Influence of food concentration on the physiological energetics and growth ofOstrea edulis larvae

Feeding, respiration and growth rates of oyster (Ostrea edulis L.) larvae reared at five food levels were measured throughout the entire larval period. Energy budgets were derived as a function of alga (Isochrysis galbana Parke) food concentration. Ingestion rate (IR, cells h^-1) and oxygen consumption rate ( , nl h^-1) were almost isometric functions of larval size [ash-free dry weight, (AFDW, g)], characterized by the equations: IR=803.9 AFDW^1.13 and =4.85 AFDW^1.09. Ingested ration was directly correlated to cell concentration up to a maximum at 200 cells l^-1, with further increases failing to support higher ingestion rates. Likewise, growth rate linearly increased with food ration up to 100 cells l^-1 (max. growth efficiency,K ₁=25%) and reached a maximum at 200 cells l^-1 (growth rate=5.6 m d^-1), with further increases in food not supporting significantly faster growth. Maintenance ration was 2 to 3% daily dry weight (DW); optimum ration increased during larval development from 5 to 20% DW; maximum ration was 20% DW. During larval rearing, an increasing feeding schedule of 50, 100 and 200 cells l^-1 from Days 0, 5 and 10, respectively, is recommended.     

Photosynthetic characteristics of nanoplankton and picoplankton from the surface mixed layer

Nanoplankton and picoplankton primary production has been studied at two oceanic stations in the Porcupine Sea-bight and at one shelf station in the Celtic Sea. At both sites, low wind conditions in June and July 1985 resulted in greatly reduced vertical turbulent mixing and a secondary, temporary thermocline developed in what is usually a well-mixed surface layer; as a result, there was physical separation of the phytoplankton within two zones of the surface mixed layer. The photosynthetic characteristics of three size fractions (>5 m, <5 to >1 m and <1 to >0.2 m) of phytoplankton populations from the two zones have been measured. Phytoplankton was more abundant at the oceanic stations and chlorophyll a values were between 1.3 and 2.2 mg chlorophyll a m^-3, compared with 0.3 to 0.6 mg chlorophyll a m^-3 at the shelf station; at both stations, numbers of cyanobacteria were slightly higher in the lower zone of the surface mixed layer. There was no effect of the temporary thermocline on the vertical profiles of primary production and most phtosynthesis occurred in the surface 10 m. Photosynthetic parameters of the three size fractions of phytoplankton have been determined; there was considerable day-to-day variation in the measured photosynthetic parameters. Assimilation number (P _m ^B ) of all >5 m phytoplankton was lower for the deeper than for the surface populations, but there was little change in initial slope (a ^B ). The small oceanic nanoplankton (<5 to >1 m) showed changes similar to the >5 m phytoplankton, but the same size fraction from the shelf station showed changes that were more like those shown by the picoplankton (<1 m) viz, little change in P _m ^B but an increase in a ^B with depth. Values of a ^B were generally greater for the picoplankton fraction than for the larger phytoplankton, but values of adaptation parameter (I _k )(=P _m ^B /) were not always less. There was little evidence to support the hypothesis that these populations of picoplankton were significantly more adapted to low light conditions than the larger phytoplankton cells. When photosynthetic parameters of the picoplankton were normalised to cell number (P _m ^C /a ^C ) rather than chlorophyll a, P _m ^C was comparable to other published data for picoplankton, but a ^C was much lower. The maximum doubling time of the picoplankton at saturating irradiance is calculated to be ca. 8.5 h for the oceanic population and ca. 6.2 h for the shelf population.     

Seasonal photosynthetic characteristics of Ascoseira mirabilis (Ascoseirales,Phaeophyceae) from King George Island,Antarctica

