Seagrass Thalassia hemprichii shoots and sediment were collected at Bise (N 26042.548’, E 127052.740’) in Okinawa Island, Japan in May 2014. After being transported to the Sesoko Station, University of the Ryukyus, each seagrass shoot was carefully washed to remove epiphytes and sediment.
Eighty juvenile sea urchins Tripneustes gratilla of the same age were obtained from Okinawa Prefectural Sea Farming Center, Okinawa Island, Japan in February 2014. The sea urchins were transported to the Sesoko Station and cultured for 4 months in 4 tanks (157 L, n=20 per tank) continuously supplied with filtered seawater (2 L min-1) and fed with Undaria pinnatifida every four days and were used as stock sea urchins for the following experiment.
Two temperature conditions; ambient temperature and high temperature (+3 °C higher than ambient) and 2 pCO2 conditions; control (300-400 µatm) and high pCO2 (900-1,000 µatm) were selected as present and year 2100 conditions according to the IPCC RCP 8.5 scenario41 (Table 1). Ambient seawater temperature fluctuated following field seawater by using flowing seawater pumped from 4-5 m depth in the front of the station. High temperature condition was controlled using heaters to be always 3 °C higher than the control. Seawater pCO2 was adjusted by bubbling seawater with air (control) or with a mixture of air and pure CO2 gas (high pCO2) controlled by mass flow controllers (Horiba Stec, SEC-E40, Japan). Both seagrass and sea urchins were acclimated for 40 days under the 2 temperatures and 2 seawater pCO2 full factorial design giving 4 experimental conditions before starting the measurements.
Just after collection, the T. hemprichii were cut into one apical shoot with two rhizome internodes and roots, and 48 shoots were planted in each of 24 aquaria (12 L) containing 5 cm sediment thickness to mimic the density of T. hemprichii at the Bise site. Six aquaria were used as replicates for each of the 4 experimental conditions. The 4 experimental seawater conditions were continuously supplied (0.5 L min-1) to each of the 6 aquaria, and T. hemprichii were cultured for 40 days under natural sunlight until conducting the following measurements.
For the sea urchin, 40 individuals (3-4 cm diameter) were randomly selected from the stock and put individually in 40 containers (900 ml) with a mesh cage cylindrical lining inside each container. Replicate 10 containers received the 4 experimental seawater conditions (0.1 L min-1) and T. gratilla were cultured for 40 days in the laboratory under 12:12 h photoperiod artificial light (100 µmol photons m-2 s-1) controlled by 2 metal-halide lamps (W039-006P, Iwasaki, Japan). Sea urchins were fed with Undaria pinnatifida during the acclimation about once every 4 days.
During the seagrass and sea urchin culture, seawater pH (NBS scale), temperature, and salinity of each aquarium and containers were measured (14:00-15:00 h) using a multiparameter portable meter (WTW Multi 3420, Germany) connected with a temperature-compensated pH electrode (SenTix 940) and conductivity electrode (TetraCon 925). For total alkalinity (TA), seawater samples were taken every 2-3 days and measured using an autoburrete titrator (Kimoto, ATT-05, Japan). Seawater pCO2 and Ωaragonite were calculated based on pH, temperature, salinity, and TA data using CO2SYS ver. 2.1 program40 with K1 and K2 dissociation constants from Mehrbach recalculated by Dickson and Millero41 (Table 1).
Seagrass leaf growth.
The leaf growth of T. hemprichii was measured by the leaf plastochrone interval (PL) method42. After all the following sea urchin feeding experiments were finished, one apical seagrass shoot was chosen randomly from each of the 24 aquaria and punched using a needle at 1 cm from the lower part of the bundle sheath. The punched shoots were replanted into the aquarium and cultured for a further 14 days under the 4 experimental conditions. Thereafter, all 24 punched seagrass shoots were recollected, and PL was calculated by dividing the number of days since marking (14 days) with the number of new leaves (unmarked leaves higher than the punch mark). Leaf growth (mg dry wt shoot-1 day-1) was calculated by dividing the dry weight measured using an electronic balance (HR-200, A&D, Japan) of the youngest mature leaf (the third leaf) dried (60 °C) for 7 days by the leaf PL.
Seagrass photo-physiological responses.
The photo-physiological responses of seagrass were measured using pulse amplitude modulated (PAM) fluorometry (Diving PAM, Walz, Germany) after the 40 days of being cultured. One apical shoot per aquarium was chosen randomly and placed in a clear container (8 L) with seawater equilibrated to the experimental condition it was previously reared at. After 15 min dark adaptation, saturation pulse (0.8 s) was applied to determine the maximum dark-adapted quantum yield of ΦPSII (Fv/Fm) measured at the third fully developed leaf. Rapid light curve (RLC) was generated from relative electron transport rate (rETR) using 8 consecutive light levels of 155, 312, 488, 724, 992, 1406, 1926, and 2922 µmol photons m-2 s-1 applied every 10 s intervals. Derived RLC photosynthetic parameters including α (photosynthetic efficiency; the initial slope of the RLC before the saturation occurred), β (slope of the RLC when the photoinhibition occurred), maximum relative electron transport rate (rETRmax), and Ek (minimum saturating irradiance) were calculated according to Platt et al.43, fitted using the Port method in the R Phytotools package44.
