Nutritional value of selected species of microalgae for larvae and early post-set juveniles of the Pacific geoduck clam, Panopea generosa

    Nutritional value of selected species of microalgae for larvae and early post-set juveniles of the Pacific geoduck clam, Panopea generosa W. Liu, C.M. Pearce, R.S. McKinley, I.P. Forster PII: DOI: Reference:

S0044-8486(15)30208-8 doi: 10.1016/j.aquaculture.2015.10.019 AQUA 631878

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

18 June 2015 15 October 2015 16 October 2015

Please cite this article as: Liu, W., Pearce, C.M., McKinley, R.S., Forster, I.P., Nutritional value of selected species of microalgae for larvae and early post-set juveniles of the Pacific geoduck clam, Panopea generosa, Aquaculture (2015), doi: 10.1016/j.aquaculture.2015.10.019

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ACCEPTED MANUSCRIPT Nutritional value of selected species of microalgae for larvae and early post-set juveniles of the

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Pacific geoduck clam, Panopea generosa

Faculty of Land and Food Systems, The University of British Columbia, Vancouver, British

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W. Liu1,2, C.M. Pearce2,*, R.S. McKinley1,3, I.P. Forster4

Columbia, Canada V6T 1Z4

Fisheries and Oceans Canada, Pacific Biological Station, Nanaimo, British Columbia, Canada

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V9T 6N7

Centre for Aquaculture and Environmental Research, The University of British Columbia /

Fisheries and Oceans Canada, West Vancouver Laboratories, West Vancouver, British

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Columbia, Canada V7V 1N6

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Fisheries and Oceans Canada, Vancouver, British Columbia, Canada V7V 1N6

* Corresponding author at: Fisheries and Oceans Canada, Pacific Biological Station, Nanaimo, British Columbia, Canada, V9T 6N7. Tel.: +1 250 756 3352; fax: +1 250 756 7053. E-mail address: [email protected] (C.M. Pearce).

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ACCEPTED MANUSCRIPT Abstract The nutritional value of eight species of microalgae for larvae and early post-set juveniles

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of the Pacific geoduck clam, Panopea generosa, was evaluated. The microalgae tested included

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two prymnesiophytes, Pavlova lutheri (PL) and Tisochrysis lutea (TL); two chlorophytes,

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Dunaliella tertiolecta (DT) and Tetraselmis suecica (TS); and four diatoms, Chaetoceros calcitrans (CC), C. muelleri (CM), Phaeodactylum tricornutum (PT), and Thallassiosira

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pseudonana (TP). These microalgae were first tested as mono-species diets on both larvae and early post-set juveniles. The resultant best-performing mono-species diet was then tested as the

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major component in mixed- (bi-, tri-, and tetra-) species diets. Further experiments examined the possibility that the best performing mixed-species diet could be improved by adding another

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algal species. The optimal diet, with the fewest number of algal species to support the best growth, was the bi-species diets of CC+TL for larvae and CM+TL for early post-set juveniles.

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The fatty-acid profiles of juveniles were influenced by the diets. Although no clear pattern between the level of any particular nutrient and growth and development of juveniles was evident, it appears that a balanced mixture of various dietary nutrients is important. Attention was given to the ratios between n-3 and n-6 fatty acids and between eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The best performing diets, including the optimal one, had a n3/n-6 ratio of 2.17–3.03 and an EPA/DHA ratio of 1.28–3.25 for larvae and 1.84–2.58 and 1.84– 2.63, respectively, for early post-set juveniles. Results of this study can be applied to improve the commercial hatchery production of geoduck seed. Statement of relevance Nutritional value of microalgae for young geoduck clams.

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ACCEPTED MANUSCRIPT Keywords Geoduck, Growth, Larvae, Microalgae, Mixed diet, Panopea generosa, Post-set juveniles

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1. Introduction

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The Pacific geoduck clam, Panopea generosa, is found from Alaska to Baja California

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(Bernard, 1983) and supports the most valuable clam fishery on the west coast of North America both in Washington state (WA), USA and British Columbia (BC), Canada (Palazzi et al., 2001;

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DFO, 2012). With concerns over the naturally low and variable recruitment rate and significant cumulative fishing pressure driven by high market prices, research was initiated to examine the

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potential for geoduck clam aquaculture and enhancement in WA in the 1970s (Beattie, 1992). The resultant hatchery culture technology was transferred to BC in the 1990s (Heath, 2005).

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Aquaculture production of geoduck clams started in WA in the mid-1990s and has increased rapidly to 612.9 t, worth US $18.5 million, in 2010 (Washington State Department of Fish and

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Wildlife, 2012). Geoduck clam farming has become common in WA throughout Puget Sound on private tidelands during the past decade (Washington State Department of Natural Resources, 2014). In BC, however, commercial-scale development of geoduck clam aquaculture and enhancement has been constrained by the lack of a reliable seed supply from hatcheries due to high larval mortality, indicating significant problems with the current production strategy. A relatively small harvest of 51.7 t farmed geoduck clams, worth CA $1.1 million, was recorded in 2010 in BC (BC Ministry of Agriculture, 2012). To establish hatchery-rearing protocols for any cultured bivalve species, it is necessary to assess such biotic and abiotic factors as diet quality and quantity, stocking density, temperature, salinity, dissolved oxygen, and pH, all of which can critically affect the early development of bivalves. Being filter-feeders, the hatchery culture of P. generosa is reliant on feeding with

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ACCEPTED MANUSCRIPT microalgae. Despite decades of hatchery practice, research on the effects of different phytoplankton species on growth, development, and survival of larval and juvenile P. generosa

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is limited and optimal algal diets for rearing this clam species have not been determined. Indeed,

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little published information is available on various aspects of the hatchery culture of P. generosa.

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Early work by Goodwin et al. (1979) examined development of larvae fed various microalgal species (i.e. Isochrysis galbana, Monochrysis lutheri, Phaeodactylum tricornutum, and

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Pseudoisochrysis paradoxa), singly or mixed, and found that development to settlement required 47 days at 14 oC. Such a prolonged planktonic larval period under laboratory conditions may

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indicate suboptimal nutrition. Goodwin and Pease (1989) recommended the use of Tahitian Isochrysis sp. (=Tisochrysis lutea) and Chaetocerous calcitrans to feed young larvae, with C.

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gracilis being added when larvae were older, but these recommendations were not based on published growth rates in response to various microalgal diets. Aside from this early dietary

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work with larvae, hatchery-related research on P. generosa has been limited: Goodwin (1973) examined the effects of salinity and temperature on embryonic development; Marshall et al. (2014a) looked at the effects of stocking density and microalgal ration on larval growth, survival, and ingestion rate; and Marshall et al. (2012, 2014b) examined the effects of temperature and feeding ration on broodstock conditioning. Lack of scientific information on the basic biology during early life stages is a major obstacle to achieving reliable hatchery production of P. generosa seed. Live microalgae are essential in the rearing of all life stages of all bivalve species (Brown, 2002; Helm and Bourne, 2004) and have been shown to play vital roles in determining growth and survival of the animals (e.g. Enright et al., 1986a,b; O'Connor et al., 1992; Gouda et al., 2006; Martínez-Fernández and Southgate, 2007; Liu et al., 2009). The nutritional value of

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ACCEPTED MANUSCRIPT microalgae for bivalves is determined by a number of factors, including ingestibility (cell size/shape), digestibility, and nutrient content (Webb and Chu, 1983; Brown et al., 1997; Knauer

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and Southgate, 1999). Much of the literature on bivalve nutrition has emphasized the important

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nutritional role of the essential polyunsaturated fatty acids (PUFAs), especially the n-3 fatty

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acids eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on bivalve growth and development (Langdon and Waldock, 1981; Thompson et al., 1996;

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Volkman and Brown, 2005; Martínez-Fernández et al., 2006). PUFAs play an important structural role in regulating cell-membrane fluidity and various cellular functions (Hazel et al.,

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1991; Hall et al., 2002). EPA and arachidonic acid (AA, 20:4n-6) also act as precursors of biologically-active metabolites, such as prostaglandins and related oxygenated fatty acids (i.e.

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C20 PUFAs), collectively known as eicosanoids, which have been shown to be of major importance in a number of physiological, behavioural, and ecological systems (Stanley-

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Samuelson, 1994; Stanley and Howard, 1998; Howard and Stanley, 1999). Bivalves have very limited or no ability to synthesize PUFAs or to elongate shorter-chained PUFAs into longer ones (Waldock and Holland, 1984; Whyte et al., 1989; Chu and Greaves, 1991; Marty et al., 1992; Delaunay et al., 1993), but require these for growth and development. No study has examined the nutritional value of microalgae for rearing larvae and juveniles of P. generosa in terms of biochemical composition. The present study examined the growth and survival of larvae and early post-set juveniles of P. generosa fed selected species of microalgae as single- and mixed-species diets and analyzed the biochemical compositions of both the microalgae and experimental animals (juveniles only) in an effort to identify optimal diets and to understand the nutritional roles of various nutrients, especially PUFAs, for successful hatchery rearing of P. generosa. Based on the

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ACCEPTED MANUSCRIPT findings of the present study examining larval/juvenile diets, Ren et al. (2014) further examined the synergistic or non-additive effects of mixed microalgal diets on juvenile geoduck growth and

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survivorship while Arney (2015a,b) explored the feasibility of substitution of live algae with

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spray-dried Schizochytrium sp. or Spirulina as well as the effects of temperature and food ration

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on juvenile geoduck growth and survivorship.

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2. Materials and methods 2.1. Microalgae

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The nutritional value of eight species of microalgae, selected from those commonly used in bivalve hatcheries (see Coutteau and Sorgeloos, 1992; Helm and Bourne, 2004), was

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evaluated by testing various single- and mixed-species diets in serial trials. The microalgae were sourced from the Provasoli-Guillard National Center for Marine Algae and Microbiota (East

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Boothbay Harbor, Maine, USA) and included: two prymnesiophytes, Pavlova lutheri (PL, CCMP1325) and Tisochrysis lutea (TL, CCMP1324); two chlorophytes, Dunaliella tertiolecta (DT, CCMP1320) and Tetraselmis suecica (TS, CCMP904); and four diatoms, Chaetoceros calcitrans (CC, CCMP1315), C. muelleri (CM, CCMP1316), Phaeodactylum tricornutum (PT, CCMP630), and Thallassiosira pseudonana (TP, CCMP1335). The microalgae were batchcultured in seawater enriched with the artificial seawater medium of Harrison et al. (1980) with a slight modification (i.e. substitution of inorganic phosphate for organic phosphate) in 20-l carboys (CM and TL) or 4-l flasks (all other species) and harvested in the late growth phase for feeding. The cultures were maintained at 18°C with continuous cool-white fluorescent lighting and bubbled with air periodically injected with CO2. Cell densities were determined using a haemocytometer.

