Characterisation of Amino Acid transport in Red Blood Cells of a Primitive Vertebrate, the Pacific Hagfish (Eptatretus Stouti)

A wide range of different amino acid transport systems has been identified in mammalian and avian red blood cells, including Na⁺-dependent systems, ASC, N, Gly, x⁻ and β, and Na⁺-independent systems, asc, y⁺, L and T (Christensen, 1979; Young, 1983; Harvey and Ellory, 1989). One function of amino acid transport in these cells is to provide intracellular precursors for glutathione (GSH) biosynthesis (Young and Tucker, 1983). In human red cells, cysteine transport for this purpose is mediated largely by the classical Na⁺-dependent system ASC (Young et al. 1979, 1983; Al-Saleh and Wheeler, 1982). In sheep and horse red cells, in contrast, cellular uptake of this amino acid occurs via a novel group of transporters designated asc(C) (Young et al. 1975, 1976; Young and Ellory, 1977; Fincham et al. 1985a, 1987a). These transporters share system /ISC’s selectivity for neutral amino acids of intermediate size, are inhibited by the Na⁺ site inhibitor harmaline, but do not require cations for activity (Young et al. 1988). Inherited deficiencies of asc are relatively common in some horse and sheep breeds and lead to GSH deficiency and decreased red cell viability (Tucker et al. 1981; Fincham et al. 1985a,b; Fisher et al. 1986).

In fish, studies of red cell amino acid transport have been limited largely to taurine, γ-amino-n-butyrate (GABA) and β-alanine. β-Amino acids are found in high concentration in both euryhaline teleost and elasmobranch red cells, where they contribute to cell volume regulation (Fugelli and Thoroed, 1986; Fincham et al. 1987b;,Goldstein and Kleinzeller, 1987). In both eel and flounder red cells, taurine uptake from plasma is mediated by a specific β-system with an apparent Na⁺/Cl“/taurine coupling ratio of 2:1:1 (Fincham et al. 1987b). A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreases the activity of the transporter. In parallel with this, low external osmolarity stimulates the activity of a Na⁺-independent taurine permeability pathway, producing net efflux of amino acid from the cells and a return towards the original cell volume (Fugelli and Thoroed, 1986; Fincham et al. 1987b). This transport route has the properties of a channel and accepts a range of α-amino acids and sugars in addition to β-amino acids. It is down-regulated by catecholamines and inhibited by loop diuretics (Wolowyk et al. 1989). Similar transport mechanisms are present in skate red cells (Goldstein and Kleinzeller, 1987). Recent studies implicate protein kinase C in the volume regulatory response (McConnell and Goldstein, 1988).

In this report we describe the first investigation of amino acid levels and amino acid transport in red cells of the Pacific hagfish (Eptatretus stouti Lockington), generally considered as amongst the most primitive of living vertebrates. In contrast to other fish species, hagfish red cells were found to contain high intracellular levels of conventional α-amino acids. The cells possessed uniquely high asc-type transport activity, but lacked the volume-sensitive amino acid channel found in other fish species.

Hagfish (Eptatretus stouti) were trapped at 165-201 m (90-110 fathoms) in Trevor Channel, Barkley Sound, Bamfield, British Columbia, and maintained in running sea water until bled from the subcutaneous sinus into heparinised tubes.

D-and L-[U-¹⁴C]-labelled amino acids, [U-¹⁴C]sucrose and ³H₂O were purchased from Amersham International pic, Amersham, Bucks, UK. Non-radioactive amino acids were obtained from Sigma Chemical Co. Ltd, Poole, Dorset, UK. When necessary, amino acid solutions were adjusted to the required pH with KOH or HC1. n-Dibutylphthalate was purchased from E. Merck, Darmstadt, FRG. All other reagents were of analytical grade.

Cells were prepared for transport experiments by washing three times with 20 vols of an incubation medium containing 500 mmol l⁻¹ NaCl, 5 mmol l⁻¹ glucose and 15 mmol l⁻¹ Mops (titrated to pH7.5 at 10°C with KOH). The buffy coat was discarded, and the washed red cells resuspended to a haematocrit of 20% in incubation medium. The water content of hagfish red cells, determined using ³H₂O with [¹⁴C]sucrose as extracellular space marker, was 68.6% (v/v).

