O A RIGINAL

1909
Advances in Environmental Biology, 6(7): 1909-1915, 2012
ISSN 1995-0756
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, 2Mhd. Ikhwanuddin
1
2
Department of Fisheries, Government of Sindh, Pakistan.
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 30oC did produce highest survival
(p<0.05) and elevated temperature stress adversely affected larvae and no survival was achieved at temperature
40oC and 45oC 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) 16-950 262, (+60) 9-668 3638, Fax: (+60) 9-668 3390.
E-mail: [email protected], [email protected].
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Adv. Environ. Biol., 6(7): 1909-1915, 2012
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.
after hatch) actively moving at surface water column
were collected and used as experimental larvae in
this study.
Materials and Methods
Oxygen Test:
Seawater for broodstock and larviculture:
During the tests the larvae were not fed, no
aeration was provided and temperature was
maintained at 28oC. 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.
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 (1o 22’ N and 103o 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 28oC using submersible heaters. Hatching
tanks were siphoned and cleaned daily accompanied
with about a 50-100% 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
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 30-40 rotifers mL−1 (Brachionus sp.)
and microalgae Nannochloropsis sp (8x105 cells mL1
). Following tests were performed;
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 45oC.
Time period was set 12 hours. Control was at
ambient temperature 28oC 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 (H2SO4) 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 28oC 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:
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Adv. Environ. Biol., 6(7): 1909-1915, 2012
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 2way 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.
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Adv. Environ. Biol., 6(7): 1909-1915, 2012
Temperature test:
No survival of larvae was achieved in
temperature groups at 40 oC and 45 oC respectively.
Thus, highest survival of Zoea 1 and Zoea 2 in
challenge groups observed at temperature 30oC were
statistically significant (p<0.05).
However, at
temperature 35 oC 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
0
a
a
30 C
12 h
20.00±0.00
19.67±0.578
19.67±5.78a
19.67±5.78a
0
b
a
c
35 C
-do11.33±1.53
19.67±0.58
10.33±0.58
20.00±0.00a
40 0C
-do0.00±0.00b
19.33±1.15a
0.00±0.00c
18.00±1.00a
45 0C
-do0.00±0.00b
19.67±0.58a
0.00±0.00c
17.33±1.53a
Note: Values showing same superscript in same row were statistically significant (p<0.05).
pH test:
Salinity 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.
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±0.00a
18.00±1.00b
0.00±0.00c
17.00±1.00b
pH 6
-do0.00±0.00a
18.00±1.00b
0.00±0.00c
17.67±1.53b
pH 8
-do18.67±1.53a
18.33±1.53a
16.67±2.08a
17.33±1.53a
pH 10
-do0.00±0.00a
17.67±0.58b
0.00±0.00a
16.00±1.73b
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±0.00a
20.00±0.00b
0.00±0.00c
20.00±0.00a
Salinity 40
-do4.67±1.15a
20.00±0.00b
5.33±1.53c
20.00±0.00b
Salinity 60
-do0.00±0.00a
19.67±0.58b
0.00±0.00a
20.00±0.00b
Salinity 80
-do0.00±0.00a
19.33±1.15b
0.00±0.00c
19.67±0.58b
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].
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Adv. Environ. Biol., 6(7): 1909-1915, 2012
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 L1
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 oC over the control 28 oC.
Temperature profoundly influenced survival of crab
larvae in this study. Survival was determined well at
temperature 30oC and in control. Change in 5 oC
resulted escalated mortalities among the larvae. 35oC
did produce almost half mortality and elevated
temperature 40 oC and 45 oC 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 8.2-8.5 [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 8.20. 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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Adv. Environ. Biol., 6(7): 1909-1915, 2012
effect on larvae of crab. Therefore, temperature and
salinity should be constant during the larval survival.
Any fluctuation will lead to destruction of rearing
system.
The results of this study demonstrate that larval
physiological condition using a
stress test
comprising, oxygen, starvation, pH, temperature and
salinity stresses, additionally it was noted that larval
capability to survive was compromised when stressor
was at certain level however, reduced and elevated
stressor effect were adverse on survivorship of crab
larvae.
7.
8.
9.
Acknowledgement
This study was supported by the Department of
Fisheries, Government of Sindh, Pakistan, under
Capacity Building Project and by Ministry of
Science, Technology, and Innovation (MOSTI)
(Science Fund), Government of Malaysia under grant
Vot. No. 52042. Authors would like to thank the
Director and the staff of the Institute of Tropical
Aquaculture (Aquatrop) and marine hatchery for
their help. Corresponding author would like to thank
to Mr. G.M. Mahar Director General Fisheries,
Government of Sindh, Pakistan, and Mr. G. M.
Wadahar Director Fisheries Sindh Inland,
Government of Sindh, Pakistan for their extended
support for the present study.
10.
11.
12.
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