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Chronic hypoxia and low salinity impair anti-predatory responses of the green-lipped mussel Perna viridis
Marine Environmental Research 77 (2012) 84e89
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Chronic hypoxia and low salinity impair anti-predatory responses of the green-lipped mussel Perna viridis Youji Wang a, *, Menghong Hu a, *, S.G. Cheung b, P.K.S. Shin b, Weiqun Lu a, Jiale Li a a b
College of Fisheries and Life Science, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 January 2012 Received in revised form 11 February 2012 Accepted 14 February 2012
The effects of chronic hypoxia and low salinity on anti-predatory responses of the green-lipped mussel Perna viridis were investigated. Dissolved oxygen concentrations ranged from hypoxic to normoxic (1.5 0.3 mg l1, 3.0 0.3 mg l1 and 6.0 0.3 mg l1), and salinities were selected within the variation during the wet season in Hong Kong coastal waters (15&, 20&, 25& and 30&). The dissolved oxygen and salinity significantly affected some anti-predatory responses of mussel, including byssus production, shell thickness and shell weight, and the adductor diameter was only significantly affected by salinity. Besides, interactive effects of dissolved oxygen and salinity on the byssus production and shell thickness were also observed. In hypoxic and low salinity conditions, P. viridis produced fewer byssal threads, thinner shell and adductor muscle, indicating that hypoxia and low salinity are severe environmental stressors for self-defence of mussel, and their interactive effects further increase the predation risk. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords: Synergistic effects Green-lipped mussel Anti-predatory trait Hypoxia Salinity Byssus Shell thickness Shell weight Adductor muscle
1. Introduction Hypoxia, namely dissolved oxygen (DO) below 2 mg l1 in seawater induced by anthropogenic, physical and/or biological factors commonly occurs in many aquatic habitats such as estuaries, lakes, bays, deep sea and coastal waters almost worldwide (Diaz and Rosenberg,1995; Gray et al., 2002; Kidwell et al., 2009). Even though some hypoxic effects in shallow marine systems are not lethal, oxygen concentrations in the range of 1e4 mg l1 are physiologically and behaviourally stressful (Rombough, 1988; Long et al., 2008). Hypoxia causes a decrease in the density of motile animals that flee hypoxic waters (Pihl et al., 1991; Bell and Eggleston, 2005; Powers et al., 2005) and a decline in the abundance, biomass, and diversity of the sessile benthic community (Rosenberg, 1977; Gaston, 1985; Dauer et al., 1992; Llansó, 1992; Long et al., 2008). Benthic organisms can make some behavioural and physiological changes to resist hypoxic stress. They usually respond by increasing oxygen supply, reducing metabolism and oxygen consumption (Stickle et al., 1989; Wu, 2002). As a result, this could * Corresponding authors. Tel./fax: þ86 21 6190 0448. E-mail addresses: [email protected] (M. J. Wang), [email protected] (M.H. Hu).
lead to diminished growth and anti-predatory behaviour of the stressed organisms (Das and Stickle, 1993; Nilsson, 1999; Condon et al., 2001; Grove and Breitburg, 2005; Long et al., 2008). For instance, bivalves and polychaetes extend siphons or palps, reduce burial depth, or even float up above the benthic boundary layer into higher DO waters (Brafield, 1963; Rosenberg et al., 1991; Taylor and Eggleston, 2000; Seitz et al., 2003; Long et al., 2008). Epifaunal organisms lower their oxygen demand by reducing feeding and movement (Sagasti et al., 2001). These responses induced by hypoxia may have effects on the food web and trophic transfer if they increase an organism’s vulnerability to its predators. For example, extending siphons and palps farther and decrease of burial depth make detected by predators more likely for species that use sediments to obtain a refuge from predation, such as the clam Macoma balthica (De Goeij et al., 2001; Long et al., 2008) and Corbicula fluminea (Saloom and Duncan, 2004). In poorly oxygenated waters, prey may be more vulnerable to predators if their responses to attack are hindered, or their ability to use habitat refuge is reduced (Moore and Townsend, 1998; Taylor and Eggleston, 2000; Tallqvist, 2001; Saloom and Duncan, 2004). Salinity is an important abiotic factor that affects physiological activities, playing an important role in determining species distributions within estuarine and coastal systems (Rowe, 2002),
0141-1136/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2012.02.006
Y. Wang et al. / Marine Environmental Research 77 (2012) 84e89
especially in shallow waters where sessile invertebrates such as mussels live. In estuarine and coastal systems salinity can dramatically change across spatial and temporal scales (Kirkpatrick and Jones, 1985; Rowe, 2002). Marine invertebrates inhabiting estuarine and coastal areas are exposed to short-term (tidal) and long-term (rain periods) changes in salinity. Low salinity undoubtedly has a significant effect on many physiological processes in marine invertebrate, such as osmoregulation, active intracellular transport, feeding rate or nutritive absorption, respiration and excretion (Kinne, 1971). Due to the salinity fluctuation in the estuaries and coastal areas, some species in such areas presumably experience osmotic stress frequently, so they have to expend additional energy to maintain the haemolymph osmolality (Harris and Aladin, 1997). However, to this date, few anti-predatory studies related to salinity in bivalves have been investigated. Environmental stresses rarely occur in isolation and the interactive stress from natural and anthropogenic activity is known to affect the physiological response of bivalves (Bussell et al., 2008). The effects of stresses are frequently studied in isolation, while their combined effects are less well known, in particular whether hypoxia increases the susceptibility of mussels to other forms of stress such as reduced salinity. In view of this, Kidwell et al. (2009) proposed the importance and necessity of quantification of the interactive effect of hypoxia with other environmental stressors. Estuaries and coastal areas are highly stressful environments, with the benefits brought by a riverine supply of inorganic and organic nutrients countered by the associated stress of reductions in salinity. Estuarine mussels typically experience conditions of lowered salinity on every tidal cycle. However, during flood events they are likely to experience much lower salinities than normal and for a longer duration. In mussels, some anti-predation responses when exposed to predators have been reported, including increase of shell thickness in the presence of crabs and whelks (Leonard et al., 1999; Smith and Jennings, 2000; Reimer and Harms-Ringdahl, 2001), size of the adductor muscle in the presence of starfish (Reimer and HarmsRingdahl, 2001) and strength of byssal threads in the presence of crabs and starfish (Cote, 1995; Leonard et al., 1999; Seed and Richardson, 1999; Reimer and Harms-Ringdahl, 2001; Cheung et al., 2004). The green-lipped mussel Perna viridis is a dominant species in tropical and subtropical shallow areas of the Indo-Pacific region (Siddall, 1980) and occurs abundantly in low-energy habitats such as harbours and sheltered bays where the water is often hypoxic and sustains large salinity variation (Thiyagarajan and Qian, 2003). On Hong Kong’s rocky shores, small individuals of P. viridis are consumed preferentially by the portunid crab Thalamita danae which lives in subtidal waters and moves with each flood tide into the intertidal zone to feed (Seed, 1990). The role of hypoxia and low salinity, in determining the outcome of anti-predatory responses in mussels is not well known. To better understand whether chronic hypoxia and low salinity affect the anti-predatory defence of the green-lipped mussel P. viridis, we examined the shell performance, adductor muscle and byssus production of the P. viridis exposed to the portunid crab T. danae under different salinities and DO levels. 2. Materials and methods 2.1. Experimental animals Juvenile mussels (shell length: 26.05 1.55 mm; dry weight: 70.0 5.0 mg) and their potential predators T. danae (60e70 mm carapace width) were collected from a sheltered bay at Lok Wo Sha, Hong Kong. Upon return to the laboratory, the mussels were detached carefully from their clumps by cutting their byssal