Monthly variation in photosynthesis, dark respiration, chlorophyll a content and carbon: nitrogen (C:N) ratios in different lamina sections of adult plants of Ascoseira mirabilis Skottsberg from King George Island, Antarctica, was investigated between September 1993 and February 1994. Light saturated net photosynthesis (P _max) showed maximum values in September (12 to 25 mol O₂ g^-1 fr wt h^-1), and decreased towards the summer to values ranging between 2.0 and 5.0 mol O₂ g^-1. In the distal section, however, a second optimum occurred in December (25 mol O₂ g^-1 fr wt h^-1). Dark respiration rates were also highest in October and November and decreased strongly in December to February (6.0 and 1.0 mol O₂ g^-1 fr wt h^-1, respectively). Gross photosynthesis exhibited high values between September and December. Concomitant with the seasonal decrease of photosynthetic efficiency () from mean values of 1.2 mol O₂ g^-1 fr wt h^-1 (mol photons cm^-2 s^-1)^-1 in September to 0.3 mol O₂ g^-1 fr wt h^-1 (mol photons cm^-2 s^-1)^-1 in January, the initial light saturating point (I _k) gradually increased from 19 to 60 mol photons m^-2 s^-1. Likewise C:N ratios were low in spring (12 to 13) and increased in summer (20). In general, the photosynthetic parameters P _max, gross photosynthesis, and Chl a concentrations were significantly higher in the distal section of the thallus. In contrast, C:N ratios were lower in the distal section of the lamina. The results show that photosynthesis obviously strongly supports growth of the alga in late winter to spring, as it does in some morphologically related brown algae from temperate and polar regions. The question whether growth is additionally powered     

Physiological ecology of picoplankton in the North Sea

The distribution of cyanobacteria in the surface waters of the North Sea was measured during July 1987. Numbers of cyanobacteria ranged from 2.5x10⁶ to 1.7x10⁸ cells 1^-1. In the majority of stations, cyanobacterial numbers were highest in the near-surface water and a subsurface maximum was found at only one station. The distribution of ¹⁴C among the end-products of photosynthesis was determined for picoplankton (<1 m) and other phytoplankton >1 m throughout the North Sea. The majority of label was found in the protein fraction of both picoplankton and >1 m phytoplankton; incorporation into lipids and polysaccharides plus nucleic acids was much lower. We interpret the large incorporation into protein to be a consequence of nutrient limitation of these natural assemblages. Photosynthetic parameters of the two size fractions were also determined. Assimilation number (P _m ^B ) and initial slope were greater for the picoplankton fraction than for phytoplankton >1 m but there was no evidence of significant photoinhibition of either fraction at irradiances up to 1 000 E m^-2 s^-1.     

Development of the pericalymma larva of Solemya reidi (Bivalvia: Cryptodonta: Solemyidae) as revealed by light and electron microscopy

Solemya reidi Bernard 1980 is a gutless protobranch bivalve known to possess intracellular chemoautotrophic bacterial symbionts in its gill. A light and electron microscope study on the embryology and larval development of S. reidi provides data for the bivalve Subclass Cryptodonta. S. reidi spontaneously spawned large eggs (271 m in diameter), which developed within individual gelatious egg capsules. The first several cleavages were equal and a distinct molluscan cross was formed at the animal pole of the embryo, features previously unreported in bivalve development. Lecithotrophic pericalymma larvae (similar to the larvae of paleotaxodont protobranch bivalves and aplacophoran molluscs) hatched at 18 to 24 h and remained in the water column for a further 5 d at 10°C. At hatching, larvae measured from 360 to 440 m in length and from 225 to 265 m in cross-sectional diameter. Definitive adult structures developed within an epithelial locomotory test entirely covered with compound cilia. The test histolysed at metamorphosis and was ingested throught the mouth into the perivisceral cavity. Length and height of the shell following metamorphosis was 433 m (±42 m, n=16) and 282 m (± 29 m, n=13), respectively. Primary data and data from the literature show that the type of larval development in both paleotaxodont and cryptodont bivalves cannot be reliably estimated from egg or prodissoconch sizes.     

Some experiments on phosphate assimilation by coastal marine plankton

A study of phosphate assimilation by coastal marine plankton revealed that both phytoplankton and microheterotrophs incorporated radioactive phosphorus (³³P). Size fractionation of the particulate matter (using 1 m pore diameter Nucleopore^® membrane filters), antibiotic treatment (using garamycin), and independent estimaties of photoautotrophic (¹⁴CO₂ uptake) and heterotrophic (³H-glucose uptake) activities were employed to separate phyto- and bacterioplankton phosphate uptake. Results indicated that phytoplankton ³³P-uptake was best estimated by the fraction of particulate matter retained on the 1 m membrane filters. Usually, less than 10% of the phytoplankton (based on chlorophyll a measurements) passed the 1 m pore-diameter filters, whereas about 90% of the heterotrophic activity passed. At least 50% of the ³³P-uptake was associated with the <1 m fraction. It may be possible to resolve the phytoplankton and bacterial contributions to ³³P-uptake by comparing the percent of total ³³P-uptake with the percent of total ³H-glucose uptake associated with the >1 m fraction.     