Uptake of [¹⁴C]-labelled amino acid (10°C) was initiated by mixing cell suspension at 10°C with an equal volume of incubation medium at the same temperature containing the appropriate concentration of radioactive permeant (typically 1 μCiml⁻¹). Incubations were stopped at pre-determined time intervals (5 s to 3 h) by transferring 0.2 ml of the cell suspension (10% haematocrit) to a microfuge tube (volume 1.5 ml) containing 0.8 ml of ice-cold incubation medium layered on top of 0.5 ml of ice-cold n-dibutylphthalate (an ‘oil tube’). The oil tube, which was positioned in the rotor of an Eppendorf 5414 microfuge, was immediately centrifuged at 15000g for 10s. The aqueous medium and n-dibutylphthalate layers were removed by suction, leaving the cell pellet at the bottom of the tube. After carefully wiping the inside of the centrifuge tube with absorbent dental roll, the cell pellet was lysed with 0.5 ml of 0.5 % (v/v) Triton X100 in water, and 0.5 ml of 5% (v/v) trichloroacetic acid was added. The precipitate was removed by centrifugation (15 000g for 2 min) and 0.9 ml of the protein-free supernatant was counted for radioactivity by liquid scintillation spectroscopy with appropriate quench correction. Correction for radioactivity trapped in the extracellular space was made by processing cell samples which had been mixed at 1°C with either [¹⁴C]sucrose or the impermeant amino acid [¹⁴C]taurine (see Fig. 1) and immediately centrifuged. Uptake values were calculated after subtraction of these ‘blank’ estimates. Back extrapolation of L-alanine (0.2 mmol l⁻¹ extracellular concentration) time courses established that the ‘dead time’ between addition of cell suspension to the oil tube and termination of the flux was Is. Initial rates of amino acid uptake, determined from 5s kicubations and expressed as μmol l⁻¹ cell water h⁻¹, were calculated to include Tnis ‘dead time’. Incubation times were measured manually using a conventional laboratory timer.

To measure efflux, washed hagfish red cells were incubated at a haematocrit of 10% for 10 min at 10°C in incubation medium containing 0.2 mmol l⁻¹ L-[¹⁴C]alanine. The cells were rapidly washed free of extracellular radioactivity (6x20 vols of ice-cold incubation medium) by centrifugation, and resuspended in ice-cold medium to a haematocrit of 20 %. Efflux of radiolabelled amino acid was initiated by mixing 1 vol. of the ice-cold cell suspension with 19 vols of pre-warmed (10°C) incubation medium or medium containing the appropriate concentration of non-radioactive amino acid. At pre-determined time intervals (10−60 s), 1ml portions of cell suspension (final haematocrit 1%) were removed into ice-cold microcentrifuge tubes containing 0.2 ml of ice-cold n-dibutylphthalate and the cells were immediately sedimented below the oil (15 000g for 20 s). A 0.5 ml sample of cell-free supernatant was removed for scintillation counting. The starting intracellular tracer level was measured by processing a sample of the 20 % ‘loaded’ cell suspension as described for amino acid influx.

Plasma and red cell samples were deproteinised with 5% (v/v) sulphosalicylic acid and analysed on an LKB 4400 automated amino acid analyser using the manufacturer’s recommended lithium buffer system for physiological fluids.

Na⁺ and K⁺ determinations were made by flame photometry using an EEL flame photometer with appropriate filters and standards. Plasma samples were diluted with deionised water, and cells were pre-washed four times with an excess of ice-cold Na⁺-and K⁺-free solution (333 mmol l⁻¹ MgCl₂ buffered with 10 mmol1⁻¹ Mops/Tris, pH7.5). Cl⁻ determinations of plasma or red cells prewashed four times with an excess of ice-cold Cl−-free solution (500 mmol l⁻¹ sodium methylsulphate buffered with 10 mmol l⁻¹ Mops/Tris, pH 7.5) were carried out by coulometric titration using a Corning model 920M chloride meter.

