Effect of Salinity on the Egg Hatching and Early Larvae of Horseshoe Crab Tachypleus gigas (Muller, 1785) in Laboratory Culture
H. Siti Hamidah,
Tachypleus gigas (Muller) the Southeast Asian horseshoe crab, is found to nest on sandy beaches in Malaysia, thus biological information about survival of this living fossil become crucial. Laboratory experiments were carried out to investigate the effect of salinity on duration to maturation and hatching success of their eggs. The effect on size, morphological changes and growth of the hatched larvae was also investigated. Eggs of T. gigas were collected from the newly nests made on breeding beach of Pekan, Pahang (Lat 3° 56.915N; Long 103°21.933 E) in Peninsular Malaysia. Triplicates of five different salinities (15, 20, 25, 30 and 35 ppt) were chosen for the experiments. A vernier caliper was used for size measurement under a stereo microscope. The morphological changes in prosomal width and length, body length, telson length, opisthosoma length and total length were recorded. Salinity range of between 25 and 35 ppt could be an optimal incubation condition. No egg hatched at salinity 15 and 20 ppt. Highest percentage of hatching was found at 30 ppt (91.11±5.57%). Hatching also occurred at 25 and 35 ppt with 65.56±1.73% and 74.4±2.31% success rate, respectively. The shortest molting time occurred at 15 ppt (25 days after hatching). Total size changes in larvae were also highest at 15 ppt but lowest at 35 ppt. There was no mortality in larvae maintained in all treatments. Early larval development would also affected by the salinity stress but its seemed tolerable.
November 14, 2010; Accepted: April 12, 2011;
Published: June 07, 2011
Horseshoe crab, a chelicerate arthropod, is an ancient animal survived from
ancient time to date, thus called as living fossil. The extraordinary defense
mechanism is due to the blood property in this animal. It has now becomes a
great contribution to the world of medical and pharmaceutical science (John
et al., 2010). Limulus polyphemus, a species endemic to Atlantic
Ocean has been reported to be over-exploited and now strictly monitored and
regulated due to its importance in the pharmaceutical industry worldwide. Only
licensed biomedical companies could purchase large crabs, which are harvested
by trawlers or by hand from spawning beaches (ASMFC, 2004).
Abundance of other horseshoe crab species is also reported to decrease tremendously
(Tanacredi, 2001; Chen et al.,
2004). The development for human need in Asia as well has extremely put
on stress to horseshoe crab found in this region, thus giving the status of
the three species found in Asia as under threat, the same as the Atlantic species
(Morton and Blackmore, 2001). Tachypleus gigas
(Muller) is listed under Data Deficient in the 2009s IUCN together with
Carcinoscorpius rotundicaula (Latreille) and Tachypleus tridentatus
T. tridentatus, T. gigas and C. rotundicaula are found
in Malaysia.They were reported under threat of unregulated harvesting for commercial
purposes. T. tridentatus is reported only in Sabah water, East Malaysia.
The other two species are commonly found in Peninsular Malaysia and could be
distinguished physically by the body size and shape of tail (Kassim
et al., 2008). T. gigas is bigger in size with triangular
in cross section of tail while C. rotundicauda being smaller in size
with cylindaric-shaped tail.
Horseshoe crab of Tachypleus is a marine species with the smaller juveniles
prefer to be close to the shore than the older juveniles (Hu
et al., 2009). Sediment texture indirectly influences horseshoe crab
egg viability and development (Jackson et al., 2005)
and Chiu and Morton (2003) suggested that the sediment
with medium-sized sand and moderately sorted condition would promote hatching
success and survival of horseshoe crab larvae on beach. Ehlinger
and Tankersley (2003) was previously noted that hydration and agitation
with sand may increase hatching rate. Apparently they have adopted a life strategy
to survive on sandy beach that is exposed to fluctuation of temperature and
salinity (Chiu and Morton, 2004). During spawning season
in Pahang, Peninsular Malaysia, T. gigas was reported to build up to
22 nests on the breeding beach and nests could contained up to 407 individual
eggs or larvae (Nur Erni et al., 2010). Although
there was fluctuation in salinity on the breeding beach, there was no correlation
found with the number of nest or eggs in their study area.
