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Effects of salinity, temperature and pH on the survival of the nemertean Procephalothrix simulus Iwata, 1952
Journal of Experimental Marine Biology and Ecology 328 (2006) 168 – 176 www.elsevier.com/locate/jembe
Effects of salinity, temperature and pH on the survival of the nemertean Procephalothrix simulus Iwata, 1952 Zhao Yanfang, Sun Shichun * Mariculture Research Lab, Ocean University of China, 5 Yushan Road, Qingdao 266003, China Received 31 March 2005; received in revised form 20 June 2005; accepted 13 July 2005
Abstract The salinity, temperature and pH tolerance of Procephalothrix simulus Iwata, 1952, were experimentally studied. In hypomedia, the nemerteans could survive 96 h in 3.3x solution at 10 8C (median lethal salinity [LS50] was not determined at this temperature), and 96 h LS50 were 7.3x and 13.5x at 20 8C and 30 8C, respectively. In hyper-media, 96 h LS50 values were 53.9x, 47.1x and 41.4x at 10 8C, 20 8C and 30 8C, respectively. The trend of body weight changes in diluted media indicated that this nemertean is a volume regulator. During a 96-h exposure in media at 0 8C, worms were thanatoid but could recover if the temperature was gradually elevated to 20 8C. In thermal tolerance experiments, the nemertean survived 96 h in seawater of 30 8C, and worms suffered high mortalities when the temperature exceeded 32 8C. Present results suggest that the interaction of temperature and salinity on the lethal effects on P. simulus is significant ( P b 0.05). Elevated temperature (range 10–30 8C) decreased the worm’s solute tolerance, and elevated salinity (range 18–38x) decreased the worm’s thermal tolerance. The survival pH level for this nemertean ranged from 5.00 to 9.20. D 2005 Elsevier B.V. All rights reserved. Keywords: pH; Procephalothrix simulus; Salinity; Temperature; Tolerance; Volume regulation
1. Introduction The ecological studies concerning nemerteans are mainly related to their feeding behavior or interactions with their prey (Gibson, 1998). Little is known about the effects of environmental factors on the life of nemerteans, and most of our knowledge about the
* Corresponding author. Tel.: +86 532 82032273; fax: +86 532 82894024. E-mail address: [email protected] (S. Sun). 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.07.003
tolerance of nemerteans to environmental factors is based on scattered remarks that are essentially nonprimary results of investigations with different objectives. This can be exemplified by the fact that most marine nemerteans were classified as stenohaline organisms (Gibson, 1972) just because field records indicated that they showed a marked dependence upon salinity. For most nemertean species, the tolerance to environmental factors has not been experimentally determined. Temperature and salinity have long been known to be among the most important environmental factors in
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The archinemertean Procephalothrix simulus Iwata, 1952, which normally inhabits in coarse sand or under stones, is one of the most common intertidal nemerteans in Qingdao and its vicinity. The abundance of individuals makes it an appropriate animal for experimental ecology studies. Experiments were carried out to test the tolerance of this nemertean to the temperature, salinity and pH.
2. Materials and Methods 2.1. Animals P. simulus were collected from coarse sand or under stones at Taiping Jiao, Qingdao, China. In the laboratory, worms were maintained for 48–72 h in ambient seawater (31x) at 20 8C without feeding. The mean F S.D. for the wet weight of the worms was 0.024 F 0.0025 (range 0.014–0.036) g. 2.2. Salinity tolerance The salinity tolerance experiment was conducted at three temperatures (10 8C, 20 8C, 30 8C). High-salinity media were prepared by adding NaCl to local seawater (31x), and diluted media were prepared by adding distilled water to local seawater. Salinity levels were determined according to the results of pretests; 7–8 high salinities and 3–9 low salinities (see Figs. 1– 6) and one control (31x) was prepared for each
100
24h 48h 72h 96h
80
Mortality (%)
the life of marine animals, and there is often a complex co-relationship between them, where temperatures can modify the effects of salinity, and salinity can modify the effects of temperature accordingly (e.g., McLeese, 1956; Kinne, 1963, 1964). In nemerteans, the salinity adaptation has received more attentions than other factors. Previous research on salinity has focused on the mechanism of osmotic adaptation or determining the responses of organs and cells to osmotic stresses (e.g., Lechenault, 1965; Ling, 1969, 1970, 1971; Ferraris, 1979a,b,c, 1984, 1985; Ferraris and Schmidt-Nielsen, 1982; Amerongen and Chia, 1983; Ferraris and Norenburg, 1988), and the capacity of osmoregulation has been documented for a few nemertean species (Lechenault, 1965; Ferraris and Schmidt-Nielsen, 1982; Amerongen and Chia, 1983; Ferraris, 1984; Ferraris and Norenburg, 1988). Though it has been noticed that most marine nemerteans are stenohaline animals (Gibson, 1972), all of the species [Lineus ruber (Mu¨ller, 1774), Procephalothrix spiralis Coe, 1930, Pseudocarcinonemertes homari Fleming and Gibson, 1981, and Pantinonemertes californiensis Gibson et al., 1982], whose salinity tolerance have been experimentally studied (Eason, unpublished, see Gibson, 1972, and Moore and Gibson, 1985; Ferraris and Norenburg, 1988; Charmantier and Charmantier-Daures, 1991; Roe, 1993), are euryhaline. Few works have been published on the responses of nemerteans to the variation of temperature and pH. Hickman (1963) demonstrated that Geonemertes australiensis Dendy, 1892, could live for 6 months if the temperature did not exceed 23 8C, but that only a few degrees warmer (27 8C) would be fatal to it. Eason’s unpublished work (see Gibson, 1972) revealed that L. ruber could survive at least 7 days when the temperature ranged from 0 8C to 30 8C and could withstand freezing for a short time; abrupt immersion in seawater of 40 8C would be fatal; different parts of the body reacted differently to the stimulation of hydrochloric acid or sodium hydroxide, the cephalic region is sensitive to pH of 1–5 and 10–14. Okazaki et al. (2001) reported that a 2-h exposure at 36 8C was lethal to Paranemertes peregrina Coe, 1901; and a 2-h exposure at 34 8C significantly elevated the levels of heat shock protein (Hsp70) and caused extremely poor physical condition in this nemertean.
169
60 40 20 0 50
52
54
56
58
60
62
64
66
68
Salinity (‰) Fig. 1. The percentage mortality (mean F S.D.) of P. simulus in high-salinity media at 10 8C.
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100
24h 48h 72h 96h
80
Mortality (%)
80
Mortality (%)
100
24h 48h 72h 96h
60 40 20
60 40 20
0 42
44
46
48
50
52
54
56
58
0 1.1
60
2.2
Salinity (‰)
3.3
Salinity (‰)
Fig. 2. The percentage mortality (mean F S.D.) of P. simulus in high-salinity media at 20 8C.
