KOSE Co., Ltd. (Headquarters: Chuo-ku, Tokyo; President: Kazutoshi Kobayashi) has established a technology to analyze coral metabolites (metabolome analysis) from just one coral polyp through joint research with the National Institute of Advanced Industrial Science and Technology, Kinki University, and Kitasato University. This makes it possible to evaluate the effects of sunscreen ingredients, etc. on coral growth from a biochemical perspective. In the future, we will apply this technology to promote the development of sunscreens that are gentle on corals.

points

・Achieves acquisition of endogenous metabolic profiles with a single small coral polyp

・The effects of chemical substances on the endogenous metabolism of corals can now be assessed quickly and easily

・Application to new development of sunscreens that do not adversely affect corals

Flow diagram for cultivating polyps during simultaneous spawning of corals and applying them to a new evaluation method. (Author: Mr. Issei Takahashi, Nagoya University)

Overview　
National Institute of Advanced Industrial Science and Technology (hereinafter “AIST”) Geological Information Research Division Chief Researcher Iguchi Jun Research Group Leader, Iijima Mariko Research Division Researcher, Kinki University Bioscience Engineering Department Professor Zaitsu Katsura (AIST Visiting Researcher), Kitasato University Marine Life Sciences Department Lecturer Yasumoto Tsuyoshi, Specially Appointed Assistant Professor Yoshikazu Ono, PhD Researcher Mizusawa Nanami, Kose Research Institute Co., Ltd. Together, we have established a new evaluation method that enables rapid environmental impact assessments for reef-building corals (hereafter, corals), which are feared to decline due to environmental changes on a global and regional scale. Furthermore, we clarified the effects of chemical substances such as sunscreen ingredients on corals from metabolic profiles, and succeeded in obtaining new environmental impact knowledge on corals. The results obtained through this research are expected to be used as an important model for environmental impact assessment methods using marine organisms such as corals.
Note, these results were published in the British Nature Publishing Group's open access magazine “Scientific Reports” on Reiwa 6/3/5 (local time).

*See [Glossary] for red text

The social context of the research
Currently, there are great concerns about adverse effects on ecosystems due to the runoff of anthropogenic chemicals into nature and the breakdown of the nutrient cycle. From the viewpoint of planetary boundaries indicating the limits of the Earth, six of the nine boundaries, including outflow of chemical substances and collapse of the nutrient cycle, have already crossed the border (Richardson et al., 2023), and awareness of efforts to regulate chemical substances in the ocean is growing, starting with microplastics. Also, attention is being drawn to ESG investments carried out by evaluating corporate environmental (environment), society (social), and governance (governance) initiatives. The final recommendations of the TNFD (Nature-related Financial Information Disclosure Task Force) to further accelerate ESG investment were announced in 2023/9, and corporate activities that take the natural environment into consideration are strongly required, and the importance of efforts aimed at realizing nature positivity is increasing. Against this background, there is a growing need for evaluation methods to clarify how chemical substances affect marine life. Coral reef ecosystems (Figure 1), known for their beautiful scenery, play an important role in various fields such as coastal protection, fishing, and tourism. However, since corals, which support this foundation, are sensitive to environmental changes on a global and regional scale, global decline is feared. Since corals are a symbol representing marine ecosystems, there is an urgent need to establish reliable environmental impact assessment methods for corals. In particular, in recent years, reports have been made that some sunscreen ingredients have an adverse effect on corals, and there is a need to establish a method to quickly and easily evaluate the effects of sunscreen ingredients on corals.

Background of the research
AIST aims to clarify the responses of marine organisms to environmental changes and construct highly reliable environmental impact assessment methods. Furthermore, research using a multifaceted approach combining breeding experiments, genetic analysis, and chemical analysis has been carried out on coral etc. (2017/1/19, 2021/3/17, 2023/11/30, 2024/2/1, 2024/2/1 press conference). Furthermore, interdisciplinary research using metabolome analysis using mass spectrometry devices: PESI/MS/MS, which has been used mainly in the medical field as a new platform for metabolite analysis (metabolome analysis) until now, has also been developed (2020/5/25, 2021/6/30).
Furthermore, this research was carried out with support from the Japan Society for the Promotion of Science Grants-in-Aid Project Challenging Research (Germination) “Challenge to Coral Exposome Research” (Representative Researcher: Ryo Iguchi; 2019-2020).

Research content
Coral larvae obtained during simultaneous spawning of corals of the genus Euglena, which is a representative group of corals (Figure 2 and 2023/3/5 AIST X video), are bottomed out, and by artificially adding symbiotic zooxanthellae, coral polyps divided into conditions for the presence or absence of zooxanthellae were cultivated. Thanks to the research group's various ingenuity, we have succeeded in increasing opportunities for experiments by securing opportunities for spawning multiple times and maintaining the obtained larvae for a relatively long time.

