The influence of environmental factors on the feeding behavior of reared Japanese eel Anguilla japonica leptocephali

The influence of environmental factors on the feeding behavior of reared Japanese eel Anguilla japonica leptocephali | 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 The influence of environmental factors on the feeding behavior of reared Japanese eel Anguilla japonica leptocephali Yuuya Shioura, Akira Kenzaki, Suguru Okunishi, Tsutomu Tomoda, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6222909/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 Artificially reared Japanese eel Anguilla japonica leptocephali (–7 mm total length, 6–7 days post-hatch) were fed picocyanobacteria ( Synechococcus sp., strain NIES-976) and their food intake was observed using autofluorescence intensity per area of the mid-hindgut used as an index of gut fullness. Time-course observations revealed that the larvae actively fed under both light and dark conditions. Food intake was significantly higher in the low-salinity group (50% seawater) than in the control group (100% seawater). Food intake did not differ significantly under photoperiods of 24-h light versus 24-h dark, indicating a light-independent diurnal feeding rhythm. A comparison of larval feeding efficiency under high and low cell densities of picocyanobacteria showed remarkably high intake of the food material by larvae in the high-density food concentration group, indicating density-dependent food ingestion. This specific feeding ecology whereby Japanese eel larvae are able to efficiently ingest suspended pico-sized food particles from seawater even in complete darkness may enable them to adapt to the oligotrophic environment. Aquaculture and Mariculture Anguilla japonica Density-dependent Feeding behavior Light-independent Photoperiod Salinity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The Japanese eel Anguilla japonica Temminck & Schlegel, 1846 is commonly consumed in Japan. The ecology of the Japanese eel remains unknown and its aquaculture still relies on natural catches for seedlings. Thus, production using artificially hatched eel larvae has not yet been achieved on a commercial scale. The number of glass eels migrating along the coast of Japan has fluctuated significantly in recent decades, but is on the decline [Fisheries Agency website, https://www.jfa.maff.go.jp/j/saibai/attach/pdf/unagi-228.pdf (in Japanese) accessed 8 March 2025]. To stabilize prices and conserve the resource, it is essential to establish sustainable, complete aquaculture techniques on a viable scale. In 2010, the National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency (FRA), succeeded in artificial completion of the lifecycle of cultured Japanese eels (Masuda et al. 2012; Tanaka 2015). The culture technology included a slurry-type diet made from shark egg powder for rearing leptocephali to the glass eel stage (Tanaka et al. 2001), but the production costs still remain high up to the glass eel stage, owing to poor growth, morphological abnormalities, and low survival rates caused by diseases, and hence mass production has not yet been realized. These constraints continue to thwart the commercialization of eel seedling production. As a substitute for conventional slurry-type diets (Tanaka et al. 2001), floating-type diets are a promising alternative, yet no floating-type diet has been developed to support the initial growth and survival of Japanese eel larvae. The development of successful floating-type food material that can be stably cultivated and produced with low environmental impacts (Wullur et al. 2013; Tomoda et al. 2015) could lower costs and allow for mass production. However, the feeding ecology of leptocephali remains largely unknown and their feeding behavior in the wild has never been observed; nonetheless, research to date has proposed several theories about their diet before they become glass eels (e.g., Otake et al. 1993; Mochioka and Iwamizu 1996; Riemann et al. 2010; Terahara et al. 2011; Miller 2009; Miller et al. 2011, 2013, 2020; Miyazaki et al. 2011; Feunteun et al. 2015; Chow et al. 2017, 2019a; Tomoda et al. 2018; Tsukamoto and Miller 2021; Watanabe et al. 2021). In developing floating-type diets and new rearing methods, information can be gleaned from the results of previous studies (Masuda et al. 2013; Furuita et al. 2014, 2024; Yamada et al. 2019) as well as basic knowledge of the physiological, ecological and functional morphological characteristics of eel larvae (Uematsu et al. 1994; Otake 1996; Tomoda and Uematsu 1996; Ohta 2008; Okamura et al. 2009; Tsukamoto et al. 2009; Yamada et al. 2019; Yoshimatsu 2011; Politis et al. 2014, 2017, 2018; Kuroki et al. 2016; Miller and Tsukamoto 2017, 2020; Watanabe 2017; Chow et al. 2019b; Matsuda et al. 2019; Kuroki 2020; Masuda et al. 2020; Knutsen et al. 2021; Miller 2023). In a previous study (Kenzaki et al. 2022) using picocyanobacteria Synechococcus spp. as a model suspended feed, thought to be one of the food source (Lundgreen et al. 2019), we confirmed feeding selectivity and density dependence due to the food environment. Understanding the environmental conditions for efficient intake of floating-type diets will contribute to better growth and survival of cultured larvae. Low-salinity rearing was shown to improve the growth, survival rate, energy metabolism and osmoregulatory capacity of larval Japanese eels (Okamura et al. 2009) and European eels Anguilla anguilla (Politis et al. 2018). Therefore, it is expected that low-salinity rearing using floating-type diets will also improve feeding efficiency, and thereby growth and survival. In this study, we investigated the feeding efficiency of Japanese eel leptocephali in relation to the environmental factors of salinity, photoperiod, and food density of the picocyanobacteria Synechococcus sp. (Kenzaki et al. 2022) as a model food material for a floating-type diet. Materials and methods Cultivation of picocyanobacteria As the food material, Synechococcus sp. strain NIES-976 was obtained from the National Institute for Environmental Studies (Japan) cryopreserved stock; this pico-sized cyanobacterium is considered a model floating food for eel larvae and was selected from among numerous picoplankton species present in the natural habitat of Japanese eels (Tomoda et al. 2018; Watanabe et al. 2021). The culture method was as reported previously in Kenzaki et al. (2022). Exponentially growing cultures (~ 2.3 × 10 8 cells ml − 1 ) of Synechococcus sp. were concentrated by centrifugation (KUBOTA centrifuge, Model 8420) at 2 280 g for 40 min. The collected cells were frozen and stored at − 78°C until the start of the feeding experiments. Experimental fish We obtained eggs of eel broodstock by induced spawning of Japanese eels raised and maintained year-round at the Shibushi Laboratory of the Fisheries Technology Institute, National Research and Development Agency, FRA, as described previously (Satoh et al. 1992; Kagawa et al. 1997, 2013; Tachiki et al. 1997; Dou et al. 2007; Unuma et al. 2011, 2012; Ohta et al. 1996, 2017; Furuita et al. 2024). Leptocephalus larvae obtained from the spawned eggs were reared without food until first feeding at 6–7 days post hatch (dph) in the feeding behavior experiments. Rearing method For the eel larviculture, we used a closed recirculation system consisting of 2.5-l plankton kreisel tanks (Yamada et al. 2019) equipped with a bimorph pump (BPH-214G, Nitto Kohki) (Kenzaki et al. 2022). This system was created to constantly float the larvae in the rearing water and to feed in Synechococcus cells into the middle layer. The larvae were reared in ultraviolet-irradiated seawater (24°C, 34 psu) supplied by a flow-type UV steriliser (Flonlizer FDL-4-SP, Chiyoda Kohan) after microfiltration through 0.5-µm cartridge filters (TCW-0.5N-PPD, Advantec Toyo Kaisha). At 6 or 7 dph, 30 or 70 eel larvae were stocked into the rearing system, with a circulating flow rate of 0.15–0.20 l min − 1 , in a thermostatic chamber maintained at 24°C. Thereafter, Synechococcus sp. cells collected by centrifugation were added to and resuspended in the rearing water. Feeding observations under different environmental conditions To confirm the degree of food intake of suspended picocyanobacteria by the leptocephali under different environmental conditions, larvae 6–7 dph, sized 6.69–7.12 mm total length (TL) and hatched from different broodstock, were collected from rearing tanks and examined. For observations of larval feeding behavior, only a Synechococcus cell resuspension was given. Experiment 1: Comparisons under different salinities and photoperiods To compare food-intake efficiency under different salinity and photoperiod conditions, four rearing groups were established by combinations of two salinity conditions (50% SW, 16 psu; 100% SW, 32 psu) and two photoperiods (continuous light, 24L; continuous darkness, 24D). At 6 dph, 30 larvae were stocked into the rearing system. The cell density of Synechococcus sp. in the rearing water for all groups of larvae was adjusted to 2.538 × 10 7 cells ml − 1 . The photon flux density in the light condition (24L) was 0.118–0.125 µmol m − 2 s − 1 ; the dark condition (24D) was complete darkness (0.000 µmol m − 2 s − 1 ). Next, to confirm the time-course of food-intake into the digestive tracts of the eel larvae at 6–7 dph, six larvae in each tank were observed three times during this experiment: at 3 h (13:00), 6 h (16:00) and 24 h (10:00 the next morning) after feeding. Experiment 2: Comparisons under different salinities and food cell densities To compare food-intake efficiency in relation to salinity and picoplankton cell density of the food environment, three larval rearing groups were established using combinations of two salinities (50% SW; 100% SW) and two food cell densities (high density, HD; low density, LD). At 7 dph, 70 larvae