Incubation and Hatching of Tachypleus gigas (Muller, 1785) Eggs in Sand and Water Media
In order to develop suitable methods to successfully hatch Tachypleus gigas eggs, a study was carried out on different salinity and culture media. The main objective for this study was to determine the effect of watering frequency, salinity and media on the incubation period and hatching of T. gigas eggs. This research consisted of three experimental studies. In the first experiment, effect of water salinities (15, 20, 25 and 30 ppt) for watering or moistening sand was studied. For the second study, effect of watering frequencies (once in 1, 3 and 6 days) on the eggs incubated in sand were investigated. As for the last experiment, effect of incubation medium (water and sand) on eggs were compared. Data collected for these experiments were eggs diameter and hatching percentages. Embryonic developments were observed and photographed during the study period. Results from experiment 1, showed that at the end of the incubation period, watering with water salinity of 25-30 ppt produced significantly larger eggs diameter (p<0.05) while percentages of hatching was the highest with 30 ppt water. In experiment 2, it was found that percentages of hatching were significantly higher (p<0.05) when watered once a day and in three days. As for experiment 3, at the end of the incubation period, there was no significant different (p>0.05) in the eggs diameter and percentage of hatching between sand and water medium. In conclusion, the most suitable salinity and watering frequency were 25-30 ppt and once in 3 days, respectively. However, both sand and water are suitable media to successfully incubate T. gigas eggs. Overall, this study showed that T. gigas eggs can hatch as early as 40 days after fertilization.
Received: July 02, 2012;
Accepted: August 07, 2012;
Published: February 11, 2013
Horseshoe crabs, horse foot or king crab are benthic bottom dwelling invertebrates
found in both estuarine and continental shelf habitats. It is the close relative
to trilobites which is still existence (Shuster, 1982).
Therefore, they are often call living fossil (Iwanaga
and Kawabata, 1998). Earth land masses have shifted dramatically, thousands
of other species have gone extinct but horseshoe crabs remained. In the whole
world, only four species remained. Those are Limulus polyphemus (in Atlantic
coast), Carcinoscorpius rotundicauda (in Indo-Pacific) the mangrove horseshoe
crab, Tachypleus gigas (in the tropical coast) and Tachypleus tridentatus
a slightly temperate species (found in the coast of Japan, China and North Borneo).
All these species can be differentiated by their physical appearance such as
size, color and shape of tail. The coastal horseshoe crab, T. gigas (diameter
up to 25 cm) is grayish in color, with triangular and serrated telson (Sekiguchi,
1988). Horseshoe crabs begin life as embryos in unshelled, greenish and
yellowish eggs. Eggs are laid in nests on the beach at a mean depth of about
15 cm beneath the sand (Rudloe, 1979). Each group of
eggs contains between 2000 and 30000 eggs (Cohen and Brockman,
1983). After fertilization, each egg consist a central yolk, an embryo and
a thick outer membrane. The embryo goes through 21 stages between two to four
weeks of development (Sekiguchi et al., 1988).
The most studied species so far is L. polyphemus. It is being used to
produce a commercial product called Limulus Amebocyte Lysate (LAL),
a derivate from its blood. This LAL is being used by pharmaceutical and bio-medical
industries to detect bacteria in drug and implantable devices before being used
on patient (Botton, 1984). Ammonia was the one major
problem in fish culture especially in fish ponds, recirculation systems and
aquaria (Chezhian et al., 2012). Laboratory culture
allows intensive study on the horseshoe crab larvae. However, suitable culture
condition needs to be developed first. Thus, the goals of this study were to
compare the incubation period and hatching percentage of T. gigas incubated
in different culture media (sand and water), sand watered at different water
salinities and sand watered at different frequencies.
MATERIALS AND METHODS
Eggs of Tachypleus gigas and sand were collected from natural spawning,
Sitiawan, Perak, Malaysia. Experimental studies were carried out at Endocrinology
Laboratory, Department of Aquaculture, Faculty of Agriculture and Marine Laboratory
at MTDC, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. This study
consisted of 3 experiments. Experiment 1 was conducted to determine the best
water salinity for watering or moistening sand where eggs were incubated. Experiment
2 was to determine the best watering frequency for eggs incubated in sand bed.
While in experiment 3, the used of sand and water as culture medium for the
incubation of eggs were compared.