Enhancement of chlorophyll a production in Gulf Stream surface seawater by synthetic versus natural rain

Rainwater concentrations of either ammonium or nitrate were sufficient to stimulate chlorophyll a (chl a) production in bioassay experiments using Gulf Stream surface water collected off North Carolina during the summer of 1991. Previous studies primarily examined inshore waters and did not address the impact of rainwater ammonium. An increase in chl a occurred within 1 d of the addition of synthetic rainwater (2 or 5% rainwater, 98 or 95% seawater) containing up to 10 M ammonium; this increase was followed by a decrease in chl a the following day. A similar response to nitrate addition (5% addition of 20 M nitrate rain) was observed. In separate experiments, natural rainwater having nitrate and ammonium concentrations less than those in the experimental synthetic rain yielded a greater chl a response than synthetic rain when added at similar dilutions (0.5 to 5.0% rain). The maximum dissolved inorganic nitrogen concentration in the enriched seawater in these bioassays was 1.8 M; prior to enrichment the maximum was < 0.4 M. Bioassay experiments begun 2 d after a major storm event (sustained NE winds with gusts to 13 m s^-1 and ca. 390 mol m^-2 inorganic nitrogen deposition from rain) showed a chl a increase in response to addition of natural rainwater, but not to synthetic rainwater with similar dissolved inorganic nitrogen concentration. These results suggest that phytoplankton stimulants, in addition to nitrate and ammonium, exist in natural rain but not in the synthetic rain used in these experiments.     

Carotenoids versus retinoids (Vitamin A) as essential growth factors in penaeid prawns (Penaeus semisulcatus)

Penaeus semisulcatus de Haan adult gravid females, eggs and non-feeding early larvae, including Protozoea I, were used to test the hypothesis that retinoid (Vitamin A) is not required in early decapod crustacean development. In the adult gravid females, retinoids were detected only in the eyes (1.56±0.23 g g^-1 wet mass), whereas there were up to 97 g g^-1 wet mass total carotenoid in digestive gland and epidermis. This was mostly esterified, except in the ovaries, where free astaxanthin predominated (30 g g^-1). No retinoids could be detected in the eggs, the naupliar stages or Protozoea I, but free astaxanthin was metabolised exponentially, falling from 19 g g^-1 in the eggs to 4 g g^-1 in Protozoea I. This suggests that retinoid is not essential in early development and that carotenoid could be taking its place. Also, including retinoids in artificial diets appears to be unnecessary, provided adequate carotenoid is supplied.     

Particulate organic carbon and nitrogen in the adjacent seas of the Pacific Ocean

Particulate organic carbon (POC) and nitrogen in sea water were measured in samples collected in the adjacent seas of the Pacific Ocean during the cruises of T. S. Oshoro-Maru (1969, 1970) and the R. V. Hakuho-Maru (KH-70-4, KH-72-1). High values were obtained in the northern North Pacific and the Bering Sea, the concentration of particulate carbon in the upper 50-m layer ranged from 35 to 550 g Cl^-1. In the deep waters of these area, values above 50 g Cl^-1 were frequently observed. The lowest values in the surface layer and deeper layers were obtained in the Japan Sea (23 gCl^-1) and in the South China Sea (7 g Cl^-1) respectively. A consistent minimum was located in the intermediate waters (100–400 m) throughout the entire region studied. Variation with depth was generally irregular with marked peak values in different layers. The POC distribution consited of these peak values and a relatively uniform background concentration. These background values slightly decreased with increasing depth and were different locally. Correlation analysis between carbon concentration and apparent oxygen utilization (AOU) of ambient water for the samples in the Japan Sea and the Sulu Sea showed that there was no systematic decrease of particulate carbon with increasing AOU. In these areas, the carbon concentration scattered in the higher AOU domain ranged from 10–100 g Cl^-1. These observations support the conception that downward transport of particulate matter from the overlying surface layer in the adjacent seas of the Pacific Ocean may be fairly rapid.     