were stocked into the rearing system. The density of Synechococcus sp. in the rearing water was adjusted to 8.075 × 10 7 cells ml − 1 in the HD group, and to 1.225 ×10 7 cells ml − 1 in the LD group. The photoperiod for all groups was 24L, and the photon flux density was the same as in Experiment 1. To confirm the time-course of food intake into the digestive tracts of larvae at 7 dph, six larvae in each tank were observed every hour between 10:00 and 17:00 (i.e. a total of eight times). Experiment 3: Confirmation of the comparisons under different salinity conditions To confirm the reproducibility of Experiment 2 and the food-intake efficiency of larvae in a higher food-cell-density environment, two rearing groups were established under different salinity conditions (50% SW; 100% SW). The cell density of Synechococcus sp. in the rearing water was adjusted to 1.233 ×10 8 cells ml − 1 . The larval stocking density and photoperiod was the same as in Experiment 2 (24L), and thereafter the feeding observations were similarly performed. Estimation of food-intake efficiency Immediately before observation, eel larvae were collected from each tank and anesthetized. The digestive tracts of six larvae were observed using an epi-fluorescence biological microscope (20x, ECLIPSE Ni, C-HGFI, Nikon) and photographed using a digital microscope camera (DS-FI3, Nikon). In addition to bright-field observation, photographs were taken under dark-field conditions with a uniform exposure time and gain through a green excitation filter. As in Kenzaki et al. (2022), the degree of intestinal fullness was evaluated based on the area of mid-hindgut with autofluorescence; however, the influence of intercellular voids and focal depth on the measured values remained as issues to consider. To resolve this, we considered using brightness (red component) as an index value as a more-accurate evaluation method. First, the area of mid-hindgut was measured from the bright-field image, and then the brightness caused by the autofluorescence of Synechococcus cells in the mid-hindgut was measured from the dark-field image (Fig. 1 ). Here, the fluorescence intensity per area of mid-hindgut ( FIG ) was defined as the obtained brightness divided by the mid-hindgut area (mm 2 ). The FIG was calculated using the following equation: FIG = B / A where B is the brightness within mid-hindgut and A is the mid-hindgut area. Image analysis software (ImageJ 1.52t; National Institutes of Health, USA) was used to measure the area and brightness of the mid-hindgut. To gauge the feeding state of individual larvae, the value of the FIG index was used to define five classes of intestinal fullness: class A = more than half-filled (200 ≤ FIG ); class B = 150 ≤ FIG < 200; class C = quarter-filled (100 ≤ FIG < 150); class D = 50 ≤ FIG < 100; class E = a small amount (0 < FIG < 50). Statistical analysis The fluorescence intensity per area of mid-hindgut ( FIG ) was obtained for each rearing group and elapsed time, and differences in mean values of the index were examined. The Steel–Dwass multiple comparison was carried out on differences in the amount of ingested food in the mid-hindgut at different time points within each rearing group. Concerning differences in food intake between rearing groups, the assumptions of normal distribution and homogeneity of variances were checked before the statistical analysis; a Student’s t -test was used if normality and homoscedasticity were observed in both groups compared, otherwise a Welch’s t -test was used. All statistical analyses were performed using Excel (Microsoft Office 365) with the add-in software Statcel 4 (OMS Publishing Inc., Japan). Results Comparison of the time-course of food intake In all experiments, the values of FIG were higher in groups reared in 50% SW than in 100% SW (Fig. 2 a–c). In addition, no significant differences were observed in food intake into the digestive tract over time in each rearing group in all experiments ( P ≥ 0.05, Steel–Dwass test). Therefore, all the time-course observation data for each rearing group were compiled and the feeding behavior (food intake) was compared between groups (see next section). Food-intake efficiency in relation to the environmental conditions tested Table 1 summarizes the food-intake efficiency of the larvae under each salinity and photoperiod condition (Experiment 1), and Fig. 3 depicts the frequency of FIG classes of intestinal fullness in the differently reared groups. Larvae with a high feeding status (class A) were observed only in the 50% SW–24L group (Fig. 3 b). Food-intake efficiency was significantly superior in larvae reared in 50% SW compared with in 100% SW under the condition of continuous light (24L) ( P = 0.0248) as well as under the condition of continuous darkness (24D) ( P = 0.0101, Table 1). However, under 24L versus under 24D and the same salinity condition, no significant difference in food-intake efficiency was observed ( P = 0.1744 and P = 0.5255, respectively; Table 1). Furthermore, food-intake efficiency was significantly higher in the 50% SW–24L group than in the 100% SW–24D group ( P = 0.0028, Table 1), and larval feeding ability appeared to be improved by low-salinity rearing (50% SW–24D) even under the dark condition ( P = 0.1792, Table 1). Table 2 summarizes the food-intake efficiency under each salinity and food cell-density condition (Experiment 2), and Fig. 4 depicts the frequency of FIG classes of intestinal fullness in the differently reared groups. The 50% SW–HD group had the highest number of larvae presenting with a class A feeding status (Fig. 4 b). With high-density feeding, the food-intake efficiency of the larvae was higher in the 50% SW group than in the 100% SW group ( P = 0.0002, Table 2). In addition, when reared in 100% SW, food-intake efficiency was significantly higher in the HD group than in the LD group ( P = 3.039 × 10 − 12 , Table 2). Furthermore, the food-intake efficiency of the 50% SW–HD group (positive control) was significantly higher ( P = 2.181 × 10 − 14 ) than in the 100% SW–LD group (negative control) (Table 2). Table 3 presents the food-intake efficiency of larvae that were kept under the two different salinity conditions but fed the same high food-cell density (Experiment 3), and Fig. 5 shows the frequency of the FIG classes of intestinal fullness in the different salinity groups. Although many of the test larvae had morphological abnormalities, such as pericardial cavity hypertrophy, lower-jaw dysostosis and intestinal hypoplasia, the group reared in 50% SW had a higher number of larvae showing class A feeding status (Fig. 5 b), thus their food-intake efficiency was significantly higher ( P = 0.0030, Table 3). Discussion The results of this study provide insights into the feeding behavior of captively reared Japanese eel larvae in response to environmental factors, namely different salinities, photoperiods and food cell densities. The results provide important information useful for developing initial food materials, which has been a bottleneck in the mass production of Japanese eel seedlings. Since there were no significant differences in the feeding status of the larvae over time in each rearing group in all experiments, it is considered that eel larvae are constantly feeding by taking in seawater (Lee et al. 2013; Ahn et al. 2015) (Fig. 2 a–c). In Experiment 1, low-salinity rearing was found to have the effect of improving food-intake efficiency, and feeding activity was shown to be constant regardless of continuously light or dark conditions (Table 1, Fig. 3 ). Experiments 2 and 3 also showed the superiority of low-salinity rearing and food-cell density dependence (Tables 2 and 3, Figs. 4 and 5 ), similar to the report of a previous study (Kenzaki et al. 2022). Okamura et al. (2009) found that Japanese eel larvae given a slurry-type diet showed better growth and survival when reared in 50% SW than in 100% SW, similar to the superiority of larvae in the low-salinity groups in this study. This is presumably because rearing in diluted seawater reduces the energy consumed for osmoregulation (Kuroki et al. 2016), thereby contributing to improved feeding activity as compared with in full-strength seawater. Anni et al. (2016) reported that low-salinity rearing (3–6 ppt) affected the osmoregulation and energy metabolism of juvenile Plata pompano Trachinotus marginatus , thereby improving fish growth when compared with rearing in 100% SW (32 ppt). Bradshaw et al. (2023) surmised that low-salinity rearing of Florida pompano T. carolinus provides an advantage in terms of the ability to treat halophilic pathogens. From these findings, the advantages of low-salinity rearing can be appreciated. Eel larvae exhibit negative phototaxis and undergo diurnal vertical migration (Yamada et al. 2019; Watanabe 2017). It is speculated that during vertical migration wild leptocephali actively and passively feed when passing through the subsurface chlorophyll maximum (SCM) depth zone, where particulate organic matter (POM) is relatively abundant (Otake et al. 1998; Watanabe et al., unpublished data); interestingly, this would match the continuous feeding behavior verified under dark conditions in this study. It has also been suggested that eel larvae identify food by olfactory function (Uematsu et al. 1994; Ohta 2008) rather than visual function (Tomoda and Uematsu 1996), which supports the results of this study. Kenzaki et al. (2022) found that food-intake efficiency was higher under twilight conditions (9L:15D) than under complete darkness (24D). Although the present study confirmed a similar trend, food intake by the larvae did not differ significantly under light and dark conditions (Table 1). The different results may be attributable to differences in feeding activity depending on the batch of experimental fish and age of the larvae in days at the start of experiments. Although