Preparation of sand and trays: Sand used in this study was autoclaved
at 121°C for 15-20 min. The size of trays used in this study were 44x33x10
cm. Eggs samples (50 eggs/basket) were placed in baskets (10x15 cm) made from
plastic net and buried in sand bed at 8 cm depth. Baskets were prepared in triplicates
for all treatments. During the watering or moistening of the sand bed, seawater
was poured until it drained through the outlet at the bottom of the tray. All
experiments were carried out at room temperature.
Conditioning of T. gigas eggs: In experiment 1 and 3, one-week
old eggs were conditioned in sea water in laboratory for five days prior to
the initiation of the experiments. These eggs were basically 12 days old. While
for experiment 2, the eggs used were three weeks old. After 5 days conditioning,
eggs were 26 days old when used for the experiment.
Experimental design: In the first experiment, water salinities of 15,
20, 25 and 30 ppt were used to water or moisten sand bed containing baskets
of T. gigas eggs. For the second experiment, watering frequencies of
once in 1, 3 and 6 days were applied on sand bed containing baskets of T.
gigas eggs. A control treatment was added, whereby eggs were incubated in
water with daily water change throughout the experimental period. As for the
last experiment, incubation medium using water and sand were investigated and
compared. In this experiment 30 ppt seawater was used. Sand medium was watered
once in 2 days while for water medium, water changed was carried out once a
Data collection: Data collected for these experiments were eggs diameter
and hatching percentages. Eggs diameter were measured using a Digital caliper
(DigiMax). Eggs samples in each net baskets were observed using dissecting microscope
(LEICA EZ4) once a week. Embryonic developments were observed and photographed
during the study period. Hatched eggs were also counted and percentage of hatching
calculated. All data from these experiment were compiled and stored using Microsoft
Office Excel. ANOVA was used to analyzed data for eggs diameter and percentages
of hatching from experiment 1 and 2. As for experiment 3, student t-test was
used to determine the significant difference between the incubation media (sand
RESULTS AND DISCUSSION
Hatching and development of T. gigas eggs incubated in sand at different
salinities: During the first week of this experiment, the recorded mean
diameter of horseshoe crab eggs was 3.65 mm and kept on increasing weekly. Maximum
mean size attained by the eggs at salinity 25 ppt was 6.80±0.04 mm. Result
showed that 25 and 30 ppt produced eggs with significantly larger (p<0.05)
diameter (Table 1). This is due to the fact that 25-30 ppt
is the same salinity as it is in natural habitat. Salinity fluctuations usually
occurs at coastal water near river mouth and that affects the growth and survival
of marine organisms including horseshoe crab (Botton and
Itow, 2009). This study showed that differences in egg diameter is possibly
due to the different salinity used to water the eggs.
Earlier study indicated the hatching of T. gigas occurs at 20-32 ppt
(Christianus and Saad, 2010). However, this study showed
that there was no significantly different (p>0.05) on the percentage of hatching
between 25 and 30 ppt. This finding has narrowed the salinity margin to 25-30
ppt. Eggs started to hatch at week 5 and the subsequent, week 6, showed the
highest percentage of eggs hatched for every treatment (Fig. 1).
At lower salinity, eggs development were rather slow but the increased in the
water content (perivitelline fluid) of the eggs may have accelerated the hatching
process of horseshoe crab eggs (Saigusa, 1996). Ehlinger
and Tankersley (2003) showed that eggs of L. polyphemus successfully
can hatch at salinity 10-70 ppt.
||Means diameter (mm) of T. gigas eggs incubated in sand
and watered with seawater of different salinities during the eight weeks
|Values with same superscripts within a column are not significantly
different at p>0.05, Eggs were 12 days old at the initiation of the experiment
||Hatching percentage of T. gigas eggs incubated in sand
and watered with seawater of different salinities during the eight weeks
period, Eggs were 12 days old at the initiation of the experiment
|| Fungal infection on T. gigas eggs
However, the results of this experiment indicated negative effect on percentage
of eggs hatching when incubated at salinity below 25 ppt. Study by Zaleha
et al. (2011) on T. gigas is in agreement with this study.
In this study, it was observed that eggs incubated with salinity of 15-20 ppt
were prone to fungal infection (Fig. 2). This kind of infection
was also observed on horseshoe crab larvae (Faizul et
al., 2011). Undeveloped eggs were distinguishable since it will turned
black or red and start to decompose.