85
threads with scissors to avoid damaging the pedal apparatus which would impair their re-secretion. They were then maintained in a fibreglass tank (500 L) equipped with a filtering system and air supply and fed with the brown alga Thalassiosira pseudonana with a concentration of 5.0 105 cells ml1 in the tank once every two days. The seawater was maintained at 21 C and 30& throughout the acclimation and individuals of P. viridis and T. danae were allowed to acclimate to laboratory conditions for one week prior to experimentation. Before the experiment, the portunid crabs T. danae were fed with mussels everyday, and male crabs that consumed the same amount of mussels were selected as predators. 2.2. Experimental system The system was mainly composed of an experimental tank (18 L) with a middle compartment (12 L) and two side compartments (3 L), a digital dissolved oxygen controller (model no. 01972-00, ColeeParmer, Illinois, USA), a cylinder of compressed nitrogen and an air pump (Fig. 1). The oxygen level in the experimental tank was monitored automatically by the oxygen probe of the controller. When the desired oxygen level deviated from the preset value, the dissolved oxygen controller would send a signal to the valves connecting to the nitrogen gas tank or air pump so as to restore the desired oxygen level by delivering either nitrogen or air into the experimental tank. Three oxygen concentrations were selected: 1.5 0.3 mg l1 O2 as severely hypoxic, 3.0 0.3 mg l1 O2 as moderately hypoxic and 6.0 0.3 mg O2 l1 as normoxic level; and 4 levels of salinity (15&, 20&, 25& and 30&) were setup at each fixed DO level for total twelve treatments. Each treatment consisted of three replicates with 25 mussels per replicate. 2.3. Experimental design Twenty five mussels were kept in the middle compartment, and two male crabs T. danae were introduced to the two side compartments, respectively, avoiding physical contact between experimental mussels and their predators, but allowing chemical cues from the crabs to be detected by the mussels (Fig. 1). Experimental crabs were put into aquariums for 3 h daily. This experiment lasted for six weeks under controlled conditions as above. At the end of the experiment, the number of byssal threads that were secreted by each mussel was counted. Mussels were dried at 80 C to calculate shell weight, and shell thickness and adductor muscle diameter were measured using digital Vernier calipers. In our experiment, the crabs mostly attacked mussels by cleaving posterior shell lip, so we measured the shell thickness 1 mm from the centre posterior shell lip (Smith and Jennings, 2000). 2.4. Statistical analysis Each individual aquarium of the same treatment was considered a replicate (i.e., N ¼ 3), and a mean value for each replicate was calculated by averaging the pooled data in each aquarium. Data on evaluated parameters were statistically analysed using two-way analysis of variance (ANOVA). When interactions between the two main factors were present, one-way ANOVAs to test for differences among levels of one factor were tested at each level of the other factor, and Student Newman Keuls (SNK) tests were performed to determine which treatments were different. Differences were considered significant at P < 0.05. Prior to the analysis, normality of the data was evaluated by using the ShapiroeWilk’s test and homogeneity of variances was checked by Levene’s test using the statistical software SPSS 16.0. The results are expressed as the means S.D. of the data.
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Y. Wang et al. / Marine Environmental Research 77 (2012) 84e89
Fig. 1. Experimental setup consists of DO controller, DO probe, air and nitrogen supplies, temperature regulator, water pump, protein skimmer and experimental tank (dimension: 40 cm 40 cm 12 cm). The oxygen level in the experimental tank was determined by the opening and closing of the valves connected to the air and nitrogen supplies.
3. Results During the experiment, only five mussels died (one at 3.0 mg O2 l1 salinity 25&, two at 1.5 mg O2 l1 salinity 20&, and two at 6.0 mg O2 l1 salinity 15&). Only adductor diameter was not affected by DO, and all other parameters were significantly affected by DO and salinity (Table 1). Significant interactions of DO and salinity on shell thickness and byssus number were observed (Table 1), and multiple comparisons between DO/salinity treatments at each fixed salinity/DO level were displayed in Fig. 3 and Fig. 5. DO showed no significant effect at salinity 30&, and salinity showed no significant effect at DO 6.0 mg l1 on shell thickness (Table 2, Fig. 3). The shell weight was gradually reduced with the drop in the DO and salinity. The lowest shell weight was observed from the DO 1.5 mg l1 salinity 15& treatment, and the highest was observed in the DO 6.0 mg l1 salinity 30& treatment (Fig. 2). The significant differences caused by DO and salinity were both detected, and the interaction between DO and salinity on shell weight was not significant (Table 1). The shell thickness varied significantly with dissolved oxygen and salinity, and interactive effect of DO and salinity was observed Table 1 Summary of two-way ANOVA results on effects of dissolved oxygen (DO) and salinity (S) on shell weight (SW), shell thickness (ST), adductor diameter (AD) and byssus number (BN). Dissolved oxygen: 1.5, 3.0 and 6.0 mg O2 l1; salinity: 15, 20, 25 and 30&. Source
df
MS
DO S DO*S Error
2 3 6 24
SW 0.047 0.111 0.005 0.005
F
P
8.794 20.629 0.901
0.001 <0.001 0.51
DO S DO*S Error
2 3 6 24
AD 0.015 0.146 0.008 0.005
3.088 31.143 1.605
0.064 <0.001 0.189
MS
F
(Table 1). Generally, the lower the salinity and dissolved oxygen, the thinner the shell thickness (Fig. 3). However, under normal DO or salinity conditions, salinity or DO could not significantly influence the shell thickness (Table 2, Fig. 3). Adductor diameter was not significantly different among DO treatments, but was significantly different among salinity treatments. The adductor diameter was greater under higher salinity treatments (Fig. 4). No interactive effect of dissolved oxygen and salinity was found on the adductor diameter (Table 1). Byssus number varied significantly with DO and salinity, and there was a significant interaction between dissolved oxygen and salinity (Table 1). Significant effects of DO were observed at each fixed salinity level and also salinity significantly affected the bysses number at each fixed DO level (Table 2). The lowest value was observed in the DO 1.5 mg l1 salinity 15& treatment, and the highest was seen in the DO 6.0 mg l1 salinity 30& treatment (Fig. 5). 4. Discussion At low salinity and DO levels, the mussels displayed poor growth in shell weight and adductor, compared to those reared at high salinity and DO levels. The low growth rate found at low salinity and DO seems to be the result of various physiological constraints
P
ST 0.005 0.01 0.001 0.000 BN 1362.661 796.036 69.308 6.29
16.714 35.387 3.44
<0.001 <0.001 0.014
216.639 126.556 11.019
<0.001 <0.001 <0.001
Fig. 2. Shell weight (SW) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM).
Y. Wang et al. / Marine Environmental Research 77 (2012) 84e89
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Fig. 3. Shell thickness (ST) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM). Different capital letters denote significant differences between three DO treatments at each salinity level, and different small letters denote significant differences between four salinity treatments at each DO level.
Fig. 5. Byssus number (BN) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM). Different capital letters denote significant differences between three DO treatments at each salinity level, and different small letters denote significant differences between four salinity treatments at each DO level.
