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1 Advances in Environmental Biology, 6(7): , 2012 ISSN This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Effects of stress tests on larvae of blue swimming crab, Portunus pelagicus (Linnaeus, 1758) 1,2 Allah Dad Talpur, 2 Mhd. Ikhwanuddin 1 Department of Fisheries, Government of Sindh, Pakistan. 2 Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia Allah Dad Talpur, Mhd. Ikhwanuddin: Effects of stress tests on larvae of blue swimming crab, Portunus pelagicus (Linnaeus, 1758) ABSTRACT Studies of stress tolerance in marine organisms are key in considerate effects on larval survival. A change between environmental factors has been assumed the first mechanism restricting survival of larvae. Therefore, Zoea I and Zoea 2 larvae of P. pelagicus were exposed to various regimes of activity stress tests such as oxygen, starvation, ph, temperature, and salinity to examine larval competency against these factors. Larval performance was affected at extreme increase or by decreases in stress activity. In oxygen test, no survival achieved in treated groups. However, only some Zoea 2 survived in starvation test. Temperature 30 o C did produce highest survival (p<0.05) and elevated temperature stress adversely affected larvae and no survival was achieved at temperature 40 o C and 45 o C respectively. Low ph 4, 6 and higher ph 10 did affect negatively, thus no survival of larvae, and only ph 8 did produce better survival (p<0.05). However, salinity greatly influenced the larval survival and only low survival 4.67±1.15% of Zoea 1 larvae and 5.33±1.53% of Zoea 2 determined at salinity 40 ppt was not significantly different (p>0.05). The significantly highest survival (p<0.05) of larvae was achieved in untreated groups (controls). The findings of this study indicate that the larval survival of P. pelagicus was compromised with certain level of stressor, elevated and low stressor had shown unfavourable effect on larval survival. Key words: Mechanism, Starvation, Performance, Negatively, Influenced, stressor, Unfavourable Introduction Blue swimming crab, P. pelagicus is one of the most common species of Malaysia, with a distribution extending from the East Malaysia to Peninsula Malaysia, which occupies a wide diversity of aquatic habitats from nearly marine to estuarine waters. The adaptation P. pelagicus to different and most often varying environments has resulted in an incline of their competency and life cycle approach. Different environmental factors directly or indirectly affect the larval survival. Oxygen demand and the limited capacity of oxygen supply to be the first device restricting survival at the limits of the thermal tolerance pane of marine organisms [1,2,3]. Starvation may, however, also an important mortality agent [4] as dietary status could influence different features of larval survival. Starved larvae with reduced energy reserves have less energy to assign to defence mechanism avoidance against pathogenic or predatory attacks or physical death. It has been reported by various researchers that starved fish larvae are more susceptible to die than fed larvae [5,6,7]. Among the environmental parameters, ph is one of the important factor the directly or indirectly influence the metabolism and other physiological processes of an organisms. Decreased in ph values result in toxicity of culture water by increasing nitrite and hydrogen sulphide contents. Therefore, stable ph levels in culture system play fundamental role. Like other environmental factors, temperature is one of key element in living life. Based on studies of thermal tolerance of adults, water temperature can strongly influence larvae by affecting survival, developmental time and growth [8,9]. However, the fact that larvae might experience temperature fluctuations and are more vulnerable to thermal and osmotic stresses than adults [10]. (Among others, one of major factors accounting for larvae stress is salinity, as osmoregulatory capability develops throughout the larval sequence of stages [11], and most newly-hatched stages are regarded as being more sensitive to low salinity [12]. Larval survival is therefore, sturdily affected by temperature and salinity [13,14], although each species tolerance will be specific for its degree of adaptation to the environmental gradients of coastal systems. The variability of water masses, which affects larvae during early development, may have a major impact on their survival and animals those are susceptible to Corresponding Author Allah Dad Talpur., Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030, Kuala Terengganu, Malaysia. Tel: (+60) , (+60) , Fax: (+60) mrtalpur@yahoo.com, ikwanuddin@umt.edu.my.

