Figs. (1A and B) show one of the samples, Halocynthia roretzi, and the categories of the tunic specimens, respectively. The samples were obtained from Research Center for Marine Biology, Graduate School of Life Sciences, Tohoku University (Aomori, Japan) and Yamanaka Inc. (Miyagi, Japan). The tunic was removed from other organs with tweezers and trimming blades (Feather trimming blade; Feather Safety Razor, Co. Ltd., Osaka, Japan), and cut into appropriate sizes. The specimen was categorized into 4 groups as Figs. (1A and B) show: Siphon, tubular parts for influx and efflux of the seawater; M1, regions with spines; M2, regions with no spine; and Bottom, regions contacting the bottom of the muscle. The artificial seawater was made by Reef Crystals (Aquarium Systems, Sarrebourg, France) and deionized water.

All the samples were put into the artificial seawater at 5˚C in the refrigerator (MRP-215FS; Panasonic Healthcare Co., Ltd., Tokyo, Japan) up to 10 days. Each day of the period is named as Day 1 and so on. The increment in the mass of the tunic (Δm_r) and increment in the mass per day (Δm_r/day) were evaluated as the section ‘Mass and Moisture Content of the Tunic’ shows. The number of the specimens in each group was as follows: Siphon, 22; M1, 27; M2, 15; Bottom, 25; Inside (inner regions of M2 and Bottom), 7; Outside (outer regions of M2 and Bottom), 6. The measurement frequency was as follows: Siphon, every day (n = 14), every day or two days (n = 6) and every day or three days (n=2); M1, every day (n = 19), every day or two days (n = 4), every day or three days (n = 3) and every day or six days (n = 1); M2, every day (n = 7); every day or two days (n = 4), every day or three days (n = 3) and every day or six days (n = 1); Bottom, every day (n = 19), every day or two days (n = 3), every day or three days (n = 3) and every day or six days (n = 1); Inside, every day (n = 7); Outside, every day (n = 6).

Fig. (1) The sample of Halocynthia roretzi(A) and tunic specimens (B). The tunic specimens were categorized into 4 groups: Siphon, tubular parts for influx and efflux of the seawater; M1, regions with spines; M2, regions without spines; Bottom, regions contacting the base of the muscle.

The mechanical stimuli given to the tunic specimen at Day 1 were tapping the outer surface of the tunic specimen by tweezers for 3 minutes in the artificial seawater, at the temperature less than 10˚C, which was kept by ice packs. After the mechanical stimuli were given to the tunic specimen, the specimen and artificial seawater were at 5˚C in the refrigerator again, up to Day 7. The increment in the mass of the tunic (Δm_r) and increment in the mass per day (Δm_r/day) were evaluated as the section ‘Mass and Moisture Content of the Tunic’ shows. The number of the specimens was as follows: Siphon, 6; M1, 6; M2, 3; Bottom, 5. The frequency for measuring the mass was as follows: Siphon, every day (n = 4) and every day or five days (n = 2); M1, every day (n = 4) and every day or five days (n = 2); M2, every day (n = 2) and every day or five days (n = 1); Bottom, every day (n = 4) and every day or five days (n = 1).

The mass of each specimen was measured by the balance (UW420S; Shimadzu Corporation, Kyoto, Japan) after removing the water at the surface of the specimen by wrapping it in paper wipers for 10 seconds (Kimwipe; Nippon Paper Crecia, Tokyo, Japan). The moisture content of the specimen was measured when the drying temperature was 200˚C by the moisture analyzer (MX-50; A&D, Tokyo, Japan) after removing the water at the sample surface by the aforementioned method and cutting the specimen into pieces by the trimming blade. The moisture content described by Equation (4) was measured when the change in the moisture content was less than 0.05%/min or 0.01%/min.

The mass of the specimen, m, was normalized by that just after making the specimen, m_o, as follows:

(1)

where m_r is the normalized mass. Hence, the increment in the mass of the tunic, Δm_r, is described as follows:

(2)

The increment in the mass of the tunic per day, Δm_r/day, is as follows:

(3)

where t_i is the time for the i^th measurement and m_ri is the normalized mass at t_i. At giving the mechanical stimuli to the specimen, Δm_r/day was calculated as follows, for convenience:

(3’)

where m_rMS and m_rpre are the normalized mass at giving the mechanical stimuli and just before providing the stimuli at the same day, respectively. At the time of the measurement next to that at giving the stimuli, t_post, Δm_r/day at t_post was calculated as follows:

(3”)

where m_rpost is the mass at t_post, and t_MS is the time at giving the stimuli.