Report on the effect of salinity on the eggs, hatching success or larval development
of horseshoe crab from tropical coast is scarce. The hatching success at beaches
in Malaysia is not known to date. Furthermore, the conservation status of horseshoe
crab in Malaysia as well as in many parts in Asia is still unknown while the
populations continuely facing threats from potential loss of prime spawning
habitat because of erosion and/or coastal development, and pollution (Botton,
2001). Pollution is reported as the main factor affecting the development
of embryos and larvae of Tachypleus gigas and Carcinoscopius rotundicauda
rather than salinity and temperature (Botton and Itow, 2009).
Despite that, a study on the larval survival and development in T. gigas
from India showed the important role of salinity (Chatterji
et al., 2004). In view of the long term needs to conserve and manage
this important species in Malaysia water, and the potential contribution of
culture success of the larval and juvernile stage for conservation, effort to
compile data on their ecology and biology should become priority as to fully
understand their biological importance. In the present study, focus is made
to investigate the effect of different salinity towards the eggs, their hatching
success and early larval growth under laboratory condition. This information
is vital for future work on conservation and beach rehabilitation program.
MATERIALS AND METHODS
Field sampling: Field sampling was carried out in day time during the full moon of August 2009 at the breeding beach of Pekan, Pahang (Latitude 3° 56.915N; Longitude 103°21.933 E) during low tide. Station for field sampling, Pekan, Pahang (Latitude 3° 56.915N, Longitude 103°21.933) was located in the East Coast of the Malay Peninsula, facing the South China Sea. The field sampling was done for two days, 5-7th of August 2009 at about noon which was coincide with the spring tide. It was during the full moon of lunar cycle where high number of horseshoe crabs could be expected to land for breeding on the beach. Field survey was started about 2hours before the time of highest tide. Adults horseshoe crabs were observed to come out to the beach following the tide.
Spawning pairs of horseshoe crab T. gigas were monitored and the nests left by the pairs were escavated carefully. During the field work, observation was made from far and the spawned females were left undisturbed or captured, thus no data on female size was collected. Horseshoe crab eggs were collected by hand. About 1500 of horseshoe crab eggs were obtained for this study. At the sampling site, the eggs were taken with sand from their nest as to ensure the sprayed sperms from male fully fertilised the eggs. The physical parameter namely the Dissolved Oxygen (DO), pH, salinity and temperature at the sampling site were measured in situ using a YSI 556 multi-parameter probe. Color of eggs was observed and recorded and eggs counting were done at the sampling site.
Sieve of 500 microns was used to discard the sand and other particles. The fertilized eggs are olive green in color with thick elastic chorion. Horseshoe crab eggs were then transferred into plastic bag containing filtered natural seawater (30 ppt). As no technical aeration was provided, the plastic bag was opened to let dissolved of oxygen from air periodically during their transportation to laboratory.
Laboratory experiment 1 and 2 was then carried out in the marine hatchery of Institute of Tropical Aquaculture, Universiti Malaysia Terengganu. Upon arrival, all eggs were transferred into containers with filtered natural seawater of 30 ppt and mild aeration. The eggs were maintained for a week before experiment in ambient temperature (26-29°C). All experiments were carried out within two months.
Effect of salinity on egg development and hatching success: Artificial
Sea Water (ASW) was prepared to get the salinity of 15, 20, 25, 30 and 35 ppt
in triplicates glass beaker container of 500 mL. Salinity was measured using
a salinity refractometer (model RHS10 ACT) with offered feature of Automatic
Temperature Compensation. The refractometer was first calibrated by placing
2 to 3 drops of distilled water on the meter, look through the eyepiece and
turn the calibration screw until the scale reads 0. To check the salinity of
the prepared beakers, 2 to 3 drops of water from each beaker were placed on
the meter before reading was made through the eyepiece. Soft aeration was given
to each container. A total of 30 fertilized eggs were carefully placed in each
beaker. Each beaker was then covered with parafilm as to reduce dessication.
