Materials
The benthic diatom Nitzschia sp. was provided by the Laboratory of Algae Biotechnology of Yantai University.
Seven kinds of macroalgae were selected as carriers for culture of benthic diatoms, including Ulva pertusa, degummed Laminaria japonica, Sargassum muticum, Chaetomorpha valida, Zostera marina, Sargassum fusiforme, and Sargassum thunbergii, which were commonly used as raw ingredients for sea cucumber feed. The dried macroalgae were provided by Qingdao Great Haidi Feed Co. Ltd, and were pulverized into ultra-fine pieces (< 75 μm), and then dried thoroughly at 80 °C.
Seawater was collected from a clean area of the Bohai Sea in the Yantai region and used after deposition treatment and filtration by grit and a 0.45-μm membrane. The treated seawater had a pH of 7.5–7.9 and salinity of approximately 32‰.
Commercial feed (formulated diets) for sea cucumber was purchased from Qingdao Great Haidi Feed Co. Ltd. and contained 15% protein, 2–5% crude fat, 10% coarse fiber, and 45% crude ash, which was used as a ference diet in sea cucumber feeding test.
Treatment of carriers
Three different methods were used to process the carriers:
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A.
0.1 g seaweed powder and 300 ml M Guillard's F/2 culture medium (M-f/2 substrate)with silicate were sterilized separately at 121 °C for 30 min, and were then mixed after cooling.
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B.
0.1 g seaweed powder was added to 300 ml M-f/2 substrate with silicate. The mixture was sterilized at 121 °C for 30 min and was used in diatom culture after cooling.
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C.
300 ml M-f/2 substrate with silicate was sterilized at 121 °C for 30 min and was then supplemented with 0.1 g dried macroalga powder after cooling.
The pH of the medium was adjusted to 7.5–7.9 before used in diatom culture.
Diatom cultivation
Culture medium (300 ml) was prepared according to the methods described above, and was added to 500-ml conical flasks. Diatom seed liquid were prepared in 500 mL sterilized flasks with M-f/2 substrate at 21–23 °C under illumination intensity of 3000 lx (42 μE m−2 s−1) for 24 h on end. When reaching at log phase, the seed liquid was inoculated with 10% (v/v) into the medium. The diatoms were cultured at 21–23 °C in an oscillating incubator under continuous shaking at 120 r/min for 24 h at continuously illumination intensity of 3000 lx.
Three parallel cultures were set for every experiment, and diatom cultures without microalgal carrier were used as control. Throughout culturing, the growth and attachment of diatoms were observed daily using regular microscopy, and culturing was ended when the diatoms began to detach from the carrier.
After cultivation, the culture solution was centrifuged at 8000 r/min for 20 min to obtain the harvest containing diatoms and carriers, which were then washed twice with deionized water. The wet weight, total nitrogen (TN), total phosphorus (TP), protein, and polysaccharide contents were determined, as well as the content of total N and total P in the water after centrifugation.
Sea cucumber feeding test
Juvenile Stichopus japonicus were provided by Oriental Ocean Technology Co. Ltd. All animals used in this experiment were of the same batch. The sea cucumbers were acclimated to laboratory conditions after placement in square concrete tanks (2 × 2 × 1.5 m) for at leat 15 days. The animals were cultured in the laboratory at room temperature (15–18 °C) until the start of the experiments and fed formulated diet every 2 days at a ratio of 3% of the animals’ total fresh weight (W/W). Individuals with adequate physical strength (good stretching ability and pointed parapodium) and similar wet weights (5.0 ± 2.0 g) were selected for feeding experiments, the selected sea cucumbers were randomly assigned to each experimental group.
The experiments were conducted in plastic aquariums (100 cm in diameter and 80 cm in height, with a depth of 60 cm water) that were sterilized with potassium permanganate (KMnO4) before the seawater was injected. A special frame with polyethylene corrugated plates was placed in each aquarium and was used for the attachment of sea cucumbers. The temperature and salinity of the water were controlled and ranged from 15–18 °C and 29–33‰, respectively. Sunshade nets were set above the aquariums in order to avoid the harm of direct sunlight to the sea cucumber, and every aquarium was continuously aerated through two air vents to maintain the dissolved oxygen value between 5.3 mg/L and 7.10 mg/L.
Four experimental feeding groups were set according to their diet: (1) diatoms cultured with Ulva carrier (group I), Ulva powder (group II), diatoms cultured without carrier (group III), and commercial feed (group IV). Three replicates were used for each treatment group, each containing 30 sea cucumbers.
Sea cucumbers were fed with feed (3% of animals’ total fresh weight, W/W) every morning and evening (9:00AM and 5:00PM). The feed was dispersed evenly into each tank after immersing with seawater. Residual feed was siphoned off, and 1/3 of the water was replaced every day to maintain water cleanliness. The frame was replaced every 15 days. The total N and P contents of the aquaculture water were measured every five days (before water replacement) throughout a 60-day feeding period. The ingestion status and mortalities of the animals were recorded daily.