2 stress tests, a correlate can be achieved between animal activity and larval capability [15,16]. The objective of this study was to develop an activity tests comprising different stressors to assess the capability of newly-hatched (Zoea 1) and four day after hatch (Zoea 2) of P. pelagicus larvae for culture activity. Additionally the competence of larvae exposed to elevated stressors was examined. At present, there is no information whether P. pelagicus larvae are competent of such fortitude. In the present study to determine the responsive behaviour of P. pelagicus larval physiological condition using a stress tests comprising, oxygen, starvation, ph, temperature and salinity stresses were conducted. To achieve this objective, a factorial designed experiment was carried out under desired conditions. Materials and Methods Seawater for broodstock and larviculture: Ultraviolet (UV) treated seawater for broodstock and larviculture was filtered through a 10 µm net and sterilise/disinfected with sodium hypochlorite for 24 hours (h). This procedure was followed by supplementation of chelating agent, Ethylenediaminetetraacetic acid (EDTA) 100 gm per ton for 12 hours ensure clarity of seawater and neutralization with sodium thiosulphate (at same concentrations of sodium hypochlorite) at the beginning of the experiment for broodstock and larviculture use. The culture water exchange began from the day second, using disinfected seawater (28 ppt). Broodstock, study site and experimental larvae: Gravid females were collected from Strait of Tebrau (1 o 22 N and 103 o 38 E), Johor, West Malaysia and transported to marine hatchery of Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, Malaysia for breeding. Females were disinfected according to Talpur et al. [17] and kept in 300 L capacity hatching tanks filled with disinfected seawater (28 ppt) provided with sand substrate and equipped with mild aeration. Temperature of hatching tanks was maintained constant to 28 o C using submersible heaters. Hatching tanks were siphoned and cleaned daily accompanied with about a % water exchange (treated). After hatching, the female crab was removed from the incubation tank, using a scoop net, and the aeration was turned off in order to settle down any debris to the bottom of the incubation tank, while the energetic zoea remained active at the surface. Newly hatched zoea were then collected from the incubation tank near the surface of the water column using a 1L glass beaker and kept in 5 L aquarium filled with sterilise seawater. Zoea 1 (Z1) and Zoea 2 (4 day 1910 after hatch) actively moving at surface water column were collected and used as experimental larvae in this study. Experimental setup: The test vessels 1L aquaria containing sterilise/disinfected seawater (28ppt), which was filled with the desired stressor and controls were with larvae and sterilise seawater without stressor. A quantity of 20 zoea were then randomly picked and individually pipetted into each of 3 replicate test vessels. Except starvation test larvae tested groups and controls were fed with a mixture of live prey composed of rotifers ml 1 (Brachionus sp.) and microalgae Nannochloropsis sp (8x10 5 cells ml - 1 ). Following tests were performed; Oxygen Test: During the tests the larvae were not fed, no aeration was provided and temperature was maintained at 28 o C. To reduce evaporation and oxygen from atmosphere, each test vessel was covered with an aluminium foil during the duration of the test. This test was conducted for 0-4 hours, controls were with larvae and sterilise seawater with aeration. Starvation test: Larvae were transferred to 1L aquaria containing sterilise seawater (28 ppt) with the help of pipette. No feeding was provided to test larvae and each aquarium was equipped with aeration. Control was given live prey feed of rotifers and microalgae Nannochloropsis sp. Test period was set for 48 h. Temperature test: Temperature range was set 30, 35, 40 and 45 o C. Time period was set 12 hours. Control was at ambient temperature 28 o C and each aquarium was equipped with aeration. ph challenges test: Different ph were used to test the larvae. ph range was 4, 6, 8 and 10. However control was at natural ph. Sulfuric acid (H 2 SO 4 ) was used to reduce the ph to acid condition while quicklime (CaO) was used to increase the ph values. Temperature of test vessels was set to 28 o C ph and reading was recorded using YSI 556 MPS multi probe meter (USA). Time period was set 24 hour after that mortality was recorded. All treated and control vessel was equipped with aeration. Salinity challenge test:

3 Salinity was maintain 0, 40, 60 and 80 ppt. Sterilise zero water was added to sterilise freshwater to achieve salinity 0 was used as 0 salinity. Hyper salinity was achieved by adding sea salt in seawater then sterilise. Control and treated groups were maintained at 28 ppt and each aquarium was equipped with aeration. Data analyses: Effects of various stressors on survival of larval stages among treatment differences were tested by 2- way factorial ANOVA and Tukey s multiple comparison tests were used to identify the significantly different mean values (P < 0.05). Percent cumulative survivals were arc sin square root transformed to approximate normality. All statistics were performed using SPSS version 16 for Windows Data were presented as means and standard deviation (SD). Results: Survival of larvae: Different challenge produced different effects on the survival of larvae when tested against respective challenge/stress test. Oxygen Test: During the oxygen test, it was observed that no survival achieved in treated groups. All larvae survived in control group were statistically significant (p<0.05) (Figure 1). Oxygen in treated tanks was < 0.5 mg L -1 and in control it was >6 mg L -1. Fig. 1: Larval survival during oxygen test of Zoea 1 and Zoea 2 of P. pelagicus. Bars showing same superscript were statistically significant (p<0.05). Results were represented as mean of triplicate observations. Starvation Test: During 48 h starvation test, no Zoea 1 survived in treated group and only 2.67% Zoea 2 survived in challenge group was not statistically significant (p>0.05). However, higher survival was achieved in controls were statistically significant (p<0.05) (Figure 2). Fig. 2: Larval survival during starvation test of Zoea 1 and Zoea 2 of P. pelagicus. Bars showing same superscript were statistically significant (p<0.05). Results were represented as mean of triplicate observations.

