Feeding ecology of anguilliform leptocephali considering the structure and proximate composition of food organisms and gut contents

Feeding ecology of anguilliform leptocephali considering the structure and proximate composition of food organisms and gut contents | 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 Feeding ecology of anguilliform leptocephali considering the structure and proximate composition of food organisms and gut contents Tsutomu Tomoda, Tsuyoshi Watanabe, Kazuaki Tadokoro, Hirofumi Furuita, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8218055/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 Scanning electron microscope (SEM) observation and component analysis were conducted out on the gut contents of wild larvae fed particulate organic matter (POM) and cultured larvae fed on available microorganisms to determine the physical characteristics and nutritional contributions of biological species that are presumed to be food elements of anguilliform leptocephali. There were no traces of food organisms, such as autofluorescence of algae and faecal pellets, or carcasses of zooplankton in the gut contents of wild larvae. The gut contents of both wild and cultured larvae were light brown sols or gels and were composed of amorphous substances consisting mainly of carbohydrates, proteins and amino acids. The structural properties of the gut contents of cultured larvae were similar to those of POM in environmental water; mucus secreted from the intestinal epithelium was also present. These results suggest that low-molecular-weight saccharides, proteins, and amino acids produced by algae and bacteria may be among the available nutritional sources of eel larvae and that the mucus layer of the intestinal epithelium contributes to a feeding mode that effectively captures microparticles from environmental water. Taken together, the results of recent surveys suggest that pico- and nanosized POMs, which are ubiquitous in environmental waters and are easily swallowed and easily digestible and absorbable, i.e., substances produced by algae and bacteria that account for a high proportion of marine biomass and dissolved organic matter (DOM), may be directly utilized by the intestinal epithelium through pinocytosis without microbial degradation. Aquaculture and Mariculture Marine and Freshwater Biology Behavioral Ecology Anguilliform leptocephali Feeding ecology Gut contents Mucus POM SEM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction In recent years, various proposals have been made to implement resource management and conservation policies for eels, and the importance of research focusing on biology and ecology has been noted as a clue to identify problems (Dekker 2016 ; Kaifu et al. 2018 ; Righton et al. 2021 , 2025 ; Shiraishi and Kaifu 2024 ). Since the existing eel farming industry depends only on natural seedlings and because aquaculture using artificial seedlings has not yet been realized on a commercial scale, the establishment of sustainable complete aquaculture technologies (Tanaka et al. 2001 ; Masuda et al. 2012 ; Tanaka 2015 ) is desired for the stabilization of seed prices and resource conservation. Therefore, it is necessary to innovate not only the feed but also the rearing form, which is a bottleneck in the expansion of production scale. An urgent task is to first clarify the feeding ecology in nature, which should be a model. Understanding the major species used as food sources and determining the feeding mode of eel larvae and the formation mechanism of the food environment will greatly contribute to understanding the conversion of seedling production forms. It will also lead to cost and labour savings and to the restructuring of the aquaculture industry, which in turn can be expected to protect natural resources and stabilize aquaculture management. Our recent feeding studies of cultured larvae confirmed the feeding selectivity and density dependence associated with the food environment (Kenzaki et al. 2022 ) and the influence of environmental factors such as salinity, food density, and photoperiod on feeding behaviour (Shioura et al. 2025 ). To develop feed and rearing methods for mass production of glass eels in the future, we believe that it is important not only to focus on the composition and nutritional value of feed (Masuda et al. 2013 ; Furuita et al. 2014 , 2024 ; Yamada et al. 2019 ) but also to develop technology based on fundamental information such as physiology, ecology and functional morphology (Uematsu et al. 1994 ; Otake 1996 ; Tomoda and Uematsu 1996 ; Ohta 2008 ; Miller 2009 ; Okamura et al. 2009 ; Tsukamoto et al. 2009 ; Yamada et al. 2009 ; Yoshimatsu 2011 ; Politis et al. 2014 , 2017 , 2018 ; Kuroki et al. 2016 ; Miller and Tsukamoto 2017 , 2020 ; Watanabe 2017 ; Chow et al. 2019a ; Matsuda et al. 2019 ; Kuroki 2020 ; Masuda et al. 2020 ; Knutsen et al. 2021 ; Miller 2023 ). There are various theories on the feeding habits of eel leptocephali, and particulate organic matter (POM), dissolved organic matter (DOM), faecal pellets and discarded houses of appendicularians have been assumed to be the most likely food sources (Otake et al. 1993 ; Mochioka and Iwamizu 1996 ). Recently, various approaches, such as microscopic observation (Govoni 2010 ; Miller et al. 2011 , 2019 ; Tomoda et al. 2018 ) and genetic analysis (Riemann et al. 2010 ; Terahara et al. 2011 ; Ayala et al. 2018 ; Chow et al. 2019b ; Watanabe et al. 2021 ; Kume et al. 2025 ) of gut contents and stable isotope ratio analysis (Miyazaki et al. 2011 ; Miller et al. 2013 ; Feunteun et al. 2015 ; Chow et al. 2017 ; Ghinter et al. 2020 ; Kimura et al. 2024 ), have been applied, and many findings have been reported (Miller 2009 ; Miller et al. 2020 ; Tsukamoto and Miller 2021 ). However, feeding behaviour in nature has not yet been observed, and the actual conditions of food utilized by wild larvae, such as the composition of biomass as a food source (Lundgreen et al. 2019 ), the contribution of each food element or abiotic fraction, and the feeding mode, are unknown. In particular, the origin of amorphous substances (Miller et al. 2011 , 2019 , 2020 ; Tomoda et al. 2018 ; Tsukamoto and Miller 2021 ), which constitute a high proportion of the gut contents, is not yet clear. Furthermore, food environment surveys have concentrated on eukaryotes, whereas few studies have focused on prokaryotes. According to the estimation of the trophic level of natural food by the amino acid nitrogen isotope ratio (Miller et al. 2013 ), the food of eel leptocephali has been identified as marine snow (Alldredge and Silver 1988 ; Lampitt et al. 1993 ; Chajwa et al. 2024 ), which originates from the carcasses of zoo- and phytoplankton; prokaryotes such as cyanobacteria and bacteria have also been suggested to be food components. Research in the field of marine ecosystem microbiology indicates that algae and bacteria account for a high proportion of biomass in the open ocean (Zhang et al. 2008 ; Lee et al. 2012 ; Flombaum et al. 2013 ) and are also components of the POM that eel leptocephali feed on, suggesting their importance in organic matter transport and contribution to food webs (Long and Azam 1996 ; Azam 1998 ; Verdugo et al. 2004 ; Kogure K (ed) 2006; Azam and Malfatti 2007 ; Tsukasaki and Tanoue 2010 ; Biller et al. 2014 , 2022 , 2023 ; Scanlan 2014 ; Zhou et al. 2016 ; Busch et al. 2017 ; Mari et al. 2017 ; Seymour et al. 2017 ; Mühlenbruch et al. 2018 ; Cirri and Pohnert 2019 ; Guo et al. 2023 ; Casillo et al. 2024 ; Chajwa et al. 2024 ). Algae, bacteria, and their products (Thornton 2018 ; Ogasawara et al. 2021 ) play roles as sources of nutrients for eel larvae, which possess specific feeding habits. The intestinal microbiota, which is composed of various bacteria, is thought to contribute to the enhancement of digestion and absorption, maintenance of homeostasis, and improvement of immune function (Yukgehnaish et al. 2020 ; Diwan et al. 2023 ; Morshed and Lee 2023 ). Other expected benefits of bacteria include probiotics (Schmidt et al. 2017 ; Yukgehnaish et al. 2020 ) and synbiotics (Fujii et al. 2024 ). Therefore, detailed observation and component analysis of the gut contents of eel larvae that ingested algae and bacteria from their habitat via environmental water may elucidate the actual food environment and feeding mode. In this study, we compared the gut contents of wild larvae of Anguilliformes collected from the subtropical western North Pacific Ocean, such as algae, bacteria and ciliates, which are components of POM, and the gut contents of cultured larvae of the Japanese eel Anguilla japonica that ingested POM originating from these microorganisms by scanning electron microscope (SEM) observation and component analysis. The aim of this study was to further clarify the feeding ecology by understanding the structural properties and nutritional contribution of species as food sources. Materials and methods Collection and identification of wild leptocephali Net sampling for anguilliform leptocephali was conducted 45 times at 22 stations from 19 August to 8 September in 2020 and 15 times at 13 stations from 1 to 13 September in 2022 during R/V Soyo-Maru cruises (SY2005 and SY2204) in the eastern waters of the Philippines (Fig. 1 , Table 1). Leptocephali were collected using an Isaacs-Kidd Midwater Trawl (IKMT) net (Isaacs and Kidd 1953 ) (IKMT; mouth opening area: 8.7 m²; mesh size: 0.5 mm) in 2020 and a Matsuda-Oozeki-Hu Trawl (MOHT) net (Oozeki et al. 2004 , 2012 ) (MOHT; mouth opening area: 5.0 m²; mesh size: 1.59 mm) in 2022. Oblique tows were performed from a depth of 200 m to the surface at night and from depths of 400–435 or 300–330 m to the surface during the day in the 2020 cruise (SY2005). In the 2022 cruise (SY2204), oblique tows were performed from a depth of 100 m to the surface at night. The leptocephali were sorted after collection and placed on a chilled Petri dish. A total of 54 leptocephali with gut contents ( A. japonica , n = 12; Ariosoma spp., n = 20; Conger spp., n = 22) were selected for bacterial isolation, SEM observation, and component analysis (Table 1). A small piece of the dorsal muscle was dissected and placed in a separate sterile 1.5-ml Eppendorf tube, and the remaining body was placed in a separate Uni-Pack (SEISANNIPPONSHA Ltd.). All the samples were kept at − 60°C and transferred to the laboratory. DNA samples from the muscle were extracted using the QuickGene DNA Tissue Kit S (KURABO, Osaka, Japan). Leptocephali were subjected to direct 18S rDNA sequence analysis. Cultivation of bacteria The results of bacterial isolation and identification will be submitted to PeerJ by the authors. Bacterial communities that were the dominant species in the gut contents and POM were selected as potential food souses in this study because they are presumed to contribute to the degradation of various organic substances constituting marine snow (Suzuki et al. 2001 ; Yoon et al. 2003 ; Heuchert et al. 2004 ; Jean et al. 2006 ; Liu et al. 2013 ). We used preserved stocks of Erythrobacter flavus (strain No. 39), Tenacibaculum mesophilum (strain No. 130), Paracoccus stylophorae (strain No. 143) and Psychrobacter sp. (strain Shi-11) from the Shibushi Field Station ( FRA ) and Thalassomonas agariperforans (strain M-M1) from the Culture Collection University of Gothenburg (CCUG). The bacterial strains were routinely cultured on Marine Agar (MA; Condalab, Spain) plates at 25°C and maintained in Marine Broth (MB; Condalab, Spain) supplemented with 16% (v/v) glycerol at − 80°C. Bacteria for the feeding experiments, SEM observations and component analysis were cultured aerobically in MB medium at 25°C for 3–4 days with stirring at 400 rpm using a magnetic stirrer (SW-400ND, NISSIN, Japan). When used in the feeding experiments and component analysis, the bacterial cells were concentrated by centrifugation (KUBOTA Model 7000, Japan) at 18,890 × g for 15 min, washed twice to remove nutrients from the culture medium, and resuspended in sterile artificial seawater (RED SEA SALT, Red Sea, Israel). Cultivation of algae The algae used for the feeding experiments, SEM observations and component analysis were cultured in this study. We used Heterocapsa niei (strain NIES-420) and Prochlorococcus spp. (strains NIES-2884, 2887) from the National Institute for Environmental Studies (NIES) preserved stocks and Isochrysis galbana (strain NRIA-0074) and Chaetoceros calcitrans (strain NRIA-0001) from the Nansei Field Station ( FRA ) preserved stocks. These cyanobacteria and microalgae are considered one of the food sources selected from among the numerous phytoplankton species in habitat areas (Tomoda et al. 2018 ; Watanabe et al. 2021 ). Prochlorococcus was incubated with PRO-99 medium (Moore et al. 2007 ) at 23°C for 7–8 days with a light intensity of 20–36 µmol photons m − 2 s − 1 on a 16-h/8-h light/dark cycle and with moderate shaking at 100 rpm (SK-O180-E, AS ONE, Japan). Heterocapsa was incubated with F/2 medium (Guillard and Ryther 1962 ) at 25°C for 14–15 days under static conditions with the same light conditions as those used for Prochlorococcus . Isochrysis and Chaetoceros were cultured with KW21 medium (DAIICHI CO., Ltd., Japan) at 20°C for 7–10 days under continuous irradiance of 100–128 µmol photons m − 2 s − 1 , and air was supplied at an aeration rate of 1.0 volume (air) −1 volume (medium) −1 minute − 1 (vvm). In addition, Gelculture (DAIICHI Co., Ltd., Japan) was supplemented with Chaetoceros culture. Artificial seawater for culture media was sterilized by autoclaving (121°C for 20 min) and filtering through a 0.22-µm filter unit (VFTB-1000, VIOLAMO, Japan). When used in the feeding experiments and component analysis, exponentially growing cultures (average density, Prochlorococcus : NIES-2884: 3.51 × 10 8 cells mL − 1 ; NIES-2887: 2.81 × 10 8 cells mL − 1 ; Heterocapsa : 3.35 × 10 5 cells mL − 1 ; Isochrysis : 2.78 × 10 7 cells mL − 1 ; Chaetoceros : 2.84 × 10 7 cells mL − 1 ) were concentrated by centrifugation (KUBOTA Model 8420, Japan) at 2,280 × g for 50 min, rewashed to remove nutrients from the culture medium, and resuspended in sterile artificial seawater. Preparation of cultured leptocephali gut contents and mucus Fish To obtain Japanese eel larvae for feeding and starvation experiments, female and male adult eels were induced to maturity using a hormonal treatment at the Minami-izu, Nansei, or Shibushi Field Station of the Fisheries Technology Institute, 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 ; Nomura et al. 2018 ; Kazeto et al. 2023 ; Suzuki et al. 2024 ). Larvae obtained from spawning eggs were stored in a 30-L or 200-L semicylindrical tank supplied with filtered seawater (10 L min − 1 ) at 25°C and then fed 5 times a day at 2 h intervals with a fishmeal-based slurry-type diet (Furuita et al. 2024 ) until the feeding or starvation experiment was conducted. Food organisms The food organism formulations used for the feeding experiments are shown in Table 2. In general, algae and ciliates are microorganisms that exist in aquatic environments (Lundgreen et al. 2019 ; Watanabe et al. 2021 ; Kume et al. 2025 ), and bacteria isolated from the gut contents of wild larvae are likely derived from POM ingested through environmental waters (Azam 1998 ; Azam and Malfatti 2007 ; Busch et al. 2017 ; Guo et al. 2023 ). Therefore, we used mainly algae and bacteria grown in the previous sections and ciliates as food organisms. Ciliates were isolated from seawater in the SCM layer (depth of 50 m) off Yamaga Bay in Kagoshima Prefecture (31°11.74' N, 130°42.81' E), where the Kuroshio Current inflows and a culture strain of Euplotes charon (strain SFS-Y2112) growing on Prochlorococcus spp. and Isochrysis galbana was obtained (Tomoda et al. manuscript in preparation). In the feeding experiment, the larvae were stored in a rearing tank in which these food source microorganisms were mixed, and rearing groups (algae alone, bacteria alone, algae + bacteria, or algae + bacteria + ciliates) were established in which the larvae were allowed to ingest environmental water. The amounts of algae (0.21–1.00 g w.w. L − 1 ) and/or bacteria (0.01–1.00 g w.w. L − 1 ) added to the rearing water were determined according to the amount (0.7–2.3 g w.w. L − 1 ) of slurry-type diet (Masuda et al. 2013 ; Furuita et al. 2014 , 2024 ) and liquid-type diet (Yamada et al. 2019 ) fed. With respect to the amount of ciliates added, the initial inoculation density was set at 15–20 individuals mL − 1 based on the assumption that the number of ciliates increases in the rearing water. Feeding experiments Feeding experiments were carried out using larvae aged 8–139 days post-hatch (dph) and with average total lengths of 7.5–36.3 mm from different hatcheries and broodstock (Table 2). For larviculture, we used recirculating aquaculture systems (RASs) (Fig. S1) consisting of a 2.5-L plankton kreisel tank (Okamura et al. 2009 ; Kenzaki et al. 2022 ) with a bimorph pump (BPH-214G, NITTO KOHKI) and 30-L cylindro conical tanks (SBF-30, EARTH CORPORATION, Japan) with an amphibious pump (RSD-10A, REI-SEA, Japan). These systems were created to constantly float eel larvae in the rearing water and to feed in POM into the middle layer. During the experiment, cultured larvae that had been starved for 18 hours were stocked into a rearing tank and allowed to ingest environmental water containing algae, bacteria and ciliates. The larvae were reared in ultraviolet-irradiated seawater (24°C, salinity 32) supplied by a flow-type UV sterilizer (Flonlizer FDL-4-SP, Chiyoda Kohan, Japan) after microfiltration through 0.5-µm cartridge filters (TCW-0.5N-PPD, Advantec Toyo Kaisha, Japan). The rearing water recirculating ratio was maintained at 65.5–70.7 cycles day − 1 . The photoperiod conditions were 9 L:15D (light period 8:00–17:00), and the photon flux density during the light period ranged from 0.118–0.125 µmol m − 2 s − 1 (dark period of 0.000 µmol m − 2 s − 1 ). We also conducted a feeding experiment using a 200-mL beaker in a static rearing system. To confirm food intake into the digestive tract, 10–15 larvae were collected from each tank 5–17 hours after the beginning of the experiment. The collected larvae were kept frozen, after which the gut contents were squeezed out and pipetted into separate sterile 1.5-mL Eppendorf tubes for SEM observation and component analysis. Starvation experiments Since the Japanese eel (Yamada and Yokote 1975 ) secretes mucus from goblet cells in the intestinal epithelium, it was considered that mucus was mixed in the gut contents. Therefore, as a comparison of the above feeding experiments, cultured larvae of two sizes (9 dph: TL 7.2 mm and 46 dph: TL 15.3 mm) raised on a fishmeal-based slurry-type diet (Furuita et al. 2024 ) were starved, and fasted individuals were collected at every timepoint, anaesthetized, and kept frozen (Table 2). For larviculture, we used a