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Enhancement of dietary effect of Nannochloropsis sp. on juvenile Ruditapes philippinarum clams by alginate hydrolysates
Aquaculture Reports 9 (2018) 31–36
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Enhancement of dietary effect of Nannochloropsis sp. on juvenile Ruditapes philippinarum clams by alginate hydrolysates
T
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Yasuhiro Yamasakia, , Keita Ishiia, Shigeru Tagab, Masanobu Kishiokab a b
Department of Applied Aquabiology, National Fisheries University, 2-7-1 Nagata-Honmachi, Shimonoseki, Yamaguchi 759-6595, Japan Yamaguchi Prefectural Fisheries Research Center, 437-77, Aiofutajima, Yamaguchi, Yamaguchi 754-0893, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Alginate hydrolysates Algal diet Dietary enhancement Juvenile clam Ruditapes philippinarum
The eustigmatophyte Nannochloropsis sp. can be produced on a large scale at low cost. However, its dietary usefulness for juvenile bivalves is less than that of other algae. Recently, we reported that growth of juveniles of the clam Ruditapes philippinarum was dramatically promoted by supplementing a diet of the diatom Chaetoceros neogracile with alginate hydrolysates (AHs) at the concentration of 4 mg L−1. In this study, we examined the effect of AHs on Nannochloropsis sp. as a clam diet. Ten-day rearing experiments in 500-mL beakers showed that AHs have a beneficial effect on clam culture regardless of water temperature. Shell growth in clams given AHs at the concentration of 4 mg L−1 along with Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 was higher than in any other test groups at 15 or 25 °C. In 20-day rearing experiments in 30-L tanks, the average shell length in the groups given AHs at the concentration of 4 mg L−1 along with Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was significantly greater than that in the groups given C. neogracile and Nannochloropsis sp. alone (P ≪ 0.05). Furthermore, the total weight of clams given AHs at the concentration of 4 mg L−1 along with Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was greater than that in the group given only Nannochloropsis sp. (P ≪ 0.05). Hence, the enhanced dietary effect of a combination of Nannochloropsis sp. and AHs will help to shorten the rearing time of R. philippinarum and to provide a stable supply of algal diet.
1. Introduction The Manila clam Ruditapes philippinarum (Adams and Reeve, 1850) is not only a commercially important bivalve but also one of the most ecologically important bivalves in the world. However, the annual catch of this species in coastal waters of Japan, which once led the world, continues to decrease drastically. Several factors have been suggested as causes of the dramatic decrease (Hamaguchi et al., 2002; Matsukawa et al., 2008; Park et al., 2006; Toba et al., 2013; Tsutsumi, 2006) in addition to overfishing, although the precise cause is as yet unknown. On the other hand, there have been a wide variety of studies aimed at conserving the clam resource and developing clam culture (Bartoli et al., 2001; Dang et al., 2010; Nizzoli et al., 2006; Paillard, 2004; Paul-Pont et al., 2010). Thus, the development of a growth-promoting factor, the development of a mass-culture method for production of the clam, and clarification of its growth mechanisms would have important implications for clam culture, and would contribute to the conservation of the clam resource in the field and a stable market supply.
Jǿrgensen (1983) reported that clams take up dissolved organic matter (DOM) in seawater through epidermal tissue in the mantle and gills. Welborn and Manahan (1990) showed that larvae of the bivalve Crassostrea gigas (Pacific oyster) can take up dissolved glucose, maltose, cellobiose, and cellotriose, but not rhamnose or maltotriose. In addition, Uchida et al. (2010) reported that the growth rate of soft tissue in R. philippinarum was significantly promoted by supplementing a diet of the diatom Chaetoceros calcitrans with glucose. Thus, certain types of sugars are potentially a good supplement for R. philippinarum growth. Furthermore, Taga et al. (2013) reported the beneficial effects of the raphidophyte Heterosigma akashiwo, known as a harmful algal species, on the diet of juvenile R. philippinarum, and suggested that certain kinds of sugars, specifically the acidic sugars in phytoplankton, might be one of the important factors determining the growth of juvenile clams. Alginate is a natural acidic linear polysaccharide that is composed of α-L-guluronate and β-D-mannuronate (uronic acids) residues, and is also known as a type of dietary fiber. Several studies have reported that the dietary administration of alginate stimulates the immune abilities of white shrimp and juvenile carp (Cheng et al., 2005; Huttenhus et al.,
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Corresponding author. E-mail addresses: yamasaky@fish-u.ac.jp (Y. Yamasaki), [email protected] (K. Ishii), [email protected] (S. Taga), [email protected] (M. Kishioka). https://doi.org/10.1016/j.aqrep.2017.11.006 Received 6 September 2017; Received in revised form 21 November 2017; Accepted 30 November 2017 2352-5134/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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2006), and the compositional ratio of α-L-guluronate and β-D-mannuronate or the degree of polymerization affects the physical properties and multiple biological activities of the alginate (Iwamoto et al., 2005; Iwasaki and Matsubara, 2000). In addition, alginate and its derivatives are currently used in a wide range of commercial enterprises, including the food, medical, and cosmetic industries. Recently, we showed that growth of R. philippinarum was dramatically promoted by supplementing a diet of the diatom Chaetoceros neogracile with alginate hydrolysates (AHs) at the concentration of 4 mg L−1 (Yamasaki et al., 2015, 2016), and metabolomics indicated that each of the states of starvation, food satiation, and sexual maturation of R. philippinarum has a characteristic pattern in the metabolite profile (Yamasaki et al., 2016). Although C. neogracile is widely used for clam culture as an algal diet, the cost of cultivating C. neogracile is far from cheap, and culture crashes of this species often occur during mass cultivation. The eustigmatophyte Nannochloropsis sp. is known to contain a high concentration of polyunsaturated fatty acids, especially eicosapentaenoic acid, and is used in aquaculture to feed a wide variety of marine organisms (Borowitzka, 1997; Rico-Villa et al., 2006; Wickfors and Onho, 2001). Moreover, the cost of producing Nannochloropsis biomass in outdoor flat-plate glass reactors (Zhang et al., 2001) is lower than in hatcheries (Benemann, 1992). Thus, Nannochloropsis sp. can be produced on a large scale at low cost. However, Nannochloropsis sp. has less dietary effect on juvenile bivalves compared with other algae diets. In this study, we examined the use of AHs to enhance the dietary effect of Nannochloropsis sp. on juvenile R. philippinarum.