the observations in the present study were made over a short period of just 8–24 h, feeding was confirmed regardless of the light or dark conditions, as shown in previous research (Kenzaki et al. 2022); hence, it is possible that the feeding mode of leptocephali is similar to that of filter feeders by drinking water (Lee et al. 2013; Ahn et al. 2015) (Fig. 2 a–c). A great advantage of this mode is that it allows eel larvae to feed under dark conditions without relying on vision, while also avoiding predation (Miller et al. 2015). In addition, the ability to feed continuously even in darkness while suppressing energy consumption at the equivalent level to the basal metabolism is also efficient for survival in the oligotrophic environment. Even so, in this study, as in the study of Kenzaki et al. (2022), it was observed that ingested cells of Synechococcus sp. circulated in the mid-hindgut for several hours before they accumulated and were excreted from the anus as feces (Fig. 1 ). It is speculated that POM in seawater is efficiently captured in the mid-hindgut by the movement of microvilli and the mucus produced in the intestinal epithelium (Yamada and Yokote 1975; Otake 1996). These observations suggest that water and the proteins and amino acids contained in POM are digested and absorbed in the rectum (Otake et al. 1993;Otake 1996; Lee et al. 2013; Hsu et al. 2015). Picoplankton and amorphous aggregates, as part of the marine snow originating from zooplankton and phytoplankton decomposed by bacteria (Miller et al. 2013; Knutsen et al. 2021; Watanabe et al. 2021), have been observed in the gut contents of anguilliform leptocephali (Tomoda et al. 2018). Kenzaki et al. (2022) speculated that feeding selectivity for Synechococcus sp. (strain NIES-976) is induced by its amount of extracellular protein, compatible with the high protein digestibility and amino acid utilization ability of eel larvae (Hsu et al. 2015). Thus, it is speculated that feeding modes like filter feeding or ciliary mucus feeding (Riisgård et al. 2000; Riisgård and Larsen 2010), whereby the animal can efficiently ingest suspended particles from seawater under various environmental conditions, such as low salinity and darkness, may enable adaptation to the oligotrophic environment. Therefore, rearing methods that accommodate this feeding mode could lead to three-dimensional utilization of the rearing tank and ultimately increase the scale of seedling production. Future studies should examine the environmental conditions, species selection, and rearing methods best for optimizing use of various food organisms (eukaryotic picophytoplankton, cyanobacteria and bacteria) that are considered as part of marine snow. Conclusions This manuscript describes important findings contributing to mass larviculture of Japanese eel. The feeding behavior of eel leptocephali, which has not been observed in nature, was observed under artificial rearing, and the influence of environmental factors was clarified. In this study, we proposed a recognition method based on fluorescence brightness to enable more accurate recognition of eel feeding behavior to suspended particles and confirmed its effectiveness. We investigated the feeding efficiency of Japanese eel leptocephali in relation to the environmental factors of salinity, photoperiod, and food density. The superiority of low-salinity environmental water and high-density food concentration, and light-independence feeding rhythm were confirmed. The specific feeding ecology whereby eel larvae are able to efficiently ingest suspended particles from seawater even in complete darkness may enable them to adapt to the oligotrophic environment. The rearing method corresponding to the feeding mode clarified in this study may lead to the three-dimensional utilization of the rearing tank and eventually to the expansion of the seedling production scale. Our findings add new knowledge to the research field on feeding ecology of eel leptocephali, and contribute to the development of aquaculture research. Declarations Acknowledgments We thank Keisuke Yamano (Director of the Glass Eel Production Division at the Fisheries Technology Institute, National Research and Development Agency, Japan Fisheries Research and Education Agency [FRA]) for his support in conducting this research. We are sincerely grateful to the staff of the Shibushi Field Station of FRA for their help providing leptocephali. The anonymous reviewers and the journal’s editor-in-chief provided many helpful suggestions. Cynthia Kulongowski with Edanz (https://jp.edanz.com/ac) edited the language of a draft of this manuscript. This research was conducted as a part of the project ‘Development of biological materials feeds’ commissioned by FRA as part of the ‘Demonstration project of a mass-production system for commercialization of eel seedlings’. Author contributions T.T., S.O. and H.M. conceived the study; all authors contributed to the design of the conceptual framework and analyses; Y.S. and T.T. cultured the picocyanobacteria and reared the leptocephali; Y.S., A.K., T.T. and S.O. conducted the laboratory experiments and completed the statistical analyses; The first draft of the manuscript was written by Y.S. and all authors were involved with drafting and editing of the manuscript. Funding This study was supported in part by grants from the Fisheries Agency, Ministry of Agriculture, Forestry and Fisheries of Japan. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethical approval All experimental fish were handled and treated in accordance with the Guidelines for Animal Experimentation at the Fisheries Technology Institute, Japan Fisheries Research and Education Agency (FRA). 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(in Japanese with English abstract). https://doi.org/10.11233/aquaculturesci.68.155 Matsuda K, Kamoshida M, Masuda Y (2019) Wavelength-specific thresholds of artificially reared Japanese eel Anguilla japonica larvae determined from negative-phototactic behaviours. J Fish Biol 95(4):1040–1045. https://doi.org/10.1111/jfb.14097 Miller MJ (2009) Ecology of Anguilliform Leptocephali: Remarkable Transparent Fish Larvae of the Ocean Surface Layer. Aqua-BioScience Monographs, 2, 1-94. Miller MJ, Otake T, Aoyama J, Wouthuyzen S, Suharti S, Sugeha HY, Tsukamoto K (2011) Observations of gut contents of leptocephali in the North Equatorial Current and Tomini Bay, Indonesia. Coastal marine science 35(1):277–288. Miller MJ, Chikaraishi Y, Ogawa NO, Yamada Y, Tsukamoto K, Ohkouchi N (2013) A low trophic position of Japanese eel larvae indicates feeding on marine snow. 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Moser’s Seminal “Morphological and Functional Aspects of Marine Fish Larvae”: The Commonalities of Leptocephali and Larvae of Other Marine Teleosts. Fishes 8(11):548. https://doi.org/10.3390/fishes8110548 Miyazaki S, Kim HY, Zenimoto K, Kitagawa T, Miller MJ, Kimura S (2011) Stable isotope analysis of two species of anguilliform leptocephali ( Anguilla japonica and Ariosoma major ) relative to their feeding depth in the North Equatorial Current region. Mar Biol 158:2555−2564. https://doi.org/10.1007/s00227-011-1756-x Mochioka N, Iwamizu M (1996) Diet of anguillid larvae: leptocephali feed selectively on larvacean houses and fecal pellets. Mar Biol 125;447–452. https://doi.org/10.1007/BF00353257 Ohta H, Kagawa H, Tanaka H, Okuzawa K, Hirose K (1996) Milt production in the Japanese eel Anguilla japonica induced by repeated injections of human chorionic gonadotropin. Fish Sci 62(1):44–49. https://doi.org/10.2331/fishsci.62.44 Ohta H, Sato Y, Imaizumi H, Kazeto Y (2017) Changes in milt volume and sperm quality with time after an injection of recombinant Japanese eel luteinizing hormone in male Japanese eels. Aquaculture 479:150–154. https://doi.org/10.1016/j.aquaculture.2017.05.044 Ohta J (2008) Physiological studies on the olfactory function of fish (master’s thesis). Available from TUMSAT-OACIS Library. (in Japanese). https://oacis.repo.nii.ac.jp/records/771. Accessed 8 March 2025. Okamura A, Yamada Y, Mikawa N, Horie N, Utoh T, Kaneko T, Tanaka S, Tsukamoto K (2009) Growth and survival of eel leptocephali ( Anguilla japonica ) in low-salinity water. Aquaculture 296(3–4):367–372. https://doi.org/10.1016/j.aquaculture.2009.08.039 Otake T, Nogami K, Maruyama K (1993) Dissolved and particulate organic matter as possible food sources for eel leptocephali. Mar Ecol Prog Ser 92:27–34. https://doi.org/10.3354/meps092027 Otake T (1996) Fine structure and function of the alimentary canal in leptocephali of the Japanese eel Anguilla japonica . Fish Sci 62(1):28–34. https://doi.org/10.2331/fishsci.62.28 Otake T, Inagaki T, Hasumoto H, Mochioka N Tsukamoto K (1998) Diel vertical distribution of Anguilla japonica leptocephali. Ichthyol Res 45:208–211. https://doi.org/10.1007/BF02678565 Politis SN, Butts IAE, Tomkiewicz J (2014) Light impacts embryonic and early larval development of the European eel, An guilla Anguilla . J Exp Mar Biol Ecol 461:407–415. https://doi.org/10.1016/j.jembe.2014.09.014 Politis SN, Mazurais D, Servili A, Infante JLZ, Miest JJ, Sørensen SR, Tomkiewicz J, Butts IAE (2017) Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae. PLoS ONE 12(8):e0182726. https://doi.org/10.1371/journal.pone.0182726 Politis SN, Mazurais D, Servili A, Infante JLZ, Miest JJ, Tomkiewicz J, Butts IAE (2018) Salinity reduction benefits European eel larvae: Insights at the morphological and molecular level. PLoS ONE 13(6):e0198294. https://doi.org/10.1371/journal.pone.0198294 Riemann L, Alfredsson H, Hansen MM, Als TD, Nielsen TG, Munk P, Aarestrup K, Maes GE, Sparholt H, Petersen MI, Bachler M, Castonguay M (2010) Qualitative assessment of the diet of European eel larvae in the Sargasso Sea resolved by DNA barcoding. Biol Lett 6:819–822. https://doi.org/10.1098/rsbl.2010.0411 Riisgård HU, Nielsen C, Larsen