FY2019 Annual Report

Physics and Biology Unit
Professor Jonathan Miller

Abstract

Several years of intense effort by unit personnel yielded a facility at the OIST Seragaki Marine Station where we closed the lifecycles of native Okinawan squid (Sepioteuthis lessoniana) and cuttlefish (Sepia pharaonis) species, so that they have been bred and raised entirely within our laboratory.  This local capacity for a consistent and reliable source of cephalopods is a prerequisite for quantitative study of the psychophysics of cephalopod camouflage and perception. The lifecycle of the deepfin reef squid, a cephalopod that we have observed to camouflage richly in the laboratory, has been closed to breed and raise multiple generations of an inbred strain. This species is also of potential interest for commercial aquaculture. These capabilities having been achieved with the close of fiscal year 2019, the unit's facility was handed over to OIST to operate as an institutional facility so that the unit can focus on data collection and analysis.

1. Staff

• Dr. Shotaro Funai, Staff Scentist
• Dr. Michael Kuba, Staff Scentist 
• Dr. Tamar Gutnick, Postdoctoral Scholar 
• Dr. Teresa Iglesias, Postdoctoral Scholar
• Keishu Asada, Technician
• Dr. Zdenek Lajbner, Technician
• Takahiro Nishibayashi, Technician 
• Dr. Reuven Pnini, Technician
• Midori Tanahara Administrative Assistant

2. Collaborations

2.1 Sepioteuthis lessoniana, Shiro-ika, OIST strain of Physics and Biology Unit.

• Type of collaboration: Joint research
• Researchers:
  • Professor Ryuta Nakajima, University of Minnesota, Duluth. Sabbatical Visitor.

2.2 Primary Orthologs from Local Sequence Context.

• Type of collaboration: Joint research
• Researchers:
  • Professor Kun Gao, School of Science, USTC.

3. Activities and Findings

3.1 Sepioteuthis lessoniana, Shiro-ika, OIST strain of Physics and Biology Unit.

Contributors: K Asada, T Gutnick, T Iglesias, M Kuba, Z Labjner, J Miller, R Nakajima, T Nishibayashi.

Shiro-ika, Sepioteuthis lessoniana sp.2, is a local Okinawan squid suitable as a model for psychophysics of cephalopood camouflage and perception, for biomedical research, and for commercial aquaculture (Lee et al. 1994). It is an important marine resource for Japanese and worldwide fisheries, but its wild population size has fluctuated, possibly reflecting climate change and rising sea temperature (Arkhipkin et al. 2015). Unlike other cephalopods, it has been bred for seven generations in captivity without indications of inbreeding depression (Walsh et al. 2002). It shares Okinawan waters with morphologically and behaviorally distinct close relatives S. lessoniana, Aka-ika (sp.1), and Kuwa-ika (sp.3) with lower commercial value. Landscape genetic data suggest that Shiro-ika inhabits the largest geographical range of the three (Cheng et al. 2014). It has played an important role in Japanese cephalopod research, and is by far the most frequently studied of the three, and we chose the Shiro-ika as the primary model species for a diverse set of experiments.

Strings of Shiro-ika eggs were collected in 2017 at a depth above 2m, off the coast of Okinawa Island, Ryukyu Archipelago, Japan. Eggs were transported to the Okinawa Institute of Science and Technology Graduate University Marine Science Station (OMSS) where we closed the squid life cycle in a flow-through system, and where we rear them continuously over multiple generations (Lajbner et al. 2020, manuscript in preparation). We are now maintaining G5 hatchlings, the 6th generation in the laboratory.

Our fifth lab-bred generation performs well in measurable reproductive success. In comparison to the only previously published multi-generation attempt at the UTMB, which exhibited less than 10% hatching rate (Walsh et al 2002), our G4 squids laid 3 clutches of eggs with the first clutch just hatched at completion with 88% hatching rate (472 live hatchlings), and the younger eggs also developing well. Over the past three years, Lajbner et al. (manuscript in preparation, 2020) modified the Sakurai/Ikeda methods and other aquaculture techniques to incorporate new methods specific to the OMSS flow-through system. Our innovations include finding the optimal number of squid per tank for mating, establishing a diversity of dietary requirements, refining egg case handling and incubation technique, and the introduction of a new artificial reef to serve as spawning substrate.