Fig. 4. The percentage mortality (mean F S.D.) of P. simulus in dilute media at 10 8C.
temperature. All treatments were triplicated with 10 worms assigned for each replicate. In another experiment, worms were transferred to distilled water to determine the response of worms in distilled water. In addition, an experiment was performed to test the capacity of volume regulation of this nemertean in diluted solutions. The volume of the nemertean is difficult to measure because of the powerful variation in body shape. Kinne (1964) indicated that volume changes were always related to changes in body weight. The volume regulation of P. simulus was thus estimated by the changes of body weight. After exposure to solutions of 7.5x and 15.0x at 20 8C, the wet weight of worms was measured at different times (0 min, 5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h). Five worms were tested for either salinity level.
2.3. Temperature tolerance
100
The pH tolerance experiment was conducted at temperature of 20 8C and at salinity of 31x. Eight
24h 48h 72h 96h
80
Mortality (%)
Mortality (%)
2.4. pH tolerance
100
24h 48h 72h 96h
80
The temperature tolerance experiment was conducted at three salinities (18x, 31x and 38x). In the high temperature tolerance test, five temperature levels (30 8C, 32 8C, 34 8C, 35 8C and 36 8C) and a control (20 8C) were designated. In the low temperature tolerance test, nemerteans were immersed into 0 8C seawater for 96 h. Each treatment was conducted in three replicates, and 10 worms were assigned for each replicate.
60 40
60 40 20
20 0 38
40
42
44
46
48
50
52
54
Salinity (‰) Fig. 3. The percentage mortality (mean F S.D.) of P. simulus in high-salinity media at 30 8C.
0 2.2
3.3
4.4
5.5
6.6
7.7
8.8
Salinity (‰) Fig. 5. The percentage mortality (mean F S.D.) of P. simulus in dilute media at 20 8C.
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24h 48h 72h 96h
100
Mortality (%)
80 60 40 20 0 5.5
6.6
7.7
8.8
9.9
11.0
12.1
13.2
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or hypo-media. In this case, the death was judged by the condition of the head. The method of McLeese (1956) was also used: worms were returned to normal seawater at 20 8C and if they did not recover in 1 h, the worms were considered dead. Median lethal salinities (LS50) were calculated on probit analysis. Two-way ANOVA ( P b 0.05) was used to test the interaction of salinity and temperature. Statistical analysis was performed by SPSS 10.0.
14.3
Salinity (‰) Fig. 6. The percentage mortality (mean F S.D.) of P. simulus in dilute media at 30 8C.
pH levels (4.50, 4.70, 5.00, 5.20, 9.20, 9.50, 9.70 and 10.00) and one control (7.95, which is the value of the local seawater) were established to assess the lethal effects of pH stress on the nemertean. Each treatment was conducted in two replicates and 10 worms were assigned for each replicate. The acidified and basified solutions were prepared by adding 1 M HCl and 1 M NaOH to the normal seawater, respectively. During the experimental period, the solutions were fully replaced every 2–3 h to keep the pH stable. 2.5. Experiment procedure, death judgment and statistical analysis All the solutions were filtered through a 3-Am cartridge filter paper. Tolerance experiments were performed in 200-mm 150-mm 150-mm glass aquaria. Each aquarium contained 500 ml solution, and a complete water exchange was performed everyday (with the exception of the previously described pH tolerance experiment). As the sudden transfer method is the easiest way for the comparison of tolerance of marine animals (Dorgelo, 1976), animals were directly transferred to the test media from the normal seawater (31x, 20 8C). During the experiments, the behavior of nemerteans was carefully observed and recorded; dead individuals were counted and removed at 1, 6, 12, 24, 48, 72 and 96 h. The criterion for determining death was the lack of response after repeated probing. Sometimes animals would autolyze when exposed to hyper-
3. Results 3.1. Salinity tolerance In the high-salinity tolerance tests, the mortality increased with the elevation of salinity and the exposure time (Figs. 1–3). Salinity tolerance was affected significantly by the temperature; elevated temperature reduced the salinity tolerance. After a 24-h exposure in media at 10 8C, 20 8C and 30 8C, the salinities of 100% survival were 60.0x, 50.0x and 43.0x, respectively; the salinities of 100% death were 68.0x, 60.0x and 53.0x, respectively (Figs. 1–3); the LS50 were 64.5x, 55.2x and 46.8x, respectively (Table 1). After a 96-h exposure in media at 10 8C, 20 8C and 30 8C, the
Table 1 The LS50 values and 95% confidence intervals (x) of P. simulus in hyper- and hypo-media at three temperatures Temperature (8C)
Time (h)
Hyper-media
Hypo-media
LS50
95% confidence intervals
LS50
95% confidence intervals
10
24 48 72 96 24 48 72 96 24 48 72 96
64.5 59.8 56.1 53.9 55.2 51.1 48.8 47.1 46.8 43.7 41.9 41.4
63.2–65.8 58.4–61.3 54.7–57.5 52.2–55.1 54.1–56.4 49.0–53.3 47.4–50.3 45.6–48.6 45.3–48.3 42.2–45.2 41.0–42.9 40.3–42.4
–a –a –a –a –a 3.5 6.3 7.3 6.9 10.4 12.8 13.5
–a –a –a –a –a 2.8–4.8 6.0–6.8 6.8–8.0 6.6–7.6 9.6–11.8 11.7–16.1 12.5–15.1
20
30
a LS50 and 95% confidence interval were not calculated because of the abrupt change of mortality between neighboring salinities.
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salinities of 100% survival decreased to 50.0x, 43.0x and 38.0x, respectively; the salinities of 100% death decreased to 60.0x, 53.0x and 48.0x, respectively (Figs. 1–3); the LS50 were 53.9x, 47.1x and 41.4x, respectively (Table 1). When immersed in the distilled water at 20 8C, worms violently writhed for about 5 min and then became motionless. After 15-min immersion in distilled water, worms would spend 1–2 min to recover in full seawater. If immersed in distilled water for 30 or 60 min, the time for the worms to restore the movability (of head) in full seawater was about 5 and 15 min, respectively. Two-hour immersion was fatal to the nemertean, which could not revive after 1h immersion in full seawater. Tolerance of the nemertean to diluted media was also related to temperatures. As in the high-salinity tests, elevated temperature reduced the salinity tolerance. The strongest tolerance was observed at 10 8C. At this temperature, the nemertean could not survive for 24 h in 1.1x medium and for 48 h in 2.2x medium, but a 96-h exposure in 3.3x medium was not lethal to it (Fig. 4). Although worms appeared thanatoid, they could recover if returned to full seawater. Because of this abrupt change in mortality, the LS50 at 10 8C was not calculated. At 20 8C and 30 8C, mortalities were greatly affected by the duration of exposure (Figs. 5 and 6). At 20 8C in 5.5x media, for example, the
mortalities were 0.0%, 3.3%, 80.0% and 100.0% after the exposure of 24, 48, 72 and 96 h, respectively (Fig. 5). The LS50 at 20 8C and 30 8C are presented in Table 1. Two-way ANOVA performed on mortality data at different times (24, 48, 72 and 96 h) showed that the interaction of salinity and temperature was statistically significant ( P b 0.05) in both high- and low-salinity tests. Profound response was observed when the nemerteans were exposed to extreme hyper- and hypo-media, worms tended to make convulsive movement and sometimes everted their proboscides immediately after being immersed into such media. As a result of osmotic transfer, the volume of the body became smaller and the body color became darker when exposed to high-salinity media. In dilute media, a contrary response happened. Animals became swollen and bleached. Wet weight of worms increased rapidly after exposure to diluted media (Fig. 7). In 7.5x and 15.0x, maximum weight increase was observed after 4 h (119.3%) and 20 min (61.5%), respectively. After then, the body weight decreased gradually, but could not restore the normal weight in 48 h (Fig. 7). The weight changes in diluted media suggested that P. simulus possessed the capability of volume regulation and performed better in 15.0x than that in 7.5x.