The moisture content of the sample, C, is described as follows:

(4)

where m_d is the mass of the dried specimen when the moisture content was measured. m_rw, the mass of the water in the specimen normalized by m_o, is described as follows:

(5)

Hence, the increment of m_rw, Δm_rw, is as follows:

(6)

The number of the specimens, which was used for evaluating Δm_rw, was 35, including 4 specimens just after receiving the mechanical stimuli at Day 2 (Tapping for 6 min at the temperature less than 10˚C), and 2 specimens 5 days after receiving the aforementioned mechanical stimuli.

The absorbance of the artificial seawater used for the tunic specimen was measured by the spectrophotometer (UV-1280; Shimadzu Corporation, Kyoto, Japan). The concentrations of nitrate and dissolved organic matter in the artificial seawater were evaluated by the absorbance at 220 nm [26Collos, Y.; Mornet, F.; Sciandra, A.; Waser, N.; Larson, A.; Harrison, P.J. An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures. J. Appl. Phycol., 1999, 11, 179-184.
[http://dx.doi.org/10.1023/A:1008046023487] ] and integrated absorbance from 250 - 350 nm [25Foster, P.; Morris, A.W. The use of ultra-violet absorption measurements for the estimation of organic pollution in inshore sea water. Water Res., 1971, 5, 19-27.
[http://dx.doi.org/10.1016/0043-1354(71)90059-5] ], respectively. The integrated absorbance, ΣA_250-350, was equal to the average of absorbance at 250 – 350 nm, Ᾱ_250-350, multiplied by the range of the wavelength, 100 nm. Hence, ΣA_250-350 is described as follows:

(7)

The absorbance at 250 – 350 nm was measured every 0.5 nm. Every absorbance was measured three times at least and averaged. Also, the artificial seawater without usage was evaluated in the same way. The number of the artificial seawater sample was as follows: without usage, n = 1; for the tunic specimen without the mechanical stimuli, n = 11; just after the mechanical stimuli were given to the tunic specimen at Day 2 (Tapping for 6 min at the temperature less than 10˚C), n = 2; 5 days after the aforementioned mechanical stimuli were given, n = 2. The tunic specimen in the artificial seawater was less than 0.2 g/ml while that was 0.1 – 0.35 g/ml when the mechanical stimuli were given.

The tissue structure of the tunic was evaluated by observing the stained specimen. The specimen was fixed in 20% formalin, which was diluted by the artificial seawater. After the fixation, the specimen was embedded in paraffin, sliced and stained by the following staining methods: Hematoxylin-Eosin stain, distribution of cells; Elastica-Masson stain, distribution of fibers; Periodic acid/Shiff reaction (PAS reaction), simple stain and Gram stain, distribution of bacteria. All the processes after the fixation were carried out by Surgical Pathology Japan, Inc. (Miyagi, Japan). The stained sample was observed by the light microscope (BX51; Olympus Corporation, Tokyo, Japan).

The difference in average between two groups and that between the periods in the same group were assessed by two sample t-test and paired t-test, respectively. In addition, the coefficient of determination, R², in the regression line was evaluated by F value which the following equation shows:

(8)

where n is the number of samples. F shows the F-distribution, whose df₁ and df₂, degrees of freedom, is 1, and n-1, respectively. The level of significance was 0.05.

Fig. (2) shows the increment in the mass, Δm_r, when the tunic was put into the artificial seawater at 5˚C, without the mechanical stimuli. While Fig. (2A) (Siphon) and Fig. (2B) (M1) show that Δm_r gradually increased as the period was longer, Fig. (2D) (Bottom) and Fig. (2E) (Inside) show the decrease in the mass. Fig. (2C) (M2) and Fig. (2F) (Outside) show that Δm_r increased as well as decreased. Fig. (3) shows the increment per day, Δm_r/day, in each group and division of the period, Day 1, Day 2-10 and Day 1-10, by mean ± SD (standard deviation). The period was divided into the three groups because the mechanical stimuli were given to the tunic at Day 1 in the other experiment. The result of the statistical analysis about Δm_r/day is mentioned in Tables 1 and 2. As Fig. (3A), Fig. (3B) and Table 1 show, Δm_r/day was significantly different between all the adjacent groups at Day 1-10: Siphon > M1; M1 > M2; M2 > Bottom; Outside > Inside. The adjacent groups except for the combination, Inside and Outside, were significantly different at Day 2-10 while the two combinations, M1 and M2, and, Inside and Outside, were at Day 1. According to these results, M1 > M2 was maintained through the period although other combinations were not. Fig. (3) and Table 2 mention that the absolute values of Δm_r/day in M1 and Inside were significantly decreased from Day 1 to Day 2-10. If the increment in the mass is caused by decreasing the stress in the tunic, compression of M1 and stretch of Inside at the initial state (Day 0) would be larger than those on other regions.

Table 1
Influence of the region on the increment in the mass per day (Δm_r/day).