Our OIST Shiro-ika strain represents a distinctive scientific resource. We are developing strategies to secure its preservation, for example by making our inbred line available for display at other institutions. In recent years, we provided our squid to the Onna village Fisheries Association and Churaumi aquarium. Both of these institutions succeeded at breeding our squid in 2018. In 2020, we are pursuing expansion of our collaboration on OIST Shiro-ika maintenance to institutions in mainland Japan and internationally. Another level of security will be established by cryopreservation of germline tissues, especially testes and sperm cells. Pioneering studies have demonstrated cryopreservation of cephalopod sperm (Robles et al. 2013). We established collaborations with two laboratories specialized in reproduction biology of aquatic organisms and tissue cryopreservation, and are developing cryopreservation protocols with them. Simultaneously, we are collaborating on developing and applying new protocols for assisted and artificial reproduction that would simplify the use of cryopreserved cells for reproduction. We are exploring xenotransplantation of cryopreserved germline that enables the reproduction of even lethal genotypes. Therefore, our squid breeding and commercialization projects do not rely solely on propagating the lineage at OIST indefinitely.

Apart from the extensive scientific use and potential of the OIST Shiro-ika strain, we are exploring opportunities for commercialization of squid aquaculture. The Shiro-ika annual catch in eight major fish markets in Japan between 1986 and 2010 was variable from year to year, ranging from 55 to 166 tonnes and the local Japanese market is distinguished by its high demand for the fresh/live Shiro-ika (Arkhipkin et al. 2015); the latter authors remark that:

“The bigfin reef squid is generally caught nearshore and landed within several hours, so it is sold fresh. In Japan, S. lessoniana is called the “king of squids” because of its good taste and the beautiful transparency of its meat in sashimi and sushi. Therefore, this squid is expensive; squid >1 kg are traded at >2000–3000 yen/kg (20–30 USD/kg) at the Tsukiji fish market in Tokyo and by wholesalers selling to fancy sushi restaurants and Japanese restaurants. Some of the catch is sold live to restaurants specializing in squid dishes.”

We identified several bottlenecks in scaling of squid production and cost reduction, and we are discussing collaboration with commercial organizations. Inbreeding speeds QTL mapping, and development of domestic strains better adapted for aquaculture may make commercializaton of our inbred line of greater interest to external investors. The experience acquired by our unit on Shiro-ika aquaculture has already been applied to closing the life cycle and maintenance of Sepia pharaonis, another native Okinawan cephalopod species with a high aquaculture/aquaponics potential. In the face of climate change and overpopulation, cephalopod aquaculture has the potential to reduce fishery pressure on native species and to provide a nutritious food source to the general public both in Okinawa and on mainland Japan. The combination of OIST's advanced technology with the experience accumulated by traditional Okinawan fisheries could faciliate one of the world's first viable models for squid aquaculture.

Drs. Lajbner, Nakajima and Miller would like to thank colleagues for their comments on this text.

Literature cited

Arkhipkin AI, Rodhouse PG, Pierce GJ, Sauer W, Sakai M, Allcock L, Arguelles J, Bower JR, Castillo G, Ceriola L, …, Chen CS (2015) World squid fisheries. Reviews in Fisheries Science & Aquaculture 23, 92-252.

Cheng SH, Anderson FE, Bergman A, Mahardika GN, Muchlisin ZA, Dang BT, Calumpong HP, Mohamed KS, Sasikumar G, Venkatesan V, Barber PH (2014) Molecular evidence for co-occurring cryptic lineages within the Sepioteuthis cf. lessoniana species complex in the Indian and Indo-West Pacific Oceans. Hydrobiologia 725, 165-188.

Lajbner Z, Nakajima R, Gutnick T, Asada K, Iglesias T, Nishibayashi T, Miller J, Kuba M (2020, in preparation) Multigenerational breeding of oval squid Sepioteuthis cf lessoniana.

Lee PG, Turk PE, Yang WT, Hanlon RT (1994) Biological characteristics and biomedical applications of the squid Sepioteuthis lessoniana cultured through multiple generations. Biological Bulletin 186, 328-341.

