with professor Tian to get some advice. She told us that qPCR is a technique commonly used for DNA quantification, which requires professional skills to operate, costing lots of time and money. The cell-free system established in our project seemed to be more inexpensive and simpler, which was more suitable for shrimp farmers. So, she also advised us to compare the detection results between qPCR and our cell-free system.
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2 Interview with Professor Dapeng Shan

Dapeng Shan
Professor Dapeng Shan:
An expert in marine microbiology.
Research field:
The utilization of Marine microbial resources.

Interview:

    In the middle of the season, many practical problems had been put in front of us, which affected the application of these tools developed in this project in the future. For example, where did our engineered strains battle with Vibrio parahaemolyticus, in water or shrimp gut? How to maintain the effective concentration of engineered strains and/or OMVs for a long time? How could biosecurity be guaranteed? Thus, we consulted with Dr. Shan about these questions, who is an expert in marine microbiology.
    He believed that compared with water, the shrimp gut was a more ideal environment for engineered strains to attack Vibrio parahaemolyticus, in which the concentration of OMVs could not be diluted by the water. After discussion, we reached an agreement that shrimp gut may provide not only nutrient substance for the proliferation of engineered strains, which provided a guarantee for the effective concentration of engineered strains and/or OMVs for a long time; but also a favorable environment for us to design kill switch to guarantee biosecurity.
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Feedback

    At the end of the season, experiments were further revised and completed based on the advice from technicists and scientists. We performed feedbacks to fishermen and enterprises. We hope that our work can bring them an innovative and effective method to deal with AHPND. 
    First, we performed a return visit to shrimp farmers to introduce more detailed information of our project and a feasible method for them when faced with AHPND. Second, we gave the Mata Ecology company a science lecture on synthetic biology and introduced innovative and effective strategies based on the results for them. At last, our integrated project got recognition from the technicist in the company. Although much more data is needed to modify our project before it can be used, we still provide a paradigm in the detection, prevention, and treatment of AHPND.
    In order to offer services to shrimp aquaculture, we will share hands with technicists, scientists, and the government to provide a series of lectures, support, and training in the future. Hpoe that we can jointly make AHPND problem prefectly solved!

Reference

      1. H.-C. Wang, S.-J. Lin, A. Mohapatra, R. Kumar, H.-C. Wang, A Review of the Functional Annotations of Important Genes in the AHPND-Causing pVA1 Plasmid. Microorganisms 8, (2020).
      2. 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).
      3. 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).