(Wang et al., 2011). Reduced anti-predatory traits were found after continued exposure to salinity 15& for six weeks, but the mussels were able to survive under low salinity. This indicates a limited euryhalinity of P. viridis and its osmoregulatory capacity could not compensate prolonged effects of low salinity on juvenile growth, which was also reported in other juvenile invertebrates (Charmantier et al., 2002; Torres et al., 2002). Unfavourably low salinity may cause a reduction of feeding or growth efficiency, presumably due to metabolic adjustments induced by osmotic stress. For instance, significant reduction in the metabolism of the Carcinus maenas exposed to low salinity caused reduced growth (Anger et al., 1998). Juvenile P. viridis can also survive six weeks under hypoxia (1.5 mg l1), highlighting its strong tolerance to hypoxia at the expense of growth. Some bivalves have efficient strategies for anaerobic energy production (De Zwaan, 1977) and may reduce their metabolic rate in response to hypoxia (Storey and Storey, 2004). Carroll and Wells (1995) reported that intertidal species like Paphies australis are well adapted to environmental hypoxia. Sessile bivalves generally show a strong resistance to hypoxia, due in part to a reduction in metabolic activity and energy utilization (Widdows, 1987). In anti-predatory studies of mussels, the byssus production, shell thickness and adductor muscle diameter are often used as robust indexes for the effects of biotic/abiotic factors (Cote, 1995; Leonard et al., 1999; Nagarajan et al., 2006, 2008; Fässler and Kaiser, 2008). Mussels are sedentary organisms attached to solid substrata by means of byssus threads. It is generally agreed that an increase in the number of the byssus threads is an anti-predatory response of the mussel exposure to predators (Cheung et al., 2004). Also the shell thickness and adductor muscle diameter of a prey item are strongly related to its resistance to predators’ attacks (Smith and Jennings, 2000; Addison, 2009). Mussels are the major diet of crabs T. danae which employ different modes of attack, depending on the overall size and relative shell thickness of the mussel (Seed,
1993). Crabs are able to cut byssal threads and shell lips using their claws and dislodge individuals from a mussel bed (Elner, 1978). Mussels with firmer attachment and thicker shell and adductor, therefore, are less likely to be dislodged and attacked by crabs and mortality is thus reduced (Lin, 1991; Hughes and Seed, 1995). In contrast, decreased adductor, shell and byssus performance, therefore, may increase predation rate by crabs (Smith and Jennings, 2000; Nagarajan et al., 2006; Farrell and Crowe, 2007). In the present study, the green-lipped mussel produced fewer byssus, thinner shell and adductor when exposed to low salinity and low DO than those in high salinity and high DO, indicating that under hypoxic and hyposaline conditions, the anti-predatory responses were impaired. Whether predators are capable of taking advantage of the stressed prey is a matter of debate, as predators may leave stressful areas (Pihl et al., 1991; Bell and
Fig. 4. Adductor diameter (AD) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM).
Table 2 Summary of one-way ANOVA results on effects of dissolved oxygen (DO) at each fixed salinity level and effects of salinity (S) at each fixed DO level, on shell thickness (ST) and byssus number (BN). Dissolved oxygen: 1.5, 3.0 and 6.0 mg O2 l1; salinity: 15, 20, 25 and 30&. df DO effect ST
S15 Error S20 Error S25 Error S30 Error Salinity effect ST DO1.5 Error DO3.0 Error DO6.0 Error DO effect BN S15 Error S20 Error S25 Error S30 Error Salinity effect BN DO1.5 Error DO3.0 Error DO6.0 Error
MS
F
P
2 6 2 6 2 6 2 6
0.003 0.000 0.003 0.000 0.002 0.000 0.000 0.000
9.619
0.013
9.601
0.013
5.414
0.045
0.944
0.440
3 8 3 8 3 8
0.004 0.000 0.007 0.000 0.001 0.000
26.694
<0.001
33.417
<0.001
2.014
0.191
2 6 2 6 2 6 2 6
71.574 3.181 221.223 2.666 669.623 2.506 608.164 16.808
22.500
0.002
82.993
<0.001
267.255
<0.001
36.184
<0.001
3 8 3 8 3 8
74.820 2.826 242.290 10.119 617.543 5.925
26.477
<0.001
23.944
<0.001
104.227
<0.001
88
Y. Wang et al. / Marine Environmental Research 77 (2012) 84e89
Eggleston, 2005). However, predator behaviour in systems that suffer from hypoxia could lead to an increase in foraging after a hypoxic episode (Bell et al., 2003; Long et al., 2008). Motile predators can rapidly reinvade areas after relaxation of hypoxic conditions (Pihl et al., 1991; Nestlerode and Diaz, 1998; Bell and Eggleston, 2005). They may take advantage of stressed benthic organisms following hypoxia. For example, the diet of T. danae includes a high percentage (53.9%) of mussel P. viridis (Cheung et al., 2006). Besides, the predators of mussel include other predators like starfish and oystercatcher (Cheung et al., 2004; Nagarajan et al., 2008), which would undoubtedly increase the predation risk of mussel under that situation. Byssal threads constitute a significant component of total production by mussels, accounting for 3e10% of annual production in P. viridis (Shafee, 1979; Cheung, 1991). At each fixed level of DO and salinity, the byssus production was significantly reduced by low salinity and low DO, respectively, and there was an interactive effect on byssus production, which indicates combination of low DO and low salinity impair the physiological activities more severely than the single stressed condition, i.e., hypoxia or hyposalinity (Fig. 5). Shell weight was reduced by low salinity and low DO independently. For salinity effect on shell weight, Nagarajan et al. (2006) explained that the calcium in the shell of marine shellfish dissolved in respond to changes of ecological conditions especially the salinity variations. Almada-Villele (1984) suggested that the processes of calcification and shell deposition were depressed at low salinities and mussels in less saline environments have thinner and weaker shells than those from higher saline habitat. Schlieper (1971) and Kautsky et al. (1990) showed that the dry weight and calcium carbonate content of mussel shells was lower in the hyposaline environment than highly saline waters. Earlier studies (Crenshaw and Neff, 1969; Akberali et al., 1977, 1983; Akberali, 1980) suggested that the reduction of salinity inflicts physiological stress of the mussels at cellular level. They close their valves and respire anaerobically under hyposaline condition, which yields the by-products of succinic acid and ammonia. To neutralise the metabolites, the mussels dissolve the calcium carbonate from their shells (Crenshaw and Neff, 1969). It is likely that respiratory switching causes acid production that dissolves away shells. In our experiments, hypoxia also resulted in low respiration and physiological constraint, and thus affected the shell performance. However, there was an interaction of DO and salinity on shell thickness, further one-way AVOVAs