Effects of the brown algae Sargassum horneri and Saccharina japonica on survival, growth and resistance of small sea urchins Strongylocentrotus intermedius

Mass mortality of the long line culture of the sea urchin Strongylocentrotus intermedius in summer, which is greatly associated with their disease, energy storage and resistant abilities, is the most serious problem for the development of the aquaculture. Here, a feeding experiment was conducted for ~ 9 weeks to investigate the survival, growth and gonadal development of small S. intermedius (~ 3 cm) fed either brown algae Sargassum horneri or Saccharina japonica. Subsequently, we assessed their resistant abilities via observing the behaviors of righting, tube feet extension and Aristotle's lantern reflex at both moderately elevated and acutely changed water temperatures. Sea urchins fed S. horneri showed significantly fewer diseased individuals and slower gonadal development than those fed S. japonica. Consistently, significantly greater Aristotle's lantern reflex occurred in sea urchins fed S. horneri at moderately elevated temperatures. These findings suggest that S. horneri has direct application potential as food for the long line culture of S. intermedius in summer because of the advantage in health, energy storage (avoid the energy loss caused by gonadal development at small body sizes) and resistance abilities. In comparison, sea urchins fed S. japonica outperformed those fed S. horneri for all experimental behaviors under the acutely changed water temperatures. These findings clearly suggest that S. intermedius fed S. japonica is more suitable for the areas with cold water mass in summer, because it can effectively avoid or reduce the negative impacts of acute changes of water temperature on sea urchins. The present study provides valuable information into the management of the long line culture of S. intermedius in summer.

. Differences in the number of survivors and diseased sea urchins between the treatments in summer. Sargassum horneri and Saccharina japonica refer to the experimental and control groups, respectively. χ 2 and P were the statistical results of the Fisher′s exact test. A probability level of P < 0.05 was considered significant. Gonadal yield. Compared with the initial conditions, no significant differences were found in wet gonad weight (t = 0.197, P = 0.846 for S. intermedius fed S. horneri; t = 0.491, P = 0.631 for individuals fed S. japonica) and gonad index (t = 0.281, P = 0.783 for S. intermedius fed S. horneri; t = 0.435, P = 0.670 for individuals fed S. japonica) in either treatment. Significant differences were not detected in wet gonad weight (0.58 ± 0.45 g for S. intermedius fed S. horneri; 0.63 ± 0.35 g for individuals fed S. japonica; t = 0.195, P = 0.850) and gonad index (5.83 ± 1.90 for S. intermedius fed S. horneri; 7.17 ± 1.40 for individuals fed S. japonica; t = 0.564, P = 0.585) in either treatment.
Gonadal development. After ~ 9 weeks, all of the gonads of S. intermedius fed S. horneri were in the growing stage (stage II). However, 83.33% and 16.67% of individuals fed S. japonica were in the premature gonads (stage III) and growing stage (stage II), respectively.
In the gonads of S. intermedius fed S. horneri, the primary oocytes only attached to the follicular wall of ovaries ( Fig. 2A) and sperms only occurred in the follicular wall of testes (Fig. 2B). Regarding the gonads of sea urchins fed S. japonica at the same period, the oocytes were detached from the wall and gradually replaced nutritive phagocytes in the follicular cavity (Fig. 2C). Consistently, the basophilic clusters of sperm with a length of ~ 2 μm occurred in both the follicular wall and follicular cavity of testes (Fig. 2D).

