Fish Sci (2010) 76:267–273 DOI 10.1007/s12562-010-0214-x
ORIGINAL ARTICLE
Biology
Effect of salinity and temperature on salinity tolerance of the sea cucumber Apostichopus japonicus Meiyan Hu • Qi Li • Li Li
Received: 16 October 2009 / Accepted: 23 December 2009 / Published online: 4 February 2010 Ó The Japanese Society of Fisheries Science 2010
Abstract Sea cucumber Apostichopus japonicus juveniles acclimated to different environmental conditions (23, 25, and 27°C combined with 25, 30, and 35 psu) were assessed for tolerance to increasing and decreasing levels of salinity at a rate of 2 psu h-1. They were also tested for the LS50 (median lethal salinity) when transferred directly into a series of higher salinity (32–46 psu) and lower salinity (9–25 psu). The CSMax (critical salinity maximum), CSMin (critical salinity minimum), USTL (upper salinity tolerance limit), and LSTL (lower salinity tolerance limit) were positively correlated to the acclimated salinity level but negatively correlated to temperature. The CSMax of A. japonicus was 6.2–10.0 psu higher than the USTL, and the CSMin was 5.5–8.5 psu lower than the LSTL, indicating that gradual changes in salinity resulted in the wide range of salinity tolerance that was observed, but that abrupt changes in salinity resulted in the narrow range of tolerance. Two-way analysis of variance revealed that salinity and temperature had a significant effect on 50% CSMax, 50% CSMin, USTL, and LSTL (P \ 0.001). The information obtained in this study will be valuable for the further development of the sea cucumber aquaculture industry in China. Keywords Apostichopus japonicus Salinity Salinity tolerance Sea cucumber Temperature
M. Hu Q. Li (&) L. Li Fisheries College, Ocean University of China, 266003 Qingdao, China e-mail:
[email protected]
Introduction The sea cucumber Apostichopus japonicus (Selenka, 1867) (Echinodermata, Holothuroidea) is an epibenthic, temperate species whose natural habitat is the waters off the coast of Asia. A. japonicus has long been exploited as an important fishery resource in Russia, China, Japan, and North and South Korea [1]. The dried sea cucumber (beche-de-mer) is of high commercial value and in great demand from China where it is believed to have aphrodisiac and curative properties. The soaring price and the benefits obtained by farmers have stimulated the development of the sea cucumber industry of China, and since the 1980s, sea cucumber farming and ranching has become an economically important part of the aquaculture sector in northern China, including Liaoning and Shandong provinces [2]. The total area of sea cucumber farming reached 73,987 ha in China, and the total output was 77,517 tons in 2007 [3]. Salinity and temperature are two of the most important abiotic factors affecting the growth and survival of aquatic organisms. It is well known that the response to these environmental parameters is species specific and that salinity and temperature may interact to influence the growth and survival of aquaculture species. In mud crab Scylla serrata, temperature and salinity as well as the interaction of the two parameters were found to significantly affect the survival of zoeal larvae [4]. The salinity tolerance of juvenile abalone Haliotis diversicolor supertexta was observed to increase directly with the acclimated salinity but was inversely related to temperature [5]. Several aspects of the biology of A. japonicus have been studied, including reproduction, osmoregulation, and circadian rhythm [6–8]. However, there is a lack of knowledge on the combined effects of various variables and
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temperature on salinity tolerance in A. japonicus. It is important to evaluate the interactions of two or more variables on the functional responses of aquatic organisms if these variables are suspected/known to interact, since such studies provide information on the adaptative and physiological potential of organisms exposed to different environmental factors. In China, most sea cucumbers are cultured in shallow intertidal ponds. The salinity of the water in these pool may suddenly drop to 20 psu following rainstorms, which occur most frequently during the summer. Low salinity during the rainy season is therefore a common cause of high mortality among cultured A. japonicus. In addition, the daily surface water temperature can rapidly increase from 20 to 30°C during the low tidal period [7]. Therefore, the effects of salinity and temperature on the tolerance, survival, and growth of A. japonicus are of primary concern. It would be of particular interest to determine if increases in water temperature (e.g., during the summer period) in aquaculture ponds with rainwater influxes have a detrimental effect on the survival of juveniles. In the study reported here, we investigated the effects of different acclimation temperatures and salinity levels on the salinity tolerance of A. japonicus.