The common jellyfish Aurelia aurita: standing stock,excretion and nutrient regeneration in the Kiel Bight,Western Baltic

The population dynamics, ammonia and inorganic phosphate excretion, and nutrient regeneration of the common jellyfish Aurelia aurita was investigated from 1982 to 1984 in the Kiel Bight, western Baltic Sea. During summer 1982, medusae abundance ranged between 14 and 23 individuals 100 m^-3, biomass was estimated at about 5 g C 100 m^-3 and the mean final diameter of individuals was 22 cm. Abundance, based on numbers, in 1983 and 1984 was an order of magnitude lower; biomass was less than 2 g C 100 m^-3 and jellyfish grew to 30 cm. During the summers of 1983 and 1984, A. aurita biomass constituted roughly 40% of that of the total zooplankton>200 m. In 1982, for which zooplankton data were lacking, it was assumed that medusae biomass was greater than that of all other zooplankton groups. Total ammonia excretion ranged between 6.5 and 36 mol h^-1 individual^-1, whereas inorganic phosphate release was 1.4 to 5.7 mol h^-1 individual^-1. Allometric equations were calculated and exponents of 0.93 for NH₄–N release and 0.87 for PO₄–P excretion were determined. Nitrogen and phosphorus turnover rates were 5.4 and 14.6% d^-1, respectively. In 1982, the medusae population released 1 100 mol NH₄–N m^-2 d^-1, about 11% of the nitrogen requirements of the phytoplankton. The inorganic phosphate excretion (150 mol m^-2 d^-1) sustained 23% of the nutrient demands of the primary producers. In the other two years the nutrient cycling of the medusae was much less important, and satisfied only 3 to 6% of the nutrient demands. It is suggested that in some years A. aurita is the second most important source of regenerated nutrients in Kiel Bight, next to sediment.     

Seston composition of the bottom waters of Great Lameshur Bay,St. John,US Virgin Islands

Seston composition [particulate organic carbon (POC), particulate nitrogen (PN), phyto- and microzooplankton numbers and biomass] was investigated in the bottom waters of Great Lameshur Bay, St. John, Virgin Islands (USA), in October, 1970 during Tektite II Mission 17–50. Mean values of 67.4 g POC · I^-1 and 7.2 g PN · I^-1 were determined. A mean phytoplankton carbon content of 42.2 g · I^-1 and zooplankton carbon content of 5.5 g · I^-1 were calculated from counts. The phytoplankton consisted mainly of dinoflagellates 71.2% phytoplankton carbon. Copepods were the dominant zooplankters (61.8% zooplankton carbon), followed by larvaceans (30.9% zooplankton carbon). Organic carbon content of counted zooplankton faecal pellets ranged between 0.4 and 1.6 g · I^-1, and amounted probably to about 15% of the total zooplankton carbon value. Plankton and detritus components as possible food for coral-reef animals are discussed. The ratio carbon: nitrogen of suspended particles is compared to that of sedimented matter.     

Estimation in situ d'une population d'Oikopleura longicauda (Appendicularia) à l'aide de deux filets de maille différente

A population of Oikopleura longicauda was sampled daily for 10 days, a period longer than their life cycle, using two nets of different mesh size (53 and 200 m). Analysis of variance revealed that the number of specimens collected by the two nets was not significantly different for individuals larger than 300 m in trunk length. A grand mean regression plotted for log of length versus log of number of specimens indicated that the number of small-sized O. longicauda (<300 m), which are not quantitatively sampled by nets, can be estimated from the collections by use of a coefficient of correction.     

Effects of nitrogen and phosphorus enrichement on in vivo symbiotic zooxanthellae of Pocillopora damicornis

The reef coral Pocillopora damicornis (Linnaeus) was grown for 8 wk in four nutrient treatments: control, consisting of ambient, unfiltered Kaneohe Bay seawater [dissolved inorganic nitrogen (DIN, 1.0 M) and dissolved inorganic phosphate (DIP, 0.3 M)]; nitrogen enrichment (15 M DIN as ammonium); phosphorus enrichment (1.2 M DIP as inorganic phosphate); and 15 M DIN+1.2 M DIP. Analyses of zooxanthellae for C, N, P and chlorophyll a after the 8 wk experiment indicated that DIN enrichment increased the cellular chlorophyll a and excess nitrogen fraction of the algae, but did not affect C cell^-1. DIP enrichment decreased both C and P cell^-1, but the decrease was proportionally less for C cell^-1. the response of cellular P to both DIN and DIP enrichment appeared to be in the same direction and could not be explained as a primary effect of external nutrient enrichment. The observed response of cellular P might be a consequence of in situ CO₂ limitation. DIN enrichment could increase the CO₂ (aq) demand by increasing the net production per unit area. DIP enrichment could slow down calcification, thus decreasing the availability of CO₂ (aq) in the coral tissue.Hawaii Institute of Marine Biology Contribution No. 920     