The experiment was carried out in room temperature (27-29°C) under natural
light with more or less of natural photoperiod. No additional lighting was given.
Physical parameters were measured every day using YSI Meter Model 550A for temperature and dissolved oxygen, a portable pH meter (Thermo Russel RL060p) for pH and a refractometer (model RHS10 ACT) for salinity. ASW was changed every other day and samples were checked daily for the presence of newly hatched larvae. A vernier caliper was used for size measurement. The egg development was observed under a stereo microscope and hatched eggs were recorded.
Effect of salinity on early larval growth: Eggs collected from field
sampling were maintained at 30 ppt in laboratory condition. Culture condition
for this experiment was as the same as in experiment 1. A total of 30 newly
hatched larvae were put into each 500 mL experimental glass beaker of 15, 20,
25, 30 and 35 ppt and softly aerated. Larvae were fed with a planktonic copepod
Apocyclops sp. at a rate of 26 copepod/day/larvae. Supply of copepods
was obtained from the established culture in the same laboratory.
The physical parameters of culture in all beakers were measured daily as in Experiment 1 for 40 days. To count the survival rate, any mortality was recorded everyday. The larvae were considered died when they were inactive (no leg or book-gill movement) and did not response to mechanical stimulation. The morphological changes were observed and described as change of size in prosomal width and length, body length, telson length, opisthosoma length and total length.
Data collected was analysed statistically to determine the effect of different salinity on development, duration of eggs to hatch and hatching success using SYSTAT 11.5, SPSS. One-way Analysis of Variance (ANOVA) was used to determine the significant difference of time needed for hatching and the larvae to molt into first instar. It is also used to test if the size changes of body parts were significantly different at different salinities.
Environmental parameters: Environmental parameters measured in situ during the field sampling is summarizes in Table 1. The values indicate the sampling area was at a normal condition of tropical water. Dissolved oxygen showed comparatively low concentration while temperature indicated as warm weather condition during the samplings.
In the laboratory, culture condition was maintained at almost the same condition
found in their nest except for temperature (Table 2).
||Salinity, Dissolved Oxygen, pH, temperature (°C) and conductivity
(mS cm-1) of overlying water at horseshoe crab nest at Pekan
||Mean value of DO (mg L-1), pH and temperature (°C)
maintained in different salinity
||Effect of salinity (ppt) on size of T. gigass
egg maintained at laboratory condition
Temperature in the laboratory was maintained at between 26.7±0.2 and
27.2±0.1°C, lower than at field.
Effect of different salinity on eggs size: Initially, at all salinities, the increment in size was slow but at day-10 eggs maintained in 30ppt increase their size drastically (Fig. 1). On the day-1, eggs were 3.58±0.06 mm in diameter. On the day-25, the egg size was bigger at salinity 30 ppt before reduced on the day-35. Maximum mean size attained by the eggs was recorded at salinity 30 ppt (6.2 mm). There was no size increase at salinity 15 and 20 ppt.
Effect of different salinity on hatching success: The analysis of variance
showed a significant different of day to hatch (p<0.05) between salinity
treatment. No eggs hatched at salinity 15 and 20 ppt.The shortest duration for
the eggs to hatch was when maintained at 30 ppt with the mean duration of about
33 days (Table 3). Number of individual to firstly hatch was
also highest at 30ppt. At this salinity, the earliest times for the eggs to
hatch was on the day-26 with 8 individuals (27%). Higher number of hatched eggs
was obtained on the day-27 with 11 individuals (37%) (Fig. 2).
