Sea urchins and experimental design
Seven hundred small S. intermedius (31.9 ± 0.4 mm of test diameter, mean ± SD) were chosen from an aquaculture farm in Changhai County, Dalian (122° 63′ N, 39° 25′ E) on 23 July 2020. They were subsequently transported to the Key Laboratory of Mariculture and Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs at Dalian Ocean University (121° 56′ N, 38° 87′ E) and maintained in a fiberglass tank (a closed culture system, length × width × height: 150 × 100 × 60 cm) with aeration for 7 days to acclimatize to laboratory conditions. The kelp Saccharina japonica, which is the most common food used for S. intermedius culture58, was fed ad libitum under the neutral photoperiod (12 h light:12 h dark). One-half of the seawater was changed daily. Water temperature, pH and salinity were 22.6 ± 0.2 °C, 7.7 ± 0.3 and 30.7 ± 0.1 ‰ (Mean ± SD) according to the daily measurement using a portable water quality monitor (YSI Incorporated, OH, USA), respectively.
The rearing space was defined as the ratio of culture volume to the number of sea urchins (cm3 ind−1). Rearing assemblage is the main factor being tested in this study. To simulate the currently used rearing assemblage in longline culture, 24 individuals were placed at plastic devices without layer divisions (length × width × height: 24.5 × 16.8 × 6 cm for culture volume; 25 holes of 0.5 cm diameter/100 cm2) as group A (the control group, 102.9 cm3 ind−1 of initial rearing space, Fig. 7a). To investigate whether multi-layer rearing assemblage improves the survival, food utilization and growth, 24 sea urchins were equally put into the cages where were evenly divided into three layers (8 sea urchins in each layer and length × width × height: 24.5 × 16.8 × 6 cm for each layer, 308.7 cm3 ind−1 of initial rearing space; 25 holes of 0.5 cm diameter/100 cm2; group B; Fig. 7b). Further, to evaluate whether eliminating interaction further contributes to the improvement of these commercially important traits of sea urchins in multi-layer rearing assemblage, 8 sea urchins were divided into eight divisions for each layer in the cages as group C (length × width × height: 8.3 × 5.9 × 6 cm for each division, 297.36 cm3 ind−1 of initial rearing space; 25 holes of 0.5 cm diameter/100 cm2; Fig. 7c). Each treatment had 8 replicates. All devices were placed in a fiberglass tank (length × width × height: 150 × 100 × 60 cm) and immersed in water for ~ 30 cm with aeration. They were easily disassembled for the experimental management.
Diagrams of the experimental cages used for the groups A (a), B (b) and C (c), the sea urchin with the spotting disease (d) and without the disease (e) and the devices used for measuring the Aristotle’s lantern reflex (f).
The experimental period was about ~ 7 weeks (from 31 July 2020 to 20 September 2020) under the neutral photoperiod (12 h light: 12 h dark). The kelp, which was regularly collected in the intertidal waters at Heishijiao, Dalian (121° 58′ E, 38° 87′ N), was daily provided to sea urchins in abundance for all the groups. The remained kelp, feces and dead sea urchins were removed daily. One-half of the seawater was replaced daily by the fresh and filtered seawater which was pumped from the coast of Heishijiao, Dalian. Water temperature was not controlled, ranging from 22.2 to 24.5 °C (the natural seasonal cycle of increasing temperature during summer in the region). Water quality parameters were measured weekly as salinity 29.3 ± 0.6 ‰, pH 7.8 ± 0.2 (mean ± SD) using a portable water quality monitor (YSI Incorporated, OH, USA).
To ensure the random sampling, sea urchins were taken out from the experimental device and placed in 24 plastic boxes (labeled from number 1 to number 24, length × width × height: 6 × 6 × 4 cm for each box). Individuals were chosen corresponding to the number (within 24) generated by the “sample” function in R studio (1.1.463). Sampling was re-conducted if the number corresponds to empty, dead or diseased sea urchins.
Mortality and morbidity
Spotting disease, which appears as spotting lesions with red, purple or blackish color on the test (Fig. 7d), is the most common lethal disease in S. intermedius aquaculture12. Sea urchin without disease is shown in Fig. 7e. Dead sea urchins were removed daily and the number of survivor and diseased sea urchins was recorded weekly for each cage during the experiment (N = 8).
Food consumption
The measurement of food consumption (g dry weight) was conducted once a week (24 h from Tuesday to Wednesday) (N = 8). The total supplied and remained diets were weighted wet by an electric balance (G & G Co., San Diego, USA) after the removal of the surface moisture. The dried weights of feces and samples of supplied and uneaten kelp were determined after 4 days at 80 °C in a convection oven (Yiheng Co., Shanghai, China).
Food consumption was calculated as follows (revised from Hu et al.9 for being more concise):
$${text{F}} = frac{{{text{A}}_{0} times frac{{{text{A}}_{1} }}{{{text{A}}_{2} }} – {text{B}}_{0} times frac{{{text{B}}_{1} }}{{{text{B}}_{2} }}}}{{text{N}}}$$
F = dry food intake per sea urchin (g ind−1 day−1), A0 = wet weight of total supplied diets (g), B0 = wet weight of total uneaten diets (g), A1 = dried weight of sample supplied diets (g), A2 = wet weight of sample supplied diets (g), B1 = dry weight of sample uneaten diets (g), B2 = wet weight of sample uneaten diets (g), N = the number of sea urchins.
