Human Practices

Introduction

With the development of social economy, environmental heavy metal pollution is becoming increasingly severe, causing significant serious, which has caused great harm to both the environment and human health. Therefore, timely and effective detection of heavy metal pollution plays an important role in guiding people towards a healthy lifestyle. However, the existing heavy metal pollution detection means are usually expensive equipment with high technical requirements that require sending samples to special laboratories for testing. On-site testing is also not suitable for promotion and large-scale application in remote and backward areas. For this reason, a low-cost, low technical requirement and high portability of heavy metal monitoring/detection technology is extremely important in these areas

To this end, we conducted a series of targeted research activities, including public questionnaires, expert interviews, and literature review. According to these research results, combined with the actual needs of remote and backward areas, we chose the development path of establishing transgenic indicator organisms. This involved selecting drosophila with low maintenance costs for genetic modification, enabling them to detect the concentration of heavy metals in the environment.

1 The current situation of heavy metal pollution in remote and backward areas

Heavy metal pollution has become a global environmental problem, especially in remote and backward areas. Environmental protection experts, Ms. Li Yongbei and S Mr. Zhou Bocheng, pointed out that these areas are often weak industrial base, lack of sound environmental protection measures, coupled with small population, lack of environmental awareness, imperfect environmental legal system, law enforcement and supervision and other reasons, resulting in heavy metal pollution is difficult to be effectively controlled.

In the actual social division of labor, especially in developing countries, remote and backward areas are often the main producing areas of food. The latest research shows that in the past 20 years, the heavy metal pollution of cultivated soil in China's main grain-producing areas has been on the rise, and the agricultural soil samples exceeded the environmental quality standard set by China Ministry of Environmental Protection increased from 7.16% to 21.49% of the total samples. Cadmium (Cd), nickel (Ni), copper (Cu), zinc (Zn) and mercury (Hg) were the main pollutants, and the heavy metal pollution of soil in the main grain-producing areas in southern China was heavier than that in northern China (Fig. 1) ( Shang EP et al.,2018 ). According to the 2019 data from the Report on the State of China's Agricultural Ecological Environment, the excessive rate of heavy metals in farmland soil in China reached 19.4%.China's annual production reduction due to heavy metal pollution of more than 10 million tons of grain, heavy metal pollution of grain up to 12 million tons a year, a total economic loss of more than 20 billion yuan (Lü HX et al., 2018). Heavy metal pollution in remote and backward areas will seriously affect people's health through food and along the food chain.

Fig. 1 Heavy metal pollution levels in soil samples from the main grain producing regions (Shang EP et al.,2018)

As for the sources of pollution, considering the characteristics of local social and economic activities, Mr. Zhou believes that the sources of heavy metals in remote and backward areas are mainly from local mining and smelting waste, excessive use of chemical fertilizers and pesticides, sewage irrigation and industrial waste.

2 Detection demand for heavy metal pollution in remote and backward areas

Because heavy metals in the environment will eventually enter the human body along the food chain, threatening human health and life safety, people are generally more concerned. According to Mr. Zhou, due to differences in information acceptance and other social reasons, people in developed areas pay more attention to heavy metal pollution, while those in backward areas pay less attention. In addition, people in different regions and different industries also have certain differences.

For the concern of heavy metal pollution, the public is most concerned about the environment and whether the heavy metal content of products will endanger health. Therefore, people's demand for monitoring/detection of heavy metal pollution is also clear. Based on the experience of Mr. Zhou and Ms. Li, there are roughly these points: comprehensive monitoring, regular detection, fast detection speed and accurate detection results to minimize pollution risks.

However, Ms. Li mentioned that due to differences in geographical environment and economic development level, there are still problems such as insufficient funds, relatively low technical level of personnel and inconvenient transportation for monitoring/detection heavy metal pollution in remote and backward areas. On the one hand, the social and economic backwardness makes the local environmental monitoring/detection departments lack sufficient funds to purchase conventional experimental equipment and introduce and maintain high-tech personnel; On the other hand, for conventional detection technologies, samples often need to be sent to the laboratory for testing, and the backward local traffic conditions seriously limit the convenience of sampling and sample delivery by technicians.

Therefore, in the demand for heavy metal pollution monitoring/detection in remote and backward areas, in addition to the above conventional needs, the low cost, low technical requirements and high portability of instruments and equipment are particularly important.