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2.2. Broodstock, larvae, and early post-set juveniles

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Broodstock were collected in the Strait of Georgia, BC, off Denman Island in October

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2010 and in Kulleet Bay, BC in 2011, and had a mean shell length and live weight of 157.7±1.9

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cm and 1.3±0.3 kg (±SE, n=47) and 149.6±2.07 cm and 1.40±0.45 kg (n=50), respectively. Approximately 25 animals were laid horizontally on the bottom of a shallow tank (L×W×H:

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1.2×0.9×0.3 m) and provided with flow-through, sand-filtered, and UV-treated seawater at 3–4 l min–1 and 8–12oC. A single-algal diet of CM or TL was drip-fed at a daily ration of 4–6×109

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cells ind.–1. Spawning was induced by adding excessive TL to the holding tank. Fertilized eggs obtained from multiple parents were hatched in shallow tanks (L×W×H: 1.2×0.9×0.3 m) at a

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density of <30 eggs ml–1 and a temperature of 12–15oC. After 48–60 h, newly developed Dlarvae were collected and used for various experiments or for further rearing to produce juveniles,

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as described below.

D-larvae were cultured in a 300-l cylindro-conical tank at an initial density of 3–8 ind. ml–1. Larvae developed into pediveligers after 18–20 d and were settled in a circular tray (D: 36 cm; area: 1,018 cm2; bottom mesh size: 200–240 µm) which floated on the water surface of the tank. Due to difficulty in rearing larvae at an early stage of the research, one batch of pediveligers was brought directly from a commercial hatchery (Island Scallops Ltd., Qualicum Beach, BC, Canada) for settlement. Juveniles were cultured at densities of <30 ind. cm−2 before they reached the size for various experiments (i.e. 1−1.6 mm mean shell length). Both larvae and juveniles were cultured under static conditions, seawater in the cylindroconical tank being moderately aerated and fully renewed every 1−2 d with 1-µm filtered and UV-treated seawater at a mean temperature of 15.5±0.4oC (±SD). During the post-set period, the

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ACCEPTED MANUSCRIPT upwelling water motion by aeration also facilitated water exchange between the inside and outside of the settlement tray. Larvae were fed predominantly a bi-algal diet of CC+TL and

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juveniles with CM+TL (both mixed at a 1:1 ratio by ash-free dry weight, AFDW) at 1–2×104 and

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2–5×104 equivalent TL cells by AFDW (E-TL) ml–1 d–1, respectively. A photoperiod of 16-h

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light and 8-h dark, using overhead cool-white fluorescent lights, was used throughout.

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2.3. Experimental designs and set-up for larvae 2.3.1. Experiment 1: Mono-species diets

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The eight species of microalgae were first tested on larvae as mono-species diets. A mixture of all eight algal species (by equal AFDW of each species) was also evaluated.

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Newly hatched D-larvae with a shell length of 124.5±1.0 µm (mean±SE, n=20) were dispensed into 20-l containers (D×H: 30.0×55.0 cm) filled with 16 l of seawater with moderate

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aeration. The initial stocking density was 3 ind. ml–1. Larvae were fed one of the experimental diets every other day, after a full water change, at 2–3×104 E-TL cells ml–1. Three replicate containers were assigned to each dietary treatment using a completely randomized design. Seawater used for the experiment was filtered to 1-µm and UV treated. Water temperature was maintained at 15±0.8oC (mean±SD) and photoperiod at 16-h light and 8-h dark. During the 26-d experiment, 30–40 ml were removed from each container, after mixing to suspend larvae, every 4 d until day 20 (at approximately the pediveliger stage) and fixed in 4% buffered formalin for later examination of larval shell length and survival rate. At the end of the experiment (day 26), the whole containers were sampled (due to very low larval survival rate). Mean shell length was obtained for each sample by measuring 20 randomly-chosen larvae, or all individuals if the sample contained fewer than 20 larvae. Measurements were taken using a dissecting microscope

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ACCEPTED MANUSCRIPT with the imaging software Motic Images Advances 3.2 for Windows (Motic Electric Group Co., Ltd., Richmond, BC, Canada). Healthy-looking larvae were counted and survival rates expressed

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as a percentage of the initial population.

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2.3.2. Experiment 2: TL-based mixed-species diets

None of the mono-species diets tested in Experiment 1 was adequate for rearing the

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larvae, but in relative terms TL, PL, CC, and CM appeared to be better ones, with TL being the best. This follow-up Experiment 2 tested TL in conjunction with the other three relatively better

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mono-species diets of PL, CC, and CM in all possible bi-, tri-, and tetra-species combinations (Fig. 2). Each mixed-algal diet tested comprised 50% (by AFDW) of TL and 50% of the other

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species (i.e. by mixing equal AFDW of the other component species, totalling 50% of the AFDW of the diet). A mono-species diet of TL was used as a control. All conditions of this experiment

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were identical to those described for Experiment 1.

2.3.3. Experiment 3: TL+CC-based mixed-species diets Results from Experiment 2 showed that, among those tested, the bi-species diet of TL+CC appeared to support the best larval growth. However, low larval survival rates were also observed across the different dietary treatments at the end. In Experiment 3 the bi-species diet of TL+CC was re-examined using alternative husbandry techniques, including complete daily water change and reduced feeding density (1×104 E-TL cells ml–1 d–1), with the aim of improving larval survival rates. In addition to the bi-algal diet of TL+CC, tri-species combinations of TL+CC with DT, PT, TP, or TS, the four species of poorest nutritional value as mono-species diets for the larvae (Section 2.3.1 and Results) but which had not been tested in mixed-species

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ACCEPTED MANUSCRIPT diets, were tested. Each of these tri-species diets contained 80% of the bi-algal diet of TL+CC (mixed at 1:1 by AFDW, totalling 80% of the AFDW of the diet) and 20% (by AFDW) of the

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third algal species. The lower (20%) inclusion of these four algae is because their poor food

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value may not render them major components in mixed-algal diets. Other experimental

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conditions were identical to those of Experiment 1.

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2.4. Experimental designs and set-up for early post-set juveniles 2.4.1. Experiment 4: Mono-species diets

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As with the larvae, the eight species of microalgae were first tested as mono-species diets for the juveniles, with a mixture of all eight species also being evaluated.

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Early post-set juveniles (grown from pediveligers produced by Island Scallops Ltd.) with an initial shell length of 1,600.0±38.0 µm (mean±SE, n=20) and dry weight of 0.19±0.01 mg

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ind.–1 (mean±SE, n=3 groups of 80 ind.) were used for the experiment. Eighty animals were placed in PVC cylinders (D×H: 10×25 cm) with a 300-µm bottom mesh. Each cylinder was suspended individually off the bottom of a 20-l plastic container (D×H: 30.0×55.0 cm) filled with 16 l of aerated seawater. Three replicate containers were assigned to each dietary treatment, according to a completely randomized design. The juveniles were fed different algal diets every two days, increasing from 2 to 5×104 E-TL cells ml –1, after a full water change. The water used was filtered to 1-µm, UV treated, and at a temperature of 15.7±0.5oC (mean±SD).

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photoperiod of 16-h light and 8-h dark was maintained throughout. On days 10, 18, and 28, the juveniles were transferred into a Petri dish. Digital images were then taken of 15–20 randomly-chosen ind. per cylinder, with shell length being measured as in the larval experiments. Dead animals were removed and counted at these times. Due to the

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ACCEPTED MANUSCRIPT low growth rate in many of the dietary treatments, the juveniles were returned to their respective cylinders on days 10 and 18 in an attempt to obtain higher final biomasses for biochemical

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analysis. Upon final sampling, animals from each PVC cylinder were left unfed overnight to

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purge gut contents, collected and counted into a vial, washed with 0.5-M ammonium formate to

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remove surface salt, and stored at -80oC for later biochemical analysis. Three initial samples of

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known numbers of juveniles were also prepared for biochemical analysis (see Section 2.5.2).

2.4.2. Experiment 5: CM-based mixed-species diets

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Results from Experiment 4 revealed that CM was the best mono-species diet for the juveniles, but its food value was deemed only moderate (see Results). In Experiment 5, CM was

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further tested in all possible bi-, tri-, and tetra-species combinations with the other three relatively better algal species of CC, PL, and TL for juvenile growth. Each mixed-species diet

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tested comprised 50% of CM (by AFDW) and 50% of the other species (i.e. by mixing equal AFDW of the other component species, totalling 50% of the AFDW of the diet). A mono-species diet of CM was used as a control. Experimental conditions were similar to those in Experiment 4, except that smaller juveniles produced from the present research were used (shell length: 841.6±15.8 µm, n=20; dry weight: 0.036±0.001 mg ind.–1; n=3 groups of 400 ind.; means±SE), 150 juveniles were added per cylinder, and destructive samplings were conducted (120 and 80 ind. remaining in each cylinder on days 10 and 18, respectively). In addition, juveniles in those dietary treatments showing the fastest growth (e.g. CM/TL) were fed daily at 5x104 E-TL cells ml–1 during the final sampling period between days 18 and 28.

2.4.3. Experiment 6: CM+TL-based mixed-species diets

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ACCEPTED MANUSCRIPT This experiment determined whether the nutritional value of the bi-species treatment of CM+TL, the best diet found for the juveniles in Experiment 5, could be improved. Each diet

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tested contained 80% of the bi-species diet of CM+TL (mixed at 1:1 by AFDW, totalling 80% of

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the AFDW of the diet) and 20% (by AFDW) of a third alga DT, TS, PT, or TP; the four species

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of evidently poorest nutritional value as mono-species diets for the juveniles (Section 2.4.1; see also Results), but which had not been tested in mixed-species diets. The lower (20%) inclusion

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of these four algae is because their poor food value may not render them as major components in mixed-algal diets. PL and CC were excluded from the test since they lacked added food value in

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the mixed diets (see Results). The bi-species diet of CM+TL was used as a control. Experimental conditions were similar to those in Experiment 4, except that juveniles in all treatments were fed

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daily at 5x104 E-TL cells ml–1 during the final sampling period between days 18 and 28, due to the faster growth across all the dietary treatments. The initial shell length of the juveniles was

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955.0±21.7 µm (mean±SE, n=20) and dry weight was 0.052±0.001 mg ind.–1 (mean±SE, n=3 groups of 400 ind.).