Fig. (2) Increment in the mass of the tunic (Δmr) for the specimen without the mechanical stimuli. a, Siphon (n = 22); b, M1 (n = 27); c, M2 (n = 15); d, Bottom (n = 25); e, Inside (n = 7); f, Outside (n = 6).

Fig. (3) Increment in the mass of the tunic per day (Δmr/day) for the specimen without the mechanical stimuli. Δmr/day was evaluated at Day 1, Day 2–10, and Day 1–10 (mean ± standard deviation (SD)). A, Siphon (n = 22), M1 (n = 27), M2 (n = 15) and Bottom (n = 25); B, Inside (n = 7) and Outside (n = 6). The results of the statistical tests were described in Tables 1 and 2.

Table 2
Influence of the period on the increment in the mass per day (Δm_r/day).

Fig. (4) shows the increment in the mass, Δm_r, when the mechanical stimuli were given to the tunic at Day 1. As Fig. (4A) (Siphon), Fig. (4B) (M1), Fig. (4C) (M2) and Fig. (4D) (Bottom) indicate, Δm_r was decreased by the mechanical stimuli in each group while Δm_r after the mechanical stimuli was diverse. The increment in the mass per day, Δm_r/day, of each group is indicated by mean ± SD in Fig. (5), where the number of the samples in M2 was small (n = 3) so that the group named as M2 & Bottom, including the samples in both the groups, was organized. In addition, the period was divided into 3 groups: Pre, before giving the mechanical stimuli to the tunic specimen (corresponding to Day 1); MS, at giving the mechanical stimuli; Post, after giving the mechanical stimuli (corresponding to Day 2-10). Tables 1 and 2 show the results of the statistical analysis about Δm_r/day. According to Fig. (5) and Table 1, Δm_r/day in M1 was significantly larger than that in M2 & Bottom at Pre and Post while Δm_r/day in Spine and M1 did not show significant difference in all the divisions of the period. At MS, there was no significant difference between all the adjacent groups. As Fig. (5) and Table 2 show, all the combinations of the period divisions in Siphon and M1 indicated the significant differences in Δm_r/day: Pre > MS, Post > MS and Pre > Post. While Δm_r/day in M2 & Bottom at MS was also significantly smaller than those of Pre and Post, the difference between Δm_r/day in Pre and Post was not significant.

Fig. (4) Increment in the mass of the tunic which the mechanical stimuli were put on (Δmr). A, Siphon (n = 6); B, M1 (n = 6); C, M2 (n = 3); D, Bottom (n = 5). The dotted lines indicate Δmr when the mechanical stimuli were applied to the specimen just after the mass was measured at Day 1.

Fig. (5) Increment in the mass per day for the specimen with the mechanical stimuli (Δmr/day). Δmr/day was calculated in each group for the following three divisions of the period: Pre, before giving the mechanical stimuli to the specimen; MS, at giving the mechanical stimuli; Post, after giving the mechanical stimuli. The results of the statistical tests were described in Tables 1 and 2. Siphon, n = 6; M1, n = 6; M2 & Bottom, n = 8.

Because that Pre and Post are corresponding to Day 1 and Day 2-10 in the tunic specimens without the mechanical stimuli, respectively, Δm_r/day in these corresponding periods was compared. As Tables 1 and 2 show, the results of the statistical tests related to Δm_r/day of Siphons were changed by the mechanical stimuli. As Table 1 shows, Δm_r/day of Siphon and M1 were significantly different at Day 2-10, but those at Post were not. Also, Δm_r/day of Siphon at Day 1 was not significantly different from that at Day 2-10, but Δm_r/day at Pre was significantly larger than that at Post. according to Table 2. Fig. (6) shows the comparison between the corresponding divisions of the period in the same regions. Δm_r/day of Siphon at Post was significantly smaller than that at Day 2-10. These results indicate that the influence of the mechanical stimuli on Siphon would be larger than those on other regions.

The increment in the mass of water at the specimen, Δm_rw, and that in the mass of the specimen, Δm_r, are shown in Fig. (7). As the coefficient of determination, R², of the regression line in Fig. (7) show, Δm_rw was significantly proportional to Δm_r. Also, the gradient of the regression line was close to 1 so that Δm_r would approximate to Δm_rw. The results indicate that change in mass of the tunic would be caused by influx and efflux of water.

Fig. (8) shows the concentrations of nitrate and dissolved organic matter in the artificial seawater, which are corresponding to the absorbance at 220 nm, A₂₂₀, and the integrated absorbance from 250 nm to 350 nm, ΣA_250-350, respectively. The coefficient of determination, R², of the regression line for all the samples shows that the concentration of dissolved organic matter was significantly proportional to that of nitrate. Change in the concentrations of nitrate and dissolved organic matter was not clear just after the tunic was mechanically stimulated, but became clear 5 days after. This result indicates that the tunic would secrete the same substances whether or not the mechanical stimuli were given to it, but the mechanical stimuli could promote the secretion slowly.