flow-through system consisting of a 10-L flat-bottom circular tank (Tanaka et al. 2001 ; Masuda et al. 2012 ). The rearing water exchange ratio was maintained at 86.4–100.8 cycles day − 1 . On a later day, the mid-hindgut was photographed using a stereoscopic microscope (SMZ1500, Nikon, Japan) attached to a photographic apparatus (DS-Fi3 and DS-L4, Nikon, Japan) to observe the mucus secretion status. In accordance with Masuda et al. ( 2010 ), the area of the mid-hindgut and the area of mucus occupied in the mid-hindgut were measured, and the area ratio was determined as mucous fullness (%). Image analysis software (NIS-Elements D ver. 5.42.06; Nikon, Japan) was used for the area measurements. The mucus was squeezed out and pipetted into separate sterile 1.5-mL Eppendorf tubes for SEM observations. SEM observations Food organisms (algae, bacteria, and ciliates) detected by genetic analysis of the gut contents (Ayala et al. 2018 ; Chow et al. 2019b ; Watanabe et al. 2021 ), food materials (POM in environmental water), and samples of the gut contents and mucus of A. japonica leptocephali (10 wild fish and 14 kinds of fed and starved cultured fish) were prepared for scanning electron microscope (SEM) imaging using the water freeze-drying method (Kuwata et al. 2018 , 2019 ) to confirm the structure of natural food. To observe the gut contents and mucus of the cultured fish, fourteen samples were pooled in batches of 1–5 individuals. The samples were coated with platinum (Pt) using an ion-sputter coater (E-1030; Hitachi, Tokyo, Japan). The coated samples were then observed using an SEM (JSM-6510LV, JEOL Ltd., Tokyo, Japan) at 100× magnification, an accelerating voltage of 15 kV, and a working distance of 11 mm. In a 4 mm × 4 mm sample, the area of 3,831 µm × 4,785 µm was divided into 12 (3 × 4) regions, and a single image of 1,920 × 2,560 pixels captured an area of 957 µm × 1,277 µm. Component analysis of food organisms and gut contents of leptocephali To estimate the nutritive value of natural food, proximate composition and amino acid analyses of food organisms and the gut contents of anguilliform leptocephali (wild Ariosoma sp., Conger sp. and 2 kinds of cultured A. japonica ) were carried out. For wild leptocephali, specimens collected in the same trawl nets as Japanese eels were used for analysis. For the analysis of the gut contents of eel larvae, twelve samples were pooled in batches of 3–11 individuals. The samples were homogenized with 5% TCA and centrifuged at 8,000 rpm (3,500 × g) for 10 min. The supernatant was collected and neutralized with sodium hydroxide, and the amino acids were determined using trinitrobenzene sulfonate (Hazra et al. 1984 ). After the precipitate was neutralized with sodium hydroxide, the protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Lipids were measured according to the methods of Holland and Gabbott ( 1971 ). After the lipids were extracted with a chloroform and methanol mixture, a portion was separated, and the solvent was removed with a nitrogen stream. The samples were then heated with sulfuric acid at 200°C for 15 minutes with a heating block and colorimetrically quantified with distilled water using a spectrophotometer. Tripalmitin was used as a standard. The carbohydrate content was determined by the phenol sulfuric acid method as described by Dubois et al. ( 1956 ). Statistical analysis The mucous fullness data expressed as percentages were first arcsine transformed; the arcsine-transformed data were then tested for significant differences between mean values. The assumptions of a normal distribution and homogeneity of variance were checked before statistical analysis; one-way ANOVA or a two-sided Student’s t test was used if normality and homoscedasticity were observed among the groups compared. When the one-way ANOVA was significant, differences among means were analysed using the Tukey‒Kramer multiple comparison test. All the statistical analyses were performed using Excel (Microsoft Office 365) with the add-in software Statcel 4 (OMS Publishing, Inc., Japan). Results Structure of food organisms and gut contents of leptocephali SEM observations of food source organisms (prochlorophytes, dinoflagellates, haptophytes, bacteria, and ciliates) revealed loose aggregation of extracellular products in algae and bacteria (Fig. 2 ). The gut contents (POM in environmental water) squeezed from anaesthetized cultured larvae were fragile and translucent gels with fluidity; however, they became light brown and increased in viscosity with time after death. When the gut contents of the wild larvae of Anguilliformes and the cultured larvae of Japanese eel were compared by stereomicroscopic observation, light microscopy revealed that the gut contents of both larvae were light brown and amorphous substances with fluidity, as previously reported (Miller et al. 2011 , 2019 , 2020 ; Tomoda et al. 2018 ; Tsukamoto and Miller 2021 ) (Fig. 3 , Fig. S2). In the gut contents of Japanese eel wild larvae (10 specimens), no traces of food organisms, such as the autofluorescence of algae or the faecal pellets and carcasses of zooplankton, were observed, and SEM observations confirmed the presence of amorphous substances, which seemed to originate mostly from polysaccharides and proteins derived from algae and bacteria (Fig. 4 ). The gut contents of Japanese eel-cultured larvae (44 samples) were similar to those of wild larvae; however, the structure differed according to the microbial composition of the ingested environmental water, the digestive ability of the larvae depending on their age (body size), or the progress of digestion and absorption (Fig. 5 ). In particular, the structural properties of the gut contents of artificial larvae, whose body sizes were the same as those of wild larvae, were very similar (Fig. 5 g, h, i; 136–139 dph). Furthermore, the structural properties of POM in environmental water containing algae and bacteria were very similar to those of the gut contents and differed slightly according to the composition of the food source organisms added (Fig. 6 ). Structure of mucus and estimation of the mucus secretion status of cultured leptocephali Mucus in the mid-hindgut squeezed from anaesthetized cultured larvae was a fragile and transparent gel with fluidity, but it became light brown and increased in viscosity with time after death, similar to the composition of the gut contents. Although individual differences were observed in fasted-cultured larvae, we confirmed that they secreted sol- or gel-like mucus similar to the gut contents of wild larvae and cultured larvae ingesting POM in environmental water (Fig. 7 a, b, c). Mucus was also observed at 72 h after fasting, and there was no difference in mucus secretion until 48–49 h after fasting in both 9 dph (TL7.2 mm) and 46 dph (TL15.3 mm) larvae (Fig. 7 d, e). However, in 9-day-old larvae, the mucous fullness at 72 h was significantly lower than those at 24 and 48 h ( P < 0.01 and P < 0.05, respectively; Fig. 7 e). At the same fasting time (48–49 h), 11-day-old larvae had significantly greater mucous fullness than 48-day-old larvae did ( P = 2.467×10 − 12 ; Fig. 7 f), but their mucous fullness was clearly lower than the intestinal fullness of wild larvae and cultured larvae ingesting POM in environmental water (Fig. 7 g). When SEM observation of the gut contents and mucus was conducted, we considered the possibility that autodigested intestinal epithelial tissues (e.g., goblet cells, cilia, and microvilli) were mixed in the squeezed contents. Therefore, the mid-hindgut of cultured larvae was dissected and examined, but the structural properties clearly differed from those of food (POM in environmental water) and mucus (Fig. S3). Proximate composition of food organisms and gut contents of leptocephali There were marked differences in the composition ratios of the 5 algal and 10 bacterial strains among the species and strains. The total ratio of proteins and amino acids to the total of the four components of proteins, amino acids, lipids, and carbohydrates was 68.4% (54.1–84.9%) on average for all 15 strains of algae and bacteria (Fig. 8 ). The total ratio of proteins and amino acids in the 5 bacterial strains (average 74.0%) was greater than those in the 10 algal strains (average 57.2%). In particular, the average abundance of 3 strains of Psychrobacter , which are the dominant genera of POM, was 83.3%, which was markedly greater than that of the body component of the cultured larvae (66.9%). Similarly, there were marked differences in larval gut contents among fish species and diets (POM in environmental water), with a greater total ratio of proteins and amino acids in cultured Japanese eel larvae (average of 47.5%) than in wild larvae of Ariosoma and Conger (average of 39.1%). Moreover, the gut contents of wild and cultured larvae contained markedly greater percentages of carbohydrates (43.1–67.2%) than their original food components did (3.4–30.7%), probably because proteins and amino acids were absorbed through digestion, and cell wall components (e.g., cellulose, hemicellulose, and peptidoglycan) remained. In addition, when the gut contents of cultured Japanese eel larvae that ingested algae, bacteria and their produced substances were compared with those of wild larvae (conger eels), no marked differences were noted in the component composition, but the ratio of amino acids in cultured larvae (average 10.6%) was twice as high as that in wild larvae (average 5.3%). Discussion Investigation of feeding habits in this study In this study, we confirmed the phenomenon in which cultured Japanese eel larvae ingest algae, bacteria and their produced substances via environmental water, resulting in the occurrence of amorphous substances similar to those of wild larvae in the intestine through digestion, absorption and metabolism. As a result, the understanding of the structural properties and components of natural food and the feeding ecology of eel larvae was improved, and the elucidation of the formation mechanism of the feeding environment was approached. Based on the results of a component analysis of food organisms and gut contents, carbohydrates, proteins and amino acids (Thornton 2018 ; Ogasawara et al. 2021 ; Biller et al. 2022 ; Casillo et al. 2024 ) produced by algae and bacteria were assumed to be among the substrates and nutrient sources of the gut contents, and these aggregates were considered to be the principal components of natural food. The results of component analysis as well as previous findings (Miller et al. 2013 ) suggest that algae and bacteria can contribute nutritionally to the growth and survival of eel larvae. In a feeding survey prior to this study, there were no significant differences in intestinal fullness (Masuda et al. 2010 ) between the algae solo feeding, algae + bacteria combined feeding, and algae + bacteria + ciliates combined feeding conditions ( P = 0.5044, ANOVA). However, significant differences in intestinal fullness were observed among the three algal feeding groups ( Prochlorococcus sp., Heterocapsa niei , Isocrysis galbana ; P = 0.0149; ANOVA), and the abundance of Prochlorococcus , the habitat-dominant species (Zhang et al. 2008 ; Flombaum et al. 2013 ; Watanabe et al. 2021 ), was significantly greater than that of Isocrysis , the coastal species ( P < 0.05; Scheffe's F test). These results revealed that the feeding ability of algae solo feeding of habitat-dominant species was comparable to that of wild larvae but suggested that the involvement of bacteria is necessary to increase nutritional supplementation, digestive absorption, and immune functions (Yukgehnaish et al. 2020 ; Patel et al. 2022 ; Diwan et al. 2023 ; Morshed and Lee 2023 ; Yamada et al. 2025 ) to improve growth and survival. However, 5 out of 33 strains of Pseudoalteromonas sp., which we obtained by culturing gut contents, showed algicidal activity (Inaba et al. 2016 ; Coyne et al. 2022 ; Wang et al. 2022 ; Zhong et al. 2023 ) against Isocrysis galbana . Therefore, these algicidal bacteria may contribute to the degradation of algae and the digestion and absorption of eel larvae (Tomoda, unpubl data). That is, compared with the cell walls of gram-positive bacteria and eukaryotic algae, the peptidoglycan layer of Prochlorococcus sp. (Ting et al. 2007 ), a habitat-dominant species, is thought to be thinner and more easily digested; thus, it may be more useful as food. According to recent feeding habit surveys in the field and observations of feeding behaviour in the laboratory, submicrometre particles (SMP) (Koike et al. 1990 ; Yamasaki et al. 1998 ) and gel-like particles (Yamada et al. 2016 ), which commonly exist in environmental waters and are easily swallowed, i.e., POM and DOM, including substances produced by algae and bacteria (TEP, CSP, EPS, MVs, and EVs) (Long and Azam 1996 ; Biller et al. 2014 , 2022 , 2023 ; Scanlan 2014 ; Busch et al. 2017 ; Decho and Gutierrez 2017 ; Mari et al. 2017 ; Thornton 2018 ; Tomoda et al. 2018 ; Daly et al. 2023 ; Casillo et al. 2024 ), constituting a high proportion of marine biomass, may be utilized directly by pinocytosis in the intestinal epithelium (Tanaka 1972 ; Watanabe 1982 ; Otake et al. 1993 ; Kurokawa et al. 1996 ; Otake 1996 ) without microbial degradation. It is assumed that the differences in the structural properties of the gut contents of cultured larvae are caused not only by differences in digestive ability and mucus secretion with days post-hatching but also by differences in the properties of the POM ingested, species of food organisms, and their products. The gut contents containing carbohydrates, proteins, and amino acids are likely derived from aggregates of TEP, CSP, and ESP present in environmental water. Thus, if the feeding habits at lower trophic levels (Miller et al. 2013 ) are able to utilize algae, bacteria, and their products, which account for a large proportion of marine biomass, it is assumed that adaptation to oligotrophic environments (Pilskaln et al. 2005 ; Onda et al. 2017 ; Ayala et al. 2018 ; Chow et al. 2019b ; Lundgreen et al. 2019 ; Watanabe et al. 2021 ) is possible. Consistency with past feeding habits surveys and feeding behaviour observations Similar to our previous feeding habit surveys of wild larvae (Tomoda et al. 2018 ; Chow et al. 2019b ; Watanabe et al. 2021 ), microscopic observations in this study revealed almost no evidence of eukaryotes (autofluorescence, faecal pellets and carcasses). In addition, feeding surveys on zoo- and phytoplankton subjected to various treatments (freezing and thawing, alkaline hydrolysis, and enzymolysis) revealed that cultured eel larvae could not ingest and digest food with hard physical properties such as cell walls, shells, and spines that retained their original forms (Tomoda, unpubl data). Consequently, feeding habits that do not utilize the organism itself as a direct source of nutrition (Terahara et al. 2011 ) have been reconfirmed. Moreover, we have recently conducted feeding surveys on 395 diets prepared from various biological materials (e.g., seaweed, thraustochytrids, Euglena , coastal microalgae, bacteria, zooplankton, and jellyfish carcasses), including microorganisms that have been identified as potential food elements (Ayala et al. 2018 ; Miller et al. 2020 ). However, no food with good feeding properties was found that could stably reproduce feeding conditions similar to those of wild larvae (Table S1). Our feeding surveys revealed that discarded appendicularian houses and zooplankton faecal pellets (Otake et al. 1993 ; Mochioka and Iwamizu 1996 ), which have been reported to be food sources, were not significantly attractive for feeding, and the intestinal fullness was also apparently low (Tomoda et al. 2015 ; Fig. S4). In particular, in the latest feeding surveys, no positive feeding behaviour was observed on zooplankton faecal pellets ( Artemia franciscana , 23 cases; Acartia tonsa , 10 cases) or jellyfish carcasses ( Bolinopsis mikado , 2 cases; Aurelia coerulea , 1 case), and both the feeding ratio and intestinal fullness were significantly low (Fig. S4; Table S1). The digestibility of zooplankton faecal pellets and phytoplankton was low, and the residual autofluorescence in the gut contents was more pronounced than that in the wild larvae (Fig. S4). Therefore, discarded appendicularian houses and the carcasses and faecal pellets of zooplankton seem not to be positively consumed by eel larvae, and our observations are also supported by those of Kume et al. ( 2025 ). However, these differences in observations may be influenced by spatiotemporal differences in food abundance between coastal and open ocean areas, dietary changes during development, and differences among species of Anguilliformes. Our findings are also supported by those of Chow et al. ( 2019b ), who reported that these biological materials are not direct food elements for eel larvae because they have no attractive properties for feeding and impair feeding and swallowing functions. Considering these findings, the needle-shaped larval teeth (Yoshimatsu 2011 ) and velum (Miller 2009 ) of anguilliform leptocephali may function as organs for screening foreign substances other than food that are difficult to eat, swallow and digest. Furthermore, recent molecular biology surveys of feeding habits (Riemann et al. 2010 ; Terahara et al. 2011 ; Ayala et al. 2018 ; Chow et al. 2019b ; Watanabe et al. 2021 ; Kume et al. 2025 ) might have indicated the detection of environmental DNA (Zhao et al. 2021 ; Feng et al. 2022 ; Sildever et al. 2023 ) by cross-contamination from the body surface and/or drinking environmental water (Lee et al. 2013 ; Ahn et al. 2015 ). That is, environmental DNA may be captured by POM via TEP, CSP, and ESP as attachment substrates and may reflect the results of analyses in which environmental DNA is ingested by larvae. Moreover, in the past, when a large portion of eel larvae were detected by 18S rRNA gene analysis of gut contents, the mucus (Yamada and Yokote 1975 ) of the intestinal epithelium and environmental DNA (Takeuchi et al. 2019 ) of eel larvae themselves may have been detected instead of food intake (POM). These results suggest that eel larvae preferentially utilize sugars, proteins, and amino acids contained in extracellular products (e.g., TEP, CSP, and ESP), which are the origin of marine snow (POM), rather than utilizing algal cell bodies or zooplankton carcasses and faecal pellets as a nutrient source. Moreover, Prochlorococcus sp., which dominate the habitat, is a prochlorophyte with remarkable attenuation and quenching of autofluorescence. Therefore, these genes may not have been detected by fluorescence microscopy or eukaryotic 18S rRNA gene analysis, and their contribution to the feeding environment may be underestimated. Alternatively, the peptidoglycan layer (Ting et al. 2007 ) and bacterial extracellular vesicles (Biller et al. 2014 , 