In situ observation of shrimp feeding process under microgravity environment

In situ observation of shrimp feeding process under microgravity environment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In situ observation of shrimp feeding process under microgravity environment Chihiro YOKOTA, Seiichi TSUMURA, Kanji KAMEYAMA, Syou MAKI, Ryosuke TADOKORO, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7308179/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract We carried out in situ observation of the feeding process of edible shrimp under microgravity environment. To realize that condition, we developed a new type of clinostat with high-speed rotation, with a maximum rotation of 130 rpm. This rotation speed is much faster than that of conventional models. Under that rotation speed, aquatic organisms such as fish and crustaceans have a suppressed capability of recovering their proper swimming form, making it possible for them to be exposed to a pseudo-weightless condition. By this method, we could successfully observe the feeding process of shrimp in that condition. We also performed a genetic analysis of shrimp exposed to microgravity for 24 h. The results of gene ontology analysis suggested that the effects of exposure to weightlessness were significant, as compared with control shrimp. We believe that the high-speed clinostat will contribute greatly to the future progress of engineering applications in microgravity research. Clinostat Centrifugal force Third water Koteki Weightlessness Microgravity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In recent years, academic reports on the utilization of the space environment have been actively published from around the world. There are several known techniques to realize a microgravity condition on Earth, e.g., parabolic flight (Pletser 2004 ; Acharya et al. 2019 ) and drop-tower (Izumi et al. 2009 ; Brungs et al. 2016 ). Weightlessness can be maintained for only a very short period of time by these techniques, however, and they are not suitable for some examinations simulating long-term behavioral and biological studies of organisms. Unique methods have been developed to actualize long periods of weightlessness, such as using sound waves (Biswas 1995 ; Santesson et al. 2003 ; Cao et al. 2012 ), electrostatic repulsion (Rhim and Chung 1991 ), and a magnetic force (Ikezoe et al. 1998 ; Maki et al. 2004 ; Maki et al. 2016 ; Maki and Hagiwara. 2022), by offsetting the gravitational force with a vertical upward external force. The weightless space achieved by these methods is very small, and they also require highly detailed adjustments to the experiment. Ideally, using International Space Station (ISS) would be the most desirable method, but this is not realistic due to the enormous cost and the limited experimental samples required. Such practical difficulties hereinabove, time-consuming research in the weightlessness has not been thoroughly investigated yet. In comparison, a random positioning machine (also commonly known as a “ clinostat ”) is a simple device for creating a pseudo-weightless condition, by which the effect of the gravity vector is invalidated by rotating a test object centrifugally (Gravite®; Loon 2007 ). Although a clinostat cannot structurally realize strict weightlessness, it is designed so that the time average of the resultant force between the centrifugal force and the vertically downward gravitational force comes to be approximately zero. Many research projects have been carried out by using machines similar to the clinostat, especially in the academic fields of stem cell research (Yuge et al. 2006 ; Kawahara et al. 2009 ; Mitsuhara et al. 2013 ; Malczyk et al. 2024 ). In particular, a clinostat was utilized to investigate the effect of microgravity on fish. The French National Institute for Ocean Science has been using a clinostat to fertilize and hatch fish eggs in a simulated weightless environment. That research, which began in 2019, is part of the Lunar Hatch Programme (Przybyla et al. 2023 ) that aims to supply fish as food for the lunar base planned by the European Space Agency in the future. In practice, however, we would like to point out that there is an essential, unsolved problem in their experimental approach. They use a conventional clinostat with a rotation speed of about 10 to 25 rpm. Such a slow speed leaves enough time for the fish or crustaceans to recover their usual swimming form, making it difficult to study the effects of weightlessness. We confirmed through our preparatory experiments that fish tend to stay at the center of rotation of the clinostat, where the effect of the outer force by the rotation becomes smallest. This means that the fish feel the bodily sensation not of 0 G, but of almost 1 G. Unless the fish is immobilized in the water or fixed onto the clinostat, there is no convincing, concrete evidence that they have been exposed to weightlessness. In view of the present situation, we developed a new type of clinostat that rotates at such a high-speed that the fish are given little time to regain their balance. This clinostat allows us to simulate the behavior of fish and crustaceans in microgravity much more accurately than conventional methods. Envisioning fish and crustacean production in outer space, there are many unknown problems lseft in our future project of “ space aquaculture ”, one of which are whether they can feed properly even in the weightless condition. In this paper, we present the performance and structure of this clinostat, and demonstrate in situ observation of shrimp feeding process. Furthermore, we also denote the biological effect of weightlessness on the shrimp. Mechanical Performance Prior to the investigation of weightlessness on aquatic organisms, we experimentally confirmed that it takes less than 0.5 seconds for fish to recover their proper swimming form in water. For that reason, we set the clinostat’s rotation speed to 120 rpm or more. The practical development and manufacturing of the device were done with the support of AES Co., Ltd (AES Co., Ltd). Figure 1 shows a photograph of the new type of high-speed rotation clinostat, named as “Small 3D Clinostat PMS-V1-R02”. It consists of the main body part (Fig. 1 (a)) that rotates samples, and the control part (Fig. 1 (b)) that sets the rotation speed. The size of the main body part is 380 mm wide, 403 mm high, and 306 mm deep. The size of the control part is 260 mm wide, 109 m high, and 230 mm deep. The Clinostat PMS-V1-R02 has a dual-axis to drive the rotation, similar to a conventional clinostat. Two different rotations along each axis are generated independently, and have dedicated complicated centrifugal forces. The resultant force between each centrifugal force works on the sample so as to cancel the effect of the gravitational force, and a pseudo-weightless condition is achieved at the center of rotation. Test samples were installed into a “rectangular sample box”, which is made of clear board (Fig. 1 (c)). As shown, the size of the box has enough space to hold two 275 mL bottles. We can reduce the number of bottles to one and, in place of the other, install a digital camera inside the box. This box is fixed to the “rotating space”, as shown in Fig. 1 (a). This space is designed to be 100 mm wide, 140 mm high, and 80 mm deep. The maximum sample weight that can be loaded into the rotating space is 1.0 kg in total. Most of the structure of the Clinostat PMS-V1-R02 is made of stainless steel. The operating temperature range is 0 to 50°C. The operating humidity range is 85% or less. When the first and second axes are named as the X - and Y -axes, respectively, the maximum rotation speed of the X - axis is 110 rpm, and that of the Y - axis is 130 rpm. Figure 2 shows the rotational acceleration performance of the Clinostat PMS-V1-R02. Figure 2 (a) shows the response curves of the three-dimensional components of the acceleration, which are resolved into a Cartesian coordinate system ( x , y , and z coordinates). As shown, each response curve is not completely zero. Owing to the fluctuation by virtue of the slight difference in those curves, the total superimposed accelerations cancel each other out and become close to zero. Figure 2 (b) characterizes the trajectory of the acceleration vectors projected over a virtual sphere. Each axis in both figures represents acceleration in units of