Discussion
The increased incidence of bacteria causes black-mouth and spotting diseases greatly decreased the production of S. intermedius aquaculture 1,4,8 . We found that S. intermedius fed S. horneri showed significantly fewer morbidity than those fed S. japonica. A reasonable explanation is that polysaccharides enriched in S. horneri 30 stimulate the innate immune system of sea urchins because cell walls of pathogenic bacteria possess polysaccharides that are identified as characteristic antigen molecules by the innate immune system [31][32][33] . This indicates that S. japonica could be an effective approach to the disease prevention in S. intermedius. Strongylocentrotus intermedius necessarily requires at least one summer to develop from fertilized eggs to adults of commercial size (> 5 cm of test diameter) in long line culture in China 4,7 . It is not essential for small sea urchins to develop gametes, but more appropriate for somatic growth in aquaculture 34 . Gonadal precocity greatly consumes the stored energy and consequently leads to poor somatic growth 35,36 and probable mortality of sea urchins. Dietary protein is the basis of gonadal developments of sea urchins 35,37 . The present study showed that S. intermedius fed S. horneri exhibited slower gonadal development than those fed S. japonica, even though no significant difference of crude protein concentration were found between the two brown algae. This indicates that gonadal development of sea urchins is probably due to other nutrient elements. Collectively, the present study indicates that S. horneri is an effective food to avoid the precocious puberty of S. intermedius and may subsequently contribute to their energy storage.
In addition, cultured S. intermedius requires higher resistance ability at adverse water temperatures in summer. Behaviors, which are realized by the coordination of neuromuscular systems 38,39 , display a strong correlation with the fitness of sea urchins 40 . Water temperature significantly affects neuromuscular activities. For example, the sea urchin Strongylocentrotus purpuratus showed a decreased adhesion when being exposed to the elevated water temperatures 41 . For the first time, the present study found that different species of brown algae have different effects on sea urchin behaviors under acutely changed and moderately elevated temperatures. Specifically, sea urchins fed S. horneri showed significantly greater Aristotle's lantern reflex than those fed S. japonica when being exposed to moderately elevated temperatures (an increase from 23.5 to 26.5 °C at a rate of 0.5 °C per day and maintained at 26.5 °C for 1 week). The Aristotle's lantern reflex represents the ability to operate sea urchin jaws to grasp a food around and is commonly used as an indicator for the food intake capacity 29,42 . Consistently, S. intermedius fed S. horneri exhibited significantly higher food consumption than those fed S. japonica. The study indicates that S. intermedius fed S. horneri displays a significantly greater capacity in thermal tolerance than those fed S. japonica. In comparison, sea urchins fed S. japonica outperformed those fed S. horneri in behaviors of righting, tube feet extension and Aristotle's lantern reflex under acutely changed water temperatures. These www.nature.com/scientificreports/ findings clearly suggest that S. intermedius fed S. japonica is more suitable for the areas with cold water mass in summer because it probably reduces the negative impacts of acute changes in water temperature on sea urchins. A possible explanation is that S. japonica contributes to the reduction of the free-radical level in organisms 43 , decreases tissue hypoxia and subsequently improves the neuromuscular activities of sea urchins.
In conclusion, S. horneri has direct application potential for the long line culture of S. intermedius in summer, because of the advantages in health, energy storage and resistance abilities. Further, S. japonica is appropriate for S. intermedius aquaculture in the areas with cold water mass where acute changes of water temperature B,E) and Aristotle's lantern reflex (mean ± SD, N = 7 for experimental group and N = 10 for control group at moderately elevated temperatures; (C) mean ± SD, N = 10 for both groups; (F) in either treatment in respond to moderately elevated or acutely changed water temperatures. Sargassum horneri and Saccharina japonica refer to the experimental and control groups, respectively. Moderately elevated water temperatures refers to seawater temperature rose from 23.5 to 26.5 °C at a rate of 0.5 °C per day and maintained for 1 week. To simulate the changes of water temperature in Haiyang island near Dalian (39° 03′ N, 123° 09′ E) where water temperature frequently fluctuates from 22 to 16 °C instantly by the cold water mass, sea urchins were transferred quickly from 23.5 to 15 °C, maintaining at 15 °C for an hour and subsequently quickly returned to 23.5 °C for another hour to finish one cycle of acutely changed temperatures. After four cycles, these behaviors were observed. The asterisks *,** and *** mean P < 0.05 and P < 0.01 mean P < 0.001. Test diameter, wet body weight and wet gonad weight were evaluated for the initial conditions of sea urchins before the experiments started (N = 20 for test diameter and wet body weight; N = 10 for wet gonad weight).

Scientific RepoRtS
Crude protein, fiber, fat and ash of S. horneri and S. japonica. Samples were taken from each dried brown alga to investigate their organic composition (crude protein, crude fiber and crude fat) and ash on 20 August 2019 (N = 3). Semi-micro Kjeldahl nitrogen was used to determine the crude protein concentration of the dried brown algae 44 . In order to measure the crude fiber concentration of brown algae, about 10 g of each sample of the dried brown algae was boiled with a mixed solution (1.25% dilute acid and dilute alkali) for 30 min and ashed at 550 °C to remove the minerals 45 . Five grams of each dried sample and 15 mL petroleum ether were added to the Soxhlet extractor and refluxed at constant temperature (45 ± 1 °C) for eight hours to assess the crude fat concentration of the brown algae 46 . To investigate the ash concentration of the brown algae, approximate two g dried samples were placed in a constant weight fritted glass and burned in a muffle furnace (M110, Thermo CO., U.S) at 550 °C for 48 h 45 .