Percentage increase in wet weight
140
140 15.0‰ 7.5‰
120
120
100
100
80
80
60
60
40
40
20
20
0
0 0
10
20
30
min
40
50
60
Time
4
8 12 16 20 24 28 32 36 40 44 48
h
Fig. 7. The percentage increase (mean F S.D.) of the wet weight of P. simulus in diluted solutions of two salinities.
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Table 2 The percentage mortality (%) (mean F S.D.) of P. simulus exposed to different temperatures for 24–96 h at three salinities Salinity (x)
Temperature (8C)
24 h
48 h
72 h
96 h
18
30 32 34 35 36 20 (control) 30 32 34 35 36 20 (control) 30 32 34 35 36 20 (control)
0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 43.3 F 5.8 96.7 F 5.8 0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 20.0 F 10.0 60.0 F 0.0 100.0 F 0.0 0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 40.0 F 10.0 100.0 F 0.0 100.0 F 0.0 0.0 F 0.0
0.0 F 0.0 3.3 F 5.8 63.3 F 5.8 96.7 F 5.8 100.0 F 0.0 0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 83.3 F 5.8 100.0 F 0.0
0.0 F 0.0 3.3 F 5.8 100.0 F 0.0 100.0 F 0.0
0.0 F 0.0 10.0 F 0.0
0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 100.0 F 0.0
0.0 F 0.0 0.0 F 0.0 3.3 F 5.8
31
38
0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 100.0 F 0.0
0.0 F 0.0 0.0 F 0.0 53.3 F 15.3
0.0 F 0.0 0.0 F 0.0 76.7 F 5.8
0.0 F 0.0
0.0 F 0.0
0.0 F 0.0
not respond severely to 32 8C. When the temperature was raised to 34 8C and 35 8C, the mortalities rose along with the salinity rising from 18x to 38x, which suggested that elevated salinity reduced the thermal tolerance of this nemertean. The combination of salinity and temperature significantly affected the survival of the nemertean ( P b 0.05).
3.2. Temperature tolerance Tolerance of P. simulus to low temperature was observed. After a 96-h exposure in medium of 0 8C, worms ceased to move, but could recover by gradually raising the temperature to 20 8C. A lethal low temperature could not be determined. Results of thermal tolerance tests are shown in Table 2. Worms were able to tolerate 30 8C for 96 h in three salinities. At 32 8C in 38x media, no death was observed during the initial 48 h, but a sudden elevation of mortality occurred after 48 h (Table 2). In 18x and 31x media, however, nemerteans did
3.3. pH tolerance In acidified seawater of pH V 4.70, all worms responded rigorously and died within 24 h. During the 96-h test, pH of 5.00–9.20 had no effects on
Table 3 The percentage mortality (%) of P. simulus immersed in media of different pH for different times (temperature is 20 8C; pH of control is 7.95; two replicates are indicated by the circled number) pH
24 h
48 h
72 h
96 h
!
"
Mean
!
"
Mean
!
"
Mean
!
"
Mean
4.50 4.70 5.00 5.20 9.20 9.50 9.70 10.00 Control
100 100 0 0 0 0 0 100 0
100 100 0 0 0 0 0 100 0
100 100 0 0 0 0 0 100 0
0 0 0 0 80
0 0 0 20 90
0 0 0 10 85
0 0 0 30 100
0 0 0 50 100
0 0 0 40 100
0 0 0 100
0 0 0 100
0 0 0 100
0
0
0
0
0
0
0
0
0
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nemertean survival. In seawater basified to pH 9.50 or higher, lethality became obvious. Medium of pH with 10.00 was immediately fatal to the nemertean (Table 3).
4. Discussion 4.1. Salinity tolerance In intertidal environments, the salinity fluctuations tend to be largely due to a combination of factors, including evaporation, rainfall, and freshwater runoff (Jansson, 1967). Dorgelo (1976) suggested that the intertidal animals were more or less euryhaline species. P. simulus displays very strong salinity tolerance. This species can survive for at least 4 days in 3.3– 50.0x at 10 8C, in 8.8–43.0x at 20 8C, and in 14.3– 38.0x at 30 8C. Other intertidal nemerteans whose salinity tolerance has been tested are also tolerant to a wide range of salinities. For example, the littoral heteronemertean L. ruber was able to survive salinity variations from 24.5x to 4.9x for up to 7 days, 1.25x for 24 h [Eason, unpublished, see Gibson, 1972; Ling, 1969 reported that L. ruber could survive 30–150% seawater (10–51x)]; and in the archinemertean P. spiralis death began to occur at 30% Frenchman Bay seawater (salinity about 10x) and all worms died within 3 days at 15% Frenchman Bay seawater (salinity about 5x) at 7 8C (Ferraris and Norenburg, 1988). As in most euryhaline animals (Kinne, 1964), all of these nemerteans possess the capacity for volume regulation at least to some extent (Lechenault, 1965; Ling, 1971; Ferraris and Schmidt-Nielsen, 1982; Ferraris, 1984; Ferraris and Norenburg, 1988; the proof for volume regulation of the present species see Fig. 7). Kinne (1964) concluded that the causes of death in high or low salinities might be primarily related to (1) osmotic phenomena such as disturbances in the water and mineral balance in body fluids and cells and (2) in extremely low or high salinities to the concomitant changes in absorption and saturation coefficients of dissolved gases, especially in severe reduction in the amount of dissolved ambient oxygen in very high salinities. In nemerteans, very little is known about the lethality mechanism of salinity stresses. Ferraris and Schmidt-Nielsen (1982) explained that the disability of volume regulation in low-salinity