Fish Sci (2010) 76:267–273
25, and 27 ± 0.5°C) were reached. Target temperatures above ambient were maintained by 100-W heaters connected to a temperature regulator provided with a thermocouple. Once the chosen salinities and experimental temperatures were reached, the juvenile sea cucumbers remained in those conditions for 3 weeks. During the acclimation period, juveniles were fed ad libitum daily at 07:00 and 17:00 with a formulated feed made from mixture of commercial feed (Liuhe Marine Technology Co, Qingdao, China). The commercial feed contains powdered Sargassum spp., sea mud, polysaccharide vitamin, and mineral premixes. The seawater in the rearing tanks was exchanged completely each day in order to maintain the desired temperature and salinity. Each aquarium was continuously aerated by an air stone, and all aquaria were kept under a 12/12/-h light/dark photoperiod. Two studies were conducted: (1) a gradual change in salinity; (2) a sudden change in salinity. The LS50 (median lethal salinity) was calculated while the test salinity was gradually being changed and when the juveniles were transferred suddenly to higher or lower salinities [9]. The experiments were repeated three times, with different individuals used each time. Tolerance to a gradual change in salinity
Materials and methods Collection and maintenance of test animals Apostichopus japonicus juveniles (wet weight 0.46 ± 0.17 g; body length 3.21 ± 0.36 mm; n = 100) were collected from the Yongshun Hatchery in Rongcheng, Shandong Province, China. The individual wet weight measurements were taken 10 s after each sea cucumber had been removed from seawater, and external water was removed from the specimens by drying on sterile gauze. The individual body length measurements take as maximum extension while the specimens freely extended and contracted. A total of 2160 juveniles were randomly selected from the original stock, evenly distributed into nine 5-L plastic aquaria, and reared in filtered aerated seawater maintained at a salinity of 35 psu and room temperature (23°C). Based on water temperature and salinity values in June–August along the coast of Shandong Province, the sea cucumbers were acclimated in nine environmental regimes (combinations of salinity at 25, 30, and 35 psu and temperature at 23, 25, and 27°C). The salinity of the seawater was adjusted 1–2 psu day-1 with dechlorinated tap water (dilution) or with artificial sea salt (increase) until the test salinities (25, 30, and 35 ± 0.5 psu) were reached. The temperature was increased 0.5–1°C day-1 until the test temperatures (23,
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Twenty juveniles in each of the nine environmental conditions were used in our study on the effects of increases in salinity, and another 20 juveniles were used to study the effects of a salinity decrease. The salinity of the water in the different environmental conditions was increased or decreased at a rate of 2 psu h-1 as described in the previous section. Salinities were measured with a portable refractometer. A. japonicus juveniles that could not re-attach to the walls of the aquarium after being touched with a glass rod were defined as dead [5]. The salinity levels at which no juveniles survived was recorded as the CSMax (critical salinity maximum) or the CSMin (critical salinity minimum), when salinity was increased or decreased at a rate of 2 psu h-1. The salinity at which all juveniles survived was recorded as the SSMax (survival salinity maximum) or the SSMin (survival salinity minimum), when salinity was increased and decreased, respectively. The 50% CSMax and 50% CSMin (the salinity at which half of the juveniles survived when the salinity was increased or decreased) was calculated by probit analysis with SPSS ver. 13.0 (SPSS, Chicago, IL) software. Tolerance to abrupt change in salinity Twenty juveniles in each of the nine environmental conditions were used to study tolerance to higher salinity, and another 20 juveniles were used to study tolerance to lower
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Statistical analysis
salinity. Juveniles who had been acclimated to a specific environmental condition were transferred directly to a 3-L plastic circular aquarium containing water at a same temperature but higher salinity 32–46 psu or lower salinity 9–25 psu water baths (Table 1). The number of juveniles that died after 96 h was recorded. The 96-h LS50 (median lethal salinity) was taken as the upper or lower salinity at which 50% of the test juveniles survived after 96 h. The USTL (upper salinity tolerance limit), LSTL (lower salinity tolerance limit) and their 95% confidence limits were calculated by probit analysis with SPSS.
