1859

The Journal of Experimental Biology 213, 1859-1867 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jeb.040030

Oral and integumental uptake of free exogenous glycine by the Japanese Panulirus japonicus phyllosoma larvae

Juan Carlos Rodriguez Souza1, Carlos Augusto Strüssmann1,*, Fumio Takashima1, Hiroo Satoh1, Shintaro Sekine2, Yasuhiro Shima2 and Hirokazu Matsuda3 1Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-Ku, Tokyo, 108-8477, , 2Minamiizu Station, National Center for Stock Enhancement, Fisheries Research Agency, Irouzaki, Minamiizu, Shizuoka, 415-0156, Japan and 3Mie Prefecture Fisheries Research Institute, 3564-3 Hamajima, Hamjima-cho, Shima, Mie, 517-0404, Japan *Author for correspondence ([email protected])

Accepted 10 February 2010

SUMMARY The possibility of direct integumental absorption of the amino acid glycine from a solution in seawater was investigated in 250–260day old (16.9–50.0mg wet mass) phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus Von Siebold 1824. The uptake of the amino acid was assessed by autoradiography and liquid scintillation counting (LSC) of larvae incubated with [2-3H]glycine and the net uptake was estimated by a time course high-performance liquid chromatography (HPLC) analysis of the concentration of glycine in the incubation medium. Autoradiography revealed the presence of labelled glycine in the cuticle, epidermis and internal tissues (digestive system, muscle, haemocytes) within 30min of the onset of incubation. Absorption through the integument was confirmed by autoradiography and LSC as glycine uptake was observed even in larvae whose mouths were artificially sealed with cyanoacrylate bond prior to incubation. Scanning electron microscopic examination of the body surface revealed no bacterial population that could have mediated the uptake. HPLC revealed a consistent net uptake (0.29–0.39molg–1 body massh–1) of glycine in larvae incubated in 6moll–1 glycine and high individual variation (e.g. absorption or release) in larvae incubated at higher concentrations (30 and 60moll–1). Thus, the results of this study provide clear confirmation that, in addition to the known mode of oral feeding on macroscopic food masses, P. japonicus phyllosoma larvae are also able to absorb nutrients directly from the surrounding medium. Key words: phyllosoma, dissolved organic matter, nutrition, lobster, integument.

INTRODUCTION In those studies, phyllosoma larvae showed improvement in Utilization of particulate organic mater (POM) and dissolved histological and cellular characteristics after incubation in solutions organic mater (DOM) has been reported in adults and larvae of a or dispersions of representative substances from the three main range of marine invertebrates (for reviews, see Stephens and classes (protein, carbohydrate, lipid). More importantly, the results Schinske, 1961; Stephens, 1981; Manahan, 1983; Preston, 1993; of the studies indicated that the absorption of POM and DOM by Ben-David-Zaslow and Benayahu, 2000; Baines et al., 2005; the Japanese spiny lobster larvae occurred not only through the gut, Grover et al., 2008) and fish larvae (Otake et al., 1993; Otake et from orally ingested water, but also through the integument al., 1995). In many marine invertebrates, the external body surface (Rodriguez Souza et al., 1999). This possibility was advanced based is considered to be a very important route and, in some cases, the on the presence of mucus-like inclusions in the integument, of pore primary route for the absorption of exogenous amino acids channels in the cuticle connecting the epidermis to the external (Preston, 1993). However, the capacity to absorb dissolved medium, and of tegumental glands in the epidermis along the cuticle. substances directly from the surrounding water has never been In addition, P. japonicus have a flat, leaf-like larval stage, with a demonstrated in any species of and the impermeable large surface area to body volume ratio, and a body that is covered exoskeleton is considered to be the major impediment to the with a soft, uncalcified cuticle, characteristics that provide development of this capacity (Stephens and Schinske, 1961; particularly favourable conditions for integumental absorption. In Anderson and Stephens, 1969; Castille and Lawrence, 1979; fact, these characteristics are commonly found in aquatic Preston, 1993). Indeed, the occasional utilization of dissolved invertebrates that have the ability to absorb nutrients through the nutrients in arthropods has often been attributed to the ingestion integument (Manahan, 1983). of exogenous bacteria attached to the body surface (Castille and The feeding ecology of Japanese spiny lobster phyllosoma larvae Lawrence, 1979; Siebers, 1979) and more recently to the ingestion is still not understood and, in spite of a century of trials on larval of water (Tonheim et al., 2000). On the other hand, Rodriguez rearing and development of feeding protocols for this species, Souza and colleagues provided preliminary evidence that the survival and growth rates during the larval stage are still insufficient phyllosoma larvae of the Japanese spiny lobster Panulirus for mass seed production (Matsuda, 2006; Matsuda and Takenouchi, japonicus Von Siebold 1824 can take up POM and DOM directly 2006). Thus, POM and DOM could provide a nutritional supplement from the surrounding medium (Rodriguez Souza, 1997; Rodriguez and be the key to the improvement of performance during larval Souza et al., 1999). rearing. In this context, the present study was designed to explore