showed that the shell thickness was not influenced by DO and salinity under normal salinity and DO, respectively, indicating the shell thickness could be only affected by salinity when DO is less than 6.0 mg l1, and affected by DO when salinity is below 30& (Fig. 2). Salinity is a vital factor determining growth and distribution of marine organisms in aquatic environment, especially in variably saline habitats such as estuaries and coastal areas (Nagarajan et al., 2006, 2008). Low salinity in the field negatively influences the growth of mussels as well as their shell performance. Westerbom et al. (2002) investigated the distribution of mussels in relation to salinity gradient in the northeastern Baltic Sea and found a marked decline in mussel size and biomass from the saline west to the less saline east, which is similar to the distribution of P. viridis in Hong Kong. Severely low oxygen condition is a problem in many coastal and estuarine ecosystems where great salinity variation frequently exists. It is well known that hypoxia and low salinity increase mortality and/or force the extirpation of many species, whereas it is not well understood how they affect complex ecological relationships such as predatoreprey interactions interactively. This laboratory investigation emphasized the importance of evaluating effects of hypoxia and salinity on antipredator responses. The results provided evidences that the
vulnerability of P. viridis to predation increases with decreasing oxygen and salinity. Fewer byssal threads, thinner shell thickness and thinner adductor diameter decrease the time needed and difficulty for crabs to dislodge the mussels from the surrounding bed (Elner, 1978). Hence, food web dynamics between P. viridis and its predators and structure of marine soft-bottom communities could be changed under hypoxic conditions in coastal and estuarine waters. Acknowledgements The work described in this paper was supported by a grant from the Scientific Research Foundation, Shanghai Ocean University [B8812-11-0195 (gs1)] and a studentship from UGC of Hong Kong. References Addison, B., 2009. Shell traits of a marine mussel mediate predation selectivity by crabs and sea stars. Journal of Shellfish Research 28, 299e303. Akberali, H.B., Marriott, K.R.M., Trueman, E.R., 1977. Calcium utilisation during anaerobiosis induced by osmotic shock in a bivalve mollusc. Nature 266, 852e853. Akberali, H.B., Brear, K., Currey, J.D., 1983. Mechanical and morphological properties of the shell of Scrobicularia plana (Da Costa) under normal and stress conditions. Journal of Molluscan Studies 49, 93e97. Akberali, H.B., 1980. Calcium uptake and dissolution in the shell of Scrobicularia plana (da Costa). Journal of Experimental Marine Biology and Ecology 43, 1e9. Almada-Villele, P.C., 1984. The effects of reduced salinity on the shell growth of small Mytilus edulis. Journal of the Marine Biological Association of the United Kingdom 64, 171e182. Anger, K., Spivak, E., Luppi, T., 1998. Effects of reduced salinities on development and bioenergetics of early larval shore crab, Carcinus maenas. Journal of Experimental Marine Biology and Ecology 220, 287e304. Bell, G.W., Eggleston, D.B., 2005. Species-specific avoidance responses by blue crabs and fish to chronic and episodic hypoxia. Marine Biology 146, 761e770. Bell, G.W., Eggleston, D.B., Wolcott, T.G., 2003. Behavioral responses of free-ranging blue crabs to episodic hypoxia. II. Feeding. Marine Ecology Progress Series 259, 227e235. Brafield, A.E., 1963. The effects of oxygen deficiency on the behavior of Macoma balthica (L.). Animal Behaviour 11, 2e3. Bussell, J.A., Gidman, E.A., Causton, D.R., Gwynn-Jones, D., Malham, S.K., Jones, M.L.M., Reynolds, B., Seed, R., 2008. Changes in the immune response and metabolic fingerprint of the mussel, Mytilus edulis (Linnaeus) in response to lowered salinity and physical stress. Journal of Experimental Marine Biology and Ecology 358, 78e85. Carroll, J.L., Wells, R.M.G., 1995. Strategies of anaerobiosis in New Zealand infaunal bivalves: adaptations to environmental and functional hypoxia. New Zealand Journal of Marine & Freshwater Research 29, 137e146. Charmantier, G., Giménez, L., Charmantier-Daurès, M., Anger, K., 2002. Ontogeny of osmoregulation, physiological plasticity, and export strategy in the grapsid crab Chasmagnathus granulate (Crustacea, Decapoda). Marine Ecology Progress Series 229, 185e194. Cheung, S.G., Tong, P.Y., Yip, K.M., Shin, P.K.S., 2004. Chemical cues from predators and damaged conspecifics affect byssus production in the Green-lipped mussel Perna viridis. Marine and Freshwater Behaviour and Physiology 37, 127e135. Cheung, S.G., Luk, K.C., Shin, P.K.S., 2006. Predator-labeling effect on byssus production in marine mussels Perna viridis (L.) and Brachidontes variabilis (Krauss). Journal of Chemical Ecology 32, 1501e1512. Cheung, S.G., 1991. Energetics of transplanted populations of the green-lipped mussel Perna viridis (Linnaeus) (Bivalvia: Mytilacea) in Hong Kong. II: Integrated energy budget. Asian Marine Biology 8, 133e147. Condon, R.H., Decker, M.B., Purcell, J.E., 2001. Effects of low dissolved oxygen on survival and asexual reproduction of scyphozoan polyps (Chrysaora quinquecerrha). Hydrobiologia 451, 89e95. Cote, I.M., 1995. Effects of predatory crab effluent on byssus production in mussels. Journal of Experimental Marine Biology and Ecology 188, 233e241. Crenshaw, M.A., Neff, M.J., 1969. Decalcification at the mantle-shell interface in molluscs. American Zoologist 9, 881e885. Das, T., Stickle, W.B., 1993. Sensitivity of crabs Callinectes sapidus and C. similis and the gastropod Stramonita haemastoma to hypoxia and anoxia. Marine Ecology Progress Series 98, 263e274. Dauer, D.M., Rodi Jr., A.J., Ranasinghe, J.A., 1992. Effects of low dissolved oxygen events on the macrobenthos of the lower Chesapeake Bay. Estuaries 15, 348e391. De Goeij, P., Luttikhuizen, P.C., van der Veer, J., Piersma, T., 2001. Facilitation on an intertidal mudflat: the effect of siphon nipping by flatfish on burying depth of the bivalve Macoma balthica. Oecologia 126, 400e506. De Zwaan, A., 1977. Anaerobic energy metabolism in bivalve molluscs. Oceanography and Marine Biology: An Annual Review 15, 103e187.