Table 1 Number of Apostichopus japonicus juveniles which died 96 h after being transferred to a series of different higher and lower salinity levels from a specific climate condition (temperature, salinity) to which they had been acclimated (n = 20 juveniles in each environmental condition)
Salinity (psu)
All data were subjected to one-way and two-way analysis of variance (ANOVA), and Tukey’s multiple comparisons test was then used to identify significant differences between treatments. All statistical significance tests were at the P \ 0.05 level. The relationships among 50% CSMax, 50% CSMin, USTL, LSTL, salinity, and temperature were tested using the generalized linear model procedure and regression procedure. All statistical analyses were conducted using SPSS software.
Temperature (°C) 23 Test salinity
25 No. died
Test salinity
27 No. died
Test salinity
No. died
Transferred to a series of higher salinity (psu) 25
30
35
32
0
32
2
32
2
34
0
34
2
34
6
36
12
36
16
36
17
38
16
38
20
38
20
40
20
40
20
40
20
34
0
34
2
34
4
36
4
36
6
36
10
38 40
10 14
38 40
12 20
38 40
15 20
42
20
42
20
42
20
38
0
38
0
38
0
40
5
40
6
40
8
42
7
42
8
42
14
44
10
44
14
44
18
46
20
46
20
46
20
Transferred to a series of lower salinity (psu) 25
30
35
20
4
20
0
20
0
17
10
17
8
17
0
14
18
14
16
14
6
11
20
11
20
11
20 20
9
20
9
20
9
23
0
23
0
23
0
20 17
6 14
20 17
4 8
20 17
2 4
14
20
14
20
14
18
11
20
11
20
11
20
25
0
25
0
25
0
23
2
23
2
23
2
20
8
20
8
20
2
17
16
17
12
17
6
14
20
14
20
14
20
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Results
Fish Sci (2010) 76:267–273
a 100
23°C
Tolerance to gradual change in salinity 25°C
Tolerance to abrupt change in salinity Table 1 shows the number of A. japonicus juveniles which died among those acclimated for 96 h to one of nine different environmental conditions and then transferred directly to a series of higher salinity or a series of lower salinity levels. The USTL of sea cucumbers acclimated in seawater of 25 and 35 psu at 23, 25, and 27°C was 35.7,
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27°C 60 40 20 0
7
11
15
19
8
12
16
20
0 11
15
19
23
23
27
31
35
39
43
b
Survival (%)
100 80 60 40 20 0
c
24
28
32
36
40
44
48
27
31
35
39
43
47
51
100 80
Survival (%)
All of the sea cucumbers acclimated in seawater of 25 psu at 23, 25, and 27°C and subjected to increasing salinity started to die at 35, 33, and 31 psu, respectively, while those acclimated in seawater of 35 psu at 23, 25, and 27°C started to die at 45, 43, and 41 psu, respectively (Fig. 1). When the salinity was decreased, sea cucumber acclimated in seawater of 25 psu at 23, 25, and 27°C started to die at 19, 17, and 15 psu, respectively, while those acclimated in seawater of 35 psu at 23, 25, and 27°C started to die at 25, 23, and 21 psu, respectively (Fig. 1). Both the SSMax and SSMin were positively correlated to salinity, but negatively correlated to temperature (Table 2). The 50% CSMax of sea cucumbers acclimated in seawater of 25, 30, and 35 psu at 23°C was significantly higher than that of sea cucumbers acclimated at the same salinity levels at 27°C (P \ 0.05). The 50% CSMax of individuals acclimated in seawater of 30 and 35 psu at 25°C was significantly higher than that of sea cucumbers acclimated at the same salinity levels at 27°C (P \ 0.05). The 50% CSMax of sea cucumbers increased significantly with increasing salinity at 23, 25, and 27°C (P \ 0.05). The 50% CSMin of sea cucumbers acclimated in seawater of 30 and 35 psu at 23°C was significantly higher than that of sea cucumbers acclimated at the same salinity levels at 27°C, and the 50% CSMin at 35 psu at 25°C was significantly higher than that at the same salinity levels at 27°C (P \ 0.05). However, in seawater of 25 psu, there was no significant difference in the 50% CSMin among individuals acclimated at the three temperatures (P [ 0.05). The 50% CSMin of sea cucumbers increased significantly with increasing salinity at 23, 25, and 27°C (P \ 0.05). The results from two-way ANOVA revealed that salinity and temperature each had a significant effect on the 50% CSMax and 50% CSMin (P \ 0.001). However, the effects of the interaction between salinity and temperature on 50% CSMax and 50% CSMin were not significant (P [ 0.05) (Table 3). The relationship among 50% CSMax, 50% CSMin, salinity (S) and temperature (T) is as follows: 50% CSMax = 32.635 ? 0.778S - 0.563T (R2 = 0.989); 50% CSMin = 9.168 ? 0.612S - 0.563T (R2 = 0.956).