2.5. Biochemical analyses 2.5.1. Microalgae

Biochemical analyses of each algal species were performed on three independent cultures. Cellular AFDW was determined by filtering known volumes and densities of algal cultures onto pre-ashed (550oC) and pre-weighed Whatman® GF/C filters and drying at 60oC to constant weight. AFDW was obtained as dry-weight loss after combustion at 500oC for 4 h. This result was used to determine the cellular AFDW and the routine feeding amount needed for each algal species in the various feeding experiments.

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ACCEPTED MANUSCRIPT Bulk algal samples were obtained by centrifuging algal cultures in 50-ml tubes at 3,000 g for 10 min. After decanting excess seawater, centrifuged cells were freeze-dried and stored at -

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80oC for later biochemical analyses. Triplicate cultures were used for each algal species. AFDW

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was determined for each bulk algal sample (~36 mg) as dry-weight loss after combustion at 500oC for 4 h. The result was used to calculate sample contents of protein, lipid, carbohydrate,

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and fatty acids on a per-unit-AFDW basis (mg g–1 AFDW).

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Protein was determined based on the protocols of Lowry et al. (1951), using the commercial kit DC Protein Assay (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada)

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with bovine serum albumin as the standard. Samples (~22 mg) were hydrolyzed in 0.5 N NaOH at 60oC for 3 h before the assay. Total carbohydrate was determined according to Dubois et al.

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(1956) after hydrolysis of samples (~9 mg) in 0.5 N H2SO4 at 100oC for 3 h. D-glucose was used as the standard. Total lipid was extracted from samples (~180 mg) using the method of Bligh and

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Dyer (1959) and determined gravimetrically after evaporation of solvents at 60oC. Fatty acid methyl esters (FAMEs) of algal samples (~53 mg) were prepared using the one-step method of Abdulkadir and Tsuchiya (2008), with nonadecanoic acid (19:0) (Nu-Chek Prep, Inc., Elysian, Minnesota, USA) as the internal standard. The antioxidant butylated hydroxytoluene was also added to the hexane solvent (75 mg l–1) to prevent lipid oxidation (Christie, 1973). The resultant FAMEs and fatty acid standards were analyzed on a Varian 3900 gas chromatograph (Agilent Technologies, Santa Clara, California, USA), equipped with a Chrompack CP-Sil 88 capillary column for FAME (50 m x 0.25 mm; 0.20-µm film thickness), an autosampler (Model: 8400), and a CP-1177 injector. The injector was maintained at 270°C. An injection volume of 1.0 µl and a split ratio of 10:1 were used. Hydrogen was used as the carrier gas at a flow rate of 1.0 ml min–1. The oven temperature was elevated to 160°C at 15°C

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ACCEPTED MANUSCRIPT min–1, after an initial holding period of 1 min at 80°C, and further to 220°C at 4°C min–1 and then held for 5 min (total run time: 26.33 min). The flame ionization detector was operated at 270°C,

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with a flow rate of 25, 30, and 300 ml min–1 for nitrogen, hydrogen, and air, respectively. A

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signal-to-noise ratio of 5:1 was applied for each run. Fatty acid peaks were identified with the

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Varian Star Chromatography Software 6.41 by cross-referring the methylated fatty acid standards (GLC-428, GLC-443, GLC-455, GLC-480, GLC-642, and GLC-643) from Nu-Chek

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Prep, Inc. Fatty acid contents were determined by comparing their peak areas with those of the internal standard and expressed as mg g–1 AFDW for each alga (Abdulkadir and Tsuchiya, 2008).

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For mixed-algal diets, proximate and fatty acid compositions were calculated based on the AFDW ratio of each component algal species in the mixed diets and expressed as mg g–1

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2.5.2. Early post-set juveniles

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AFDW.

Due to limited sample biomass, only body fatty acid profiles of the juveniles were analysed. Animals from each treatment replicate, including the initial samples, were freeze-dried and weighed collectively to determine mean individual dry weight (mg ind.–1). A small portion (2–16 mg) of each replicate sample was used to determine AFDW as dry-weight loss after combustion at 500oC for 4 h. FAME preparation (12–44 mg samples) and fatty acid analysis and expression (mg g–1 AFDW) were performed in the same manner as for the algae, except that an injection volume of 5.0 µl was used. As mentioned previously, the low juvenile growth in the mono-species diet experiment (Section 2.3.1) led to too little sample biomass in many of the dietary treatments for biochemical analyses., For each dietary treatment, therefore, juveniles of the three replicates were pooled to

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ACCEPTED MANUSCRIPT form a single sample (12–38 mg), with fatty acid analysis run as duplicate on each sample and a mean value calculated and expressed as per unit juvenile dry weight (mg g–1 DW; i.e. AFDW

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was not measured).

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2.6. Statistical analysis

Dietary effects on mean shell length, dry weight, and survival rate were evaluated by one-

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way ANOVA. When the effect was significant (P<0.05), a Newman-Keuls (NK) test was performed to detect significant differences among treatment means. Each rearing container was

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treated as a replicate unit (i.e. individual shell length and weight means were calculated for each container and used as replicate values). Analyses of growth and survival rates were carried out

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on the final data obtained, except for Experiment 1 (Section 2.3.1), in which only data obtained on day 8 were analysed because the low survival rate rendered too few larvae for statistical

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analyses. Biochemical compositions of the algal diets and the juveniles were also examined with one-way ANOVA, followed by a NK test, where significant. This included analyses on proximate compositions, individual fatty acids, sum of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), n-3 polyunsaturated fatty acids (PUFA), and n-6 PUFA, as well as ratios of EPA/DHA and n-3/n-6 fatty acids. Data were tested for normality and homogeneity of variances by the Kolmogorov-Smirnov and Levene’s test, respectively, prior to analyses. All statistical analyses were conducted with the assistance of computer software NCSS 2007 (NCSS LLC, Kaysville, Utah, USA). All data are presented as means±SE (n=3) unless specified.

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3. Results

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3.1. Growth and survival of larvae 3.1.1. Experiment 1: Mono-species diets

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On day 8, the mixture of all eight algal species and the three mono-species diets of CC, TL, and PL resulted in significantly greater shell-length means (167.8−181.8 µm) than DT, TS,

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PT, CM, and TP (148.8−153.3 µm) (Fig. 1A). Mean shell length of larvae fed the mixture of all eight algal species at day 8 (181.8±1.9 µm) was significantly greater than that of those given TL

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(168.9±3.6 µm) and PL (167.8±1.5 µm), but not those fed CC (176.4±2.1 µm). No significant differences were found in shell-length means among the three best mono-species diets (CC, TL,

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and PL) or among the five poorest ones (DT, TS, PT, CM, and TP). Survival to day 8 ranged from 13.0% to 31.0% (Fig. 1B), with no significant difference between dietary treatments. After day 8, larvae fed the mixture of all eight algal species or TL continued to grow, the fewer survivors (0.4% and 0.2%, respectively) at the end of the experiment (day 26) reaching mean shell lengths of 374.0±22.9 µm (pediveligers) and 329.7±6.5 µm (late-umbo larvae), respectively. Larval cultures collapsed (sudden, high mortality) by day 16 when fed CC and by day 20 when given PL. Larvae fed DT, TS, PT, and TP maintained slower growth rates after day 8, but nearly all had died by day 20. Larvae fed CM showed faster growth after day 8 and caught up in size with those fed TL by day 20 (Fig. 1A). The results indicate that none of the mono-species diets tested were adequate for rearing larval P. generosa. But in relative terms, taking both growth and survival into account, CC, PL,

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ACCEPTED MANUSCRIPT and TL appeared to be better diets, TL being the best. CM appeared to be a better diet for 8-day and older larvae. DT, PT, TP, and TS were poor mono-species diets for larval P. generosa, as

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they supported only limited growth. Microscopic examination revealed that D-larvae fed CC, PL,

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and TL developed coloured digestive glands within 24 h of feeding, which was not observed

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3.1.2. Experiment 2: TL-based mixed-species diets

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with those algal species of poor nutritional value (DT, PT, TP and TS, and CM before day 8).

At the end of the experiment (day 26), the greatest mean shell length (494.9±9.9 µm) was

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associated with the bi-species diet of TL+CC, but it was not significantly different than those of TL+CC as tri- and tetra-species diets (TL+CC+CM; TL+CC+PL; TL+CC+CM+PL)

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(444.9−483.3 µm) and that of TL+CM+ PL (443.5±27.6 µm) (Fig. 2A). Final samples in these five dietary treatments consisted of 22.5−55.0% metamorphosed seed at day 26. Shell-length

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means at day 26 (376.8−400.3 µm, pediveligers) were lower in the diets of TL+CM, TL+PL, and TL alone, but the differences were significant only when compared to the bi-species diet of TL+CC and the tetra-species diet of TL+CC+CM+PL (Fig. 2A). Survival of the larvae was low (0.2−3.0%) by the end of the experiment, no significant dietary effects being found (Fig. 2B). The low survival rates appeared to be associated with filament-like structures (a suspected fungal infection) which began to develop after day 16 and slowly spread on larval shell surfaces. As a result, survival was reduced sharply during the last two sampling periods from days 16 to 26.

3.1.3. Experiment 3: TL+CC-based mixed-species diets

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ACCEPTED MANUSCRIPT When a third algal species was included in the bi-species diet of TL+CC, shell-length means (379.7−399.3 µm, pediveligers) and survival (6.4−21.3%) were statistically similar

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among different dietary treatments at the end of the experiment (Fig. 3A,B). The previously-

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described filament-like structures were not found in this experiment, although the resultant

likely due to the reduced feeding density applied.

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larvae were smaller than those in the TL-based mixed-species diets (Section 3.1.2) at the end,

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The bi-species treatment of TL+CC was, therefore, considered practically as the optimal diet for rearing larvae of P. generosa, since it supported the best larval growth and yet had the

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fewest number of algal species among the different multi-species diets tested. This optimal diet has subsequently been validated under commercial hatchery production conditions, with

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reproducible results among different batches (data not shown), larvae generally having a planktonic stage of 16−20 days at a rearing temperature of 15oC and an overall survival rate of

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40−90% from day 4 to pediveligers. It should also be noted that it has been the general acceptance of the industry that P. generosa is very susceptible to infestations of various pathogens during the larval stage. This has been observed in commercial hatcheries in both BC and WA (even given the relative greater success of WA-based hatcheries).

3.2. Growth and survival of early post-set juveniles 3.2.1. Experiment 4: Mono-species diets After 28 days, the shell-length and dry-weight means (3090.3±43.9 µm and 1.29±0.09 mg ind.–1, respectively) were significantly greater in juveniles fed the mixed diet of all eight algal species than those fed any of the mono-species diets tested (1791.9–2800.6 µm and 0.23– 0.87 mg ind.–1, respectively) (Fig. 4A,B).