media might be a result of cellular damage caused by overhydration. 4.2. Temperature tolerance Benthic animals from cold-water environments are reported to survive lower temperatures better than animals living in warmer climates; whereas warmwater animals did better at elevated thermal points (Vernberg, 1962). Animals exposed to a widely ranging seasonal thermal regime typically are better able to survive a wider range of temperatures than organisms living in a region of either relatively constant high or low temperatures (Vernberg, 1981). The yearround temperature of the coastal waters of Qingdao is about 3–27 8C. At the high tide zone, much lower and much higher temperatures should occur in winter and summer, respectively (the extreme air temperature may be lower than 10 8C in winter and higher than 35 8C in summer). Thus, P. simulus, which is commonly collected from the high tide zone, has to experience a wide range of temperatures and develops the capability of eurythermal tolerance. It could survive 0–30 8C (lethal low temperature was not determined) for at least 4 days in solutions of 18–38x. Our field observations suggested that the microdistribution of this species varied with the alternation of seasons. In late spring (May and June) and autumn (October), a large number of individuals were found to aggregate in coarse sand or under stones at high tide zone. In summer and winter when the temperature is high or low, much fewer worms were found from the same habitats. They were presumed to have migrated downwards and dispersed to avoid the hypo- or hyperthermal stress in the high tide zone, although the aggregation of worms in later spring might also be related to their reproductive strategy. 4.3. pH tolerance In marine and estuarine waters, water is naturally buffered by the CO2–carbonate equilibrium (Whitfield and Turner, 1986). The pH stress of animals living in natural waters is thus not as rigorous as the stresses of temperature and salinity. Studies revealed that pH level of 6.5 was deleterious to many marine and estuary animals (e.g., Bamber, 1987, 1990; Davies, 1991; Batien and Bamber, 1996). The only data about
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the response of nemerteans to acid and alkali indicated that the cephalic region of L. ruber was sensitive to pH levels of 1–5 and 10–14, but did not respond to the pH of 6–9 (Eason, unpublished, see Gibson, 1972). Present work suggested that the pH of 5.00–9.20 brought no lethality to P. simulus within short periods, and that pH lower than 5.00 or higher than 9.20 was fatal to it. However, whether nemerteans are more tolerant to acid and alkali stresses than other marine animals cannot be claimed. As the effects of pH stress on marine animals may be greatly affected by the temperature and the timescale assigned to experiments (Bamber, 1987, 1990; Batien and Bamber, 1996), comparison of data obtained from experiments that have been carried out at different conditions may be unreliable.
temperature and the salinity is statistically significant ( P b 0.05). Elevated temperature decreases the salinity tolerance and high salinity negatively affects the temperature tolerance.
4.4. Interaction of multiple environmental factors
Amerongen, H.M., Chia, F.S., 1983. The role of nemertean cerebral organs in salinity stress tolerance re-examined in Paranemertes peregrina Coe (Hoplonemertea: Monostilifera). Mar. Behav. Physiol. 10, 1 – 22. Bamber, R.N., 1987. The effects of acidic seawater on young carpet-shell clams Venerupis decussate (L.) (Mollusca: Veneracea). J. Exp. Mar. Biol. Ecol. 108, 241 – 260. Bamber, R.N., 1990. The effects of acidic seawater on three species of lamellibranch mollusc. J. Exp. Mar. Biol. Ecol. 143, 181 – 191. Batien, S.D., Bamber, R.N., 1996. The effects of acidified seawater on the polychaete Nereis virens Sars, 1835. Mar. Pollut. Bull. 32, 283 – 287. Charmantier, G., Charmantier-Daures, M., 1991. Salinity tolerance and osmoregulation in the nemertean Pseudocarcinonemertes homari, an egg predator of American lobster, Homarus americanus. Can. J. Fish. Aquat. Sci. 48, 209 – 214. Davies, J.K., 1991. Reactions of sand smelt to low pH seawater. Mar. Pollut. Bull. 22, 74 – 77. Dorgelo, J., 1976. Salt tolerance in Crustacea and the influence of temperature upon it. Biol. Rev. Camb. Philos. Soc. 51, 255 – 290. Ferraris, J.D., 1979a. Histological study of secretory structures of nemerteans subjected to stress. I. Neurosecretory systems. Gen. Comp. Endocrinol. 39, 423 – 433. Ferraris, J.D., 1979b. Histological study of secretory structures of nemerteans subjected to stress: II. Cerebral organs. Gen. Comp. Endocrinol. 39, 434 – 450. Ferraris, J.D., 1979c. Histological study of secretory structures of nemerteans subjected to stress: III. Cephalic glands. Gen. Comp. Endocrinol. 39, 451 – 466. Ferraris, J.D., 1984. Volume regulation in intertidal Procephalothrix spiralis (Nemertina) and Clitellio arenarius (Oligochaeta). II. Effects of decerebration under fluctuating salinity conditions. J. Comp. Physiol. 154B, 125 – 137. Ferraris, J.D., 1985. Putative neuroendocrine devices in the Nemertina. An over-view of structure and function. Am. Zool. 25, 73 – 85.
Multiple factor interactions on the lethality of marine animals have been proved by many studies (e.g., McLeese, 1956; Kinne, 1964; Wallis, 1975). McLeese (1956) demonstrated that a sublethal but stressful exposure to one factor might become lethal when an animal was exposed simultaneously to a second sublethal but stressful factor, and the net result is a reduction in the size of the zone of compatibility. In P. simulus, the salinity and thermal tolerance are clearly affected by each other. Both the high and low salinity tolerance significantly decreased when the temperature was elevated from 10 8C to 30 8C. The thermal limits decreased while the salinity rose from 18x to 38x. Additional data about the combined effects of temperature and salinity on the lethality of nemerteans were provided by Charmantier and Charmantier-Daures (1991), who found that the salinity tolerance of the egg predator P. homari decreased if the temperature was raised from 7 8C to 14.5 8C.