Survival (%)
80
60 40 20
Salinity (psu) Fig. 1 Percentage survival of Apostichopus japonicus juveniles acclimated to different temperatures (23, 25, and 27°C, respectively) in seawater of 25 psu (a), 30 psu (b), and 35 psu (c) when subjected to increasing or decreasing salinity at a rate of 2 psu h-1
35.1, and 34.8 psu and 44.0, 42.7, and 40.6 psu, respectively. The LSTL of sea cucumbers acclimated in seawater of 25 and 35 psu at 23, 25, and 27°C was 17.0, 16.3, and 13.8 psu and 19.3, 18.5, and 16.1 psu, respectively (Table 4). Both the USTL and LSTL were positively correlated to salinity, but negatively correlated to temperature. The USTL of sea cucumbers acclimated in seawater of 30 and 35 psu at 23°C was significantly higher than that of sea cucumbers acclimated in seawater of the same salinity levels at 27°C (P \ 0.05). The USTL of sea cucumbers in seawater of 35 psu at 25°C was also significantly higher
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271
Table 2 CSMax, 50% CSMax, SSMax, CSMin, 50% CSMin, SSMin and their 95% confidence limits of A. japonicus juveniles maintained in seawater of 25, 30, and 35 psu at 20, 25, and 30°C Salinity (psu)
Temperature (°C)
CSMax (psu)
50% CSMax (psu)
SSMax (psu)
CSMin (psu)
50% CSMin (psu)
SSMin (psu)
25
23
43.0 (41.6, 44.4)
39.0 (38.4, 39.6)
35.0 (33.6, 36.4)
11.0 (9.6, 12.4)
11.5 (10.9, 12.1)
19.0 (17.6, 20.4)
25
43.0 (41.6, 44.4)
37.8 (37.2, 38.4)
33.0 (31.6, 34.4)
9.0 (7.6, 12.4)
10.8 (10.2, 11.4)
17.0 (15.6, 18.4)
27 23
41.0 (39.6, 42.4) 48.0 (46.6, 49.4)
37.5 (36.9, 38.1) 43.3 (42.7, 43.9)
31.0 (29.6, 32.4) 38.0 (36.6, 39.4)
7.0 (5.6, 8.4) 10.0 (8.6, 11.4)
10.5 (9.9, 11.1) 14.0 (13.4, 14.6)
15.0 (13.6, 16.4) 22.0 (20.6, 23.4)
25
46.0 (44.6, 47.4)
42.4 (41.8, 43.0)
36.0 (34.6, 37.4)
8.0 (6.6, 9.4)
13.3 (12.7, 13.9)
20.0 (18.6, 21.4)
27
44.0 (42.6, 45.4)
41.0 (40.4, 41.6)
34.0 (32.6, 35.4)
8.0 (6.6, 9.4)
12.0 (11.4, 12.6)
18.0 (16.6, 19.4)
23
51.0 (49.6, 52.4)
47.5 (46.9, 48.1)
45.0 (43.6, 46.4)
13.0 (11.6, 14.4)
18.0 (17.4, 18.6)
25.0 (23.6, 26.4)
25
49.0 (47.6, 50.4)
45.9 (45.3, 46.5)
43.0 (41.6, 44.4)
11.0 (9.6, 12.4)
17.0 (16.4, 17.6)
23.0 (21.6, 24.4)
27
47.0 (45.6, 48.4)
44.6 (44.0, 45.2)
41.0 (39.6, 42.4)
11.0 (9.6, 12.4)
15.0 (14.7, 16.0)
21.0 (19.6, 22.4)
30
35
CSMax, Critical salinity maximum; CSMin, critical salinity minimum; SSMax, survival salinity maximum; SSMin, survival salinity minimum; 50% CSMax, 50% CSMin, salinity at which half of the juveniles survived when the salinity was increased or decreased, respectively The 95% confidence limits are given in parenthesis
than that of sea cucumbers acclimated in the same salinity levels at 27°C (P \ 0.05). However, there was no significant difference in the USTL among individuals acclimated in seawater of 25 psu at the three temperatures (P [ 0.05). The USTL increased significantly with increasing salinity at 23 and 25°C (P \ 0.05), but at 27°C, the USTL of sea cucumbers in seawater of 35 psu was significantly higher than that in sea cucumbers in seawater of 25 and 30 psu (P \ 0.05). The LSTL of sea cucumber acclimated in seawater of 25, 30, and 35 psu at 23 and 25°C was significantly higher than that in sea cucumber at the same salinity levels at 27°C (P \ 0.05). However, there was no significant difference in the LSTL among individuals acclimated at 23 and 25°C at the three salinity levels (P [ 0.05). The LSTL of sea cucumbers in seawater of 35 psu at 23, 25, and 27°C was significantly higher than that in sea cucumbers at same temperature levels in seawater of 25 psu (P \ 0.05). The LSTL of sea cucumbers in seawater of 30 psu at 23 and 27°C was significantly higher than that of sea cucumbers at 25 psu at same temperature levels (P \ 0.05). Two-way ANOVA revealed that salinity and temperature had a significant effect on 50% USTL and LSTL (P \ 0.001). However, the effects of the interaction between salinity and temperature were not significant (P [ 0.05) (Table 3). The relationship among USTL, LSTL, salinity (S) and temperature (T) is as follows: USTL = 29.669 ? 0.723S - 0.525T (R2 = 0.918); LSTL = 29.222 ? 0.227S 0.767T (R2 = 0.945).