2022 , 2023 ; Scanlan 2014 ) of Prochlorococcus sp. may be more physically fragile than the cell walls of eukaryotic algae are and thus may be easier to digest and absorb by pinocytosis. Therefore, we can fully understand why in many previous reports, traces of food organisms (e.g., cell walls, shells, and autofluorescence) were rarely found in the gut contents, and spherical picoplankton (Tomoda et al. 2018 ) were frequently found. Many biological materials with physical properties that leptocephali cannot digest and absorb are also present in POM at the depth of their habitat; accidental ingestion of these materials has been confirmed to lead to deaths from intestinal obstruction and intestinal necrosis (Chow et al. 2017 ; Tomoda, unpubl data). As mentioned above, not only the needle-shaped larval teeth and velum but also the olfactory characteristics (Watanabe 2017 ) and feeding selectivity (Kenzaki et al. 2022 ) of leptocephali may play a role in preventing unexpected deaths due to the accidental ingestion of these foreign substances. Furthermore, artificial digestion experiments using digestive enzymes purified from the intestine of cultured larvae (over 200 dph) revealed that the gut contents squeezed out from wild larvae were not digested (Tomoda, unpubl data). Therefore, the gut contents, which had been regarded as undigested food just after feeding, may have been close to the state of faeces; that is, the residues were completely digested and absorbed by the larvae after being decomposed by algicidal bacteria and intestinal bacteria. Structural properties of the gut contents The structural properties of the gut contents of wild larvae were similar to those of POM in environmental water where algae and bacteria coexist, and they were in a sol- or gel-like state with a high proportion of carbohydrates. These findings reflect previous findings that leptocephali have a high ability to digest proteins but a low ability to digest carbohydrates (Kurokawa et al. 1996 , 2002 , 2004 ; Pedersen et al. 2003 ; Hsu et al. 2015 ). The results of the component analysis of food organisms and gut contents also suggest that low-molecular-weight saccharides, proteins, and amino acids produced extracellularly by algae and bacteria may be used as nutrient sources. Eel larvae may grow and survive by constantly drinking seawater containing these produced substances, taking up nutrients through pinocytosis in the intestinal epithelium. In other words, algal and bacterial products are presumed to be the source of the substrate structure and nutrition of the gut contents, and these aggregates may form a food web as a major component of natural food. The mechanism by which gel-like substances accumulate in the intestinal tract through the ingestion of environmental water containing algae and bacteria is assumed to indicate that low-molecular-weight saccharides, proteins, amino acids, and water are digested and absorbed, whereas carbohydrates derived from cell walls and produced polysaccharides remain undigested. The structural properties of the gut contents were considered to differ depending on factors such as the species of food organisms added to the environmental water, the difference in carbohydrate and protein contents with the added ratio, the difference in digestive abilities and the progress of digestion and absorption with the age of the larvae. To improve food intake and retention time in the intestine, the presence of fine particles (Furuita, unpublished data) and substrates that adsorb organic matter (Tomoda et al. 2015 ) is necessary. Additionally, the inclusion of proteins on the surface of substrates and particles increases food intake (Kenzaki et al. 2022 ). This finding was also confirmed by surveys of the feeding habits of wild larvae (Tomoda et al. 2018 ). In other words, because fine particles are less likely to be retained in the intestinal tract in the case of liquids with low viscosity, it has been found empirically that fragile gels such as marine snow (Alldredge and Silver 1988 ; Lampitt et al. 1993 ; Chajwa et al. 2024 ) or sols consisting of colloidal particles or SMP are preferable in terms of feeding efficiency. In addition, a matrix structure with high porosity is advantageous for maintaining the buoyancy of food, and organic matter decomposition is promoted by the presence of an attached substrate that provides a habitat for bacteria (Kajihara 1987 ; Shanks and Edmondson 1989 ; Yamada et al. 2016 ; Hou et al. 2025 ). Recently, the availability of colloid-type (Masuda et al. 2010 ) and liquid-type (Okamura et al. 2019 ; Yamada et al. 2019 ) diets that simulate such physical properties has been reported. Furthermore, prolonged encounters with a floating-type diet under dark conditions have been shown to enable stable midwater feeding (Shioura et al. 2025 ). Therefore, the current standard mode of rearing, which uses high-viscosity and high-gravity artificial feed (Furuita et al. 2024 ; Jinbo et al. 2025 ; Sudo et al. 2025 ), has room for improvement even to expand the production scale. Mucus function The European eel Anguilla anguilla (Domeneghini et al. 2005 ), the Japanese eel Anguilla japonica (Yamada and Yokote 1975 ), and the Whitespotted conger Conger Myriaster (Takiue and Akiyoshi 2014 ) are known to secrete both acidic and neutral mucus. Mucus secreted on the mucosal epithelial surface of the digestive tract, together with microvilli, has been implicated in digestion and absorption (Grau et al. 1992 ; Otake et al. 1993 ; Otake 1996 ; Tibbets 1997). In this study, amorphous substances such as the gut contents of wild larvae were detected in the mid-hindgut of fasted cultured larvae. These are considered to comprise the mucus secreted from the intestinal epithelium, and there is a high possibility that mucin, which is considered to be the main component of mucus, was also intermingled in previous surveys of the feeding habits of wild larvae. The gut contents (mucus and POM) of both wild and cultured larvae observed after sampling were likewise all light brown, suggesting that metabolic and physiological functions in the intestine may be involved. In this study, we revealed individual differences in the mucous fullness of cultured larvae. However, this may be due to changes in digestive enzymes and mucus secretion and the intestinal microbiome by not only environmental factors (Morshed and Lee 2023 ) but also developmental stages and starvation conditions (Xia et al. 2014 ) of the larvae. The greater amount of mucus secretion in 10-day-old larvae observed in this study may indicate a greater feeding ability (POM trapping efficiency) in the early larval stage, and the lower amount of mucus secretion in 47-day-old larvae may indicate an improvement in starvation resistance after mass mortality events at the early larval stage. It is speculated that prolonged mucus secretion under starvation conditions efficiently and effectively traps and retains fine POM in the intestinal tract, thereby prolonging the opportunity for nutrient absorption. That is, mucus secretion in anguilliform leptocephali is considered to contribute greatly to feeding behaviour in oligotrophic oceans. During the ingestion of environmental water, POM and DOM may be trapped in the mucus layer of the intestinal epithelium or on the surface of cilia and microvilli and absorbed by pinocytosis. This feeding mode seems to resemble suspension feeding (Riisgård and Larsen 2010 ), such as the mucous mesh feeding of pelagic tunicates and thecosome pteropods (Hiebert et al. 2025 ), or the ciliary filter feeding of bivalves and polychaetes (Riisgård et al. 2000 ; Okutani and Kurokawa 2020 ). This hypothesis is supported by previous reports (Kenzaki et al. 2022 ; Shioura et al. 2025 ). That is, the phenomenon in which picocyanobacteria are fed through environmental water ingestion, circulate in the mid-hindgut by ciliary movement, and remain and aggregate in the intestine for a certain period of time is considered to be a result of capturing fine particles in the mucus layer of the intestinal epithelium, as well as on the surfaces and gaps of cilia and microvilli (Fig. S3). Mucus secretion in the intestinal epithelium (Yamada and Yokote 1975 ) is likely closely involved in the feeding habits that make POM, DOM, and SMP (Koike et al. 1990 ; Otake et al. 1993 ; Yamasaki et al. 1998 ) available and may be a shortcut in the food chain pathway. Additionally, aggregates of bacterial strains ( Erythrobacter flavus ) from POM and picocyanobacteria ( Synechococcus sp.) were digested and absorbed in the posterior mid-hindgut of cultured larvae during microscopic observation (Inaba et al. unpubl data; KY1704 cruise), suggesting pinocytosis. Thus, a feeding mode in which pico- and nanosized POM and DOM can be retained and digested in the mid-hindgut is considered to be an efficient and effective survival strategy for eel larvae in depth zones where food is scarce and oligotrophic. These findings indicate that the improvements in growth and survival associated with a low-viscosity liquid-type diet (Okamura et al. 2019 ; Yamada et al. 2019 ) may reflect the abovementioned advantages of the feeding mode. Furthermore, the mucus layer not only provides nutritional glycoproteins (Bakke et al. 2010 ) and habitats for intestinal bacteria but also contributes to biological defence against pathogenic microorganisms (Jutfelt 2011 ). Although we cannot deny the possibility that some mucus containing glycoproteins or digestive enzymes was involved when the gut contents were squeezed, all the amorphous substances found in all individuals were unlikely to be mucus derived from eel larvae. Factors contributing to the establishment of the feeding environment Previous evidence has suggested that marine snow, which mainly consists of eukaryotes, might be a food source for leptocephali (i.e., carcasses and faecal pellets of zoo- and phytoplankton that have been decomposed by microorganisms into sizes and physical properties that can be swallowed by eel larvae) (Chow et al. 2019b ; Miller et al. 2020 ; Tsukamoto and Miller 2021 ; Watanabe et al. 2021 ; Kume et al. 2025 ). However, the results of this study suggest that food webs are formed in which prokaryotes, such as cyanobacteria as primary producers and bacteria as decomposers as well as consumers, are the main food sources. As mentioned above, Prochlorococcus sp. and bacteria are known to dominate habitat biomass (Zhang et al. 2008 ; Watanabe et al. 2021 ) and represent a high proportion of POM (Watanabe et al. submit to PeerJ). The bacterial extracellular vesicles (EVs) (Biller et al. 2014 , 2023 ; Casillo et al. 2024 ) released by these prokaryotes contain proteins and nucleic acids. Recently, reported effects of nucleic acid supplementation (Jinbo et al. 2025 ) on the survival, growth, and metamorphosis of cultured larvae may indicate that EVs contribute to the feeding environment of wild leptocephali. In particular, bacteria influence food web dynamics (Long and Azam 1996 ; Azam 1998 ; Azam and Malfatti 2007 ; Koshikawa et al. 1999 ; Kogure 2006 ; Kato and Masuda 2014 ; Patel et al. 2022 ; Yamada et al. 2025 ), suggesting that algal–bacterial interactions in the phycosphere contribute to organic matter transport and energy transfer to higher trophic level organisms (Zhou et al. 2016 ; Seymour et al. 2017 ; Mühlenbruch et al. 2018 ; Cirri and Pohnert 2019 ). The components of POM and DOM, such as marine snow, are known to include monosaccharides (Skoog et al. 2008 ) as well as amino sugars and peptidoglycans (Benner and Kaiser 2003 ). Extracellular polymeric substances (EPS) are the origin of marine snow and are produced through interactions between algae and bacteria (Decho and Gutierrez 2017 ; Daly et al. 2023 ). These substances include not only polysaccharides and proteins but also monosaccharides that are available as nutrients for eel larvae (Okamura et al. 2014 ). Additionally, the carbon‒nitrogen stable isotope ratio of POM-dominant bacteria is close to the trophic level of marine snow (Watanabe et al. submit to PeerJ), and the results of the component analysis in this study also indicate sufficient nutritional value for food. Therefore, the microbial loop (Azam 1998 ; Azam and Malfatti 2007 ) and phycosphere (Zhou et al. 2016 ; Seymour et al. 2017 ; Mühlenbruch et al. 2018 ; Cirri and Pohnert 2019 ) seem to contribute as a feeding ground to provide highly nutritious POM and DOM for eel larvae. Recently, we confirmed that there is enough POM just below the subsurface chlorophyll maximum (SCM) layer for eel larvae to satiate in a short time (Watanabe et al. unpubl data). The SCM layer is densely dotted with “hot spots” (Azam 1998 ) of biogenic SMP and POM, and eel larvae may both passively and aggressively utilize these organic substances during diel vertical migration (Kudo 2001 ; Chang et al. 2015 , 2018 ). Future perspectives The results of this study suggest the following possibilities. The gut contents include mucus secreted from the intestinal epithelium, as well as substances produced by algae and bacteria. The mucus layer of the intestinal epithelium contributes to efficiently trap particles from environmental water for feeding. Eel larvae utilize mainly low-molecular-weight saccharides, proteins, and amino acids contained in algal and bacterial products. The carcasses and faecal pellets of zoo- and phytoplankton are not the main food for eel larvae. POM and DOM are directly utilized by pinocytosis in the intestinal epithelium without microbial degradation. In previous studies of feeding habits based on microscopic observation and molecular biology, a food web has been proposed in which aggregates (POM) of the carcasses and faecal pellets of zoo- and phytoplankton degraded by microorganisms are the main food. However, a combination of relevant findings and our ten-year field and laboratory feeding surveys suggest that the feeding ecology of eel larvae in the mesopelagic zone of low-latitude oceans (Rodgers et al. 2024 ) may be directly supported by a lower trophic level ecosystem that underlies the microbial loop and phycosphere rather than by a food web, as previously assumed (Watanabe et al. 2021 ) (Fig. 9 ). To conserve natural resources and stabilize the aquaculture industry, we must not only manage resources through industry–government–academia cooperation but also establish glass eel mass production technology and realize true social implementation. To achieve this goal, the development of innovative feed and the transformation of seedling production forms are urgent issues. In particular, to eliminate bottlenecks in cost and labour savings and expand production scale, we need to create a foraging environment with a low environmental load, such as production forms using food organisms (Hagiwara et al. 2001 ; Tomoda et al. 2011 , 2016 ), by utilizing the elucidation of feeding ecology at the leptocephalus stage. However, in the current eel research field, almost no research has been conducted on microbial ecology (Lampitt et al. 1993 ; Kogure K (ed) 2006; Azam and Malfatti 2007 ) to elucidate the feeding environment or on microbial engineering to produce marine snow (Kajihara 1987 ; Shanks and Edmondson 1989 ; Yamada et al. 2016 ; Hou et al. 2025 ) as food. In the future, basic research should continue to elucidate the entire feeding ecology of leptocephalus in terms of its nutritional physiological function (capture of POM and DOM by the mucus and pinocytosis of POM and DOM in the intestinal epithelium), its habitat, and the establishment mechanism of the feeding environment based on the lower trophic level ecosystem. Additionally, advancements should be made in the development of feed that effectively utilizes the potential of microorganisms for the mass production of seedlings. Conclusions Although many reports have shown that leptocephali consume mainly POM, the mechanism underlying how they effectively capture, ingest, digest, and absorb small POM has not been elucidated. In this study, we obtained new information about the feeding ecology of anguilliform leptocephali, which has been a mystery for many years, by comparing the gut contents of wild and cultured larvae. The gut contents of eel larvae and food organisms were observed via light and scanning electron microscopy observations to clarify the morphological characteristics and component composition and to elucidate the feeding ecology of eel larvae. In particular, the close relationship between mucus secretion and the feeding mode in the intestine, which has not been previously reported, seems to be related to not only survival strategies in oligotrophic oceans but also adaptive evolution to the environment. Importantly, this paper clarified the possibility that mucus secretory function contributes to the feeding mode. Another interesting observation is the possibility that the difference in mucus secretion at the developmental stage leads to the improvement of starvation resistance. In considering the feeding ecology of anguilliform leptocephali in nature through laboratory feeding surveys, it is difficult to comprehensively grasp the morphological and biochemical knowledge of the food organisms, gut contents, and mucus focused on in this study. However, this study provides a basic understanding of how digestive and immune organs developed during the evolution of leptocephali. Our findings promote research on the feeding ecology of anguilliform leptocephali and thus contribute to the development of aquaculture research. Declarations Supplementary Information The online version contains supplementary material available at https://doi.org/ Acknowledgments We thank Keisuke Yamano (former Director of the Aquaculture Research Department at the Fisheries Technology Institute, FRA ), Hiroaki Kurogi (Director of the Socio-Ecological Systems Division at the Fisheries Resources Institute, FRA ) and Daisuke Hasegawa (Shiogama Field Station, FRA ) for their support in conducting this study. We would also like to extend our thanks to the helpful crew of R/V Soyo-Maru for performing the collection of the leptocephali. We are sincerely grateful to the staff of the Minami-izu, Nansei, and Shibushi Field Stations of FRA for their assistance with providing cultured leptocephali. We also thank the staff of Hatsukaichi and Momoshima Field Stations of FRA for sharing Heterocapsa niei and microbial flocks and Hideaki Matsui (National Fisheries University, FRA ) and Tomoya Kotani (Faculty of Fisheries, Kagoshima University) for sharing faecal pellets of the copepods Acartia tonsa and Masahiro Hayashi (Faculty of Agriculture, University of Miyazaki) for sharing Thraustochytrids and Euglena with us. We sincerely appreciate the reviewers for their suggestions and comments. This study utilized samples collected through the Fisheries Resource Survey and Evaluation Promotion Project commissioned by the Fisheries Agency, Ministry of Agriculture, Forestry and Fisheries of Japan. Author contributions TT, TW, KT, SN and MU conceived the study. All authors contributed to the design of the conceptual framework and analyses. DA, NF, KH, YN and YS conducted ship surveys and collected the leptocephali. TT, TW, KT, SN and MU conducted the laboratory experiments and observation. HF, TY and SN conducted the analyses. TT wrote the first draft of the manuscript. All authors have contributed to the revision and improvements of the manuscript. Funding This study was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (The Special Scheme Project on Advanced Research and Development for Next-generation Technology) and the project "Development of biological material feeds" commissioned by FRA as part of the "Demonstration project of a mass-production system for commercialisation of eel seedlings" from the Ministry of Agriculture, Forestry and Fisheries of Japan. Data availability The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author, upon reasonable request. 