Seagrass carbon and nitrogen content.
Two shoots of seagrass that were not used for the above experiments were taken from each aquarium after the 40 days of culture. Epiphytes were scraped off of the seagrass leaves, and then they were divided into the above-ground part (leaves) and below-ground part (rhizomes and roots). Thereafter, all samples were dried (60 °C) for 7 days and the above- and below-ground parts of each of the two shoots were ground with a mortar and pestle into a homogenized fine powder. Ten mg of powder was weighed using an electronic balance (HR-202i, Japan) from each sample, and the carbon and nitrogen were measured the using CN analyzer (Sumigraph NC-22A, Japan).
Sea urchin feeding and fecal production rate.
To evaluate the sea urchins and seagrass interactive effects, feeding and fecal production rate of the sea urchins fed with the 2 seagrass treatments (experimental and control seagrass) were measured. All sea urchins were starved for 5 days after 35 days acclimation under the 4 experimental conditions. After starvation and taking all feces from each container, sea urchins in each of the 4 experimental conditions were fed with seagrass leaves that were cultured for 40 days under the same conditions as the sea urchins were cultured (experimental seagrass). Seagrass leaves (3.5 g, blot dried) were added to each container with the sea urchins. After 2 days, all the remnant leaves were collected, blotted dry, and weighed to calculate the feeding rate (g leaves ind-1 day-1). Additionally, all feces were collected from each container by filtering the seawater using pre-combusted (550 °C, 4 h) and pre-weighed fiberglass filter (Whatman GF/C). After removing all small remnant leaves using tweezers, each filter was dried at 60 °C until constant weight. The fecal production rate was calculated by subtracting the weight of filter containing feces with the filter weight (g dry feces ind-1 day-1). Additionally, to evaluate the carbon and nitrogen absorption efficiency, the dried feces were ground into a powder, and ten mg samples were weighed and fecal carbon and nitrogen content were measured with CN analyzer (Sumigraph NC-22A, Japan). Absorption efficiencies of carbon and nitrogen by sea urchin were calculated by the following formula:
After the feeding experiment of experimental seagrass and the following respiration and ammonium excretion measurements detailed below, the same sea urchins were starved again for another 5 days. Thereafter all sea urchins were fed with the seagrass leaves cultured under the control condition (control seagrass). Two days later, the same procedure as above was repeated to measure the feeding and fecal production rate.
Sea urchin respiration and ammonium (NH4+) excretion rate.
Respiration and ammonium (NH4+) excretion rates of the sea urchins were measured just after the experimental and control seagrass feeding experiments, respectively. The next day after the feeding experiment, sea urchins were placed individually in 450 ml glass containers with a magnetic stirrer. After 24 h acclimation in continuously flowing experimental seawater, each glass container was closed tightly without headspace, and oxygen concentrations were measured 3 times at 0, 30, 60 min using FIBOX fiberoptic oxygen meter (Presens GmbH, Germany). Sea urchin respiration rate (µmol O2 L-1 h-1 g-1) was calculated by dividing the oxygen concentration change with seawater volume, incubation time, and wet weight (HR-200, A&D, Japan) of the sea urchin.
Concurrently with the respiration measurement, the ammonium (NH4+) excretion rate was measured by sampling seawater (1 ml) just before closing and just after opening each glass container containing sea urchins. Working reagent (250 µl) which consisted of borate buffer, sodium sulfite, and orthophthaldialdehyde (OPA) solution was added to each sample and incubated (2h) in the dark (following Holmes et al.45). The NH4+ amount was measured colorimetrically (360 nm, UV-1800, Shimadzu, Japan), and the ammonium excretion rate (nmol NH4+ h-1 g-1) was calculated from the change of NH4+ concentration between the end and initial concentration, divided by seawater volume and wet weight of the sea urchin. After the ammonium excretion measurement, the sea urchins were starved to conduct the control seagrass feeding experiment, and then the same procedure was conducted again.
All statistical analyses were calculated using R-Studio version 1.3.95946. All the data were checked for normality with the Shapiro-Wilk test and homogeneity of variances with the Levene’s test. Seagrass leaf growth, photo-physiological parameters, carbon and nitrogen content, and leaf C:N ratio were analyzed using two-way ANOVA with pCO2 and temperature as fixed factors. Data were transformed to meet assumptions of normality such as Fv/Fm (x^4 transformed), β (square-root(x) transformed), and Ek (log10(x) transformed). Sea urchin fecal production, respiration (square-root(x) transformed), and ammonium (NH4+) excretion rate were analyzed using three-way ANOVA with pCO2, temperature, and leaf sources as fixed factors. Data were further analyzed using Tukey’s HSD post-hoc test when the result of ANOVA test showed a significant interaction between the factors.
Data of seagrass leaf plastochrone interval (PL) and sea urchin feeding rate was analyzed using Generalized Linear Model (GLM). Inverse Gaussian was used to analyzed PL with pCO2, temperature and their interaction were used as model variables. Quasi-Poisson was used to analyze sea urchin feeding rate with pCO2, temperature, leaf sources, and their interactions were used as model variables. When the interaction between independent variables was found, multiple comparisons were applied using the multcomp package47.