The pH of the water was checked daily, and there were no significant pH changes in any of the rearing tests. In experiments 1 (25 °C) and 2 (15 °C), we investigated the optimum feeding concentration and water temperature for growth of juvenile clams. For each rearing test, 50 clams [initial average shell length: 0.790 ± 0.137 mm (25 °C), 1.129 ± 0.121 mm (15 °C)] were reared for 10 d in 0.5 L filtered seawater (i.e. unfed), a 0.5-L suspension of C. neogracile (8 × 104 cells mL−1), a 0.5-L suspension of Nannochloropsis sp. (20–80 × 104 cells mL−1), and a 0.5 L suspension of Nannochloropsis sp. (20–80 × 104 cells mL−1) with AHs added at the concentration of 4 mg L−1. There were three replicate beakers for each treatment. The rearing water along with any algal diet or AHs was exchanged daily (Table 1). In addition, all beakers were cleaned every 5 days to prevent bacterial growth and to ensure clam survival. After the rearing tests, all shell lengths were measured and the survival rates were determined. In experiment 3 (25 °C), we examined the enhancement of the dietary effect of Nannochloropsis sp. on juvenile clams by AHs at a larger scale and for a longer period. For each rearing test, 2000 clams (initial average shell length: 1.088 ± 0.244 mm) were reared for 20 d in a 20L suspension of C. neogracile (8 × 104 cells mL−1), a 20-L suspension of Nannochloropsis sp. (30 × 104 cells mL−1), and a 20-L suspension of Nannochloropsis sp. (30 × 104 cells mL−1) with AHs added at the concentration of 4 mg L−1. There were three replicate tanks for each treatment. The rearing water along with any algal diet or AHs was exchanged daily (Table 1). In addition, all tanks were cleaned every 10 days to prevent bacterial growth and to ensure clam survival. After the rearing test, the total wet weight includes the shells of all clams and the shell lengths of randomly selected individuals (approximately 10% of the total wet weight) from each tank were measured.
2. Materials and methods 2.1. Preparation of alginate hydrolysates
2.4. Growth of Nannochloropsis sp. with and without alginate hydrolysates
We prepared AHs using sodium alginate (SKAT-ULV, KIMICA Co., Tokyo, Japan) in accordance with previous studies (Yamasaki et al., 2012, 2015, 2016). Samples of AHs were stored at −30 °C until use. Yamasaki et al. (2012) reported that the primary oligomers in AHs were a monomer (176 Da), a dimer (352 Da), and a trimer (528 Da), with no significant quantities of larger oligomers.
To determine whether AHs affected the growth of Nannochloropsis sp., we conducted culture experiments in 0.5-L beakers containing 0.5 L of medium as follows. As a test group, a 0.5-L suspension of Nannochloropsis sp. (20 × 104 cells mL−1) with AHs at the concentration of 4 mg L−1 was incubated for 24 h. As a control, a 0.5-L suspension of Nannochloropsis sp. (20 × 104 cells mL−1) without added AHs was incubated for 24 h. There were three replicate beakers for each treatment, and aerated cultures were maintained at 25 °C in the dark. Cells were counted under a microscope in 1-mL subsamples collected 0 and 24 h after the start of the experiment. For all experiments, cells were counted five times for each sample.
2.2. Microalgal diet and growth conditions The microalgae Nannochloropsis sp. and C. neogracile were grown in 30-L tanks containing 20 L of filtered seawater enriched with KW21 marine alga growth medium (Daiichi Seimo Co. Ltd., Kumamoto, Japan). Aerated cultures were maintained at 20 °C under continuous light.
2.5. Statistical analysis 2.3. Clam rearing tests The data from the clam rearing experiments were analyzed by oneway analysis of variance followed by Tukey’s post hoc test. In addition, the data from the growth experiment with Nannochloropsis sp. with and without AHs were compared using a paired t-test. The analyses were performed using SPSS for Windows (SPSS version 19.0; SPSS, Inc., Chicago, IL, USA) using a significance level of P ≪ 0.05.
Rearing tests were conducted as shown in Table 1 in aerated, filtered natural seawater (pore size 1 μm) with mean salinity of 30–31‰. Table 1 Experimental conditions for each rearing test.
Average shell length (mm) Rearing density (individuals L−1) Rearing period (d) Mean water temperature (°C) Quantity of rearing water (L) Concentration of AHs (mg L−1) Feed dosage of C. neogracile ( ×104 cells mL−1 d−1) Feed dosage of Nannochloropsis sp. ( ×104 cells mL−1 d−1) Exchange frequency of rearing water
Experiment 1
Experiment 2
Experiment 3
0.79 ± 0.14 100 10 25 0.5 4 8
1.13 ± 0.12 100 10 15 0.5 4 8
1.09 ± 0.24 100 20 23–25 20 4 8
20–80
20–80
30
Everyday
Everyday
Everyday
3. Results 3.1. Dietary effects of Nannochloropsis sp. supplemented with AHs on growth of clams at 25 °C The survival of clams in all groups grown at 25 °C ranged from 91% to 100% (Fig. 1). Growth (as measured by final shell length) of the groups given Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 (with and without AHs) and at the concentration of 40 × 104 cells mL−1 (with AHs) and growth of the group given C. neogracile were virtually the same, whereas the groups given Nannochloropsis sp. at the concentration of 40 × 104 (without AHs) and at the concentration of 32
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Fig. 1. Effects of Nannochloropsis sp. in the presence or absence of AHs (4 mg L−1) on the growth, as measured by shell length, and survival of juvenile Manila clams, R. philippinarum, at 25 °C. Rearing tests were conducted as described in the text and Table 1. Data are means ± SD of the average shell length of clams in each test group (n = 3) after 10 days. Bars with different lowercase letters are significantly different at P ≪ 0.05 (oneway analysis of variance [ANOVA] followed by Tukey’s post hoc test).
Fig. 2. Effects of Nannochloropsis sp. in the presence or absence of AHs (4 mg L−1) on the growth, as measured by shell length, and survival of juvenile Manila clams, R. philippinarum, at 15 °C. Rearing tests were conducted as described in the text and Table 1. Data are means ± SD of the average shell length of clams in each test group (n = 3) after 10 days. Bars with different lowercase letters are significantly different at P ≪ 0.05 (oneway analysis of variance [ANOVA] followed by Tukey’s post hoc test).
80 × 104 cells mL−1 (with and without AHs) showed a significantly lower shell-length growth as compared with the group given C. neogracile (P ≪ 0.05, Fig. 1). Although supplementing the diet of Nannochloropsis sp. with AHs at the concentration of 4 mg L−1 tended to promote growth, there was no significant increase in growth as compared with the groups given C. neogracile (Fig. 1). The mean, median, minimum and maximum shell lengths for each test group are shown in Table 2. The mean and median for each test group were virtually the same except for the groups given Nannochloropsis sp. at the concentration of 40 × 104 (with AHs) and at the concentration of 80 × 104 cells mL−1 (with AHs). In addition, the minimum and maximum values in the groups with high growth tended to be higher than in the groups with low growth.
