Metamorphosis Induction of the Dog Conch Strombus canarium
(Gastropoda: Strombidae) Using Cues Associated with Conch Nursery Habitat
Strombus canarium is a commercially important gastropod that has great potential for advancement into aquaculture. In this study, the metamorphosis response of Strombus canarium larvae to various metamorphosis cues associated with conch nursery habitat and to KCl and GABA, were tested. Bioassays were run as static, no choice experiment and adopting a continuous exposure approach. Strombus canarium larvae showed strong metamorphosis responses when sediment (i.e., conch nursery habitat sediment/SD-NU) and detrital substrata (i.e., Thalassia detritus leachate/T-LC) from their nursery habitat were used (p<0.05). There was no metamorphosis in treatments using sterilized conch nursery habitat sediment (SD-ST) and sediment taken from outside conch nursery habitat (SD-OT). Experiments using fresh macrophyte blades of Enhalus acoroides (EA), Thalassia hemprichii (TH), Halophila ovalis (HA) and Ulva (UL) and adult conditioned seawater (SD-SW) also showed negative respond. Conch larvae demonstrate active habitat selection during metamorphosis and no spontaneous metamorphosis was observed. Settlement in S. canarium is associative in nature where epibionts associated with conch nursery habitat could be the cue for the metamorphosis. However, the specific epibionts/inducers and mechanisms underlining the process were not studied and therefore are subjected to more detailed investigation. The use of KCl was comparable with treatments using natural inducers (SD-NU and T-LC), thus was suggested for application in hatchery spat production of the species.
Metamorphosis in marine invertebrates involved significant morphological and
physiological changes that facilitate the transformation of the larvae into
young juvenile. Major ecological changes occurred, which include a shift from
pelagic, encapsulated or brooded existence to that of an independent benthic
individual (Gosselin, 1997; Bryan
and Qian, 1998). The transformation process involved lots of energy due
to significant increased in metabolic demand during the metamorphosis (Shilling
et al., 1996; Marsh et al., 1999;
Bryan, 2004). Successful metamorphosis is therefore,
one of the most critical parts in supplying new recruits for the natural populations.
It is also important for advancement of commercially important invertebrate
species into aquaculture. The percentage of successful metamorphosis is considered
to be of primary importance and critical for aquaculture (Garcia-Lavandeira
et al., 2005) in order to maintain a steady supply and reliable source
Metamorphosis in marine invertebrate is an active process and rarely occurred
spontaneously. Upon reaching the metamorphosis competence stage invertebrate
larvae will actively in search for these cues. In most cases the cues are very
specific and generally reflects suitable habitat for the post-metamorphic life
phase (Stoner et al., 1996; Williams
and Degnan, 2009). Abundant literature has shown that settling marine invertebrate
larvae are influenced by specific physical, chemical and biological cues derived
from a variety of sources in the marine environment (Manriquez
et al., 2004; Sawatpeera et al., 2004;
Holmes et al., 2005; Xing
et al., 2008; Hayakawa et al., 2009;
Williams et al., 2009).
Strombus canarium Linnaeus, 1758 is a widely distributed conch species
within the tropical waters and is highly associated with muddy bottom and seagrass
bed areas (Purchon and Purchon, 1981; Poutiers,
1998; Cob et al., 2009a). It is a commercially
important conch species within the Southeast Asian region (Erlambang
and Siregar, 1995; Cob et al., 2008, 2009a,
b). It has traditionally been fished and contributes
to the economic of the local fishermen (Purchon and Purchon,
1981; Erlambang and Siregar, 1995).
Strombus canarium is a gonochoristic gastropod with separate sexes.
Fertilization of egg is internal and large egg masses are laid by attaching
them to seagrasses during spawning season from November to March. The egg mass
is creamy-white in color and can easily be collected from the seagrass bed during
low tides throughout the spawning season (Cob et al.,
2009d). The species showed high fecundity, which ranged from 48,000 to 71,000
eggs in a single egg mass (Cob et al., 2009c).
The larval stages of this conch have recently been described (Cob
et al., 2009c, 2009d) and it shared great
similarity with other Strombus species (e.g., Davis
et al., 1993; De Navarrete et al., 2007).
The species certainly has great aquaculture potential. However, more information
is needed, particularly regarding the settlement and metamorphosis of the species.
Recent study showed that there is specific nursery habitat for S. canarium
population at Merambong Shoal, in the Straits of Johor, Malaysia (Cob
et al., 2009b). The purpose of the investigation was to determine
parameters within the conch nursery habitat that might contribute as natural
cues for conch larvae settlement and metamorphosis. Therefore, we tested and
analyzed the effect of various natural cues associated with the nursery habitat.
Apart from that the effect of K+ ion elevation and neurotransmitter
GABA on S. canarium larvae were also conducted. The result is practically
very important for management and conservation of the resources and hopefully
lead to advancements in aquaculture of the species.
