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LABORATORY SCALE CULTURE OF EARLY-STAGE KURUMA SHRIMP MARSUPENAEUS JAPONICUS LARVAE FED ON THRAUSTOCHYTRIDS AURANTIOCHYTRIUM AND PARIETICHYTRIUM.

INTRODUCTION

The n-3 highly unsaturated fatty acids are important nutritional components for the growth and physiological function of marine animals (Sargent et al. 1997, Glencross 2009). For example, 22:6n-3 docosahexaenoic acid (DHA) plays an important role in optimizing nerve tissue and the visual system in marine species, and DHA has been reported to enhance growth rate in marine fishes and crustaceans (e.g., Watanabe et al. 1989, Kanazawa et al. 1979d). Many marine crustaceans, however, have a limited ability to synthesize sufficient n-3 highly unsaturated fatty acids to meet their metabolic requirements (Kanazawa et al. 1979d, Mercian & Shim 1996). Because these crustaceans must obtain DHA from their diets, feeds for marine crustacean aquaculture, including rotifer, artemia, and artificial mixed feeds, should be enriched in DHA.

Docosahexaenoic acid for industrial use is generally obtained from marine fish and fish oil products, but both the amount and price of fish and fish oils are destabilized by the variability in natural marine fishery resources. Because of emerging concerns over the sustainability of natural marine fishery resources, efforts have been made to identify alternative sources of DHA. Microbial production of DHA is one possibility, with autotrophic microbes, such as microalgae, considered a promising source of DHA (Carvalho & Malcata 2005). The Thraustochytrids are a group of heterotrophic protists, frequently found in marine and estuarine waters, and include several genera, such as Aurantiochytrium, Parietichytrium, Schizochytrium, and Thraustochytrium (Yokoyama & Honda 2007). Because the Thraustochytrids produce and accumulate large quantities of n-3 highly unsaturated fatty acids, especially DHA (Nakahara et al. 1996), they may be good alternative commercial sources of oils enriched in DHA (Ratledge 2004). One genus, Aurantiochytrium, is already in use in the commercial production of DHA-rich oils because it exhibits a high rate of proliferation, producing biomasses with relatively high yields of DHA (Fan et al. 2007). Dried powders of Aurantiochytrium-derived oils are currently available as sources of DHA in foods, feeds, and nutritional supplements (Fan et al. 2007).

The Kuruma shrimp Marsupenaeus japonicus is a major species of farmed marine crustacean in Japan. During aquaculture, early stage larvae of M. japonicus are fed up to mysis stages on microalgae, including Chaetoceros calcitrans, Chaetoceros gracilis, and/or Tetraselmis tetrathele, with diameters ranging from 3 to 9 [micro]m, 2 to 6 urn, and 6 to 15 [micro]m, respectively (Okauchi & Fukusho 1984, Olenina et al. 2006). Indoor microalgal proliferation is becoming relatively common, whereas outdoor microalgal proliferation is still common. The outdoor microalgal proliferation is affected by weather conditions (e.g., sunshine and air temperature), making stable and systematic proliferation of large amounts of microalgae difficult. Because the Thraustochytrids are heterotrophic and multiply rapidly in artificially controlled environments, their culture in large amounts is easy, stable, and systematic. Furthermore, vegetative cells of some Thraustochytrids, such as Aurantiochytrium and Parietichytrium, are spherical, with diameters of approximately 3-20 [micro]m (e.g., Yokoyama et al. 2007, Kaya et al. 2011, Nakazawa et al. 2012, Gao et al. 2013, Fig. 1). These characteristics suggested that Thraustochytrids may be reliably used, in place of microalgae, in diets for the mass culture of M. japonicus larvae. To our knowledge, however, vegetative, unprocessed cells of Thraustochytrids have never been used in artificial diets of early stage larvae of marine crustaceans.

This study was designed to determine whether two genera of Thraustochytrids, Aurantiochytrium and Parietichytrium, could be used instead of microalgae in diets for early stage shrimp Marsupenaeus japonicus larvae. Larvae of M. japonicus in laboratory culture were fed different diets, containing Chaetoceros calcitrans or strains of Aurantiochytrium or Parietichytrium, and the survival, development, and growth of these larvae were compared, as were the fatty acid compositions of these diets.