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ACCEPTED MANUSCRIPT Juveniles fed the various mono-species diets fell into two distinct growth groupings at the end of the experiment. Those fed TL, CM, PL, and CC had significantly greater shell-length and

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dry-weight means (2439.5−2800.6 µm and 0.67–0.87 mg ind.–1, respectively) than those fed TP

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TS, PT, or DT (1791.9−1994.6 µm and 0.23–0.32 mg ind.–1, respectively); juveniles fed the

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latter four algae showed limited growth over time. Of the four better-performing diets, shelllength and dry-weight means of TL and CM treatments were significantly greater than those of

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PL and CC (Fig. 4A,B). Moreover, the digestive organs of juveniles fed the poor algal diets of

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DT, PT, TP, and TS soon faded from the initial dark-brown colour upon feeding, becoming pale or light within two days, as if under starvation conditions (the observation was possible due to

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the translucent shell of young juvenile P. generosa).

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Feeding the CM diet resulted in significantly higher survival at day 28 (53.5%) than any other mono-species diet identified for better juvenile growth (26.4−32.8% for TL, PL, and CC),

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but it did not significantly differ from the diets identified as being poor for juvenile growth (53.1−59.7% for TP, PT, and TS) or the mixture of all eight algal species (56.5%) (Fig. 4C). The above findings show that CM was the best mono-species diet among those tested for juvenile P. generosa in terms of both growth and survival, but its nutritional value was moderate at best, due to the better-performing mixture of all eight algal species.

3.2.2. Experiment 5: CM-based mixed-species diets After 28 days, shell-length and dry-weight means (3927.2±49.2 µm and 2.64±0.11 mg ind.–1, respectively) of juveniles fed the bi-species diet of CM+TL were significantly higher than those fed any other diets tested, including the CM control (2616.1±73.1 µm and 0.68±0.03 mg ind.–1, respectively) (Fig. 5A,B). CM in combination with TL as tri-species diets (CM+CC+TL

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ACCEPTED MANUSCRIPT and CM+PL+TL) also led to significantly greater shell-length means (3291.1 and 3338.5 µm, respectively) and dry-weight means (1.40 and 1.49 mg ind.–1, respectively) than the control CM.

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When CM was combined with PL and/or CC, no significant increase in shell-length or dry-

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weight means (1933.4–2686.2 µm and 0.33–0.85 mg ind.–1, respectively) was found for any

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dietary combination when compared to the CM+TL combination; the length and dry-weight means were even reduced significantly in the bi-species diet of CM and CC (Fig. 5A,B). This

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suggests that the overall nutritional value was generally reduced when PL and/or CC were mixed into the diets. Survival in the various dietary treatments at day 28 varied between 76.7% and

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97.2%, with no significant dietary effect being found.

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3.2.3. Experiment 6: CM+TL-based mixed-species diets When a third algal species was included in the bi-species diet of CM+TL, juveniles

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showed similar growth rates for all diets tested, reaching a shell length mean of 4,032.7–4,139.7 mm and dry weight mean of 2.62–2.96 mg ind.–1 at the end. No significant difference was found between either mean (Fig. 6A,B). Survival was high among all treatments (97.5–98.9%) and the dietary effect was not significant.

In practical consideration of the above results, the bi-species diet of CM+TL was identified as the optimal diet among those tested for rearing juvenile P. generosa, as this supported the best juvenile growth with the fewest number of algal species among the different multi-species diets tested.

3.3. Biochemical compositions 3.3.1. Microalgal diets

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ACCEPTED MANUSCRIPT The results of analysis of the mono-species diets used for feeding the larvae and juveniles (Table 1) revealed that protein varied between 190.3 and 343.3 mg g–1, with TS being

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significantly lower than CC, PL, TP, and TL. Lipid contents ranged from 128.1 to 283.4 mg g–1,

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with DT and TS being significantly lower than all other treatments. DT and TS contained significantly higher levels of carbohydrate (586.4 and 683.7 mg g–1, respectively) than all the

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other species of microalgae (291.4–453.5 mg g–1), except for CC.

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The two prymnesiophytes (PL and TL) contained significantly higher levels of DHA than

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all the other algal species. TL also contained significantly higher levels of 18:1n-9, 20:1n-9, and 18:2n-6 than most of the other algal species. The fatty acid profiles of the two chlorophytes (DT

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and TS) were comprised mainly of 16:0, 18:1n-9, and 18:3n-3. Other PUFAs (e.g. EPA and

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DHA) were generally low or lacking in these two algal species, except for a high level of 18:2n-6 in TS. The four diatoms (CC, CM, PT, and TP) were similar in major fatty acid compositions, all

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containing higher levels of 14:0, 16:0, 16:1, and EPA. A higher level of EPA was also observed in PL. The sums of SFA and MUFA were relatively low in DT and TS, while relatively high sums of n-6 PUFA were present in TL and TS, and relatively high EPA/DHA ratios were observed among the diatoms. Higher levels of n-3 PUFA and n-3/n-6 ratios were found in PL and diatoms.

Biochemical components of the various mixed-algal diets used for feeding the larvae and juveniles generally lacked too-high or too-low values as observed in the mono-species diets (Tables 2–5). For instance, the optimal diet for larvae with the mixture of TL and CC had a moderate level of EPA of 13.63 mg g–1, compared to that of 1.54 and 25.71 mg g–1 in the monospecies diets of TL and CC, respectively (Tables 1 and 2). Since each species of microalga tested can be rich in a particular nutrient and yet none of them were demonstrated as being optimal

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ACCEPTED MANUSCRIPT diets, there is no clear pattern between a particular nutrient and its nutritional value in supporting fast growth and development during the early life stage of hatchery-reared geoduck clams.

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The optimal diet for larvae (TL+CC) had an EPA/DHA ratio of 2.43 and a n-3/n-6 ratio

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of 2.59 (Table 2). Moreover, diets resulting in the best larval growth (those of TL+CC as tri- and

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tetra-algal diets, and TL+CM+PL) were characterized with an EPA/DHA ratio of 1.28–3.25 and a n-3/n-6 ratio of 2.17–3.03 (Tables 2 and 3). The EPA/DHA and n-3/n-6 ratios of low

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performing diets generally were outside these particular ratio ranges (Tables 1, 2, and 3).

3.3.2. Early post-set juveniles

Dietary effects were generally reflected in the juvenile bodies, especially on the major

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fatty acid compositions. With the mono-species diets, a higher level of EPA (0.61–1.98 mg g–1) was seen in the juveniles fed the EPA-rich diatoms, a higher level of DHA (1.57 mg g–1) in those

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fed the DHA-rich TL, and a higher level of EPA and DHA (0.8 and 1.03 mg g–1, respectively) in those fed PL which was rich in both fatty acids. Higher dietary levels of 18:3n-3 in DT and TS were also reflected in the juveniles (0.35 and 0.23 mg g–1, respectively) (Table 6). In the juveniles fed the CM-based diets, dietary inclusion of the DHA-rich TL resulted in significantly elevated levels of DHA relative to those fed the EPA-rich CM (2.96–3.98 vs. 1.17 mg g–1) (Table 7). In the juveniles fed the CM+TL-based diets, inclusion of a third algal species by only 20% caused significant differences in several fatty acid compositions (e.g. a higher 18:3n-3 level of 3.93 mg g–1 with the inclusion of DT rich in it) (Table 8). The major fatty acids in the juveniles fed the optimal diet (CM+TL) included 16:0, 18:0, 16:1, 18:1n9, EPA, and DHA, with an EPA/DHA ratio of 1.15–1.16 and a n-3/n-6 ratio of 3.16– 3.44 (Tables 7 and 8). Other fast growing juveniles (those fed a third algal species; Table 8), had

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ACCEPTED MANUSCRIPT an EPA/DHA ratio of 1.05–1.71 and a n-3/n-6 ratio of 3.06–4.11. The respective ratios of the slower growing juveniles generally did not fall within the range (Tables 6 and 7). Furthermore,

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the optimal diet for juveniles (CM+TL) had an EPA/DHA ratio of 1.84 and a n-3/n-6 ratio of

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2.11 (Table 4). Other top-performing diets (those fed a third algal species; Table 5) had an

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EPA/DHA ratio of 1.84–2.63 and a n-3/n-6 ratio of 1.84–2.58. As with the larvae, those low performing diets generally did not have their EPA/DHA and n-3/n-6 ratios both falling within

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these particular ratio ranges (Tables 1, 4, and 5).

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4. Discussion 4.1. Microalgae

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This study examined the nutritional value of eight species of microalgae commonly used in bivalve hatcheries for the culture of larvae and early post-set juveniles of P. generosa. None of

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the mono-species diets was found to be adequate in supporting good larval and juvenile growth/survival. The bi-species diet of TL+CC was shown to be optimal for larvae and CM+TL for juveniles among those tested. Ontogenetic changes in food preference have been reported for other bivalve species (Walker et al., 1998; Martínez-Fernández and Southgate, 2007; Liu et al., 2009). However, CC and CM appeared to share a similar nutritional profile, as did their respective mixtures with TL (Tables 1 and 2). The present finding may therefore not be due to ontogenetic change in nutritional requirements of P. generosa, but largely to important anatomical and physiological phases of the feeding organs (Beninger et al., 1994; Chaparro et al., 2001, Veniot et al., 2003; Cannuel and Beninger, 2006), resulting in differing ingestion rates with varying food-particle size. Its long spines may render CM cells in the diets difficult for

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ACCEPTED MANUSCRIPT younger larvae of P. generosa to handle, since the cells could reach over 50 µm in length, with the spines, in the present study (Liu, unpublished data).