PS (2000) Downstream collecting in ciliary suspension feeders: the catch-up principle. Mar Ecol Prog Ser 207:33–51. https://doi.org/10.3354/meps207033 Riisgård HU, Larsen PS (2010) Particle capture mechanisms in suspension-feeding invertebrates. Mar Ecol Prog Ser 418:255–293. https://doi.org/10.3354/meps08755 Satoh H, Yamamori K, Hibiya T (1992) Induced spawning of the Japanese eel. Nippon Suisan Gakkaishi 58(5):825–832. https://doi.org/10.2331/suisan.58.825 Tachiki H, Nakagawa T, Tamura K, Hirose K (1997) Effects of oral administration of estradiol-17ß to young on gonadal sex and growth of Japanese eel Anguilla ja ponica . Aquacult Sci 45(1):61–66. https://doi.org/10.11233/aquaculturesci1953.45.61 Tanaka H, Kagawa H, Ohta H (2001) Production of leptocephali of Japanese eel ( Anguilla japonica ) in captivity. Aquaculture 201(1–2):51–60. https://doi.org/10.1016/S0044-8486(01)00553-1 Tanaka H (2015) Progression in artificial seedling production of Japanese eel Anguilla japonica . Fish Sci 81：11–19. https://doi.org/10.1007/s12562-014-0821-z Terahara T, Chow S, Kurogi H, Lee SH, Tsukamoto K, Mochioka N, Tanaka H, Takeyama H (2011) Efficiency of peptide nucleic acid-directed PCR clamping and its application in the investigation of natural diets of the Japanese eel leptocephali. PLoS ONE 6(11):e25715. https://doi.org/10.1371/journal.pone.0025715 Tomoda H, Uematsu K (1996) Morphogenesis of the brain in larval and juvenile Japanese eels, Anguilla japonic a . Brain Behav Evol 47(1):33–41. https://doi.org/10.1159/000113227 Tomoda T, Kurogi H, Okauchi M, Kamoshida M, Imaizumi H, Jinbo T, Nomura K, Furuita H, Tanaka H (2015) Hatchery-reared Japanese eel Anguilla japonica larvae ingest various organic matters formed part of marine snow. Nippon Suisan Gakkaishi 81(4):715–721. (in Japanese with English abstract). https://doi.org/10.2331/suisan.81.715 Tomoda T, Chow S, Kurogi H, Okazaki M, Ambe D, Furuita H, Matsunari H, Nagai S, Yokouchi K, Sawayama S, Nomura K, Tanaka H, Sudou R, Hasegawa D, Inaba N (2018) Observations of gut contents of anguilliform leptocephali collected in the western North Pacific. Nippon Suisan Gakkaishi 84(1):34–44. (in Japanese with English abstract). https://doi.org/10.2331/suisan.17-00025 Tsukamoto K, Yamada Y, Okamura A, Kaneko T, Tanaka H, Miller MJ, Horie N, Mikawa N, Utoh T, Tanaka S (2009) Positive buoyancy in eel leptocephali: an adaptation for life in the ocean surface layer. Mar Biol 156:835–846. https://doi.org/10.1007/s00227-008-1123-8 Tsukamoto K, Miller MJ (2021) The mysterious feeding ecology of leptocephali: a unique strategy of consuming marine snow materials. Fish Sci 87:11–29. https://doi.org/10.1007/s12562-020-01477-3 Uematsu K, Tomoda H, Omura Y (1994) Brain and sensory organs of eel leptocephali. Kaiyo Monthly 26:282–287. (In Japanese) Unuma T, Hasegawa N, Sawaguchi S, Tanaka T, Matsubara T, Nomura K, Tanaka H (2011) Fusion of lipid droplets in Japanese eel oocytes: stage classification and its use as a biomarker for induction of final oocyte maturation and ovulation. Aquaculture 322–323:142–148. https://doi.org/10.1016/j.aquaculture.2011.10.001 Unuma T, Sawaguchi S, Hasegawa N, Tsuda N, Tanaka T, Nomura K, Tanaka H (2012) Optimum temperature of rearing water during artificial induction of ovulation in Japanese eel. Aquaculture 358–359:216–223. https://doi.org/10.1016/j.aquaculture.2012.07.004 Watanabe S (2017) Trials of improved feeding techniques for Japanese eel larvae based on its olfaction characteristics. Fiscal Year Final Research Report KAKEN No.16K14967. (in Japanese with English abstract). https://kaken.nii.ac.jp/en/file/KAKENHI-PROJECT-16K14967/16K14967seika.pdf. Accessed 8 March 2025. Watanabe T, Nagai S, Kawakami Y, Asakura T, Kikuchi J, Inaba N, Taniuchi Y, Kurogi H, Chow S, Tomoda T, Ambe D, Hasegawa D (2021) 18S rRNA gene sequences of leptocephalus gut contents, particulate organic matter, and biological oceanographic conditions in the western North Pacific. Sci Rep 11:5488. https://doi.org/10.1038/s41598-021-84532-y Wullur S, Yoshimatsu T, Tanaka H, Ohtani M, Sakakura Y, Kim HJ, Hagiwara A (2013) Ingestion by Japanese Eel Anguilla japonica Larvae on Various Minute Zooplanktons. Aquaculture Sci 61(4):341–347. https://doi.org/10.11233/aquaculturesci.61.341 Yamada Y, Okamura A, Mikawa N, Horie N, Tsukamoto K (2019) A new liquid-type diet for leptocephali in mass production of artificial glass eels. Fish Sci 85;545–551. https://doi.org/10.1007/s12562-019-01295-2 Yamada K, Yokote M (1975) Morphochemical analysis of mucosubstances in some epithelial tissues of the eel ( Anguilla japonica ) Histochemistry 43:161–172. https://doi.org/10.1007/BF00492444 Yoshimatsu T (2011) Early Developrnent of Preleptocephalus Larvae of the Japanese Eel in Captivity with Special Reference to the Organs for Larval Feeding. Bull Graduate School of Bioresources Mie Univ 37:11–18. https://citeseerx.ist.psu.edu/document?repid=rep1&type=pdf&doi=4198b718f475140999d4c75b7f536453f1bcced4. Accessed 8 March 2025. Tables Tables are available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. 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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-6222909","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":428582362,"identity":"93bc31b5-09a4-4321-81aa-326527c8695b","order_by":0,"name":"Yuuya Shioura","email":"","orcid":"","institution":"Shibushi Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency","correspondingAuthor":false,"prefix":"","firstName":"Yuuya","middleName":"","lastName":"Shioura","suffix":""},{"id":428582363,"identity":"b7b7e4df-730c-4d2c-8124-5f040a40d49a","order_by":1,"name":"Akira Kenzaki","email":"","orcid":"","institution":"Graduate School of Agriculture, Forestry and Fisheries, Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Kenzaki","suffix":""},{"id":428582364,"identity":"f3dda7ae-0c39-46aa-b84b-ed906e71de24","order_by":2,"name":"Suguru Okunishi","email":"","orcid":"","institution":"Faculty of Fisheries, Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Suguru","middleName":"","lastName":"Okunishi","suffix":""},{"id":428582365,"identity":"023c0368-ba72-484f-b771-36cdd35714d4","order_by":3,"name":"Tsutomu Tomoda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYNACNgY59gYQw0YOKsKDWzEPG0SLMc8BECPNmHgtiT2oWvAAe/nmoxs+lNml90gffsDwI8Egn7+B/eIDBpk7eGxhS7s541xybg9fmgFjT4KB5YwDPMUGDDzP8GjhMbvN28acu5+HwYCB98cfA6DyNAkGnsOEtNSn8/Cwf2D8k2BAtJbDCTw8PAbMPGAt7MfwazmWBvLLccMeHp6CwzJALRKHeZgNEvD4hb358LEbH8qq5YEO2/jwDVALf3v7wwcfe3CHGAo4ACaZeQxg0UQ0YH/AwPCDNC2jYBSMglEwrAEAyTNKp8nuiLcAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0003-4607-3141","institution":"Shibushi Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency","correspondingAuthor":true,"prefix":"","firstName":"Tsutomu","middleName":"","lastName":"Tomoda","suffix":""},{"id":428582366,"identity":"b97a091c-2629-4033-b3b6-b98b831cbd66","order_by":4,"name":"Hiroto Maeda","email":"","orcid":"","institution":"Faculty of Fisheries, Kagoshima University","correspondingAuthor":false,"prefix":"","firstName":"Hiroto","middleName":"","lastName":"Maeda","suffix":""}],"badges":[],"createdAt":"2025-03-14 00:38:26","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6222909/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6222909/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79155478,"identity":"e1a22996-7642-4d2c-99fd-864746cb868a","added_by":"auto","created_at":"2025-03-25 06:07:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":420539,"visible":true,"origin":"","legend":"\u003cp\u003ePicocyanobacteria cells (\u003cem\u003eSynechococcus\u003c/em\u003e sp., strain NIES-976) in the mid-hindgut of an artificially reared \u003cem\u003eAnguilla japonica\u003c/em\u003elarva, observed under an epi-fluorescence microscope using fluorescence observation (x20, green excitation filter). The cells were circulated and accumulated in the intestinal lumen, gradually solidified in the rectum, and excreted from the anus as feces. The area outlined in white marks the mid-hindgut of a larva reared in 50% SW, seen (\u003cstrong\u003ea\u003c/strong\u003e) 3 h after feeding (before excretion: the whole mid-hindgut is full of food), and (\u003cstrong\u003eb\u003c/strong\u003e) 6 h after feeding (some food was excreted from the posterior end of the mid-hindgut to the rectum, and some feces remained near the anus)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6222909/v1/7268687b9ea6f0840e9dd433.png"},{"id":79156428,"identity":"6aaa2480-e7ac-446f-b9a4-d1039e59e751","added_by":"auto","created_at":"2025-03-25 06:23:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205495,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of time-course food-intake efficiency of \u003cem\u003eAnguilla japonica\u003c/em\u003elarvae under different environmental conditions. The fluorescence intensity per area of mid-hindgut (\u003cem\u003eFIG\u003c/em\u003e) as an index of intestinal fullness was calculated by dividing the autofluorescence portion of picocyanobacteria cells by the area of mid-hindgut in the larva. Estimated food-intake efficiency in response to different (\u003cstrong\u003ea)\u003c/strong\u003e salinity and photoperiod conditions at 6–7 dph (Experiment 1); \u003cstrong\u003eb\u003c/strong\u003e salinity and cell-density conditions at 7 dph (Experiment 2); and (\u003cstrong\u003ec\u003c/strong\u003e) salinity conditions under high-cell-density feeding at 7 dph (Experiment 3). No significant differences were observed over the time-course within each group (Steel–Dwass test; \u003cem\u003en\u003c/em\u003e = 6 larvae examined at each time point per group)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6222909/v1/58e7d50468dfe931a916fbcb.png"},{"id":79155486,"identity":"151fe464-49c1-418f-8743-0bd2cb900b5b","added_by":"auto","created_at":"2025-03-25 06:07:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":228017,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of