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Rosenberg, R., Hellman, B., Johansson, B., 1991. Hypoxic tolerance of marine benthic fauna. Marine Ecology Progress Series 79, 127e131. Rosenberg, R., 1977. Benthic macrofaunal dynamics, production, and dispersion in an oxygen-deficient estuary of west Sweden. Journal of Experimental Marine Biology and Ecology 26, 107e133. Rowe, C.L., 2002. Differences in maintenance energy expenditure by two estuarine shrimp (Palaemonetes pugio and P. vulgaris) that may permit partitioning of habitats by salinity. Comparative Biochemistry and Physiology A 132, 341e351. Sagasti, A., Schaffner, L.C., Duffy, J.E., 2001. Effects of periodic hypoxia on mortality, feeding and predation in an estuarine epifaunal community. Journal of Experimental Marine Biology and Ecology 258, 257e283. Saloom, M.E., Duncan, R.S., 2004. Low dissolved oxygen levels reduce antipredation behaviours of the freshwater clam Corbicula fluminea. Freshwater Biology 50, 1233e1238. Schlieper, C., 1971. Physiology of brackish water. In: Remane, A., Schlieper, C. (Eds.), Biology of Brackish Water. Wiley Interscience, New York, pp. 211e350. Seed, R., Richardson, C.A., 1999. Evolutionary traits in Perna viridis (Linnaeus) and Septifer virgatus (Wiegmann) (Bivalvia: Mytilidae). Journal of Experimental Marine Biology and Ecology 239, 273e287. Seed, R., 1990. Predatoreprey relationships between the swimming crab Thalamita danae Stimpson (Decapoda: Portunidae) and the mussels Perna viridis (L.) and Brachidontes variabilis (Krauss). In: Morton, B. (Ed.), Proceedings of the Second International Marine Biological Workshop: The Marine Flora and Fauna of Hong Kong and southern China. Hong Kong University Press, Hong Kong, pp. 993e1013. Seed, R., 1993. Invertebrate predators and their role in structuring coastal and estuarine populations of filter feeding bivalves. In: Dame, R.F. (Ed.), Bivalve Filter Feeders in Estuarine and Coastal Ecosystem Processes. Springer-Verlag, Berlin, pp. 149e194. Seitz, R.D., Marshall, L.S., Hines, A.H., Clark, K.L., 2003. Effects of hypoxia on predatoreprey dynamics of the blue crab Callinectes sapidus and the Baltic clam Macoma balthica in Chesapeake Bay. Marine Ecology Progress Series 257, 179e188. Shafee, M.S., 1979. Ecological energy requirements of the green mussel, Perna viridis Linnaeus from the Ennore Estuary, Madras. Oceanologica Acta 2, 69e74. Siddall, S.E., 1980. A clarification of the genus Perna (Mytilidae). Bulletin of Marine Science 30, 858e870. Smith, L.D., Jennings, J.A., 2000. Induced defensive responses by the bivalve Mytilus edulis to predators with different attach modes. Marine Biology 136, 461e469. Stickle, W.B., Kapper, M.A., Liu, L.L., Gnaiger, E., Wang, S.Y., 1989. Metabolic adaptations of several species of crustaceans and mollusks to hypoxia: tolerance and microcalorimetric studies. Biological Bulletin 177, 303e312. Storey, K.B., Storey, J.M., 2004. Metabolic rate depression in animals: transcriptional and translational controls. Biological Reviews 79, 207e233. Tallqvist, M., 2001. Burrowing behaviour of the Baltic clam Macoma balthica: effects of sediment type, hypoxia and predator presence. Marine Ecology Progress Series 212, 183e191. Taylor, D.L., Eggleston, D.B., 2000. Effects of hypoxia on an estuarine predatoreprey interaction: mutual interference and foraging behavior of the blue crab (Callinectes sapidus) on infaunal clam prey (Mya arenaria). Marine Ecology Progress Series 196, 221e237. Thiyagarajan, V., Qian, P.Y., 2003. Effect of temperature, salinity and delayed attachment on development of the solitary ascidian Styela plicata (Lesueur). Journal of Experimental Marine Biology and Ecology 290, 133e146. Torres, G., Giménez, L., Anger, K., 2002. Effects of reduced salinity on the biochemical composition (lipid, protein) of Zoea 1 decapod crustacean larvae. Journal of Experimental Marine Biology and Ecology 277, 43e60. Wang, Y.J., Hu, M.H., Wong, W.H., Shin, P.K.S., Cheung, S.G., 2011. The combined effects of oxygen availability and salinity on physiological responses and scope for growth in the green-lipped mussel Perna viridis. Marine Pollution Bulletin 63, 255e261. Westerbom, M., Kilpi, M., Mustonen, O., 2002. Blue mussels, Mytilus edulis, at the edge of the range: population structure, growth, and biomass along a salinity gradient in the north-eastern Baltic Sea. Marine Biology 140, 991e999. Widdows, J., 1987. Application of calorimetric methods in ecological studies. In: James, A.M. (Ed.), Thermal and Energetic Studies of Cellular Biological Studies. Wright, Bristol, pp. 182e215. Wu, R.S.S., 2002. Hypoxia: from molecular responses to ecosystem responses. Marine Pollution Bulletin 45, 35e45.
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Chronic hypoxia and low salinity impair anti-predatory responses of the green-lipped mussel Perna viridis Youji Wang a, *, Menghong Hu a, *, S.G. Cheung b, P.K.S. Shin b, Weiqun Lu a, Jiale Li a a b
College of Fisheries and Life Science, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai 201306, China Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 January 2012 Received in revised form 11 February 2012 Accepted 14 February 2012
The effects of chronic hypoxia and low salinity on anti-predatory responses of the green-lipped mussel Perna viridis were investigated. Dissolved oxygen concentrations ranged from hypoxic to normoxic (1.5 0.3 mg l1, 3.0 0.3 mg l1 and 6.0 0.3 mg l1), and salinities were selected within the variation during the wet season in Hong Kong coastal waters (15&, 20&, 25& and 30&). The dissolved oxygen and salinity significantly affected some anti-predatory responses of mussel, including byssus production, shell thickness and shell weight, and the adductor diameter was only significantly affected by salinity. Besides, interactive effects of dissolved oxygen and salinity on the byssus production and shell thickness were also observed. In hypoxic and low salinity conditions, P. viridis produced fewer byssal threads, thinner shell and adductor muscle, indicating that hypoxia and low salinity are severe environmental stressors for self-defence of mussel, and their interactive effects further increase the predation risk. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords: Synergistic effects Green-lipped mussel Anti-predatory trait Hypoxia Salinity Byssus Shell thickness Shell weight Adductor muscle
1. Introduction Hypoxia, namely dissolved oxygen (DO) below 2 mg l1 in seawater induced by anthropogenic, physical and/or biological factors commonly occurs in many aquatic habitats such as estuaries, lakes, bays, deep sea and coastal waters almost worldwide (Diaz and Rosenberg,1995; Gray et al., 2002; Kidwell et al., 2009). Even though some hypoxic effects in shallow marine systems are not lethal, oxygen concentrations in the range of 1e4 mg l1 are physiologically and behaviourally stressful (Rombough, 1988; Long et al., 2008). Hypoxia causes a decrease in the density of motile animals that flee hypoxic waters (Pihl et al., 1991; Bell and Eggleston, 2005; Powers et al., 2005) and a decline in the abundance, biomass, and diversity of the sessile benthic community (Rosenberg, 1977; Gaston, 1985; Dauer et al., 1992; Llansó, 1992; Long et al., 2008). Benthic organisms can make some behavioural and physiological changes to resist hypoxic stress. They usually respond by increasing oxygen supply, reducing metabolism and oxygen consumption (Stickle et al., 1989; Wu, 2002). As a result, this could * Corresponding authors. Tel./fax: þ86 21 6190 0448. E-mail addresses: [email protected] (M. J. Wang), [email protected] (M.H. Hu).