THE JOURNAL OF EXPERIMENTAL BIOLOGY 1860 J. C. Rodriguez Souza and others further the absorption capacity of the Japanese spiny lobster also observed without staining to identify possible artifacts from phyllosoma larvae. Experiments were conducted to verify the the staining procedure (Peter and Ashley, 1967). The number of uptake of the amino acid glycine and, in particular, to clarify its silver grains per unit area (mm2) was determined in selected route of absorption and internal distribution using histology and structures (cuticle, epidermis, muscle, haemolymph, haemocytes, autoradiography as well as to quantify its uptake by high midgut gland cells and lumen) of glycine-treated larvae from the performance liquid chromatography (HPLC) and liquid scintillation second experiment. Counts were made by digital image analysis counting (LSC). of about 20 fields of 10m2 for each type of structure per larvae using the ‘count object’ feature of ImagePro Plus (Media MATERIALS AND METHODS Cybernetics Inc., Bethesda, MD, USA). Correction for background Source of materials and general experimental conditions labelling artifacts (based on control larvae) was not necessary as Phyllosoma larvae were obtained through natural spawning of they were negligible. broodstock maintained at the Minamiizu Station, National Center for Stock Enhancement, Fisheries Research Agency, and the Mie High performance liquid chromatography Prefecture Fisheries Research Institute, Japan. A total of 65 larvae The net uptake of glycine by the larvae was determined by HPLC were used in this study. The specimens were 250–260days old analysis of the changes in glycine concentration in the medium with (13.0–20.7mm carapace length, 16.9–50.0mg wet mass) and were incubation time. For this purpose, 13 larvae were incubated reared to this age using standard rearing practices as described individually in 100ml of 6, 30 and 60moll–1 solutions of inactive previously (Rodriguez Souza et al., 1996). Among these, 11 larvae glycine, including 2 larvae with sealed mouths at 6moll–1 and 2 had their mouths sealed with cyanoacrylate bond to permit at 60moll–1. Bottles without larvae served as controls for possible distinction between the oral and integumental uptake of glycine. natural (bacterial) decay of glycine. Aliquots of 1ml were collected Sealing was carried out under a stereomicroscope by gently touching in triplicate from each of the bottles at 0, 2, 5 and 10h of incubation, the mouth-parts with the tip of a bond-coated copper wire. immediately filtered through a 0.2m Millipore filter, and stored were rinsed 3 times in 0.45m pore-filtered, ultraviolet light- or at –80°C. HPLC analysis was performed using a post-column autoclave-sterilized artificial sea water (Aquamarin, Yashima derivatization and fluorescence detection method developed by Chemicals Inc., Asagi, Japan) containing 100gml–1 streptomycin Shimadzu Corporation (manufacturer’s instructions; Shimadzu and 100Uml–1 penicillin, and maintained in similar medium without Corporation, Kyoto, Japan) that enables selective detection of food for 24h prior to the experiments. For the experiments, larvae amino acids with high sensitivity (detection limit of the order of were incubated individually or in groups of 3–4 in 100–300ml of fmol). The method uses O-phthalaldehyde (OPA) as the deriving artificial sea water or glycine–sea water media (see description reagent for amino acid detection. Analyses were performed in a below) at temperatures between 23 and 25°C. All rearing media Shimadzu LC-10AS amino acid analysis system (Shimadzu were prepared with sterilized artificial sea