Sample collection and measurement
After the feeding experiment, all of the sea cucumbers were subject to a starvation period of 48 h, and then all individuals were removed to a clean cystosepiment to stretch naturally. Their body length and body mass were measured, and the animals were dissected in order to collect their body wall, intestines, and coelomic fluid. The weight of the body wall and the weight and length of the intestines as well as their biochemical compositions were determined to calculate the weight gain rate, specific growth rate, feed efficiency, ratio of visceral weight to body wall weight, ratio of intestine weight to body wall weight, and ratio of intestine length to body length. After drying at 65 °C to a consistent weight, the biochemical composition of the body wall was also analyzed.
The activities of acid phosphatase (ACP), alkaline phosphatase (AKP), and superoxide dismutase (SOD) in the coelomic fluid were determined, and the intestinal tract was immediate deep frozen and preserved at − 80 °C until the activities of amylase (AMS), cellulase, and protease were determined.
Determination methods
The Kjeldahl method was applied to determine the total nitrogen (N) content in the algae harvest which was expressed in terms of the weight of nitrogen per gram of harvest (mg/g wt). Total phosphorus (P) in the havest was detected using ammonium molybdate spectrophotometric method after digested with H2SO4 and was expressed as the weight of phosphorus per gram of havest (mg/g wt).
The total nitrogen and total phosphorus contents in the water were measured according to the technical specifications in the manual “Criterion of Ocean Investigation” [17].
The increase of N and P (\(\Delta\)N and \(\Delta\)P, g) in the algal harvest were calculated as follows:
$$\Delta N = {TN_1} \times {m_1} - {\text{TN}}_2 \times {m_2}$$
$$\Delta P = {TP_1} \times {m_1} - {\text{TP}}_2 \times {m_2}$$
where TN1 and TP1 are the contents of total nitrogen and total phosphorus in the harvest after culture (mg/g wt), TN2 and TP2 are the total nitrogen and total phosphorus contents in the carrier materials (mg/g dry weight), m1 is the wet weight (g) of the havest obtained after culture, and m2 is the dry weight (g) of the seaweed carrier.
A Vario EL III Element Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany) was used to measure the nitrogen content in the body walls, and the protein content was calculated as N × 6.25. Fat concentration was determined using the Soxhlet extraction method, and ash content was assayed after heated for 10 h in an SX-2.5-10 N muffle furnace (Shanghai, China) at 400 °C.
Growth indices of sea cucumbers were calculated as follows:
The final weight: \(W_{f} (g) = \frac{{T_{w} }}{{N_{f} }}\), where Tw is the final total weight of each individual, and Nf is the number that survived at the end of the experiment.
Survival rate (SR): \(Sr (\% ) = \frac{{100 \times N_{f} }}{{N_{i} }}\), where Nf and Ni are the final and initial numbers of sea cucumbers, respectively.
$${\text{Weight}}\;{\text{ growth }}\;{\text{rate }}\left( {WGR} \right):WGR(\% ) = \, \frac{{W_{t} - W_{0} }}{{W_{0} }} \times 100$$
$${\text{Special growth rate }}\left( {SGR; \, \% {\text{ d}}^{{ - {1}}} } \right):SGR=\frac{{100 \times ({\text{In}}W_{t} - {\text{In}}W_{0} )}}{t}$$
$${\text{Feed efficiency }}\left( {FE} \right):FE(\% ) \, = 100 \times \frac{{W_{t} - W_{0} }}{F}$$
$${\text{Ratio of visceral weight to body wall weight }}\left( {VBR} \right):VBR(\% ) \, = \, \frac{{W_{v} }}{{W_{b} }} \times 100$$
$${\text{Ratio of intestine weight to body wall weight }}\left( {IBR} \right):IBR(\% ) \, = \, \frac{{W_{i} }}{{W_{b} }} \times 100$$
$${\text{Ratio of intestine length to body length }}\left( {IBL} \right):IBL(\% ) \, = \, \frac{{L_{i} }}{{L_{b} }} \times 100$$
where Wt and W0 are the average body wet weight at the end and beginning of the experiment, respectively, and t is the duration of the experiment. F is the quantity of feed. Wv, Wb and Wi are the wet weights of the viscera, body wall, and intestines, respectively. Li and Lb are the lengths of the intestines and bodies, respectively, of the sea cucumbers.
The activities of ACP, AKP, SOD, amylase, protease, and cellulase in coelomic fluid as well as protein contents were detected with corresponding kits purchased from the Nanjing Jiancheng Bioengineering Institute, these included an acid phosphatase assay kit, an alkaline phosphatase assay kit, a superoxide dismutase (SOD) assay kit, an amylase assay kit, a protease assay kit, and a cellulase (CL) test kit.
Statistical analyses
SPSS 17.0 software was used for all statistical analyses. One-way ANOVAs and T-tests were used for the comparative analyses. P values less than 0.05 were considered statistically significant.