lead to diminished growth and anti-predatory behaviour of the stressed organisms (Das and Stickle, 1993; Nilsson, 1999; Condon et al., 2001; Grove and Breitburg, 2005; Long et al., 2008). For instance, bivalves and polychaetes extend siphons or palps, reduce burial depth, or even float up above the benthic boundary layer into higher DO waters (Brafield, 1963; Rosenberg et al., 1991; Taylor and Eggleston, 2000; Seitz et al., 2003; Long et al., 2008). Epifaunal organisms lower their oxygen demand by reducing feeding and movement (Sagasti et al., 2001). These responses induced by hypoxia may have effects on the food web and trophic transfer if they increase an organism’s vulnerability to its predators. For example, extending siphons and palps farther and decrease of burial depth make detected by predators more likely for species that use sediments to obtain a refuge from predation, such as the clam Macoma balthica (De Goeij et al., 2001; Long et al., 2008) and Corbicula fluminea (Saloom and Duncan, 2004). In poorly oxygenated waters, prey may be more vulnerable to predators if their responses to attack are hindered, or their ability to use habitat refuge is reduced (Moore and Townsend, 1998; Taylor and Eggleston, 2000; Tallqvist, 2001; Saloom and Duncan, 2004). Salinity is an important abiotic factor that affects physiological activities, playing an important role in determining species distributions within estuarine and coastal systems (Rowe, 2002),
0141-1136/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2012.02.006
Y. Wang et al. / Marine Environmental Research 77 (2012) 84e89
especially in shallow waters where sessile invertebrates such as mussels live. In estuarine and coastal systems salinity can dramatically change across spatial and temporal scales (Kirkpatrick and Jones, 1985; Rowe, 2002). Marine invertebrates inhabiting estuarine and coastal areas are exposed to short-term (tidal) and long-term (rain periods) changes in salinity. Low salinity undoubtedly has a significant effect on many physiological processes in marine invertebrate, such as osmoregulation, active intracellular transport, feeding rate or nutritive absorption, respiration and excretion (Kinne, 1971). Due to the salinity fluctuation in the estuaries and coastal areas, some species in such areas presumably experience osmotic stress frequently, so they have to expend additional energy to maintain the haemolymph osmolality (Harris and Aladin, 1997). However, to this date, few anti-predatory studies related to salinity in bivalves have been investigated. Environmental stresses rarely occur in isolation and the interactive stress from natural and anthropogenic activity is known to affect the physiological response of bivalves (Bussell et al., 2008). The effects of stresses are frequently studied in isolation, while their combined effects are less well known, in particular whether hypoxia increases the susceptibility of mussels to other forms of stress such as reduced salinity. In view of this, Kidwell et al. (2009) proposed the importance and necessity of quantification of the interactive effect of hypoxia with other environmental stressors. Estuaries and coastal areas are highly stressful environments, with the benefits brought by a riverine supply of inorganic and organic nutrients countered by the associated stress of reductions in salinity. Estuarine mussels typically experience conditions of lowered salinity on every tidal cycle. However, during flood events they are likely to experience much lower salinities than normal and for a longer duration. In mussels, some anti-predation responses when exposed to predators have been reported, including increase of shell thickness in the presence of crabs and whelks (Leonard et al., 1999; Smith and Jennings, 2000; Reimer and Harms-Ringdahl, 2001), size of the adductor muscle in the presence of starfish (Reimer and HarmsRingdahl, 2001) and strength of byssal threads in the presence of crabs and starfish (Cote, 1995; Leonard et al., 1999; Seed and Richardson, 1999; Reimer and Harms-Ringdahl, 2001; Cheung et al., 2004). The green-lipped mussel Perna viridis is a dominant species in tropical and subtropical shallow areas of the Indo-Pacific region (Siddall, 1980) and occurs abundantly in low-energy habitats such as harbours and sheltered bays where the water is often hypoxic and sustains large salinity variation (Thiyagarajan and Qian, 2003). On Hong Kong’s rocky shores, small individuals of P. viridis are consumed preferentially by the portunid crab Thalamita danae which lives in subtidal waters and moves with each flood tide into the intertidal zone to feed (Seed, 1990). The role of hypoxia and low salinity, in determining the outcome of anti-predatory responses in mussels is not well known. To better understand whether chronic hypoxia and low salinity affect the anti-predatory defence of the green-lipped mussel P. viridis, we examined the shell performance, adductor muscle and byssus production of the P. viridis exposed to the portunid crab T. danae under different salinities and DO levels. 2. Materials and methods 2.1. Experimental animals Juvenile mussels (shell length: 26.05 1.55 mm; dry weight: 70.0 5.0 mg) and their potential predators T. danae (60e70 mm carapace width) were collected from a sheltered bay at Lok Wo Sha, Hong Kong. Upon return to the laboratory, the mussels were detached carefully from their clumps by cutting their byssal
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threads with scissors to avoid damaging the pedal apparatus which would impair their re-secretion. They were then maintained in a fibreglass tank (500 L) equipped with a filtering system and air supply and fed with the brown alga Thalassiosira pseudonana with a concentration of 5.0 105 cells ml1 in the tank once every two days. The seawater was maintained at 21 C and 30& throughout the acclimation and individuals of P. viridis and T. danae were allowed to acclimate to laboratory conditions for one week prior to experimentation. Before the experiment, the portunid crabs T. danae were fed with mussels everyday, and male crabs that consumed the same amount of mussels were selected as predators. 2.2. Experimental system The system was mainly composed of an experimental tank (18 L) with a middle compartment (12 L) and two side compartments (3 L), a digital dissolved oxygen controller (model no. 01972-00, ColeeParmer, Illinois, USA), a cylinder of compressed nitrogen and an air pump (Fig. 1). The oxygen level in the experimental tank was monitored automatically by the oxygen probe of the controller. When the desired oxygen level deviated from the preset value, the dissolved oxygen controller would send a signal to the valves connecting to the nitrogen gas tank or air pump so as to restore the desired oxygen level by delivering either nitrogen or air into the experimental tank. Three oxygen concentrations were selected: 1.5 0.3 mg l1 O2 as severely hypoxic, 3.0 0.3 mg l1 O2 as moderately hypoxic and 6.0 0.3 mg O2 l1 as normoxic level; and 4 levels of salinity (15&, 20&, 25& and 30&) were setup at each fixed DO level for total twelve treatments. Each treatment consisted of three replicates with 25 mussels per replicate. 2.3. Experimental design Twenty five mussels were kept in the middle compartment, and two male crabs T. danae were introduced to the two side compartments, respectively, avoiding physical contact between experimental mussels and their predators, but allowing chemical cues from the crabs to be detected by the mussels (Fig. 1). Experimental crabs were put into aquariums for 3 h daily. This experiment lasted for six weeks under controlled conditions as above. At the end of the experiment, the number of byssal threads that were secreted by each mussel was counted. Mussels were dried at 80 C to calculate shell weight, and shell thickness and adductor muscle diameter were measured using digital Vernier calipers. In our experiment, the crabs mostly attacked mussels by cleaving posterior shell lip, so we measured the shell thickness 1 mm from the centre posterior shell lip (Smith and Jennings, 2000). 2.4. Statistical analysis Each individual aquarium of the same treatment was considered a replicate (i.e., N ¼ 3), and a mean value for each replicate was calculated by averaging the pooled data in each aquarium. Data on evaluated parameters were statistically analysed using two-way analysis of variance (ANOVA). When interactions between the two main factors were present, one-way ANOVAs to test for differences among levels of one factor were tested at each level of the other factor, and Student Newman Keuls (SNK) tests were performed to determine which treatments were different. Differences were considered significant at P < 0.05. Prior to the analysis, normality of the data was evaluated by using the ShapiroeWilk’s test and homogeneity of variances was checked by Levene’s test using the statistical software SPSS 16.0. The results are expressed as the means S.D. of the data.
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Fig. 1. Experimental setup consists of DO controller, DO probe, air and nitrogen supplies, temperature regulator, water pump, protein skimmer and experimental tank (dimension: 40 cm 40 cm 12 cm). The oxygen level in the experimental tank was determined by the opening and closing of the valves connected to the air and nitrogen supplies.
3. Results During the experiment, only five mussels died (one at 3.0 mg O2 l1 salinity 25&, two at 1.5 mg O2 l1 salinity 20&, and two at 6.0 mg O2 l1 salinity 15&). Only adductor diameter was not affected by DO, and all other parameters were significantly affected by DO and salinity (Table 1). Significant interactions of DO and salinity on shell thickness and byssus number were observed (Table 1), and multiple comparisons between DO/salinity treatments at each fixed salinity/DO level were displayed in Fig. 3 and Fig. 5. DO showed no significant effect at salinity 30&, and salinity showed no significant effect at DO 6.0 mg l1 on shell thickness (Table 2, Fig. 3). The shell weight was gradually reduced with the drop in the DO and salinity. The lowest shell weight was observed from the DO 1.5 mg l1 salinity 15& treatment, and the highest was observed in the DO 6.0 mg l1 salinity 30& treatment (Fig. 2). The significant differences caused by DO and salinity were both detected, and the interaction between DO and salinity on shell weight was not significant (Table 1). The shell thickness varied significantly with dissolved oxygen and salinity, and interactive effect of DO and salinity was observed Table 1 Summary of two-way ANOVA results on effects of dissolved oxygen (DO) and salinity (S) on shell weight (SW), shell thickness (ST), adductor diameter (AD) and byssus number (BN). Dissolved oxygen: 1.5, 3.0 and 6.0 mg O2 l1; salinity: 15, 20, 25 and 30&. Source
df
MS
DO S DO*S Error
2 3 6 24
SW 0.047 0.111 0.005 0.005
F
P
8.794 20.629 0.901
0.001 <0.001 0.51
DO S DO*S Error
2 3 6 24
AD 0.015 0.146 0.008 0.005
3.088 31.143 1.605
0.064 <0.001 0.189
MS
F
(Table 1). Generally, the lower the salinity and dissolved oxygen, the thinner the shell thickness (Fig. 3). However, under normal DO or salinity conditions, salinity or DO could not significantly influence the shell thickness (Table 2, Fig. 3). Adductor diameter was not significantly different among DO treatments, but was significantly different among salinity treatments. The adductor diameter was greater under higher salinity treatments (Fig. 4). No interactive effect of dissolved oxygen and salinity was found on the adductor diameter (Table 1). Byssus number varied significantly with DO and salinity, and there was a significant interaction between dissolved oxygen and salinity (Table 1). Significant effects of DO were observed at each fixed salinity level and also salinity significantly affected the bysses number at each fixed DO level (Table 2). The lowest value was observed in the DO 1.5 mg l1 salinity 15& treatment, and the highest was seen in the DO 6.0 mg l1 salinity 30& treatment (Fig. 5). 4. Discussion At low salinity and DO levels, the mussels displayed poor growth in shell weight and adductor, compared to those reared at high salinity and DO levels. The low growth rate found at low salinity and DO seems to be the result of various physiological constraints
P
ST 0.005 0.01 0.001 0.000 BN 1362.661 796.036 69.308 6.29
16.714 35.387 3.44
<0.001 <0.001 0.014
216.639 126.556 11.019
<0.001 <0.001 <0.001
Fig. 2. Shell weight (SW) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM).