with the group given C. neogracile (P ≪ 0.05, Fig. 2). The mean, median, minimum and maximum shell lengths for each test group are shown in Table 2. The mean and median in each test group were virtually the same. In addition, there was no pronounced variation in minimum and maximum values between any test groups. 3.3. Dietary effects of Nannochloropsis sp. supplemented with AHs on growth of clams at a larger scale and for a longer period For the clams grown in larger cultures for a longer time, growth, as measured by shell-length, was dramatically promoted by supplementing a diet of Nannochloropsis sp. with AHs at the concentration of 4 mg L−1. The average shell length in the groups given AHs in addition to Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was significantly greater than that in the groups given C. neogracile and Nannochloropsis sp. (P ≪ 0.05, Fig. 3A). Also, the total wet weight of clams in the group given AHs in addition to Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was greater than that in the group given only Nannochloropsis sp. (P ≪ 0.05, Fig. 3B), whereas there was no significant difference in total weight as compared with the group given C. neogracile (Fig. 3B). Length–frequency histograms of each group show the growth-promoting effect of AHs as a supplement to Nannochloropsis sp. on the clams (Fig. 4). The mode of shell length in the group given Nannochloropsis sp. was 1.6–1.8 mm (Fig. 4A) and in the group given AHs plus Nannochloropsis sp. was 2.0–2.2 mm (Fig. 4B). In addition, the mode shell length in the group given C. neogracile was 2.0–2.2 mm (Fig. 4C). Furthermore, the minimum shell lengths in the group given Nannochloropsis sp., the group given AHs plus Nannochloropsis sp., and the group given C. neogracile were 1.136, 1.227, and 1.111 mm,
3.2. Dietary effects of Nannochloropsis sp. supplemented with AHs on growth of clams at 15 °C The survival of clams in all groups grown at 15 °C ranged from 85% to 100% (Fig. 2). Growth as measured by shell-length increase was promoted by supplementing a diet of Nannochloropsis sp. with AHs at the concentration of 4 mg L−1. The average shell length in the groups given AHs in addition to Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 was significantly greater than in the groups given C. neogracile and Nannochloropsis sp. (P ≪ 0.05, Fig. 2). In addition, growth in the groups given Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 (without AHs), 40 × 104 cells mL−1 (with and without AHs), and 80 × 104 cells mL−1 (with AHs) and growth in the group given C. neogracile were virtually the same, whereas there was significantly lower growth in the groups given Nannochloropsis sp. at the concentration of 80 × 104 cells mL−1 (without AHs) compared Table 2 Mean, median, minimum and maximum values of shell length in each test group. Experimental group
Unfed C. neogracile (8 × 104 cells mL−1) Nannochloropsis sp. (20 × 104 cells Nannochloropsis sp. (40 × 104 cells Nannochloropsis sp. (80 × 104 cells Nannochloropsis sp. (20 × 104 cells Nannochloropsis sp. (40 × 104 cells Nannochloropsis sp. (80 × 104 cells
Experiment 1: Shell length (mm)
−1
mL ) mL−1) mL−1) mL−1) + AHs mL−1) + AHs mL−1) + AHs
Experiment 2: Shell length (mm)
Mean
Median
Minimum
Maximum
Mean
Median
Minimum
Maximum
0.952 1.439 1.394 1.284 1.196 1.465 1.374 1.246
0.967 1.402 1.355 1.246 1.170 1.435 1.297 1.192
0.514 0.719 0.503 0.536 0.634 0.696 0.667 0.569
1.488 2.525 2.472 2.280 2.069 2.405 2.545 2.166
1.158 1.312 1.299 1.291 1.238 1.396 1.310 1.317
1.139 1.316 1.297 1.279 1.226 1.379 1.306 1.306
0.827 0.912 0.959 1.017 0.922 1.031 0.873 1.048
1.450 1.613 1.742 1.637 1.653 1.764 1.604 1.746
33
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Fig. 4. Length–frequency histograms of juvenile Manila clams, R. philippinarum, (A) fed only Nannochloropsis sp., (B) fed Nannochloropsis sp. supplemented with AHs (4 mg L−1), and (C) fed only C. neogracile. Rearing tests were conducted as described in the text.
Fig. 3. Effects of Nannochloropsis sp. in the presence or absence of AHs (4 mg L−1) on (A) the growth, as measured by shell length, and (B) total wet weight of juvenile Manila clams, R. philippinarum, at 25 °C. Rearing tests were conducted as described in the text and Table 1. Data are means ± SD of the average shell length of clams in each test group (n = 3) after 20 days. Bars with different lowercase letters are significantly different at P ≪ 0.05 (one-way analysis of variance [ANOVA] followed by Tukey’s post hoc test).
respectively. In contrast, maximum shell lengths in the group given Nannochloropsis sp., the group given AHs plus Nannochloropsis sp., and the group given C. neogracile were 2.707, 3.806, and 3.617 mm, respectively. 3.4. Effect of AHs on the growth of Nannochloropsis sp. Culture experiments tested whether AHs affected the growth of Nannochloropsis sp. There were no significant differences in cell density of Nannochloropsis sp. cultures with or without AHs or between the densities at the start and at the end of the culture experiment (Fig. 5). Fig. 5. Cell density of Nannochloropsis sp. cultures in the presence or absence of AHs (4 mg L−1) after 0 and 24 h of culture. Data are means ± SD (cells mL−1) of triplicate measurements.
4. Discussion Our results show that shell length and total weight of clams were significantly promoted by supplementing a diet of Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 with AHs at the concentration of 4 mg L−1 as compared with the groups given Nannochloropsis sp. only (Figs. 3 and 4). The main benefit of our findings is that AHs can enhance the dietary effect of Nannochloropsis sp., which can be produced on a large scale at low cost. Although this species is a less sufficient diet for clams as compared with other algae, the enhanced benefits from AH supplementation may lead to a shorter rearing time for R. philippinarum and a more reliable supply of algal diet. The other useful outcome is that the effective amount of added AHs is lower than that previously reported for glucose (Uchida et al., 2010). The acidic polysaccharide alginate, known as a dietary fiber, has little stimulating effect on bacterial growth in the rearing water compared
with glucose, which is generally known as an energy source. Furthermore, the alginate used in this study is food-grade, the same as in our previous studies (Yamasaki et al., 2015, 2016), and its availability ensures a reliable supply of a safe diet for clams at low cost to the aquaculture industry. Thus, from a practical perspective, AHs can have a noteworthy effect on clam culture. Dong et al. (2000) examined the effect of temperature on filtration rate, clearance rate and absorption efficiency of R. philippinarum at 9, 16, 22, and 26 °C. They reported that all three metrics increased with a rise in temperature and reached maxima at 22 °C, but then decreased slightly at the highest temperature (Dong et al., 2000). Thus, growth of R. philippinarum decreases with a drop in temperature. Interestingly, although in our study clams reared at 25 °C grew faster than those 34
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Nannochloropsis sp. on juvenile R. philippinarum clams. The enhanced dietary effect of a combination of Nannochloropsis sp. and AHs will help to shorten the rearing time of R. philippinarum and to stabilize supplies of algal diet because Nannochloropsis sp. can be produced on a large scale at low cost. Further studies are needed to translate the utilization of AHs into practical applications in clam culture.