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CC, PL, and TL are the most common microalgal species used to feed larval, early

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juvenile, and broodstock stages of bivalve molluscs (Brown, 1997). TL does not possess a cell

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wall and is readily ingested and digested by bivalve larvae (Zhu, 1997; Liu and Lin, 2001). The high nutritional value of Chaetoceros spp., including CC and CM, has been reported for larvae

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and juveniles of other bivalve species (Enright et al., 1986a; Laing and Millican, 1986; Nell and O’Connor, 1991; O’Connor and Heasman, 1997; Taylor et al., 1997; Rivero-Rodríguez et al.,

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2007; Liu et al., 2009). Martínez-Fernández et al. (2004) examined ingestion and digestion by pearl oyster (Pteria sterna) larvae of ten species of microalgae, similar to those employed in the

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present study, and found that only PL and TL were ingested/digested. No ingestion or digestion was found with any other species tested throughout larval development except for ingestion of

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the much smaller Nannochloris sp. However, in contrast to reports in the literature, MartínezFernández et al. (2004) also found that pearl oyster larvae are unable to handle CC. Easy digestion of CC, PL, and TL by larval P. generosa is suggested by the present study. D-larvae fed these three algal species singly developed coloured digestive glands within 24 h after feeding. This was not the case with the other algal species (CM, DT, PT, TP, and TS) tested, indicating poor digestion if not poor ingestion of these microalgae associated with poor larval performance (Fig. 1). However, good digestion does not necessarily mean good nutrition, as CC, PL, and TL supported better larval growth only during the earlier developmental days. Poor digestion of DT, PT, TP, and TS seemed to persist in the juveniles since the colour of their digestive organs soon faded from the initial dark-brown colour (fed the optimal diet of CM+TL) after feeding, becoming pale or light in two days, as if the juveniles were under

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ACCEPTED MANUSCRIPT starvation conditions. Juvenile P. generosa are able to readily ingest the tested microalgal species, which are all commonly used in bivalve hatcheries. Bivalve digestive organs, as a rule,

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take on the colour of the food they contain (Loosanoff and Davis, 1963). The observed colour

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patterns of digestive glands throughout larval/juvenile development suggest inefficient digestion

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and utilization of these poor performing diets (DT, PT, TP, and TS) and little ontogenetic change in digestive ability in the early life of hatchery-reared geoduck clams. Poor digestion of PT and

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TS has been noted for juveniles of other bivalve species, thick cell walls often being given as the explanation for the latter species (Epifanio, 1979; Romberger and Epifanio, 1981; Rivero-

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Rodríguez et al., 2007). Larvae of the Sydney rock oyster, Saccostrea commercialis, grow better when provided with concentrated and stored PT than on fresh cells (Nell and O’Connor, 1991).

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Likely the centrifugation process disrupted the cell walls, rendering them more susceptible to digestion. The cell-wall lacking DT has a surface coating, possibly with an ability to withstand

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certain extreme environmental changes (Oliveira and Bisalputra, 1980). It has poor food value for larvae and juveniles of various bivalve species (Enright et al., 1986a; Delaunay et al., 1993; Caers et al., 1998; Nevejan et al., 2003). In contrast, TP is a diet of moderate food value for bivalves (Enright et al., 1986a; Thompson and Harrison, 1992; O'Connor et al., 1992; Liu et al., 2009). It is worth noting that wild P. generosa can live in water as deep as 110 m, well below the photic zone. Clams below this zone probably feed on live phytoplankton, carried by winddriven or tidal currents, or dead plankton and bacteria (marine snow) settling from the photic zone (Goodwin and Pease, 1989). Marine snow is characterized by dense bacterial colonization, and associated microbial processes, such as enzyme activity, are often high (Alldredge et al., 1986; Smith et al., 1992; Grossart et al., 2003). It is not known how the elevated extracellular

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ACCEPTED MANUSCRIPT enzyme activity (e.g. Brock, 1989) would affect the digestive physiology of P. generosa as a result of adaptation.

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Although the body fatty-acid profile of juvenile P. generosa was basically dependent on

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the diets, the lower inclusion of major fatty acids in individuals fed those poor-performing diets

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(DT, PT, TP, and TS) would suggest lower lipid digestion of these algae: 1) the lower level of 18:3n-3 in juveniles fed DT (0.35 mg g-1) and TS (0.23 mg g-1) than in those fed TL (0.99 mg g-1;

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Table 6) although the former two were much richer in this fatty acid than the latter (Table 1); 2) the lower level of EPA in juveniles fed PT (0.79 mg g-1) and TP (0.61 mg g-1) than in those fed

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CC (1.98 mg g-1) and CM (1.27 mg g-1; Table 6) although all four diatoms were equally rich in EPA (Table 1). Poor overall digestion may also be linked to poor protein digestion in particular –

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for example certain proteases were only present in juvenile oysters, Crassostrea gigas, with an effective growth response (Babuin, 1999). Digestive enzyme activity can also be affected by

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food type (Tizon et al., 2013). Our further trial (Ren et al., 2014) showed that juvenile P. generosa fed the bi-species diet of PT+TL performed nearly the same as those fed CM+TL, suggesting that PT became more digestible in the presence of TL. In the present study, inclusion of a third alga of poor food value in the most favourable diet did not improve the performance of larval and juvenile P. generosa. This could be because these algae were still difficult to digest even in the presence of a good diet (activating normal digestive enzyme activity) or, if digested, added little to the optimal mixtures. Further studies on digestive enzyme activity of P. generosa with different diets would help understand the digestive physiology and food utilization of this species to the benefit of improved hatchery production (e.g. Crosby and Reid, 1971).

4.2. Biochemical compositions

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ACCEPTED MANUSCRIPT Microalgae used for mariculture typically contain 30–40% protein, 10–20% lipid, and 5– 15% carbohydrate in terms of dry weight (Brown et al., 1997; Brown, 2002). The species used in

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the present study appeared to have a much higher carbohydrate content (29–68% AFDW) (Table

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1). Higher carbohydrate contents of microalgal species have been reported in other studies (Ben-

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Amotz et al., 1987; Albentosa et al., 1993) and this variation among studies may be caused by differences in culture conditions (Enright et al., 1986b; Thompson and Harrison, 1992). Fatty

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acid compositions of the algal species used in the present study are consistent with those reported

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in the published literature: i.e. TL is rich in DHA, PL is rich in both EPA and DHA, the diatoms are rich in EPA, and TS has only EPA (Volkman et al., 1989; Thompson and Harrison, 1992;

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Martínez-Fernández et al., 2006; Rivero-Rodríguez et al., 2007). Moreover, DT contains almost

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no n-3 PUFAs other than 18:3n-3 (Caers et al., 1998; Nevejan et al., 2003). Much of the literature on bivalve nutrition has emphasized the important role of PUFAs,

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especially the n-3 fatty acids EPA and DHA on bivalve growth and development (Langdon and Waldock, 1981; Brown, 2002; Volkman and Brown, 2005; Martínez-Fernández et al., 2006). In the present study, P. generosa performed best on the bi-species diet of TL+CC for larvae and CM+TL for juveniles, both of which contained only moderate levels of EPA and DHA (and other nutrients) as a result of mixing (Tables 2–5). This suggests that the quantity of essential fatty acids (and other nutrients) in algal diets required need not be high but must be sufficient to allow adequate accumulation above a specific threshold level (Whyte et al., 1990a); neither dietary amount of EPA (i.e. CC or CM), DHA (i.e. TL), nor EPA and DHA combined (i.e. PL) can serve as exclusive factors in determining the nutritional value of an alga for P. generosa. This is consistent with the present observation that contents of EPA and DHA in the juveniles did not seem to explain their observed growth response. Another factor of greater importance

27

ACCEPTED MANUSCRIPT may be the n-3/n-6 ratio. Webb and Chu (1983) reported that most good algal diets for larval oysters, Crassostrea virginica, had a n-3/n-6 ratio of 2–3. This ratio was 2.1–3.2 with the best

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performing diets for post-larval and juvenile scallops, Argopecten irradians (Milke et al., 2006)

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and 2.9 for juvenile oysters, Crassostrea corteziensis (Rivero-Rodríguez et al., 2007). The

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present research showed that the top performing diets for P. generosa had n3/n-6 ratios in the range of 2.17 to 3.03 for larvae (Section 3.3.1; Tables 2 and 3) and 1.84 to 2.58 for juveniles

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(Section 3.3.2; Tables 4 and 5). Such a general agreement amongst studies and species emphasizes the important role of the n-3/n-6 ratio in bivalve nutrition, although Volkman et al.

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(1989) argued that the n-3/n-6 ratio did not account for the actual fatty acids present and was of limited value.

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The fatty acid profile of bivalve tissues generally reflects that of the diets supplied (e.g. Albentosa et al., 1994; Whyte et al., 1990a; Knauer and Southgate, 1997). The present study of P.

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generosa is not an exception. However, the EPA/DHA ratios were substantially lower in the juvenile tissues than in the diets in all cases except for the DHA-rich TL (Tables 1 and 6; Tables 4 and 7). Such a preferential incorporation of dietary DHA into body tissues has been reported for other bivalve species (Nevejan et al., 2003; Milke et al., 2004, 2006) and interpreted as indications of a more energetic role of EPA and a more structural role of DHA (Whyte et al., 1990b; Coutteau et al., 1996). The EPA/DHA ratio in juvenile P. generosa fed the optimal diet of CM+TL was 1.16 (Table 7), close to the value of 1 reported for wild geoduck mantles and siphons (Oliveira et al., 2011). If an EPA/DHA ratio of 1 approximates the ideal condition of P. generosa tissues then the higher catabolic nature of EPA would require an EPA/DHA ratio greater than 1 for the top performing diets which was indeed the case: 1.28–3.25 for the larvae (Section 3.3.1; Tables 2 and 3) and 1.84–2.63 for the juveniles (Section 3.3.2; Tables 4 and 5).

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ACCEPTED MANUSCRIPT For other bivalve species the best performing diet was found to have an EPA/DHA ratio of 4.7 [post-larval and juvenile scallops, Argopecten irradians (Milke et al., 1996)] and 9.8 [juvenile

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oysters, C. corteziensis (Rivero-Rodríguez et al., 2007)] and does not appear to have a general

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consensus among species other than the requirements of a higher dietary EPA/DHA ratio relative

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to bivalve body tissue.

The ratio of EPA and AA may also be important for bivalve nutrition. EPA and AA are

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both precursors of eicosanoids (Stanley-Samuelson, 1994; Stanley and Howard, 1998). In mammals and fish, EPA competes with AA in eicosanoid production, eicosanoids derived from

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the latter being more biologically active than those produced from the former. Thus, eicosanoid actions are determined by the cellular ratio of EPA/AA which, in turn, is determined by the

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dietary intake of these two fatty acids. An imbalanced ratio of EPA/AA appears to be damaging (Tocher, 2003). A minimum EPA/AA ratio of 4.2 was recommended for finfish larvae by

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Sargent et al. (1999). In the present study, the EPA/AA ratios with the various diets tested (Tables 1, 2, and 4), including the optimal ones, were far greater than this recommended value for fish larvae. In contrast, Milke et al. (2006) found that this recommended ratio was greatly exceeded with the poorest diets tested and higher than those of the best-performing diets for the scallop Argopecten irradians. Nevejan et al. (2007), however, observed that the contents of AA in seed mussels, Mytilus galloprovincialis, remained constant (0.4 mg g–1), independent of diet, and explained this as an indication that the mussels had developed a mechanism to regulate the content of this fatty acid. A similar trend was found in the present study for juvenile P. generosa fed the various mixed-algal diets (Tables 7 and 8), but the possibility that the dietary range of the contents of AA (Tables 4 and 5) was too narrow to exert any significant effect cannot be ruled out.