Concentrations of amino acids in hagfish plasma were within the range normally encountered in mammalian and other higher vertebrate species (Table 1). In marked contrast, the mean red cell content of amino acids in three fish was approximately 100 mmol l⁻¹ cell water. The intracellular amino acid pool was composed largely of neutral amino acids (87%), with only minor contributions from acidic (5 %) or dibasic amino acids (8 %). Cell:plasma distribution ratios of individual amino acids ranged from 219, 209 and 173 for alanine, α-aminon butyrate and proline, respectively, to 11 and 13 for lysine and arginine. Unlike teleost and elasmobranch red cells, those from the hagfish contained only small amounts of GABA and no detectable taurine or β-alanine.

Hagfish plasma contained 400 mmol l⁻¹ Na⁺ and 7 mmol l⁻¹ K⁺, compared with intracellular levels of 17 and 148 mmol l⁻¹ cell water, respectively (Table 2). These concentrations give almost identical transmembrane ratios of 23 (out:in) for Na⁺, and 21 (in:out) for K⁺. In contrast to red blood cells from higher vertebrate species, a large (threefold) transmembrane Cl⁻ gradient was also present.

Fig. 1 shows time courses of uptake by hagfish red cells of a representative series tof [¹⁴C]-labelled amino acids, measured at 10°C in NaCl medium and at an initial Extracellular concentration of 0.2 mmol l⁻¹. Cells were most permeable to L-alanine and L-leucine, uptake within the first 3min. of the incubation period corresponding to the majority of available extracellular amino acid. Alanine transport was partially stereospecific, the D-isomer reaching the same final intracellular level as L-alanine, but with a slower time course. Uptake rates for the other amino acids tested were in the order glycine>L-lysine>GABA>L-gluta-mate>taurine. After 2h of incubation, uptake values for the first three of these amino acids were significantly above the starting extracellular concentration of 0.2 10 mmol l⁻¹. Detailed time courses of L-[¹⁴C]alanine uptake measured at three different initial extracellular concentrations (0.1, 1.0 and 10 mmol l⁻¹) are presented in Fig. 2. At the lowest permeant concentration, uptake proceeded rapidly, and at 15 s there was a one-third depletion of extracellular radioactivity. At steady state, the celkmedium [¹⁴C] distribution ratio was 320. Corresponding distribution ratios at the two higher L-alanine concentrations were 96 (1.0 mmoll⁻¹) and 13 (10mmol1⁻¹), with L-[¹⁴C]alanine uptake values of 12.6 ancr 66 mmol l⁻¹ cell water, respectively.

To test for Na⁺-dependence of amino acid uptake, the initial rate (5 s flux) and 10min uptake values for selected amino acids (0.2 mmol l⁻¹ initial extracellular concentration) were measured both in NaCl medium and in medium where NaCl was iso-osmotically replaced by choline chloride (Table 3). Initial rates of transport for L-alanine, L-glutamine, L-leucine and L-histidine were in the range 66-153 mmoll⁻¹cellwaterh⁻¹, with no indication of Na⁺-dependence. In agreement with the results presented in Fig. 1, glycine exhibited a lower rate of transport. This was significantly decreased (24 %) in choline chloride medium, but sholine substitution did not affect the 10 min uptake value. L-Proline transport was slow and highly Na⁺-dependent, uptake in NaCl medium being 36 times greater than in choline chloride medium.

For L-alanine, we also investigated transport in media where NaCl was replaced by LiCl or (N-methyl-D-glucamine chloride and under cation-free conditions (NaCl replaced by iso-osmotic mannitol). None of these substitutions significantly influenced the ability of hagfish red cells to accumulate L-alanine (data not shown). We also considered the possibility that L-alanine uptake by the cells might be proton-dependent. Pre-treatment of cells in NaCl medium for 1 min at 10°C with the proton conductors CCCP (carbonylcyanide-m-chlorophenylhydrazone) and FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) (10 μmol l⁻¹, 10% haematocrit) had no significant effect on 5 s or 10 min L-alanine uptake values (data not shown).

Subsequent experiments in the present study focused on the properties of the L-alanine transport mechanism(s) in hagfish red cells.