reared at 15 °C, the utilization of AHs was effective regardless of water temperature (Figs. 1 and 2). In experiment 1 (25 °C), the mean and median shell lengths in the groups given Nannochloropsis sp. at the concentration of 20 × 104 (with AHs) were the highest of all test groups (Table 1), although shell growth was not significantly higher than that in the groups given C. neogracile (P = 0.998, Fig. 1). In experiment 2 (15 °C), shell growth in the groups given AHs in addition to Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 was significantly higher than that in any other groups (P ≪ 0.05, Fig. 2), and the mean, median, and maximum shell lengths in this group were the highest of all test groups (Table 1). Therefore, supplementation with AHs would have a beneficial effect on clam culture not only during early summer but also in winter. Several studies reported that bivalves change filtration rate depending on the concentration of diet algae in order to increase assimilation efficiency (Navarro and Winter, 1982; Winter, 1978). Toba and Miyama (1993) reported that the filtration rate of juvenile R. philippinarum feeding on the haptophyte Pavlova lutheri was highest at the concentration of 5000 cells mL−1 and continuously declined with increasing algal density. In fact, our results showed that growth as measured by shell-length increase tended to decrease with increasing density of algal diet (Figs. 1 and 2, Table 2); that is, our results suggest a decrease in ingestion rate and/or assimilation efficiency with increases in algal density. In the present study, however, growth of clams in all groups given AHs along with Nannochloropsis sp. tended to be higher as compared with the groups given only Nannochloropsis sp. (Figs. 1 and 2, Table 2). It is unlikely that the AHs are used as a source of energy because a previous study showed that shell-length growth was significantly inhibited in R. philippinarum given AHs only (Yamasaki et al., 2015). Therefore, AHs likely affect shell-length growth of R. philippinarum not by acting as an energy source but rather by enhancing the dietary effect of Nannochloropsis sp. We have not yet identified the mechanisms that explain the dietary enhancement of AHs on Nannochloropsis sp. for clams. A likely explanation is that the growth-promoting effect of AHs results from their stimulating an increase in Nannochloropsis sp. biomass. For example, growth of the green alga Chlamydomonas reinhardtii was significantly promoted by an alginate oligomer mixture (AOM) prepared by enzymatic degradation (Yamasaki et al., 2012). However, growth of the alga was not affected by an AOM prepared by acid hydrolysis and other saccharides (Yamasaki et al., 2012). Furthermore, culture experiments were conducted in the dark as well as under the conditions of the rearing experiments to determine whether AHs affected the growth of C. neogracile (Yamasaki et al., 2016). In this experiment, the biomass of C. neogracile was not affected by AHs after a 24-h incubation (Yamasaki et al., 2016). As in this previous study (Yamasaki et al., 2016), we found in the present study that the biomass of Nannochloropsis sp. was not affected by AHs after a 24-h incubation (Fig. 5). Thus, it is not likely that AHs affected the biomass of Nannochloropsis sp. within 24 h under continuous dark conditions. Another possible explanation is that the growth-promoting effect of AHs results from the broad bioactivity of alginate, as shown in previous studies (Chaki et al., 2007; Iwasaki and Matsubara, 2000; Xu et al., 2003; Yokose et al., 2009). Jǿrgensen (1983) reported that uptake of DOM by mussels occurs through epidermal tissue located in the mantle and gills. On the other hand, Yamasaki et al. (2016) tried to detect glucose uptake in R. philippinarum using a fluorescent D-glucose (2NBDG), and found that much of the 2-NBDG was immediately taken up through the incurrent siphon and accumulated in the alimentary canal. These reports suggest that the clams immediately take up AHs dissolved in seawater, and thus the growth of R. philippinarum may be affected through the bioactivity of the alginate.
Acknowledgment This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 15K14796. References Bartoli, M., Nizzoli, D., Viaroli, P., Turolla, E., Castaldelli, G., Fano, E.A., Rossi, R., 2001. Impact of Tapes philippinarum farming on nutrient dynamics and benthic respiration in the Sacca di Goro. Hydrobiol. 455, 203–212. Benemann, J.R., 1992. Microalgae aquaculture feeds. J. Appl. Phycol. 4, 233–245. Borowitzka, M.A., 1997. Microalgae for aquaculture: opportunities and constraints. J. Appl. Phycol. 9, 393–401. Chaki, T., Kajimoto, N., Ogawa, H., Baba, T., Hiura, N., 2007. Metabolism and calcium antagonism of sodium alginate oligosaccharides. Biosci. Biotechnol. Biochem. 71, 1819–1825. Cheng, W., Liu, C., Kuo, C., Chen, J., 2005. Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeous vannamei and its resistance against Vibrio alginolyticus. Fish Shellfish Immunol. 18, 1–12. Dang, C., de Montaudouin, X., Gam, M., Paroissin, C., Bru, N., Caill-Milly, N., 2010. The Manila clam population in Arcachon Bay (SW France): can it be kept sustainable? J. Sea Res. 63, 108–118. Dong, B., Xue, Q., Li, J., 2000. The effect of temperature on the filtration rate, clearance rate and absorption efficiency of Manila clam, Ruditapes philippinarum. Mar. Fish. Res. 21, 37–42 (in Chinese with English abstract). Hamaguchi, M., Sasaki, M., Usuki, H., 2002. Prevalence of a Perkinsus protozoan in the clam Ruditapes philippinarum in Japan. Jpn. J. Benthol. 57, 168–176 (in Japanese with English abstract). Huttenhus, H., Ribeiro, A., Bowden, T., Bavel, C., Taverne-Thiela, A., Rombout, J., 2006. The effect of oral immuno-stimulation in juvenile carp (Cyprinus carpio L.). Fish Shellfish Immunol. 21, 261–271. Iwamoto, M., Kurachi, M., Nakashima, T., Kim, D., Yamaguchi, K., Oda, T., Iwamoto, Y., Muramatsu, T., 2005. Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells. FEBS Lett. 579, 4423–4429. Iwasaki, K., Matsubara, Y., 2000. Purification of alginate oligosaccharides with root growth-promoting activity toward lettuce. Biosci. Biotechnol. Biochem. 64, 1067–1070. Jǿrgensen, C.B., 1983. Patterns of uptake of dissolved amino acids in Mussels (Mytilus edulis). Mar. Biol. 73, 177–182. Matsukawa, Y., Cho, N., Katayama, S., Kamio, K., 2008. Factors responsible for the drastic catch decline of the Manila clam Ruditapes philippinarum in Japan. Nippon Suisan Gakk. 74, 137–143 (in Japanese with English abstract). Navarro, J.M., Winter, J.E., 1982. Ingestion rate, assimilation efficiency and energy balance in Mytilus chilensis in relation to body size and different algal concentrations. Mar. Biol. 67, 255–266. Nizzoli, D., Bartoli, M., Viaroli, P., 2006. Nitrogen and phosphorous budgets during a farming cycle of the Manila clam Ruditapes philippinarum: an in situ experiment. Aquaculture 261, 98–108. Paillard, C., 2004. A short review of brown ring disease, a vibriosis affecting clams, Ruditapes philippinarum and Ruditapes decussatus. Aquat. Living Resour. 