Robles V, Martínez-Pastor F, Petroni G, Riesco M F, Bozzano A, Villanueva R (2013) Cryobiology of cephalopod (Illex coindetii) spermatophores. Cryobiology 66, 288-294.

Walsh LS, Turk PE, Forsythe JW, Lee PG (2002) Mariculture of the loliginid squid Sepioteuthis lessoniana through seven successive generations. Aquaculture 212, 245-262.

3.2 Primary Orthologs from Local Sequence Context

Contributors: K Gao, USTC; J Miller, OIST. BMC Bioinformatics 2020 21:48

Background

The evolutionary history of genes serves as a cornerstone of contemporary biology. Most conserved sequences in mammalian genomes don’t code for proteins, yielding a need to infer evolutionary history of sequences irrespective of what kind of functional element they may encode. Thus, sequence-, as opposed to gene-, centric modes of inferring paths of sequence evolution are increasingly relevant. Customarily, homologous sequences derived from the same direct ancestor, whose ancestral position in two genomes is usually conserved, are termed “primary” (or “positional”) orthologs. Methods based solely on similarity don’t reliably distinguish primary orthologs from other homologs; for this, genomic context is often essential. Context-dependent identification of orthologs traditionally relies on genomic context over length scales characteristic of conserved gene order or whole-genome sequence alignment, and can be computationally intensive.

Results

We demonstrate that short-range sequence context—as short as a single “maximal” match— distinguishes primary orthologs from other homologs across whole genomes. On mammalian whole genomes not preprocessed by repeat-masker, potential orthologs are extracted by genome intersection as “non-nested maximal matches:” maximal matches that are not nested into other maximal matches. It emerges that on both nucleotide and gene scales, non-nested maximal matches recapitulate primary or positional orthologs with high precision and high recall, while the corresponding computation consumes less than one thirtieth of the computation time required by commonly applied whole-genome alignment methods. In regions of genomes that would be masked by repeat-masker, non-nested maximal matches recover orthologs that are inaccessible to Lastz net alignment, for which repeat-masking is a prerequisite. mmRBHs, reciprocal best hits of genes containing non-nested maximal matches, yield novel putative orthologs, e.g. around 1000 pairs of genes for human-chimpanzee.

Conclusions

We developed a sequence intersection method to infer evolutionary history of a sequence based on short-range genomic sequence context. Significant advantages of our method over others with similar purposes are that it relies neither on repeat-masking nor alignment, and applies to sequences that are not necessarily known to be genes. The method is based on an extension of concepts of non-nested maximal sequence matches previously developed in the unit for characterizing sequence duplications within a single genome. Ortholog identification based on non-nested maximal matches is parameter-free, and less computationally intensive than many alignment-based methods. It is especially suitable for genome-wide identification of orthologs, and may be applicable to unassembled genomes.

 

4. Publications

4.1 Journals

1. K Gao, J Miller. Primary orthologs from local sequence context. BMC Bioinformatics (2020) https://doi.org/10.1186/s12859-020-3384-2. 
2. Gutnick T, Weissenbacher A & Kuba MJ. (2019) The Underestimated Giants. Operant conditioning, visual discrimination and long-term memory in giant tortoises. Animal Cognition  https://doi.org/10.1007/s10071-019-01326-6,
3. Shotaro Shiba Funai and Hirotaka Sugawara, “Flavor Moonshine”, Progress of Theoretical and Experimental Physics, Volume 2020, Issue 1, January 2020, 013B03. DOI: 10.1093/ptep/ptz137
4. Iglesias TL, Boal JG, Frank MG, Zeil J, Hanlon RT (2019) Cyclic nature of the REM sleep-like state in the cuttlefish Sepia officinalis. Journal of experimental biology 222:jeb174862 https://doi.org/10.1242/jeb.174862
5. Warren DL, Matzke NJ, Iglesias TL (2020) Evaluating presence‐only species distribution models with discrimination accuracy is uninformative for many applications. Journal of Biogeography 47:167-80 https://doi.org/10.1111/jbi.13705

4.2 Books and other one-time publications

1. 船井正太郎. 機械学習と繰り込み群 in 『物理学者、機械学習を使う(橋本幸士)』朝倉書店. pp. 142-153. Section 10. ISBN: 978-4-254-13129-1 / Shotaro Funai. Machine Learning and Renormalization Group in “Physicists Use Machine Learning” (Koji Hashimoto)” Asakura Publishing Co., Ltd. pp. 142-153. Section 10. ISBN: 978-4-254-13129-1.  