The remarkable ability of hagfish red cells to achieve cation-independent accumulation of L-alanine from the extracellular medium was confirmed by direct amino acid analysis. In the experiment summarised in Table 4, cells were incubated in the presence and in the absence of 10 mmol l⁻¹ extracellular L-[¹⁴C]alanine and the cellular content of individual amino acids and radiolabelled L-alanine were monitored as a function of time. In cells incubated with extracellular L-[¹⁴C]alanine, cellular [alanine] rose from 5.7mmol l⁻¹ cell water at time zero to 59.3 mmol l⁻¹ cell water after lh, an increase of 53.6 mmol l⁻¹ cell water. The corresponding uptake of L-[¹⁴C]alanine was 58.5 mmol l⁻¹ cell water. As shown in Table 4, the large net uptake of alanine was accompanied by a corresponding efflux of other amino acids from the cells, such that the total intracellular amino acid content of 117 mmol l⁻¹ cell water remained unchanged during the 1 h incubation period. During the initial phase of L-alanine uptake, the exchanged amino acids were largely made up of threonine, serine, glutamine, valine and alanine (Fig. 3). Additional amino acids exchanged for extracellular L-alanine during the later stages of the incubation included glycine, methionine and leucine. Cells incubated for 1 h in the absence of extracellular L-alanine retained 97 % of their intracellular amino acid pool.

Efflux of L-[¹⁴C]alanine from pre-loaded cells was used as an alternative way to examine the properties of L-alanine transport in hagfish red cells. Fig. 4 shows that efflux of tracer, measured at 10°C in NaCl medium, was very slow in the absence of extracellular amino acid, but rapid in the presence of external non-radioactive alanine (10 mmol l⁻⁻ extracellular concentration), the L-isomer causing a greater stimulation of transport than the D-isomer. In another experiment, the initial rate of L-alanine efflux (30 s flux) was measured in the presence of different extracellular concentrations of L-and D-alanine in the range 0.05−10 mmol l⁻¹ (Fig. 5). Stimulation of L-[¹⁴C]alanine efflux by extracellular L-alanine was saturable, the maximum response representing a 9.3-fold increase in efflux rate over the control value (efflux measured in the absence of extracellular alanine). The external L-alanine concentration required to give half-maximal stimultion of efflux was 0.25 mmol l⁻¹. Stimulation of L-[¹⁴C]alanine efflux by D-alanine was less effective, with a considerably lower apparent affinity compared with the L-isomer.

The ability of hagfish red cells to participate in exchange reactions was exploited to investigate the substrate specificity of the L-alanine transporter in these cells (Table 5). Extracellular amino acid concentrations of 1 mmol l⁻¹ were used. In certain cases, a higher concentration of 10 mmol l⁻¹ was employed in attempts to detect amino acids with low, but significant, transporter affinities. Neutral amino acids most effective in stimulating L-alanine efflux (30 s incubation) were L-serine, the L-cysteine analogue L-α-amino-n-butyrate and L-alanine followed, in order of effectiveness, by L-glutamine, L-valine, glycine, D-alanine, D-α-amino-n-butyrate, L-leucine, L-methionine and α-amino-iso-butyrate. A modest stimulatory effect (60 % at 10 mmol l⁻¹ extracellular concentration) was also observed for the dibasic amino acid L-arginine. α,β-Diaminopropionate, in contrast, caused marked stimulation of L-alanine efflux. At the experimental pH of 7.5, this amino acid (pK₂=6.7) is likely to act largely (86%) as a neutral substrate. The acidic amino acid L-glutamate did not stimulate L-alanine efflux.

To test for the presence of a volume-sensitive amino acid channel in hagfish red cells, cells were pre-loaded with radioactive L-alanine, as described in Materials and methods, and incubated at 10°C for either 30 s or 10 min in 500 mmol l⁻¹ isotonic NaCl incubation medium or in a series of hypotonic media prepared by 10−50% (v/v) dilution of 500 mmol l⁻¹ NaCl medium with water. In contrast to the large and progressive increase in α-and β-amino acid permeability seen in other fish species, L-alanine efflux from hagfish red cells was unresponsive to changes in medium osmolarity. For example, at an intermediate medium dilution of 25 % (relative cell volume 1.19), L-alanine effluxes were 92 % (30 s incubation) and 103 % (10 min incubation) of control (efflux under isotonic conditions, mean of duplicate determinations). Corresponding values at the maximum medium dilution of 50 % were: relative cell volume 1.45, L-alanine effluxes 98 and 112 % of control, respectively. In separate experiments, it was found that hypotonic cell swelling had no significant effect on the low taurine permeability of hagfish red cells seen in Fig. 1 (data not shown).