17, 467–475. Park, K.-I., Figueras, A., Choi, K.-S., 2006. Application of enzyme-linked immunosorbent assay (ELISA) for the study of reproduction in the Manila clam Ruditapes philippinarum (Mollusca: bivalvia): II. Impacts of Perkinsus olseni on clam reproduction. Aquaculture 251, 182–191. Paul-Pont, I., de Montaudouin, X., Gonzalez, P., Soudant, P., Baudrimont, M., 2010. How life history contributes to stress response in the Manila clam Ruditapes philippinarum. Environ. Sci. Pollut. Res. 17, 987–998. Rico-Villa, B., Le Coz, J.R., Mingant, C., Robert, R., 2006. Influence of phytoplankton diet mixtures on microalgae consumption, larval development and settlement of the Pacific oyster Crassostrea gigas (Thunberg). Aquaculture 256, 377–388. Taga, S., Yamasaki, Y., Kishioka, M., 2013. Dietary effects of the red-tide raphidophyte Heterosigma akashiwo on growth of juvenile Manila clams, Ruditapes philippinarum. Plankton Benthos Res. 8, 102–105. Toba, M., Miyama, Y., 1993. Gross growth efficiency in juvenile Manila clam Ruditapes philippinarum fed different levels of Pavlova lutheri. Bull. Chiba Pref. Fish. Exp. Sta. 51, 29–36 (in Japanese with English abstract). Toba, M., Yamakawa, H., Shoji, N., Kobayashi, Y., 2013. Spatial distribution of Manila clam Ruditapes philippinarum larvae characterized through tidal-cycle observations at Banzu coast, Tokyo Bay, in summer. Nippon Suisan Gakk. 79, 355–371 (in Japanese with English abstract). Tsutsumi, H., 2006. Critical events in the Ariake Bay ecosystem clam population collapse, red tides, and hypoxic bottom water. Plankton Benthos Res. 1, 3–25. Uchida, M., Kanematsu, M., Miyoshi, T., 2010. Growth promotion of the juvenile clam,
5. Conclusions Our results demonstrate that AHs can enhance the dietary effect of 35
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of Chlamydomonas reinhardtii. J. Biosci. Bioeng. 113, 112–116. Yamasaki, Y., Taga, S., Kishioka, M., 2015. Preliminary observation of growth-promoting effects of alginate hydrolysates on juvenile Manila clams, Ruditapes philippinarum. Aquac. Res. 46, 1013–1017. Yamasaki, Y., Taga, S., Kishioka, M., Kawano, S., 2016. A metabolic profile in Ruditapes philippinarum associate with growth-promoting effects of alginate hydrolysates. Sci. Rep. 6, 29923. Yokose, T., Nishikawa, T., Yamamoto, Y., Yamasaki, Y., Yamaguchi, K., Oda, T., 2009. Growth-promoting effect of alginate oligosaccharides on a unicellular marine microalga, Nannochloropsis oculata. Biosci. Biotechnol. Biochem. 73, 450–453. Zhang, C.W., Zmora, O., Kopel, R., Richmond, A., 2001. An industrial-size flat plate glass reactor for mass production of Nannochloropsis sp. (Eustigmatophyceae). Aquaculture 195, 35–49.
Ruditapes philippinarum, on sugars supplemented to the rearing water. Aquaculture 302, 243–247. Welborn, J.R., Manahan, D.T., 1990. Direct measurements of sugar uptake from seawater into molluscan larvae. Mar. Ecol. Prog. Ser. 65, 233–239. Wickfors, G.H., Onho, M., 2001. Impact of algal research in aquaculture. J. Phycol. 37, 968–974. Winter, J.E., 1978. A review of the knowledge of suspension feeding in lamellibranchiate bivalves, with special reference to artificial aquaculture systems. Aquaculture 13, 1–33. Xu, X., Iwamoto, Y., Kitamura, Y., Oda, T., Muramatsu, T., 2003. Root growth-promoting activity of unsaturated oligomeric urinates from alginate on carrot and rice plants. Biosci. Biotechnol. Biochem. 67, 2022–2025. Yamasaki, Y., Yokose, T., Nishikawa, T., Kim, D., Jiang, Z., Yamaguchi, K., Oda, T., 2012. Effects of alginate oligosaccharide mixtures on the growth and fatty acid composition
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Enhancement of dietary effect of Nannochloropsis sp. on juvenile Ruditapes philippinarum clams by alginate hydrolysates
T
⁎
Yasuhiro Yamasakia, , Keita Ishiia, Shigeru Tagab, Masanobu Kishiokab a b
Department of Applied Aquabiology, National Fisheries University, 2-7-1 Nagata-Honmachi, Shimonoseki, Yamaguchi 759-6595, Japan Yamaguchi Prefectural Fisheries Research Center, 437-77, Aiofutajima, Yamaguchi, Yamaguchi 754-0893, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Alginate hydrolysates Algal diet Dietary enhancement Juvenile clam Ruditapes philippinarum
The eustigmatophyte Nannochloropsis sp. can be produced on a large scale at low cost. However, its dietary usefulness for juvenile bivalves is less than that of other algae. Recently, we reported that growth of juveniles of the clam Ruditapes philippinarum was dramatically promoted by supplementing a diet of the diatom Chaetoceros neogracile with alginate hydrolysates (AHs) at the concentration of 4 mg L−1. In this study, we examined the effect of AHs on Nannochloropsis sp. as a clam diet. Ten-day rearing experiments in 500-mL beakers showed that AHs have a beneficial effect on clam culture regardless of water temperature. Shell growth in clams given AHs at the concentration of 4 mg L−1 along with Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 was higher than in any other test groups at 15 or 25 °C. In 20-day rearing experiments in 30-L tanks, the average shell length in the groups given AHs at the concentration of 4 mg L−1 along with Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was significantly greater than that in the groups given C. neogracile and Nannochloropsis sp. alone (P ≪ 0.05). Furthermore, the total weight of clams given AHs at the concentration of 4 mg L−1 along with Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was greater than that in the group given only Nannochloropsis sp. (P ≪ 0.05). Hence, the enhanced dietary effect of a combination of Nannochloropsis sp. and AHs will help to shorten the rearing time of R. philippinarum and to provide a stable supply of algal diet.
1. Introduction The Manila clam Ruditapes philippinarum (Adams and Reeve, 1850) is not only a commercially important bivalve but also one of the most ecologically important bivalves in the world. However, the annual catch of this species in coastal waters of Japan, which once led the world, continues to decrease drastically. Several factors have been suggested as causes of the dramatic decrease (Hamaguchi et al., 2002; Matsukawa et al., 2008; Park et al., 2006; Toba et al., 2013; Tsutsumi, 2006) in addition to overfishing, although the precise cause is as yet unknown. On the other hand, there have been a wide variety of studies aimed at conserving the clam resource and developing clam culture (Bartoli et al., 2001; Dang et al., 2010; Nizzoli et al., 2006; Paillard, 2004; Paul-Pont et al., 2010). Thus, the development of a growth-promoting factor, the development of a mass-culture method for production of the clam, and clarification of its growth mechanisms would have important implications for clam culture, and would contribute to the conservation of the clam resource in the field and a stable market supply.