At salinity 35 ppt, horseshoe crab eggs started to hatch on the day-28 with
7 individuals (23%) and at salinity 25 ppt it hatched on the day-30 with 9 individuals
||Effect of salinity (ppt) on number of hatch per day and day
to hatch of T. gigass egg maintained in laboratory
||Effect of salinity (ppt) on hatching success of T. gigass
egg maintained in laboratory condition
|| Mean number of days for eggs to hatch at different salinities
Effect of salinity on total percentage of hatching success is summarizes in Fig. 3. Highest percentage was found at 30 ppt (91.11±5.57%) while at 35 and 25 ppt the hatching success was at 74.4±2.31% and 65.56±1.73%, respectively. No hatching was observed in both the 15 and 20 ppt and eggs had already covered by fungi. After day-35, most of horseshoe crab eggs in other salinities became rotten eggs and the color turned red or black.
Effect of salinity on growth of larvae: Trilobites hatched from the
eggs was monitored for their growth within 40 days and the result on the changes
in different morphometric data is summarizes in Fig. 4. Salinities
of 15 to 25 ppt showed stronger effect on the size changes if compared to at
30 and 35 ppt.
||Size changes as shown by different morphometric data of trilobites
of T. gigas maintained in different salinities
||Effect of salinity (ppt) on molting time of trilobites of
T. gigas maintained in laboratory condition
||Effect of salinity (ppt) on molting success (%) of trilobites
of T. gigas maintained in laboratory condition
Major size change for every treatment was shown by the prosomal width followed
by the body length while the least was by telson length.
The time needed by the trilobites to molt into instars increases significantly
(p<0.05) with the increase of salinity. Due to their fast increase in size,
the larvae at 15 ppt took the shortest time to molt into first instar which
was only 25 days after hatching (Fig. 5). At 20 and 25 ppt,
it took 30 and 28 days, respectively to molt while at 30 ppt, molting occurred
on day-34. No molting larvae were observed within 40 days experiment at 35 ppt.
Percentage of molting success in different salinity is shown in Fig. 6. Obviously the molting success decreases to less than 50% if cultures were maintained at higher salinity between 30 and 35 ppt. In contrast, higher molting percentage was found when trilobites were maintained at salinity 15 and 25 ppt.
Effect of different salinity on egg size and hatching: Salinity fluctuations
are common in beaches and it is known to influence survival and growth of marine
invertebrates including horseshoe crab (Botton et al.,
2006). Its influences many physiological functions and important in regulating
the distribution of estuarine and marine organisms (Laughlin
and French, 1989; Goncalves et al., 1995).
Ehlinger and Tankersley (2004) found that embryo in
the eggs of L. polyphemus could develop into larval stage and successfully
hatch in salinities between 10 and 70 ppt. In contrast, eggs of T. gigas
failed to increase their size or hatch at salinity of 15 and 20 ppt in the present
study which could be related to the small tolerant limit and salinity fluctuation
in this tropical species.
The change in inner fluid osmolality in response to outer medium osmalality
had been reported in crustacean such as homarid lobsters (Charmantier
et al., 2001). This possibly true also for horseshoe crab due to
the nature of horseshoe crabs embryonic membrane which was reported as
very permeable to water, chloride and sodium ions (Laughlin,
1981). Hannan and Evans (1973) already proved the
ability of Limulus to change it permeability when maintained at low salinity
medium, a strategy of invertebrate to regulate osmotic pressure in relation
to the external environment (Prusch, 1983). Ehlinger
and Tankersley (2004) related the hyperosmotic condition of internal fluid
during development to the osmotic shock condition as a result of certain factor
such as dessiccation. Nevertheless, there was no shocking condition applied
to the eggs in the present study and size increment was only occurred at salinities
25, 30 and 35 ppt. The size increase must be related to the increase in the
embryo development which occur at it salinity tolerence limit (25-35 ppt) and
salinity 15 and 20 ppt could be beyond the tolerant limit for T. gigass
eggs in this study. This is shown by the highest percent of hatching found at
30 ppt while less number of hatching occurred at 25 and 35 ppt. This results
were in agreement with Kosaku et al. (2000) where
salinity of higher than 19 ppt was reported as suitable condition for T.
tridentatus eggs to hatch, thus support the idea of a different salinity
tolerant limit for different crab species.