In the present series of experiments, we have demonstrated the occurrence of exceptionally high neutral amino acid transport activity and high intracellular levels of amino acids in red cells of the Pacific hagfish (Eptatretus stouti), one of the most primitive of living vertebrates. The total intracellular amino acid pool (approx. 100 mmol l⁻¹ cell water) was 50-fold greater than that of plasma, but in the same range as reported previously for parietal muscle from the closely related Atlantic hagfish Myxine glutinosa (Cholette and Gagnon, 1973). In both hagfish cell types, the dominant intracellular amino acid was proline, followed by the other neutral amino acids alanine, threonine, valine and leucine. Plasma:red cell distribution ratios were highest for proline, alanine and α-amino-n-butyrate. Of the amino acids tested, only L-proline and glycine exhibited evidence of Na⁺-dependent transport activity. In other vertebrate species, cation-dependent uptake of these amino acids is mediated by the Na⁺-dependent system ASC and by the Na⁺-and Cl⁻-dependent system Gly (Christensen, 1979; Young, 1983; Harvey and Ellory, 1989). Hagfish red cells have a large (20-fold) inwardly directed transmembrane Na⁺ gradient and also a threefold transmembrane Cl⁻ gradient (Table 2).

For alanine, uptake by the hagfish red cell was stereoselective for the L-isomer and tightly coupled to amino acid efflux (1:1 stoichiometry), L-Alanine homoexchange efflux proceeded as expected from simple Michaelis-Menten kinetics, half-maximal stimulation of tracer L-alanine efflux occurring at an extracellular L-alanine concentration of 0.25 mmol l⁻¹. As judged by trans-acceleration exper-g iments, the L-alanine transport mechanism exhibited a substrate preference for neutral amino acids of intermediate size. This feature, together with the system’s lack of Na⁺-dependence, identifies it as an asc-type transporter similar to systems characterised initially by us in horse and sheep red cells (Young and Ellory, 1977; Fincham et al. 1985a, 1987a) and subsequently by others in avian red cells (Vadgama and Christensen, 1985a), mammalian exocrine pancreas (Mann and Peran, 1986) and erythroid cells from foetal rat liver (Vadgama et al. 1987).

It has been established that Na⁺ binding to the human red cell ASC-system is competitively inhibited by the halucinogenic alkaloid harmaline and that a topographically equivalent harmaline binding site is also present on horse red cell asc (Young et al. 1988). In contrast to ASC, however, the asc site exhibits no measurable affinity for Na⁺. In preliminary inhibition studies, we have confirmed that harmaline blocks L-alanine uptake (0.2 mmol l⁻¹ extracellular concentration, 10°C) by hagfish red cells, the concentration required for 50% inhibition (3.5 mmol l⁻¹) comparing favourably with K_i values in the range 1-3 mmol l⁻¹ for harmaline inhibition of human red cell ASC and horse red cell asc (Young et al. 1988). For ASC, Na⁺ and amino acid bind in close juxtaposition, such that the combined (Na⁺/neutral amino acid) permeation site can be occupied by a dibasic amino acid (Thomas and Christensen, 1970, 1971). Parallel interactions of dibasic amino acids with mammalian asc transporters have also been observed (Young et al. 1976; Young and Ellory, 1977; Fincham et al. 1988). In contrast to harmaline inhibition, this property is poorly expressed in hagfish, as judged by the relative rates of L-alanine and L-lysine uptake by the cells (Fig. 1, Table 3) and by the small effects of dibasic amino acids on L-alanine efflux (Table 5). Studies reported by Vadgama and Christensen (1985b) indicate that dibasic amino acids also interact weakly with the avian red cell asc-type transport mechanism.

Based upon the initial rate of L-alanine uptake in Table 3, asc-type transport activity in hagfish red cells is 10⁴-fold higher than that found in horse and sheep red cells at the same temperature and is correspondingly higher than the ASC transport capacity of human and avian red cells (Eavenson and Christensen, 1967; Young et al. 1976, 1983; Fincham et al. 1987a). Uptake rates for L-leucine and L-histidine were even higher (Table 3). Studies to be published elsewhere indicate that these amino acids are transported by a separate high-affinity L-type exchange mechanism. We consider it likely, therefore, that amino acid transporters represent major intrinsic membrane components of hagfish red cell membranes. Since hagfish red cells have minimal numbers of the band 3 anion-exchange transporter (Ellory et al. 1987), normally the most abundant red cell membrane protein, these cells represent an ideal model system in which to explore molecular aspects of Na⁺-independent amino acid transporters. As a first step in this direction, we have established that the hagfish L-alanine transporter, in common with the sheep asc system (Young, 1980), possesses a p-chloromercuribenzenesul-phonate (PCMBS)-reactive thiol group(s) located within its exofacial permeant binding site (D. A. Fincham, M. W. Wolowyk and J. D. Young, unpublished observation). It is anticipated that this cysteine residue will facilitate identification and subsequent isolation of the transporter.

