Press Release

February 2, 2022
Keio University

Utilizing the “Stealth” of the Invasive Ascidian‐Towards establishing a new model organism for bioimaging‐
A research team including students and faculty at the Keio University has taken the firststep toward making Ascidiella aspersa (also known as the European sea squirt) a new modelorganism for bioimaging research by defining their developmental stages and constructing athree-dimensional embryological image resource. The team included Haruka M. Funakoshiand Takumi T. Shito, second-year master’s students at Keio’s Graduate School of Science andTechnology along with Professor Kotaro Oka and Associate Professor Kohji Hotta of theFaculty of Science and Technology.

A. aspersa is an ascidian belonging to the class of chordates—the closest relatives ofvertebrates. A. aspersa is a well-known invasive species that reproduces invasively not onlyin Japan but around the world, inflicting damage to shellfish aquaculture operations byattaching themselves to scallops. Despite this, embryos of this species are so transparent thatthey can transmit 90% of visible light, making scientists investigate their potential as apossible model organism for bioimaging research. However, until now there has been nostandard developmental table for this species, an essential element in furthering suchresearch.
As a first step to establishing A. aspersa as a model organism, researchers referenced theworld standard developmental table of Ciona intestinalis,1 defining 28 different
developmental stages for A. aspersa that span from fertilized egg to hatched larva.
Additionally, they developed a web-based three-dimensional embryological image of thedevelopmental table viewable by anyone in the world. This resource contains an astounding
collection of over 3,000 cross-sectional and 3D images of the organism taken via confocal laser
scanning microscopy.
The A. aspersa 3D image resource constructed in this study is indispensable for connecting
different omics data to each developmental stage’s spatiotemporal hierarchy, and is expected
to aid in forming a system-level understanding of the embryonic development and phylogenic
evolution of chordates—a classification that includes humans. If research on this species
continues to expand the research foundation for A. aspersa, scientists believe that it could
connect to potential measures to prevent these ascidians from attaching to and harming
scallops.
The results of this study were published in the online edition of the Swiss scientific journal

Frontiers in Cell and Developmental Biology on December 17, 2021.

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1. Research Background
Bioimaging—a technique that visualizes the dynamics of various molecules in living organisms
and is facilitated by the remarkable development of science and technologies such as microscopes
and various fluorescent probes—has contributed greatly to life science research. The embryonictransparency of laboratory organisms is incredibly important, especially when conducting imagestudies using living organisms. This is because if an embryo is opaque, it is difficult to capture thedepth of the embryo, leading to an underutilization of imaging technology.
Ascidians of the genus Phallusia have transparent embryos and are already being used aslaboratory organisms in Europe. They are not, however, found in other regions such as Japan andthe United States. For this study, researchers searched the Japanese coast comprehensively forspecies with transparent embryos suitable for imaging. As a result, they found that the ascidianwith the clearest embryo in Japan was the non-native A. aspersa, reported to be attached in large
numbers to farmed scallops, seriously damaging the scallop aquaculture industry (Kanamori,2016; Shito et al., 2020).
Several factors attest to the viability of A. aspersa as a world-class model organism. One is that,in addition to their high embryological transparency in the visible light range (light transmittanceof about 90% or equivalent to that of a glass window), this species can translate mRNA prior toegg fertilization, allowing researchers to use florescent protein probes to monitor early stages ofdevelopment. Another factor is that, as it is an invasive species, it can be found living in areas
uninhabited by ascidians of the genus Phallusia, making it readily available worldwide.
For these reasons, this research group aims to establish this species as a new model organism
suitable for imaging by taking advantage of the embryonic transparency, or "stealth," of A. aspersa.
The study began by first defining what would be established as the world standard developmental
table for A. aspersa.

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Figure 1: Ascidiella aspersa (triangles) on a scallop (arrow)
As pictured, various organisms, including ascidians and mussels, attach to farmed scallops,inhibiting their growth. A. aspersa can be identified by their distinctive reddish coat.

Figure 2: Ascidiella aspersa and Ciona intestinalis larvae photographed under bright-field andpolarized light (from Shito et al., 2020).
Ciona are visible in all lighting conditions, whereas, in bright-field lighting, A. aspersa larvae(left) are “stealthy” in that only the eye (triangle) is visible.