MATERIALS AND METHODS

Study Species

The Marsupenaeus japonicus is widely distributed in the Indian and Western Pacific Oceans and is one of the most important crustaceans for fisheries and aquaculture (Hayashi 1992). Their minimum cephalothorax length at sexual maturity is approximately 37-41 mm (Minagawa et al. 2000). This species spawns from mid-May to September/October in inland seas of western Japan, including the Seto Inland Sea, the Ariake Sea, and Tachibana Bay (Yatsuyanagi & Maekawa 1955, Minagawa et al. 2000). Females spawn repeatedly during the reproductive season, although little is known about the number of spawnings per season in their natural habitats. The estimated number of eggs per spawning increases with increasing female body size, with the number estimated at approximately 0.4-1.4 million (Suitoh 2014). Spawned eggs are released and dispersed into the water column. Hatched larvae pass through six nauplii stages, three zoea stages, and three mysis stages until reaching the postlarval stage (Hudinaga 1942). Nauplius larvae feed on their own yolks. Zoea larvae are herbivorous and feed on diatoms, whereas mysis larvae are omnivorous and start to feed on zooplankton (Hudinaga and Kittaka 1966).

Laboratory Rearing Experiment

Two laboratory rearing experiments were performed, the first starting on 25 June 2016 and the second on 13 August 2016. Females in imminent spawning condition were collected in the Bungo-suido Channel, located between Oita and Ehime Prefectures in Japan, by small-scale trawl fishing, on 23 June and 11 August 2016. Collected prawns were transferred to the Research Center for Marine Invertebrates, National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency (34[degrees] 37'N, 133[degrees] 27'E), on Momoshima Island, Hiroshima Prefecture, Japan. The females were kept individually in a net, measuring 65 cm X 65 cm X 70 cm and of mesh size 122 urn, in a 40,000-L tank filled with filtered and aerated seawater and maintained at 25[degrees]C. Prawns were not fed during the experiment to prevent larvae from being contaminated with food or feces. Six females (individual identification numbers, F1-6), three in each experiment, spawned until morning (9:00 AM) of the days following each transfer. Spawned females were removed from the net and their CLs measured to the nearest 0.01 mm using digital calipers (CD-20PMX; Mitutoyo Corporation). The CLs of F1-F3, which spawned during the first rearing experiment, were 55.9, 56.5, and 52.4 mm, respectively, whereas the CLs of F4-F6, which spawned during the second rearing experiment, were 58.2, 52.6, and 52.7 mm, respectively. Approximately 300 spawned eggs of each female were collected from the net using a pipette and transferred to a 500-mL beaker filled with 500 mL seawater that had been filtered with a hollow fiber filtration membrane (0.2 urn). Three beakers containing eggs from three different females were incubated in a temperature-controlled incubator (MIR-252; Sanyo Electric Co. Ltd., Osaka, Japan) preset to 18[degrees]C and a light:dark cycle of 14:10 h to collect larvae just after hatching the next morning (9:00 AM).

To compare the effects of diets containing Chaetoceros calcitrans and strains of Aurantiochytrium and Parietichytrium on larval survival, development, and growth, four treatment groups of larvae from females F1 to F3 in the first rearing experiment and three treatment groups of larvae from females F4 to F6 in the second rearing experiment were prepared. Each group was prepared in triplicate, resulting in 12 groups during the first rearing experiment and nine during the second. Multiple groups were prepared because maternal influences on larval qualities are known in this species (Sato et al. 2017).

Seventy-five hatched nauplius larvae from each female were randomly collected using a pipette and divided into three groups of 25 larvae each. Each group was placed in a 500-mL beaker filled with 500 mL seawater that had been filtered with a hollow fiber filtration membrane (0.2 [micro]m). In the first rearing experiment, three groups of larvae were each fed diets containing 2 X [10.sup.5] cells m[L.sup.-1] of (1) Chaetoceros calcitrans (I.S.C. Co., Ltd, Fukuoka, Japan); (2) the NIES-3737 strain of Awantiochytrium limacinum; (3) the NYH1 strain of Aurantiochytrium mangrovei; or (4) the KOU10 strain of Parietichytrium sp. (Table 1). In the second rearing experiment, three groups of larvae were each fed diets containing 2 X [10.sup.5] cells m[L.sup.-1] per day of (1) C. calcitrans; (2) the KOU14 strain of Parietichytrium sp.; or (3) the KOU101 strain of Parietichytrium sp. (Table 1).