food-intake efficiency in \u003cem\u003eAnguilla japonica\u003c/em\u003e larvae reared under different salinity and photoperiod conditions at 6–7 dph (Experiment 1); \u003cstrong\u003ea\u003c/strong\u003eThe fluorescence intensity per area of mid-hindgut (\u003cem\u003eFIG\u003c/em\u003e) as an index of intestinal fullness was calculated by dividing the autofluorescence portion of picocyanobacteria cells by the area of mid-hindgut in the larva (\u003cem\u003en\u003c/em\u003e = 18 larvae examined per group); \u003cstrong\u003eb\u003c/strong\u003e Intestinal fullness was classified according to the value of \u003cem\u003eFIG\u003c/em\u003e: class A = more than half-filled (200 ≤ \u003cem\u003eFIG\u003c/em\u003e); class B = 150 ≤ \u003cem\u003eFIG\u003c/em\u003e \u0026lt; 200; class C = quarter-filled (100 ≤ \u003cem\u003eFIG\u003c/em\u003e \u0026lt; 150); class D = 50 ≤ \u003cem\u003eFIG\u003c/em\u003e \u0026lt; 100; class E = a small amount (0 \u0026lt; \u003cem\u003eFIG\u003c/em\u003e \u0026lt; 50)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6222909/v1/562783f022343b8879450d3c.png"},{"id":79155483,"identity":"d56ab1bc-86e9-4f99-8c58-6059ad3c8845","added_by":"auto","created_at":"2025-03-25 06:07:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":131201,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of food-intake efficiency in \u003cem\u003eAnguilla japonica\u003c/em\u003e larvae reared under different salinity and food cell-density conditions at 7 dph (Experiment 2). See footnote of Fig. 3. concerning the fluorescence intensity per area of mid-hindgut (\u003cem\u003eFIG\u003c/em\u003e) as an index of intestinal fullness (\u003cem\u003en\u003c/em\u003e= 48 larvae examined per group)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6222909/v1/78e2e172b5efaba33421fd47.png"},{"id":79155985,"identity":"90d477c6-5eca-496c-b50b-2b728fd27fa3","added_by":"auto","created_at":"2025-03-25 06:15:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":111889,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of food-intake efficiency in \u003cem\u003eAnguilla japonica\u003c/em\u003e larvae reared under different salinity conditions under high-cell-density feeding at 7 dph (Experiment 3). See footnote in Fig. 3 concerning the fluorescence intensity per area of mid-hindgut (\u003cem\u003eFIG\u003c/em\u003e) as an index of intestinal fullness (\u003cem\u003en\u003c/em\u003e = 48 larvae examined per group)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6222909/v1/b9986ea35f3a727f66100ddb.png"},{"id":79156435,"identity":"4a6c8d81-561a-4092-889e-592ce18d7c90","added_by":"auto","created_at":"2025-03-25 06:23:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1700747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6222909/v1/2f78afd7-4446-4e0d-a810-f154f1ad9ee7.pdf"},{"id":79155477,"identity":"25637f3a-82fc-4812-bde3-686bb9877d8e","added_by":"auto","created_at":"2025-03-25 06:07:03","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18451,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6222909/v1/4e26d89d044d860cb8976e18.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eThe influence of environmental factors on the feeding behavior of reared Japanese eel \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Japanese eel \u003cem\u003eAnguilla japonica\u003c/em\u003e Temminck \u0026amp; Schlegel, 1846 is commonly consumed in Japan. The ecology of the Japanese eel remains unknown and its aquaculture still relies on natural catches for seedlings. Thus, production using artificially hatched eel larvae has not yet been achieved on a commercial scale. The number of glass eels migrating along the coast of Japan has fluctuated significantly in recent decades, but is on the decline [Fisheries Agency website, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jfa.maff.go.jp/j/saibai/attach/pdf/unagi-228.pdf\u003c/span\u003e\u003cspan address=\"https://www.jfa.maff.go.jp/j/saibai/attach/pdf/unagi-228.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (in Japanese) accessed 8 March 2025]. To stabilize prices and conserve the resource, it is essential to establish sustainable, complete aquaculture techniques on a viable scale. In 2010, the National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency (FRA), succeeded in artificial completion of the lifecycle of cultured Japanese eels (Masuda et al. 2012; Tanaka 2015). The culture technology included a slurry-type diet made from shark egg powder for rearing leptocephali to the glass eel stage (Tanaka et al. 2001), but the production costs still remain high up to the glass eel stage, owing to poor growth, morphological abnormalities, and low survival rates caused by diseases, and hence mass production has not yet been realized. These constraints continue to thwart the commercialization of eel seedling production.\u003c/p\u003e \u003cp\u003eAs a substitute for conventional slurry-type diets (Tanaka et al. 2001), floating-type diets are a promising alternative, yet no floating-type diet has been developed to support the initial growth and survival of Japanese eel larvae. The development of successful floating-type food material that can be stably cultivated and produced with low environmental impacts (Wullur et al. 2013; Tomoda et al. 2015) could lower costs and allow for mass production. However, the feeding ecology of leptocephali remains largely unknown and their feeding behavior in the wild has never been observed; nonetheless, research to date has proposed several theories about their diet before they become glass eels (e.g., Otake et al. 1993; Mochioka and Iwamizu 1996; Riemann et al. 2010; Terahara et al. 2011; Miller 2009; Miller et al. 2011, 2013, 2020; Miyazaki et al. 2011; Feunteun et al. 2015; Chow et al. 2017, 2019a; Tomoda et al. 2018; Tsukamoto and Miller 2021; Watanabe et al. 2021). In developing floating-type diets and new rearing methods, information can be gleaned from the results of previous studies (Masuda et al. 2013; Furuita et al. 2014, 2024; Yamada et al. 2019) as well as basic knowledge of the physiological, ecological and functional morphological characteristics of eel larvae (Uematsu et al. 1994; Otake 1996; Tomoda and Uematsu 1996; Ohta 2008; Okamura et al. 2009; Tsukamoto et al. 2009; Yamada et al. 2019; Yoshimatsu 2011; Politis et al. 2014, 2017, 2018; Kuroki et al. 2016; Miller and Tsukamoto 2017, 2020; Watanabe 2017; Chow et al. 2019b; Matsuda et al. 2019; Kuroki 2020; Masuda et al. 2020; Knutsen et al. 2021; Miller 2023).\u003c/p\u003e \u003cp\u003eIn a previous study (Kenzaki et al. 2022) using picocyanobacteria \u003cem\u003eSynechococcus\u003c/em\u003e spp. as a model suspended feed, thought to be one of the food source (Lundgreen et al. 2019), we confirmed feeding selectivity and density dependence due to the food environment. Understanding the environmental conditions for efficient intake of floating-type diets will contribute to better growth and survival of cultured larvae. Low-salinity rearing was shown to improve the growth, survival rate, energy metabolism and osmoregulatory capacity of larval Japanese eels (Okamura et al. 2009) and European eels \u003cem\u003eAnguilla anguilla\u003c/em\u003e (Politis et al. 2018). Therefore, it is expected that low-salinity rearing using floating-type diets will also improve feeding efficiency, and thereby growth and survival. In this study, we investigated the feeding efficiency of Japanese eel leptocephali in relation to the environmental factors of salinity, photoperiod, and food density of the picocyanobacteria \u003cem\u003eSynechococcus\u003c/em\u003e sp. (Kenzaki et al. 2022) as a model food material for a floating-type diet.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCultivation of picocyanobacteria\u003c/h2\u003e \u003cp\u003eAs the food material, \u003cem\u003eSynechococcus\u003c/em\u003e sp. strain NIES-976 was obtained from the National Institute for Environmental Studies (Japan) cryopreserved stock; this pico-sized cyanobacterium is considered a model floating food for eel larvae and was selected from among numerous picoplankton species present in the natural habitat of Japanese eels (Tomoda et al. 2018; Watanabe et al. 2021). The culture method was as reported previously in Kenzaki et al. (2022). Exponentially growing cultures (~\u0026thinsp;2.3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of \u003cem\u003eSynechococcus\u003c/em\u003e sp. were concentrated by centrifugation (KUBOTA centrifuge, Model 8420) at 2 280 \u003cem\u003eg\u003c/em\u003e for 40 min. The collected cells were frozen and stored at \u0026minus;\u0026thinsp;78\u0026deg;C until the start of the feeding experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental fish\u003c/h3\u003e\n\u003cp\u003eWe obtained eggs of eel broodstock by induced spawning of Japanese eels raised and maintained year-round at the Shibushi Laboratory of the Fisheries Technology Institute, National Research and Development Agency, FRA, as described previously (Satoh et al. 1992; Kagawa et al. 1997, 2013; Tachiki et al. 1997; Dou et al. 2007; Unuma et al. 2011, 2012; Ohta et al. 1996, 2017; Furuita et al. 2024). Leptocephalus larvae obtained from the spawned eggs were reared without food until first feeding at 6\u0026ndash;7 days post hatch (dph) in the feeding behavior experiments.