G. Figure 2 (b) represents that the trajectory of the resultant acceleration is adjusted so that it does not follow the same orbit. That adjustment is due to creating a condition that is as pseudo-weightless as possible. The rotation speed of both axes is controllable via the control part and a computer. The power supply is AC100V, and the input range is AC85 to 265 V (47 to 63Hz). The operating temperature range is 0 to 40°C. The operating humidity range is 20 to 85%. We can optimize the balance of force of the rotated samples by intentionally changing the rotation speeds (i.e., frequency). When the rotation speed of one axis is set at zero, a hypergravity condition can be realized by the centrifugal force of the single-axis rotation. The Clinostat PMS-V1-R02 has some safety measure functions. When starting the rotation, it takes approximately 3 minutes until the set rotation speed is reached. Similarly, when stopping the rotation, it takes approximately 3 minutes from the command to stop the operation until the rotation actually stops. It is also equipped with an emergency stop function in case of contact, enabling stopping within 3 seconds. Other Attachments and Accessories Figure 3 exhibits other attachments and accessories of the Clinostat PMS-V1-R02. A rectangular sample box (Fig. 3 (a)), the size of which is 99.5 mm long, 140 mm wide, and 80 mm high, is made of 5 mm thick acrylic and is processed to fit in perfect accord with the rotating space. A digital camera can be stuffed inside that space (Fig. 3 (a)). A small sample container is located in the position indicated by the pale blue circle in Fig. 3 (a), in contact with the camera, and there is also a light that is set at the location of the hatched square, allowing us to can record real-time movies of the samples during rotation. There is a small window in the backside of the rectangular sample box, so that the LCD screen or some switches behind the camera can be operated from outside the box. Figure 3 (b) shows a top view of the small sample container. This container is cylindrical and transparent with a diameter of 60 mm and a thickness that can be changed from 10 to 20 mm. Samples of aquatic organisms, e.g., fish or shrimp, can be confined inside the container. In the experiment, the items mentioned (sample container, a camera, and a light) are installed together into the rectangular sample box, and they are rigorously fixed with an acrylic board and screws, as shown in Fig. 3 (c), in order to endure the high-speed rotation. A plastic net (Fig. 3 (d)) is arranged in the sample container to gently immobilize the fish or crustaceans. The volume of the small sample container is about 50 mL, and one side of it is covered with a fluororesin porous film (TEMISH™, Nitto Denko Co., Ltd), as shown by the mark A in Fig. 3 (e). This film does not leak water but ensures breathability, effectively preventing the fish and crustaceans from becoming oxygen-deprived during long-term experiments. There is a protective acrylic barrier for when the rotation is in operation, as shown in Fig. 3 (f). Experiments We used high-speed rotation of the Clinostat PMS-VI-R02 to examine the effects of a pseudo-weightless condition on samples of living aquatic organisms. We used juvenile shrimp of the species Marsupenaeus japonicus as a test sample. They were nauplii larvae when we purchased them (MBC Development Co., Ltd.), and were reared for several days in “ third water ” just before the experiment. The third water is known as “ Koteki-Kankyo-Sui ” in Japanese (Yamamoto No.6056949; Yamamoto No. 5487378; Yamamoto No. 5487377; Yamamoto No. 5364874; Yamamoto No. 5578401; Yamamoto No. 5062550; Yamamoto No. 4665258; Yamamoto No. 4665252). This water (“Koteki Water” hereafter) has many advantages in carrying out recirculating aquaculture systems, especially in closed systems. This is a versatile and highly reliable technology that can be employed even in developing countries (JICA Final Report 2013 ; JICA REPORT 2015 ; JICA Final Report 2015 ). We have currently been directing a far-reaching project to cultivate edible large fish and crustaceans in outer space or on the lunar surface, and we would like to apply this technology for materializing a “ space aquaculture ” in future (Yokota 2024; Yamamoto 2025 ). This is why we utilized Koteki Water in this experiment. There is very little research on space aquaculture at present (Przybyla et al. 2021 ). We hope that proactive proposals using Koteki Water will be made for the introduction of future approaches, particularly in the engineering field of RAS. In the present experiment, Koteki Water was adjusted to the same level as standard seawater, with a density of 1.008 [g/cm³]. The reason we used shrimp was that, since they could stick to the plastic net, we thought that they would be less affected than fish by the complex flows caused by the rotation of the clinostat. Other reasons are that they can survive in highly dissolved nitrogen concentrations better than fish, and they can survive for the long duration of the experiment. We conducted two types of experiments. One was an in situ observation of a short-time application of pseudo-weightless condition to the shrimp. We observed their behavior and feeding process directly under the clinostat rotation. The other was a long-time experiment of exposure of the shrimp to microgravity. The biological influence on the shrimp was examined by the use of gene expression analysis. In Situ Observation One juvenile shrimp ( Marsupenaeus japonicus , 132 dah, n = 1) was confined in the small sample container, and three shrimp feed pellets (Sigma P-2 for kuruma shrimp cultivation, Hayashikane Sangyo Co., Ltd.) were also installed in the container (see Fig. 3 (b)). This was set into the rectangular sample box, and a smart digital camera (Stylus tg-2 tough, Olympus Co., Ltd.) was fixed beneath the container in order to observe the shrimp's behavior through the transparent container bottom (see Fig. 3 (c)). All the shrimp we used were not fed before the experiment. Figure 4 shows a sample shrimp confined in the container. All of the images show the shrimp’s behavior in an ongoing situation of high-speed rotation by the Clinostat PMS-VI-R02. We carried out the experiment two times, and the rotation was operated for 15 minutes. The length of a short movie of the first experiment is 11 min 52 s. The length of a movie of the second experiment is 13 min 14 s. Both experiments were carried out when the temperature was 20±1°C. Figures 4 (a)-(d) exhibit the moment of the shrimp’s feeding in the first experiment. Figures 4 (e)-(h) are the same moment seen in the second experiment. In the first experiment, the rotation speed was set to 55 rpm on the X -axis and 65 rpm on the Y -axis, respectively. The moment during the rotation when the shrimp tried to feed was captured twice. The shrimp picked up pellets and ate them when they happened to be brought in front of the shrimp. The shrimp continued to eat a food pellet while holding it from 3 min 51 s to 9 min 27 s, and from 9 min 51 s to 10 min 52. In the second experiment, the speed was increased to 110 rpm on the X -axis and 130 rpm on the Y -axis, respectively. The shrimp’s feeding behavior was captured twice this time, too. The shrimp ate a pellet from 4 min 46 s to 6 min 16 s, and another one from 6 min 36 s to 12 min 59. The green triangular arrows in the figures indicate instructive moments when the shrimp picks up a food pellet and brings it to its mouth. Thus, the shrimp’s behavior and feeding process were perfectly recorded while being filmed by the camera. Two short movies of that process are available on the website. https://www.youtube.com/watch?v=CRqHW7jbMO4 ; https://www.youtube.com/watch?v=2ma-vsk5rmk Gene Expression Analysis Gene expression analysis was conducted on the shrimp exposed to long-time weightlessness. To begin with, one juvenile shrimp ( Marsupenaeus japonicus , 158 dah, n = 3) was captured in each of three small sample containers. The three containers were then placed in the rectangular sample box, as shown in Fig. 3 (e). Next, the box was set in the Clinostat PMS-VI-R02, and was continuously rotated for 24 hours. The room light was turned off during the rotation in both experiments (Fig. 3 (f)). After that, the shrimp were removed from the container. Since one of three shrimp had died, we used the two living shrimp to conduct the gene expression analysis. A control experiment was performed simultaneously using the same species of shrimp of a similar size. For the purpose of oxygen supply to the control shrimp, a magnetic stirrer was packed into the container and the water was stirred for 24 hours. RNA extraction was performed as described below. To begin with, the shrimp were homogenized at room temperature (about 20°C) using a 20 mL glass teflon homogenizer, to which was added 20 mL of RNA extraction reagent RNAzol®RT. The homogenized shrimp were transferred to a centrifuge tube (Labcon, SuperClear 50 mL) and centrifuged for 15 minutes. 