Embryo of L. polymephus hatched into trilobites in 28 days before entered
water during high tide (Penn and Brockmann, 1994). Trilobite
larvae of T. gigas in India was reported to hatch out 40-45 days after
fertilization (Chatterji et al., 2004). Nevertheless,
by maintaining salinity at 30ppt at constant temperature in the present study,
hatching occurred as early as 26th day after fertilization. This results were
contradicting with those previous report most likely due to the different environmental
setting for the study. Constant temperature maintained in the laboratory had
reduced the possible thermal effect as usually experienced by the eggs at the
beaches (Chiu and Morton, 2004). On the beach, the timing
of hatching may be influenced by a counteracting influence of low salinity on
embryonic development rate and hatching, particularly with the effect of freshwater
run-off. At low salinities, embryos must have developed more slowly, but later
the enhanced water content of the eggs might have accelerated the hatching process
Effects of salinity on larval growth and molting: Morphometric data
such as prosomal width and length is usually the practical way to measure growth
in larval and juveniles of horseshoe crab (Lee and Morton,
2005). It will give important data such as the age of the horseshoe crab
(Sekiguchi et al., 1988). Embryo and larval
growth in L. polymephus showed that salinity influenced the rate of crab
development, although only the extreme condition has an effect on survival (Ehlinger
and Tankersley, 2004). They also indicated that the time from hatching to
molting to the first juvenile instar decreased significantly with increasing
salinity. Sekiguchi et al. (1988) found that
growth of trilobite larvae and juveniles was slightly delayed at 35 ppt. These
reports were in agreement with the present study where lower salinity of 15
and 25 ppt had initiate the increase in size of larvae particularly the prosomal
width and body length. Giménez and Anger (2003)
also noted on the same effect of incubation in low salinity to enhance the development
of esturine crab larvae of Chasmagnathus granulata. They related the
intraspecific variability in maternal energy investment into offspring production
may play a significant role for the chances of larval survival and development.
The earliest molting period which was found at 15 and 30 ppt in the present
study contradicting the report of Chatterji et al.
(2004). In their study, at 40 ppt the growth rate was faster and moulting
occurred on the 35th day of the experimental period, whereas moulting in other
salinities (10, 20, 30 ppt) was recorded on the 42nd day. A recent report on
the respiratory metabolism of trilobites of T. gigas indicating insignificant
influence of salinity (10-40 ppt) on the oxygen consumption by the larvae (Suniza
et al., 2011). This might indicate the role of other environmental
parameters such as oxygen and temperature which affect larval growth. On the
other hand, diet could be a factor that promote early molting. Jegla
and Costlow (1970) maintained newly hatched trilobite larval culture of
Limulus in seawater at constant temperature of 25°C and fed with
newly hatched Artemia salina. Chatterji et al.
(2004) did not offer any diet to their larval culture. There was no report
on the effect of early diet to trilobite larvae in culture experiment but the
copepod given during the experiment could be a contributor to the fast grow
of the larvae. Copepods is known to have higher nutrient content than Artemia
(Nanton and Castell, 1998; Carli
et al., 1995). Further study is needed to confirm this effect. It
was reported that in natural environment, the larvae usually bury themselves
in the sand during high tide and emerge from it and begin to feed on the larger
elements such as insect larvae or polychaete larvae in the surface sediments
during low tide (Chiu and Morton, 2004).
The data collected in the present study adds new information on the incubation period of eggs of T.gigas in laboratory culture. Maintaining the eggs in constant salinity of its tolerence limit between 25 and 35 ppt particularly at 30 ppt could induce early hatching, as early as 26 days after fertilization. Trilobites reconditioned at low salinity of 15 ppt after hatching could increase their tendency to molt early, while higher salinities such as 35 ppt could delay molting process.
The authors are indebted to the Ministry of Agriculture and Agrobased Industry Malaysia (MOA) for the financial support through e-science fund granted to one of the authors.
