Horseshoe crabs are extremely valuable to the medical research community. Currently,
one major biomedical product is Limulus Amoebocyte Lysate (LAL), a cloting agent
in horseshoe crab (Limulus polyphemus) blood that is used to detect human
pathogens in medical equipment (Karl, 2004). The western
countries have led the biomedical research and product development since the
early 1900s. This crustacean is also used for vision studies because of similarity
of its complex eye structure to the human eye. Until today, researches on horseshoe
crabs continue. So far, products derived from horseshoe crabs have good potential
for commercialization. There are only four existing species of horseshoe crabs
today. They are Limulus polyphemus, Tachypleus gigas, T. tridentatus
and Carcinoscorpius rotundicauda (Brusca and Brusca,
1990). In spite of their commercial and medical importance, horseshoe crab
populations are threatened by the loss of habitats. Human impact is be the major
cause for this problem. The coastal changes and development in response to rapid
population growth led to deterioration of water quality and habitat. For example,
even though horseshoe crabs are able to tolerate the changes in environment,
high concentration of pollutants (i.e., heavy metals) can cause detrimental
effect on the growth of this invertebrate. Their populations declined have caused
international interact in the conservation of this species.
In the US, L. polyphemus captured for blood extraction (LAL production)
has approximately ten percent greater mortality than those in the wild (Karl,
2004). A more serious and immediate threat may be the recent, dramatic increase
in horseshoe crabs harvests for bait for the eel and whelk fisheries. In Malaysia,
horseshoe crabs are left to die in the sun when trapped in fishermans
fishing nets (Hunaini, 2007). Horseshoe crabs are also
harvested and processed as a feed additive for poultry and livestock and as
a compound fertilizers (Shuster, 2003). Horseshoe crabs
take a long time to grow and it takes about 9-12 years to reach maturity. Horseshoe
crabs are characterized by high fecundity but with high eggs and larval mortalities
(Loveland et al., 1996). There are very few researches
on the Asian Horseshoe crabs except by Sekiguchi et al.
(1988) in particularly the embryonic development. During its formative years,
the horseshoe crab molts, to accommodate its growing body. The new carapace
then hardens. Juvenile horseshoe crabs may molt several times in the first and
second years, then only once annually. However, in the laboratory where environmental
temperatures remain constant and are often elevated above natural cyclic temperatures,
juvenile horseshoe crabs were found to molt three to five times in the first
year and then later once or even twice a year (Stephen and
Berkson, 2005). In culture condition, molting problem was observed when
the larvae when the larvae move out of the shell, legs, or telson, get stucked
and this resulted in the mortality (Stephen and Berkson,
2005). Furthermore, during this molting process, these soft-shelled horseshoe
crabs are highly susceptible to predation. Uncontrolled water quality such as
ammonia toxicity, pH extremes, gas supersaturation and high turbidity have negative
impact on their growth and survival.
Horseshoe crabs survive better in the natural environment. Few researchers
have successfully used both natural seawater and commercially synthetic marine
salt formulations to maintain horseshoe crabs for long period of time (Stephen
and Berkson, 2005). The present study use seawater from Port Dickson. In
order to reduce maintenance cost particularly in the cost for seawater, this
study compared two methods (conventional and recirculating aquaculture system)
for the culture of horseshoe crabs larvae in laboratory condition. Therefore
this study to compare the effect of two culture methods on the prosomal width
and weight increment and molting frequency of T. gigas larvae from the
age of 6 to 11 month.
MATERIALS AND METHODS
Materials: The specimen and materials used in this study were 6 month-old T. gigas larvae. The larvae were produced from trough artificial fertilization using T. gigas brood stock captured from the wild. Other materials used were Recirculating Aquaculture System (RAS), rearing trays, micrometer and analytic balance, water testing kits, Artemia nauplii and seawater.
Samples and rearing trays: In this study, approximately 300 individuals
of sixth month-old T. gigas larvae were used. Three trays for conventional
rearing methods and another three for RAS. Each of these tray contained 50 T.
gigas larvae. Seawater was used as the rearing media. Larval rearing was
carried out in laboratory condition. The average prosomal width and weight measurements
were taken at the initial stage of this study and once a month thereafter until
larvae reach 11 month-old.
Recirculating aquaculture system, RAS: Generally, this system involved the water recirculation from the rearing trays into the mechanical and biological filters and the sterilized through UV light before being return to the rearing tray. In RAS, the mechanical and biological filter both functioned to filter to solid waste and remove ammonia from the system. Polyethylene (PE) tray were arranged to resemble drawers which can be pulled out for monitoring and maintenance purposes.
Feeding: Artemia nauplii was used as live feed for the horseshoe crab larvae. In this study, about 2.5 to 3.5 g of Artemia eggs were hatch everyday. Seawater with 18-20 ppt was used as a media for the culture. Artemia cysts were incubated for 24 h and the resulted nauplii were fed to horseshoe crabs larvae. Larvae was feed to station twice a day.
Water testing kit: There were five main water parameters measured in this study. The kits or apparatus used were; the refractometer (salinity), pH meter (pH), DO meter (dissolved oxygen and temperature) and ammonia kit (ammonia-nitrogen). Measurements were taken twice a week.
Size and weight increment: The larval growth was accomplished by molting their exoskeleton. Size and weight were measured after each molt. This measurement was carried out using a digital caliper (prosomal width measurement) and an analytical balance (body weight measurement). Length and weight measurements were carried once a month throughout out the culture period. Larvae were monitored daily to observe mortality and molting.