0.55 mL of the supernatant was dispensed into a 2.0 mL PP microtube using a micropipette. 240 µL of purified water was added to this and centrifuged at 12,000 G for 15 minutes. 1.0 mL of the supernatant was transferred to another microtube. 3.15 µL of 4-bromoanisole (TCI, B0547) was added to the 1 mL of the transferred supernatant, left to stand for 3 minutes, and centrifuged at 12,000 G for 10 minutes. 570 µL of the supernatant and 570 µL of isopropanol were mixed in a 2 mL PP microtube, left to stand for 10 minutes, and centrifuged at 12,000 G for 10 minutes. The RNA pellet was mixed twice with 100 µL of 75% ethanol in a 2 mL PP microtube, and centrifuged at 6,000 G for 3 minutes. The RNA pellet was dissolved in RNase-free water and vortexed at room temperature for 5 minutes. A quality check was performed using the extracted sample of RNA. Finally, gene expression analysis of this RNA was outsourced to the research laboratory of a private company (Novogene Corp. Inc.). Results and Discussion Shrimp Behavior in the Weightless Condition We noticed from in situ observation that high-speed rotation caused turbulent flows in the container. The feed pellets were vigorously stirred in such a situation, and the shrimp seldom chased them. The shrimp were observed to be clinging to the net. When a pellet happened to be brought in front of the shrimp did they pick up and eat it, as shown in Fig. 4 . Similar feeding behavior was observed many times in the video. The shrimp's behavior was clearly affected by the flow but their appetite was voracious. The finding of the capability of shrimp to eat some feed even in a weightless condition is one of the most distinctive features in this experiment. This finding suggests that if shrimp are to be farmed in space, they may be transported live, rather than as eggs, into space. We repeated the same experiment many times, and confirmed the similar feeding process. In addition, microscopic observation showed evidence that the feed had accumulated in the intestine as digested matter. We also confirmed in every case that most of the shrimp began to eat the pellets as soon as the flows were stopped. Hence, we think that environmental factors such as strong flows would play a large role in their feeding behavior, especially in a weightless condition. We can deduce that, when we cultivate shrimp in a weightless environment like outer space, we should stop flows or make it weak inside the container so as to allow the shrimp to catch their feed more easily. These findings will be extremely beneficial for the advancement of space aquaculture. Gene Ontology Analysis The results of gene ontology (GO) analysis are presented in Fig. 5 and Fig. 6 . These quantitative analyses were carried out by a research company (Novogene Corp. Inc.). Figure 5 is the gene expression map visualized by levels. The contour colors of this map reflect the magnitude of the expression level, and the arrangement of the pattern corresponds to the results of the cluster analysis of the genes. The left column in Fig. 5 (a) shows the group of the shrimp exposed to weightlessness, and the right column in Fig. 5 (b) shows the control group. The red contours in the map indicate high gene expression, and the green contours indicate low gene expression. As shown, the red contours were limited to some small areas only, and most areas were covered in green or black. Judging from the difference in the contour patterns between the exposed group and the control, we found that the expression levels were different from each other. Figure 6 shows the results of GO enrichment analysis. There are three categories classified by molecular function (MF), cellular component (CC), and biological process (BP). The column charts of BP, CC, and MF are colored with red, green, and blue, respectively. As regards the BP categories, “chitin metabolic process”, “amino sugar metabolic process”, “glucosamine-containing compound metabolic process”, “aminoglycan metabolic process”, and “drug metabolic process”, specifically, scored high. As regards the CC categories, only “extracellular region” was notable, and “actin cytoskeleton” was almost zero. As regards the MF categories, the increase in the “structural constituent of cuticle” was the most remarkable, and “structural molecule activity” and “chitin binding” were also clearly distinguished. It is noteworthy that the BP categories were associated with the chitin metabolic process and the MF categories were related to the structural constituent of cuticle. These categories are suggestive of the function of the shrimp’s movement. That is, shrimp exposed to a weightless environment might be some tangible, especially in the categories of the organ of locomotion. Similar findings were also confirmed in a study of the function of osteoclasts in Medaka fish conducted on the International Space Station (Asaka et al. 2010 ). In summary, we can say that the GO analyses offered valuable suggestions with respect to the influence of exposure to the weightless condition on the shrimp. The details, however, are still unknown, and further study is in progress. Conclusions We observed feeding process of shrimp under the weightless condition. To realize that condition, we developed a new type of clinostat with high-speed rotation, with a maximum rotation of 130 rpm. We could successfully record the shrimp’s feeding process. We have proven the capability of the shrimp to eat some feed even in a weightless condition. This finding suggests that when cultivating shrimp in space. This may be possible to transport them live, rather than as eggs. That could be useful information for the advancement of space aquaculture. In addition, we also performed gene ontology analysis of the shrimp exposed to the microgravity environment for 24 h. Those results showed the possibility that the exposure to weightlessness might be affectable as compared with the control shrimp, especially in the functions and genes related to the shrimp exoskeleton system. We suspect that environmental factors such as the flows by high-speed rotation played a large role in those effects. The details, however, are still unknown yet. Further study should be carried out in the ISS. In this research, we represented a high-speed rotating clinostat has a great impact on the progress of various kinds of experiments that were not possible with a conventional low-speed clinostat. We believe that a high-speed clinostat will greatly contribute to the progress of biological research and its application over the future microgravity science. Declarations Acknowledgements We received gracious cooperation from the members of the Design and Manufacturing Center of Okayama University of Science for the development of the experimental equipment. A new type of random positioning machine,“Small 3D Clinostat PMS-V1-R02”, was designed and developed with the support of AES Co., Ltd, which is the most famous manufacturing company of a clinostat in Japan. We are very grateful to this company for all the mechanical support and advice. We were given great assistance by Mr. Kazuo Odakami, who is senior staff of the Head Audit Office, Kake Educational Institution, Okayama University of Science. This paper was proofread in English by Prof. Christopher Carman of the University of Occupational and Environmental Health. All the mentioned support was essentially important to the progress of our research project. We would like to express our deepest gratitude. Conflict of Interest Every author declares no competing interests. Author Contributions Chihiro Yokota: Experiment, Data analysis, Seiichi Tsumura: Experiment, Data analysis, Kanji Kameyama: Technical support, Development of the system, Syou Maki : Writing, Investigation, Design of the system, Conceptualization, Ryosuke Tadokoro: Experiment, Investigation, Gene ontology analysis, Review, Toshimasa Yamamoto: Experiment, Conceptualization, Review and editing. Data Availability All the data in this study are available upon reasonable request. Please contact the research institute where the corresponding author (Syou Maki) is employed. 