water as described above; Corporation). Glycine concentrations in the medium are shown as glassware and tools used to handle and rear the animals were the percentage remaining in the medium compared with the initial autoclaved for 20min at 120°C. concentration. Values represent the means of triplicate measurements for each individual per observation period. The rate of net uptake Histology and autoradiography for each individual was calculated using the first order depletion The route of uptake and the distribution of exogenous glycine in constant K, derived from the slope of a least-squares linear regression the larval body as well as the histological characteristics of glycine- of ln-transformed concentration values (Segel, 1976; Jaeckle and treated larvae were examined in two experiments using a total of Manahan, 1989), and the initial concentration. Net uptake rates were 28 larvae, including 4 larvae in the first experiment whose mouths expressed as mol of glycineg–1 body mass h–1. were sealed with the cyanoacrylate bond as described above. Groups of 2 larvae were incubated in 300ml of 0.04moll–1 [2- Liquid scintillation counting 3H]glycine with a specific activity of 18.6Cimmoll–1 in the first The uptake of glycine from the medium was analysed by LSC using experiment and in 200ml of media containing 250Ci of [2- 19 larvae incubated under the conditions described above for the 3H]glycine (specific activity 14.9Cimmoll–1) in combination with second autoradiography experiment, including 3 larvae whose inactive glycine to produce a final concentration of 6moll–1 in the mouths were sealed. Samples were collected and initially processed second experiment. Larvae incubated in glycine were sampled at as for autoradiography. Specimens were subsequently digested in 0.5, 2, 5, 12 and 24h. Control larvae incubated in clean sea water 0.5ml of Soluene (Packard Instrument Company, Meriden, CT, were sampled at 0, 5 and 12h. USA) tissue solubilizer for 72h at 60°C. After solubilization, 10ml After each incubation period, the larvae were rinsed 3 times in of scintillation cocktail was added and scintillation counting was artificial seawater alone (first experiment) or seawater containing carried out in a Packard 2550 TR/LL liquid scintillation analyser inactive glycine (second experiment) and then fixed for 24h in for 10min. Values measured by LSC were corrected for the change Bouin’s solution. Specimens were then dehydrated in an ethyl in the specific activity of glycine following addition of the inactive alcohol series, cleared in xylene, and embedded in tissue carrier for representation as mol per larvae. The uptake rates, embedding media (Paraplast Plus; Sigma-Aldrich Co., St Louis, expressed as molg–1 body mass h–1, were calculated using mass MO, USA). Saggital histological sections of the cephalic shield data of individual larvae measured immediately after the experiment and thorax were cut (5–6m) and mounted on glass slides. The (nearest 0.1mg). slides were deparaffinized, re-hydrated, coated with Konica NR- M2 autoradiographic emulsion in a dark room, air dried, and Scanning electron microscopy exposed for 6months in a light-tight desiccator at 4°C. Probing of the body surface for attached bacteria by scanning Autoradiographs were developed after exposure and the tissue was electron microscopy (SEM) was performed in 5 larvae incubated stained with Mayer’s haematoxylin and mounted for microscopic in 6moll–1 inactive glycine for 10–12h. Larvae were fixed in 2.5% observation. Adjacent portions of the stained tissue sections were glutaraldehyde in cacodylate buffer (pH7.2) for 2h, washed