Hagfish plasma is in osmotic equilibrium with the external environment under a wide range of osmotic pressures (Cholette and Gagnon, 1973). In fish adapted to different salinities, intracellular isosmotic regulation in muscle, and presumably also in red cells, relies, in part, on compensating changes in the intracellular amino acid pool (Cholette and Gagnon, 1973). As detailed in the Introduction, intracellular amino acids also participate in cell volume regulation in elasmobranchs and euryhaline teleosts, but the mechanisms involved appear to differ. For example, the dominant amino acids in elasmobranch (Goldstein and Kleinzeller, 1987) and teleost red cells (Fugelli and Thoroed, 1986; Fincham et al. 1981b) are β-amino acids, compared with conventional neutral amino acids in hagfish. Also, as reported here, a volume-activated amino acid channel present in these species does not appear to be present in hagfish red cells, exposure to hypotonic medium having no measureable effect on either taurine or L-alanine permeability.

With regard to cellular accumulation of amino acids and their participation in cell volume regulation, it is important to emphasise that both the hagfish asc and L transporters are Na⁺-independent and function predominantly in an exchange mode. Thus, the dramatic ability of the hagfish asc system to accumulate L-alanine from the extracellular medium occurs at the expense of intracellular amino acids. Possible sources of these amino acids include Na⁺-dependent uptake from plasma (see above) and, as occurs in invertebrates, intracellular metabolism (Pierce, 1982). In preliminary experiments, we have found that Na⁺-dependent uptake of L-[¹⁴C]proline from the extracellular medium is highly concentrative; the cells achieve a 50-fold transmembrane distribution ratio over a 5 h incubation period (initial extracellular L-proline concentration 0.2 mmol l⁻¹). Competition experiments identified the transport mechanism as an ASC-type system with an unusually high affinity for imino acids. To exclude the unlikely possibility that hagfish red cells accumulate amino acids in exchange for non-amino-acid organic solutes such as sugars, keto acids or nucleosides, we have tested a wide range of more than 50 membrane transport inhibitors for their ability to block L-alanine uptake. With the exception of thiol reagents (see above), none was an effective inhibitor of L-alanine transport (D. A. Fincham, M. W. Wolowyk and J. D. Young, unpublished data). Both Na⁺-dependent uptake from plasma and intracellular amino acid production/degradation are potential sites for the regulation of intracellular amino acid levels in response to changes in extracellular osmolarity. In this context, asc and L would facilitate the rapid exchange of amino acids between cells and plasma, serving to modulate the amino acid composition of the intracellular pool and perhaps allowing red cells to participate in inter-organ transport of amino acids (Christensen, 1982). As occurs in mammalian red cells (Young et al. 1975; Fisher et al. 1986; Fincham et al. 1981a), the hagfish red cell asc transporter may also function to make available plasma cysteine for intracellular glutathione biosynthesis.

In summary, the present series of experiments demonstrates the presence of high concentrations of neutral amino acids and correspondingly high rates of amino acid transport in red cells of the Pacific hagfish. For L-alanine, transport was mediated by a Na⁺-independent asc-type mechanism, indicating that transporters of this type pre-date, or arose at an early stage of, vertebrate evolution. A volumesensitive amino acid channel found in higher fish species was not present in hagfish red cells.

We are grateful to the staff of the Bamfield Marine Station for providing laboratory and other facilities. This research was funded by project grants from the Natural Sciences and Engineering Research Council of Canada, The Royal Hong Kong Jockey Club and the University and Polytechnics Grants Committee, Hong Kong. In 1988, DAF was supported as a Research Associate of the Western Canadian Universities Marine Biological Society. JDY is a Heritage Medical Scientist.