Jǿrgensen (1983) reported that clams take up dissolved organic matter (DOM) in seawater through epidermal tissue in the mantle and gills. Welborn and Manahan (1990) showed that larvae of the bivalve Crassostrea gigas (Pacific oyster) can take up dissolved glucose, maltose, cellobiose, and cellotriose, but not rhamnose or maltotriose. In addition, Uchida et al. (2010) reported that the growth rate of soft tissue in R. philippinarum was significantly promoted by supplementing a diet of the diatom Chaetoceros calcitrans with glucose. Thus, certain types of sugars are potentially a good supplement for R. philippinarum growth. Furthermore, Taga et al. (2013) reported the beneficial effects of the raphidophyte Heterosigma akashiwo, known as a harmful algal species, on the diet of juvenile R. philippinarum, and suggested that certain kinds of sugars, specifically the acidic sugars in phytoplankton, might be one of the important factors determining the growth of juvenile clams. Alginate is a natural acidic linear polysaccharide that is composed of α-L-guluronate and β-D-mannuronate (uronic acids) residues, and is also known as a type of dietary fiber. Several studies have reported that the dietary administration of alginate stimulates the immune abilities of white shrimp and juvenile carp (Cheng et al., 2005; Huttenhus et al.,
⁎
Corresponding author. E-mail addresses: yamasaky@fish-u.ac.jp (Y. Yamasaki), [email protected] (K. Ishii), [email protected] (S. Taga), [email protected] (M. Kishioka). https://doi.org/10.1016/j.aqrep.2017.11.006 Received 6 September 2017; Received in revised form 21 November 2017; Accepted 30 November 2017 2352-5134/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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2006), and the compositional ratio of α-L-guluronate and β-D-mannuronate or the degree of polymerization affects the physical properties and multiple biological activities of the alginate (Iwamoto et al., 2005; Iwasaki and Matsubara, 2000). In addition, alginate and its derivatives are currently used in a wide range of commercial enterprises, including the food, medical, and cosmetic industries. Recently, we showed that growth of R. philippinarum was dramatically promoted by supplementing a diet of the diatom Chaetoceros neogracile with alginate hydrolysates (AHs) at the concentration of 4 mg L−1 (Yamasaki et al., 2015, 2016), and metabolomics indicated that each of the states of starvation, food satiation, and sexual maturation of R. philippinarum has a characteristic pattern in the metabolite profile (Yamasaki et al., 2016). Although C. neogracile is widely used for clam culture as an algal diet, the cost of cultivating C. neogracile is far from cheap, and culture crashes of this species often occur during mass cultivation. The eustigmatophyte Nannochloropsis sp. is known to contain a high concentration of polyunsaturated fatty acids, especially eicosapentaenoic acid, and is used in aquaculture to feed a wide variety of marine organisms (Borowitzka, 1997; Rico-Villa et al., 2006; Wickfors and Onho, 2001). Moreover, the cost of producing Nannochloropsis biomass in outdoor flat-plate glass reactors (Zhang et al., 2001) is lower than in hatcheries (Benemann, 1992). Thus, Nannochloropsis sp. can be produced on a large scale at low cost. However, Nannochloropsis sp. has less dietary effect on juvenile bivalves compared with other algae diets. In this study, we examined the use of AHs to enhance the dietary effect of Nannochloropsis sp. on juvenile R. philippinarum.
The pH of the water was checked daily, and there were no significant pH changes in any of the rearing tests. In experiments 1 (25 °C) and 2 (15 °C), we investigated the optimum feeding concentration and water temperature for growth of juvenile clams. For each rearing test, 50 clams [initial average shell length: 0.790 ± 0.137 mm (25 °C), 1.129 ± 0.121 mm (15 °C)] were reared for 10 d in 0.5 L filtered seawater (i.e. unfed), a 0.5-L suspension of C. neogracile (8 × 104 cells mL−1), a 0.5-L suspension of Nannochloropsis sp. (20–80 × 104 cells mL−1), and a 0.5 L suspension of Nannochloropsis sp. (20–80 × 104 cells mL−1) with AHs added at the concentration of 4 mg L−1. There were three replicate beakers for each treatment. The rearing water along with any algal diet or AHs was exchanged daily (Table 1). In addition, all beakers were cleaned every 5 days to prevent bacterial growth and to ensure clam survival. After the rearing tests, all shell lengths were measured and the survival rates were determined. In experiment 3 (25 °C), we examined the enhancement of the dietary effect of Nannochloropsis sp. on juvenile clams by AHs at a larger scale and for a longer period. For each rearing test, 2000 clams (initial average shell length: 1.088 ± 0.244 mm) were reared for 20 d in a 20L suspension of C. neogracile (8 × 104 cells mL−1), a 20-L suspension of Nannochloropsis sp. (30 × 104 cells mL−1), and a 20-L suspension of Nannochloropsis sp. (30 × 104 cells mL−1) with AHs added at the concentration of 4 mg L−1. There were three replicate tanks for each treatment. The rearing water along with any algal diet or AHs was exchanged daily (Table 1). In addition, all tanks were cleaned every 10 days to prevent bacterial growth and to ensure clam survival. After the rearing test, the total wet weight includes the shells of all clams and the shell lengths of randomly selected individuals (approximately 10% of the total wet weight) from each tank were measured.
2. Materials and methods 2.1. Preparation of alginate hydrolysates
2.4. Growth of Nannochloropsis sp. with and without alginate hydrolysates
We prepared AHs using sodium alginate (SKAT-ULV, KIMICA Co., Tokyo, Japan) in accordance with previous studies (Yamasaki et al., 2012, 2015, 2016). Samples of AHs were stored at −30 °C until use. Yamasaki et al. (2012) reported that the primary oligomers in AHs were a monomer (176 Da), a dimer (352 Da), and a trimer (528 Da), with no significant quantities of larger oligomers.
To determine whether AHs affected the growth of Nannochloropsis sp., we conducted culture experiments in 0.5-L beakers containing 0.5 L of medium as follows. As a test group, a 0.5-L suspension of Nannochloropsis sp. (20 × 104 cells mL−1) with AHs at the concentration of 4 mg L−1 was incubated for 24 h. As a control, a 0.5-L suspension of Nannochloropsis sp. (20 × 104 cells mL−1) without added AHs was incubated for 24 h. There were three replicate beakers for each treatment, and aerated cultures were maintained at 25 °C in the dark. Cells were counted under a microscope in 1-mL subsamples collected 0 and 24 h after the start of the experiment. For all experiments, cells were counted five times for each sample.
2.2. Microalgal diet and growth conditions The microalgae Nannochloropsis sp. and C. neogracile were grown in 30-L tanks containing 20 L of filtered seawater enriched with KW21 marine alga growth medium (Daiichi Seimo Co. Ltd., Kumamoto, Japan). Aerated cultures were maintained at 20 °C under continuous light.
2.5. Statistical analysis 2.3. Clam rearing tests The data from the clam rearing experiments were analyzed by oneway analysis of variance followed by Tukey’s post hoc test. In addition, the data from the growth experiment with Nannochloropsis sp. with and without AHs were compared using a paired t-test. The analyses were performed using SPSS for Windows (SPSS version 19.0; SPSS, Inc., Chicago, IL, USA) using a significance level of P ≪ 0.05.