Discussion Salinity and temperature affect the physiological responses of aquatic organisms and partly determine the distribution
and survival of coastal organisms [10]. Temperature is a direct and controlling factor of an aquatic organism’s activity, while salinity is a indirect factor that modifies numerous physiological responses, such as metabolism, growth, life cycle, nutrition and intra- and interspecific relationships [11]. In China, sea cucumber culture ponds are usually shallow (2–3 m), making them susceptible to changes in environmental conditions. The water salinity in these ponds may drop to below 20 psu in the summer during the rainy reason and when typhoons strike, and the water temperature may jump to as high as 30°C. Therefore, a basic knowledge on the salinity tolerance of A. japonicus at different temperatures and salinities is important in terms of optimal aquacultural practices. In this study, the CSMax, CSMin, SSMax, and SSMin of A. japonicus juveniles were positively correlated to acclimated salinity, but negatively correlated to acclimated temperature. When the salinity of the seawater was raised or decreased at a rate of 2 psu h-1, A. japonicus juveniles were able to tolerate a salinity of 25–45 psu following acclimation to 35 psu at 23°C, and a salinity of 15–31 psu following acclimation to 25 psu at 27°C, respectively. These results indicate that the salinity tolerance level of our A. japonicus juvenile population depended on the salinity and temperature experienced by the juveniles during the acclimation process. Dong et al. [7] found that A. japonicus was an osmotic conformer and that the activity of Na?/K?-ATPase had an adaptive response to changes in ambient salinity. The increasing salinity tolerance seen in our study may be attributed to the osmoregulation ability of A. japonicus. Similar results have also been reported for the abalone H. diversicolor supertexta: when the salinity was raised or decreased, H. diversicolor supertexta juveniles that had been acclimated in seawater of 25 psu at 30°C survived a salinity of
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Fish Sci (2010) 76:267–273
Table 3 Two-way ANOVA table of 50%CSMax, 50%CSMin, USTL, and LSTL for A. japonicus juveniles maintained under different salinity (S) and temperature (T) conditions
Table 4 The USTL, LSTL and their 95% confidence limits for A. japonicus juveniles maintained under different salinity and temperature conditions
Source
Salinity (psu)
Temperature (°C)
USTL (psu)
LSTL (psu)
25
23
35.7 (35.0, 36.4)
17.0 (16.1, 17.9)
df
Sum of squares
Mean squares
F ratio
P value
50% CSMax Model
8
198.181
24.773
262.299
\0.001
25
35.1 (34.4, 35.8)
16.3 (15.4, 17.2)
T
2
15.188
7.594
80.406
\0.001
27
34.8 (34.1, 35.5)
13.8 (12.9, 14.7)
S
2
181.888
90.944
962.935
\0.001
23
38.0 (37.3, 38.7)
18.5 (17.6, 19.4)
T9S
4
1.106
0.276
2.926
25
37.3 (36.6, 38.0)
16.5 (15.7, 17.2)
Error
9
0.850
0.0944
27
36.0 (35.3, 36.7)
15.7 (14.9, 16.4)
Total
18
31833.540
Corrected total
17
199.031
Model
8
131.584
16.448
86.065
\0.001
T
2
15.288
7.644
39.997
\0.001
S
2
113.974
56.987
298.189
T9S
4
2.322
0.581
3.038
Error Total
9 18
1.720 3392.240
0.191
Corrected total