Strains of Aurantiochytrium and Parietichytrium were maintained and passaged in the laboratory of the University of Tsukuba. Each strain was cultured for 48 h in 100 mL medium containing 5% glucose, 1% fish extract (Kyokuto Pharmaceutical Industries, Tokyo, Japan), and 0.5% yeast extract (BSP-B, Oriental Yeast co. Ltd., Tokyo, Japan) in 80% seawater in a 500-mL Erlenmeyer flask at 25[degrees]C on a reciprocal shaker (Taitec Corp., Saitama, Japan) at 100 rpm. The cells were harvested by centrifugation at 1,300 X g for 10 min, washed three times with sterile seawater, suspended at [10.sup.6] cells m[L.sup.-1] in sterile seawater, and stored at 4[degrees]C until use.

Beakers containing Marsupenaeus japonicus larvae were incubated in a temperature-controlled incubator (MIR-252; Sanyo Electric Co. Ltd., Osaka, Japan) preset to 25[degrees]C and a light:dark cycle of 14:10 h (approximately 4,000-5,400 lux) with a light emitting diode (MIR-252; ESCO, Ltd., Yamanashi, Japan). Each beaker was aerated (approximately 55 mL/min) and the seawater and diets were renewed daily. Dihydrostreptomycin (Tamura-seiyaku Corporation, Tokyo, Japan), at a concentration of 10 ppm, was added to the water used for cultivation to prevent bacterial attachment to the larvae.

Larval survival and stages of all surviving larvae were monitored every morning. Larvae were considered dead when no heartbeat was observed under a dissecting microscope (Eclipse 55i; Nikon Corporation, Tokyo, Japan) at X 100 magnification. In both rearing experiments, reared nauplius larvae reached first-stage mysis at least 12 days after hatching (DAH), except for a group fed the NIES-3737 strain of Aurantiochytrium limacinum, in which all larvae died before reaching mysis stage. Larval stage was determined by observation under a stereomicroscope (SMZ-U; Nikon Corporation, Tokyo, Japan). Larval stage index (LSI) was calculated using the formula LSI = (absolute value X number of larvae)/number of surviving individuals (Millamena & Quinitio 2000). Individuals observed to be in nauplius stage; first-, second-, and third-stage zoea; and first-stage mysis were assigned absolute values of 1-5, respectively. The number of days required to reach first-stage mysis was recorded. Rearing experiments were terminated when all larvae reached first-stage mysis or died. The body length of first-stage mysis larvae, from the eyespot to the tip of the telson, was measured to the nearest 0.001 mm using the profile projector (V-12A; Nikon Corporation) and digital calipers.

Generalized linear mixed-effects models (GLMMs), without considering any interactions, were used to examine the effects of different diets on (1) larval survival until first-stage mysis, (2) the number of days required to reach first-stage mysis, (3) the relationship between LSI and larval age (DAH), and (4) larval body length at first-stage mysis. Error distributions, response variables, and explanatory variables in the GLMMs are shown in Table 2. The effects of beaker, mother, and time of the rearing experiment (first or second) were treated as random effects in the GLMMs. Because all larvae fed on NIES-3737 died before reaching third-stage zoea, data on these groups were excluded from all analyses, except for analysis of larval survival. The glmmADMB package (Bolker et al. 2012) was used for modeling in R v.3.3.2 (R Development Core Team 2016). The statistical significances of explanatory variables in GLMMs were evaluated with the F test (type II) using Anova function implemented in the car package (Fox & Weisberg 2011). Differences among treatments were evaluated with the Tukey method using the glht function implemented in the multcomp package (Hothorn et al. 2008).