4.3 Oral and Poster Presentations

[Oral Presentations]

[Posters]

1. Teresa L. Iglesias, Fabienne Ziadi, Tamar Gutnick, Zdenek Lajbner, Jonathan Miller, Michael Kuba, Preliminary observations of the ‘sleep-like’ state in the broadclub cuttlefish. Evolution 2019: The joint conference of the American Society of Naturalists, the Society for the Study of Evolution, and the Society of Systematic Biologists. Providence, RI, USA. June 22, 2019. 

[Seminars]

1. Shotaro Shiba Funai, “Feature extraction by machine learning and its related physics”, Southampton University, January 17, 2020
2. Zdenek Lajbner. “Research on cephalopods and other news from OIST, Okinawa, Japan.” 2020/01/03 Institute of Animal Physiology and Genetics, Czech Academy of Science; 2020/01/06 University of South Bohemia; 2020/01/08 Charles University; 2020/01/09 Institute of Organic Chemistry and Biochemistry, Czech Academy of Science; 2020/01/10 Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University.

3.  船井正太郎/短歌の“良さ”を表現できるか. 生理学研究所. 2019年10月24日. / Shotaro Funai. Can “goodness” of tanka (Japanese verse) be expressed? National Institute of Physiological Sciences. October 24, 2019

4. Jonathan Miller. Psychophysics of cephalopod camouflage. KEK Colloquium talk, Tsukuba. Oct 2^nd, 2019
5. 船井（柴）正太郎. 外積型”word2vec. TKP秋葉原カンファレンスセンター. 2019年7月16日 / Shotaro Shiba Funai. Word2vec of “external type. TKP Akihabara Conference Center. July 16, 2019

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events

6.1 Seminar "Building a quantum computer using silicon quantum dots"

• Date: February 4, 2020
• Venue: C209, Center Bldg, OIST
• Speaker: Prof. Susan Coppersmith, The University of New South Wales

6.2 Seminar "Circadian rhythm and genetics of Bipolar disorder"

• Date: January 20, 2020 
• Venue: C700, Lab3, OIST
• Speaker: Prof. Mary Waye, The Chinese University of Hong Kong

6.3 Seminar "How can we predict efficiently?"

• Date: January 8, 2020 
• Venue: C700, Lab3, OIST
• Speaker: Prof. Sarah Marzen, Claremont McKenna College

6.3 Seminar "One of the Biggest Mysteries in the Standard Model: Revealing Neutron Star Matter with Deep Learning"

• Date: October 15, 2019
• Venue: C700, Lab3, OIST
• Speaker: Prof. Kenji Fukushima, University of Tokyo

6.4 Outreach activities "Children's School of Science 2019 - 3rd-4th Grade Class A: Sea Animals" 

• Date: September 19-21 & 23, 2019
• Venue: Onna-son Civic Center & OIST Marine Station 
• Presenters: Takahiro NIshibayashi, Keishu Asada, Tamar Gitnick, Zdenek Lajbner (Miller Group, OIST) & Liu Yung-Chieh (OIST Ph.D. student)
• DAY1: Talk "Camouflage works (color changing and chromatophores)", DAY2: Seragaki Marine Station Tour, DAY3: Reef Walking, DAY 4: Talk "Animals in habitats"

6.5 Seminar "The development of the information-theoretical method for the classification of texts and its application to the definition of authorship"

• Date: September 18, 2019
• Venue: C016, Lab1
• Speaker:  Dr. Nadezhda Savina,  Novosibirsk State University

6.6 Seminar "Time-universal data compression and prediction"

• Date: September 17, 2019
• Venue: C700, Lab3, OIST
• Speaker: Prof. Boris Ryabko, ICT, Siberian Branch of RAS.

6.7  Seminar "Work of Resaerch Unit Technician" - Lecture for junior high school students in Onna / "技術員の仕事" - 恩納村内中学生への講義

• Date: June 27, 2019
• Venue: OIST Marine Science Station
• Speaker: Takahiro Nishibayashi (OIST)

 

7. Other

Nothing to report.