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Supplementary Files FigS1.tiff Figure S1 Recirculating aquaculture systems (RASs) for rearing Anguilla japonica leptocephali. (a, b) A 2.5-L plankton kreisel tank with a bimorph pump; (c, d) 30-L cylindro conical tanks with an amphibious pump FigS1.tiff Figure S1 Recirculating aquaculture systems (RASs) for rearing Anguilla japonica leptocephali. (a, b) A 2.5-L plankton kreisel tank with a bimorph pump; (c, d) 30-L cylindro conical tanks with an amphibious pump FigS3.tiff Figure S3 Cross-sectional SEM micrographs of the mid-hindgut of cultured Anguilla japonica leptocephali. (a) Intestinal epithelial tissue (76 dph: TL 25.6 mm), (b) enlarged view. Scale bars (a): 20 μm, (b): 10 μm FigS3.tiff Figure S3 Cross-sectional SEM micrographs of the mid-hindgut of cultured Anguilla japonica leptocephali. (a) Intestinal epithelial tissue (76 dph: TL 25.6 mm), (b) enlarged view. Scale bars (a): 20 μm, (b): 10 μm TableS1.docx Table S1 Biological material feeds that had been poorly fed to cultured Anguilla japonica leptocephali Tables12.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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(\u003cstrong\u003ea\u003c/strong\u003e) The grey square indicates our research area, (\u003cstrong\u003eb\u003c/strong\u003e) An enlarged view of the research area. Circles (◯) and triangles (△) indicate the sampling stations in 2020 (SY2005 survey) and 2022 (SY2204 survey), respectively\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/4698096f58c675ba7c656168.png"},{"id":97137928,"identity":"81af5a5d-be79-4ade-9c8b-6e1c45ef2a75","added_by":"auto","created_at":"2025-12-01 09:58:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1465981,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of food source organisms. (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eProchlorococcus\u003c/em\u003esp. (NIES-2884), (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eHeterocapsa niei\u003c/em\u003e (NIES-420), (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eIsocrysis galbana\u003c/em\u003e (NRIA-0074), (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eTenacibaculum mesophilum\u003c/em\u003e (No. 114), (\u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eParacoccus stylophorae\u003c/em\u003e, (No. 143), (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eThalassomonas agariperforans\u003c/em\u003e (M-M1), (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eErythrobacter flavus\u003c/em\u003e (No. 39), (\u003cstrong\u003eh\u003c/strong\u003e) \u003cem\u003ePsychrobacter faecalis\u003c/em\u003e (No. 84), and (\u003cstrong\u003ei\u003c/strong\u003e) \u003cem\u003eEuplotes charon\u003c/em\u003e(Ciliata). Scale bars (\u003cstrong\u003ea, e, g, h\u003c/strong\u003e): 1 μm, (\u003cstrong\u003ec, d, f\u003c/strong\u003e): 2 μm, (\u003cstrong\u003eb, i\u003c/strong\u003e): 10 μm\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/e8d2d0edaeee445705b2b8bd.png"},{"id":96969878,"identity":"9ab57200-9950-457d-912e-d773ed17976c","added_by":"auto","created_at":"2025-11-28 07:11:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10776411,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the gut contents of wild and cultured eel leptocephali under light microscopy. Wild leptocephali fed POM: (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eAnguilla japonica\u003c/em\u003e (SY2204), (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eAriosoma\u003c/em\u003esp. (KY1903), and (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eGnathophis\u003c/em\u003e sp. (KY1903). Cultured \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali: (\u003cstrong\u003ed\u003c/strong\u003e) 3 algae and 4 bacteria and ciliate feeding (8 dph: TL 7.5 mm), (\u003cstrong\u003ee\u003c/strong\u003e) 1 alga and 1 bacterium feeding (9 dph: TL 7.5 mm), (\u003cstrong\u003ef\u003c/strong\u003e) 1 bacterium feeding (36 dph: TL 12.1 mm), (\u003cstrong\u003eg\u003c/strong\u003e) 1 alga feeding (36 dph: TL 12.1 mm), (\u003cstrong\u003eh\u003c/strong\u003e) 3 bacteria feeding (36 dph: TL 12.1 mm), and (\u003cstrong\u003ei\u003c/strong\u003e) 1 alga and 3 bacteria feeding (36 dph: TL 12.1 mm). Scale bars (\u003cstrong\u003ea, b\u003c/strong\u003e): 2 mm, (\u003cstrong\u003ec, d, e, f, g, h, i\u003c/strong\u003e): 200 μm\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/0434fdb4f8c33806a1896772.png"},{"id":97137985,"identity":"58047880-fe6a-4f00-bf10-1925a1b2c6f6","added_by":"auto","created_at":"2025-12-01 09:58:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8089239,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of the gut contents of wild \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali (SY2005). (\u003cstrong\u003ea\u003c/strong\u003e) No. 23 (TL 36.1 mm), (\u003cstrong\u003eb\u003c/strong\u003e) No. 24 (TL 37.9 mm), (\u003cstrong\u003ec\u003c/strong\u003e) No. 35 (TL 40.6 mm), (\u003cstrong\u003ed\u003c/strong\u003e) No. 36 (TL 41.2 mm), (\u003cstrong\u003ee\u003c/strong\u003e) No. 76 (TL 37.8 mm), (\u003cstrong\u003ef\u003c/strong\u003e) No. 82 (TL 38.0 mm), (\u003cstrong\u003eg\u003c/strong\u003e) No. 392 (TL 38.0 mm), (\u003cstrong\u003eh\u003c/strong\u003e) No. 394 (TL 38.5 mm), and (\u003cstrong\u003ei\u003c/strong\u003e) No. 403 (TL 32.6 mm). Scale bars: 10 μm, except for (\u003cstrong\u003ee\u003c/strong\u003e): 20 μm\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/965909c198a280533b526091.png"},{"id":97138032,"identity":"7ace1e0b-1521-4acd-ac4c-40e190852f6b","added_by":"auto","created_at":"2025-12-01 09:58:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9027214,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of the gut contents of cultured \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali: (\u003cstrong\u003ea\u003c/strong\u003e) 1 bacterium feeding (36 dph: TL 12.1 mm), (\u003cstrong\u003eb\u003c/strong\u003e) 3 bacteria feeding (36 dph: TL 12.1 mm), (\u003cstrong\u003ec\u003c/strong\u003e) 3 bacteria feeding (41 dph: TL 13.4 mm), (\u003cstrong\u003ed\u003c/strong\u003e) 1 bacterium feeding (41 dph: TL 13.4 mm), (\u003cstrong\u003ee\u003c/strong\u003e) 1 alga feeding (42 dph: TL 13.4 mm), (\u003cstrong\u003ef\u003c/strong\u003e) 3 algae and 4 bacteria and ciliate feeding (110 dph: TL 24.4 mm), (\u003cstrong\u003eg\u003c/strong\u003e) 3 algae and 4 bacteria feeding (136 dph: TL 36.3 mm), (\u003cstrong\u003eh\u003c/strong\u003e) 3 algae feeding (136 dph: TL 36.3 mm), and (\u003cstrong\u003ei\u003c/strong\u003e) 3 algae and 4 bacteria and ciliate feeding (139 dph: TL 36.3 mm). Scale bars: 10 μm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/3433bad95cbb64099d8858b7.png"},{"id":96969901,"identity":"8c8eee24-cd0a-48de-888c-109c0bed4a16","added_by":"auto","created_at":"2025-11-28 07:11:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4422216,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of POM in environmental water containing algae and bacteria. (\u003cstrong\u003ea, d\u003c/strong\u003e) Suspended particles composed of 3 algae and 4 bacteria, (\u003cstrong\u003eb, e\u003c/strong\u003e) suspended particles composed of 3 algae, and (\u003cstrong\u003ec, f\u003c/strong\u003e) suspended particles of 1 alga. The lower parts (\u003cstrong\u003ed, e, f\u003c/strong\u003e) show enlarged views. Scale bars (upper): 10 μm, (lower): 2 μm\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/a9378b86c99e5b6bfae60a61.png"},{"id":96969895,"identity":"23d454d1-304a-40fa-a2f1-0a4cd90adb56","added_by":"auto","created_at":"2025-11-28 07:11:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6284784,"visible":true,"origin":"","legend":"\u003cp\u003eMucus secretion in cultured \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali under starvation conditions. (\u003cstrong\u003ea\u003c/strong\u003e) 48 dph larva (49-hour fasted state) and (\u003cstrong\u003eb\u003c/strong\u003e) 11 dph larva (48-hour fasted state). (\u003cstrong\u003ec\u003c/strong\u003e) SEM micrographs of the mucus of 47 and 48 dph larvae (upper panel; 25-hour and lower panel; 39-hour fasted state). (\u003cstrong\u003ed\u003c/strong\u003e) Comparison of the time-course mucous fullness of 46-day-old fasted larvae. No significant differences were observed over the time course (ANOVA; n = 14–15 larvae examined at each time point). (\u003cstrong\u003ee\u003c/strong\u003e) Comparison of the time-course mucous fullness of 9-day-old fasted larvae. Significant differences were observed among the time courses (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Tukey–Kramer test; n = 30 larvae examined at each time point). (\u003cstrong\u003ef\u003c/strong\u003e) Comparison of the mucous fullness of 11 and 48 dph larvae (48- and 49-h fasted state). Significant differences were observed between the two groups (***; \u003cem\u003eP\u003c/em\u003e = 2.467 × 10\u003csup\u003e-12\u003c/sup\u003e; Student’s \u003cem\u003et\u003c/em\u003e test; n = 15 and 30 larvae examined, respectively). (\u003cstrong\u003eg\u003c/strong\u003e) Larvae fed POM consisting of algae and bacteria (upper panel; 36 dph and lower panel; 54 dph). Scale bars (\u003cstrong\u003ea\u003c/strong\u003e): 0.8 mm, (\u003cstrong\u003eb\u003c/strong\u003e): 0.3 mm, (\u003cstrong\u003ec\u003c/strong\u003e): 10 μm, (\u003cstrong\u003eg\u003c/strong\u003e): 0.5 mm (upper) and 0.8 mm (lower). Boxplots (\u003cstrong\u003ed, e, f\u003c/strong\u003e) represent the 10th, 25th, 50th (median), 75th and 90th percentiles, respectively, with open circle plots indicating the outliers\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/102f13f651bf46e02801991b.png"},{"id":96969904,"identity":"20ccbb5e-1002-4dbd-b955-76ca8e64513a","added_by":"auto","created_at":"2025-11-28 07:11:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":582352,"visible":true,"origin":"","legend":"\u003cp\u003eProximate composition of food organisms and gut contents of eel leptocephali. Data represent the mean. Food organism samples (n = 2) except for \u003cem\u003eThalassomonas\u003c/em\u003e (n = 4). The strain IDs of the algae and bacteria are indicated on the horizontal axis. Gut content samples of cultured larvae (\u003cem\u003eAnguilla japonica\u003c/em\u003e; n = 2) and wild larvae (\u003cem\u003eAriosoma\u003c/em\u003e sp.; n = 5; \u003cem\u003eConger\u003c/em\u003e sp.; n = 3). The parenthesis of the gut content sample indicates the food ingested by the larva\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/b80da559d9b0a0528eeb8367.png"},{"id":97137934,"identity":"92345289-d6fd-4820-ba9a-e96175daf64d","added_by":"auto","created_at":"2025-12-01 09:58:20","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":346942,"visible":true,"origin":"","legend":"\u003cp\u003eFood web of anguilliform leptocephali. (\u003cstrong\u003ea\u003c/strong\u003e) Estimated food web. POM, in which zoo- and phytoplankton carcasses and faecal pellets are decomposed by bacteria, is utilized as food for eel leptocephali. (\u003cstrong\u003eb\u003c/strong\u003e) New hypotheses of the food web: possibility that leptocephali utilize low-molecular-weight saccharides, proteins, and amino acids contained in the produced substances and possibility of direct utilization of POM and DOM by pinocytosis in the intestine without undergoing the microbial decomposition process. The feeding ecology of eel leptocephali may be supported mainly by a “phycosphere” composed of algae and bacteria\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/e62ce8ce0808824912c54942.png"},{"id":97249477,"identity":"335d2fcb-39e8-4de7-9e88-10acc0e44b9c","added_by":"auto","created_at":"2025-12-02 13:12:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":43229529,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/b88cbf88-7fbf-401a-accf-483aef627651.pdf"},{"id":96969882,"identity":"81f95b1c-94cb-46be-bc05-6a60a5c94a16","added_by":"auto","created_at":"2025-11-28 07:11:55","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2934656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e Recirculating aquaculture systems (RASs) for rearing \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali. (\u003cstrong\u003ea, b\u003c/strong\u003e) A 2.5-L plankton kreisel tank with a bimorph pump; (\u003cstrong\u003ec, d\u003c/strong\u003e) 30-L cylindro conical tanks with an amphibious pump\u003c/p\u003e","description":"","filename":"FigS1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/e90d92323b1ff87c43e5afa7.tiff"},{"id":97138077,"identity":"2ba5b7f1-d10d-453a-87e9-5d9f57820186","added_by":"auto","created_at":"2025-12-01 09:58:28","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2934656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e Recirculating aquaculture systems (RASs) for rearing \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali. (\u003cstrong\u003ea, b\u003c/strong\u003e) A 2.5-L plankton kreisel tank with a bimorph pump; (\u003cstrong\u003ec, d\u003c/strong\u003e) 30-L cylindro conical tanks with an amphibious pump\u003c/p\u003e","description":"","filename":"FigS1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/2ebe9a6c297968db80ca5fdb.tiff"},{"id":97137548,"identity":"321847a7-8906-44cb-803e-836344bbae1e","added_by":"auto","created_at":"2025-12-01 09:57:53","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2938120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3\u003c/strong\u003e Cross-sectional SEM micrographs of the mid-hindgut of cultured \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali. (\u003cstrong\u003ea\u003c/strong\u003e) Intestinal epithelial tissue (76 dph: TL 25.6 mm), (\u003cstrong\u003eb\u003c/strong\u003e) enlarged view. Scale bars (\u003cstrong\u003ea\u003c/strong\u003e): 20 μm, (\u003cstrong\u003eb\u003c/strong\u003e): 10 μm\u003c/p\u003e","description":"","filename":"FigS3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/47655a38912729bb9b0b6816.tiff"},{"id":96969891,"identity":"d7b30fde-5c6b-4ecd-92fc-f1e587f0d4ba","added_by":"auto","created_at":"2025-11-28 07:11:55","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2938120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3\u003c/strong\u003e Cross-sectional SEM micrographs of the mid-hindgut of cultured \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali. (\u003cstrong\u003ea\u003c/strong\u003e) Intestinal epithelial tissue (76 dph: TL 25.6 mm), (\u003cstrong\u003eb\u003c/strong\u003e) enlarged view. Scale bars (\u003cstrong\u003ea\u003c/strong\u003e): 20 μm, (\u003cstrong\u003eb\u003c/strong\u003e): 10 μm\u003c/p\u003e","description":"","filename":"FigS3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/8a19e24695fbebb7fb8c985e.tiff"},{"id":96969883,"identity":"4c6667c3-abb8-4466-b90e-0da337c66bc4","added_by":"auto","created_at":"2025-11-28 07:11:55","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":17944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e Biological material feeds that had been poorly fed to cultured \u003cem\u003eAnguilla japonica\u003c/em\u003e leptocephali\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/2e156fe8110881fe98ec9a0a.docx"},{"id":96969892,"identity":"48c947e6-89be-4b6e-8550-e9c3d3806843","added_by":"auto","created_at":"2025-11-28 07:11:56","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":27150,"visible":true,"origin":"","legend":"","description":"","filename":"Tables12.docx","url":"https://assets-eu.researchsquare.com/files/rs-8218055/v1/ac36fa3ffa5f3360aeb9b937.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eFeeding ecology of anguilliform leptocephali considering the structure and proximate composition of food organisms and gut contents\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, various proposals have been made to implement resource management and conservation policies for eels, and the importance of research focusing on biology and ecology has been noted as a clue to identify problems (Dekker \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kaifu et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Righton et al. \u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Shiraishi and Kaifu \u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Since the existing eel farming industry depends only on natural seedlings and because aquaculture using artificial seedlings has not yet been realized on a commercial scale, the establishment of sustainable complete aquaculture technologies (Tanaka et al. \u003cspan citationid=\"CR146\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Masuda et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tanaka \u003cspan citationid=\"CR145\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) is desired for the stabilization of seed prices and resource conservation. Therefore, it is necessary to innovate not only the feed but also the rearing form, which is a bottleneck in the expansion of production scale. An urgent task is to first clarify the feeding ecology in nature, which should be a model. Understanding the major species used as food sources and determining the feeding mode of eel larvae and the formation mechanism of the food environment will greatly contribute to understanding the conversion of seedling production forms. It will also lead to cost and labour savings and to the restructuring of the aquaculture industry, which in turn can be expected to protect natural resources and stabilize aquaculture management.