Fatty Acid Analysis

We lyophilized and ground Aurantiochytrium and Parietichytrium, and the powder (ca 100 mg) was weighed precisely. Fatty acids were methyl esterified directly by adding 5% methanolic HC1 (Tokyo Kasei, Tokyo, Japan) and incubated at 90[degrees]C for 90 min under an [N.sub.2] atmosphere. Fatty acid methyl esters were extracted with n-hexane, and the extracts were dried and resuspended in hexane. Each suspension was applied to a Sep-Pak Plus Silica cartridge (Waters) preconditioned in 10 mL n-hexane, and the cartridges were washed with n-hexane (5 mL) to remove hydrocarbons. Fatty acid methyl esters retained in the cartridges were eluted with 10% diethyl ether in hexane (5 mL). The eluates were dried in vacuo, and fatty acid methyl esters were analyzed by gas chromatography as described (Ishihara et al. 1998). Nonadecanoic acid (1 mg) was added to each sample as an internal standard. Peaks were identified by comparisons with the retention times of known standards (Supelco PUFA3, Supelco, PA) and cochromatography with authentic standards.

RESULTS

Laboratory Rearing Experiments

Except for larvae fed the NIES-3737 strain of Aurantiochytrium limacinum, all of which died by 9 DAH, all larvae reached first-stage mysis by 12 DAH (Fig. 2). The differences in diets significantly affected the number of surviving larvae even before they reached first-stage mysis (Table 3). The number of surviving larvae was significantly higher in those fed the KOU10 strain of Parietichytrium sp. than in those fed the NYH1 strain of Aurantiochytrium mangrovei or the NIES-3737 strain of A. limacinum (Tukey method, P< 0.05). In addition, the number of surviving larvae was significantly lower in those fed the NIES-3737 strain of A. limacinum than in all other groups (Tukey method, P < 0.05). More than half of the larvae fed the NYH1 strain of A. mangrovei died before reaching first-stage mysis (Fig. 2, Table 4).

Differences in diets also significantly affected the rate of larval development, with the number of days required to reach first-stage mysis differing significantly (Table 3). The larvae fed the KOU10 strain of Parietichytrium sp. required significantly fewer days to reach first-stage mysis than larvae fed the NYH 1 strain of Aurantiochytrium mangrovei (Tukey method, P < 0.05), with all larvae in the former group reaching first-stage mysis by 8 DAH (Fig. 3, Table 4). The LSI was also significantly affected by diet (Table 3), being significantly higher in larvae fed the KOU10 strain of Parietichytrium sp. and Chaetoceros calcitrans than in those fed the NYH1 strain of A. mangrovei (Tukey method, P < 0.05; Fig. 3). Moreover, LSI tended to be higher in larvae fed the KOU10 strain of Parietichytrium sp. than in those fed the KOU14 (Tukey method, P = 0.052) and KOU101 (Tukey method, P = 0.065) strains, and tended to be lower in larvae fed the NYH1 strain of A. mangrovei than in those fed the KOU14 (Tukey method, P = 0.057) and KOU101 (Tukey method, P = 0.067) strains of Parietichytrium sp.

Larval body length at first-stage mysis also differed by diet (Table 3). Body size was significantly larger in larvae fed the KOU10 strain of Parietichytrium sp. than in all other groups (Tukey method, P < 0.05; Fig. 4).

Fatty Acid Analysis

Fifteen fatty acids were identified in Chaetoceros calcitrans, with the 14:0, 16:ln-7, and 20:5n-3 fatty acids dominant (Table 5) and small amounts of 18:2n-6, 18:3n-3, and 22:6n-3 fatty acids (Table 6). The amount of 20:5n-3 fatty acids was greater in C. calcitrans than in any of the strains of Aurantiochytrium and Parietichytrium. The low amount of 22:6n-3 fatty acids and the high amount of 20:5n-3 fatty acids resulted in a very low DHA to eicosapentaenoic acid (EPA) ratio in C. calcitrans (Table 6).