29

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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT Acknowledgements The project was funded by the Aquaculture Collaborative Research and Development

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Program of Fisheries and Oceans Canada (DFO) (Project number: P10-01-007) and the Klahoose

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Shellfish Limited Partnership. We thank Laurie Keddy (DFO, Pacific Biological Station) for

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microalgal culturing and Bruce Clapp, Tracy Scott (West Coast Geoduck Research Corporation), and Sean Williams (Abrupt Shellfish Incorporated) for broodstock collection. We also thank

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Yingyi Chen (Island Scallops Ltd.) and Dr. Abayomi Alabi (Seed Science Ltd.) for valuable

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discussions. Island Scallops Ltd. is acknowledged for providing geoduck larvae.

References

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Abdulkadir,S. and Tsuchiya,M. 2008. One-step method for quantitative and qualitative analysis of fatty acids in marine animal samples. J. Exp. Mar. Biol. Ecol. 354, 1–8.

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Albentosa, M., Perez-Camacho, A., Labarta, U., Beiras, R., Ferna´ndez-Reiriz, M.J., 1993. Nutritional value of algal diets to clam spat Venerupis pullastra. Mar. Ecol., Prog. Ser. 97, 261–269. Albentosa, M., Labarta, U., Pérez-Camacho, A., Fernandez-Reiriz, M.J., Beiras, R., 1994. Fatty acid composition of Venerupis pullastra spat fed on different microalgae diets. Comp. Biochem. Physiol. Part A: Physiol. 108, 639–648. Alldredge, A.L., Cole, J.J., Caron, D.A., 1986. Production of heterotrophic bacteria inhabiting macroscopic organic aggregates (marine snow) from surface waters. Limnol. Oceanogr. 31, 68– 78. Arney, B., Liu, W., Forster, I.P., McKinley, R.S. Pearce, C.M., 2015a. Feasibility of dietary substitution of live microalgae with spray-dried Schizochytrium sp. or Spirulina in the hatchery culture of juveniles of the Pacific geoduck clam (Panopea generosa). Aquaculture 444, 117–133.

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ACCEPTED MANUSCRIPT Arney, B., Liu, W., Forster, I., McKinley, R.S., Pearce, C.M. 2015b. Temperature and food-ration optimization in the hatchery culture of juveniles of the Pacific geoduck Panopea generosa. J.

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Table 1 Proximate and fatty acid compositions (mg g-1 ash-free dry weight) of the eight species of microalgae used for feeding larvae and early post-set juveniles of Panopea generosa CC 297.2±34.2 ab 259.5±15.0 a 453.5±96.9 bc

CM 248.7±9.9 bc 280.7±18.9 a 336.8±5.9 c

DT 249.8±12.0 bc 160.2±3.4 b 586.4±60.5 ab

PL 294.3±13.0 ab 263.1±11.1 a 291.4±37.0 c

PT 320.6±26.7 ab 236.6±16.7 a 389.6±64.5 c

Fatty acids 14:0 16:0 18:0 14:1 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) 18:2n6 18:3n6 20:3n6 20:4n6 (AA) 22:4n6 22:5n6

67.78±14.81 a 19.24±0.46 bc 0.11±0.06 b 0.27±0.16 47.88±8.50 b 0.18±0.11 b 0.56±0.07 b --0.10±0.05 c 25.71±4.95 a --1.38±0.25 b 3.48±1.58 b 0.69±0.11 b --0.05±0.05 -----

29.58±3.46 b 55.14±16.88 b 1.38±0.61 a --63.78±16.06 b 2.55±1.02 b ------21.14±0.95 a --2.50±0.5 b 1.00±0.24 b 3.68±0.57 a 0.47±0.23 0.95±0.48 -----

0.12±0.02 c 12.24±1.32 c 0.16±0.02 b --0.05±0.01 d 5.40±0.81 b ----14.75±0.69 a ------2.53±0.19 b 1.58±0.04 b ---------

29.16±6.58 b 25.23±2.50 bc 0.26±0.02 ab 0.17±0.04 33.02±3.86 b 2.77±0.47 b 0.11±0.07 b --0.78±0.13 c 20.60±1.48 a --12.94±2.70 a 1.09±0.24 b 0.24±0.03 b --0.18±0.05 --1.02±0.29

27.85±0.81 b 88.72±12.23 a 1.21±0.16 ab --120.05±12.01 a 9.41±1.91 b 0.33±0.05 b 0.30±0.05 0.11±0.06 c 23.64±3.05 a 0.35±0.11 1.57±0.49 b 2.06±0.11 b 1.23±0.09 b --0.31±0.05 -----

Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

87.13±15.30 ab 48.89±8.61 bc 27.19±5.24 ab 4.23±1.46 b 18.54±0.50 a 7.11±1.07 b

86.09±20.17 ab 66.33±16.91 b 23.64±1.40 ab 6.09±1.38 b 8.94±1.21 c 4.38±1.15 bc

12.51±1.36 c 5.45±0.82 d 14.77±0.67 bc 4.16±0.25 b N/A 3.56±0.11 bc

54.66±9.05 b 36.07±3.93 bcd 34.33±4.13 a 2.52±0.60 b 1.70±0.26 d 14.38±1.79 a

117.79±12.55 a 130.09±13.82 a 25.67±3.47 ab 3.60±0.15 b 15.66±1.38 b 7.10±0.75 b

TP 343.3±13.4 a 283.4±14.0 a 345.3±27.6 c

TS 190.3±17.0 c 128.1±8.9 b 683.7±13.8 a

All 263.2 235.2 434.6

51.28±6.93 ab 27.84±2.21 bc 0.73±0.42 ab 0.21±0.04 4.94±1.40 c 25.85±6.17 a 3.22±0.68 a 0.14±0.08 4.59±0.37 b 1.54±0.81 b 0.08±0.04 9.85±0.38 a 9.69±1.66 a 0.77±0.14 b --0.18±0.03 0.27±0.02 1.58±0.04

22.61±5.16 b 34.53±11.07 bc 0.91±0.38 ab --39.94±8.29 b 1.67±0.75 b ----0.04±0.04 c 19.69±3.49 a 0.50±0.50 3.08±0.74 b --2.11±1.07 b ---------

--13.15±0.60 c ------9.84±0.59 b ----5.16±0.18 b 1.89±0.01 b ----9.63±0.32 a 0.12±0.07 b ---------

28.55 34.51 0.59 0.08 38.71 7.99 0.53 0.05 3.19 14.28 0.12 3.92 3.69 1.21 0.06 0.17 0.03 0.33

79.84±9.36 ab 34.36±5.26 bcd 16.07±0.72 bc 12.49±1.51 a 0.16±0.08 d 1.33±0.19 c

58.05±13.18 b 41.61±8.50b cd 23.31±3.88 ab 3.04±0.54 b 6.84±1.19 c 8.16±1.17 b

13.15±0.60 c 9.84±0.59 d 7.04±0.18 c 9.74±0.26 a N/A 0.72±0.01 c

63.65 46.43 21.51 5.50 3.64 3.91

RI

SC

NU

MA

PT ED

CE

AC

TL 261.1±18.6 ab 280.9±21.2 a 390.2±17.8 c

PT

Protein Lipid Carbohydrate

Values are means±SE (n=3). Within a row, means with the same or no letter do not differ significantly (P>0.05). CC: Chaetoceros calcitrans; CM: C. muelleri; DT: Dunaliella tertiolecta; PL: Pavlova lutheri; PT: Phaeodactylum tricornutum TL: Tisochrysis lutea; TP: Thallassiosira pseudonana; TS: Tetraselmis suecica; All: mixture of all eight algal species, the values of which were calculated based on the ratio of ash-free dry weight of each component algal species in the mixture. ---: not detectable. N/A: not applicable.

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Table 2 Proximate and fatty acid compositions (mg g-1 ash-free dry weight) of TL-based mixed-algal diets used for feeding larvae of Panopea generosa TL 261.2 280.9 390.2

TL+CC* 279.2 265.2 421.9

TL+CM 254.9 280.8 363.5

TL+PL 277.7 272.0 340.8

TL+CC+CM 267.0 273.0 392.7

TL+CC+PL 278.4 268.6 381.3

TL+CM+PL 266.3 276.4 352.1

TL+CC+CM+PL 270.6 272.7 375.4

Fatty acids 14:0 16:0 18:0 14:1 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) 18:2n6 18:3n6 20:3n6 20:4n6 (AA) 22:4n6 22:5n6

51.28 27.84 0.73 0.21 4.94 25.85 3.22 0.14 4.59 1.54 0.08 9.85 9.69 0.77 --0.18 0.27 1.58

59.53 23.54 0.42 0.24 26.41 13.01 1.89 0.07 2.35 13.63 0.04 5.61 6.59 0.73 --0.12 0.14 0.79

40.43 41.49 1.05 0.10 34.36 14.20 1.61 0.07 2.30 11.34 0.04 6.17 5.35 2.22 0.23 0.56 0.14 0.79

40.22 26.54 0.49 0.19 18.98 14.31 1.67 0.07 2.69 11.07 0.06 11.39 5.39 0.50 --0.18 0.14 1.30

49.98 32.52 0.73 0.17 30.39 13.61 1.75 0.07 2.32 12.48 0.04 5.89 5.97 1.48 0.12 0.34 0.14 0.79

49.87 25.04 0.46 0.22 22.70 13.66 1.78 0.07 2.52 12.35 0.05 8.50 5.99 0.62 --0.15 0.14 1.04

40.32 34.01 0.77 0.15 26.67 14.25 1.64 0.07 2.49 11.21 0.05 8.78 5.37 1.36 0.12 0.37 0.14 1.04

46.73 30.52 0.65 0.18 26.58 13.84 1.72 0.07 2.44 12.01 0.05 7.73 5.78 1.15 0.08 0.29 0.14 0.96

Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

79.84 34.36 16.07 12.49 0.16 1.33

83.5 41.6 21.6 8.36 2.43 2.59

82.97 50.35 19.85 9.43 1.84 2.11

83.2 46.0 20.7 8.83 2.12 2.35

75.4 38.4 23.4 7.93 1.45 2.95

75.1 42.8 22.5 8.40 1.28 2.68

77.9 42.4 22.2 8.39 1.56 2.65

AC

CE

PT ED

MA

NU

SC

RI

PT

Protein Lipid Carbohydrate

67.3 35.2 25.2 7.51 1.0 3.36

Values were calculated based on the ratio of ash-free dry weight of each component algal species in the mixtures. CC: Chaetoceros calcitrans; CM: C. muelleri; PL: Pavlova lutheri; TL: Tisochrysis lutea. *: optimal diet for larvae. ---: not detectable.