In-situ studies on the life cycle of Diastylis rathkei (Cumacea: Crustacea)

Aspects of the life cycle of the cumacean Diastylis rathkei Kröyer, the most important food item of demersal fishes in the Western Baltic Sea, were investigated from the Underwater Laboratory (UWL) Helgoland in Lübeck Bay. In August the population density was 10.1±3.6 individuals 100 cm^-2 in fine-medium sand (Md=292 m), and 3.6±2.4 individuals in coarser medium sand (Md=470 m). In both substrata, abundance values decreased until October, at each station to a different extent. Particular attention was paid to vertical migrations of D. rathkei. Plankton catches and numerous direct observations provided evidence that the pelagic phase, observed at night, is always combined with ecdysis. During short periods of high swarming and moulting activities, the population structure changed considerably: juveniles of the 3rd stage dominated in early August; thereafter they were replaced by prematures, the last juvenile stage. The first mature individuals were observed in October. Some data are given on growth rate.     

Thallium in marine plankton

Concentrations of thallium in phytoplankton (0.02 to 0.8 g g^–1), zooplankton (0.03 to 0.5 g g^–1) and ichthyoplankton (0.1 g g^–1) from the central Pacific were comparable , as were the atomic ratios of thallium to calcium (3x10^–6) and to potassium (1x10^–6) in those organisms. These relatively constant ratios, plus the biounlimited ocean profile of thallium, indicate that it is rapidly cycled through plankton in the same manner as potassium, its principal biogeochemical analogue. The higher atomic ratios of thallium to potassium in pelagic clays (6x10^–6) and ferromanganese nodules (4x10^–3) suggest that both biological transport processes and abiotic transport processes influence this trace element's oceanic cycle.     

Effect of inhibitors of carbonic anhydrase activity on photosynthesis in the red alga Soliera filiformis (Gigartinales: Rhodophyta)

The effect of light intensity, pH and carbonic anhydrase (CA) inhibitors on photosynthesis of the red marine macroalgae Solieria filiformis (Kützing) Gabrielson, collected from Taliarte (Gran Canaria, Canary Islands) in 1991, has been investigated. Plants taken from the sea (wild phenotype) developed spherical morphology (ball phenotype) after 2 mo culture in aerated tanks. The photosynthetic oxygen evolution in the wild phenotype was saturated at 100 mol photons m^-2s^-1, while the ball phenotype displayed saturation at 200 mol photons m^-2s^-1. The inhibitors of total CA activity (6-ethoxizolamide) and extracellular CA activity (dextran-bound sulfonamide) inhibited photosynthesis at pH 8.2, to 90 and 50% respectively, in both phenotypes. No inhibition of the photosynthetic oxygen evolution was detected at pH 6.5. CA activity was associated with both supernatant and pellet fractions of crude extracts of S. filiformis. The rate of alkalization of the medium by the algae was dependent on light intensity. We suggest that carbon dioxide is the general form of inorganic carbon transported across the plasmamembrane in S. filiformis. HCO₃ transport into the cell takes place simultaneously by an indirect mechanism (dehydration to CO₂ catalyzed by CAext) and by direct uptake. Extracellular (CAext) and intracellular (CAint) CAs are involved in the mechanisms of inorganic carbon assimilation by S. filiformis.     