5. Conclusion P. simulus is a eurytopic animal and possesses the ability of volume regulation. The worm can survive for at least 4 days in 3.3–50.0x at 10 8C, in 8.8– 43.0x at 20 8C, in 14.3–38.0x at 30 8C, and can endure 0 8C to 30 8C in media of 18–38x, and is not sensitive to pH of 5.20–9.20. The interaction between
Acknowledgments The study is supported by the National Natural Science Foundation of China (30270235). We are grateful to Dr. Wang Fang, Professor Zeng Xiaoqi, and Miss Wang Haiyan for their kind help in field and laboratory work. [PH]
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Ferraris, J.D., Norenburg, J.L., 1988. Water and solute regulation in Procephalothrix spiralis Coe and Clitellio arenarius (Mu¨ller). Long-term acclimation to diluted seawaters and effect of putative neuroendocrine structures. Hydrobiologia 156, 161 – 173. Ferraris, J.D., Schmidt-Nielsen, B., 1982. Volume regulation in an intertidal nemertine, Procephalothrix spiralis Coe. I. Short-term effects and independence on decerebration. J. Exp. Zool. 224, 307 – 319. Gibson, R., 1972. Nemerteans. Hutchinson University Library, London. Gibson, R., 1998. Epilogue—one hundred years of nemertean research: Bu¨rger (1895) to the present. Hydrobiologia 368, 301 – 310. Hickman, V.V., 1963. The occurrence in Tasmania of the land nemertine, Geonemertes australieusis Dendy, with some account of its distribution, habits, variations and development. Pap. Proc. R. Soc. Tasman. 97, 63 – 75. Jansson, B.O., 1967. The importance of tolerance and preference experiments for interpretation of mesopsmmon field experiments. Helgol. Wiss. Meeresunters. 15, 41 – 58. Kinne, O., 1963. The effect of temperature and salinity on marine and brackish water animals. I. temperature. Oceanogr. Mar. Biol. Ann. Rev. 1, 301 – 340. Kinne, O., 1964. The effects of temperature and salinity on marine and brackish water animals. II. Salinity and temperature–salinity combinations. Oceanogr. Mar. Biol. Ann. Rev. 2, 281 – 339. Lechenault, H.M., 1965. Neurose´cre´tion et osmoregulation chez les Lineidae (He´te´rone´mertes). C. R. Hebd. Se´ances Acad. Sci., Paris, Ser. D 261, 4868 – 4871.
Ling, E.A., 1969. The structure and function of the cephalic organ of a nemertine Lineus ruber. Tissue Cell 1, 503 – 524. Ling, E.A., 1970. Further investigations on the structure and function of cephalic organ of a nemertine Lineus ruber. Tissue Cell 2, 569 – 588. Ling, E.A., 1971. The proboscis apparatus of the nemertine Lineus ruber. Philos. Trans. R. Soc., Ser. B 262, 1 – 22. McLeese, D.W., 1956. Effects of temperature, salinity and oxygen on the survival of the American lobster. J. Fish. Res. Board Can. 13, 247 – 272. Moore, J., Gibson, R., 1985. The evolution and comparative physiology of terrestrial and freshwater nemerteans. Biol. Rev. 60, 257 – 312. Okazaki, R.K., Ye, W., Berthelemy-Okazaki, N.J., Chang, E.S., 2001. Preliminary study of heat shock proteins in nemerteans. Hydrobiologia 456, 211 – 219. Roe, P., 1993. Aspects of the biology of Pantinonemertes californiensis, a high intertidal nemertean. Hydrobiologia 266, 29 – 44. Vernberg, F.J., 1962. Latitudinal effects on physiological properties of animal populations. Annu. Rev. Physiol. 24, 517 – 546. Vernberg, F.J., 1981. Benthic macrofauna. In: Vernberg, F.J., Vernberg, W.B. (Eds.), Functional Adaptations of Marine Organisms. Academic Press, New York, pp. 179 – 230. Wallis, R.L., 1975. Thermal tolerance of Mytilus edulis of eastern Australia. Mar. Biol. 30, 183 – 191. Whitfield, M., Turner, D.R., 1986. The carbonate-dioxide system in estuarines—an inorganic perspective. Sci. Total Environ. 49, 235 – 255.
Effects of salinity, temperature and pH on the survival of the nemertean Procephalothrix simulus Iwata, 1952 Zhao Yanfang, Sun Shichun * Mariculture Research Lab, Ocean University of China, 5 Yushan Road, Qingdao 266003, China Received 31 March 2005; received in revised form 20 June 2005; accepted 13 July 2005
Abstract The salinity, temperature and pH tolerance of Procephalothrix simulus Iwata, 1952, were experimentally studied. In hypomedia, the nemerteans could survive 96 h in 3.3x solution at 10 8C (median lethal salinity [LS50] was not determined at this temperature), and 96 h LS50 were 7.3x and 13.5x at 20 8C and 30 8C, respectively. In hyper-media, 96 h LS50 values were 53.9x, 47.1x and 41.4x at 10 8C, 20 8C and 30 8C, respectively. The trend of body weight changes in diluted media indicated that this nemertean is a volume regulator. During a 96-h exposure in media at 0 8C, worms were thanatoid but could recover if the temperature was gradually elevated to 20 8C. In thermal tolerance experiments, the nemertean survived 96 h in seawater of 30 8C, and worms suffered high mortalities when the temperature exceeded 32 8C. Present results suggest that the interaction of temperature and salinity on the lethal effects on P. simulus is significant ( P b 0.05). Elevated temperature (range 10–30 8C) decreased the worm’s solute tolerance, and elevated salinity (range 18–38x) decreased the worm’s thermal tolerance. The survival pH level for this nemertean ranged from 5.00 to 9.20. D 2005 Elsevier B.V. All rights reserved. Keywords: pH; Procephalothrix simulus; Salinity; Temperature; Tolerance; Volume regulation
1. Introduction The ecological studies concerning nemerteans are mainly related to their feeding behavior or interactions with their prey (Gibson, 1998). Little is known about the effects of environmental factors on the life of nemerteans, and most of our knowledge about the
* Corresponding author. Tel.: +86 532 82032273; fax: +86 532 82894024. E-mail address: [email protected] (S. Sun). 0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2005.07.003
tolerance of nemerteans to environmental factors is based on scattered remarks that are essentially nonprimary results of investigations with different objectives. This can be exemplified by the fact that most marine nemerteans were classified as stenohaline organisms (Gibson, 1972) just because field records indicated that they showed a marked dependence upon salinity. For most nemertean species, the tolerance to environmental factors has not been experimentally determined. Temperature and salinity have long been known to be among the most important environmental factors in
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The archinemertean Procephalothrix simulus Iwata, 1952, which normally inhabits in coarse sand or under stones, is one of the most common intertidal nemerteans in Qingdao and its vicinity. The abundance of individuals makes it an appropriate animal for experimental ecology studies. Experiments were carried out to test the tolerance of this nemertean to the temperature, salinity and pH.