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Yokota, C., Maki, S., Tsumura, S., Yamamoto, T., Yamaguchi T.: Development of a closed circulation system for rearing aquatic organisms for the International Space Station. Annual Meeting of the Japanese Society of Fisheries Science, Abstracts p.261, Kyoto Univ., 24-27, Sep. 2024, in Japanese . Yuge, L., Kajiume, T., Tahara, H., Kawahara, Y., Umeda, C., Yoshimoto, R., Wu, S. L., Yawaoka, K., Asashima, M., Kataoka, K., Ide, T.: Microgravity potentiates stem cell proliferation while sustaining the capability of differentiation. Stem Cells Develop. 15 , 921–929 (2006). DOI: 10.1089/scd.2006.15.921 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7308179","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509567474,"identity":"5535c8fe-692e-4a97-b881-cda821c51ee6","order_by":0,"name":"Chihiro YOKOTA","email":"","orcid":"","institution":"Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University of Science","correspondingAuthor":false,"prefix":"","firstName":"Chihiro","middleName":"","lastName":"YOKOTA","suffix":""},{"id":509567483,"identity":"a45d69c8-3050-497c-9e43-4e27aae5ff4b","order_by":1,"name":"Seiichi TSUMURA","email":"","orcid":"","institution":"Education and Research Center for Organisms Production, Organization for Research Development and Outreach, Okayama University of Science","correspondingAuthor":false,"prefix":"","firstName":"Seiichi","middleName":"","lastName":"TSUMURA","suffix":""},{"id":509567484,"identity":"c2187899-c73c-4d45-8eec-f0517a3ba63a","order_by":2,"name":"Kanji KAMEYAMA","email":"","orcid":"","institution":"Research Support Office Joint Facilities Management, Okayama University of Science","correspondingAuthor":false,"prefix":"","firstName":"Kanji","middleName":"","lastName":"KAMEYAMA","suffix":""},{"id":509567486,"identity":"0874a98c-7d76-4303-b881-478ab6bc3df6","order_by":3,"name":"Syou MAKI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFACNoYDIIofxmdsIFaLJEwlUVrAwOAAsc7SbT+WePBnm03i5uM9xh8YauwYmGcTsMbsTNqBw7xtaYnbzpwxk2A4lszAOIeAfWYH0hsOM7YdTtx2I8cM6MoDDIwzEghoOf+8Aeiww4mbZ+QAHfaPGC030g4c4AVq2SCRYyDB2EaUlmcJh3nOpRnPOHOsTCKxL5mHsF/Opxl//FFmI9vf3rz5w4dvdnKGhEIMDBihccMAdBKP4QwidDAw/EFiy0sQpWUUjIJRMApGEAAAENVK24n4B60AAAAASUVORK5CYII=","orcid":"","institution":"Institute of Frontier Science and Technology, Okayama University of Science","correspondingAuthor":true,"prefix":"","firstName":"Syou","middleName":"","lastName":"MAKI","suffix":""},{"id":509567487,"identity":"51fd4415-9fc6-4157-897e-5f497e12e827","order_by":4,"name":"Ryosuke TADOKORO","email":"","orcid":"","institution":"Department of Bioscience, Faculty of Life Science, Okayama University of Science","correspondingAuthor":false,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"TADOKORO","suffix":""},{"id":509567488,"identity":"86ec6b29-6555-4124-a8e3-749c6eba0b35","order_by":5,"name":"Toshimasa YAMAMOTO","email":"","orcid":"","institution":"Department of Bioscience, Faculty of Life Science, Okayama University of Science","correspondingAuthor":false,"prefix":"","firstName":"Toshimasa","middleName":"","lastName":"YAMAMOTO","suffix":""}],"badges":[],"createdAt":"2025-08-06 09:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7308179/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7308179/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90629526,"identity":"0980af1e-5153-4f71-867a-3d82add87a27","added_by":"auto","created_at":"2025-09-05 01:56:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":286999,"visible":true,"origin":"","legend":"\u003cp\u003e“Small 3D Clinostat PMS-V1-R02”, a new type of high-speed rotation clinostat. (a) The main body part, the size of which is 380 mm wide, 403 mm high, and 306 mm deep, to rotate the samples. We can see that there is dual-axis to drive the rotation. Two different rotations along each axis are independently generated, and have dedicated complicated centrifugal forces. The maximum rotation speed of the \u003cem\u003eX\u003c/em\u003e-axis is 110 rpm and that of the \u003cem\u003eY\u003c/em\u003e- axis is 130 rpm. (b) The control part for setting up the rotation speed. (c) The rectangular sample box, which has enough space to arrange two 275 mL bottles. This box is fixed to the “rotating space” of the clinostat, as shown in (a). The size of this space is 100 mm wide, 140 mm high, and 80 mm deep. The maximum sample weight that can be loaded into the rotating space is 1.0 kg in total.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7308179/v1/2746c5f8af10ed57df26046d.png"},{"id":90629530,"identity":"5e0e89b5-fe66-49f6-ab18-4214fa13f093","added_by":"auto","created_at":"2025-09-05 01:56:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120980,"visible":true,"origin":"","legend":"\u003cp\u003eThe rotational acceleration performance of Clinostat PMS-V1-R02. (a) The response curves of three-dimensional components of the acceleration, resolved into a Cartesian coordinate system (\u003cem\u003ex\u003c/em\u003e, \u003cem\u003ey\u003c/em\u003e, and \u003cem\u003ez\u003c/em\u003e coordinates). (b) The trajectory of the resultant acceleration vector projected over a virtual sphere. Each axis in both figures represents acceleration in units of G.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7308179/v1/20cdf55ed630d46b114bde01.png"},{"id":90629656,"identity":"fd4b5a2f-b0da-4063-91c7-af1568d999c0","added_by":"auto","created_at":"2025-09-05 02:04:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":285684,"visible":true,"origin":"","legend":"\u003cp\u003ePhotos of the other attachments and accessories of the Clinostat PMS-V1-R02. (a) The rectangular sample box and a digital camera. The small sample container is located in the position indicated by the light blue circle. A light is also equipped at the location of the hatched square. (b) The top view of the small sample container. (c) The rectangular sample box that includes a sample container, a light, and a camera. These items are installed together into the rectangular sample box, and they are rigorously fixed with an acrylic board and screws in order to endure the high-speed rotation. (d) The plastic net in the small sample container to gently immobilize the fish or crustaceans. (e) Three containers placed in the rectangular sample box. The mark A shows a fluororesin porous film. (f) We used the protective acrylic barrier when operating the rotation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7308179/v1/fe2f6a0d6df94741d2d4c6f6.png"},{"id":90629657,"identity":"dffa6ee3-8683-4619-9634-535f92c3e2ef","added_by":"auto","created_at":"2025-09-05 02:04:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":293145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e observation of shrimp behavior and feeding process during the rotation. One shrimp was confined to each small sample container. All of the images demonstrate the shrimp’s behavior in an ongoing situation of high-speed rotation by the Clinostat PMS-VI-R02. (a)-(d) show the moment of the shrimp’s feeding in the first experiment. The rotation speed was set to 55 rpm on the \u003cem\u003eX\u003c/em\u003e-axis and 65 rpm on the \u003cem\u003eY\u003c/em\u003e-axis, respectively. During the rotation, the moment when the shrimp tried to feed was captured twice. The shrimp continued to eat a food pellet while holding it from 3 min 51 s to 9 min 27 s, and from 9 min 51 s to 10 min 52. (e)-(h) display the same moment in the second experiment. The rotation speed was increased to 110 rpm on the \u003cem\u003eX\u003c/em\u003e-axis and 130 rpm on the \u003cem\u003eY\u003c/em\u003e-axis, respectively. The shrimp’s feeding behavior was captured twice this time, too. The shrimp ate a pellet from 4 min 46 s to 6 min 16 s, and from 6 min 36 s to 12 min 59. The green triangular arrows in the figures indicate instructive moments when the shrimp picks up a food pellet and brings it to its mouth. Thus, the shrimp’s behavior and feeding process were perfectly recorded while being filmed by the camera.