Experiment I. Experimental design.
Diet was the experimental factor, either S. horneri or S. japonica. Fresh S. horneri were collected from a farm in Huangnichuan Dalian (121° 45′ N, 38° 82′ E) and S. japonica from Dalian Bay (120° 37′ E 38° 56′ N) in July 2019. Individuals were fed dried S. horneri (experimental group) and dried S. japonica (control group) ad libitum for ~ 9 weeks during the experiment (from 23 July 2019 to 25 September 2019). One large fiberglass tank was used for each experimental treatment. One hundred sea urchins were haphazardly chosen and put into 100 individual cylindrical cages (length × width × height: 10 × 10 × 20 cm; 1.5 cm of mesh size) in each tank (length × width × height: 150 × 100 × 60 cm) of the recirculating system (Huixin Co., Dalian, China) with aeration, according to the experimental design. Diseased sea urchins were removed timely from the tanks to avoid the potential spread of infectious diseases in experimental treatments and were transported into new tanks (length × width × height: 75 × 45 × 35 cm) for individual culture and observation following with the previous management.
Water temperature was not controlled, ranging from 21.3 to 25.6 °C during the experiment. Water quality parameters were measured weekly as pH 7.59-7.85 and salinity 32.69-32.13. One-half of the seawater was renewed daily.
Number of survived and diseased sea urchins. Black-mouth disease refers to the perioral membrane turns black ( Fig. 4A) with the decreased ability of attaching and feeding in sea urchins 47 . Sea urchin with spotting disease is indicated by the spotting lesions with red, purple or blackish color on the body wall followed by the detachment of local spines 48 (Fig. 4B). The enlarging spotting lesions commonly cause ulceration on the body wall and finally result in death 8 . Sea urchin without disease performance is shown in Fig. 4C. The number of survived and diseased sea urchins (either black-mouth or spotting diseased) was recorded during the experiment.
Dried food consumption. The measurement of food consumption was conducted for six consecutive days (from 7 August 2019 to 12 August 2019). The total supplemented and remained diets were weighed (G & G Co., San Diego, USA) after removing the water on their surface. The samples of uneaten diets were collected, weighed and dried for 4 days at 80 °C and then reweighed (N = 5). To avoid the loss of uneaten food, a fine silk net (mesh size 260 μm) was set outside the cage to collect the fragments of uneaten brown algae 7 .
Dried food consumption was calculated as follows (according to Zhao et al. 49 with some revisions): F = dried food consumption (g), W 1 = wet weight of total supplement diets (g), W 2 = wet weight of total uneaten diets (g), B s = wet weight of sample supplemented diets (g), B u = dry weight of sample supplemented diets (g), C s = wet weight of sample uneaten diets (g), C u = dry weight of sample uneaten diets (g).
Growth. Test diameters, Aristotle's lantern length were measured using a digital vernier caliper (Mahr Co., Ruhr, Germany). Body, Aristotle's lantern and gut were weighted wet using an electric balance (G & G Co., San Diego, USA) on 25 September 2019 (N = 27 for test diameter and wet body weight; N = 6 for Aristotle's lantern length, wet weight of Aristotle's lantern and gut).   www.nature.com/scientificreports/ Subsequently, to investigate whether S. horneri and S. japonica contribute to the resistance abilities of small S. intermedius at moderately elevated temperatures, 40 individuals were haphazardly chosen from each treatment and placed into 40 cylindrical cages (5 × 10 × 10 cm) in each tank (length × width × height: 77.5 × 47.0 × 37.5 cm) of a temperature-controlled system (Huixin Co., Dalian, China) with aeration for both groups on 9 October 2019. They were subsequently exposed to the moderately elevated temperatures (rose from 23.5 to 26.5 °C at a rate of 0.5 °C per day and maintained at 26.5 °C for 1 week), according to the records of water temperature in Heishijiao sea area (~ 2 m water depth, 38° 51′ N, 121° 33′ E) in the summer of 2017 and 2018 (Fig. 5). Righting behavior, tube feet extension and Aristotle's lantern reflex were assessed on 23 October 2019.
Similarly, to explore the effects of S. horneri and S. japonica on the resistance abilities of small S. intermedius under acute changes of water temperature, another 40 individuals were randomly selected and placed into 40 cylindrical cages (5 × 10 × 10 cm) in each tank (length × width × height: 77.5 × 47.0 × 37.5 cm) of the temperaturecontrolled system (Huixin Co., Dalian, China) with aeration for both treatments on 9 October 2019. The water temperature was set at 23.5 °C. A tank of seawater was prepared at 15 °C. To simulate the changes of water temperature in Haiyang island near Dalian (39° 03′ N, 123° 09′ E) where water temperature frequently fluctuates from 22 to 16 °C instantly by the cold water mass 17 , sea urchins were transferred directly from 23.5 to 15 °C, maintained at 15 °C for an hour and subsequently quickly returned to 23.5 °C for another hour to finish one cycle of the acute change of water temperature. After four cycles, righting behavior, tube feet extension and Aristotle's lantern reflex of sea urchins were observed.
Righting behavior. Sea urchins were placed with the aboral side down on the bottom of an experimental tank (length × width × height: 60 × 40 × 16 cm, Fig. 4D). Righting response time is the time required for individuals in the inverted posture to right themselves with the aboral side up 22 . The righting response time in seconds was recorded during 10 min. If individuals did not right themselves within 10 min, the time was counted as 600 s (N = 15).
Tube feet extension. The method of assessing tube feet extension was established according to You et al. 27 , with some revisions. Sea urchins were maintained in a tank (length × width × height: 12 × 10 × 10 cm) with fresh seawater for ~ 5 min before the observation (N = 15). The subjective assessment of tube feet extension was evaluated by a well-trained team (5 persons) that was familiar with tube feet extension analysis of sea urchins. The ranking method was quantified based on the quantity and length of tube foot.