Rearing tests were conducted as shown in Table 1 in aerated, filtered natural seawater (pore size 1 μm) with mean salinity of 30–31‰. Table 1 Experimental conditions for each rearing test.
Average shell length (mm) Rearing density (individuals L−1) Rearing period (d) Mean water temperature (°C) Quantity of rearing water (L) Concentration of AHs (mg L−1) Feed dosage of C. neogracile ( ×104 cells mL−1 d−1) Feed dosage of Nannochloropsis sp. ( ×104 cells mL−1 d−1) Exchange frequency of rearing water
Experiment 1
Experiment 2
Experiment 3
0.79 ± 0.14 100 10 25 0.5 4 8
1.13 ± 0.12 100 10 15 0.5 4 8
1.09 ± 0.24 100 20 23–25 20 4 8
20–80
20–80
30
Everyday
Everyday
Everyday
3. Results 3.1. Dietary effects of Nannochloropsis sp. supplemented with AHs on growth of clams at 25 °C The survival of clams in all groups grown at 25 °C ranged from 91% to 100% (Fig. 1). Growth (as measured by final shell length) of the groups given Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 (with and without AHs) and at the concentration of 40 × 104 cells mL−1 (with AHs) and growth of the group given C. neogracile were virtually the same, whereas the groups given Nannochloropsis sp. at the concentration of 40 × 104 (without AHs) and at the concentration of 32
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Fig. 1. Effects of Nannochloropsis sp. in the presence or absence of AHs (4 mg L−1) on the growth, as measured by shell length, and survival of juvenile Manila clams, R. philippinarum, at 25 °C. Rearing tests were conducted as described in the text and Table 1. Data are means ± SD of the average shell length of clams in each test group (n = 3) after 10 days. Bars with different lowercase letters are significantly different at P ≪ 0.05 (oneway analysis of variance [ANOVA] followed by Tukey’s post hoc test).
Fig. 2. Effects of Nannochloropsis sp. in the presence or absence of AHs (4 mg L−1) on the growth, as measured by shell length, and survival of juvenile Manila clams, R. philippinarum, at 15 °C. Rearing tests were conducted as described in the text and Table 1. Data are means ± SD of the average shell length of clams in each test group (n = 3) after 10 days. Bars with different lowercase letters are significantly different at P ≪ 0.05 (oneway analysis of variance [ANOVA] followed by Tukey’s post hoc test).
80 × 104 cells mL−1 (with and without AHs) showed a significantly lower shell-length growth as compared with the group given C. neogracile (P ≪ 0.05, Fig. 1). Although supplementing the diet of Nannochloropsis sp. with AHs at the concentration of 4 mg L−1 tended to promote growth, there was no significant increase in growth as compared with the groups given C. neogracile (Fig. 1). The mean, median, minimum and maximum shell lengths for each test group are shown in Table 2. The mean and median for each test group were virtually the same except for the groups given Nannochloropsis sp. at the concentration of 40 × 104 (with AHs) and at the concentration of 80 × 104 cells mL−1 (with AHs). In addition, the minimum and maximum values in the groups with high growth tended to be higher than in the groups with low growth.
with the group given C. neogracile (P ≪ 0.05, Fig. 2). The mean, median, minimum and maximum shell lengths for each test group are shown in Table 2. The mean and median in each test group were virtually the same. In addition, there was no pronounced variation in minimum and maximum values between any test groups. 3.3. Dietary effects of Nannochloropsis sp. supplemented with AHs on growth of clams at a larger scale and for a longer period For the clams grown in larger cultures for a longer time, growth, as measured by shell-length, was dramatically promoted by supplementing a diet of Nannochloropsis sp. with AHs at the concentration of 4 mg L−1. The average shell length in the groups given AHs in addition to Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was significantly greater than that in the groups given C. neogracile and Nannochloropsis sp. (P ≪ 0.05, Fig. 3A). Also, the total wet weight of clams in the group given AHs in addition to Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 was greater than that in the group given only Nannochloropsis sp. (P ≪ 0.05, Fig. 3B), whereas there was no significant difference in total weight as compared with the group given C. neogracile (Fig. 3B). Length–frequency histograms of each group show the growth-promoting effect of AHs as a supplement to Nannochloropsis sp. on the clams (Fig. 4). The mode of shell length in the group given Nannochloropsis sp. was 1.6–1.8 mm (Fig. 4A) and in the group given AHs plus Nannochloropsis sp. was 2.0–2.2 mm (Fig. 4B). In addition, the mode shell length in the group given C. neogracile was 2.0–2.2 mm (Fig. 4C). Furthermore, the minimum shell lengths in the group given Nannochloropsis sp., the group given AHs plus Nannochloropsis sp., and the group given C. neogracile were 1.136, 1.227, and 1.111 mm,
3.2. Dietary effects of Nannochloropsis sp. supplemented with AHs on growth of clams at 15 °C The survival of clams in all groups grown at 15 °C ranged from 85% to 100% (Fig. 2). Growth as measured by shell-length increase was promoted by supplementing a diet of Nannochloropsis sp. with AHs at the concentration of 4 mg L−1. The average shell length in the groups given AHs in addition to Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 was significantly greater than in the groups given C. neogracile and Nannochloropsis sp. (P ≪ 0.05, Fig. 2). In addition, growth in the groups given Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 (without AHs), 40 × 104 cells mL−1 (with and without AHs), and 80 × 104 cells mL−1 (with AHs) and growth in the group given C. neogracile were virtually the same, whereas there was significantly lower growth in the groups given Nannochloropsis sp. at the concentration of 80 × 104 cells mL−1 (without AHs) compared Table 2 Mean, median, minimum and maximum values of shell length in each test group. Experimental group
Unfed C. neogracile (8 × 104 cells mL−1) Nannochloropsis sp. (20 × 104 cells Nannochloropsis sp. (40 × 104 cells Nannochloropsis sp. (80 × 104 cells Nannochloropsis sp. (20 × 104 cells Nannochloropsis sp. (40 × 104 cells Nannochloropsis sp. (80 × 104 cells
Experiment 1: Shell length (mm)
−1
mL ) mL−1) mL−1) mL−1) + AHs mL−1) + AHs mL−1) + AHs
Experiment 2: Shell length (mm)
Mean
Median
Minimum
Maximum
Mean
Median
Minimum
Maximum
0.952 1.439 1.394 1.284 1.196 1.465 1.374 1.246
0.967 1.402 1.355 1.246 1.170 1.435 1.297 1.192
0.514 0.719 0.503 0.536 0.634 0.696 0.667 0.569
1.488 2.525 2.472 2.280 2.069 2.405 2.545 2.166
1.158 1.312 1.299 1.291 1.238 1.396 1.310 1.317
1.139 1.316 1.297 1.279 1.226 1.379 1.306 1.306
0.827 0.912 0.959 1.017 0.922 1.031 0.873 1.048
1.450 1.613 1.742 1.637 1.653 1.764 1.604 1.746
33
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Fig. 4. Length–frequency histograms of juvenile Manila clams, R. philippinarum, (A) fed only Nannochloropsis sp., (B) fed Nannochloropsis sp. supplemented with AHs (4 mg L−1), and (C) fed only C. neogracile. Rearing tests were conducted as described in the text.