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7308179/v1/4da7e137677467e8086c775c.png"},{"id":90629661,"identity":"63f70d5c-013f-43bf-af26-418c6853709f","added_by":"auto","created_at":"2025-09-05 02:04:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42406,"visible":true,"origin":"","legend":"\u003cp\u003eThe gene expression map visualized by the level. The contour colors of this map reflect the magnitude of the expression level, and their arrangement pattern corresponds to the results of cluster analysis of the genes. (a) Group exposed to the weightlessness. (b) Control group. Red contours indicate high gene expression, and green contours indicate low gene expression. As shown, we found that the expression levels are different from each other.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7308179/v1/437d9a70239fee345b833263.png"},{"id":90629535,"identity":"c25dc37d-86fe-44b0-97df-62ff50a8358d","added_by":"auto","created_at":"2025-09-05 01:56:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60187,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of GO enrichment analysis. There are three categories classified by molecular function (MF), cellular component (CC), and biological process (BP). The column charts of BP, CC, and MF are colored with red, green, and blue, respectively. Those results offered valuable suggestions with respect to the influence on the shrimp of exposure to the pseudo-weightless condition.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7308179/v1/66d67901e757581c1a365bca.png"},{"id":91316726,"identity":"b4286549-f9c5-4bbb-b1b8-7f5ce8258ff2","added_by":"auto","created_at":"2025-09-15 08:24:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1745168,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7308179/v1/c52bef7d-d0e1-4036-8aab-a1f533910be4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In situ observation of shrimp feeding process under microgravity environment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, academic reports on the utilization of the space environment have been actively published from around the world. There are several known techniques to realize a microgravity condition on Earth, e.g., parabolic flight (Pletser \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Acharya et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and drop-tower (Izumi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Brungs et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Weightlessness can be maintained for only a very short period of time by these techniques, however, and they are not suitable for some examinations simulating long-term behavioral and biological studies of organisms. Unique methods have been developed to actualize long periods of weightlessness, such as using sound waves (Biswas \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Santesson et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), electrostatic repulsion (Rhim and Chung \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), and a magnetic force (Ikezoe et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Maki et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Maki et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Maki and Hagiwara. 2022), by offsetting the gravitational force with a vertical upward external force. The weightless space achieved by these methods is very small, and they also require highly detailed adjustments to the experiment. Ideally, using International Space Station (ISS) would be the most desirable method, but this is not realistic due to the enormous cost and the limited experimental samples required. Such practical difficulties hereinabove, time-consuming research in the weightlessness has not been thoroughly investigated yet.\u003c/p\u003e\u003cp\u003eIn comparison, a random positioning machine (also commonly known as a \u0026ldquo;\u003cem\u003eclinostat\u003c/em\u003e\u0026rdquo;) is a simple device for creating a pseudo-weightless condition, by which the effect of the gravity vector is invalidated by rotating a test object centrifugally (Gravite\u0026reg;; Loon \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Although a clinostat cannot structurally realize strict weightlessness, it is designed so that the time average of the resultant force between the centrifugal force and the vertically downward gravitational force comes to be approximately zero. Many research projects have been carried out by using machines similar to the clinostat, especially in the academic fields of stem cell research (Yuge et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kawahara et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mitsuhara et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Malczyk et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In particular, a clinostat was utilized to investigate the effect of microgravity on fish. The French National Institute for Ocean Science has been using a clinostat to fertilize and hatch fish eggs in a simulated weightless environment. That research, which began in 2019, is part of the Lunar Hatch Programme (Przybyla et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) that aims to supply fish as food for the lunar base planned by the European Space Agency in the future. In practice, however, we would like to point out that there is an essential, unsolved problem in their experimental approach. They use a conventional clinostat with a rotation speed of about 10 to 25 rpm. Such a slow speed leaves enough time for the fish or crustaceans to recover their usual swimming form, making it difficult to study the effects of weightlessness. We confirmed through our preparatory experiments that fish tend to stay at the center of rotation of the clinostat, where the effect of the outer force by the rotation becomes smallest. This means that the fish feel the bodily sensation not of 0 G, but of almost 1 G. Unless the fish is immobilized in the water or fixed onto the clinostat, there is no convincing, concrete evidence that they have been exposed to weightlessness. In view of the present situation, we developed a new type of clinostat that rotates at such a high-speed that the fish are given little time to regain their balance. This clinostat allows us to simulate the behavior of fish and crustaceans in microgravity much more accurately than conventional methods. Envisioning fish and crustacean production in outer space, there are many unknown problems lseft in our future project of \u0026ldquo;\u003cem\u003espace aquaculture\u003c/em\u003e\u0026rdquo;, one of which are whether they can feed properly even in the weightless condition. In this paper, we present the performance and structure of this clinostat, and demonstrate \u003cem\u003ein situ\u003c/em\u003e observation of shrimp feeding process. Furthermore, we also denote the biological effect of weightlessness on the shrimp.\u003c/p\u003e\n\u003ch3\u003eMechanical Performance\u003c/h3\u003e\n\u003cp\u003ePrior to the investigation of weightlessness on aquatic organisms, we experimentally confirmed that it takes less than 0.5 seconds for fish to recover their proper swimming form in water. For that reason, we set the clinostat\u0026rsquo;s rotation speed to 120 rpm or more. The practical development and manufacturing of the device were done with the support of AES Co., Ltd (AES Co., Ltd).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows a photograph of the new type of high-speed rotation clinostat, named as \u0026ldquo;Small 3D Clinostat PMS-V1-R02\u0026rdquo;. It consists of the main body part (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)) that rotates samples, and the control part (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)) that sets the rotation speed. The size of the main body part is 380 mm wide, 403 mm high, and 306 mm deep. The size of the control part is 260 mm wide, 109 m high, and 230 mm deep. The Clinostat PMS-V1-R02 has a dual-axis to drive the rotation, similar to a conventional clinostat. Two different rotations along each axis are generated independently, and have dedicated complicated centrifugal forces. The resultant force between each centrifugal force works on the sample so as to cancel the effect of the gravitational force, and a pseudo-weightless condition is achieved at the center of rotation.