Using grown coral polyps, we prepared samples exposed to oxybenzone-3, which is one of the sunscreen ingredients, and nutrients (ammonium and nitrate) that affect the environmental sensitivity of coral-zooxanthellae symbionts. However, the diameter of coral polyps is very small, only about 2 mm, and when applying conventional metabolome analysis, it was necessary to mix multiple coral polyps and perform pre-treatment such as extraction operations to ensure sample volume. In this case, in addition to the need to grow a large amount of coral polyps, there was a disadvantage that the results obtained were also averaged. Therefore, in this study, a new metabolic analysis platform using PESI/MS/MS capable of analyzing fine samples: pITmap (Zaitsu, Iguchi et al., 2020) was applied to coral polyps (video 1). As a result, we have newly discovered that metabolic profiles can be obtained from just one coral polyp.

*Figures from the original paper are quoted and modified. Creative Commons license (show 4.0 International)

In particular, in this study, we succeeded in observing metabolites constituting glycolysis, citric acid cycle, urea cycle, pentose phosphate pathways, glutathione metabolism, and methionine pathways for each coral polyp. By applying the PiTmap platform to the obtained metabolome data, multivariate analysis is also performed automatically. As a result of applying latent structure projection discrimination analysis (PLS-DA), which is one type of multivariate analysis, significant changes in metabolic profiles were observed in coral polyps without zooxanthellae, such as a decrease in several amino acids due to oxybenzone-3 (Figure 3 above). Meanwhile, no changes in metabolic profile due to oxybenzone-3 exposure were seen in coral polyps with zooxanthellae (Figure 3 below). Similar trends were also confirmed in ammonium-exposed samples. This indicates that symbiotic zooxanthellae may remove the adverse effects of exposed substances on coral bodies.
The evaluation method established this time does not require any complicated preprocessing operations required for conventional metabolome analysis. In general, pretreatment operations for metabolome analysis required about 1 to 2 days, but using this method, it is now possible to analyze endogenous metabolites from just one coral polyp in about 3 minutes.

Figure 3. PLS-DA score plot using oxybenzone-3 exposure samples. The horizontal axis indicates the first principal component, and the vertical axis indicates the second principal component. The figure above shows the results of coral polyps without zooxanthellae, and the figure below shows the results of coral polyps with zooxanthellae. Each point is equivalent to 1 coral polyp. The dashed line and dotted line indicate 95% and 99% confidence intervals for all data, respectively. The red and light blue ellipses indicate 95% confidence intervals for each group.
*Figures from the original paper are quoted and modified. Creative Commons license (show 4.0 International)

Future plans
The new evaluation method established this time is expected to be widely used for environmental impact assessments of corals where needs are growing. It can also be used as a quick method to search for alternative substances to reduce the effects of anthropogenic substances. Furthermore, it is expected to be used not only for risk assessments of chemical substances, etc., but also for positive impact assessments such as increased growth and promotion of metabolism. AIST will cooperate with other research institutes in the future to consider applying this method to marine organisms other than corals. KOSE Co., Ltd., which is one of the joint research partners, has been collaborating with coral aquaculture experts to conduct visual evaluations of the effects of sunscreen and its ingredients on coral growth (2022/2/7, 2022/4/13 KOSE News Release), and by utilizing this evaluation method, we will continue to work on product development that takes the marine environment into consideration.

Paper information
Published in: Scientific Reports
Paper title: Single-polyP metabolomics for coral health assessment
Authors: Akira Iguchi, Mariko Iijima, Nanami Mizusawa, Yoshikazu Ohno, Ko Yasumoto, Atsushi Suzuki, Shunichi Suga, Ken Tanaka, Kei Zaitsu.
DOI: 10.1038/s41598-024-53294-8

Bibliography
Richardson, K. et al. (2023). Earth beyond six of nine planetary boundaries. Science Advances, 9 (37), eadh2458.

Explanation of terms
planetary boundaries
A concept showing the boundaries of the global environment that must be crossed in order for humans to survive sustainably on Earth.

Nature positive
A concept that aims to stop and restore natural ecosystem loss due to social and economic activities.

metabolome analysis
A method for comprehensively analyzing the increase or decrease in metabolome, which is the sum total of metabolites (metabolites).

PESI/MS/MS
Probe electrospray ionization tandem mass spectrometry (probe electrospray ionization tandem mass spectrometry). A method of puncturing (scanning) a sample using a fine metal needle with a tip diameter of 700 nm, and ionizing biocomponents adsorbed on the needle surface for analysis.

Metabolic analysis platform: pITmap
It is a new platform for metabolic analysis jointly developed by Senior Researcher Iguchi and Professor Zaitsu in 2020. Extremely quick metabolome analysis was implemented by combining metabolome analysis using PESI/MS/MS described above with an automatic bioinformatics execution processing function using the statistical analysis language R.

Latent structure projection discrimination analysis (Pls-DA)
It is one of the multivariate analysis methods, and it is a method for grasping data trends and profiles by providing group information on multivariate data (multidimensional data), reducing the dimensions.