\u003c/p\u003e\u003cp\u003eOur recent feeding studies of cultured larvae confirmed the feeding selectivity and density dependence associated with the food environment (Kenzaki et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and the influence of environmental factors such as salinity, food density, and photoperiod on feeding behaviour (Shioura et al. \u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To develop feed and rearing methods for mass production of glass eels in the future, we believe that it is important not only to focus on the composition and nutritional value of feed (Masuda et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Furuita et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR173\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) but also to develop technology based on fundamental information such as physiology, ecology and functional morphology (Uematsu et al. \u003cspan citationid=\"CR161\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Otake \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Tomoda and Uematsu \u003cspan citationid=\"CR152\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Ohta \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Miller \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Okamura et al. \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tsukamoto et al. \u003cspan citationid=\"CR157\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR171\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yoshimatsu \u003cspan citationid=\"CR177\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Politis et al. \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kuroki et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Miller and Tsukamoto \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Watanabe \u003cspan citationid=\"CR166\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chow et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Matsuda et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kuroki \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Masuda et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Knutsen et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Miller \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThere are various theories on the feeding habits of eel leptocephali, and particulate organic matter (POM), dissolved organic matter (DOM), faecal pellets and discarded houses of appendicularians have been assumed to be the most likely food sources (Otake et al. \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Mochioka and Iwamizu \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Recently, various approaches, such as microscopic observation (Govoni \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Miller et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and genetic analysis (Riemann et al. \u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Terahara et al. \u003cspan citationid=\"CR148\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ayala et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chow et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kume et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) of gut contents and stable isotope ratio analysis (Miyazaki et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Miller et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Feunteun et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chow et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ghinter et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kimura et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), have been applied, and many findings have been reported (Miller \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Miller et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tsukamoto and Miller \u003cspan citationid=\"CR158\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, feeding behaviour in nature has not yet been observed, and the actual conditions of food utilized by wild larvae, such as the composition of biomass as a food source (Lundgreen et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the contribution of each food element or abiotic fraction, and the feeding mode, are unknown. In particular, the origin of amorphous substances (Miller et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tsukamoto and Miller \u003cspan citationid=\"CR158\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which constitute a high proportion of the gut contents, is not yet clear. Furthermore, food environment surveys have concentrated on eukaryotes, whereas few studies have focused on prokaryotes. According to the estimation of the trophic level of natural food by the amino acid nitrogen isotope ratio (Miller et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), the food of eel leptocephali has been identified as marine snow (Alldredge and Silver \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Lampitt et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Chajwa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which originates from the carcasses of zoo- and phytoplankton; prokaryotes such as cyanobacteria and bacteria have also been suggested to be food components.\u003c/p\u003e\u003cp\u003eResearch in the field of marine ecosystem microbiology indicates that algae and bacteria account for a high proportion of biomass in the open ocean (Zhang et al. \u003cspan citationid=\"CR179\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Flombaum et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and are also components of the POM that eel leptocephali feed on, suggesting their importance in organic matter transport and contribution to food webs (Long and Azam \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Azam \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Verdugo et al. \u003cspan citationid=\"CR164\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kogure K (ed) 2006; Azam and Malfatti \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Tsukasaki and Tanoue \u003cspan citationid=\"CR159\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Biller et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Scanlan \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR182\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Busch et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mari et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Seymour et al. \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; M\u0026uuml;hlenbruch et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cirri and Pohnert \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Casillo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Chajwa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Algae, bacteria, and their products (Thornton \u003cspan citationid=\"CR149\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ogasawara et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) play roles as sources of nutrients for eel larvae, which possess specific feeding habits. The intestinal microbiota, which is composed of various bacteria, is thought to contribute to the enhancement of digestion and absorption, maintenance of homeostasis, and improvement of immune function (Yukgehnaish et al. \u003cspan citationid=\"CR178\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Diwan et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Morshed and Lee \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Other expected benefits of bacteria include probiotics (Schmidt et al. \u003cspan citationid=\"CR132\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yukgehnaish et al. \u003cspan citationid=\"CR178\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and synbiotics (Fujii et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, detailed observation and component analysis of the gut contents of eel larvae that ingested algae and bacteria from their habitat via environmental water may elucidate the actual food environment and feeding mode.\u003c/p\u003e\u003cp\u003eIn this study, we compared the gut contents of wild larvae of Anguilliformes collected from the subtropical western North Pacific Ocean, such as algae, bacteria and ciliates, which are components of POM, and the gut contents of cultured larvae of the Japanese eel \u003cem\u003eAnguilla japonica\u003c/em\u003e that ingested POM originating from these microorganisms by scanning electron microscope (SEM) observation and component analysis. The aim of this study was to further clarify the feeding ecology by understanding the structural properties and nutritional contribution of species as food sources.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCollection and identification of wild leptocephali\u003c/h2\u003e\u003cp\u003eNet sampling for anguilliform leptocephali was conducted 45 times at 22 stations from 19 August to 8 September in 2020 and 15 times at 13 stations from 1 to 13 September in 2022 during R/V \u003cem\u003eSoyo-Maru\u003c/em\u003e cruises (SY2005 and SY2204) in the eastern waters of the Philippines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;1). Leptocephali were collected using an Isaacs-Kidd Midwater Trawl (IKMT) net (Isaacs and Kidd \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1953\u003c/span\u003e) (IKMT; mouth opening area: 8.7 m\u0026sup2;; mesh size: 0.5 mm) in 2020 and a Matsuda-Oozeki-Hu Trawl (MOHT) net (Oozeki et al. \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (MOHT; mouth opening area: 5.0 m\u0026sup2;; mesh size: 1.59 mm) in 2022. Oblique tows were performed from a depth of 200 m to the surface at night and from depths of 400\u0026ndash;435 or 300\u0026ndash;330 m to the surface during the day in the 2020 cruise (SY2005). In the 2022 cruise (SY2204), oblique tows were performed from a depth of 100 m to the surface at night. The leptocephali were sorted after collection and placed on a chilled Petri dish. A total of 54 leptocephali with gut contents (\u003cem\u003eA. japonica\u003c/em\u003e, n\u0026thinsp;=\u0026thinsp;12; \u003cem\u003eAriosoma\u003c/em\u003e spp., n\u0026thinsp;=\u0026thinsp;20; \u003cem\u003eConger\u003c/em\u003e spp., n\u0026thinsp;=\u0026thinsp;22) were selected for bacterial isolation, SEM observation, and component analysis (Table\u0026nbsp;1). A small piece of the dorsal muscle was dissected and placed in a separate sterile 1.5-ml Eppendorf tube, and the remaining body was placed in a separate Uni-Pack (SEISANNIPPONSHA Ltd.). All the samples were kept at \u0026minus;\u0026thinsp;60\u0026deg;C and transferred to the laboratory. DNA samples from the muscle were extracted using the QuickGene DNA Tissue Kit S (KURABO, Osaka, Japan). Leptocephali were subjected to direct 18S rDNA sequence analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCultivation of bacteria\u003c/h3\u003e\n\u003cp\u003eThe results of bacterial isolation and identification will be submitted to PeerJ by the authors. Bacterial communities that were the dominant species in the gut contents and POM were selected as potential food souses in this study because they are presumed to contribute to the degradation of various organic substances constituting marine snow (Suzuki et al. \u003cspan citationid=\"CR140\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Yoon et al. \u003cspan citationid=\"CR176\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Heuchert et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Jean et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). We used preserved stocks of \u003cem\u003eErythrobacter flavus\u003c/em\u003e (strain No. 39), \u003cem\u003eTenacibaculum mesophilum\u003c/em\u003e (strain No. 130), \u003cem\u003eParacoccus stylophorae\u003c/em\u003e (strain No. 143) and \u003cem\u003ePsychrobacter\u003c/em\u003e sp. (strain Shi-11) from the Shibushi Field Station (\u003cem\u003eFRA\u003c/em\u003e) and \u003cem\u003eThalassomonas agariperforans\u003c/em\u003e (strain M-M1) from the Culture Collection University of Gothenburg (CCUG). The bacterial strains were routinely cultured on Marine Agar (MA; Condalab, Spain) plates at 25\u0026deg;C and maintained in Marine Broth (MB; Condalab, Spain) supplemented with 16% (v/v) glycerol at \u0026minus;\u0026thinsp;80\u0026deg;C. Bacteria for the feeding experiments, SEM observations and component analysis were cultured aerobically in MB medium at 25\u0026deg;C for 3\u0026ndash;4 days with stirring at 400 rpm using a magnetic stirrer (SW-400ND, NISSIN, Japan). When used in the feeding experiments and component analysis, the bacterial cells were concentrated by centrifugation (KUBOTA Model 7000, Japan) at 18,890 \u0026times; g for 15 min, washed twice to remove nutrients from the culture medium, and resuspended in sterile artificial seawater (RED SEA SALT, Red Sea, Israel).\u003c/p\u003e\n\u003ch3\u003eCultivation of algae\u003c/h3\u003e\n\u003cp\u003eThe algae used for the feeding experiments, SEM observations and component analysis were cultured in this study. We used \u003cem\u003eHeterocapsa niei\u003c/em\u003e (strain NIES-420) and \u003cem\u003eProchlorococcus\u003c/em\u003e spp. (strains NIES-2884, 2887) from the National Institute for Environmental Studies (NIES) preserved stocks and \u003cem\u003eIsochrysis galbana\u003c/em\u003e (strain NRIA-0074) and \u003cem\u003eChaetoceros calcitrans\u003c/em\u003e (strain NRIA-0001) from the Nansei Field Station (\u003cem\u003eFRA\u003c/em\u003e) preserved stocks. These cyanobacteria and microalgae are considered one of the food sources selected from among the numerous phytoplankton species in habitat areas (Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eProchlorococcus\u003c/em\u003e was incubated with PRO-99 medium (Moore et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) at 23\u0026deg;C for 7\u0026ndash;8 days with a light intensity of 20\u0026ndash;36 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on a 16-h/8-h light/dark cycle and with moderate shaking at 100 rpm (SK-O180-E, AS ONE, Japan). \u003cem\u003eHeterocapsa\u003c/em\u003e was incubated with F/2 medium (Guillard and Ryther \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) at 25\u0026deg;C for 14\u0026ndash;15 days under static conditions with the same light conditions as those used for \u003cem\u003eProchlorococcus\u003c/em\u003e. \u003cem\u003eIsochrysis\u003c/em\u003e and \u003cem\u003eChaetoceros\u003c/em\u003e were cultured with KW21 medium (DAIICHI CO., Ltd., Japan) at 20\u0026deg;C for 7\u0026ndash;10 days under continuous irradiance of 100\u0026ndash;128 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and air was supplied at an aeration rate of 1.0 volume (air)\u003csup\u003e\u0026minus;1\u003c/sup\u003e volume (medium)\u003csup\u003e\u0026minus;1\u003c/sup\u003e minute\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (vvm). In addition, Gelculture (DAIICHI Co., Ltd., Japan) was supplemented with \u003cem\u003eChaetoceros\u003c/em\u003e culture. Artificial seawater for culture media was sterilized by autoclaving (121\u0026deg;C for 20 min) and filtering through a 0.22-\u0026micro;m filter unit (VFTB-1000, VIOLAMO, Japan). When used in the feeding experiments and component analysis, exponentially growing cultures (average density, \u003cem\u003eProchlorococcus\u003c/em\u003e: NIES-2884: 3.51 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; NIES-2887: 2.81 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eHeterocapsa\u003c/em\u003e: 3.35 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eIsochrysis\u003c/em\u003e: 2.78 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003eChaetoceros\u003c/em\u003e: 2.84 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were concentrated by centrifugation (KUBOTA Model 8420, Japan) at 2,280 \u0026times; g for 50 min, rewashed to remove nutrients from the culture medium, and resuspended in sterile artificial seawater.\u003c/p\u003e\n\u003ch3\u003ePreparation of cultured leptocephali gut contents and mucus\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eFish\u003c/h2\u003e\u003cp\u003eTo obtain Japanese eel larvae for feeding and starvation experiments, female and male adult eels were induced to maturity using a hormonal treatment at the Minami-izu, Nansei, or Shibushi Field Station of the Fisheries Technology Institute, \u003cem\u003eFRA\u003c/em\u003e, as described previously (Satoh et al. \u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Kagawa et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tachiki et al. \u003cspan citationid=\"CR142\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Dou et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Unuma et al. \u003cspan citationid=\"CR162\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR163\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ohta et al. \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nomura et al. \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kazeto et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Suzuki et al. \u003cspan citationid=\"CR141\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Larvae obtained from spawning eggs were stored in a 30-L or 200-L semicylindrical tank supplied with filtered seawater (10 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 25\u0026deg;C and then fed 5 times a day at 2 h intervals with a fishmeal-based slurry-type diet (Furuita et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) until the feeding or starvation experiment was conducted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFood organisms\u003c/h2\u003e\u003cp\u003eThe food organism formulations used for the feeding experiments are shown in Table\u0026nbsp;2. In general, algae and ciliates are microorganisms that exist in aquatic environments (Lundgreen et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kume et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and bacteria isolated from the gut contents of wild larvae are likely derived from POM ingested through environmental waters (Azam \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Azam and Malfatti \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Busch et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, we used mainly algae and bacteria grown in the previous sections and ciliates as food organisms. Ciliates were isolated from seawater in the SCM layer (depth of 50 m) off Yamaga Bay in Kagoshima Prefecture (31\u0026deg;11.74' N, 130\u0026deg;42.81' E), where the Kuroshio Current inflows and a culture strain of \u003cem\u003eEuplotes charon\u003c/em\u003e (strain SFS-Y2112) growing on \u003cem\u003eProchlorococcus\u003c/em\u003e spp. and \u003cem\u003eIsochrysis galbana\u003c/em\u003e was obtained (Tomoda et al. manuscript in preparation). In the feeding experiment, the larvae were stored in a rearing tank in which these food source microorganisms were mixed, and rearing groups (algae alone, bacteria alone, algae\u0026thinsp;+\u0026thinsp;bacteria, or algae\u0026thinsp;+\u0026thinsp;bacteria\u0026thinsp;+\u0026thinsp;ciliates) were established in which the larvae were allowed to ingest environmental water. The amounts of algae (0.21\u0026ndash;1.00 g w.w. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and/or bacteria (0.01\u0026ndash;1.00 g w.w. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) added to the rearing water were determined according to the amount (0.7\u0026ndash;2.3 g w.w. L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of slurry-type diet (Masuda et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Furuita et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and liquid-type diet (Yamada et al. \u003cspan citationid=\"CR173\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) fed. With respect to the amount of ciliates added, the initial inoculation density was set at 15\u0026ndash;20 individuals mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e based on the assumption that the number of ciliates increases in the rearing water.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFeeding experiments\u003c/h3\u003e\n\u003cp\u003eFeeding experiments were carried out using larvae aged 8\u0026ndash;139 days post-hatch (dph) and with average total lengths of 7.5\u0026ndash;36.3 mm from different hatcheries and broodstock (Table\u0026nbsp;2). For larviculture, we used recirculating aquaculture systems (RASs) (Fig. S1) consisting of a 2.5-L plankton kreisel tank (Okamura et al. \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kenzaki et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with a bimorph pump (BPH-214G, NITTO KOHKI) and 30-L cylindro conical tanks (SBF-30, EARTH CORPORATION, Japan) with an amphibious pump (RSD-10A, REI-SEA, Japan). These systems were created to constantly float eel larvae in the rearing water and to feed in POM into the middle layer. During the experiment, cultured larvae that had been starved for 18 hours were stocked into a rearing tank and allowed to ingest environmental water containing algae, bacteria and ciliates. The larvae were reared in ultraviolet-irradiated seawater (24\u0026deg;C, salinity 32) supplied by a flow-type UV sterilizer (Flonlizer FDL-4-SP, Chiyoda Kohan, Japan) after microfiltration through 0.5-\u0026micro;m cartridge filters (TCW-0.5N-PPD, Advantec Toyo Kaisha, Japan). The rearing water recirculating ratio was maintained at 65.5\u0026ndash;70.7 cycles day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The photoperiod conditions were 9 L:15D (light period 8:00\u0026ndash;17:00), and the photon flux density during the light period ranged from 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 (dark period of 0.000 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). We also conducted a feeding experiment using a 200-mL beaker in a static rearing system. To confirm food intake into the digestive tract, 10\u0026ndash;15 larvae were collected from each tank 5\u0026ndash;17 hours after the beginning of the experiment. The collected larvae were kept frozen, after which the gut contents were squeezed out and pipetted into separate sterile 1.5-mL Eppendorf tubes for SEM observation and component analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eStarvation experiments\u003c/h3\u003e\n\u003cp\u003eSince the Japanese eel (Yamada and Yokote \u003cspan citationid=\"CR170\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) secretes mucus from goblet cells in the intestinal epithelium, it was considered that mucus was mixed in the gut contents. Therefore, as a comparison of the above feeding experiments, cultured larvae of two sizes (9 dph: TL 7.2 mm and 46 dph: TL 15.3 mm) raised on a fishmeal-based slurry-type diet (Furuita et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) were starved, and fasted individuals were collected at every timepoint, anaesthetized, and kept frozen (Table\u0026nbsp;2). For larviculture, we used a flow-through system consisting of a 10-L flat-bottom circular tank (Tanaka et al. \u003cspan citationid=\"CR146\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Masuda et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The rearing water exchange ratio was maintained at 86.4\u0026ndash;100.8 cycles day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. On a later day, the mid-hindgut was photographed using a stereoscopic microscope (SMZ1500, Nikon, Japan) attached to a photographic apparatus (DS-Fi3 and DS-L4, Nikon, Japan) to observe the mucus secretion status. In accordance with Masuda et al. (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), the area of the mid-hindgut and the area of mucus occupied in the mid-hindgut were measured, and the area ratio was determined as mucous fullness (%). Image analysis software (NIS-Elements D ver. 5.42.06; Nikon, Japan) was used for the area measurements. The mucus was squeezed out and pipetted into separate sterile 1.5-mL Eppendorf tubes for SEM observations.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSEM observations\u003c/h2\u003e\u003cp\u003eFood organisms (algae, bacteria, and ciliates) detected by genetic analysis of the gut contents (Ayala et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chow et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), food materials (POM in environmental water), and samples of the gut contents and mucus of \u003cem\u003eA. japonica\u003c/em\u003e leptocephali (10 wild fish and 14 kinds of fed and starved cultured fish) were prepared for scanning electron microscope (SEM) imaging using the water freeze-drying method (Kuwata et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to confirm the structure of natural food. To observe the gut contents and mucus of the cultured fish, fourteen samples were pooled in batches of 1\u0026ndash;5 individuals. The samples were coated with platinum (Pt) using an ion-sputter coater (E-1030; Hitachi, Tokyo, Japan). The coated samples were then observed using an SEM (JSM-6510LV, JEOL Ltd., Tokyo, Japan) at 100\u0026times; magnification, an accelerating voltage of 15 kV, and a working distance of 11 mm. In a 4 mm \u0026times; 4 mm sample, the area of 3,831 \u0026micro;m \u0026times; 4,785 \u0026micro;m was divided into 12 (3 \u0026times; 4) regions, and a single image of 1,920 \u0026times; 2,560 pixels captured an area of 957 \u0026micro;m \u0026times; 1,277 \u0026micro;m.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eComponent analysis of food organisms and gut contents of leptocephali\u003c/h2\u003e\u003cp\u003eTo estimate the nutritive value of natural food, proximate composition and amino acid analyses of food organisms and the gut contents of anguilliform leptocephali (wild \u003cem\u003eAriosoma\u003c/em\u003e sp., \u003cem\u003eConger\u003c/em\u003e sp. and 2 kinds of cultured \u003cem\u003eA. japonica\u003c/em\u003e) were carried out. For wild leptocephali, specimens collected in the same trawl nets as Japanese eels were used for analysis. For the analysis of the gut contents of eel larvae, twelve samples were pooled in batches of 3\u0026ndash;11 individuals. The samples were homogenized with 5% TCA and centrifuged at 8,000 rpm (3,500 \u0026times; g) for 10 min. The supernatant was collected and neutralized with sodium hydroxide, and the amino acids were determined using trinitrobenzene sulfonate (Hazra et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). After the precipitate was neutralized with sodium hydroxide, the protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Lipids were measured according to the methods of Holland and Gabbott (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). After the lipids were extracted with a chloroform and methanol mixture, a portion was separated, and the solvent was removed with a nitrogen stream. The samples were then heated with sulfuric acid at 200\u0026deg;C for 15 minutes with a heating block and colorimetrically quantified with distilled water using a spectrophotometer. Tripalmitin was used as a standard. The carbohydrate content was determined by the phenol sulfuric acid method as described by Dubois et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1956\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe mucous fullness data expressed as percentages were first arcsine transformed; the arcsine-transformed data were then tested for significant differences between mean values. The assumptions of a normal distribution and homogeneity of variance were checked before statistical analysis; one-way ANOVA or a two-sided Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test was used if normality and homoscedasticity were observed among the groups compared. When the one-way ANOVA was significant, differences among means were analysed using the Tukey‒Kramer multiple comparison test. All the 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=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStructure of food organisms and gut contents of leptocephali\u003c/h2\u003e\u003cp\u003eSEM observations of food source organisms (prochlorophytes, dinoflagellates, haptophytes, bacteria, and ciliates) revealed loose aggregation of extracellular products in algae and bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The gut contents (POM in environmental water) squeezed from anaesthetized cultured larvae were fragile and translucent gels with fluidity; however, they became light brown and increased in viscosity with time after death. When the gut contents of the wild larvae of Anguilliformes and the cultured larvae of Japanese eel were compared by stereomicroscopic observation, light microscopy revealed that the gut contents of both larvae were light brown and amorphous substances with fluidity, as previously reported (Miller et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tsukamoto and Miller \u003cspan citationid=\"CR158\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig. S2). In the gut contents of Japanese eel wild larvae (10 specimens), no traces of food organisms, such as the autofluorescence of algae or the faecal pellets and carcasses of zooplankton, were observed, and SEM observations confirmed the presence of amorphous substances, which seemed to originate mostly from polysaccharides and proteins derived from algae and bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The gut contents of Japanese eel-cultured larvae (44 samples) were similar to those of wild larvae; however, the structure differed according to the microbial composition of the ingested environmental water, the digestive ability of the larvae depending on their age (body size), or the progress of digestion and absorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In particular, the structural properties of the gut contents of artificial larvae, whose body sizes were the same as those of wild larvae, were very similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, h, i; 136\u0026ndash;139 dph). Furthermore, the structural properties of POM in environmental water containing algae and bacteria were very similar to those of the gut contents and differed slightly according to the composition of the food source organisms added (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eStructure of mucus and estimation of the mucus secretion status of cultured leptocephali\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eMucus in the mid-hindgut squeezed from anaesthetized cultured larvae was a fragile and transparent gel with fluidity, but it became light brown and increased in viscosity with time after death, similar to the composition of the gut contents. Although individual differences were observed in fasted-cultured larvae, we confirmed that they secreted sol- or gel-like mucus similar to the gut contents of wild larvae and cultured larvae ingesting POM in environmental water (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b, c). Mucus was also observed at 72 h after fasting, and there was no difference in mucus secretion until 48\u0026ndash;49 h after fasting in both 9 dph (TL7.2 mm) and 46 dph (TL15.3 mm) larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, e). However, in 9-day-old larvae, the mucous fullness at 72 h was significantly lower than those at 24 and 48 h (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). At the same fasting time (48\u0026ndash;49 h), 11-day-old larvae had significantly greater mucous fullness than 48-day-old larvae did (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.467\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003ef), but their mucous fullness was clearly lower than the intestinal fullness of wild larvae and cultured larvae ingesting POM in environmental water (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). When SEM observation of the gut contents and mucus was conducted, we considered the possibility that autodigested intestinal epithelial tissues (e.g., goblet cells, cilia, and microvilli) were mixed in the squeezed contents. Therefore, the mid-hindgut of cultured larvae was dissected and examined, but the structural properties clearly differed from those of food (POM in environmental water) and mucus (Fig. S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eProximate composition of food organisms and gut contents of leptocephali\u003c/h2\u003e\u003cp\u003eThere were marked differences in the composition ratios of the 5 algal and 10 bacterial strains among the species and strains. The total ratio of proteins and amino acids to the total of the four components of proteins, amino acids, lipids, and carbohydrates was 68.4% (54.1\u0026ndash;84.9%) on average for all 15 strains of algae and bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The total ratio of proteins and amino acids in the 5 bacterial strains (average 74.0%) was greater than those in the 10 algal strains (average 57.2%). In particular, the average abundance of 3 strains of \u003cem\u003ePsychrobacter\u003c/em\u003e, which are the dominant genera of POM, was 83.3%, which was markedly greater than that of the body component of the cultured larvae (66.9%). Similarly, there were marked differences in larval gut contents among fish species and diets (POM in environmental water), with a greater total ratio of proteins and amino acids in cultured Japanese eel larvae (average of 47.5%) than in wild larvae of \u003cem\u003eAriosoma\u003c/em\u003e and \u003cem\u003eConger\u003c/em\u003e (average of 39.1%). Moreover, the gut contents of wild and cultured larvae contained markedly greater percentages of carbohydrates (43.1\u0026ndash;67.2%) than their original food components did (3.4\u0026ndash;30.7%), probably because proteins and amino acids were absorbed through digestion, and cell wall components (e.g., cellulose, hemicellulose, and peptidoglycan) remained. In addition, when the gut contents of cultured Japanese eel larvae that ingested algae, bacteria and their produced substances were compared with those of wild larvae (conger eels), no marked differences were noted in the component composition, but the ratio of amino acids in cultured larvae (average 10.6%) was twice as high as that in wild larvae (average 5.3%).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eInvestigation of feeding habits in this study\u003c/h2\u003e\u003cp\u003eIn this study, we confirmed the phenomenon in which cultured Japanese eel larvae ingest algae, bacteria and their produced substances via environmental water, resulting in the occurrence of amorphous substances similar to those of wild larvae in the intestine through digestion, absorption and metabolism. As a result, the understanding of the structural properties and components of natural food and the feeding ecology of eel larvae was improved, and the elucidation of the formation mechanism of the feeding environment was approached. Based on the results of a component analysis of food organisms and gut contents, carbohydrates, proteins and amino acids (Thornton \u003cspan citationid=\"CR149\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ogasawara et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Biller et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Casillo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) produced by algae and bacteria were assumed to be among the substrates and nutrient sources of the gut contents, and these aggregates were considered to be the principal components of natural food. The results of component analysis as well as previous findings (Miller et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) suggest that algae and bacteria can contribute nutritionally to the growth and survival of eel larvae.\u003c/p\u003e\u003cp\u003eIn a feeding survey prior to this study, there were no significant differences in intestinal fullness (Masuda et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) between the algae solo feeding, algae\u0026thinsp;+\u0026thinsp;bacteria combined feeding, and algae\u0026thinsp;+\u0026thinsp;bacteria\u0026thinsp;+\u0026thinsp;ciliates combined feeding conditions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5044, ANOVA). However, significant differences in intestinal fullness were observed among the three algal feeding groups (\u003cem\u003eProchlorococcus\u003c/em\u003e sp., \u003cem\u003eHeterocapsa niei\u003c/em\u003e, \u003cem\u003eIsocrysis galbana\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0149; ANOVA), and the abundance of \u003cem\u003eProchlorococcus\u003c/em\u003e, the habitat-dominant species (Zhang et al. \u003cspan citationid=\"CR179\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Flombaum et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), was significantly greater than that of \u003cem\u003eIsocrysis\u003c/em\u003e, the coastal species (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Scheffe's \u003cem\u003eF\u003c/em\u003e test). These results revealed that the feeding ability of algae solo feeding of habitat-dominant species was comparable to that of wild larvae but suggested that the involvement of bacteria is necessary to increase nutritional supplementation, digestive absorption, and immune functions (Yukgehnaish et al. \u003cspan citationid=\"CR178\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Diwan et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Morshed and Lee \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR174\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) to improve growth and survival.\u003c/p\u003e\u003cp\u003eHowever, 5 out of 33 strains of \u003cem\u003ePseudoalteromonas\u003c/em\u003e sp., which we obtained by culturing gut contents, showed algicidal activity (Inaba et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Coyne et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR165\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR181\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) against \u003cem\u003eIsocrysis galbana\u003c/em\u003e. Therefore, these algicidal bacteria may contribute to the degradation of algae and the digestion and absorption of eel larvae (Tomoda, unpubl data). That is, compared with the cell walls of gram-positive bacteria and eukaryotic algae, the peptidoglycan layer of \u003cem\u003eProchlorococcus\u003c/em\u003e sp. (Ting et al. \u003cspan citationid=\"CR151\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), a habitat-dominant species, is thought to be thinner and more easily digested; thus, it may be more useful as food.