THE JOURNAL OF EXPERIMENTAL BIOLOGY Absorption of glycine by lobster larvae 1861 overnight in the same buffer, and post-fixed in 1.3% osmium label could be seen in areas adjacent to those covered by the bond tetraoxide for 2h. Specimens were dehydrated in an ascending ethyl (Fig.4). The temporal changes in the density of label in each alcohol series and transferred to isoamyl acetate. Samples were then structure during incubation are summarized in Fig.5. Labelled critical point-dried, mounted on aluminium plates, coated with glycine showed higher accumulation early (0.5–2h) and then platinum/palladium (about 10nm) with an ion sputter (E102, decreased with time in the epidermis, muscle and haemolymph. Hitachi, Tokyo, Japan), and observed with a Hitachi S4000 scanning In contrast, accumulation increased with incubation time in the electron microscope. cuticle, haemocytes and midgut gland cells. The density of labelling in the lumen of the midgut gland was relatively constant RESULTS between the first (0.5h) and last (24h) samplings. Histology and autoradiography A number of structural variations were evident in the larvae High performance liquid chromatography immersed in glycine solutions compared with the controls. After Measurement by HPLC of the glycine concentration in the medium 12h, relative to controls, glycine-treated larvae had thicker for each individual showed that the response of the larvae was highly epidermis and muscles, larger tegumental glands, numerous and uniform at the lowest concentration (6moll–1) but markedly large haemocytes in the haemolymph space and, except for the E- irregular at the higher concentrations (30 and 60moll–1; Fig.6). cells at the distal portion, presented larger midgut gland cells At 6moll–1 (larval mass range 40.8–49.4mg), all larvae showed (Fig.1). Autoradiography showed that after 30min the labelled consistent net uptake throughout incubation, as can be inferred from amino acid had already been absorbed and retained in many the continuous decrease in the concentration of glycine in the structures such as the cuticle, epidermis, muscle, haemocytes and medium. At 30moll–1 (mass range 40.6–50.0mg), although all tegumental glands (Figs2–4). Portions of the integument showed larvae eventually showed net uptake after 5h, two of them showed label forming dense lines across the cuticle (Fig.2A). Substantial temporary net losses of glycine. At 60moll–1 (mass range accumulation of labelled glycine was observed in the semi- 16.9–48.6mg), the results varied greatly with larval body size: the enclosed chamber formed by the labrum and paragnaths; label was 48.6mg larva showed steady net uptake of glycine whereas larvae found attached to the cuticle and denticles, and expanding into the ranging from 16.9 to 22.5mg had continued net losses. The inner layers of the cuticle and tissues adjacent to the mouth of intermediate size larva (30.4mg) showed first net uptake until 5h intact larvae (Fig.3). In the same animals, faint labelling was and then loss at 10h. The net uptake rates for individual larvae at detected in the alimentary tract (e.g. proventriculus, foregut, 6moll–1 varied between 0.29 and 0.39molg–1h–1; the rates midgut, hindgut and midgut gland). In contrast, only weak or no apparently were not affected by the presence of cyanoacrylate bond labelling was found around the mouth and in the alimentary tract covering the mouth but were markedly affected by body mass of larvae whose mouths were sealed, although in these animals (Fig.7A). At 30moll–1, larvae showed net uptake rates between

