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

Sea urchins

Experimental sea urchins were produced in November 2018, fed Ulva pertusa ad libitum for ~ 2 months until the test diameter reached 0.3–0.4 cm diameter and subsequently fed S. japonica for culture^1,4. Three hundred healthy S. intermedius (~ 3 cm of test diameter) were randomly selected from an aquaculture farm in Huangnichuan, Dalian (121° 45′ N, 38° 82′ E) and then were transported to the Key Laboratory of Mariculture and Stock Enhancement in North China’s Sea, Ministry of Agriculture at Dalian Ocean University (121° 56′ N, 38° 87′ E) on 9 July 2019. Sea urchins were maintained in a large fiberglass tank (length × width × height: 180 × 100 × 80 cm) of the recirculating system (Huixin Co., Dalian, China) to acclimatize to laboratory conditions and fed S. japonica ad libitum for 1 week with aeration. Water quality parameters were measured daily. Water temperature was 23.55 ± 0.07 °C, pH 7.72 ± 0.02 and salinity 33.76 ± 0.04. They were then fasted for another week until the experiment began.

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).

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 algae⁴⁴. 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 minerals⁴⁵. 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 algae⁴⁶. 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 h⁴⁵.

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 urchins⁴⁷. 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 spines⁴⁸ (Fig. 4B). The enlarging spotting lesions commonly cause ulceration on the body wall and finally result in death⁸. 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 algae⁷.

Dried food consumption was calculated as follows (according to Zhao et al.⁴⁹ with some revisions):

F = dried food consumption (g), W₁ = wet weight of total supplement diets (g), W₂ = 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).

Specific growth rate (SGR) was calculated according to the following formula:

SGR = specific growth rate, P₂ = final wet body weight, P₁ = initial wet body weight, D = experimental duration.

Gonad yield

Gonads were carefully collected from each treatment and weighed using an electric balance (G & G Co., San Diego, USA) on 25 September 2019 (N = 6). Gonad index was calculated according to the following formula:

GI = gonad index, GW = wet gonad weight, BW = wet body weight.

Gonadal development

One of five pieces of each gonad was preserved in the Bouin’s solution (saturated picric acid solution: formaldehyde: glacial acetic acid = 15: 5: 1) for 48 h between the treatments (N = 6). Standard histology technique, including embedment, infiltration, section and stain, was performed to make the gonad tissue slices⁵⁰. Sections were classified according to the stage of development of germinal cells and nutritive phagocytes: stage I, recovering; stage II, growing; stage III, premature; stage IV, mature; stage V, partly spawned; stage VI, spent^51,52,53.

Experiment II

Experimental design

Experiment II lasted for 4 weeks (from 25 September 2019 to 23 October 2019). Eighty healthy sea urchins were haphazardly selected from each treatment at the end of experiment I. They were then distributed into 80 cylindrical cages (5 × 10 × 10 cm) in each fiberglass tanks (length × width × height: 77.5 × 47.0 × 37.5 cm) of the temperature-controlled system (Huixin Co., Dalian, China) with aeration in both treatments. Sea urchins were maintained at 23.5 °C for 2 weeks (the average water temperature of experiment I) to eliminate the past thermal history, following the previous diet strategy of experiment I. Water quality was recorded daily as pH 7.83–7.85 and salinity 32.44–32.62. One-third of the seawater was renewed daily.

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 temperature-controlled 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 mass¹⁷, 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 up²². 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.

Tube feet extension (rating 1–5):

• 1 = extremely abnormal (not extending)

• 2 = severe abnormality (extremely low quantity and extremely short length)

• 3 = moderate anomaly (low quantity and short length)

• 4 = mild abnormality (slight decrease in quantity and length)

• 5 = normal (normal quantity and length)

Aristotle’s lantern reflex

A simple device, which has two small compartments (length × width × height: 4.8 × 5.6 × 4.5 cm) with a food film on the bottom, was used to measure Aristotle’s lantern reflex according to our previous study²⁹. Food film was made by a mixture of ~ 2.5 g agar and 50 ml seawater in order to avoid the potential impacts of the food palatability on sea urchins. The number of Aristotle’s lantern reflex were counted within 5 min using a digital camera (Canon Co., Shenzhen, China) under the device (N = 7 for sea urchins fed S. horneri and N = 10 for individuals fed S. japonica under moderately elevated temperatures; N = 10 for both groups under acutely changed temperatures; Fig. 4E).

Statistical analysis

Normal distribution and homogeneity of variance of the data were analyzed using the Kolmogorov–Smirnov test and Levene test, respectively. The number of survived and diseased S. intermedius were compared using the Fisher′s exact test. Food consumption was analyzed using one-way repeated measured ANOVA. Kruskal–Wallis test was performed to compare the difference of righting behavior and tube feet extension between the treatments and also used to analyze Aristotle’s lantern reflex under acutely changed temperatures. Independent-samples t test was carried out to compare the difference between the final and initial conditions of sea urchins. Test diameter, wet body weight, SGR, Aristotle’s lantern length, wet Aristotle’s lantern weight, wet gut weight, gonad index and Aristotle’s lantern reflex (under moderately elevated temperatures) were analyzed using the independent-samples t 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