The dominant fatty acids in the strains of Aurantiochytrium and Parietichytrium tested were the 16:0, 22:5n-6, and 22:6n-3 fatty acids (Table 5), with 18:0 fatty acids also dominant in the KOU10 and KOU14 strains of Parietichytrium sp. Total fatty acid contents were greater in the NYH1 strain of Aurantiochytrium mangrovei and the KOU14 strain of Parietichytrium sp. than in Chaetoceros calcitrans and other strains of Aurantiochytrium and Parietichytrium (Table 6). All strains of Aurantiochytrium and Parietichytrium contained low quantities of 18:2n-6, 18:3n-3, and 20:5n-3 fatty acids. The amounts of 22:6n-3 fatty acids in the NIES-3737 strain of Aurantiochytrium limacinum and the NYH1 strain of A. mangrovei were larger than in all others tested, resulting in larger total amounts of n-3PUFA and markedly higher DHA to EPA ratios (Table 6).

DISCUSSION

This study showed that diet had an important effect on the growth of early stage Marsupenaeus japonicus larvae. Except for those fed the NIES-3737 strain of Aurantiochytrium limacinum, all of which died before reaching first-stage mysis, these larvae grew well whereas feeding only on strains of Aurantiochytrium or Parietichytrium. Furthermore, larval survival, development, and growth were significantly greater when M. japonicus larvae were fed the KOU10 strain of Parietichytrium sp. than when fed all other diets tested, including Chaetoceros calcitrans. To our knowledge, this study is the first to show that Parietichytrium and Aurantiochytrium can be used, instead of microalgae, as a diet for early stage larvae of a marine crustacean. Use of Parietichytrium especially may result in the stable and systematic production of herbivorous larvae of marine animals, regardless of weather conditions.

The nutritional values for larvae of Marsupenaeus japonicus varied among strains of Aurantiochytrium and Parietichytrium, with statistically significant differences in rates of survival, development, and growth. Survival rates were low in larvae fed the NIES-3737 strain of Aurantiochytrium limacinum or the NYH1 strain of Aurantiochytrium mangrovei, with all larvae fed the former diet dying by 9 DAH. Larvae fed the NYH1 strain of A. mangrovei had a slow development rate, as shown by its LSI and number of days to reach first-stage mysis. Both of these strains contained small amounts of 18:2n-6, 18:3n-3, and 20:5n-3 EPA fatty acids (Table 6), which are considered essential fatty acids for M. japonicus (Kanazawa et al. 1979b), although both contain sufficient amounts of 22:6n-3 essential fatty acids. Conversely, Chaetoceros calcitrans also contains small amounts of 18:2w-6 and 18:3n-3, and the amount of 20:5n-3 in the KOU10 strain of Parietichytrium was comparable with that in the NYH1 strain of A. mangrovei although mean survival rates of M. japonicus larvae fed both were high (89.5% and 94%, respectively, Fig. 2), and the rate of development of larvae fed the KOU10 strain of Parietichytrium was rapid. These results indicate that the amounts of 18:2n-6, 18:3n-3, and 20:5n-3 fatty acids in the diet were not associated with the survival rate or development of M. japonic us larvae, at least through first-stage mysis. The low amount of 20:5n-3 fatty acids in the NIES-3737 strain of A. limacinum (0.07 g/100 g of dried diet, Table 6) may have led to the death before mysis stage of all larvae fed this diet.

The low survival and slow development rates observed in Marsupenaeus japonicus larvae fed certain diets may have been due to a high ratio of 22:6n-3-20:5n-3 fatty acids (DHA/EPA) in the diet. HUFAs, especially 22:6n-3 and 20:5n-3 fatty acids, are important for the survival and growth of marine crustaceans (Read 1981, Limet al. 1997), including M. japonicus (Kanazawa et al. 1979c). An abnormally high DHA/EPA ratio, however, can result in poor survival and growth of marine animal larvae (e.g., Watanabe et al. 1989, Rodriguez et al. 1997). The DHA/EPA ratio of tissue phospholipids is thought to be closely related with DHA and EPA contents in membrane phospholipids and to affect the physicochemical properties and biochemical functions of these membranes, which may affect animal growth and survival (Ibeas et al. 1997). The survival and growth performance of swimming crab (Portunus trituberculatus) juveniles have been reported to correlate with the DHA/EPA ratio in their diets, with both survival and growth decreasing as DHA/EPA ratio increases (Hu et al. 2017). The DHA/EPA ratios were found to be extremely high in the NIES-3737 strain of A. limacinum and the NYH1 strain of A. mangrovei because both contain large quantities of DHA, suggesting that these high ratios were responsible for the low survival and slow development rates of M. japonicus larvae fed these strains. Little is known about the effects of dietary DHA/EPA ratios on the survival and development of crustaceans, indicating a need for further studies of the effects of dietary DHA/EPA ratio on larval growth.