Fig.1. Autoradiographs showing structural variations observed in the midgut gland area (sagittal sections) of a control (starved) and a glycine-treated larva. (A)Starved control larva. (B)Larva incubated for 12h in glycine medium. Cu: cuticle; Ep: epidermis; Hl: haemolymph space; Lu: midgut gland lumen; Mg: midgut gland tubules; Mu: muscle; arrow: haemocytes. Scale bars, 100m.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 1862 J. C. Rodriguez Souza and others

Fig.2. Autoradiographs showing label in different structures of glycine-treated larvae. (A)Cuticle and adjacent regions of larva incubated for 30min in glycine medium; note label in the pore channels of the cuticle (arrowheads). (B)Distal portion of the cephalothorax in larvae after incubation for 24h (the midgut gland cells observed are E-cells). Cu: cuticle; Ep: epidermis; Hl: haemolymph space; Lu: midgut gland lumen; Mg: midgut gland tubules; Mu: muscle; arrows: haemocytes. Scale bars: A, 50m; B, 100m.

0.12 and 1.31molg–1h–1 and these rates were also positively DISCUSSION correlated with body mass. At 60moll–1, the largest larva showed This study provides evidence from multiple sources that Panulirus a net uptake of 0.42molg–1h–1 whereas the smaller larvae showed japonicus phyllosoma larvae are able to absorb nutrients directly losses ranging from 0.07 to 1.81molg–1h–1. from the surrounding water. The results confirm and support our previous histological findings, which pointed to the possibility of Liquid scintillation counting oral and integumental absorption of nutrients (Rodriguez Souza et The uptake rates for individual larvae determined by LSC analysis al., 1999). Strong labelling was present in the cuticle, epidermis and varied between 0.04 and 0.29molg–1h–1 and were positively muscle, and labelling was comparatively weaker in the various parts correlated with body size (Fig.7B). After 12h of incubation, larvae of the digestive tract. As revealed by the results of label counting accumulated an average of 2.72molg–1 (N3; s.d.0.64) of glycine in autoradiographs, the most significant accumulation up to 2h was (results not shown). As for the determination using HPLC, there observed in the epidermis and muscles. At 5h an apparent reduction was no obvious difference in the accumulation of labelled glycine in the density of label was observed in these structures, presumably and the uptake rates between intact larvae and those that had their caused by utilization of amino acids in response to metabolic needs. mouths sealed with bond (Fig.7B). A reduction in the density of label with time was also observed in the haemolymph space. In the cuticle, haemocytes and midgut gland Scanning electron microscopy cells, the accumulation seemed to be steady. The decrease in label Observation by SEM revealed that bacteria attached to the body density in the epidermis and muscles concurrently with its increase surface of the individuals after glycine treatment were few and were in inner structures suggests that the amino acid was being transferred therefore unlikely to significantly affect the uptake of glycine or to from the integument to the internal organs. That this transfer is not cause its depletion from the rearing water (Fig.8). primarily dependent on the digestive tube seems to be borne out by

THE JOURNAL OF EXPERIMENTAL BIOLOGY Absorption of glycine by lobster larvae 1863

Fig.3. Autoradiographs of the mouth of glycine-treated larvae after 30min. (A)Sagittal section of the mouth region including a portion of the proventriculus; note accumulation of label in the semi-enclosed chamber and tegumental glands in the paragnaths. (B)Detail of the area indicated by the upper box in A; note the label attached to the cuticle and denticles (arrowhead), and label in the cuticle and epidermis (arrows). (C)Detail of the proventriculus (extension of area indicated by the lower box in A); note the presence of faint, but widespread labelling. Cu: cuticle; Ep: epidermis; Pg: paragnaths; Pv: proventriculus; Sc: semi-enclosed chamber; Fp: filter press; Tg: tegumental glands; Lu: lumen. Scale bars: A, 200m; B, 50m; C, 100m.

the low and almost invariable density of label in the midgut gland examined larvae indicates that the contribution to the uptake of lumen. glycine by heterotrophic organisms is negligible. Rodriguez Souza Large accumulations of labelled material seemed to be externally and colleagues reported the presence of pore channels in extensive retained in the mouthparts as well, probably adhering to mucus areas of the cuticle providing apparent connection between the secreted by the tegumental glands in the paragnaths. The adjacent external medium and the epidermis, and suggested that these inner sections of the cuticle and contiguous tissues in this region structures could be implicated in the absorptive process with the were also strongly labelled. This observation and the low aid of mucus secreted by the tegumental glands in the epidermis concentration of label found in subsequent parts of the digestive (Rodriguez Souza et al., 1999). The possibility of absorption system indicate that a significant portion of the orally ingested amino through the pores was supported in this study by the observation acids may be absorbed through the cuticle in the semi-enclosed of label forming dense lines across the cuticle in areas corresponding chamber formed by the labrum and paragnaths. In larvae with a to the location of the pores. sealed mouth no label was observed in the semi-enclosed chamber The net uptake obtained by HPLC and uptake obtained by LSC and subsequent portions of the digestive tract; however, labelling strongly supported the autoradiographic observations. Both in other structures presented similar characteristics to those observed analytical methods also showed that sealing the mouth of the larvae in regular larvae, indicating that the integumental mode of absorption did not affect the response of the larvae to the treatment, a fact may account for the presence of label in these structures. Moreover, which further points to the integument as a primary site for amino the absence of bacterial populations over the body surface of the acid uptake. The concentration of free amino acids in the cells of