\u003c/p\u003e\u003cp\u003e According to recent feeding habit surveys in the field and observations of feeding behaviour in the laboratory, submicrometre particles (SMP) (Koike et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Yamasaki et al. \u003cspan citationid=\"CR175\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and gel-like particles (Yamada et al. \u003cspan citationid=\"CR172\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which commonly exist in environmental waters and are easily swallowed, i.e., POM and DOM, including substances produced by algae and bacteria (TEP, CSP, EPS, MVs, and EVs) (Long and Azam \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Biller et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Scanlan \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Busch et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Decho and Gutierrez \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mari et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Thornton \u003cspan citationid=\"CR149\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Daly et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Casillo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), constituting a high proportion of marine biomass, may be utilized directly by pinocytosis in the intestinal epithelium (Tanaka \u003cspan citationid=\"CR147\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Watanabe \u003cspan citationid=\"CR168\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Otake et al. \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Kurokawa et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Otake \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) without microbial degradation. It is assumed that the differences in the structural properties of the gut contents of cultured larvae are caused not only by differences in digestive ability and mucus secretion with days post-hatching but also by differences in the properties of the POM ingested, species of food organisms, and their products. The gut contents containing carbohydrates, proteins, and amino acids are likely derived from aggregates of TEP, CSP, and ESP present in environmental water. Thus, if the feeding habits at lower trophic levels (Miller et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) are able to utilize algae, bacteria, and their products, which account for a large proportion of marine biomass, it is assumed that adaptation to oligotrophic environments (Pilskaln et al. \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Onda et al. \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ayala et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chow et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Lundgreen et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) is possible.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eConsistency with past feeding habits surveys and feeding behaviour observations\u003c/h2\u003e\u003cp\u003eSimilar to our previous feeding habit surveys of wild larvae (Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chow et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), microscopic observations in this study revealed almost no evidence of eukaryotes (autofluorescence, faecal pellets and carcasses). In addition, feeding surveys on zoo- and phytoplankton subjected to various treatments (freezing and thawing, alkaline hydrolysis, and enzymolysis) revealed that cultured eel larvae could not ingest and digest food with hard physical properties such as cell walls, shells, and spines that retained their original forms (Tomoda, unpubl data). Consequently, feeding habits that do not utilize the organism itself as a direct source of nutrition (Terahara et al. \u003cspan citationid=\"CR148\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) have been reconfirmed. Moreover, we have recently conducted feeding surveys on 395 diets prepared from various biological materials (e.g., seaweed, thraustochytrids, \u003cem\u003eEuglena\u003c/em\u003e, coastal microalgae, bacteria, zooplankton, and jellyfish carcasses), including microorganisms that have been identified as potential food elements (Ayala et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Miller et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, no food with good feeding properties was found that could stably reproduce feeding conditions similar to those of wild larvae (Table S1). Our feeding surveys revealed that discarded appendicularian houses and zooplankton faecal pellets (Otake et al. \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Mochioka and Iwamizu \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), which have been reported to be food sources, were not significantly attractive for feeding, and the intestinal fullness was also apparently low (Tomoda et al. \u003cspan citationid=\"CR154\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Fig. S4). In particular, in the latest feeding surveys, no positive feeding behaviour was observed on zooplankton faecal pellets (\u003cem\u003eArtemia franciscana\u003c/em\u003e, 23 cases; \u003cem\u003eAcartia tonsa\u003c/em\u003e, 10 cases) or jellyfish carcasses (\u003cem\u003eBolinopsis mikado\u003c/em\u003e, 2 cases; \u003cem\u003eAurelia coerulea\u003c/em\u003e, 1 case), and both the feeding ratio and intestinal fullness were significantly low (Fig. S4; Table S1). The digestibility of zooplankton faecal pellets and phytoplankton was low, and the residual autofluorescence in the gut contents was more pronounced than that in the wild larvae (Fig. S4). Therefore, discarded appendicularian houses and the carcasses and faecal pellets of zooplankton seem not to be positively consumed by eel larvae, and our observations are also supported by those of Kume et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, these differences in observations may be influenced by spatiotemporal differences in food abundance between coastal and open ocean areas, dietary changes during development, and differences among species of Anguilliformes. Our findings are also supported by those of Chow et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e), who reported that these biological materials are not direct food elements for eel larvae because they have no attractive properties for feeding and impair feeding and swallowing functions. Considering these findings, the needle-shaped larval teeth (Yoshimatsu \u003cspan citationid=\"CR177\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and velum (Miller \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) of anguilliform leptocephali may function as organs for screening foreign substances other than food that are difficult to eat, swallow and digest.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, recent molecular biology surveys of feeding habits (Riemann et al. \u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Terahara et al. \u003cspan citationid=\"CR148\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ayala et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chow et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kume et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) might have indicated the detection of environmental DNA (Zhao et al. \u003cspan citationid=\"CR180\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sildever et al. \u003cspan citationid=\"CR137\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) by cross-contamination from the body surface and/or drinking environmental water (Lee et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ahn et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). That is, environmental DNA may be captured by POM via TEP, CSP, and ESP as attachment substrates and may reflect the results of analyses in which environmental DNA is ingested by larvae. Moreover, in the past, when a large portion of eel larvae were detected by 18S rRNA gene analysis of gut contents, the mucus (Yamada and Yokote \u003cspan citationid=\"CR170\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) of the intestinal epithelium and environmental DNA (Takeuchi et al. \u003cspan citationid=\"CR143\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) of eel larvae themselves may have been detected instead of food intake (POM). These results suggest that eel larvae preferentially utilize sugars, proteins, and amino acids contained in extracellular products (e.g., TEP, CSP, and ESP), which are the origin of marine snow (POM), rather than utilizing algal cell bodies or zooplankton carcasses and faecal pellets as a nutrient source. Moreover, \u003cem\u003eProchlorococcus\u003c/em\u003e sp., which dominate the habitat, is a prochlorophyte with remarkable attenuation and quenching of autofluorescence. Therefore, these genes may not have been detected by fluorescence microscopy or eukaryotic 18S rRNA gene analysis, and their contribution to the feeding environment may be underestimated. Alternatively, the peptidoglycan layer (Ting et al. \u003cspan citationid=\"CR151\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and bacterial extracellular vesicles (Biller et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Scanlan \u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) of \u003cem\u003eProchlorococcus\u003c/em\u003e sp. may be more physically fragile than the cell walls of eukaryotic algae are and thus may be easier to digest and absorb by pinocytosis. Therefore, we can fully understand why in many previous reports, traces of food organisms (e.g., cell walls, shells, and autofluorescence) were rarely found in the gut contents, and spherical picoplankton (Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) were frequently found.\u003c/p\u003e\u003cp\u003eMany biological materials with physical properties that leptocephali cannot digest and absorb are also present in POM at the depth of their habitat; accidental ingestion of these materials has been confirmed to lead to deaths from intestinal obstruction and intestinal necrosis (Chow et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tomoda, unpubl data). As mentioned above, not only the needle-shaped larval teeth and velum but also the olfactory characteristics (Watanabe \u003cspan citationid=\"CR166\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and feeding selectivity (Kenzaki et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) of leptocephali may play a role in preventing unexpected deaths due to the accidental ingestion of these foreign substances.\u003c/p\u003e\u003cp\u003eFurthermore, artificial digestion experiments using digestive enzymes purified from the intestine of cultured larvae (over 200 dph) revealed that the gut contents squeezed out from wild larvae were not digested (Tomoda, unpubl data). Therefore, the gut contents, which had been regarded as undigested food just after feeding, may have been close to the state of faeces; that is, the residues were completely digested and absorbed by the larvae after being decomposed by algicidal bacteria and intestinal bacteria.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eStructural properties of the gut contents\u003c/h2\u003e\u003cp\u003eThe structural properties of the gut contents of wild larvae were similar to those of POM in environmental water where algae and bacteria coexist, and they were in a sol- or gel-like state with a high proportion of carbohydrates. These findings reflect previous findings that leptocephali have a high ability to digest proteins but a low ability to digest carbohydrates (Kurokawa et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pedersen et al. \u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hsu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The results of the component analysis of food organisms and gut contents also suggest that low-molecular-weight saccharides, proteins, and amino acids produced extracellularly by algae and bacteria may be used as nutrient sources. Eel larvae may grow and survive by constantly drinking seawater containing these produced substances, taking up nutrients through pinocytosis in the intestinal epithelium. In other words, algal and bacterial products are presumed to be the source of the substrate structure and nutrition of the gut contents, and these aggregates may form a food web as a major component of natural food. The mechanism by which gel-like substances accumulate in the intestinal tract through the ingestion of environmental water containing algae and bacteria is assumed to indicate that low-molecular-weight saccharides, proteins, amino acids, and water are digested and absorbed, whereas carbohydrates derived from cell walls and produced polysaccharides remain undigested. The structural properties of the gut contents were considered to differ depending on factors such as the species of food organisms added to the environmental water, the difference in carbohydrate and protein contents with the added ratio, the difference in digestive abilities and the progress of digestion and absorption with the age of the larvae.\u003c/p\u003e\u003cp\u003eTo improve food intake and retention time in the intestine, the presence of fine particles (Furuita, unpublished data) and substrates that adsorb organic matter (Tomoda et al. \u003cspan citationid=\"CR154\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) is necessary. Additionally, the inclusion of proteins on the surface of substrates and particles increases food intake (Kenzaki et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This finding was also confirmed by surveys of the feeding habits of wild larvae (Tomoda et al. \u003cspan citationid=\"CR156\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In other words, because fine particles are less likely to be retained in the intestinal tract in the case of liquids with low viscosity, it has been found empirically that fragile gels such as marine snow (Alldredge and Silver \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Lampitt et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Chajwa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) or sols consisting of colloidal particles or SMP are preferable in terms of feeding efficiency. In addition, a matrix structure with high porosity is advantageous for maintaining the buoyancy of food, and organic matter decomposition is promoted by the presence of an attached substrate that provides a habitat for bacteria (Kajihara \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Shanks and Edmondson \u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR172\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hou et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Recently, the availability of colloid-type (Masuda et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and liquid-type (Okamura et al. \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR173\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) diets that simulate such physical properties has been reported. Furthermore, prolonged encounters with a floating-type diet under dark conditions have been shown to enable stable midwater feeding (Shioura et al. \u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, the current standard mode of rearing, which uses high-viscosity and high-gravity artificial feed (Furuita et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Jinbo et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sudo et al. \u003cspan citationid=\"CR139\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), has room for improvement even to expand the production scale.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eMucus function\u003c/h2\u003e\u003cp\u003eThe European eel \u003cem\u003eAnguilla anguilla\u003c/em\u003e (Domeneghini et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), the Japanese eel \u003cem\u003eAnguilla japonica\u003c/em\u003e (Yamada and Yokote \u003cspan citationid=\"CR170\" class=\"CitationRef\"\u003e1975\u003c/span\u003e), and the Whitespotted conger \u003cem\u003eConger Myriaster\u003c/em\u003e (Takiue and Akiyoshi \u003cspan citationid=\"CR144\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) are known to secrete both acidic and neutral mucus. Mucus secreted on the mucosal epithelial surface of the digestive tract, together with microvilli, has been implicated in digestion and absorption (Grau et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Otake et al. \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Otake \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Tibbets 1997). In this study, amorphous substances such as the gut contents of wild larvae were detected in the mid-hindgut of fasted cultured larvae. These are considered to comprise the mucus secreted from the intestinal epithelium, and there is a high possibility that mucin, which is considered to be the main component of mucus, was also intermingled in previous surveys of the feeding habits of wild larvae. The gut contents (mucus and POM) of both wild and cultured larvae observed after sampling were likewise all light brown, suggesting that metabolic and physiological functions in the intestine may be involved. In this study, we revealed individual differences in the mucous fullness of cultured larvae. However, this may be due to changes in digestive enzymes and mucus secretion and the intestinal microbiome by not only environmental factors (Morshed and Lee \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) but also developmental stages and starvation conditions (Xia et al. \u003cspan citationid=\"CR169\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) of the larvae.