Y. Wang et al. / Marine Environmental Research 77 (2012) 84e89
87
Fig. 3. Shell thickness (ST) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM). Different capital letters denote significant differences between three DO treatments at each salinity level, and different small letters denote significant differences between four salinity treatments at each DO level.
Fig. 5. Byssus number (BN) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM). Different capital letters denote significant differences between three DO treatments at each salinity level, and different small letters denote significant differences between four salinity treatments at each DO level.
(Wang et al., 2011). Reduced anti-predatory traits were found after continued exposure to salinity 15& for six weeks, but the mussels were able to survive under low salinity. This indicates a limited euryhalinity of P. viridis and its osmoregulatory capacity could not compensate prolonged effects of low salinity on juvenile growth, which was also reported in other juvenile invertebrates (Charmantier et al., 2002; Torres et al., 2002). Unfavourably low salinity may cause a reduction of feeding or growth efficiency, presumably due to metabolic adjustments induced by osmotic stress. For instance, significant reduction in the metabolism of the Carcinus maenas exposed to low salinity caused reduced growth (Anger et al., 1998). Juvenile P. viridis can also survive six weeks under hypoxia (1.5 mg l1), highlighting its strong tolerance to hypoxia at the expense of growth. Some bivalves have efficient strategies for anaerobic energy production (De Zwaan, 1977) and may reduce their metabolic rate in response to hypoxia (Storey and Storey, 2004). Carroll and Wells (1995) reported that intertidal species like Paphies australis are well adapted to environmental hypoxia. Sessile bivalves generally show a strong resistance to hypoxia, due in part to a reduction in metabolic activity and energy utilization (Widdows, 1987). In anti-predatory studies of mussels, the byssus production, shell thickness and adductor muscle diameter are often used as robust indexes for the effects of biotic/abiotic factors (Cote, 1995; Leonard et al., 1999; Nagarajan et al., 2006, 2008; Fässler and Kaiser, 2008). Mussels are sedentary organisms attached to solid substrata by means of byssus threads. It is generally agreed that an increase in the number of the byssus threads is an anti-predatory response of the mussel exposure to predators (Cheung et al., 2004). Also the shell thickness and adductor muscle diameter of a prey item are strongly related to its resistance to predators’ attacks (Smith and Jennings, 2000; Addison, 2009). Mussels are the major diet of crabs T. danae which employ different modes of attack, depending on the overall size and relative shell thickness of the mussel (Seed,
1993). Crabs are able to cut byssal threads and shell lips using their claws and dislodge individuals from a mussel bed (Elner, 1978). Mussels with firmer attachment and thicker shell and adductor, therefore, are less likely to be dislodged and attacked by crabs and mortality is thus reduced (Lin, 1991; Hughes and Seed, 1995). In contrast, decreased adductor, shell and byssus performance, therefore, may increase predation rate by crabs (Smith and Jennings, 2000; Nagarajan et al., 2006; Farrell and Crowe, 2007). In the present study, the green-lipped mussel produced fewer byssus, thinner shell and adductor when exposed to low salinity and low DO than those in high salinity and high DO, indicating that under hypoxic and hyposaline conditions, the anti-predatory responses were impaired. Whether predators are capable of taking advantage of the stressed prey is a matter of debate, as predators may leave stressful areas (Pihl et al., 1991; Bell and
Fig. 4. Adductor diameter (AD) of P. viridis exposed to three different dissolved oxygen concentrations and four salinities for six weeks. Vertical whiskers indicate standard deviation of mean (SDM).
Table 2 Summary of one-way ANOVA results on effects of dissolved oxygen (DO) at each fixed salinity level and effects of salinity (S) at each fixed DO level, on shell thickness (ST) and byssus number (BN). Dissolved oxygen: 1.5, 3.0 and 6.0 mg O2 l1; salinity: 15, 20, 25 and 30&. df DO effect ST
S15 Error S20 Error S25 Error S30 Error Salinity effect ST DO1.5 Error DO3.0 Error DO6.0 Error DO effect BN S15 Error S20 Error S25 Error S30 Error Salinity effect BN DO1.5 Error DO3.0 Error DO6.0 Error
MS
F
P
2 6 2 6 2 6 2 6
0.003 0.000 0.003 0.000 0.002 0.000 0.000 0.000
9.619
0.013
9.601
0.013
5.414
0.045
0.944
0.440
3 8 3 8 3 8
0.004 0.000 0.007 0.000 0.001 0.000
26.694
<0.001
33.417
<0.001
2.014
0.191
2 6 2 6 2 6 2 6
71.574 3.181 221.223 2.666 669.623 2.506 608.164 16.808
22.500
0.002
82.993
<0.001
267.255
<0.001
36.184
<0.001
3 8 3 8 3 8
74.820 2.826 242.290 10.119 617.543 5.925
26.477
<0.001
23.944
<0.001
104.227
<0.001
88
Y. Wang et al. / Marine Environmental Research 77 (2012) 84e89
Eggleston, 2005). However, predator behaviour in systems that suffer from hypoxia could lead to an increase in foraging after a hypoxic episode (Bell et al., 2003; Long et al., 2008). Motile predators can rapidly reinvade areas after relaxation of hypoxic conditions (Pihl et al., 1991; Nestlerode and Diaz, 1998; Bell and Eggleston, 2005). They may take advantage of stressed benthic organisms following hypoxia. For example, the diet of T. danae includes a high percentage (53.9%) of mussel P. viridis (Cheung et al., 2006). Besides, the predators of mussel include other predators like starfish and oystercatcher (Cheung et al., 2004; Nagarajan et al., 2008), which would undoubtedly increase the predation risk of mussel under that situation. Byssal threads constitute a significant component of total production by mussels, accounting for 3e10% of annual production in P. viridis (Shafee, 1979; Cheung, 1991). At each fixed level of DO and salinity, the byssus production was significantly reduced by low salinity and low DO, respectively, and there was an interactive effect on byssus production, which indicates combination of low DO and low salinity impair the physiological activities more severely than the single stressed condition, i.e., hypoxia or hyposalinity (Fig. 5). Shell weight was reduced by low salinity and low DO independently. For salinity effect on shell weight, Nagarajan et al. (2006) explained that the calcium in the shell of marine shellfish dissolved in respond to changes of ecological conditions especially the salinity variations. Almada-Villele (1984) suggested that the processes of calcification and shell deposition were depressed at low salinities and mussels in less saline environments have thinner and weaker shells than those from higher saline habitat. Schlieper (1971) and Kautsky et al. (1990) showed that the dry weight and calcium carbonate content of mussel shells was lower in the hyposaline environment than highly saline waters. Earlier studies (Crenshaw and Neff, 1969; Akberali et