2. Materials and Methods 2.1. Animals P. simulus were collected from coarse sand or under stones at Taiping Jiao, Qingdao, China. In the laboratory, worms were maintained for 48–72 h in ambient seawater (31x) at 20 8C without feeding. The mean F S.D. for the wet weight of the worms was 0.024 F 0.0025 (range 0.014–0.036) g. 2.2. Salinity tolerance The salinity tolerance experiment was conducted at three temperatures (10 8C, 20 8C, 30 8C). High-salinity media were prepared by adding NaCl to local seawater (31x), and diluted media were prepared by adding distilled water to local seawater. Salinity levels were determined according to the results of pretests; 7–8 high salinities and 3–9 low salinities (see Figs. 1– 6) and one control (31x) was prepared for each
100
24h 48h 72h 96h
80
Mortality (%)
the life of marine animals, and there is often a complex co-relationship between them, where temperatures can modify the effects of salinity, and salinity can modify the effects of temperature accordingly (e.g., McLeese, 1956; Kinne, 1963, 1964). In nemerteans, the salinity adaptation has received more attentions than other factors. Previous research on salinity has focused on the mechanism of osmotic adaptation or determining the responses of organs and cells to osmotic stresses (e.g., Lechenault, 1965; Ling, 1969, 1970, 1971; Ferraris, 1979a,b,c, 1984, 1985; Ferraris and Schmidt-Nielsen, 1982; Amerongen and Chia, 1983; Ferraris and Norenburg, 1988), and the capacity of osmoregulation has been documented for a few nemertean species (Lechenault, 1965; Ferraris and Schmidt-Nielsen, 1982; Amerongen and Chia, 1983; Ferraris, 1984; Ferraris and Norenburg, 1988). Though it has been noticed that most marine nemerteans are stenohaline animals (Gibson, 1972), all of the species [Lineus ruber (Mu¨ller, 1774), Procephalothrix spiralis Coe, 1930, Pseudocarcinonemertes homari Fleming and Gibson, 1981, and Pantinonemertes californiensis Gibson et al., 1982], whose salinity tolerance have been experimentally studied (Eason, unpublished, see Gibson, 1972, and Moore and Gibson, 1985; Ferraris and Norenburg, 1988; Charmantier and Charmantier-Daures, 1991; Roe, 1993), are euryhaline. Few works have been published on the responses of nemerteans to the variation of temperature and pH. Hickman (1963) demonstrated that Geonemertes australiensis Dendy, 1892, could live for 6 months if the temperature did not exceed 23 8C, but that only a few degrees warmer (27 8C) would be fatal to it. Eason’s unpublished work (see Gibson, 1972) revealed that L. ruber could survive at least 7 days when the temperature ranged from 0 8C to 30 8C and could withstand freezing for a short time; abrupt immersion in seawater of 40 8C would be fatal; different parts of the body reacted differently to the stimulation of hydrochloric acid or sodium hydroxide, the cephalic region is sensitive to pH of 1–5 and 10–14. Okazaki et al. (2001) reported that a 2-h exposure at 36 8C was lethal to Paranemertes peregrina Coe, 1901; and a 2-h exposure at 34 8C significantly elevated the levels of heat shock protein (Hsp70) and caused extremely poor physical condition in this nemertean.
169
60 40 20 0 50
52
54
56
58
60
62
64
66
68
Salinity (‰) Fig. 1. The percentage mortality (mean F S.D.) of P. simulus in high-salinity media at 10 8C.
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100
24h 48h 72h 96h
80
Mortality (%)
80
Mortality (%)
100
24h 48h 72h 96h
60 40 20
60 40 20
0 42
44
46
48
50
52
54
56
58
0 1.1
60
2.2
Salinity (‰)
3.3
Salinity (‰)
Fig. 2. The percentage mortality (mean F S.D.) of P. simulus in high-salinity media at 20 8C.
Fig. 4. The percentage mortality (mean F S.D.) of P. simulus in dilute media at 10 8C.
temperature. All treatments were triplicated with 10 worms assigned for each replicate. In another experiment, worms were transferred to distilled water to determine the response of worms in distilled water. In addition, an experiment was performed to test the capacity of volume regulation of this nemertean in diluted solutions. The volume of the nemertean is difficult to measure because of the powerful variation in body shape. Kinne (1964) indicated that volume changes were always related to changes in body weight. The volume regulation of P. simulus was thus estimated by the changes of body weight. After exposure to solutions of 7.5x and 15.0x at 20 8C, the wet weight of worms was measured at different times (0 min, 5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h). Five worms were tested for either salinity level.
2.3. Temperature tolerance
100
The pH tolerance experiment was conducted at temperature of 20 8C and at salinity of 31x. Eight
24h 48h 72h 96h
80
Mortality (%)
Mortality (%)
2.4. pH tolerance
100
24h 48h 72h 96h
80
The temperature tolerance experiment was conducted at three salinities (18x, 31x and 38x). In the high temperature tolerance test, five temperature levels (30 8C, 32 8C, 34 8C, 35 8C and 36 8C) and a control (20 8C) were designated. In the low temperature tolerance test, nemerteans were immersed into 0 8C seawater for 96 h. Each treatment was conducted in three replicates, and 10 worms were assigned for each replicate.
60 40
60 40 20
20 0 38
40
42
44
46
48
50
52
54
Salinity (‰) Fig. 3. The percentage mortality (mean F S.D.) of P. simulus in high-salinity media at 30 8C.
0 2.2
3.3
4.4
5.5
6.6
7.7
8.8
Salinity (‰) Fig. 5. The percentage mortality (mean F S.D.) of P. simulus in dilute media at 20 8C.
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24h 48h 72h 96h
100
Mortality (%)
80 60 40 20 0 5.5
6.6
7.7
8.8
9.9
11.0
12.1
13.2
171
or hypo-media. In this case, the death was judged by the condition of the head. The method of McLeese (1956) was also used: worms were returned to normal seawater at 20 8C and if they did not recover in 1 h, the worms were considered dead. Median lethal salinities (LS50) were calculated on probit analysis. Two-way ANOVA ( P b 0.05) was used to test the interaction of salinity and temperature. Statistical analysis was performed by SPSS 10.0.
14.3
Salinity (‰) Fig. 6. The percentage mortality (mean F S.D.) of P. simulus in dilute media at 30 8C.
pH levels (4.50, 4.70, 5.00, 5.20, 9.20, 9.50, 9.70 and 10.00) and one control (7.95, which is the value of the local seawater) were established to assess the lethal effects of pH stress on the nemertean. Each treatment was conducted in two replicates and 10 worms were assigned for each replicate. The acidified and basified solutions were prepared by adding 1 M HCl and 1 M NaOH to the normal seawater, respectively. During the experimental period, the solutions were fully replaced every 2–3 h to keep the pH stable. 2.5. Experiment procedure, death judgment and statistical analysis All the solutions were filtered through a 3-Am cartridge filter paper. Tolerance experiments were performed in 200-mm 150-mm 150-mm glass aquaria. Each aquarium contained 500 ml solution, and a complete water exchange was performed everyday (with the exception of the previously described pH tolerance experiment). As the sudden transfer method is the easiest way for the comparison of tolerance of marine animals (Dorgelo, 1976), animals were directly transferred to the test media from the normal seawater (31x, 20 8C). During the experiments, the behavior of nemerteans was carefully observed and recorded; dead individuals were counted and removed at 1, 6, 12, 24, 48, 72 and 96 h. The criterion for determining death was the lack of response after repeated probing. Sometimes animals would autolyze when exposed to hyper-
3. Results 3.1. Salinity tolerance In the high-salinity tolerance tests, the mortality increased with the elevation of salinity and the exposure time (Figs. 1–3). Salinity tolerance was affected significantly by the temperature; elevated temperature reduced the salinity tolerance. After a 24-h exposure in media at 10 8C, 20 8C and 30 8C, the salinities of 100% survival were 60.0x, 50.0x and 43.0x, respectively; the salinities of 100% death were 68.0x, 60.0x and 53.0x, respectively (Figs. 1–3); the LS50 were 64.5x, 55.2x and 46.8x, respectively (Table 1). After a 96-h exposure in media at 10 8C, 20 8C and 30 8C, the
Table 1 The LS50 values and 95% confidence intervals (x) of P. simulus in hyper- and hypo-media at three temperatures Temperature (8C)
Time (h)
Hyper-media
Hypo-media
LS50
95% confidence intervals
LS50
95% confidence intervals
10
24 48 72 96 24 48 72 96 24 48 72 96
64.5 59.8 56.1 53.9 55.2 51.1 48.8 47.1 46.8 43.7 41.9 41.4
63.2–65.8 58.4–61.3 54.7–57.5 52.2–55.1 54.1–56.4 49.0–53.3 47.4–50.3 45.6–48.6 45.3–48.3 42.2–45.2 41.0–42.9 40.3–42.4
–a –a –a –a –a 3.5 6.3 7.3 6.9 10.4 12.8 13.5
–a –a –a –a –a 2.8–4.8 6.0–6.8 6.8–8.0 6.6–7.6 9.6–11.8 11.7–16.1 12.5–15.1
20
30
a LS50 and 95% confidence interval were not calculated because of the abrupt change of mortality between neighboring salinities.