Molting: In order to grow, the larvae of T. gigas molt many times during their lifetime. The molting process depends on the relative growth. After molting, the size of T. gigas larvae becomes larger than before. Prosomal width and weight on horseshoe crab larvae were measured after each molting.
Statistical analysis: Data for prosomal width and weight increments by using t-test to compare the two rearing methods used.
RESULTS AND DISCUSSION
In this study, only prosomal width and weight were measured since the width
considered as being the most reliable indication of the size increment and yields
the smallest error in measurement (Sekiguchi et al.,
1988). In this study, it was found the T. gigas larvae molt 3 times
during the 6 month-old culture period. Three stages of body size increments
for T. gigas larvae were observed for both RAS and conventional method.
The result were as shown in Table 1.
|| Means data collected and result statistical analysis t-test
for weight (g) and prosomal width (mm)
|Mean±SD of body sizes (mm) of T. gigas larvae
reared in RAS and conventional method. RAS: Recirculating aquaculture system,
C: Conventional method. S: Significant different (p<0.05)
|| Effect of RAS on total weight increments compared to conventional
The weight and prosomal width of T. gigas larvae for both systems were
significantly different (p<0.05). This indicated that the different rearing
methods namely the RAS and conventional method influence the increments in the
prosomal width and weight of the larvae.
Figure 1 showed the final prosomal width achieved for T. gigas larvae cultured in RAS and conventional method was 27.99 and 23.50 mm, respectively. The final weight increment achieved for T. gigas larvae cultured in RAS was 0.92 and 0.61 g for conventional method (Fig. 2). The first molt occurred when larvae reached 187 days old, second at 250 days old and third at 278 days old for both RAS and conventional method. Therefore, RAS resulted in significantly better width and weight increments as compared to the conventional method. However, there was no different in molting frequency of T. gigas larvae cultured using these two methods using.
Estimation of molting frequency of T. gigas: Similar to other
arthropods, horseshoe crabs must molt in order to grow. When an individual is
ready to molt, a new soft and folded exoskeleton begins to form beneath the
old one. The old carapace splits along the forward edge, allowing the animal
crawl out. After molting, the size of T. gigas larvae is larger than
before. In this study, it was found that the T. gigas larvae molted 3
times during the 6 months cultured period. T-test results in Table
2 showed that the molting frequency for different molting stages for T.
gigas larvae cultured for 6 month using RAS and conventional method was
not significantly different (p>0.05). The animal will be stress in higher
or lower solenoids so it will delays the deposition of calcium on the exoskeleton
(Soundarapandian and Raja, 2008).
|| Effect of RAS on prosomal width increments compared to conventional
|| T-test for the molting frequency at different molting stages
for T. gigas larvae
|Mean±SD (standard deviations) of molting frequency
of T. gigas larvae reared in RAS and conventional method. RAS: Recirculating
aquaculture system, C: Conventional method. NS: No. of significant different
|| Range of molting frequency of different molting stages for
|RAS: Recirculating aquaculture system, C: Conventional method
Both RAS and conventional culture method have no effect on the molting frequency
at different molting stages for T. gigas reared from 6 to 11 moth-old.
The range of molting frequency of different molting stages for RAS and conventional
method is shown in Table 3.
Generally, the molting frequency of horseshoe crab larvae was observed to decrease
against their age. The decreased in the molting frequency is basically due to
the longer time needed for the larvae to grow until the exoskeleton could not
longer contain them and then the molting occurs. The bigger larvae grow, the
longer is the time taken for it to molt. During the molting process, some T.
gigas larvae were unable to move out from their older shell, thus resulted
in the mortalities of the larvae. Great amount of energy during the molting
process may have caused this mortality. Overfeeding can be detrimental to the
larvae. Artemia given as food will compete with the larvae for oxygen
especially in the conventional method. Poor water quality is also a factor to
be considered contributing to detrimental effect on the larvae. Particularly,
the DO and ammonia in the culture water quite different between the two systems.
Advantage of using RAS as compared to conventional method: This study
showed the advantages and disadvantages of using RAS and conventional method
for the culture of T. gigas larvae. In RAS, optimal water quality is
being maintained and this provides a suitable condition for the culture of the
horseshoe crab larvae. Although the initial cost of setting the system is higher
but in the long term, it is more advantages and cost saving as compared to the
conventional method, whereby the frequent water change will incur high cost.
For the optimum growth and increasing biofilter efficiency and temperature are
important in RAS (Rahman et al., 2012). During
cultured period, it was observed that the T. gigas larvae cultured using
the conventional method gets infected with fungus as compared to RAS (Fig.
3, 4). Ultraviolet installed in RAS sterilized and eliminated
pathogens ultheaviolet, therefore reducing reduced disease problem for the T.
||T. gigas larvae reared in Recirculating Aquaculture
System (RAS) appeared free from infection
|| T. gigas larvae reared in the conventional method
showing fungal infection on shell
Based on the finding of this study, it can be concluded that culture method using RAS produced significantly better growth of T. gigas larvae as compared to the conventional method T. gigas larvae were generally easier to maintain under RAS in the laboratory as compared to the conventional method. RAS provides a solution for water quality problems such as dissolved oxygen DO and ammonia. Overall, RAS has more advantages, especially in reducing water wastage, human recourses and space requirements.
The authors thank to Fundamental Research Grant Scheme (FRGS) phase 2/2010 Ministry of Higher Education Malaysia (MOHE) for providing this fund. Special thanks also to the Universiti Putra Malaysia (UPM) for facilities provided for this research.