The positive effects of HUFAs on Marsupenaeus japonicus growth have been reported to be in the order of 20:5n-3 > 22:6n-3 > 18:3n-3 > 18:2n-6 (Kanazawa et al. 1978). Body size at first-stage mysis was, however, found to be larger in larvae fed the KOU10 strain of Parietichytrium than in those fed Chaetoceros calcitrans, despite the latter containing larger quantities of 20:5n-3 fatty acids than the other organisms tested. Optimum growth of M. japonicus has been reported when 1 % of the diet contains a combination of 20:5n-3 and 22:6n-3 fatty acids (Kanazawa et al. 1979a). This finding is consistent with results of this study, showing a relationship between body size at first-stage mysis and total amount of 20:5n-3 and 22:6n-3 in diets containing C. calcitrans (2.08%) and the KOUT0 strain of Parietichytrium (1.28%) (Table 6). The larvae fed the KOU14 strain of Parietichytrium (total amount of 20:5M-3 and 22:6n-3, 1.33%), however, did not show superior growth compared with larvae fed C. calcitrans. This finding indicates that the growth of larval M. japonicus was determined not only by fatty acid components but by other dietary contents. Studies of nutritional requirements for M. japonicus have included assessments of proteins (Deshimaru & Yone 1978a), phospholipids (Teshima 1997), cholesterols (Teshima et al. 1997), amino acids (Teshima et al. 2002), vitamins (Kanazawa 2001), minerals (Deshimaru & Yone 1978b), and carbohydrates (Kanazawa 2001), as well as fatty acids, although the appropriate ratio of dietary DHA/EPA has not been determined. The mechanism underlying the superior growth performance of M. japonicus larvae fed the KOU10 strain of Parietichytrium sp. remains undetermined. Stable and systematic culture of M. japonicus larvae using Parietichytrium sp. instead of microalgae, which are difficult to culture stably, requires a comprehensive survey of the nutritional value of Parietichytrium sp.

ACKNOWLEDGMENTS

This study was financially supported in part by Cross-ministerial Strategic Innovation Promotion Program (SIP) of Cabinet Office, Government of Japan. This work was made possible thanks to the generous cooperation of Mr. T. Nakaya, Matsumoto Suisan Corporation, in collection of prawns. We would like to give our grateful acknowledgment to Prof. D. Honda (Konan University, Japan) who kindly provided Parietichytrium strains. We deeply thank the members of the Research Center for Marine Invertebrates, National Research Institute of Fisheries and Environment of Inland Sea, for much support during the present study which complied with the current laws in Japan.

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TAKU SATO, (1*) KENJI ISHIHARA, (2) TOMOHITO SHIMIZU, (3) JURI AOYA (4) AND MASAKI YOSHIDA (5)

(1) Research Center for Marine Invertebrates, National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 1760 Momoshima, Onomichi, Hiroshima 722-0061, Japan; (2) Research Center for Biochemistry and Food Technology, National Research Institute for Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan; (3) Research Center for Subtropical Fisheries, Seikai National Fisheries Research Institute, Fisheries Research Agency, 148 Fukaiota, Ishigaki, Okinawa 907-0451, Japan; (4) Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan; (5) Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

(*) Corresponding author. E-mail: takusato@affrc.go.jp

DOI: 10.2983/035.037.0310
TABLE 1. Characteristics of Aurantiochytrium and Parietichytrium
strains used in the laboratory rearing experiments of Marsupenaeus
japonicus larvae.

                                         Cell diameter
Strain name  Species                     (mean [+ or -] SD, [micro]m)

NIES-3737    Aurantiochytrium limacinum  8.4 [+ or -] 2.8
NYH1         Aurantiochytrium mangrovei  6.0 [+ or -] 1.1
KOU10        Parietichytrium sp.         5.2 [+ or -] 1.2
KOU14        Parietichytrium sp.         7.3 [+ or -] 2.2
KOU101       Parietichytrium sp.         6.9 [+ or -] 2.0

TABLE 2. Details of GLMM used to evaluate the effects of diets in
Marsupenaeus japonicus larvae.