Hatching of T. gigas eggs incubated in sand and watered at different
frequencies: Results of experiment 2 showed the percentage of hatching of
T. gigas eggs incubated in sand and watered at different frequencies
during the six weeks period (Table 2). Eggs incubated in sand
and watered daily (T1) and once in 3 days (T2) started
to hatch on week 2 with hatching 90 and 80%, respectively were observed at week
4. Eggs incubated in sand and watered once in 6 days (T3) and control
(T4) started hatching only at week 3 and 4, respectively. There was
no significant differences (p>0.05) in the hatching percentage between T1
and T2 from week 1-3. However, T1 produced the most significant
(p<0.05) hatching percentage on week 4 as compared to all the other treatments.
This experiment showed that the incubation period is shorter when eggs are incubated
in sand with watering frequency of once in 1-3 day times. In this study, it
was observed that T. gigas eggs started to hatch at week 2 (in treatment
1 and 2 of experiment 2). Eggs were 26 days old at the initiation of the experiment,
therefore at week 2 of this experiment, eggs were 40 days old. This result falls
within the 40-45 days range reported by Chatterji et
al. (2004). Comparatively, this incubation period for T. gigas
eggs is much longer than the 28 days of L. polyphemus eggs as reported
by Penn and Brockmann (1994). Recorded temperature throughout
this study was between 29-34°C. According to Karl (2004),
horseshoe crabs are able to tolerate a wide range of temperatures and low oxygen
environments. In this study, some eggs failed to hatch even with extended time.
Morton and Blackmore (2001) stated that eggs may not
hatch due to unsuitable environment and chemical processes in the sand of its
spawning site. The abundance of aquatic organism is very much affected by development,
industrialization and agriculture activities (Sasi, 2011)
at the surrounding areas.
Hatching and development of T. gigas eggs incubated in sand and water
media: In this experiment, water medium gave the higher hatching percentage
at week 5 and 6. However, from week 7-8, water and sand media were not significantly
different (Table 3). Egg diameter increased drastically from
week 2-6. After week 6, the diameter increased at slower pace due to the fact
that the eggs are near hatching. This study showed that there is no significant
different in the percentage of T. gigas eggs hatched in both water and
sand medium. In this experiment, eggs of T. gigas started to hatch at
week 5 with a diameter of 5.90 and 5.92 mm in water and sand medium, respectively.
However these eggs can increase to a maximum diameter of 7.06 and 6.73 mm in
water and sand medium, respectively.
|| Hatching of T. gigas eggs incubated in sand and watered
at different intervals during the six weeks period
|Values with same superscripts within a column are not significantly
different at p>0.05, Eggs were 26 days old at the initiation of the experiment
|| Means diameter and hatching of T. gigas eggs incubated
using different media during the eight weeks period
|NS: Nonsignificant at p>0.05, S: Significant at p<0.05,
Eggs were 12 days old at the initiation of the experiment
|| Embryonic development of T. gigas eggs after fertilization,
(a) One week old, olive green colored eggs, (b) Three weeks old, shedding
of the elastic green colored chorion membrane, (c) Four weeks old, undifferentiated
small sized embryo visible though the transparent membrane, (d) Five weeks
old, embryo with appendages is visible, (e) Six weeks old, morphology of
embryo becomes more prominent, a lateral eyes (black dots) on the side can
be seen and rotating vigorously in the perivitelline fluid and (f) Seven
weeks old, embryo with prominent morphology ready to hatch
The eggs at the initiation of the experiment were 12 days old, therefore at
5th week, eggs were actually 47 days old. Thus, this period is a bit longer
when compared to the 40 days observed in experiment 2 (of the current study)
and also the 40-45 days as reported by Chatterji et al.
(2004). Observation on the embryonic development of T. gigas from
1-7 weeks after fertilization is as shown in Fig. 3. Some
eggs start to shed its elastic olive colored chorion membrane on week 3, thereby
making the embryo visible. From week 4 onwards, the appendages of the embryo
in the perivitelline fluid was visible through the transparent membrane. Embryo
continues to grow and it changes color from yellowish green to olive green.
During the last 2 weeks (week 6 and 7) of development, morphology of the embryo
becomes more prominent with visible pair of lateral eyes and rotating vigorously
in the perivitelline fluid. At this time the embryo is ready to hatch.
In conclusion, the most suitable salinity and watering frequency were 25-30
ppt and once in 3 days, respectively. Both media, water (liquid medium) and
sand (solid medium) are equally suitable to be used as incubation media for
T. gigas eggs. Overall, this study showed that T. gigas eggs can
hatch as early as 40 days after fertilization depending on its incubation conditions.
The authors would like to thank to the Government of Malaysia for providing
fund through the Fundamental Research Grant Scheme (Grant No: 10201 vot. 5524074)
phase 2/2010 and to Universiti Putra Malaysia for providing the facilities for
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