\u003c/p\u003e\n\u003ch3\u003eRearing method\u003c/h3\u003e\n\u003cp\u003eFor the eel larviculture, we used a closed recirculation system consisting of 2.5-l plankton kreisel tanks (Yamada et al. 2019) equipped with a bimorph pump (BPH-214G, Nitto Kohki) (Kenzaki et al. 2022). This system was created to constantly float the larvae in the rearing water and to feed in \u003cem\u003eSynechococcus\u003c/em\u003e cells into the middle layer. The larvae were reared in ultraviolet-irradiated seawater (24\u0026deg;C, 34 psu) supplied by a flow-type UV steriliser (Flonlizer FDL-4-SP, Chiyoda Kohan) after microfiltration through 0.5-\u0026micro;m cartridge filters (TCW-0.5N-PPD, Advantec Toyo Kaisha). At 6 or 7 dph, 30 or 70 eel larvae were stocked into the rearing system, with a circulating flow rate of 0.15\u0026ndash;0.20 l min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, in a thermostatic chamber maintained at 24\u0026deg;C. Thereafter, \u003cem\u003eSynechococcus\u003c/em\u003e sp. cells collected by centrifugation were added to and resuspended in the rearing water.\u003c/p\u003e\n\u003ch3\u003eFeeding observations under different environmental conditions\u003c/h3\u003e\n\u003cp\u003eTo confirm the degree of food intake of suspended picocyanobacteria by the leptocephali under different environmental conditions, larvae 6\u0026ndash;7 dph, sized 6.69\u0026ndash;7.12 mm total length (TL) and hatched from different broodstock, were collected from rearing tanks and examined. For observations of larval feeding behavior, only a \u003cem\u003eSynechococcus\u003c/em\u003e cell resuspension was given.\u003c/p\u003e\n\u003ch3\u003eExperiment 1: Comparisons under different salinities and photoperiods\u003c/h3\u003e\n\u003cp\u003eTo compare food-intake efficiency under different salinity and photoperiod conditions, four rearing groups were established by combinations of two salinity conditions (50% SW, 16 psu; 100% SW, 32 psu) and two photoperiods (continuous light, 24L; continuous darkness, 24D). At 6 dph, 30 larvae were stocked into the rearing system. The cell density of \u003cem\u003eSynechococcus\u003c/em\u003e sp. in the rearing water for all groups of larvae was adjusted to 2.538 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The photon flux density in the light condition (24L) was 0.118\u0026ndash;0.125 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; the dark condition (24D) was complete darkness (0.000 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Next, to confirm the time-course of food-intake into the digestive tracts of the eel larvae at 6\u0026ndash;7 dph, six larvae in each tank were observed three times during this experiment: at 3 h (13:00), 6 h (16:00) and 24 h (10:00 the next morning) after feeding.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eExperiment 2: Comparisons under different salinities and food cell densities\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo compare food-intake efficiency in relation to salinity and picoplankton cell density of the food environment, three larval rearing groups were established using combinations of two salinities (50% SW; 100% SW) and two food cell densities (high density, HD; low density, LD). At 7 dph, 70 larvae were stocked into the rearing system. The density of \u003cem\u003eSynechococcus\u003c/em\u003e sp. in the rearing water was adjusted to 8.075 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the HD group, and to 1.225 \u0026times;10\u003csup\u003e7\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the LD group. The photoperiod for all groups was 24L, and the photon flux density was the same as in Experiment 1. To confirm the time-course of food intake into the digestive tracts of larvae at 7 dph, six larvae in each tank were observed every hour between 10:00 and 17:00 (i.e. a total of eight times).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperiment 3: Confirmation of the comparisons under different salinity conditions\u003c/h3\u003e\n\u003cp\u003eTo confirm the reproducibility of Experiment 2 and the food-intake efficiency of larvae in a higher food-cell-density environment, two rearing groups were established under different salinity conditions (50% SW; 100% SW). The cell density of \u003cem\u003eSynechococcus\u003c/em\u003e sp. in the rearing water was adjusted to 1.233 \u0026times;10\u003csup\u003e8\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The larval stocking density and photoperiod was the same as in Experiment 2 (24L), and thereafter the feeding observations were similarly performed.\u003c/p\u003e\n\u003ch3\u003eEstimation of food-intake efficiency\u003c/h3\u003e\n\u003cp\u003eImmediately before observation, eel larvae were collected from each tank and anesthetized. The digestive tracts of six larvae were observed using an epi-fluorescence biological microscope (20x, ECLIPSE Ni, C-HGFI, Nikon) and photographed using a digital microscope camera (DS-FI3, Nikon). In addition to bright-field observation, photographs were taken under dark-field conditions with a uniform exposure time and gain through a green excitation filter. As in Kenzaki et al. (2022), the degree of intestinal fullness was evaluated based on the area of mid-hindgut with autofluorescence; however, the influence of intercellular voids and focal depth on the measured values remained as issues to consider. To resolve this, we considered using brightness (red component) as an index value as a more-accurate evaluation method. First, the area of mid-hindgut was measured from the bright-field image, and then the brightness caused by the autofluorescence of \u003cem\u003eSynechococcus\u003c/em\u003e cells in the mid-hindgut was measured from the dark-field image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Here, the fluorescence intensity per area of mid-hindgut (\u003cem\u003eFIG\u003c/em\u003e) was defined as the obtained brightness divided by the mid-hindgut area (mm\u003csup\u003e2\u003c/sup\u003e). The \u003cem\u003eFIG\u003c/em\u003e was calculated using the following equation:\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFIG\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eB\u003c/em\u003e / \u003cem\u003eA\u003c/em\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eB\u003c/em\u003e is the brightness within mid-hindgut and \u003cem\u003eA\u003c/em\u003e is the mid-hindgut area.\u003c/p\u003e \u003cp\u003eImage analysis software (ImageJ 1.52t; National Institutes of Health, USA) was used to measure the area and brightness of the mid-hindgut. To gauge the feeding state of individual larvae, the value of the \u003cem\u003eFIG\u003c/em\u003e index was used to define five classes of intestinal fullness: class A\u0026thinsp;=\u0026thinsp;more than half-filled (200\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eFIG\u003c/em\u003e); class B\u0026thinsp;=\u0026thinsp;150\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eFIG\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;200; class C\u0026thinsp;=\u0026thinsp;quarter-filled (100\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eFIG\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;150); class D\u0026thinsp;=\u0026thinsp;50\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eFIG\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;100; class E\u0026thinsp;=\u0026thinsp;a small amount (0\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eFIG\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;50).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe fluorescence intensity per area of mid-hindgut (\u003cem\u003eFIG\u003c/em\u003e) was obtained for each rearing group and elapsed time, and differences in mean values of the index were examined. The Steel\u0026ndash;Dwass multiple comparison was carried out on differences in the amount of ingested food in the mid-hindgut at different time points within each rearing group. Concerning differences in food intake between rearing groups, the assumptions of normal distribution and homogeneity of variances were checked before the statistical analysis; a Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used if normality and homoscedasticity were observed in both groups compared, otherwise a Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used. All statistical analyses were performed using Excel (Microsoft Office 365) with the add-in software Statcel 4 (OMS Publishing Inc., Japan).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComparison of the time-course of food intake\u003c/h2\u003e \u003cp\u003eIn all experiments, the values of \u003cem\u003eFIG\u003c/em\u003e were higher in groups reared in 50% SW than in 100% SW (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). In addition, no significant differences were observed in food intake into the digestive tract over time in each rearing group in all experiments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.05, Steel\u0026ndash;Dwass test). Therefore, all the time-course observation data for each rearing group were compiled and the feeding behavior (food intake) was compared between groups (see next section).