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Table 3

TL+CC* 279.2 265.2 42.2

TL+CC+DT 273.2 244.0 45.5

TL+CC+PT 287.3 259.3 41.6

Fatty acids 14:0 16:0 18:0 14:1 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) 18:2n6 18:3n6 20:3n6 20:4n6 (AA) 22:4n6 22:5n6

59.53 23.54 0.42 0.24 26.41 13.01 1.89 0.07 2.35 13.63 0.04 5.61 6.59 0.73 --0.12 0.14 0.79

47.65 21.28 0.37 0.19 21.14 11.49 1.52 0.05 4.83 10.90 0.03 4.49 5.78 0.90 --0.09 0.11 0.64

53.19 36.58 0.58 0.19 45.14 12.29 1.58 0.11 1.90 15.63 0.10 4.81 5.68 0.83 0.06 0.09 0.11 0.63

Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

83.49 41.63 21.63 8.36 2.43 2.59

SC

NU MA

PT ED

CE AC

69.29 34.39 20.26 7.52 2.43 2.69

RI

Protein Lipid Carbohydrate

90.35 59.32 22.44 7.41 3.25 3.03

PT

Proximate and fatty acid compositions (mg g-1 ash-free dry weight) of TL+CC-based mixed-algal diets used for feeding larvae of Panopea generosa TL+CC+TP 271.9 268.7 40.7

TL+CC+TS 261.3 237.6 47.4

52.14 25.74 0.52 0.19 29.12 10.74 1.52 0.05 1.89 14.84 0.13 5.11 5.27 1.19 --0.09 0.11 0.63

47.62 21.46 0.33 0.19 21.13 12.38 1.52 0.05 2.91 11.28 0.03 4.49 7.20 0.61 --0.09 0.11 0.63

78.40 41.62 21.97 7.29 2.91 3.01

69.42 35.27 18.71 8.64 2.51 2.17

Values were calculated based on the ratio of ash-free dry weight of each component algal species in the mixtures. CC: Chaetoceros calcitrans; DT: Dunaliella tertiolecta; PT: Phaeodactylum tricornutum TL: Tisochrysis lutea; TP: Thallassiosira pseudonana; TS: Tetraselmis suecica. *: optimal diet for larvae. ---: not detectable.

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Table 4 Proximate and fatty acid compositions (mg g-1 ash-free dry weight) of CM-based mixed-algal diets used for feeding early post-set juveniles of Panopea generosa CM 248.7 280.7 336.8

CM+CC 272.9 265.1 395.2

CM+PL 271.5 271.9 314.1

CM+TL* 254.9 280.8 363.5

CM+CC+PL 272.2 268.5 354.7

Fatty acids 16:0 18:0 14:1 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) 18:2n6 18:3n6 20:3n6 20:4n6 (AA) 22:4n6 22:5n6

29.58 55.14 1.38 --63.78 2.55 ------21.14 --2.50 1.00 3.68 0.47 0.95 -----

48.68 37.19 0.74 0.14 55.83 1.36 0.28 --0.05 23.42 --1.94 2.24 2.18 0.23 0.50 -----

29.37 40.19 0.82 0.09 48.40 2.66 0.06 --0.39 20.87 0.02 7.72 1.04 1.96 0.23 0.56 --0.51

40.43 41.49 1.05 0.10 34.36 14.20 1.61 0.07 2.30 11.34 0.04 6.17 5.35 2.22 0.23 0.56 0.14 0.79

Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

86.09 66.33 23.64 6.09 8.94 4.38

86.61 57.61 25.41 5.16 12.08 4.92

70.38 51.20 29.01 4.35 2.70 6.66

CM+PL+TL 263.2 276.4 338.8

CM+CC+PL+TL 266.4 272.6 357.6

39.02 38.69 0.78 0.11 52.11 2.01 0.17 --0.22 22.15 0.01 4.83 1.64 2.07 0.23 0.53 --0.26

44.55 39.34 0.90 0.12 45.09 7.78 0.95 0.03 1.17 17.38 0.02 4.05 3.79 2.20 0.23 0.53 0.07 0.39

34.90 40.84 0.93 0.10 41.38 8.43 0.83 0.03 1.34 16.11 0.03 6.94 3.20 2.09 0.23 0.56 0.07 0.65

39.49 39.62 0.87 0.11 46.20 6.08 0.65 0.02 0.91 18.55 0.02 5.28 2.88 2.12 0.23 0.54 0.05 0.43

78.50 54.41 27.21 4.76 4.59 5.72

84.79 53.98 22.63 7.29 4.29 3.10

76.67 50.77 24.43 6.89 2.32 3.55

79.99 53.05 24.76 6.31 3.52 3.92

RI

SC NU

MA

PT ED

CE

AC

CM+CC+TL 263.9 273.0 379.3

PT

Protein Lipid Carbohydrate

82.97 50.35 19.85 9.43 1.84 2.11

Values were calculated based on the ratio of ash-free dry weight of each component algal species in the mixtures. CC: Chaetoceros calcitrans; CM: C. muelleri; PL: Pavlova lutheri; TL: Tisochrysis lutea. *: optimal diet for early post-set juveniles. ---: not detectable.

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Table 5 Proximate and fatty acid compositions (mg g-1 ash-free dry weight) of CM+TL-based mixed-algal diets used for feeding early post-set juveniles of Panopea generosa CM+TL* 254.9 280.8 363.5

CM+TL+DT 253.9 256.8 408.1

CM+TL+PT 252.6 281.5 359.9

Fatty acids 14:0 16:0 18:0 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 (DPA) 22:6n3 (DHA) 18:2n6 18:3n6 20:3n6 20:4n6 (AA) 22:4n6 22:5n6 (DPA)

40.43 41.49 1.05 34.36 14.20 1.61 0.07 2.30 11.34 0.04 6.17 5.35 2.22 0.23 0.56 0.14 0.79

32.37 35.64 0.87 27.50 12.44 1.29 0.05 4.79 9.07 0.03 4.94 4.78 2.09 0.19 0.45 0.11 0.64

37.91 50.94 1.08 51.50 13.24 1.36 0.11 1.86 13.80 0.10 5.25 4.69 2.03 0.25 0.45 0.11 0.63

Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

82.97 50.35 19.85 9.43 1.84 2.11

68.88 41.28 18.83 8.27 1.84 2.28

SC NU MA

PT ED

CE AC

RI

PT

Protein Lipid Carbohydrate

89.93 66.21 21.01 8.15 2.63 2.58

CM+TL+TP 268.0 272.1 368.7

CM+TL+TS 242.0 250.4 427.5

36.86 40.10 1.02 35.48 11.69 1.29 0.05 1.85 13.01 0.13 5.55 4.28 2.39 0.19 0.45 0.11 0.63

32.34 35.82 0.84 27.49 13.33 1.29 0.05 2.87 9.45 0.03 4.94 6.20 1.80 0.19 0.45 0.11 0.63

77.98 48.51 20.54 8.04 2.34 2.55

69.01 42.16 17.29 9.38 1.91 1.84

Values were calculated based on the ratio of ash-free dry weight of each component algal species in the mixtures. CM: Chaetoceros muelleri; DT: Dunaliella tertiolecta; PT: Phaeodactylum tricornutum; TL: Tisochrysis lutea; TP: Thallassiosira pseudonana; TS: Tetraselmis suecica. *: optimal diet for early post-set juveniles. ---: not detectable.

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Table 6 Fatty acid compositions (mg g-1 dry weight) of early post-set juveniles of Panopea generosa fed various mono-species algal diets for 28 days

7.97 5.28 1.65 0.68 4.12 2.43

3.58 1.15 0.86 0.59 1.03 1.45

4.69 2.58 2.02 0.62 0.78 3.27

TL 0.52 4.29 1.32 0.13 0.71 0.58 0.06 --0.99 0.23 0.03 1.57 0.89 0.05 0.34 0.27 0.06 0.31

TP 0.48 1.49 1.35 0.12 0.84 0.51 0.02 --0.09 0.61 0.07 0.38 --0.03 0.07 0.13 0.05 0.04

TS 0.71 1.10 0.88 0.10 0.38 0.42 ----0.23 0.31 0.04 0.23 0.06 0.09 0.10 0.21 0.06 0.05

All 0.56 4.36 1.94 0.16 2.93 0.77 0.06 --0.55 1.24 0.08 0.69 0.16 0.07 0.15 0.18 0.04 0.07

3.56 2.19 1.28 0.41 2.59 3.08

6.13 1.49 2.82 1.91 0.15 1.41

3.32 1.49 1.14 0.32 1.61 3.52

2.69 0.90 0.81 0.56 1.34 1.45

6.85 3.91 2.57 0.67 1.79 3.82

PT

5.88 4.13 2.55 0.31 4.36 8.16

PT 0.72 1.70 1.14 0.16 1.25 0.74 0.04 --0.08 0.79 0.10 0.30 --0.07 0.06 0.18 0.06 0.05

RI

14.6 5.17 4.17 0.87 1.53 4.33

PL 0.37 2.78 1.54 0.14 1.49 0.91 0.03 --0.11 0.80 0.07 1.03 0.03 0.03 0.08 0.23 0.08 0.17

SC

Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

DT 1.50 1.22 0.86 0.13 0.37 0.65 ----0.35 0.26 --0.25 --0.19 0.08 0.22 0.05 0.05

NU

0.05 0.10 0.15 --0.03

CM 1.07 5.04 1.85 0.15 4.12 0.89 0.12 --0.07 1.27 --0.31 0.04 0.20 0.05 0.28 0.10 ---

MA

CC 0.47 3.74 1.67 0.15 2.84 1.05 0.09 --0.12 1.98 --0.45

PT ED

Initial 6.78 5.61 2.22 0.22 3.35 1.45 0.13 0.03 0.72 1.99 0.16 0.30 0.22 0.01 0.14 0.29 --0.21

CE

Fatty acids 14:0 16:0 18:0 14:1 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 (DPA) 22:6n3 (DHA) 18:2n6 18:3n6 20:3n6 20:4n6 (AA) 22:4n6 22:5n6 (DPA)

AC

Values are the average of duplicate runs of a pooled, single sample for each dietary treatment. CC: Chaetoceros calcitrans; CM: C. muelleri; DT: Dunaliella tertiolecta; PL: Pavlova lutheri; PT: Phaeodactylum tricornutum; TL: Tisochrysis lutea; TP: Thallassiosira pseudonana; TS: Tetraselmis suecica; All: mixture of all eight algal species. ---: not detectable.