Fig. (6) Increment in the mass per day (Δmr/day) before or after the mechanical stimuli were given. While Δmr/day in each group at Day 1 and Pre (A) did not show significant difference, those in Siphon at Day 2-10 and Post (B) were significantly different (p < 0.05).

The distribution of bacteria in the tunic at Day 5 was examined by Hematoxylin-Eosin stain (Fig. (9A)), PAS reaction (Fig. (9B)), simple stain (Fig. (9C)) and Gram stain (Figs. (9D and E)). Fig. (9A) shows that the tunic specimen had cells because cytoplasm (pink) and nuclei (purple) were observed. The distribution of the region positive for PAS reaction in Fig. (9B) is similar to that for cytoplasm and nuclei in Fig. (9A). Figs. (9C and D) show that the region positive for simple stain and Gram stain was not observed. However, the positive regions, which would be corresponding to not bacteria, but phagocytes, were found in the area contacting the muscle as shown in Fig. (9E). These characteristics were the same as those of the control group (Day 0) so that the propagation of bacteria would be prevented in the tunic specimen.

Fig. (7) Increments in the mass of the tunic (Δmr) and mass of water in the tunic (Δmrw). Δmr was significantly proportional to Δmrw (n = 35, p < 0.05). R2, coefficient of determination.

Fig. (8) Nitrate and dissolved organic matter in the artificial seawater used for the tunic specimen. The absorbance at 220 nm (A220) and the integrated absorbance at 250 – 350 nm (ΣA250-350) are corresponding to the concentrations of nitrate and dissolved organic matter, respectively. The following samples were evaluated: No stimulus, for the specimen without the mechanical stimuli (n = 11); MS, for the specimen when the mechanical stimuli were given to it (n = 2); Post, for the specimen after the mechanical stimuli were given (n = 2); ASW, not used for the tunic specimen (n = 1). The regression line for all the samples is indicated (p < 0.05).

Fig. (9) Bacteria in the tunic of Halocynthia roretzi at Day 5. The specimen was stained by Hematoxylin-Eosin stain (A), Periodic acid/Schiff reaction (PAS reaction) (B), simple stain (C) and Gram stain (D and E).

Fig. (10) shows the outside of the tunic in each region at Day 0 (control) and Day 2-10 (Siphon, Day 5; other regions, Day 10), made by Hematoxylin-Eosin stain. Siphon was divided into two groups: Inner tunic and Outer tunic because the tunic covers the inner and outer surfaces of the tubular muscle. Uneven surfaces were observed in every region at both the divisions of the period (Day 0 and Day 2-10). The characteristics of the layer positive for Hematoxylin-Eosin stain at the outermost surface were diverse in each group but kept at both Day 0 and Day 2-10. The layer at Inner tunic of Siphon was scarcely observed while that at Outer tunic was distributed at the protrusion. At M1, the thin layer at the protrusion and the cells at the dent were observed. The layer at Bottom was relatively thick and distributed entirely. M2 also had the layer, whose characteristics were between those of M1 and Bottom. Fig. (11) shows the characteristic patterns on the inside of the tunic: layers, almost parallel to the outside and winding in some cases, and a blood vessel. The pattern of layers was observed in every region while blood vessels were observed mainly in Bottom, but also in M1 and M2. The distribution of blood vessels agreed with the previous reports [2Berill, N.J. The tunicata, 1950, -4Das, S.M. On the structure and function of the ascidian test. J. Morphol., 1936, 59(3), 589-600.
[http://dx.doi.org/10.1002/jmor.1050590308] ]. These characteristics of the outside and inside tunic were also observed in the specimen receiving the mechanical stimuli at Day 2, whose mass was decreased just after the stimuli were given.

Fig. (10) Histological characteristics of the tunic outside at Day 0 and Day 2-10. All the specimens were made by Hematoxylin-Eosin stain. In Day 2-10, Siphon, including Inner tunic and Outer tunic, was at Day 5, and other regions were at Day 10.

Fig. (11) Histological characteristics of the tunic inside. The characteristic patterns observed in the tunic inside, layers and a blood vessel, are indicated.

According to Fig. 3 and Table 1, the increment in the mass of the tunic per day, Δm_r/day, was different in each region at Day 1-10 and Day 2-10: Siphon > M1; M1 > M2; M2 > Bottom. These characteristics would be reasonable if the layer positive for Hematoxylin-Eosin stain is hard so that it would limit the increase in mass of the inside, or if water hardly penetrates the layer so that influx and efflux of water in the tunic would be limited. Assuming that the blood vessels in the tunic could be collapsed by the change of the mechanical environment, it would be reasonable that Δm_r/day at Bottom and Inside were negative.