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFood-intake efficiency in relation to the environmental conditions tested\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;1 summarizes the food-intake efficiency of the larvae under each salinity and photoperiod condition (Experiment 1), and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e depicts the frequency of \u003cem\u003eFIG\u003c/em\u003e classes of intestinal fullness in the differently reared groups. Larvae with a high feeding status (class A) were observed only in the 50% SW\u0026ndash;24L group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Food-intake efficiency was significantly superior in larvae reared in 50% SW compared with in 100% SW under the condition of continuous light (24L) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0248) as well as under the condition of continuous darkness (24D) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0101, Table\u0026nbsp;1). However, under 24L versus under 24D and the same salinity condition, no significant difference in food-intake efficiency was observed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1744 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5255, respectively; Table\u0026nbsp;1). Furthermore, food-intake efficiency was significantly higher in the 50% SW\u0026ndash;24L group than in the 100% SW\u0026ndash;24D group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0028, Table\u0026nbsp;1), and larval feeding ability appeared to be improved by low-salinity rearing (50% SW\u0026ndash;24D) even under the dark condition (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1792, Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;2 summarizes the food-intake efficiency under each salinity and food cell-density condition (Experiment 2), and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depicts the frequency of \u003cem\u003eFIG\u003c/em\u003e classes of intestinal fullness in the differently reared groups. The 50% SW\u0026ndash;HD group had the highest number of larvae presenting with a class A feeding status (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). With high-density feeding, the food-intake efficiency of the larvae was higher in the 50% SW group than in the 100% SW group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002, Table\u0026nbsp;2). In addition, when reared in 100% SW, food-intake efficiency was significantly higher in the HD group than in the LD group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.039 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e, Table\u0026nbsp;2). Furthermore, the food-intake efficiency of the 50% SW\u0026ndash;HD group (positive control) was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.181 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e) than in the 100% SW\u0026ndash;LD group (negative control) (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;3 presents the food-intake efficiency of larvae that were kept under the two different salinity conditions but fed the same high food-cell density (Experiment 3), and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the frequency of the \u003cem\u003eFIG\u003c/em\u003e classes of intestinal fullness in the different salinity groups. Although many of the test larvae had morphological abnormalities, such as pericardial cavity hypertrophy, lower-jaw dysostosis and intestinal hypoplasia, the group reared in 50% SW had a higher number of larvae showing class A feeding status (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), thus their food-intake efficiency was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0030, Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study provide insights into the feeding behavior of captively reared Japanese eel larvae in response to environmental factors, namely different salinities, photoperiods and food cell densities. The results provide important information useful for developing initial food materials, which has been a bottleneck in the mass production of Japanese eel seedlings. Since there were no significant differences in the feeding status of the larvae over time in each rearing group in all experiments, it is considered that eel larvae are constantly feeding by taking in seawater (Lee et al. 2013; Ahn et al. 2015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). In Experiment 1, low-salinity rearing was found to have the effect of improving food-intake efficiency, and feeding activity was shown to be constant regardless of continuously light or dark conditions (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Experiments 2 and 3 also showed the superiority of low-salinity rearing and food-cell density dependence (Tables\u0026nbsp;2 and 3, Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), similar to the report of a previous study (Kenzaki et al. 2022). Okamura et al. (2009) found that Japanese eel larvae given a slurry-type diet showed better growth and survival when reared in 50% SW than in 100% SW, similar to the superiority of larvae in the low-salinity groups in this study. This is presumably because rearing in diluted seawater reduces the energy consumed for osmoregulation (Kuroki et al. 2016), thereby contributing to improved feeding activity as compared with in full-strength seawater. Anni et al. (2016) reported that low-salinity rearing (3\u0026ndash;6 ppt) affected the osmoregulation and energy metabolism of juvenile Plata pompano \u003cem\u003eTrachinotus marginatus\u003c/em\u003e, thereby improving fish growth when compared with rearing in 100% SW (32 ppt). Bradshaw et al. (2023) surmised that low-salinity rearing of Florida pompano \u003cem\u003eT. carolinus\u003c/em\u003e provides an advantage in terms of the ability to treat halophilic pathogens. From these findings, the advantages of low-salinity rearing can be appreciated.\u003c/p\u003e \u003cp\u003eEel larvae exhibit negative phototaxis and undergo diurnal vertical migration (Yamada et al. 2019; Watanabe 2017). It is speculated that during vertical migration wild leptocephali actively and passively feed when passing through the subsurface chlorophyll maximum (SCM) depth zone, where particulate organic matter (POM) is relatively abundant (Otake et al. 1998; Watanabe et al., unpublished data); interestingly, this would match the continuous feeding behavior verified under dark conditions in this study. It has also been suggested that eel larvae identify food by olfactory function (Uematsu et al. 1994; Ohta 2008) rather than visual function (Tomoda and Uematsu 1996), which supports the results of this study. Kenzaki et al. (2022) found that food-intake efficiency was higher under twilight conditions (9L:15D) than under complete darkness (24D). Although the present study confirmed a similar trend, food intake by the larvae did not differ significantly under light and dark conditions (Table\u0026nbsp;1). The different results may be attributable to differences in feeding activity depending on the batch of experimental fish and age of the larvae in days at the start of experiments. Although the observations in the present study were made over a short period of just 8\u0026ndash;24 h, feeding was confirmed regardless of the light or dark conditions, as shown in previous research (Kenzaki et al. 2022); hence, it is possible that the feeding mode of leptocephali is similar to that of filter feeders by drinking water (Lee et al. 2013; Ahn et al. 2015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). A great advantage of this mode is that it allows eel larvae to feed under dark conditions without relying on vision, while also avoiding predation (Miller et al. 2015). In addition, the ability to feed continuously even in darkness while suppressing energy consumption at the equivalent level to the basal metabolism is also efficient for survival in the oligotrophic environment.\u003c/p\u003e \u003cp\u003eEven so, in this study, as in the study of Kenzaki et al. (2022), it was observed that ingested cells of \u003cem\u003eSynechococcus\u003c/em\u003e sp. circulated in the mid-hindgut for several hours before they accumulated and were excreted from the anus as feces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It is speculated that POM in seawater is efficiently captured in the mid-hindgut by the movement of microvilli and the mucus produced in the intestinal epithelium (Yamada and Yokote 1975; Otake 1996). These observations suggest that water and the proteins and amino acids contained in POM are digested and absorbed in the rectum (Otake et al. 1993;Otake 1996; Lee et al. 2013; Hsu et al. 2015). Picoplankton and amorphous aggregates, as part of the marine snow originating from zooplankton and phytoplankton decomposed by bacteria (Miller et al. 2013; Knutsen et al. 2021; Watanabe et al. 2021), have been observed in the gut contents of anguilliform leptocephali (Tomoda et al. 2018). Kenzaki et al. (2022) speculated that feeding selectivity for \u003cem\u003eSynechococcus\u003c/em\u003e sp. (strain NIES-976) is induced by its amount of extracellular protein, compatible with the high protein digestibility and amino acid utilization ability of eel larvae (Hsu et al. 2015).\u003c/p\u003e \u003cp\u003eThus, it is speculated that feeding modes like filter feeding or ciliary mucus feeding (Riisg\u0026aring;rd et al. 2000; Riisg\u0026aring;rd and Larsen 2010), whereby the animal can efficiently ingest suspended particles from seawater under various environmental conditions, such as low salinity and darkness, may enable adaptation to the oligotrophic environment. Therefore, rearing methods that accommodate this feeding mode could lead to three-dimensional utilization of the rearing tank and ultimately increase the scale of seedling production. Future studies should examine the environmental conditions, species selection, and rearing methods best for optimizing use of various food organisms (eukaryotic picophytoplankton, cyanobacteria and bacteria) that are considered as part of marine snow.