THE JOURNAL OF EXPERIMENTAL BIOLOGY 1864 J. C. Rodriguez Souza and others

Fig.4. Autoradiographs of the mouth and adjacent regions for larvae with their mouths sealed with cyanoacrylate bond, incubated in glycine media. (A)Mouth region: note the absence of label in the semi-enclosed chamber (lines and smudges observed across the figures are unavoidable cracks in the film and bubbles produced because of the presence of the hard bond when the cover glass was set over the sections on the glass slide). (B)Sagittal sections of the cuticle in regions adjacent to the mouth: note the absence of label in the cuticle immediately underneath the bond but its presence in the muscle. Cu: cuticle; Ep: epidermis; Mu: muscle; Bd: bond; Os: oesophagus; Pg: paragnaths; Sc: semi-enclosed chamber. Scale bars: A, 100m; B, 20m.

invertebrate tissues ranges from 200 to 500mmoll–1, exceeding have been observed in larval stages of other marine invertebrates 200mmoll–1 for a single amino acid in some species (Preston, 1993). such as the sea urchin (Davis and Stephens, 1984) and oyster On the other hand, the concentration of glycine in natural marine (Manahan et al., 1989). For example, Davis and Stephens described environments is between 0.6moll–1 in surface waters and 6moll–1 losses of serine when the larvae were pre-incubated in high near the sediment–seawater interface (Henrichs and Farrington, concentrations of the same amino acid (Davis and Stephens, 1984). 1979; Manahan, 1983). Therefore, the integumental mode of They concluded that this would be an adaptive mechanism by the absorption in individuals exposed to environmentally relevant (that larvae to keep the internal pool of free amino acids. Thus, it is likely is, low) concentrations probably occurs against a concentration that spiny lobster phyllosoma larvae would also show increasing gradient. The transport mechanism of free amino acids through the glycine uptake rates with increasing concentration until a limit is body surface in marine invertebrates has been reported to be a reached, whereby further increases could lead to subsequent sodium-dependent co-transport (Preston and Stevens, 1982; Preston, saturation and efflux of the amino acid. 1993) that follows Michaelis–Menten kinetics for enzyme transport. The results of this study also suggested that the capacity of amino Although a comprehensive analysis of the kinetics of glycine acid retention by phyllosoma larvae is related to larval size (mass). transport was not undertaken in this study, the characteristics of the Thus, at both concentrations that produced net uptake in all larvae uptake strongly indicate that a similar mechanism may account for (6 and 30moll–1), uptake rates increased with increasing larval the absorption of glycine in the phyllosoma larvae. Thus, glycine mass. Interestingly, only the largest larva incubated at the highest at the lowest concentration of (6moll–1) was efficiently absorbed glycine concentration (60moll–1) showed net uptake whereas all and retained by the phyllosoma larvae whereas exposure to other (smaller) larvae at the same concentration showed net losses increasing concentrations of glycine produced transient (e.g. at of the amino acid. Manahan and colleagues studied the ontogenic 30moll–1) and/or marked and steady (e.g. at 60moll–1) effluxes changes in the rates of integumental amino acid transport in of the amino acid from the larvae to the medium. Similar responses Echinoderm larvae and observed that the metabolic rates increased

THE JOURNAL OF EXPERIMENTAL BIOLOGY Absorption of glycine by lobster larvae 1865

2,500,000 0.5 h 2 h 5 h 12 h 24 h Incubation time

–2 2,000,000 a in s mm 1,500,000

1,000,000 ilver gr N u m b er of s ilver

500,000

0 Cuticle Epidermis Muscle Haemolymph Haemocytes Cells Lumen Midgut gland Tissue/cells

Fig.5. Changes in the density of label (means ± s.d., N2) in different structures (tissue or cells) of larvae incubated in a solution containing 6moll–1 glycine as a function of the incubation period (determined from autoradiographs).