Analysis object   Error         Link      Response variable
                  distribution  function

Laval survival    Poisson       Logit     Number of surving individuals

Development rate  Poisson       Logit     Number of days to
                                          reach first-stage mysis
                  Poisson       Logit     Larval stage index

Larval growth     Gamma         Logit     Body length at first-stage
                                          mysis

Analysis object   Explanatory variable  Random effect


Laval survival    Diet and larval age   Beaker, mother, and time of
                                        the rearing experiment
Development rate  Diet                  Beaker, mother, and time of
                                        the rearing experiment
                  Diet, and larval age  Beaker, mother, and time of
                                        the rearing experiment
Larval growth     Diet                  Beaker, mother, and time of
                                        the rearing experiment

Larval age is number of days after hatching.

TABLE 3. The effects of explanatory variables in the GLMM used to
evaluate the effect of diets in Marsupenaeus japonicus larvae,
evaluated with the F test (type II).

Response variable           Explanatory
                            variable     df         F      P

Number of survivng          Diet         5,217      10.69  <0.01
individuals                 Larval age   1,217      71.32  <0.01
Number of days to           Diet         4,361       2.92   0.02
reach first-stage mysis
Larval stage index          Diet         4,3976     17.19  <0.01
                            Larval age   1,3976  2,120.79  <0.01
Body length of first-stage  Diet         4,360      59.79  <0.01
mysis larvae

Larval age is number of days alter hatching.

TABLE 4. Mean survival rates at first-stage mysis and numbers of days
required to reach first-stage mysis in Marsupenaeus japonicus larvae
fed different diets (Chaetceros calcitrans, the NYH1 strain of
Aurantiochytrium mangrovei, the KOU10 strain of Parietichytrium sp.,
the KOU14 strain of Parietichytrium sp., the KOU101 strain of
Parietichytrium sp.) in the laboratory rearing experiments.

                       Survival rate          Number of days
                       at first-stage         required to reach
                       mysis                  first-stage
Diet                   (mean [+ or -] SD, %)  mysis (mean [+ or -] SD)

Chaetceros calcitrans  90.0 [+ or -] 7.5      8.4 [+ or -] 0.4
NYH1                   60.0 [+ or -] 12.0     9.9 [+ or -] 1.0
KOU 10                 97.3 [+ or -] 2.3      7.3 [+ or -] 0.2
KOU14                  74.7 [+ or -] 6.1      8.6 [+ or -] 0.1
KOU101                 81.3 [+ or -] 12.2     8.4 [+ or -] 0.2

TABLE 5. Fatty acid compositions (%) of Chaeticeros calcitrans and
Aurantiochytrium and Parietichytrium strains.

                 Aurantiochytrium and Parietichytrium strains
                 Chaetceros
                 calcitrans  NIES-3737   NYH1  KOU10  KOU14  KOU101

Fatty acid
composition (%)
 C14:0           14.03        0.72       9.18   3.47   3.20   1.29
 C15:0            0.38        1.59       3.43   0.00   0.00   0.00
 C16:0            2.96       29.86      33.23  29.15  32.53  32.25
 C17:0            0.00        0.94       1.00   0.29   0.29   0.56
 C18:0            0.00        0.62       0.82  15.28  19.69   6.63
  Total of SFA   17.37       33.73      47.66  48.19  55.71  40.73
 C16:ln-7        30.36        0.00       0.00   0.52   0.51   0.47
 C18:ln-9         0.00        0.00       0.00  11.58   7.57   2.67
 C18:ln-7         0.35        0.34       0.00   0.17   0.23   0.19
  Total of MUFA  30.72        0.34       0.00  12.27   8.31   3.32
 C16:2n-4         4.21        0.00       0.00   0.00   0.00   0.00
 C16:3n-4         8.40        0.00       0.00   0.00   0.00   0.00
 C18:2n-6         0.21        0.00       0.00   4.64   5.64   1.82
 C18:3n-3         0.00        0.49       0.00   0.00   0.00   0.00
 C18:4n-3         0.49        0.34       0.00   0.00   0.00   0.00
 C20:3n-3         0.00        0.00       0.00   0.91   0.65   0.65
 C20:4n-6         0.25        0.52       0.37   3.25   3.11   5.45
 C20:4n-3         0.28        0.65       0.70   0.00   0.00   0.00
 C20:5n-3        21.41        0.86       1.34   2.60   1.75   8.23
 C22:4n-3         0.00        0.00       0.11   3.52   4.32   2.52
 C22:5n-6         0.00       11.49      10.39  10.67   8.22  12.29
 C22:5n-3         0.00        0.65       0.58   0.94   1.30   1.12
 C22:6n-3         0.94       50.56      35.78  11.92   9.37  22.91
  Total of PUFA  36.19       65.57      49.26  38.44  34.35  54.98
  Unidentified   15.73        0.36       3.08   1.10   1.63   0.96
 M-3PUFA         23.12       53.56      38.50  19.88  17.38  35.42
 H-6PUFA          0.46       12.01      10.76  18.56  16.98  19.56