Growth
Test diameter and lantern length were measured using a digital vernier caliper (Mahr Co., Ruhr, Germany). Body, lantern and gut were weighted wet using an electric balance (G & G Co., San Diego, USA). Test diameter and body weight were evaluated every Wednesday. The average value of the three individuals was considered as the trait value for each replicate (N = 8). Lantern length, wet lantern weight and wet gut weight were recorded in week 4 (29 August 2020) and week 7 (20 September 2020) (N = 8).
Aristotle’s lantern reflex
Aristotle’s lantern reflex, which refers to one cycle from the opening to the closing of the teeth59, was measured using a simple device according to the method of Ding et al.38. There were small compartments (length × width × height: 4.8 × 5.6 × 4.5 cm) with a film (made by 3 g agar and 2 g kelp powder) on the bottom of the device38 (Fig. 7f). The frequency of Aristotle’s lantern reflex was counted within 5 min using a digital camera (Canon Co., Shenzhen, China) under the device in week 4 (29 August 2020) and week 7 (20 September 2020). The average value of all the 5 individuals was considered as Aristotle’s lantern reflex for each replicate (N = 8).
5-HT concentration
The 5-HT is a signaling molecule, playing an important role in regulating feeding behavior52. To evaluate whether 5-HT is involved in Aristotle’s lantern reflex, 5-HT concentration of muscle in lantern was measured for each treatment in week 4 and week 7. 5-HT concentration was considered as the average value of all the 3 healthy individuals for each replicate (N = 8).
The concentration of 5-HT was measured using ELISA kits (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China) according to the instructions of the manufacturer. After adding the enzyme-labeled antibody, the substrate became a colored product that was directly related to the amount of the substance tested. The concentrations of 5-HT were calculated by comparing the optical density (O.D.) value of the samples to the standard curve and calculated according to the following formula (according to the kit’s instructions):
$${text{Y}} = frac{1}{{({text{a }} + {text{bx}}^{{text{c}}} )}}$$
Y = the concentration of 5-HT (ng mL−1), x = the O.D. value of the samples, a = 0.00027, b = 0.12086, c = 1.36806.
Pepsin activity
Pepsin is important for sea urchins to digest protein-rich algae40,60. Pepsin activity was analyzed using the pepsin kits (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China) in week 4 and week 7, following the instructions of the manufacturer. The average value of all the 3 individuals was considered as the pepsin activity for each replicate (N = 8). The procedures include enzyme reaction and color development reaction39. The temperature of reaction was 37 °C and pepsin activities were counted as U mg protein−1. The formula of pepsin activity is shown as follows (according to the kit’s instructions):
$${text{P}} = frac{{{text{M}}_{0} – {text{M}}_{1} }}{{{text{M}}_{2} – {text{M}}_{3} }} times frac{{{text{S}}_{0} }}{{{text{S}}_{1} }} times frac{{{text{V}}_{1} times {text{V}}_{2} }}{{{text{V}}_{3} }}$$
P = pepsin activity (U/mg prot), M0 = the O.D. value of the sample, M1 = the O.D. value of comparison, M2 = the standard O.D. value, M3 = blank O.D. value, S0 = the standard concentration (50 μg mL−1), S1 = reaction time (10 min), V1 = total volume of reaction solution (0.64 mL), V2 = sample protein concentration (0.04 mL), V3 = sampling volume (mg prot/mL).
Gut morphological examination
After sea urchins were dissected on week 4 and week 7, all gut tissue samples (~ 1 g for each sample) were fixed in Bouin’s solution (glacial acetic acid: formaldehyde: saturated picric acid solution = 1:5:15) according to the method of Wu et al.61. They were subsequently transferred for gradient dehydration, embedding, cutting, staining and observation62 (N = 24).
Statistical analysis
Kolmogorov–Smirnov test and Levene test were used to analyze the normal distribution and homogeneity of the data, respectively. Rearing assemblage was set as the main factor in the one-way ANOVA with three levels: the control system without layer divisions (group A), a second system with divisions in the cages to simulate the three layers cages (group B) and a third system with individual divisions for each sea urchin (group C). One-way ANOVA was used to analyze the mortality (in weeks 3, 4, 5, 6, 7), morbidity (in weeks 3, 6, 7), food consumption (in weeks 2, 5, 7), test diameter (in weeks 1, 2, 3, 4, 5, 6), body weight (in weeks 1, 4, 5, 7), 5-HT, pepsin activity, lantern length, lantern weight and gut weight. Duncan multiple comparison analysis was performed when significant differences were found in the one-way ANOVA. Kruskal–Wallis test was carried out to compare the differences of mortality (weeks 1, 2), morbidity (weeks 1, 2, 4, 5), food consumption (weeks 1, 3, 4, 6), test diameter (week 7), body weight (weeks 2, 3, 6) and Aristotle’s lantern reflex, because of non-normal distribution and/or heterogeneity of variance. A non-parametric post-hoc test was carried out when significant differences were found in the Kruskal–Wallis test. All data analyses were performed using SPSS 19.0 statistical software. A probability level of P < 0.05 was considered significant.
Source: Ecology - nature.com