3 The current situation and shortcomings of heavy metal pollution monitoring/detection technology

3.1 Physical and chemical monitoring/monitoring technology

At present, there are many technologies commonly used for heavy metal pollution monitoring/monitoring on the market, and the most widely used are spectral detection technologies, which include: Ultraviolet and Visible Spectrophotometer (UV), X-ray Fluorescence Spectrometer, (XRF), Atomic Absorption Spectrometer (AAS), Atomic Fluorescence Spectrometer (AFS), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and so on. The spectroscopic method is relatively mature and can detect and analyze the heavy metal content in the sample with high sensitivity, which is the commonly used detection method and a series of national standard detection methods.

However, the equipment of spectroscopy is usually expensive (especially AAS, ICP-OES, ICP-MS), the sample pretreatment process is time-consuming, the technical requirements are high, the operation is complex, and the analysis cost is high. Except for some portable equipment of XRF, they cannot be used for rapid detection on site (Yu T et al., 2021). This is also mentioned by environmental experts Ms. L, Ms. Tang bo and Mr. Zhou.(Fig. 2)

Fig. 2 Prices of some heavy metal spectral detection devices on online sales platforms in China (Aicaigou, 2023)

Obviously, due to the high cost and the inability to determine on site, conventional spectral detection technology cannot meet the needs of heavy metal pollution monitoring/monitoring in remote and backward areas.

3.2 Biological monitoring/monitoring technology

Through literature review, we found that there are two main categories of biotechnology due to heavy metal pollution monitoring/monitoring:

The first category is the biological indicator testing. Some animals and plants (usually mussels, oysters, glazed oysters, emerald mussels, mussels, mussels and other bivalve mollusks), which are naturally sensitive to heavy metals, are directly applied as indicator organisms for long-term experimental monitoring of heavy metals in the environment. The method of directly applying is relatively simple, but there is a general applicability problem of indicator organisms -- It is difficult to quantitatively describe the degree of pollution, the monitoring indicators are not clear, and there is no complete national standard (Li ZT et Zeng Y, 2015).

The second category is some enzyme analysis and immunoassays based on enzyme chemistry and immunology technology, as well as some fusion methods that combine biotechnology with other technologies, including biosensors and nanotechnology. These technologies are easy to operate, only require small equipment, low requirements for laboratories and technicians, low testing costs, and can achieve high-throughput testing, but there are still several shortcomings (Liu Q et al., 2014):

  1. The pre-processing of samples is complicated and time-consuming, which affects the development of detection, and thus limits the development of biological detection methods (Liu Q et al., 2014).
  2. Heavy metal ions themselves lack biological properties and need to be combined with other compounds to be detected by biotechnology (Liu Q et al., 2014).
  3. Heavy metal pollution is generally caused by a variety of heavy metal ions, and the use of biotechnology detection is generally aimed at a certain heavy metal ion or the whole, and the concentration of each heavy metal ion cannot be quickly detected at one time (Liu Q et al., 2014).
  4. Although the equipment required for enzyme chemistry technology and immunology technology is not as complex or expensive as spectral detection technology, the requirements for laboratory conditions are relatively high. These include a temperature range of 18-28℃, humidity between 45% and 70%, and air conditioning configuration. Additionally, these labs must meet secondary biosafety laboratory standards (Jiuhe, 2019).

3.3 Summary

Through the above research, we found that the existing methods for heavy metal monitoring/detection, whether traditional spectral detection technology or emerging biological detection technology, are difficult to meet the monitoring/detection needs for heavy metal pollution in remote and backward areas.

So, is there any other better technology that can be applied?

4 Application of transgenic drosophila in monitoring heavy metal pollution

4.1 Inspiration

From a technical standpoint, it is clearly impossible to transform current common spectral detection technology equipment into something that reduces cost, lowers technical requirements, and increases portability for meeting heavy metal monitoring/detection needs in remote and underdeveloped areas. Is there something that we can do to improve biological methods?

Enzyme chemistry and immunoassay methods are not suitable for remote areas with relatively backward social and economic levels due to their high laboratory requirements. In light of this, can we consider beginning with the biological indicator method? However, one significant limitation of this method is its inability to quantitatively detect heavy metals. As a solution, we propose utilizing transgenic and biosynthesis technology to genetically modify a particular organism, thereby creating an innovative engineering indicator capable of quantitatively analyzing heavy metal levels in the environment.

4.2 The basis of genetic engineering transformation of drosophila

In the interview with bioengineering expert Dr Zhang Shiping and literature research, we learned that Drosophila melanogaster is a potential engineering indicator organism.