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2. Research details
Ascidians are the closest relatives to vertebrates and are a valuable model organism forunderstanding vertebrate development and evolution due to their small number of constituent
cells and well-explored cell lineage. Among them, the species primarily used in research are
Ciona intestinalis and Halocynthia roretzi. Comparatively, invasive Ascidiella aspersa havenearly transparent embryos (Shito et al., 2020), pointing to their promise as a model organism
for bioimaging research. However, scientists had not completed a standardized developmental
table had yet to be defined and lacked in anatomical knowledge. Thus, as the first step toestablishing A. aspersa as a model organism, researchers referenced the standardized
developmental table of Ciona intestinalis type A (Hotta et al., 2007) to define its standard
developmental stages (Fig. 3). The developmental process from fertilized egg to hatched larva
was classified into 28 developmental stages (stages 1-26).2

Figure 3: Defining the developmental stages of the Ascidiella aspersa embryo, which transmits
90% of visible light.

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Researchers then photographed A. aspersa embryos at each of the 28 defined developmental
stages using confocal laser scanning microscopy. Based on more than 3,000 cross-sectional
images, they created a 3D image that enables observation of the detailed structure of the embryo
from any angle (Fig. 4). These cross-sectional and 3D images also contain additional informationsuch as cell lineage, tail length, trunk length, and other criteria for defining developmentalstages.

Figure 4: Embryological images of each developmental stage constructed in 3D from more than
3,000 confocal microscopy laser scanning images

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All images, definitions of developmental stages, and annotation information obtained in thisstudy have been compiled into an online digital image resource RAMNe (Resources of Ascidiellaaspersa Morphology Network-based), which can be viewed by anyone from anywhere in theworld (Fig. 5).

Figure 5: Top page of the Ascidiella aspersa 3D database “RAMNe” developed in this study
URL: https://www.bpni.bio.keio.ac.jp/RAMNe/latest/index.html
Researchers also compared cleavage patterns, embryonic morphology, and developmental rates
between Ascidiella aspersa and Ciona intestinalis in a cross-species analysis. The results showedthat cleavage patterns and the developmental time up to the neurula period in A. aspersa wereremarkably conserved in comparison to Ciona. On the other hand, the ratio of the trunk and taillength in the tailbud period were smaller than Ciona, indicating a relatively short tail. This may
be related to the fact that the number of cells that make up the caudal epidermis of A. aspersa islower than that of Ciona. Researchers also found that the timing of the tail bend in A. aspersa isearlier than in Ciona. These differences may form the basis for A. aspera’s invasive nature.
Finally, researchers performed two imaging experiments taking advantage of the areas in which
A. aspera excels: its embryological transparency and early translation of mRNA. In the firstexperiment, researchers stained the cell membranes of A. aspera, and demonstrated that it ispossible to capture 3D images of the entire embryo while it is still alive.3 The second experimentdemonstrated the noteworthy ability of A. aspera to translate foreign mRNA in early stages ofdevelopment. (Fig. 6). The experiment also showed that with A. aspera, fluorescent protein sensors
can be used to observe the wave-like propagation of the increase in Ca2+ concentration at themoment of fertilization.4

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Figure 6: Injection of GCaMP6s mRNA results in two Ca2+ oscillations at fertilization
A: First Ca2+ oscillation directly following fertilization, B: Second Ca2+ oscillation, C: Graphshowing change in brightness during oscillations

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3. Future Developments
In the future, in order to make Ascidiella aspersa easier to utilize in experiments, theresearchers from this project hope to consolidate the information required for life science
research such as its genome and transcriptome data. In addition, interspecific comparisons with
Ciona intestinalis revealed that A. aspersa has a relatively short tail and a different
arrangement of adhesive organs.5 These characteristics may be connected to the invasive natureof this species against scallops and other marine life handled in aquaculture operations. While
this study deals with the lifecycle up to hatched larvae, scientists are also considering defining
further developmental stages for swimming larvae, metamorphosis, and even juveniles in thehopes that further research into such stages will help mitigate damage to aquaculture
operations.
In addition to these potential avenues, researchers will continue to provide embryological andanatomical information on A. aspersa to other researchers around the world through the 3D
embryological image resource RAMNe. The team also hopes that this fascinating organism will
become the new model organism of choice for understanding development and evolution at
cellular and systematic levels.
4. Acknowledgments
Ascidiella aspersa specimens were obtained with the aid of the following individuals.
 Professor Minoru Ikeda and Captain Toyokazu Hiratsuka, Onagawa Field Center, Facultyof Agriculture, Graduate School of Agricultural Science, Tohoku University
 Dr. Makoto Kanamori, Hakodate Fisheries Research Institute, Fisheries ResearchDepartment, Hokkaido Research Organization
 Mr. Akio Takiya, Dr. Takaaki Kayaba, and Dr. Motohito Yamaguchi, Central Fisheries
Research Institute, Fisheries Research Department, Hokkaido Research Organization
 Professor Gaku Kumano, Graduate School of Life Sciences, Tohoku University
This research was conducted with the support from the following organizations and grants.
 JSPS Grant-in-Aid for Scientific Research (KAKENHI): 21H00440
 Research and Education Center for Natural Sciences, Keio University Keio Leading-edge Laboratory of Science and Technology (KLL) Research Project Keio University Education and Research Adjusted Budget
 Grant-in-Aid for Fostering Research at the Research Institute of Marine Invertebrates:
IKU2021-02