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salinities of 100% survival decreased to 50.0x, 43.0x and 38.0x, respectively; the salinities of 100% death decreased to 60.0x, 53.0x and 48.0x, respectively (Figs. 1–3); the LS50 were 53.9x, 47.1x and 41.4x, respectively (Table 1). When immersed in the distilled water at 20 8C, worms violently writhed for about 5 min and then became motionless. After 15-min immersion in distilled water, worms would spend 1–2 min to recover in full seawater. If immersed in distilled water for 30 or 60 min, the time for the worms to restore the movability (of head) in full seawater was about 5 and 15 min, respectively. Two-hour immersion was fatal to the nemertean, which could not revive after 1h immersion in full seawater. Tolerance of the nemertean to diluted media was also related to temperatures. As in the high-salinity tests, elevated temperature reduced the salinity tolerance. The strongest tolerance was observed at 10 8C. At this temperature, the nemertean could not survive for 24 h in 1.1x medium and for 48 h in 2.2x medium, but a 96-h exposure in 3.3x medium was not lethal to it (Fig. 4). Although worms appeared thanatoid, they could recover if returned to full seawater. Because of this abrupt change in mortality, the LS50 at 10 8C was not calculated. At 20 8C and 30 8C, mortalities were greatly affected by the duration of exposure (Figs. 5 and 6). At 20 8C in 5.5x media, for example, the
mortalities were 0.0%, 3.3%, 80.0% and 100.0% after the exposure of 24, 48, 72 and 96 h, respectively (Fig. 5). The LS50 at 20 8C and 30 8C are presented in Table 1. Two-way ANOVA performed on mortality data at different times (24, 48, 72 and 96 h) showed that the interaction of salinity and temperature was statistically significant ( P b 0.05) in both high- and low-salinity tests. Profound response was observed when the nemerteans were exposed to extreme hyper- and hypo-media, worms tended to make convulsive movement and sometimes everted their proboscides immediately after being immersed into such media. As a result of osmotic transfer, the volume of the body became smaller and the body color became darker when exposed to high-salinity media. In dilute media, a contrary response happened. Animals became swollen and bleached. Wet weight of worms increased rapidly after exposure to diluted media (Fig. 7). In 7.5x and 15.0x, maximum weight increase was observed after 4 h (119.3%) and 20 min (61.5%), respectively. After then, the body weight decreased gradually, but could not restore the normal weight in 48 h (Fig. 7). The weight changes in diluted media suggested that P. simulus possessed the capability of volume regulation and performed better in 15.0x than that in 7.5x.
Percentage increase in wet weight
140
140 15.0‰ 7.5‰
120
120
100
100
80
80
60
60
40
40
20
20
0
0 0
10
20
30
min
40
50
60
Time
4
8 12 16 20 24 28 32 36 40 44 48
h
Fig. 7. The percentage increase (mean F S.D.) of the wet weight of P. simulus in diluted solutions of two salinities.
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Table 2 The percentage mortality (%) (mean F S.D.) of P. simulus exposed to different temperatures for 24–96 h at three salinities Salinity (x)
Temperature (8C)
24 h
48 h
72 h
96 h
18
30 32 34 35 36 20 (control) 30 32 34 35 36 20 (control) 30 32 34 35 36 20 (control)
0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 43.3 F 5.8 96.7 F 5.8 0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 20.0 F 10.0 60.0 F 0.0 100.0 F 0.0 0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 40.0 F 10.0 100.0 F 0.0 100.0 F 0.0 0.0 F 0.0
0.0 F 0.0 3.3 F 5.8 63.3 F 5.8 96.7 F 5.8 100.0 F 0.0 0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 83.3 F 5.8 100.0 F 0.0
0.0 F 0.0 3.3 F 5.8 100.0 F 0.0 100.0 F 0.0
0.0 F 0.0 10.0 F 0.0
0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 100.0 F 0.0
0.0 F 0.0 0.0 F 0.0 3.3 F 5.8
31
38
0.0 F 0.0 0.0 F 0.0 0.0 F 0.0 100.0 F 0.0
0.0 F 0.0 0.0 F 0.0 53.3 F 15.3
0.0 F 0.0 0.0 F 0.0 76.7 F 5.8
0.0 F 0.0
0.0 F 0.0
0.0 F 0.0
not respond severely to 32 8C. When the temperature was raised to 34 8C and 35 8C, the mortalities rose along with the salinity rising from 18x to 38x, which suggested that elevated salinity reduced the thermal tolerance of this nemertean. The combination of salinity and temperature significantly affected the survival of the nemertean ( P b 0.05).
3.2. Temperature tolerance Tolerance of P. simulus to low temperature was observed. After a 96-h exposure in medium of 0 8C, worms ceased to move, but could recover by gradually raising the temperature to 20 8C. A lethal low temperature could not be determined. Results of thermal tolerance tests are shown in Table 2. Worms were able to tolerate 30 8C for 96 h in three salinities. At 32 8C in 38x media, no death was observed during the initial 48 h, but a sudden elevation of mortality occurred after 48 h (Table 2). In 18x and 31x media, however, nemerteans did
3.3. pH tolerance In acidified seawater of pH V 4.70, all worms responded rigorously and died within 24 h. During the 96-h test, pH of 5.00–9.20 had no effects on
Table 3 The percentage mortality (%) of P. simulus immersed in media of different pH for different times (temperature is 20 8C; pH of control is 7.95; two replicates are indicated by the circled number) pH
24 h
48 h
72 h
96 h
!