4 Temperature test: No survival of larvae was achieved in temperature groups at 40 o C and 45 o C respectively. Thus, highest survival of Zoea 1 and Zoea 2 in challenge groups observed at temperature 30 o C were 1912 statistically significant (p<0.05). However, at temperature 35 o C half of Zoea 2 died and only 11.33% Zoea 1 did survive during the challenge assay were not statistically significant (p>0.05). Highest survival was achieved in controls were statistically significant (p<0.05) Table 1. Table 1: Survival of P. pelagicus larvae exposed to temperature stress. Results were represented as Mean ± SD (standard deviation) of triplicate observations. Temperature Test Test Time Zoea 1 Zoea 2 Treated Control Treated Control 30 0 C 12 h 20.00±0.00 a 19.67±0.578 a 19.67±5.78 a 19.67±5.78 a 35 0 C -do ±1.53 b 19.67±0.58 a 10.33±0.58 c 20.00±0.00 a 40 0 C -do- 0.00±0.00 b 19.33±1.15 a 0.00±0.00 c 18.00±1.00 a 45 0 C -do- 0.00±0.00 b 19.67±0.58 a 0.00±0.00 c 17.33±1.53 a Note: Values showing same superscript in same row were statistically significant (p<0.05). ph test: No survival of Zoea1 and Zoea 2 determined in challenge groups at ph 4, 6 and 10 respectively. Only ph 8 did produce survival 18.67% of Zoea 1 and 16.67% of Zoea 2 respectively were statistically significant (p<0.05). Control (ph 8.20) did produce highest survival than challenge groups were also statistically significant (p<0.05) Table 2. Salinity Test: No survival of larvae was observed in challenge groups treated at salinity 0, 60 and 80 ppt respectively. However, salinity 40 ppt did produce low survival 4.67% of Zoea 1 and 5.33% of Zoea 2 respectively during challenge assay were statistically significant (p>0.05).. Survival in controls was not majorly affected were statistically significant (p<0.05) Table 3. Table 2: Survival of P. pelagicus larvae exposed to ph stress test. Results were represented as Mean ± SD (standard deviation) of triplicate observations. ph Test Test Time Zoea 1 Zoea 2 Treated Control Treated Control ph 4 24h 0.00±0.00 a 18.00±1.00 b 0.00±0.00 c 17.00±1.00 b ph 6 -do- 0.00±0.00 a 18.00±1.00 b 0.00±0.00 c 17.67±1.53 b ph 8 -do ±1.53 a 18.33±1.53 a 16.67±2.08 a 17.33±1.53 a ph 10 -do- 0.00±0.00 a 17.67±0.58 b 0.00±0.00 a 16.00±1.73 b Note: Values showing same superscript in same row were statistically significant (p<0.05). Table 3: Survival of P. pelagicus larvae exposed to Salinity stress test. Results were represented as Mean ± SD (standard deviation) of triplicate observations. Salinity Test Test Time Zoea 1 Zoea 2 Treated Control Treated Control salinity 0 2h 0.00±0.00 a 20.00±0.00 b 0.00±0.00 c 20.00±0.00 a Salinity 40 -do- 4.67±1.15 a 20.00±0.00 b 5.33±1.53 c 20.00±0.00 b Salinity 60 -do- 0.00±0.00 a 19.67±0.58 b 0.00±0.00 a 20.00±0.00 b Salinity 80 -do- 0.00±0.00 a 19.33±1.15 b 0.00±0.00 c 19.67±0.58 b Note: Values showing same superscript in same row were statistically significant (p<0.05). Discussion: Stress is known to weaken the immune mechanism in fish [18]. Stressed organism, as a result of stressors, witness many physiological changes which can lead to metabolic discrepancy, increases in protein hydrolysis, increase released of cortisol from adrenal tissue with attendant biochemical exhaustion and immune suppression and in rearing conditions are susceptible to pathogens [19]. The Japanese scientists Watanabe et al., [20] developed the concept of instantaneously evaluating larval competency using an activity test. It has subsequently been adapted for use in crustacean and finfish hatcheries to ascertain the susceptibility of young animals to stress [15, 16]. While salinity is the primary stress used in many activity tests [15, 16], in this study other stress were also incorporated to accentuate stress effect. Although temperature manipulation has the ability to extend larval availability, it is not known whether larval competency relating to growth and survival is also altered [21]. Challenge tests were proposed as meaningful tools for assessing fish quality in the aquaculture industry, environmental resources management and in research [22]. The concept is based on the presumptions that stress loading above the acclimation capacity of an organism will weaken it and reduce performance in growth, survival and reproduction, and that the reduction in performance can be quantified by assessing tolerance to reference stressors [23]. Stress challenges have been widely used in crustacean aquaculture as a quality control measure [24].