On the one hand, Drosophila is relatively small in size, easy to carry and low in cost. Drosophila has a short life span and is replaced quickly. Compared to microorganisms, we can observe some changes in the phenotype of Drosophila with the naked eye. Compared to plants, Drosophila shows changes more quickly.

On the other hand, the GAL4/UAS system in Drosophila could serve as an excellent entry point for genetic modification. The Drosophila GAL4 /UAS system is one of the commonly used gene expression tools in Drosophila genetics studies, and Brand and Perrimon first introduced yeast GAL4 /UAS sequences into Drosophila in 1993 (Ma PP et al., 2014).They have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. To generate transgenic lines expressing GAL4 in numerous cell- and tissue-specific patterns, the GAL4 gene is inserted randomly into the genome, driving GAL4 expression from numerous different genomic enhancers. A GAL4-dependent target gene can then be constructed by subcloning any sequence behind GAL4 binding sites. The target gene is silent in the absence of GAL4. To activate the target gene in a cell- or tissue-specific pattern, flies carrying the target (UAS-Gene X) are crossed to flies expressing GAL4 (Enhancer Trap GAL4). In the progeny of this cross, it is possible to activate UAS-Gene X in cells where GAL4 is expressed and to observe the effect of this directed misexpression on development (Fig3) (Brand AH et Perrimon N, 1993)

Fig3. The GAL4/UAS expression system in Drosophila (Brand AH et Perrimon N, 1993)

According to Dr Zhang, we can use the GAL4/UAS expression system to modify drosophila, so that it can have different phenotypic responses to different concentrations of heavy metals.

4.3 The basic idea to establish heavy-metal sensitive Drosophila

According to Dr. Zhang, we can combine MTF-1 expression with the eye or wing development via the GAL4/UAS system and regulate the expression of gene Hid by the metal-activated MTF-1 protein. In response to heavy metals, MTF-1 is activated in the developing eyes or wings in our engineered Drosophila lines and activates the expression of Hid, which results in the apoptosis of eye or wing cells and shrinkage of the eyes or wings. As a result, we can grow the lines in the environment and observe their sizes of eyes or wings to determine the level of heavy metal pollution.

4.4 Evaluation and suggestion on heavy-metal monitoring with engineered Drosophila

As a plan provider, Dr. Zhang believes that our goal is relatively easy to achieve. However, due to the preparation process of engineering Drosophila in the early stage, it requires a certain period of time. Additionally, he clearly pointed out that because we are using live detection instead of conventional chemical methods, the accuracy will be lower.

In interviews with other experts in the field of environmental protection, they recognized our plan to modify the genes of Drosophila and apply engineering Drosophila to detect heavy metal pollution. They provided many valuable suggestions.

As for the overall application effect of the drosophila visual heavy metal pollution monitoring system planned to be developed by our project, Ms. Tang believes that it is an excellent civilian heavy metal pollution monitoring technology for remote and backward areas. This can well solve the problem of insufficient local environmental monitoring (Since the government monitoring agency only goes to the countryside once a month to collect sample data, it is therefore difficult to fully estimate all aspects of local life). Ms. Li also believes that our project has certain economic and social benefits from the perspective of promoting monitoring and preventing heavy metal pollution in remote and backward areas.

Similar to Dr Zhang's view, the two environmental protection experts agreed that due to the influence of physiological factors of the organism itself, the monitoring/detection effect of our products cannot break through the existing spectrum detection technology in the near future. The experts also provided us with several suggestions to enhance the efficiency of testing, increase convenience, and boost the effectiveness of our products. Here's a roundup of the two experts' advice:

  1. Determine the specific detection parameters. There are dozens of parameters and indicators related to heavy metal pollution. We need to establish specific indicators for the determination of specific metals. For example, the concentration of specific heavy metals can be selected as a parameter, so that the specific concentration of a certain metal can be directly interpreted through the phenotype of engineered Drosophila.
  2. Develop reference standards. In the process of testing, we can compare the test results with standard curves or standard numerical tables to quickly interpret the results.
  3. Use Drosophila phenotypes with more intuitive results as the basis for evaluation. As far as possible, select some phenotypes that can quickly present results, similar to pH test strips, which can achieve rapid detection. For example, it can directly determine the type of heavy metals based on the different color changes of a certain organ of the drosophila.
  4. Determine the maximum tolerance of Drosophila to a specific metal, that is, the maximum tolerance concentration of a specific metal, in order to facilitate targeted application and avoid exceeding the "range".