"
Mean
!
"
Mean
!
"
Mean
!
"
Mean
4.50 4.70 5.00 5.20 9.20 9.50 9.70 10.00 Control
100 100 0 0 0 0 0 100 0
100 100 0 0 0 0 0 100 0
100 100 0 0 0 0 0 100 0
0 0 0 0 80
0 0 0 20 90
0 0 0 10 85
0 0 0 30 100
0 0 0 50 100
0 0 0 40 100
0 0 0 100
0 0 0 100
0 0 0 100
0
0
0
0
0
0
0
0
0
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nemertean survival. In seawater basified to pH 9.50 or higher, lethality became obvious. Medium of pH with 10.00 was immediately fatal to the nemertean (Table 3).
4. Discussion 4.1. Salinity tolerance In intertidal environments, the salinity fluctuations tend to be largely due to a combination of factors, including evaporation, rainfall, and freshwater runoff (Jansson, 1967). Dorgelo (1976) suggested that the intertidal animals were more or less euryhaline species. P. simulus displays very strong salinity tolerance. This species can survive for at least 4 days in 3.3– 50.0x at 10 8C, in 8.8–43.0x at 20 8C, and in 14.3– 38.0x at 30 8C. Other intertidal nemerteans whose salinity tolerance has been tested are also tolerant to a wide range of salinities. For example, the littoral heteronemertean L. ruber was able to survive salinity variations from 24.5x to 4.9x for up to 7 days, 1.25x for 24 h [Eason, unpublished, see Gibson, 1972; Ling, 1969 reported that L. ruber could survive 30–150% seawater (10–51x)]; and in the archinemertean P. spiralis death began to occur at 30% Frenchman Bay seawater (salinity about 10x) and all worms died within 3 days at 15% Frenchman Bay seawater (salinity about 5x) at 7 8C (Ferraris and Norenburg, 1988). As in most euryhaline animals (Kinne, 1964), all of these nemerteans possess the capacity for volume regulation at least to some extent (Lechenault, 1965; Ling, 1971; Ferraris and Schmidt-Nielsen, 1982; Ferraris, 1984; Ferraris and Norenburg, 1988; the proof for volume regulation of the present species see Fig. 7). Kinne (1964) concluded that the causes of death in high or low salinities might be primarily related to (1) osmotic phenomena such as disturbances in the water and mineral balance in body fluids and cells and (2) in extremely low or high salinities to the concomitant changes in absorption and saturation coefficients of dissolved gases, especially in severe reduction in the amount of dissolved ambient oxygen in very high salinities. In nemerteans, very little is known about the lethality mechanism of salinity stresses. Ferraris and Schmidt-Nielsen (1982) explained that the disability of volume regulation in low-salinity
media might be a result of cellular damage caused by overhydration. 4.2. Temperature tolerance Benthic animals from cold-water environments are reported to survive lower temperatures better than animals living in warmer climates; whereas warmwater animals did better at elevated thermal points (Vernberg, 1962). Animals exposed to a widely ranging seasonal thermal regime typically are better able to survive a wider range of temperatures than organisms living in a region of either relatively constant high or low temperatures (Vernberg, 1981). The yearround temperature of the coastal waters of Qingdao is about 3–27 8C. At the high tide zone, much lower and much higher temperatures should occur in winter and summer, respectively (the extreme air temperature may be lower than 10 8C in winter and higher than 35 8C in summer). Thus, P. simulus, which is commonly collected from the high tide zone, has to experience a wide range of temperatures and develops the capability of eurythermal tolerance. It could survive 0–30 8C (lethal low temperature was not determined) for at least 4 days in solutions of 18–38x. Our field observations suggested that the microdistribution of this species varied with the alternation of seasons. In late spring (May and June) and autumn (October), a large number of individuals were found to aggregate in coarse sand or under stones at high tide zone. In summer and winter when the temperature is high or low, much fewer worms were found from the same habitats. They were presumed to have migrated downwards and dispersed to avoid the hypo- or hyperthermal stress in the high tide zone, although the aggregation of worms in later spring might also be related to their reproductive strategy. 4.3. pH tolerance In marine and estuarine waters, water is naturally buffered by the CO2–carbonate equilibrium (Whitfield and Turner, 1986). The pH stress of animals living in natural waters is thus not as rigorous as the stresses of temperature and salinity. Studies revealed that pH level of 6.5 was deleterious to many marine and estuary animals (e.g., Bamber, 1987, 1990; Davies, 1991; Batien and Bamber, 1996). The only data about
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the response of nemerteans to acid and alkali indicated that the cephalic region of L. ruber was sensitive to pH levels of 1–5 and 10–14, but did not respond to the pH of 6–9 (Eason, unpublished, see Gibson, 1972). Present work suggested that the pH of 5.00–9.20 brought no lethality to P. simulus within short periods, and that pH lower than 5.00 or higher than 9.20 was fatal to it. However, whether nemerteans are more tolerant to acid and alkali stresses than other marine animals cannot be claimed. As the effects of pH stress on marine animals may be greatly affected by the temperature and the timescale assigned to experiments (Bamber, 1987, 1990; Batien and Bamber, 1996), comparison of data obtained from experiments that have been carried out at different conditions may be unreliable.
temperature and the salinity is statistically significant ( P b 0.05). Elevated temperature decreases the salinity tolerance and high salinity negatively affects the temperature tolerance.
4.4. Interaction of multiple environmental factors
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Multiple factor interactions on the lethality of marine animals have been proved by many studies (e.g., McLeese, 1956; Kinne, 1964; Wallis, 1975). McLeese (1956) demonstrated that a sublethal but stressful exposure to one factor might become lethal when an animal was exposed simultaneously to a second sublethal but stressful factor, and the net result is a reduction in the size of the zone of compatibility. In P. simulus, the salinity and thermal tolerance are clearly affected by each other. Both the high and low salinity tolerance significantly decreased when the temperature was elevated from 10 8C to 30 8C. The thermal limits decreased while the salinity rose from 18x to 38x. Additional data about the combined effects of temperature and salinity on the lethality of nemerteans were provided by Charmantier and Charmantier-Daures (1991), who found that the salinity tolerance of the egg predator P. homari decreased if the temperature was raised from 7 8C to 14.5 8C.
5. Conclusion P. simulus is a eurytopic animal and possesses the ability of volume regulation. The worm can survive for at least 4 days in 3.3–50.0x at 10 8C, in 8.8– 43.0x at 20 8C, in 14.3–38.0x at 30 8C, and can endure 0 8C to 30 8C in media of 18–38x, and is not sensitive to pH of 5.20–9.20. The interaction between
Acknowledgments The study is supported by the National Natural Science Foundation of China (30270235). We are grateful to Dr. Wang Fang, Professor Zeng Xiaoqi, and Miss Wang Haiyan for their kind help in field and laboratory work. [PH]
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