in direct proportion to larval mass in this group (Manahan et al., all spiny lobster phyllosoma larvae used in the current experiments 1989). They also noted that all species examined showed an were of approximately the same age and within a relatively narrow increase in the capacity of amino acid transport with development range of body sizes. Therefore, and given the long duration of the and increased metabolic demands. However, it must be noted that larval stage of P. japonicus (i.e. about 1year) (Matsuda, 2006), it would be premature to conclude that this relationship between body size and uptake rate would also be valid for animals of other ages. The contribution of amino acids absorbed through the integument 100 to the nutritional budget of marine invertebrates has been examined 6 µmol l–1 SM – 40.8 mg OM – 40.0 mg by earlier investigators (Stephens, 1972; Stephens, 1981; Stephens, 90 OM – 44.6 mg OM – 45.4 mg 1988; Jorgensen, 1976; Wright, 1985; Wright, 1988; Wright and S M – 49.4 mg ge Average Manahan, 1989; Preston, 1993; Baines et al., 2005; Grover et al., 80 2008). They noted that this mode of nutrient acquisition may account not only for the nutritional requirements of the epithelial tissues but 70 also in some species for a significant fraction of the requirements of the entire organism. They also emphasized the particular 105 a tion (%) 30 µmol l–1 OM – 40.6 mg 100 OM – 45.0 mg 0.4

OM – 50.0 mg )

ge Average –1 A

95 h –1 0.3 B 90 HPLC (R2=0.8317) 0.2 110 tive glycine concentr Rel a tive a te ( µ mol g 60 µmol l–1 S M – 16.9 mg 0.1 105 OM – 22.4 mg OM – 22.5 mg 2 S M – 30.4 mg LSC (R =0.9845) OM – 48.6 mg Upt a ke r 0 100 Average 2530 355 40 45 0 Larval mass (mg) 95 02468 10 Fig.7. Uptake rates of glycine by phyllosoma larvae incubated in a solution Incubation period (h) containing 6moll–1 of the amino acid as a function of larval mass. (A)Data set obtained by high performance liquid chromatography (HPLC; Fig.6. Changes in the relative concentration of glycine in the medium R20.8317). (B)Data set obtained by liquid scintillation counting (LSC; during incubation of individual larvae in solutions containing 6, 30 and R20.9845). Filled and open symbols indicate larvae whose mouths were 60moll–1 of the amino acid. OM, open mouth; SM, sealed mouth. sealed with the cyanoacrylate bond and intact larvae, respectively.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 1866 J. C. Rodriguez Souza and others

‘ecosystem culture method’ of Kittaka (Kittaka, 1997)], experiments have always been performed by feeding larvae exclusively with large food masses. It may be that exclusive utilization of one mode of feeding may not be enough to meet the nutritional requirements of the larvae, at least during critical stages. Essential nutrients such as fatty acids and vitamins are also found dissolved in seawater, and even in small quantities may contribute to the nutrition of marine invertebrate larvae (Jorgensen, 1966). Therefore, it is suggested that the absorption of dissolved and particulate nutrients from the surrounding seawater may be concurrent and complementary to feeding on large food masses, and that it may contribute to the normal development of P. japonicus phyllosoma larvae. Ongoing studies should clarify the extent of this contribution in reared larvae, their nutritional requirements, and the occurrence of this mode of feeding in wild specimens.

ACKNOWLEDGEMENTS The authors would like to thank the staff of Minamiizu, National Center for Stock Enhancement, Fisheries Research Agency and the Mie Prefecture Fisheries Research Institute, Japan, for broodstock care, hospitality, and access to facilities. This study was supported by a scholarship from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to the senior author and by a grant from the Tokyo University of Marine Science and Technology to C.A.S.

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