ASMFC, 2004. Horseshoe crab 2004 stock: Assessment report. Atlantic State Marine Fisheries Commision.
Botton, M.L. and T. Itow, 2009. The Effects of Water Quality on Horseshoe Crab Embryos and Larvae. In: Biology and Conservation of Horseshoe Crabs, Tanacredi, J.T., M.L. Botton and D.R. Smith (Eds.). Springer, New York, USA., pp: 439-454.
Botton, M.L., 2001. The Conservation of Horseshoe Crabs: What can We learn from the Japanese Experience?. Kluwer Academic/Plenum Publishers, New York.
Botton, M.L.M. Pogorzelska, L. Smoral, A. Shehata and M.G. Hamilton, 2006. Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins. J. Exp. Mar. Biol. Ecol., 336: 65-73.
Carli, A., G.L. Mariottini and L. Pane, 1995. Influence of nutrition on fecundity and survival in Tigriopus fulvus fischer (Copepoda: Harpacticoida). Aquaculture, 134: 113-119.
CrossRef | Direct Link |
Charmantier, G., C. Haond, J. Ligno and M. Charmantier-Daures, 2001. Ecophysiological adaptation to salinity throughout a life cycle: A review in homarid lobsters. J. Exp. Biol., 204: 967-977.
Chatterji, A., S. Kotnala and R. Mathew, 2004. Effect of salinity on larval growth of horseshoe crab, Tachypleus gigas (Muller). Curr. Sci., 87: 248-249.
Direct Link |
Chen, C.P., H.Y. Yeh and P.F. Lin, 2004. Conservation of the horseshoe crab at Kinmen Taiwan: Strategies and practices. Biodiversity Conservat., 13: 1889-1904.
Direct Link |
Chiu, H.M.C. and B. Morton, 2003. The sediment and hydrographic characteristics of three horseshoe crab nursery beaches in Hong Kong. J. Ocean Univ. Qingdao, 2: 35-43.
Direct Link |
Chiu, H.M.C. and B. Morton, 2004. The behaviour of juvenile horseshoe crabs, Tachypleus tridentatus (Xiphosura), on a nursery beach at Shui Hau Wan, Hong Kong. Hydrobiologia, 523: 29-35.
Ehlinger, G.S. and R.A. Tankersley, 2003. Larval hatching in the horseshoe crab, Limulus polyphemus: Facilitation by environmental cues. J. Exp. Mar. Biol. Ecol., 292: 199-212.
Ehlinger, G.S. and R.A.Tankersley, 2004. Survival and development of horseshoe crab (Limulus polyphemus) embryos and larvae in hypersaline conditions. Biol. Bull., 206: 87-94.
Direct Link |
Gimenez , L. and K. Anger, 2003. Larval performance in an estuarine crab, Chasmagnathus granulata, is a consequence of both larval and embryonic experience. Mar. Ecol. Prog. Ser., 249: 251-264.
Direct Link |
Goncalves, F., R. Ribeiro and A.M.V.M. Soares, 1995. Laboratory study of effects of temperature and salinity on survival and larval development of a population of Rhithropanopeus harrisii from the Mondego River estuary, Portugal. Mar. Biol., 121: 639-645.
Hannan, J.V. and D.H. Evans, 1973. Water permeability in some euryhaline decapods and Limulus polyphemus. Comp. Biochem. Physiol. A: Physiol., 44: 1199-1213.
Hu, M., Y. Wang, Y. Chen, S.G. Cheung, P.K.S. Shin and Q. Li, 2009. Summer distribution and abundance of juvenile chinese horseshoe crabs Tachypleus tridentatus along an intertidal zone in southern China. Aquat. Biol., 7: 107-112.
Jackson, N.L., D.R. Smith and K.F. Nordstrom, 2005. Comparison of sediment grain size characteristics on nourished and un-nourished estuarine beaches and impacts on horseshoe crab habitat, Delaware Bay, New Jersey. Zeitschrift fur Geomorphologie, 141: 31-45.