\u003c/p\u003e\u003cp\u003eThe greater amount of mucus secretion in 10-day-old larvae observed in this study may indicate a greater feeding ability (POM trapping efficiency) in the early larval stage, and the lower amount of mucus secretion in 47-day-old larvae may indicate an improvement in starvation resistance after mass mortality events at the early larval stage. It is speculated that prolonged mucus secretion under starvation conditions efficiently and effectively traps and retains fine POM in the intestinal tract, thereby prolonging the opportunity for nutrient absorption. That is, mucus secretion in anguilliform leptocephali is considered to contribute greatly to feeding behaviour in oligotrophic oceans. During the ingestion of environmental water, POM and DOM may be trapped in the mucus layer of the intestinal epithelium or on the surface of cilia and microvilli and absorbed by pinocytosis. This feeding mode seems to resemble suspension feeding (Riisg\u0026aring;rd and Larsen \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), such as the mucous mesh feeding of pelagic tunicates and thecosome pteropods (Hiebert et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), or the ciliary filter feeding of bivalves and polychaetes (Riisg\u0026aring;rd et al. \u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Okutani and Kurokawa \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This hypothesis is supported by previous reports (Kenzaki et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shioura et al. \u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). That is, the phenomenon in which picocyanobacteria are fed through environmental water ingestion, circulate in the mid-hindgut by ciliary movement, and remain and aggregate in the intestine for a certain period of time is considered to be a result of capturing fine particles in the mucus layer of the intestinal epithelium, as well as on the surfaces and gaps of cilia and microvilli (Fig. S3). Mucus secretion in the intestinal epithelium (Yamada and Yokote \u003cspan citationid=\"CR170\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) is likely closely involved in the feeding habits that make POM, DOM, and SMP (Koike et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Otake et al. \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Yamasaki et al. \u003cspan citationid=\"CR175\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) available and may be a shortcut in the food chain pathway. Additionally, aggregates of bacterial strains (\u003cem\u003eErythrobacter flavus\u003c/em\u003e) from POM and picocyanobacteria (\u003cem\u003eSynechococcus\u003c/em\u003e sp.) were digested and absorbed in the posterior mid-hindgut of cultured larvae during microscopic observation (Inaba et al. unpubl data; KY1704 cruise), suggesting pinocytosis. Thus, a feeding mode in which pico- and nanosized POM and DOM can be retained and digested in the mid-hindgut is considered to be an efficient and effective survival strategy for eel larvae in depth zones where food is scarce and oligotrophic. These findings indicate that the improvements in growth and survival associated with a low-viscosity liquid-type diet (Okamura et al. \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR173\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) may reflect the abovementioned advantages of the feeding mode. Furthermore, the mucus layer not only provides nutritional glycoproteins (Bakke et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and habitats for intestinal bacteria but also contributes to biological defence against pathogenic microorganisms (Jutfelt \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough we cannot deny the possibility that some mucus containing glycoproteins or digestive enzymes was involved when the gut contents were squeezed, all the amorphous substances found in all individuals were unlikely to be mucus derived from eel larvae.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eFactors contributing to the establishment of the feeding environment\u003c/h2\u003e\u003cp\u003ePrevious evidence has suggested that marine snow, which mainly consists of eukaryotes, might be a food source for leptocephali (i.e., carcasses and faecal pellets of zoo- and phytoplankton that have been decomposed by microorganisms into sizes and physical properties that can be swallowed by eel larvae) (Chow et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Miller et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tsukamoto and Miller \u003cspan citationid=\"CR158\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kume et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, the results of this study suggest that food webs are formed in which prokaryotes, such as cyanobacteria as primary producers and bacteria as decomposers as well as consumers, are the main food sources. As mentioned above, \u003cem\u003eProchlorococcus\u003c/em\u003e sp. and bacteria are known to dominate habitat biomass (Zhang et al. \u003cspan citationid=\"CR179\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and represent a high proportion of POM (Watanabe et al. submit to PeerJ). The bacterial extracellular vesicles (EVs) (Biller et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Casillo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) released by these prokaryotes contain proteins and nucleic acids. Recently, reported effects of nucleic acid supplementation (Jinbo et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) on the survival, growth, and metamorphosis of cultured larvae may indicate that EVs contribute to the feeding environment of wild leptocephali. In particular, bacteria influence food web dynamics (Long and Azam \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Azam \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Azam and Malfatti \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Koshikawa et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Kogure \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kato and Masuda \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR174\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), suggesting that algal\u0026ndash;bacterial interactions in the phycosphere contribute to organic matter transport and energy transfer to higher trophic level organisms (Zhou et al. \u003cspan citationid=\"CR182\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Seymour et al. \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; M\u0026uuml;hlenbruch et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cirri and Pohnert \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The components of POM and DOM, such as marine snow, are known to include monosaccharides (Skoog et al. \u003cspan citationid=\"CR138\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) as well as amino sugars and peptidoglycans (Benner and Kaiser \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Extracellular polymeric substances (EPS) are the origin of marine snow and are produced through interactions between algae and bacteria (Decho and Gutierrez \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Daly et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These substances include not only polysaccharides and proteins but also monosaccharides that are available as nutrients for eel larvae (Okamura et al. \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, the carbon‒nitrogen stable isotope ratio of POM-dominant bacteria is close to the trophic level of marine snow (Watanabe et al. submit to PeerJ), and the results of the component analysis in this study also indicate sufficient nutritional value for food. Therefore, the microbial loop (Azam \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Azam and Malfatti \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and phycosphere (Zhou et al. \u003cspan citationid=\"CR182\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Seymour et al. \u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; M\u0026uuml;hlenbruch et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cirri and Pohnert \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) seem to contribute as a feeding ground to provide highly nutritious POM and DOM for eel larvae. Recently, we confirmed that there is enough POM just below the subsurface chlorophyll maximum (SCM) layer for eel larvae to satiate in a short time (Watanabe et al. unpubl data). The SCM layer is densely dotted with \u0026ldquo;hot spots\u0026rdquo; (Azam \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) of biogenic SMP and POM, and eel larvae may both passively and aggressively utilize these organic substances during diel vertical migration (Kudo \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Chang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eFuture perspectives\u003c/h2\u003e\u003cp\u003eThe results of this study suggest the following possibilities.\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe gut contents include mucus secreted from the intestinal epithelium, as well as substances produced by algae and bacteria.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe mucus layer of the intestinal epithelium contributes to efficiently trap particles from environmental water for feeding.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eEel larvae utilize mainly low-molecular-weight saccharides, proteins, and amino acids contained in algal and bacterial products.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe carcasses and faecal pellets of zoo- and phytoplankton are not the main food for eel larvae.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ePOM and DOM are directly utilized by pinocytosis in the intestinal epithelium without microbial degradation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eIn previous studies of feeding habits based on microscopic observation and molecular biology, a food web has been proposed in which aggregates (POM) of the carcasses and faecal pellets of zoo- and phytoplankton degraded by microorganisms are the main food. However, a combination of relevant findings and our ten-year field and laboratory feeding surveys suggest that the feeding ecology of eel larvae in the mesopelagic zone of low-latitude oceans (Rodgers et al. \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) may be directly supported by a lower trophic level ecosystem that underlies the microbial loop and phycosphere rather than by a food web, as previously assumed (Watanabe et al. \u003cspan citationid=\"CR167\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo conserve natural resources and stabilize the aquaculture industry, we must not only manage resources through industry\u0026ndash;government\u0026ndash;academia cooperation but also establish glass eel mass production technology and realize true social implementation. To achieve this goal, the development of innovative feed and the transformation of seedling production forms are urgent issues. In particular, to eliminate bottlenecks in cost and labour savings and expand production scale, we need to create a foraging environment with a low environmental load, such as production forms using food organisms (Hagiwara et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Tomoda et al. \u003cspan citationid=\"CR153\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR155\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), by utilizing the elucidation of feeding ecology at the leptocephalus stage. However, in the current eel research field, almost no research has been conducted on microbial ecology (Lampitt et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Kogure K (ed) 2006; Azam and Malfatti \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) to elucidate the feeding environment or on microbial engineering to produce marine snow (Kajihara \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Shanks and Edmondson \u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Yamada et al. \u003cspan citationid=\"CR172\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hou et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) as food. In the future, basic research should continue to elucidate the entire feeding ecology of leptocephalus in terms of its nutritional physiological function (capture of POM and DOM by the mucus and pinocytosis of POM and DOM in the intestinal epithelium), its habitat, and the establishment mechanism of the feeding environment based on the lower trophic level ecosystem. Additionally, advancements should be made in the development of feed that effectively utilizes the potential of microorganisms for the mass production of seedlings.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAlthough many reports have shown that leptocephali consume mainly POM, the mechanism underlying how they effectively capture, ingest, digest, and absorb small POM has not been elucidated. In this study, we obtained new information about the feeding ecology of anguilliform leptocephali, which has been a mystery for many years, by comparing the gut contents of wild and cultured larvae. The gut contents of eel larvae and food organisms were observed via light and scanning electron microscopy observations to clarify the morphological characteristics and component composition and to elucidate the feeding ecology of eel larvae. In particular, the close relationship between mucus secretion and the feeding mode in the intestine, which has not been previously reported, seems to be related to not only survival strategies in oligotrophic oceans but also adaptive evolution to the environment. Importantly, this paper clarified the possibility that mucus secretory function contributes to the feeding mode. Another interesting observation is the possibility that the difference in mucus secretion at the developmental stage leads to the improvement of starvation resistance. In considering the feeding ecology of anguilliform leptocephali in nature through laboratory feeding surveys, it is difficult to comprehensively grasp the morphological and biochemical knowledge of the food organisms, gut contents, and mucus focused on in this study. However, this study provides a basic understanding of how digestive and immune organs developed during the evolution of leptocephali. Our findings promote research on the feeding ecology of anguilliform leptocephali and thus contribute to the development of aquaculture research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at https://doi.org/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e We thank Keisuke Yamano (former Director of the Aquaculture Research Department at the Fisheries Technology Institute, \u003cem\u003eFRA\u003c/em\u003e), Hiroaki Kurogi (Director of the Socio-Ecological Systems Division at the Fisheries Resources Institute, \u003cem\u003eFRA\u003c/em\u003e) and Daisuke Hasegawa (Shiogama Field Station, \u003cem\u003eFRA\u003c/em\u003e) for their support in conducting this study. We would also like to extend our thanks to the helpful crew of R/V \u003cem\u003eSoyo-Maru\u003c/em\u003e for performing the collection of the leptocephali. We are sincerely grateful to the staff of the Minami-izu, Nansei, and Shibushi Field Stations of \u003cem\u003eFRA\u003c/em\u003e for their assistance with providing cultured leptocephali. We also thank the staff of Hatsukaichi and Momoshima Field Stations of \u003cem\u003eFRA\u003c/em\u003e for sharing \u003cem\u003eHeterocapsa niei\u003c/em\u003e and microbial flocks and Hideaki Matsui (National Fisheries University, \u003cem\u003eFRA\u003c/em\u003e) and Tomoya Kotani (Faculty of Fisheries, Kagoshima University) for sharing faecal pellets of the copepods \u003cem\u003eAcartia tonsa\u003c/em\u003e and Masahiro Hayashi (Faculty of Agriculture, University of Miyazaki) for sharing Thraustochytrids and \u003cem\u003eEuglena\u003c/em\u003e with us. We sincerely appreciate the reviewers for their suggestions and comments. This study utilized samples collected through the Fisheries Resource Survey and Evaluation Promotion Project commissioned by the Fisheries Agency, Ministry of Agriculture, Forestry and Fisheries of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e TT, TW, KT, SN and MU conceived the study. All authors contributed to the design of the conceptual framework and analyses. DA, NF, KH, YN and YS conducted ship surveys and collected the leptocephali. TT, TW, KT, SN and MU conducted the laboratory experiments and observation. HF, TY and SN conducted the analyses. TT wrote the first draft of the manuscript. All authors have contributed to the revision and improvements of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (The Special Scheme Project on Advanced Research and Development for Next-generation Technology) and the project \u0026quot;Development of biological material feeds\u0026quot; commissioned by \u003cem\u003eFRA\u003c/em\u003e as part of the \u0026quot;Demonstration project of a mass-production system for commercialisation of eel seedlings\u0026quot; from the Ministry of Agriculture, Forestry and Fisheries of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e The larval samples captured with plankton nets deployed from research vessels were dead on retrieval and sampled at this time, and all plankton net operations were carried out in high seas outside the Exclusive Economic Zone. Therefore, the approval of coastal states was not required under the United Nations Convention on the Law of the Sea (UNCLOS). 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 (\u003cem\u003eFRA\u003c/em\u003e). All experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of the \u003cem\u003eFRA\u003c/em\u003e (permission code: 24023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhn H, Yamada Y, Okamura A, Tsukamoto K, Kaneko T, Watanabe S (2013) Intestinal expression of peptide transporter 1 (PEPT1) at different life stages of Japanese eel, \u003cem\u003eAnguilla japonica\u003c/em\u003e. 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There were no traces of food organisms, such as autofluorescence of algae and faecal pellets, or carcasses of zooplankton in the gut contents of wild larvae. The gut contents of both wild and cultured larvae were light brown sols or gels and were composed of amorphous substances consisting mainly of carbohydrates, proteins and amino acids. The structural properties of the gut contents of cultured larvae were similar to those of POM in environmental water; mucus secreted from the intestinal epithelium was also present. These results suggest that low-molecular-weight saccharides, proteins, and amino acids produced by algae and bacteria may be among the available nutritional sources of eel larvae and that the mucus layer of the intestinal epithelium contributes to a feeding mode that effectively captures microparticles from environmental water. Taken together, the results of recent surveys suggest that pico- and nanosized POMs, which are ubiquitous in environmental waters and are easily swallowed and easily digestible and absorbable, i.e., substances produced by algae and bacteria that account for a high proportion of marine biomass and dissolved organic matter (DOM), may be directly utilized by the intestinal epithelium through pinocytosis without microbial degradation.\u003c/p\u003e","manuscriptTitle":"Feeding ecology of anguilliform leptocephali considering the structure and proximate composition of food organisms and gut contents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 07:11:50","doi":"10.21203/rs.3.rs-8218055/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":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58688899,"name":"Aquaculture and Mariculture"},{"id":58688900,"name":"Marine and Freshwater Biology"},{"id":58688901,"name":"Behavioral Ecology"}],"tags":[],"updatedAt":"2025-11-29T01:10:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-28 07:11:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8218055","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8218055","identity":"rs-8218055","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}