Details of Journal Article
Title: Developmental Table and Three-Dimensional Embryological Image Resource of theAscidian Ascidiella aspersa
Authors: Haruka M. Funakoshi†, Takumi T. Shito†, Kotaro Oka and Kohji Hotta* († = equallycontributed)
Journal of publication: Frontiers in Cell and Developmental Biology
Paper URL: https://www.frontiersin.org/articles/10.3389/fcell.2021.789046/fullDOI: 10.3389/fcell.2021.789046

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Database Information
Resources of Ascidiella aspersa Morphology Network-based; RAMNe
https://www.bpni.bio.keio.ac.jp/RAMNe/latest/index.html
References
Makoto Kanamori, Katsuhisa Baba, Yasuko Konda, Seiji Goshima (2014). Distribution of theinvasive ascidian Ascidiella aspersa (Müller, 1776) (Urochordata, Ascidiacea) in Hokkaido.
Japanese Journal of Benthology, p. 23-31, issue I, volume 69)
https://doi.org/10.5179/benthos.69.23
Shito, T. T., Hasegawa, N., Oka, K., and Hotta, K. (2020). Phylogenetic Comparison of Egg
Transparency in Ascidians by Hyperspectral Imaging. Sci. Rep. 10, 1–12.
https://doi.org/10.1038/s41598-020-77585-y
Hotta, K., Mitsuhara, K., Takahashi, H., Inaba, K., Oka, K., Gojobori, T. and Ikeo, K. (2007), A
web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D realimage embryo reconstructions: I. From fertilized egg to hatching larva. Dev. Dyn., 236: 1790-1805.
https://doi.org/10.1002/dvdy.21188
Explanation of Terms and Reference Materials
1 Ciona intestinalisOne of the ascidian model organisms used in research worldwide
Scientific name: Ciona intestinalis type A (Ciona robusta)

2 Developmental process from fertilized egg to hatched larva (video)

https://figshare.com/articles/media/Video1_Developmental_Table_and_ThreeDimensional_Embryological_Image_Resource_of_the_Ascidian_Ascidiella_aspersa_MP4/172
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3 3D shot of an Ascidiella aspersa embryo (video)

https://figshare.com/articles/media/Video2_Developmental_Table_and_ThreeDimensional_Embryological_Image_Resource_of_the_Ascidian_Ascidiella_aspersa_MP4/17255249
4 Ca2+ oscillation during fertilization (video)

https://figshare.com/articles/media/Video3_Developmental_Table_and_ThreeDimensional_Embryological_Image_Resource_of_the_Ascidian_Ascidiella_aspersa_MP4/172
55252
5 Adhesive organs

An organ used by ascidian larvae to attach to rocks and other substrates and metamorphose

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*Please direct any requests or inquiries on press coverage to the contact information provided
below in advance.
・Inquiries about research
Keio University Faculty of Science and Technology, Department of Biosciences and Informatics ,Associate Professor Kohji Hotta
Tel: +81-45-566-1700 Fax: +81-45-566-1789 E-mail: khotta@bio.keio.ac.jp
・Inquiries about press release
Keio University Office of Communications and Public Relations (Mr. Sawano)
Tel: +81-3-5427-1541 Fax: +81-3-5441-7640 Email: m-pr@adst.keio.ac.jp
https://www.keio.ac.jp/

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