\u003c/p\u003e\u003cp\u003eTest samples were installed into a \u0026ldquo;rectangular sample box\u0026rdquo;, which is made of clear board (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c)). As shown, the size of the box has enough space to hold two 275 mL bottles. We can reduce the number of bottles to one and, in place of the other, install a digital camera inside the box. This box is fixed to the \u0026ldquo;rotating space\u0026rdquo;, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a). This space is designed to be 100 mm wide, 140 mm high, and 80 mm deep. The maximum sample weight that can be loaded into the rotating space is 1.0 kg in total.\u003c/p\u003e\u003cp\u003eMost of the structure of the Clinostat PMS-V1-R02 is made of stainless steel. The operating temperature range is 0 to 50\u0026deg;C. The operating humidity range is 85% or less. When the first and second axes are named as the \u003cem\u003eX\u003c/em\u003e- and \u003cem\u003eY\u003c/em\u003e-axes, respectively, the maximum rotation speed of the \u003cem\u003eX\u003c/em\u003e- axis is 110 rpm, and that of the \u003cem\u003eY\u003c/em\u003e- axis is 130 rpm.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the rotational acceleration performance of the Clinostat PMS-V1-R02. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a) shows the response curves of the three-dimensional components of the acceleration, which are resolved into a Cartesian coordinate system (\u003cem\u003ex\u003c/em\u003e, \u003cem\u003ey\u003c/em\u003e, and \u003cem\u003ez\u003c/em\u003e coordinates). As shown, each response curve is not completely zero. Owing to the fluctuation by virtue of the slight difference in those curves, the total superimposed accelerations cancel each other out and become close to zero. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b) characterizes the trajectory of the acceleration vectors projected over a virtual sphere. Each axis in both figures represents acceleration in units of G. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b) represents that the trajectory of the resultant acceleration is adjusted so that it does not follow the same orbit. That adjustment is due to creating a condition that is as pseudo-weightless as possible.\u003c/p\u003e\u003cp\u003eThe rotation speed of both axes is controllable via the control part and a computer. The power supply is AC100V, and the input range is AC85 to 265 V (47 to 63Hz). The operating temperature range is 0 to 40\u0026deg;C. The operating humidity range is 20 to 85%. We can optimize the balance of force of the rotated samples by intentionally changing the rotation speeds (i.e., frequency). When the rotation speed of one axis is set at zero, a hypergravity condition can be realized by the centrifugal force of the single-axis rotation.\u003c/p\u003e\u003cp\u003eThe Clinostat PMS-V1-R02 has some safety measure functions. When starting the rotation, it takes approximately 3 minutes until the set rotation speed is reached. Similarly, when stopping the rotation, it takes approximately 3 minutes from the command to stop the operation until the rotation actually stops. It is also equipped with an emergency stop function in case of contact, enabling stopping within 3 seconds.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eOther Attachments and Accessories\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e exhibits other attachments and accessories of the Clinostat PMS-V1-R02. A rectangular sample box (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)), the size of which is 99.5 mm long, 140 mm wide, and 80 mm high, is made of 5 mm thick acrylic and is processed to fit in perfect accord with the rotating space. A digital camera can be stuffed inside that space (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)). A small sample container is located in the position indicated by the pale blue circle in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a), in contact with the camera, and there is also a light that is set at the location of the hatched square, allowing us to can record real-time movies of the samples during rotation. There is a small window in the backside of the rectangular sample box, so that the LCD screen or some switches behind the camera can be operated from outside the box.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b) shows a top view of the small sample container. This container is cylindrical and transparent with a diameter of 60 mm and a thickness that can be changed from 10 to 20 mm. Samples of aquatic organisms, e.g., fish or shrimp, can be confined inside the container. In the experiment, the items mentioned (sample container, a camera, and a light) are installed together into the rectangular sample box, and they are rigorously fixed with an acrylic board and screws, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c), in order to endure the high-speed rotation.\u003c/p\u003e\u003cp\u003eA plastic net (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(d)) is arranged in the sample container to gently immobilize the fish or crustaceans. The volume of the small sample container is about 50 mL, and one side of it is covered with a fluororesin porous film (TEMISH\u0026trade;, Nitto Denko Co., Ltd), as shown by the mark A in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(e). This film does not leak water but ensures breathability, effectively preventing the fish and crustaceans from becoming oxygen-deprived during long-term experiments. There is a protective acrylic barrier for when the rotation is in operation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(f).\u003c/p\u003e\u003c/div\u003e"},{"header":"Experiments","content":"\u003cp\u003eWe used high-speed rotation of the Clinostat PMS-VI-R02 to examine the effects of a pseudo-weightless condition on samples of living aquatic organisms. We used juvenile shrimp of the species \u003cem\u003eMarsupenaeus japonicus\u003c/em\u003e as a test sample. They were nauplii larvae when we purchased them (MBC Development Co., Ltd.), and were reared for several days in \u0026ldquo;\u003cem\u003ethird water\u003c/em\u003e\u0026rdquo; just before the experiment. The third water is known as \u0026ldquo;\u003cem\u003eKoteki-Kankyo-Sui\u003c/em\u003e\u0026rdquo; in Japanese (Yamamoto No.6056949; Yamamoto No. 5487378; Yamamoto No. 5487377; Yamamoto No. 5364874; Yamamoto No. 5578401; Yamamoto No. 5062550; Yamamoto No. 4665258; Yamamoto No. 4665252). This water (\u0026ldquo;Koteki Water\u0026rdquo; hereafter) has many advantages in carrying out recirculating aquaculture systems, especially in closed systems. This is a versatile and highly reliable technology that can be employed even in developing countries (JICA Final Report \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; JICA REPORT \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; JICA Final Report \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We have currently been directing a far-reaching project to cultivate edible large fish and crustaceans in outer space or on the lunar surface, and we would like to apply this technology for materializing a \u0026ldquo;\u003cem\u003espace aquaculture\u003c/em\u003e\u0026rdquo; in future (Yokota 2024; Yamamoto \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This is why we utilized Koteki Water in this experiment. There is very little research on space aquaculture at present (Przybyla et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We hope that proactive proposals using Koteki Water will be made for the introduction of future approaches, particularly in the engineering field of RAS.\u003c/p\u003e\u003cp\u003eIn the present experiment, Koteki Water was adjusted to the same level as standard seawater, with a density of 1.008 [g/cm\u0026sup3;]. The reason we used shrimp was that, since they could stick to the plastic net, we thought that they would be less affected than fish by the complex flows caused by the rotation of the clinostat. Other reasons are that they can survive in highly dissolved nitrogen concentrations better than fish, and they can survive for the long duration of the experiment.\u003c/p\u003e\u003cp\u003eWe conducted two types of experiments. One was an \u003cem\u003ein situ\u003c/em\u003e observation of a short-time application of pseudo-weightless condition to the shrimp. We observed their behavior and feeding process directly under the clinostat rotation. The other was a long-time experiment of exposure of the shrimp to microgravity. The biological influence on the shrimp was examined by the use of gene expression analysis.