al., 1977, 1983; Akberali, 1980) suggested that the reduction of salinity inflicts physiological stress of the mussels at cellular level. They close their valves and respire anaerobically under hyposaline condition, which yields the by-products of succinic acid and ammonia. To neutralise the metabolites, the mussels dissolve the calcium carbonate from their shells (Crenshaw and Neff, 1969). It is likely that respiratory switching causes acid production that dissolves away shells. In our experiments, hypoxia also resulted in low respiration and physiological constraint, and thus affected the shell performance. However, there was an interaction of DO and salinity on shell thickness, further one-way AVOVAs showed that the shell thickness was not influenced by DO and salinity under normal salinity and DO, respectively, indicating the shell thickness could be only affected by salinity when DO is less than 6.0 mg l1, and affected by DO when salinity is below 30& (Fig. 2). Salinity is a vital factor determining growth and distribution of marine organisms in aquatic environment, especially in variably saline habitats such as estuaries and coastal areas (Nagarajan et al., 2006, 2008). Low salinity in the field negatively influences the growth of mussels as well as their shell performance. Westerbom et al. (2002) investigated the distribution of mussels in relation to salinity gradient in the northeastern Baltic Sea and found a marked decline in mussel size and biomass from the saline west to the less saline east, which is similar to the distribution of P. viridis in Hong Kong. Severely low oxygen condition is a problem in many coastal and estuarine ecosystems where great salinity variation frequently exists. It is well known that hypoxia and low salinity increase mortality and/or force the extirpation of many species, whereas it is not well understood how they affect complex ecological relationships such as predatoreprey interactions interactively. This laboratory investigation emphasized the importance of evaluating effects of hypoxia and salinity on antipredator responses. The results provided evidences that the
vulnerability of P. viridis to predation increases with decreasing oxygen and salinity. Fewer byssal threads, thinner shell thickness and thinner adductor diameter decrease the time needed and difficulty for crabs to dislodge the mussels from the surrounding bed (Elner, 1978). Hence, food web dynamics between P. viridis and its predators and structure of marine soft-bottom communities could be changed under hypoxic conditions in coastal and estuarine waters. Acknowledgements The work described in this paper was supported by a grant from the Scientific Research Foundation, Shanghai Ocean University [B8812-11-0195 (gs1)] and a studentship from UGC of Hong Kong. References Addison, B., 2009. Shell traits of a marine mussel mediate predation selectivity by crabs and sea stars. Journal of Shellfish Research 28, 299e303. Akberali, H.B., Marriott, K.R.M., Trueman, E.R., 1977. Calcium utilisation during anaerobiosis induced by osmotic shock in a bivalve mollusc. Nature 266, 852e853. Akberali, H.B., Brear, K., Currey, J.D., 1983. Mechanical and morphological properties of the shell of Scrobicularia plana (Da Costa) under normal and stress conditions. Journal of Molluscan Studies 49, 93e97. Akberali, H.B., 1980. Calcium uptake and dissolution in the shell of Scrobicularia plana (da Costa). Journal of Experimental Marine Biology and Ecology 43, 1e9. Almada-Villele, P.C., 1984. The effects of reduced salinity on the shell growth of small Mytilus edulis. Journal of the Marine Biological Association of the United Kingdom 64, 171e182. Anger, K., Spivak, E., Luppi, T., 1998. Effects of reduced salinities on development and bioenergetics of early larval shore crab, Carcinus maenas. Journal of Experimental Marine Biology and Ecology 220, 287e304. Bell, G.W., Eggleston, D.B., 2005. Species-specific avoidance responses by blue crabs and fish to chronic and episodic hypoxia. Marine Biology 146, 761e770. Bell, G.W., Eggleston, D.B., Wolcott, T.G., 2003. Behavioral responses of free-ranging blue crabs to episodic hypoxia. II. Feeding. Marine Ecology Progress Series 259, 227e235. Brafield, A.E., 1963. The effects of oxygen deficiency on the behavior of Macoma balthica (L.). Animal Behaviour 11, 2e3. Bussell, J.A., Gidman, E.A., Causton, D.R., Gwynn-Jones, D., Malham, S.K., Jones, M.L.M., Reynolds, B., Seed, R., 2008. Changes in the immune response and metabolic fingerprint of the mussel, Mytilus edulis (Linnaeus) in response to lowered salinity and physical stress. Journal of Experimental Marine Biology and Ecology 358, 78e85. Carroll, J.L., Wells, R.M.G., 1995. Strategies of anaerobiosis in New Zealand infaunal bivalves: adaptations to environmental and functional hypoxia. New Zealand Journal of Marine & Freshwater Research 29, 137e146. Charmantier, G., Giménez, L., Charmantier-Daurès, M., Anger, K., 2002. Ontogeny of osmoregulation, physiological plasticity, and export strategy in the grapsid crab Chasmagnathus granulate (Crustacea, Decapoda). Marine Ecology Progress Series 229, 185e194. Cheung, S.G., Tong, P.Y., Yip, K.M., Shin, P.K.S., 2004. Chemical cues from predators and damaged conspecifics affect byssus production in the Green-lipped mussel Perna viridis. Marine and Freshwater Behaviour and Physiology 37, 127e135. Cheung, S.G., Luk, K.C., Shin, P.K.S., 2006. Predator-labeling effect on byssus production in marine mussels Perna viridis (L.) and Brachidontes variabilis (Krauss). Journal of Chemical Ecology 32, 1501e1512. Cheung, S.G., 1991. Energetics of transplanted populations of the green-lipped mussel Perna viridis (Linnaeus) (Bivalvia: Mytilacea) in Hong Kong. II: Integrated energy budget. Asian Marine Biology 8, 133e147. Condon, R.H., Decker, M.B., Purcell, J.E., 2001. Effects of low dissolved oxygen on survival and asexual reproduction of scyphozoan polyps (Chrysaora quinquecerrha). Hydrobiologia 451, 89e95. Cote, I.M., 1995. Effects of predatory crab effluent on byssus production in mussels. Journal of Experimental Marine Biology and Ecology 188, 233e241. Crenshaw, M.A., Neff, M.J., 1969. Decalcification at the mantle-shell interface in molluscs. American Zoologist 9, 881e885. Das, T., Stickle, W.B., 1993. Sensitivity of crabs Callinectes sapidus and C. similis and the gastropod Stramonita haemastoma to hypoxia and anoxia. Marine Ecology Progress Series 98, 263e274. Dauer, D.M., Rodi Jr., A.J., Ranasinghe, J.A., 1992. Effects of low dissolved oxygen events on the macrobenthos of the lower Chesapeake Bay. Estuaries 15, 348e391. De Goeij, P., Luttikhuizen, P.C., van der Veer, J., Piersma, T., 2001. Facilitation on an intertidal mudflat: the effect of siphon nipping by flatfish on burying depth of the bivalve Macoma balthica. Oecologia 126, 400e506. De Zwaan, A., 1977. Anaerobic energy metabolism in bivalve molluscs. Oceanography and Marine Biology: An Annual Review 15, 103e187.
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