17
133.304
Model
8
174.604
21.826
121.253
\0.001
T
2
20.168
10.084
56.022
\0.001
S
2
152.788
76.394
424.410
T9S
4
1.649
0.412
2.290
[0.05
Error
9
1.620
0.180
Total
18
26381.460
Corrected total
17
176.224
Model
8
45.030
5.629
21.695
\0.001
T
2
25.903
12.952
49.921
\0.001
S
2
45.030
7.145
27.540
\0.001
T9S Error
4 9
4.837 2.335
1.209 0.259
4.661
[0.05
Total
18
5077.410
Corrected total
17
47.365
[0.05 35
50% CSMin
23
44.0 (43.3, 44.7)
19.3 (18.6, 20.1)
25
42.7 (42.0, 43.4)
18.5 (17.8, 19.2)
27
40.6 (39.9, 41.3)
16.1 (15.4, 16.8)
The 95% confidence limits are given in parenthesis
\0.001 [0.05
USTL
\0.001
LSTL
USTL Upper salinity tolerance limit, LSTL lower salinity tolerance limit, ANOVA analysis of variance
14–33 psu, and those acclimated at 35 psu at 20°C survived a salinity of 20–45 psu [5]. The USTL of A. japonicus was 6.2–10.0 psu lower than the values of CSMax, and the LSTL was 5.5-8.5 psu higher than the values of CSMin. A. japonicus juveniles that had been acclimated to 25 psu at 27°C exhibited a 50% CSMin as low as 10.5 psu when the salinity was gradually decreased, but they exhibited a LSTL as low as 13.8 psu when the salinity was suddenly decreased. This difference indicates that abrupt changes in salinity resulted in the narrow range of salinity tolerance and that gradual changes
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30
in salinity was better tolerated by the sea cucumber juveniles. These results agree with those from previous studies on salinity tolerance of tilapia [12], abalone [5], yellowfin sea bream [13], and crucifix crab [14], suggesting that when an animal is gradually acclimated to the experimental conditions, the tolerance range tends to be broader [15]. Our approach of examining sudden changes in temperatures and salinities provided the animals with no opportunity to acclimate physiologically to the new condition. Rainstorms in summer can also cause sudden salinity changes in shallow sea cucumber ponds. Therefore, the results obtained here provide useful information on the sea cucumber’ responses to abrupt salinity changes in the culture ponds in northern China. The two-way ANOVA revealed that temperature and salinity had a significant effect on the 50% CSMax, 50% CSMin, USTL and LSTL (P \ 0.001) of A. japonicus juveniles, indicating that the salinity tolerance was not only determined by salinity but also by temperature. Variations in salinity and temperature may change water quality parameters, such as dissolved oxygen and CO2 levels, NH3 to NH4? ratio, and pH [16], and the osmotic pressure of the coelomic fluid changes with changing ambient water parameters [7]. The salinity tolerance limit may be reached when the osmotic pressure exceeds the ability for osmoregulation. In conclusion, the salinity tolerance of the A. japonicus juveniles tested in our study depended on a prior acclimation to salinity and temperature and varied depending on whether the transfer was gradual or abrupt. A knowledge of the salinity requirements of a commercially valuable species, such as A. japonicus, is useful in terms of selecting the best locations for intensive aquaculture and adapting environmental conditions for better growth and a lower mortality.
Fish Sci (2010) 76:267–273 Acknowledgments We would like to express our appreciation to Mr. Hang Xu from Yongshun Hatchery in Rongcheng City for his assistance in the experiment. The research was funded by the grants from Scientific and Technical Supporting Program (2006BAD09A01) and Shandong Province (2009GG10005013).
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8.
9.
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