Jegla, T.C. and J.D. Costlow, 1982. Temperature and Salinity Effects on Developmental and Early Post-hatch Stages of Limulus. In: Physiology and Biology of Horseshoe Crabs: Studies on Normal and Environmentally Stressed Animals, Bonaventura, J., C. Bonaventura and S. Tesh (Eds.). Alan R. Liss Inc., New York, USA., pp: 103-113.
John, B.A., K.C.A. Jalal, Y.B. Kamaruzzaman and K. Zaleha, 2010. Mechanism in the clot formation of horseshoe crab blood during bacterial endotoxin invasion. J. Applied Sci., 10: 1930-1936.
CrossRef | Direct Link |
Kassim, Z., H. Shahuddin, F. Shaharom and A. Chatterji, 2008. Abundance of three species of the horseshoe crab along the coast of Malaysia. J. Bombay Nat. Hist. Soc., 105: 209-211.
Kosaku, M., S. Satoko, N. Shigetomo and H. Asuka, 2000. Ecology of hatched larvae of the horseshoe crab Tachypleus tridentatus (Leach) in relation to the physical environment. Jap. J. Benthol., 55: 15-24.
Direct Link |
Laughlin, R.B. and W. French, 1989. Differences in responses to factorial combinations of temperature and salinity by zoeae from two geographically isolated populations of the mud crab Rhithropanopeus harrisii. Mar. Biol., 102: 387-395.
Laughlin, R.B., 1981. Sodium, chloride and water exchange in selected developmental stages of the horseshoe crab Limulus polyphemus (Linnaeus). J. Exp. Mar. Biol. Ecol., 52: 135-146.
Lee, C.N. and B. Morton, 2005. Experimentally derived estimates of growth by juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda (Xiphosura) from nursery beaches in Hong Kong. J. Exp. Mar. Biol. Ecol., 318: 39-49.
Morton, B. and G. Blackmore, 2001. South China Sea. Mar. Pollut. Bull., 42: 1236-1263.
Nanton, D.A. and J.D. Castell, 1998. The effects of dietary fatty acids on the fatty acid composition of harpacticoid copepods, Tisbe sp., for use as a live food for marine fish larvae. Aquacult., 163: 251-261.
Nur Erni, A.H.N., K. Kamaliah, M.O. Luthfi, A. Chatterji, B.Y. Kamaruzzaman and K. Zaleha, 2010. Variation in distribution of horseshoe crab nest along pahang coastal area. Proceedings of the University Malaysia Terengganu International Annual Symposium Science and Management 2010, May 5-8, Kuala Terengganu, pp: 540-544.
Penn, D. and H.J. Brockmann, 1994. Nest-site selection in the horseshoe crab, Limulus polyphemus. Biol. Bull., 187: 373-384.
Direct Link |
Prusch, R.D., 1983. Evolution of invertebrate homeostasis: Osmotic and ionic regulation. Comp. Biochem. Physiol. A: Physiol., 76: 753-761.
Saigusa, M., 1996. Two kinds of active factor in crab hatch water: Ovigerous-hair stripping substance ZOHSS and a proteinase. Biol. Bull., 191: 234-240.
Sekiguchi, K., H. Seshimo and H. Sugita, 1988. Post-embryonis development of the horseshoe crab. Biol. Bull., 174: 337-345.
Direct Link |
Suniza, A.M., K. Zaleha and A. Chatterji, 2011. Effects of different environmental parameters on the respiratory metabolism of the larvae of Malaysian Horseshoe crab, Tachypleus gigas (Muller). Pertanika J. Sci. Technol., 19: 1-9.
Direct Link |
Tanacredi, J.T., 2001. Horseshoe Crabs Imperiled: The Fate of a Species 350 Million Years in the Making. In: Limulus in the Limelight. A Species 350 Million Years in the Making and in Peril. Tanacredi, J.T. (Ed.). Kluwer Academic/Plenum Publishers, New York, USA.