\u003c/p\u003e\n\u003ch3\u003eIn Situ Observation\u003c/h3\u003e\n\u003cp\u003eOne juvenile shrimp (\u003cem\u003eMarsupenaeus japonicus\u003c/em\u003e, 132 dah, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1) was confined in the small sample container, and three shrimp feed pellets (Sigma P-2 for \u003cem\u003ekuruma shrimp\u003c/em\u003e cultivation, Hayashikane Sangyo Co., Ltd.) were also installed in the container (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b)). This was set into the rectangular sample box, and a smart digital camera (Stylus tg-2 tough, Olympus Co., Ltd.) was fixed beneath the container in order to observe the shrimp's behavior through the transparent container bottom (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c)). All the shrimp we used were not fed before the experiment.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows a sample shrimp confined in the container. All of the images show the shrimp\u0026rsquo;s behavior in an ongoing situation of high-speed rotation by the Clinostat PMS-VI-R02. We carried out the experiment two times, and the rotation was operated for 15 minutes. The length of a short movie of the first experiment is 11 min 52 s. The length of a movie of the second experiment is 13 min 14 s. Both experiments were carried out when the temperature was 20\u0026plusmn;1\u0026deg;C.\u003c/p\u003e\u003cp\u003eFigures \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a)-(d) exhibit the moment of the shrimp\u0026rsquo;s feeding in the first experiment. Figures\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e(e)-(h) are the same moment seen in the second experiment. In the first experiment, the rotation speed was set to 55 rpm on the \u003cem\u003eX\u003c/em\u003e-axis and 65 rpm on the \u003cem\u003eY\u003c/em\u003e-axis, respectively. The moment during the rotation when the shrimp tried to feed was captured twice. The shrimp picked up pellets and ate them when they happened to be brought in front of the shrimp. The shrimp continued to eat a food pellet while holding it from 3 min 51 s to 9 min 27 s, and from 9 min 51 s to 10 min 52. In the second experiment, the speed was increased to 110 rpm on the \u003cem\u003eX\u003c/em\u003e-axis and 130 rpm on the \u003cem\u003eY\u003c/em\u003e-axis, respectively. The shrimp\u0026rsquo;s feeding behavior was captured twice this time, too. The shrimp ate a pellet from 4 min 46 s to 6 min 16 s, and another one from 6 min 36 s to 12 min 59. The green triangular arrows in the figures indicate instructive moments when the shrimp picks up a food pellet and brings it to its mouth. Thus, the shrimp\u0026rsquo;s behavior and feeding process were perfectly recorded while being filmed by the camera. Two short movies of that process are available on the website.\u003c/p\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.youtube.com/watch?v=CRqHW7jbMO4\u003c/span\u003e\u003cspan address=\"https://www.youtube.com/watch?v=CRqHW7jbMO4\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.youtube.com/watch?v=2ma-vsk5rmk\u003c/span\u003e\u003cspan address=\"https://www.youtube.com/watch?v=2ma-vsk5rmk\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ch3\u003eGene Expression Analysis\u003c/h3\u003e\n\u003cp\u003eGene expression analysis was conducted on the shrimp exposed to long-time weightlessness. To begin with, one juvenile shrimp (\u003cem\u003eMarsupenaeus japonicus\u003c/em\u003e, 158 dah, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) was captured in each of three small sample containers. The three containers were then placed in the rectangular sample box, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(e). Next, the box was set in the Clinostat PMS-VI-R02, and was continuously rotated for 24 hours. The room light was turned off during the rotation in both experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e(f)). After that, the shrimp were removed from the container. Since one of three shrimp had died, we used the two living shrimp to conduct the gene expression analysis. A control experiment was performed simultaneously using the same species of shrimp of a similar size. For the purpose of oxygen supply to the control shrimp, a magnetic stirrer was packed into the container and the water was stirred for 24 hours.\u003c/p\u003e\u003cp\u003eRNA extraction was performed as described below. To begin with, the shrimp were homogenized at room temperature (about 20\u0026deg;C) using a 20 mL glass teflon homogenizer, to which was added 20 mL of RNA extraction reagent RNAzol\u0026reg;RT. The homogenized shrimp were transferred to a centrifuge tube (Labcon, SuperClear 50 mL) and centrifuged for 15 minutes. 0.55 mL of the supernatant was dispensed into a 2.0 mL PP microtube using a micropipette. 240 \u0026micro;L of purified water was added to this and centrifuged at 12,000 G for 15 minutes. 1.0 mL of the supernatant was transferred to another microtube. 3.15 \u0026micro;L of 4-bromoanisole (TCI, B0547) was added to the 1 mL of the transferred supernatant, left to stand for 3 minutes, and centrifuged at 12,000 G for 10 minutes. 570 \u0026micro;L of the supernatant and 570 \u0026micro;L of isopropanol were mixed in a 2 mL PP microtube, left to stand for 10 minutes, and centrifuged at 12,000 G for 10 minutes. The RNA pellet was mixed twice with 100 \u0026micro;L of 75% ethanol in a 2 mL PP microtube, and centrifuged at 6,000 G for 3 minutes. The RNA pellet was dissolved in RNase-free water and vortexed at room temperature for 5 minutes. A quality check was performed using the extracted sample of RNA. Finally, gene expression analysis of this RNA was outsourced to the research laboratory of a private company (Novogene Corp. Inc.).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eShrimp Behavior in the Weightless Condition\u003c/h2\u003e\u003cp\u003eWe noticed from \u003cem\u003ein situ\u003c/em\u003e observation that high-speed rotation caused turbulent flows in the container. The feed pellets were vigorously stirred in such a situation, and the shrimp seldom chased them. The shrimp were observed to be clinging to the net. When a pellet happened to be brought in front of the shrimp did they pick up and eat it, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Similar feeding behavior was observed many times in the video. The shrimp's behavior was clearly affected by the flow but their appetite was voracious. The finding of the capability of shrimp to eat some feed even in a weightless condition is one of the most distinctive features in this experiment. This finding suggests that if shrimp are to be farmed in space, they may be transported live, rather than as eggs, into space.\u003c/p\u003e\u003cp\u003eWe repeated the same experiment many times, and confirmed the similar feeding process. In addition, microscopic observation showed evidence that the feed had accumulated in the intestine as digested matter. We also confirmed in every case that most of the shrimp began to eat the pellets as soon as the flows were stopped. Hence, we think that environmental factors such as strong flows would play a large role in their feeding behavior, especially in a weightless condition. We can deduce that, when we cultivate shrimp in a weightless environment like outer space, we should stop flows or make it weak inside the container so as to allow the shrimp to catch their feed more easily. These findings will be extremely beneficial for the advancement of space aquaculture.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGene Ontology Analysis\u003c/h3\u003e\n\u003cp\u003eThe results of gene ontology (GO) analysis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e. These quantitative analyses were carried out by a research company (Novogene Corp. Inc.). Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e is the gene expression map visualized by levels. The contour colors of this map reflect the magnitude of the expression level, and the arrangement of the pattern corresponds to the results of the cluster analysis of the genes. The left column in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a) shows the group of the shrimp exposed to weightlessness, and the right column in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b) shows the control group. The red contours in the map indicate high gene expression, and the green contours indicate low gene expression. As shown, the red contours were limited to some small areas only, and most areas were covered in green or black. Judging from the difference in the contour patterns between the exposed group and the control, we found that the expression levels were different from each other.