Phytoplankton exudate release as an energy source for the growth of pelagic bacteria

The release of dissolved organic carbon (DOC) from phytolankton during photosynthesis, and the utilization of this carbon by planktonic bacteria, was studied using ¹⁴CO₂ and selective filtration. Natural sea water samples from a coastal area of the Northern Baltic Sea were incubated in the laboratory for detailed studies, and in situ for estimation of annual dynamics. In a laboratory incubation (at +1°C) the concentration of ¹⁴C-labelled dissolved organic carbon increased for about 2 h and then reached a steady state, representing about 0. 1% of the total DOC. Labelled organic carbon in the phytoplankton and bacterial fractions continued to increase almost linearly. The continuous increase in the bacterial fraction is thought to represent almost instantaneous utilization of the DOC released from the phytoplankton during photosynthesis. As an annual average, in 4 h in situ incubations, about 65% of the labelled organic carbon was found in the phytoplankton fraction (>3 m), about 27% in the bacterial fraction (0.2 to 3 m) and the remaining 8% as DOC (<0.2 m). Large variations in these percentages were recorded. The measured annual primary production was 93 g C m^-2 (March to December), and the estimated bacterial production due to phytoplankton exudates 29 g C m^-2. This represents a release of DOC of about 45% of the corrected annual primary production of 110 g C m^-2 (assuming a bacterial growth efficiency of 0.6).     

The functions of octopine dehydrogenase and D-lactate dehydrogenase in the pedal retractor muscle of thedog whelk Nassarius coronatus (Gastropoda: Nassariidae)

The pedal retractor muscle of Nassarius coronatus, unlike most mollusc muscles, contains high activities of both octopine dehydrogenase (159 IU g^-1 wet wt muscle) and D-lactate dehydrogenase (40 IU g^-1 wet wt muscle). Pedal retractor muscles show accumulation of octopine (increase of 4.7 mol g^-1 wet wt muscle), and a fall in arginine phosphate concentration (decrease of 4.1 mol g^-1 wet wt musscle) after exercise. During recovery from exercise, octopine returns to resting levels after about 120 min, D-lactate accumulates (5.1 mol g^-1 wet wt muscle), and arginine phosphate returns to resting levels within 30 min. D-lactate (2.0 mol g^-1 wet wt muscle) accumulates during long-term exposure to air. The results show that octopine dehydrogenase and D-lactate dehydrogenase can function to catalyze the terminal step of glycolysis during muscle anoxia associated with these different physiological states.     

Interactions between nutritional status and long-term responses to ultraviolet-B radiation stress in a marine diatom

Influences of nutritional status on the photoinhibitory effects of ultraviolet-B radiation (UVBR: 290 to 320 nm) on the specific growth rates (_obs) and biomass of Phaeodactylum tricornutum were determined using nutrient-replete batch cultures and nutrient-limited continuous cultures. P. tricornutum cultures were exposed to UVBR doses representative of current mid-latitude and ozone-depletion intensities. Specific growth rates and biomass were inhibited from 2 to 16% by UVBR during nutrient-replete growth. However, no effect of UVBR on _obs or biomass was detectable when nutrient limitation exceeded the potential for limitation by UVBR. Thus, a competitive interaction appears to occur between macronutrient stress and UVBR stress, such that _obs and biomass will be determined by the most limiting factor. Our results suggest that measurable decreases in phytoplankton _obs and biomass from UVBR are most likely in nutrient-rich areas of the ocean, while these parameters may not be appropriate for measuring UVBR stress in regions of nutrient limitation.     

Spring and summer growth rates of subarctic Pacific phytoplankton assemblages determined from carbon uptake and cell volumes estimated using epifluorescence microscopy

In order to determine whether phytoplankton growth rates were normal or depressed, total plant carbon (g l^–1) and in situ production rates (g C l^–1 d^–1) were measured for phytoplankton assemblages at Weathership Station P (50°N; 145°W) and at 53°N; 145°W in the subarctic Pacific in May and August 1984. Plant carbon, estimated from cell volumes determined using epifluorescence microscopy, was distributed as follow: 28% in the <2 m fraction, 38% in the 2 to 5 m size fraction, and the remainder in size classes >5 m. Carbon-specific growth rates (k), as doublings d^–1, were calculated for the phytoplankton assemblages as a whole at each sampling depth down to 100 m for three days in May and for four days in August. The populations in the upper part of the euphotic zone showed average doubling rates of 1 d^–1 and thus appeared to be growing at rates normally expected for the prevailing conditions of light and temperature. The low chlorophyll concentrations (0.3 to 0.4 mg chl a m^–3) characteristically found in this oceanic region do not seem to be due to very slow growth of algal populations.Contribution No. 1695 of the School of Oceanography, University of Washington, Seattle, Washington 98195, USA