Fig. 3. Effects of Nannochloropsis sp. in the presence or absence of AHs (4 mg L−1) on (A) the growth, as measured by shell length, and (B) total wet weight of juvenile Manila clams, R. philippinarum, at 25 °C. Rearing tests were conducted as described in the text and Table 1. Data are means ± SD of the average shell length of clams in each test group (n = 3) after 20 days. Bars with different lowercase letters are significantly different at P ≪ 0.05 (one-way analysis of variance [ANOVA] followed by Tukey’s post hoc test).
respectively. In contrast, maximum shell lengths in the group given Nannochloropsis sp., the group given AHs plus Nannochloropsis sp., and the group given C. neogracile were 2.707, 3.806, and 3.617 mm, respectively. 3.4. Effect of AHs on the growth of Nannochloropsis sp. Culture experiments tested whether AHs affected the growth of Nannochloropsis sp. There were no significant differences in cell density of Nannochloropsis sp. cultures with or without AHs or between the densities at the start and at the end of the culture experiment (Fig. 5). Fig. 5. Cell density of Nannochloropsis sp. cultures in the presence or absence of AHs (4 mg L−1) after 0 and 24 h of culture. Data are means ± SD (cells mL−1) of triplicate measurements.
4. Discussion Our results show that shell length and total weight of clams were significantly promoted by supplementing a diet of Nannochloropsis sp. at the concentration of 30 × 104 cells mL−1 with AHs at the concentration of 4 mg L−1 as compared with the groups given Nannochloropsis sp. only (Figs. 3 and 4). The main benefit of our findings is that AHs can enhance the dietary effect of Nannochloropsis sp., which can be produced on a large scale at low cost. Although this species is a less sufficient diet for clams as compared with other algae, the enhanced benefits from AH supplementation may lead to a shorter rearing time for R. philippinarum and a more reliable supply of algal diet. The other useful outcome is that the effective amount of added AHs is lower than that previously reported for glucose (Uchida et al., 2010). The acidic polysaccharide alginate, known as a dietary fiber, has little stimulating effect on bacterial growth in the rearing water compared
with glucose, which is generally known as an energy source. Furthermore, the alginate used in this study is food-grade, the same as in our previous studies (Yamasaki et al., 2015, 2016), and its availability ensures a reliable supply of a safe diet for clams at low cost to the aquaculture industry. Thus, from a practical perspective, AHs can have a noteworthy effect on clam culture. Dong et al. (2000) examined the effect of temperature on filtration rate, clearance rate and absorption efficiency of R. philippinarum at 9, 16, 22, and 26 °C. They reported that all three metrics increased with a rise in temperature and reached maxima at 22 °C, but then decreased slightly at the highest temperature (Dong et al., 2000). Thus, growth of R. philippinarum decreases with a drop in temperature. Interestingly, although in our study clams reared at 25 °C grew faster than those 34
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Nannochloropsis sp. on juvenile R. philippinarum clams. The enhanced dietary effect of a combination of Nannochloropsis sp. and AHs will help to shorten the rearing time of R. philippinarum and to stabilize supplies of algal diet because Nannochloropsis sp. can be produced on a large scale at low cost. Further studies are needed to translate the utilization of AHs into practical applications in clam culture.
reared at 15 °C, the utilization of AHs was effective regardless of water temperature (Figs. 1 and 2). In experiment 1 (25 °C), the mean and median shell lengths in the groups given Nannochloropsis sp. at the concentration of 20 × 104 (with AHs) were the highest of all test groups (Table 1), although shell growth was not significantly higher than that in the groups given C. neogracile (P = 0.998, Fig. 1). In experiment 2 (15 °C), shell growth in the groups given AHs in addition to Nannochloropsis sp. at the concentration of 20 × 104 cells mL−1 was significantly higher than that in any other groups (P ≪ 0.05, Fig. 2), and the mean, median, and maximum shell lengths in this group were the highest of all test groups (Table 1). Therefore, supplementation with AHs would have a beneficial effect on clam culture not only during early summer but also in winter. Several studies reported that bivalves change filtration rate depending on the concentration of diet algae in order to increase assimilation efficiency (Navarro and Winter, 1982; Winter, 1978). Toba and Miyama (1993) reported that the filtration rate of juvenile R. philippinarum feeding on the haptophyte Pavlova lutheri was highest at the concentration of 5000 cells mL−1 and continuously declined with increasing algal density. In fact, our results showed that growth as measured by shell-length increase tended to decrease with increasing density of algal diet (Figs. 1 and 2, Table 2); that is, our results suggest a decrease in ingestion rate and/or assimilation efficiency with increases in algal density. In the present study, however, growth of clams in all groups given AHs along with Nannochloropsis sp. tended to be higher as compared with the groups given only Nannochloropsis sp. (Figs. 1 and 2, Table 2). It is unlikely that the AHs are used as a source of energy because a previous study showed that shell-length growth was significantly inhibited in R. philippinarum given AHs only (Yamasaki et al., 2015). Therefore, AHs likely affect shell-length growth of R. philippinarum not by acting as an energy source but rather by enhancing the dietary effect of Nannochloropsis sp. We have not yet identified the mechanisms that explain the dietary enhancement of AHs on Nannochloropsis sp. for clams. A likely explanation is that the growth-promoting effect of AHs results from their stimulating an increase in Nannochloropsis sp. biomass. For example, growth of the green alga Chlamydomonas reinhardtii was significantly promoted by an alginate oligomer mixture (AOM) prepared by enzymatic degradation (Yamasaki et al., 2012). However, growth of the alga was not affected by an AOM prepared by acid hydrolysis and other saccharides (Yamasaki et al., 2012). Furthermore, culture experiments were conducted in the dark as well as under the conditions of the rearing experiments to determine whether AHs affected the growth of C. neogracile (Yamasaki et al., 2016). In this experiment, the biomass of C. neogracile was not affected by AHs after a 24-h incubation (Yamasaki et al., 2016). As in this previous study (Yamasaki et al., 2016), we found in the present study that the biomass of Nannochloropsis sp. was not affected by AHs after a 24-h incubation (Fig. 5). Thus, it is not likely that AHs affected the biomass of Nannochloropsis sp. within 24 h under continuous dark conditions. Another possible explanation is that the growth-promoting effect of AHs results from the broad bioactivity of alginate, as shown in previous studies (Chaki et al., 2007; Iwasaki and Matsubara, 2000; Xu et al., 2003; Yokose et al., 2009). Jǿrgensen (1983) reported that uptake of DOM by mussels occurs through epidermal tissue located in the mantle and gills. On the other hand, Yamasaki et al. (2016) tried to detect glucose uptake in R. philippinarum using a fluorescent D-glucose (2NBDG), and found that much of the 2-NBDG was immediately taken up through the incurrent siphon and accumulated in the alimentary canal. These reports suggest that the clams immediately take up AHs dissolved in seawater, and thus the growth of R. philippinarum may be affected through the bioactivity of the alginate.
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