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the results of GO enrichment analysis. There are three categories classified by molecular function (MF), cellular component (CC), and biological process (BP). The column charts of BP, CC, and MF are colored with red, green, and blue, respectively. As regards the BP categories, \u0026ldquo;chitin metabolic process\u0026rdquo;, \u0026ldquo;amino sugar metabolic process\u0026rdquo;, \u0026ldquo;glucosamine-containing compound metabolic process\u0026rdquo;, \u0026ldquo;aminoglycan metabolic process\u0026rdquo;, and \u0026ldquo;drug metabolic process\u0026rdquo;, specifically, scored high. As regards the CC categories, only \u0026ldquo;extracellular region\u0026rdquo; was notable, and \u0026ldquo;actin cytoskeleton\u0026rdquo; was almost zero. As regards the MF categories, the increase in the \u0026ldquo;structural constituent of cuticle\u0026rdquo; was the most remarkable, and \u0026ldquo;structural molecule activity\u0026rdquo; and \u0026ldquo;chitin binding\u0026rdquo; were also clearly distinguished. It is noteworthy that the BP categories were associated with the chitin metabolic process and the MF categories were related to the structural constituent of cuticle. These categories are suggestive of the function of the shrimp\u0026rsquo;s movement. That is, shrimp exposed to a weightless environment might be some tangible, especially in the categories of the organ of locomotion. Similar findings were also confirmed in a study of the function of osteoclasts in \u003cem\u003eMedaka\u003c/em\u003e fish conducted on the International Space Station (Asaka et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In summary, we can say that the GO analyses offered valuable suggestions with respect to the influence of exposure to the weightless condition on the shrimp. The details, however, are still unknown, and further study is in progress.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe observed feeding process of shrimp under the weightless condition. To realize that condition, we developed a new type of clinostat with high-speed rotation, with a maximum rotation of 130 rpm. We could successfully record the shrimp\u0026rsquo;s feeding process. We have proven the capability of the shrimp to eat some feed even in a weightless condition. This finding suggests that when cultivating shrimp in space. This may be possible to transport them live, rather than as eggs. That could be useful information for the advancement of space aquaculture. In addition, we also performed gene ontology analysis of the shrimp exposed to the microgravity environment for 24 h. Those results showed the possibility that the exposure to weightlessness might be affectable as compared with the control shrimp, especially in the functions and genes related to the shrimp exoskeleton system. We suspect that environmental factors such as the flows by high-speed rotation played a large role in those effects. The details, however, are still unknown yet. Further study should be carried out in the ISS.\u003c/p\u003e\u003cp\u003eIn this research, we represented a high-speed rotating clinostat has a great impact on the progress of various kinds of experiments that were not possible with a conventional low-speed clinostat. We believe that a high-speed clinostat will greatly contribute to the progress of biological research and its application over the future microgravity science.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe received gracious cooperation from the members of the Design and Manufacturing Center of Okayama University of Science for the development of the experimental equipment. A new type of random positioning machine,\u0026ldquo;Small 3D Clinostat PMS-V1-R02\u0026rdquo;, was designed and developed with the support of AES Co., Ltd, which is the most famous manufacturing company of a clinostat in Japan. We are very grateful to this company for all the mechanical support and advice. We were given great assistance by Mr. Kazuo Odakami, who is senior staff of the Head Audit Office, Kake Educational Institution, Okayama University of Science. This paper was proofread in English by Prof. Christopher Carman of the University of Occupational and Environmental Health. All the mentioned support was essentially important to the progress of our research project. We would like to express our deepest gratitude.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvery author declares no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChihiro Yokota:\u003c/strong\u003e Experiment, Data analysis, \u003cstrong\u003eSeiichi Tsumura:\u003c/strong\u003e Experiment, Data analysis, \u003cstrong\u003eKanji Kameyama:\u003c/strong\u003e Technical support, Development of the system, \u003cstrong\u003eSyou Maki\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing, Investigation, Design of the system, Conceptualization, \u003cstrong\u003eRyosuke Tadokoro:\u003c/strong\u003e Experiment, Investigation, Gene ontology analysis, Review, \u003cstrong\u003eToshimasa Yamamoto:\u003c/strong\u003e Experiment, Conceptualization, Review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data in this study are available upon reasonable request. Please contact the research institute where the corresponding author (Syou Maki) is employed. Two types of short movies will be available on the website.\u003c/p\u003e\n\u003cp\u003ehttps://www.youtube.com/watch?v=CRqHW7jbMO4\u003c/p\u003e\n\u003cp\u003ehttps://www.youtube.com/watch?v=2ma-vsk5rmk\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Project Research Promotion Program 2024 (OUS-RP-24-1) of Okayama University of Science.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/strong\u003e: not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcharya, A., Brungs, S., Lichterfeld, Y., Hescheler, J., Hemmersbach, R., Boeuf, H., Sachinidis, A.: Parabolic flight-induced acute hypergravity and microgravity effects on the beating rate of human cardiomyocytes. 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DOI: 10.1089/scd.2006.15.921\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Clinostat, Centrifugal force, Third water, Koteki, Weightlessness, Microgravity","lastPublishedDoi":"10.21203/rs.3.rs-7308179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7308179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe carried out \u003cem\u003ein situ\u003c/em\u003e observation of the feeding process of edible shrimp under microgravity environment. To realize that condition, we developed a new type of clinostat with high-speed rotation, with a maximum rotation of 130 rpm. This rotation speed is much faster than that of conventional models. Under that rotation speed, aquatic organisms such as fish and crustaceans have a suppressed capability of recovering their proper swimming form, making it possible for them to be exposed to a pseudo-weightless condition. By this method, we could successfully observe the feeding process of shrimp in that condition. We also performed a genetic analysis of shrimp exposed to microgravity for 24 h. The results of gene ontology analysis suggested that the effects of exposure to weightlessness were significant, as compared with control shrimp. We believe that the high-speed clinostat will contribute greatly to the future progress of engineering applications in microgravity research.\u003c/p\u003e","manuscriptTitle":"In situ observation of shrimp feeding process under microgravity environment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-05 01:56:36","doi":"10.21203/rs.3.rs-7308179/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1022ffa3-efc6-4a58-b5ef-729fa5ab546d","owner":[],"postedDate":"September 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T07:54:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-05 01:56:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7308179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7308179","identity":"rs-7308179","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}