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis manuscript describes important findings contributing to mass larviculture of Japanese eel. The feeding behavior of eel leptocephali, which has not been observed in nature, was observed under artificial rearing, and the influence of environmental factors was clarified. In this study, we proposed a recognition method based on fluorescence brightness to enable more accurate recognition of eel feeding behavior to suspended particles and confirmed its effectiveness. We investigated the feeding efficiency of Japanese eel leptocephali in relation to the environmental factors of salinity, photoperiod, and food density. The superiority of low-salinity environmental water and high-density food concentration, and light-independence feeding rhythm were confirmed. The specific feeding ecology whereby eel larvae are able to efficiently ingest suspended particles from seawater even in complete darkness may enable them to adapt to the oligotrophic environment. The rearing method corresponding to the feeding mode clarified in this study may lead to the three-dimensional utilization of the rearing tank and eventually to the expansion of the seedling production scale. Our findings add new knowledge to the research field on feeding ecology of eel leptocephali, and contribute to the development of aquaculture research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eWe thank Keisuke Yamano (Director of the Glass Eel Production Division at the Fisheries Technology Institute, National Research and Development Agency, Japan Fisheries Research and Education Agency [FRA]) for his support in conducting this research. We are sincerely grateful to the staff of the Shibushi Field Station of FRA for their help providing leptocephali. The anonymous reviewers and the journal\u0026rsquo;s editor-in-chief provided many helpful suggestions. Cynthia Kulongowski with Edanz (https://jp.edanz.com/ac) edited the language of a draft of this manuscript. This research was conducted as a part of the project \u0026lsquo;Development of biological materials feeds\u0026rsquo; commissioned by FRA as part of the \u0026lsquo;Demonstration project of a mass-production system for commercialization of eel seedlings\u0026rsquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eT.T., S.O. and H.M. conceived the study; all authors contributed to the design of the conceptual framework and analyses; Y.S. and T.T. cultured the picocyanobacteria and reared the leptocephali; Y.S., A.K., T.T. and S.O. conducted the laboratory experiments and completed the statistical analyses; The first draft of the manuscript was written by Y.S. and all authors were involved with drafting and editing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported in part by grants from the Fisheries Agency, Ministry of Agriculture, Forestry and Fisheries of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eAll experimental fish were handled and treated in accordance with the Guidelines for Animal Experimentation at the Fisheries Technology Institute, Japan Fisheries Research and Education Agency (FRA). All experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of FRA (permission code: 24023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhn H, Lee KM, Inokuchi M, Watanabe S, Okamura A, Tsukamoto K, Kaneko T (2015) Observations of initial water ingestion and ion absorption in the digestive tract of Japanese eel larvae. Fish Sci 81:283\u0026ndash;290. https://doi.org/10.1007/s12562-014-0841-8\u003c/li\u003e\n\u003cli\u003eAnni ISA, Bianchini A, Barcarolli IF, Varela Junior AS, Robaldo RB, Tesser MB, Sampaio LA (2016) Salinity influence on growth, osmoregulation and energy turnover in juvenile pompano \u003cem\u003eTrachinotus marginatus\u003c/em\u003e Cuvier 1832. 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Fish Sci 81：11\u0026ndash;19. https://doi.org/10.1007/s12562-014-0821-z\u003c/li\u003e\n\u003cli\u003eTerahara T, Chow S, Kurogi H, Lee SH, Tsukamoto K, Mochioka N, Tanaka H, Takeyama H (2011) Efficiency of peptide nucleic acid-directed PCR clamping and its application in the investigation of natural diets of the Japanese eel leptocephali. PLoS ONE 6(11):e25715. https://doi.org/10.1371/journal.pone.0025715\u003c/li\u003e\n\u003cli\u003eTomoda H, Uematsu K (1996) Morphogenesis of the brain in larval and juvenile Japanese eels, \u003cem\u003eAnguilla japonic\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e. Brain Behav Evol 47(1):33\u0026ndash;41. https://doi.org/10.1159/000113227\u003c/li\u003e\n\u003cli\u003eTomoda T, Kurogi H, Okauchi M, Kamoshida M, Imaizumi H, Jinbo T, Nomura K, Furuita H, Tanaka H (2015) Hatchery-reared Japanese eel \u003cem\u003eAnguilla japonica\u003c/em\u003e larvae ingest various organic matters formed part of marine snow. Nippon Suisan Gakkaishi 81(4):715\u0026ndash;721. (in Japanese with English abstract). https://doi.org/10.2331/suisan.81.715\u003c/li\u003e\n\u003cli\u003eTomoda T, Chow S, Kurogi H, Okazaki M, Ambe D, Furuita H, Matsunari H, Nagai S, Yokouchi K, Sawayama S, Nomura K, Tanaka H, Sudou R, Hasegawa D, Inaba N (2018) Observations of gut contents of anguilliform leptocephali collected in the western North Pacific. Nippon Suisan Gakkaishi 84(1):34\u0026ndash;44. (in Japanese with English abstract). https://doi.org/10.2331/suisan.17-00025\u003c/li\u003e\n\u003cli\u003eTsukamoto K, Yamada Y, Okamura A, Kaneko T, Tanaka H, Miller MJ, Horie N, Mikawa N, Utoh T, Tanaka S (2009) Positive buoyancy in eel leptocephali: an adaptation for life in the ocean surface layer. Mar Biol 156:835\u0026ndash;846. https://doi.org/10.1007/s00227-008-1123-8\u003c/li\u003e\n\u003cli\u003eTsukamoto K, Miller MJ (2021) The mysterious feeding ecology of leptocephali: a unique strategy of consuming marine snow materials. Fish Sci 87:11\u0026ndash;29. https://doi.org/10.1007/s12562-020-01477-3\u003c/li\u003e\n\u003cli\u003eUematsu K, Tomoda H, Omura Y (1994) Brain and sensory organs of eel leptocephali. Kaiyo Monthly 26:282\u0026ndash;287. (In Japanese)\u003c/li\u003e\n\u003cli\u003eUnuma T, Hasegawa N, Sawaguchi S, Tanaka T, Matsubara T, Nomura K, Tanaka H (2011) Fusion of lipid droplets in Japanese eel oocytes: stage classification and its use as a biomarker for induction of final oocyte maturation and ovulation. Aquaculture 322\u0026ndash;323:142\u0026ndash;148. https://doi.org/10.1016/j.aquaculture.2011.10.001\u003c/li\u003e\n\u003cli\u003eUnuma T, Sawaguchi S, Hasegawa N, Tsuda N, Tanaka T, Nomura K, Tanaka H (2012) Optimum temperature of rearing water during artificial induction of ovulation in Japanese eel. Aquaculture 358\u0026ndash;359:216\u0026ndash;223. https://doi.org/10.1016/j.aquaculture.2012.07.004\u003c/li\u003e\n\u003cli\u003eWatanabe S (2017) Trials of improved feeding techniques for Japanese eel larvae based on its olfaction characteristics. Fiscal Year Final Research Report KAKEN No.16K14967. (in Japanese with English abstract). https://kaken.nii.ac.jp/en/file/KAKENHI-PROJECT-16K14967/16K14967seika.pdf. Accessed 8 March 2025.\u003c/li\u003e\n\u003cli\u003eWatanabe T, Nagai S, Kawakami Y, Asakura T, Kikuchi J, Inaba N, Taniuchi Y, Kurogi H, Chow S, Tomoda T, Ambe D, Hasegawa D (2021) 18S rRNA gene sequences of leptocephalus gut contents, particulate organic matter, and biological oceanographic conditions in the western North Pacific. Sci Rep 11:5488. https://doi.org/10.1038/s41598-021-84532-y\u003c/li\u003e\n\u003cli\u003eWullur S, Yoshimatsu T, Tanaka H, Ohtani M, Sakakura Y, Kim HJ, Hagiwara A (2013) Ingestion by Japanese Eel \u003cem\u003eAnguilla japonica\u003c/em\u003e Larvae on Various Minute Zooplanktons. Aquaculture Sci 61(4):341\u0026ndash;347. https://doi.org/10.11233/aquaculturesci.61.341\u003c/li\u003e\n\u003cli\u003eYamada Y, Okamura A, Mikawa N, Horie N, Tsukamoto K (2019) A new liquid-type diet for leptocephali in mass production of artificial glass eels. Fish Sci 85;545\u0026ndash;551. https://doi.org/10.1007/s12562-019-01295-2\u003c/li\u003e\n\u003cli\u003eYamada K, Yokote M (1975) Morphochemical analysis of mucosubstances in some epithelial tissues of the eel (\u003cem\u003eAnguilla japonica\u003c/em\u003e) Histochemistry 43:161\u0026ndash;172. https://doi.org/10.1007/BF00492444\u003c/li\u003e\n\u003cli\u003eYoshimatsu T (2011) Early Developrnent of Preleptocephalus Larvae of the Japanese Eel in Captivity with Special Reference to the Organs for Larval Feeding. Bull Graduate School of Bioresources Mie Univ 37:11\u0026ndash;18. https://citeseerx.ist.psu.edu/document?repid=rep1\u0026amp;type=pdf\u0026amp;doi=4198b718f475140999d4c75b7f536453f1bcced4. Accessed 8 March 2025.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Fisheries Agency, Ministry of Agriculture, Forestry and Fisheries of Japan","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":"Anguilla japonica, Density-dependent, Feeding behavior, Light-independent, Photoperiod, Salinity","lastPublishedDoi":"10.21203/rs.3.rs-6222909/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6222909/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArtificially reared Japanese eel \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali (\u0026ndash;7 mm total length, 6\u0026ndash;7 days post-hatch) were fed picocyanobacteria (\u003cem\u003eSynechococcus\u003c/em\u003e sp., strain NIES-976) and their food intake was observed using autofluorescence intensity per area of the mid-hindgut used as an index of gut fullness. Time-course observations revealed that the larvae actively fed under both light and dark conditions. Food intake was significantly higher in the low-salinity group (50% seawater) than in the control group (100% seawater). Food intake did not differ significantly under photoperiods of 24-h light versus 24-h dark, indicating a light-independent diurnal feeding rhythm. A comparison of larval feeding efficiency under high and low cell densities of picocyanobacteria showed remarkably high intake of the food material by larvae in the high-density food concentration group, indicating density-dependent food ingestion. This specific feeding ecology whereby Japanese eel larvae are able to efficiently ingest suspended pico-sized food particles from seawater even in complete darkness may enable them to adapt to the oligotrophic environment.\u003c/p\u003e","manuscriptTitle":"The influence of environmental factors on the feeding behavior of reared Japanese eel Anguilla japonica leptocephali","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-25 06:06:58","doi":"10.21203/rs.3.rs-6222909/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":"1a43efb5-f3c0-4d29-803e-515498d38dec","owner":[],"postedDate":"March 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45662597,"name":"Aquaculture and Mariculture"}],"tags":[],"updatedAt":"2025-03-25T06:06:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-25 06:06:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6222909","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6222909","identity":"rs-6222909","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}