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Table 7 Fatty acid compositions (mg g-1 ash-free dry weight) of early post-set juveniles of Panopea generosa fed CM-based mixed-algal diets for 28 days

27.18±2.28 11.31±0.20 b 8.00±0.34 b 1.18±0.04 e 4.19±0.30 b 6.79±0.08 a

19.86±1.10 10.93±0.93 b 8.57±0.11 ab 1.70±0.08 cd 1.72±0.06 cd 5.06±0.32 b

CM+CC+TL 1.54±0.17 18.89±3.27 a 7.59±0.84 ab 0.67±0.04 14.34±2.34 a 2.77±0.46 b 0.45±0.09 ----6.20±0.45 0.22±0.01 c 2.96±0.08 b 0.47±0.15 b 0.35±0.06 a 0.32±0.03 a 0.93±0.10 0.27±0.06 ab 1.54±0.17 a

CM+PL+TL 1.94±0.55 17.21±0.37 ab 7.44±0.57 ab 0.63±0.10 11.29±0.27 ab 2.67±0.29 b 0.31±0.06 --0.22±0.01 b 5.24±0.21 0.20±0.01 c 3.81±0.14 a 0.37±0.05 b 0.27±0.01 ab --0.93±0.05 0.17±0.01 abc 0.56±0.01 bc

CM+CC+PL+TL 1.72±0.98 16.36±0.60 ab 6.83±0.89 ab 0.55±0.10 12.41±0.47 ab 2.50±0.07 b 0.40±0.07 --0.25±0.02 b 5.86±0.30 0.23±0.01 c 3.11±0.25 b 0.31±0.06 b 0.30±0.02 ab 0.24±0.02 ab 0.83±0.05 0.18±0.01 abc 0.38±0.03 c

22.22±2.87 14.65±1.37 ab 9.81±1.10 ab 1.88±0.08 cd 2.13±0.04 c 5.20±0.38 b

28.02±4.11 18.23±2.93 a 9.38±0.53 ab 3.88±0.41 a 2.09±0.10 c 2.44±0.14 e

26.59±1.46 14.90±0.19 ab 9.48±0.35 ab 2.29±0.09 c 1.38±0.04 de 4.14±0.04 c

24.91±2.21 15.86±0.43 ab 9.45±0.55 ab 2.24±0.16 c 1.89±0.07 c 4.23±0.09 c

PT

20.29±0.57 12.32±1.28 ab 7.80±0.47 b 1.35±0.06 de 5.06±0.02 a 5.76±0.17 b

CM+CC+PL 1.90±0.51 13.68±1.33 ab 6.64±1.11 b 0.60±0.08 10.96±1.10 ab 2.67±0.16 b 0.43±0.04 --0.36±0.03 b 6.20±0.68 0.32±0.04 b 2.93±0.36 b 0.17±0.06 b 0.26±0.03 ab 0.16±0.03 b 0.80±0.05 0.23±0.02 abc 0.27±0.03 c

RI

48.72±0.31 17.29±0.65 13.98±0.66 3.23±0.13 1.53±0.07 4.33±0.04

CM+TL* 2.93±2.24 17.68±1.07 ab 6.14±0.38 b 0.43±0.13 11.34±0.46 ab 4.96±0.17 a 0.34±0.02 --2.22±0.06 a 4.57±0.20 0.17±0.01 d 3.98±0.37 a 0.73±0.05 a 0.32±0.01 a 0.33±0.01 a 0.85±0.03 0.16±0.02 c 0.78±0.08 b

SC

Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

CM+PL 1.57±0.28 11.72±0.32 b 6.57±0.57 b 0.55±0.04 8.80±0.25 b 1.32±0.77 bc 0.27±0.04 --0.17±0.06 b 5.15±0.14 0.24±0.01 c 3.00±0.03 b 0.13±0.05 b 0.18±0.01 b 0.15±0.04 b 0.75±0.03 0.18±0.01 bc 0.32±0.01 c

NU

CM+CC 5.82±2.38 11.41±0.28 b 9.96±1.12 a 0.61±0.02 7.58±0.33 b 2.72±0.10 b 0.41±0.03 --0.27±0.05 b 5.91±0.54 0.40±0.01 a 1.41±0.01 c ------0.89±0.05 0.28±0.01 a ---

MA

CM 1.19±0.23 13.13±0.71ab 5.97±0.50 b 0.44±0.06 10.79±1.01ab 0.64±0.40 c 0.45±0.07 --0.29±0.03 b 5.94±0.34 0.40±0.02 a 1.17±0.07 c --0.31±0.02 a --0.76±0.04 0.28±0.01 a ---

PT ED

Initial 22.49±0.03 18.77±0.46 7.46±0.27 0.73±0.40 12.11±0.49 4.84±0.12 0.43±0.02 0.10±0.10 2.41±0.04 6.66±0.25 0.53±0.04 4.37±0.34 0.74±0.02 0.32±0.08 0.48±0.03 0.97±0.02 --0.72±0.06

CE

Fatty acids 14:0 16:0 18:0 14:1 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) 18:2n6 18:3n6 20:2n6 20:4n6 (AA) 22:4n6 22:5n6

26.74±3.62 17.07±0.69 ab 10.94±0.61 a 3.18±0.10 b 1.16±0.06 e 3.44±0.09 d

Values are means±SE (n=3). Within a row, means with the same or no letter do not differ significantly (P>0.05). CC: Chaetoceros calcitrans; CM: C. muelleri; PL: Pavlova

AC

lutheri; TL: Tisochrysis lutea. *: optimal diet for early post-set juveniles. ---: not detectable.

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Table 8 Fatty acid compositions (mg g-1 ash-free dry weight) of early post-set juveniles of Panopea generosa fed CM and TL-based mixed-algal diets for 28 days

39.87±1.61 14.56±0.86 b 12.92±0.49 4.23±0.25 a 1.05±0.03 b 3.06±0.13 b

CM+TL+TP 16.17±1.28 17.65±2.14 6.07±0.23 0.11±0.06 9.89±1.76 b 4.81±0.61 0.31±0.02 b --2.04±0.04 c 5.10±0.15 ab 0.42±0.18 4.18±0.41 0.67±0.07 b 0.20±0.08 b 0.44±0.01 0.78±0.05 0.20±0.03 0.56±0.10

CM+TL+TS 13.06±1.72 15.17±1.35 5.48±0.27 0.13±0.01 6.35±1.18 b 5.82±0.67 0.36±0.02 b --2.67±0.03 b 4.83±0.29 b 0.40±0.20 4.36±0.22 0.88±0.09 ab 0.27±0.21 ab 0.54±0.06 1.00±0.06 0.23±0.01 0.73±0.05

43.58±2.26 22.54±1.40 a 11.52±0.29 3.36±0.11 bc 1.71±0.11 a 3.43±0.10 b

39.89±3.24 15.12±2.35 b 11.74±0.60 2.85±0.04 c 1.24±0.10 b 4.11±0.22 a

33.71±2.98 12.65±1.40 b 12.26±0.52 3.66±0.27 b 1.11±0.01 b 3.36±0.11 b

PT

CM+TL+PT 16.17±0.90 21.22±1.30 6.19±0.26 0.18±0.03 15.91±1.76 a 6.12±0.19 0.34±0.02 b --2.24±0.03 bc 5.68±0.25 a 0.27±0.04 3.33±0.09 0.84±0.03 ab 0.33±0.09 a 0.58±0.06 0.87±0.04 0.20±0.01 0.55±0.03

RI

42.81±4.27 15.94±1.56 b 11.07±0.53 3.50±0.12 bc 1.15±0.04 b 3.16±0.29 b

SC

48.72±0.31 17.29±0.65 13.98±0.66 3.23±0.13 1.53±0.07 4.33±0.04

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Sum SFA Sum MUFA Sum n-3 PUFA Sum n-6 PUFA EPA/DHA n-3/n-6

CM+TL+DT 17.10±1.60 16.88±0.05 5.88±0.01 0.38±0.17 7.14±0.71 b 6.60±0.34 0.45±0.02 a --3.93±0.01 a 4.28±0.15 b 0.60±0.20 4.10±0.21 1.09±0.04 a 0.38±0.07 a 0.98±0.31 0.88±0.02 0.21±0.02 0.70±0.06

MA

CM+TL* 19.01±1.90 17.98±2.05 5.81±1.92 0.23±0.07 8.42±0.35 b 6.96±0.62 0.33±0.01 b --2.68±0.25 b 4.29±0.15 b 0.36±0.17 3.75±0.03 1.02±0.09 a 0.32±0.88 a 0.51±0.02 0.88±0.04 0.17±0.01 0.60±0.01

PT ED

Initial 22.49±0.03 18.77±0.46 7.46±0.27 0.73±0.40 12.11±0.49 4.84±0.12 0.43±0.02 0.10±0.10 2.41±0.04 6.66±0.25 0.53±0.04 4.37±0.34 0.74±0.02 0.32±0.08 0.48±0.03 0.97±0.02 --0.72±0.06

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Fatty acids 14:0 16:0 18:0 14:1 16:1 18:1n9 20:1n9 22:1n9 18:3n3 20:5n3 (EPA) 22:5n3 22:6n3 (DHA) 18:2n6 18:3n6 20:2n6 20:4n6 (AA) 22:4n6 22:5n6

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Values are means±SE (n=3). Within a row, means with the same or no letter do not differ significantly (P>0.05). CM: Chaetoceros muelleri; DT: Dunaliella tertiolecta; PT: Phaeodactylum tricornutum; TL: Tisochrysis lutea; TP: Thallassiosira pseudonana; TS: Tetraselmis suecica. *: optimal diet for early post-set juveniles. ---: not detected.

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This paper examines the effect of various microalgae (presented as mono-species and mixed-species diets) on the growth, survival, and fatty-acid composition of larvae and early juveniles of the Pacific geoduck clam (Panopea generosa). The optimal diet, with the fewest number of algal species supporting the best growth, was the bi-species diets of Chaetoceros calcitrans + Tisochrysis lutea for larvae and Chaetoceros muelleri + T. lutea for juveniles. The best performing diets, including the optimal one, had a n-3/n-6 ratio of 2.17–3.03 and an EPA/DHA ratio of 1.28–3.25 for larvae and 1.84–2.58 and 1.84–2.63, respectively, for early post-set juveniles.

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