TABLE 6. Fatty acid contents (g/100 g of the dried diet) of Chaetceros
calcitrans and Aurantiochytrium and Parietichytrium strains.

                   Aurantiochytrium and Parietichytrium strains
                 Chaetceros
                 calcitrans  NIES-3737  NYH1   KOU10  KOU14  KOU101

Total fatty acid contents (g/100 g of the dried algae)
                 9.28         7.93      15.74  8.80   12.00  5.60
Fatty acid contents (g/100 g of the dried diet)
 C14:0           1.30         0.06       1.44  0.31    0.38  0.07
 C15:0           0.04         0.13       0.54  0.00    0.00  0.00
 C16:0           0.27         2.37       5.23  2.56    3.90  1.81
 C17:0           0.00         0.07       0.16  0.03    0.03  0.03
 C18:0           0.00         0.05       0.13  1.34    2.36  0.37
  Total of SFA   1.61         2.67       7.50  4.24    6.68  2.28
 C16:ln-7        2.82         0.00       0.00  0.05    0.06  0.03
 C18:ln-9        0.00         0.00       0.00  1.02    0.91  0.15
 C18:ln-7        0.03         0.03       0.00  0.01    0.03  0.01
  Total of MUFA  2.85         0.03       0.00  1.08    1.00  0.19
 C16:2n-4        0.39         0.00       0.00  0.00    0.00  0.00
 C16:3n-4        0.78         0.00       0.00  0.00    0.00  0.00
 C18:2n-6        0.02         0.00       0.00  0.41    0.68  0.10
 C18:3n-3        0.00         0.04       0.00  0.00    0.00  0.00
 C18:4n-3        0.05         0.03       0.00  0.00    0.00  0.00
 C20:3n-3        0.00         0.00       0.00  0.08    0.08  0.04
 C20:4n-6        0.02         0.04       0.06  0.29    0.37  0.31
 C20:4n-3        0.03         0.05       0.11  0.00    0.00  0.00
 C20:5n-3        1.99         0.07       0.21  0.23    0.21  0.46
 C22:4n-3        0.00         0.00       0.02  0.31    0.52  0.14
 C22:5n-6        0.00         0.91       1.63  0.94    0.99  0.69
 C22:5n-3        0.00         0.05       0.09  0.08    0.16  0.06
 C22:6n-3        0.09         4.01       5.63  1.05    1.12  1.28
  Total of PUFA  3.36         5.20       7.75  3.38    4.12  3.08
  H-3PUFA        2.14         4.25       6.06  1.75    2.08  1.98
  H-6PUFA        0.04         0.95       1.69  1.63    2.04  1.10
  DHA/EPA        0.04        58.81      26.75  4.58    5.37  2.79
  EPA + DHA      2.08         4.08       5.84  1.28    1.33  1.74
Unidentified     1.46         0.03       0.48  0.10    0.20  0.05
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Author:Sato, Taku; Ishihara, Kenji; Shimizu, Tomohito; Aoya, Juri; Yoshida, Masaki
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9JAPA
Date:Aug 1, 2018
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