4.5 Risk control of engineered Drosophila

There is a risk of gene overflow in genetically modified organisms. Whether it is in the preparation stage of engineered Drosophila in the laboratory or in the subsequent monitoring/detection stage of heavy metal contamination, in order to prevent gene spillover of transgenic Drosophila, Dr Zhang suggests that we must ensure the sealed management of engineering flies. During the Drosophila culturing, it is necessary to use tape or mesh to seal the Drosophila culture tube; If any flies accidentally run out, they need to be killed with tools in time.

5 Integration and our work

5.1 At Precent

Based on the situation that the existing heavy metal pollution monitoring/detection technology cannot meet the needs of remote and backward areas, after studying and judging the improvement space of various existing technical means, we have made a transfer indicator organism and applied it to the monitoring/detection of heavy metal pollution in remote and backward areas.

After discussing with Dr. Zhang, a bioengineering expert, and reviewing various literature and resources, we determined to establish a transgenic Drosophila model through the GAL4/UAS system, incorporating the gene of gene of metal response element-binding transcription factor-1 (MTF-1) and the gene of head involution defective (Hid) in order to monitor and detect heavy metal concentrations in the environment. We plan to insert MTF-1 into the genome of Drosophila group A at the same time as inserting Hid behind genes that control eye or wing expression in Drosophila group B. Subsequently, the two groups of flies were crossbred, and the expressed traits of the eyes or wings of the resulting offspring could be combined with the expression products of MTF-1.And we cultured the hybrid offspring flies in an environment with different concentrations of heavy metal ions. Based on the response of the MTF-1 factor to heavy metals, the flies display differential expression in terms of eye or wing size, providing us with a basis for interpreting the concentration of heavy metals in the environment.

In the application stage, Based on the recommendations of Ms. Li and Ms. Tang, we will measure the eye size or wing size of the engineered fruit flies under different heavy metal concentration gradients. We will establish mathematical models based on the obtained data and scientifically predict the possible testing effects. Additionally, we will establish standard curves based on the changes in eye area or wing area of engineered fruit flies under different concentration gradient stresses. This way, we can quickly and accurately interpret the heavy metal concentration data during the subsequent actual detection process.

Meanwhile, as per Dr. Zhang's recommendations, during the implementation of the project, we will strictly do a good job in the sealing culture of engineering Drosophila to prevent the occurrence of gene spillage.

5.2 In the Future

According to the experts, our initial heavy metal detection system has limitations in terms of detection rate and effectiveness in detecting heavy metals. After completing this project, we plan to evaluate more functionality improvements by incorporating suggestions from other experts. Our aim is to develop a Drosophila-based monitoring system for heavy metal pollution that t delivers a more straightforward phenotype and quicker response.

6 Appendix:

(1)Interviewees

    Li, Yongbei: Chief engineer, Guilin Ecological Environment Monitoring Center

    Tang, Bo: Senior engineer, Guilin Municipal Institute of Environmental Protection Science

    Zhang, Shiping: Ph. D. Assistant researcher, Shanghai Tech University

    Zhou, Bocheng: Quality director, Chongqing Zhengcheng Standard Research Engineering Testing Co., Ltd.

(2)Literature reference：

    Brand A.H., Perrimon N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, 118 (2): 401–415.Li ZT et Zeng Y. Application of Heavy Metal Bio - indicator in Water Environment (in Chinese). Environmental science and management. 2015, 40 (11): 74-76.

    Liu Q, Chen GW, Zhang C et al. (2014) Application of Biological Technique in Heavy Metal Detection. Journal of Food Science and Biotechnology, 33 (9): 897-902

    Lü HX, Mo CH, Zhao HM, et al. (2018) Soil contamination and sources of phthalates and its health risk in China：a review. Environmental Research, 64: 417-429.

    Ma PP, Su XR et Chen L. (2014) Application of Drosophila GAL4 /UAS system in undergraduate genetics experiment. Journal of Yuncheng University, 32 (5): 67-69.

    Shang EP, Xu EQ, Zhang HQ et al. (2018) Spatial-temporal trends and pollution source analysis for heavy metal contamination of cultivated soils in five major grain producing regions of China. Environmental Science, (10)：4670-4683。

    Yu T, Jiang TY, Liu X et al. (2021) Research progress in current status of soil heavy metal pollution and analysis technology (in Chinese with English abstract). 2021. Geology in China, 48(2):460-476

(3)Website reference：