5 Dissolving of oxygen in the culture water not only used for respiration purpose by aquatic organisms but it also maintained required chemical and hygienic environment of the rearing water. It controls many of the oxidation reactions and maintains aerobic conditions in water. It is believed that low oxygen level produce the anaerobic conditions exist; which usually cause the nitrate into toxic ammonia and increases the ph. Moreover, reduction in oxygen level impedes metabolic activities of larvae, could reduce growth, moulting process and grounds for mortality [25]. No oxygen was provided in treated groups during present study, which resulted total life loss. However, continuous aeration was supplied to control; therefore, the oxygen level did not vary significantly was >6 mg L - 1 and no mortality observed. The results of present study suggest that oxygen level less then <0.5 mg ml -1 in treated water was not sufficient for larvae of P. pelagicus crab, hence died. Starvation has important effects on early development of fish. It determines the survival and growth of fish larvae, plays an important role in the dynamics of fish population and fisheries recruitment. During food deprivation, larvae of many fish species reach a stage of physical worsening, which mostly led them to death. Although some unfed zoea 2 larvae in the present study were alive and less active but in Zoea 1 groups no larvae survived at the end of the experiment, which prove that Zoea 1 larvae were not resistant to starvation because of their less nutritional reserves, therefore they required food for their survival. First-feeding larvae, resistance to food deprivation is proportional to the amount of energy reserves available i.e yolk sac [26]. The most likely explanation for this could be that either starved Zoea 1 utilised the egg yolk or the nutritional reserves were insufficient to survive further. Another possible explanation could be the predation in starved Zoea 1 that might affect predation mortality or injured larvae died due to pathogenic attack. Studies of yolk sac larvae have shown that starvation increases vulnerability to predation [27, 28, 29]. Low survival in Zoea 2 treatment was owing to appetite and predation (cannibalism). Therefore, it could be possible that predation and less nutritional reserves might have affected the survival of P. pelagicus larvae in treated groups. Among the environmental variables, water temperature is most likely important factor for larval rearing, because it directly affects metabolism, oxygen consumption, growth, feeding rate, morphology, size, incidence of deformity development, survival, elemental and biochemical composition of larvae during their early life history [30, 31, 32, 33, 34]. In general, a sudden change of temperature affects the larval immune system. The temperature stress treatments in the present study were between 30 ºC to 45 o C over the control 28 o C Temperature profoundly influenced survival of crab larvae in this study. Survival was determined well at temperature 30 o C and in control. Change in 5 o C resulted escalated mortalities among the larvae. 35 o C did produce almost half mortality and elevated temperature 40 o C and 45 o C resulted zero survival. The study demonstrated that temperature is one of the most environmental factor that direct influences the larval survival. The optimum temperature for larval survival of P. pelagicus from the study was between 28 C (control) and 30 C in treated group. Higher temperatures caused detrimental effect on larval survival. ph of the culture medium have vital role in metabolism and other physiological processes of an organisms. In rearing system of organisms, it changes owing to residual feed, and excreta of organisms. Toxicity of nitrite and hydrogen sulphide increased when ph decreased. The required range of ph for crustacean larval culture is [35]. In the present study, the ph level was ambient in the control tank and stressor ph was in treated tanks. The results did show that ph in the experimental tanks as stressor was not helpful in larval survival. Lower ph range from 4-6 contributed no survival both in Zoea 1 and Zoea 2, however higher ph 10 was also detrimental to larvae produced zero survival. Larvae were survived well at ph 8 and in control Results of present study demonstrated that acidic ph and higher alkaline ph have adverse effect on the larval survival and is one of cause among the mortality of crab larvae. Fluctuation in ph will adversely effect on larval survival, therefore stable ph range should be ensured for the survival of larvae. Salinity is the most important factor influencing many functional responses of the organisms as metabolism, growth, migration, osmotic behaviour, reproduction etc. Marine organisms maintain their blood and body fluids salt concentration by osmoregulation. They need considerable energy for osmoregulation to maintain their internal salt balance in relation to the external medium in which they are living. The application of salinity test to crustacean larvae is a common method exposing animal to an adverse condition to respond their competency [ [16, 24, 36]. The results of present study suggested that larvae exposed to salinity 0, 60 and 80 ppt did not survive. However, a low survival of Zoea 1 and Zoea 2 was obtained at salinity 40 ppt. The selection of salinity and temperature stressors were considered appropriate to determine a capability response and the perception behind the stress test was that larvae that are more efficient would have a better ability to maintain osmotic performances under the additional pressure of elevated salinity, temperature and other stressors. It was clear from the study that temperature and salinity have profound effect on larval survival. Elevated temperature and salinity exerted adverse

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