MATERIALS AND METHODS
Culture of Strombus canarium larvae: Strombus canarium
egg masses were collected from Merambong Shoal, Johor Straits, Malaysia (01°19.778N,
103°35.798E). The egg masses were gently washed with 0.22 μm
filtered seawater (FSW) to remove sand and debris and incubated in individual
containers filled with FSW, at salinity 30±1 PSU and temperature 29±1°C
until hatch. Larvae were held at concentrations of 1-2 larvae mL-1
in 2 L round bottom glass container of 30±1 PSU and 29±1°C
and fed daily with the algae Isochrysis galbana at 1000 cells mL-1
(Cob et al., 2009c). The culture medium was totally
replaced every 2 days with freshly filtered FSW. After 16 days, larvae that
exhibit exploration and swim-crawl behavior as well as other physical characteristics
that indicates metamorphic competence such as the presence of dark-green pigmentation
on shell, foot and mantle margin, development of proboscis etc. (Cob
et al., 2009c, d), were utilized for the
Design of settlement experiments: Metamorphosis assays were run as static, no-choice experiments, with three replicates per treatment. Ten competent larvae were used in each replicate, which were taken from the same egg mass to reduce variability and all experiments were tested simultaneously. Larvae were introduced into a well of a 6-well plate (sterile Nunclon® 6-well polystyrene, 35 mm diameter, 15 mL volume), containing the respective settlement cues in 10 mL of FSW. The well plates (with lid on) were kept in an incubator at 29±1°C and salinity 30±1 PSU, under a 12L:12D light regimen. Larvae were subjected to continuous exposure to the metamorphosis inducers. A treatment with only FSW was used as a negative control, which also act as a test of spontaneous metamorphosis.
Larvae behavior was observed under a dissecting microscope. The larvae were
considered have successfully metamorphosed when they lost their velar lobes
and began to crawl using their foot (Cob et al., 2009c). The conch juvenile
showed active feeding activities immediately after metamorphosed (Cob et al., 2009c). Percentage of metamorphosis was determined at 24, 48, 72 and
96 h after the initiation of the experiment. Abnormal behavior of the newly
settled juvenile due to incomplete metamorphosis e.g., slow moving, inactive
and/or not using foot to crawl (Boettcher and Targett, 1998;
Cob et al., 2009c) were also noted.
Metamorphosis assays Settlement in response to fresh macrophyte blades: The larval settlement response to fresh macrophyte blades i.e., Thalasia hemprichii (TH), Halophila ovalis (HA), Enhalus acoroides (EA) and Ulva sp. (UL) were tested. The macrophytes were taken from conch nursery habitat during extreme low tide when the seagrass bed was exposed. The macrophytes were gently rinsed with FSW and their surfaces were carefully scraped to remove detritus, sediment and other epibionts. A small fragment (ca 1 cm2) of the macrophyte blades were introduced into the respective experimental well.
Settlement in response to seagrass detritus: Senescent and partially
decomposed seagrass blades (i.e., macro-detritus) of Thalassia hemprichii
(T-DET) and Halophila ovalis (H-DET) were collected from the conch nursery
habitat during extreme low tide. The macro detritus was gently rinsed with FSW
to remove sediment, but the epibiota were kept intact (not scraped). Fragments
of about 1 cm2 of T-DET and H-DET were introduced into the respective
Settlement in response to aqueous extract of seagrass detritus: Extract from partially decomposed leaf fragments (macro-detritus) of Thalassia hemprichii (T-LC) and Halophila ovalis (H-LC) were tested. The macro-detritus was gently rinsed in FSW to remove sand and unwanted debris and was further shredded and ground using pestle and mortar. FSW was added at a ratio of ~0.5 g detritus to 1 mL FSW. The solution was kept frozen for 1 week to lyse the cells. Prior the experiment the solution was allowed to thaw overnight and filtered using a qualitative whatman-1 filter paper. Assay concentrations of 10 mL aqueous extracts per 1 L FSW were used in the settlement experiment.
Settlement in response to surface sediment: The uppermost layer (5 mm) of top sediment was used for this experiment. The treatment for this experiment includes: sediment taken from conch nursery habitat (SD-NU), sediment taken from conch nursery habitat but further autoclaved (SD-ST) and sediment taken from outside conch nursery habitat (SD-OT) (i.e., from dense Enhalus bed of more than 80% coverage). The macro-detritus was manually removed from the sediment using forceps and smaller detritus was removed by decanting method using FSW. The sediments were transferred into the experimental wells to about 3-4 mm in thickness. Ten milliliter of FSW was added and the assays were conditioned for 24 h with mild aeration prior to the introduction of competent larvae.
Surface sediment conditioned seawater (SD-SW): Uppermost (5 mm) layer of sediment were taken from conch nursery habitat during extreme low tide. The macro-detritus was manually removed and FSW was added at a ratio of 0.5 g sediment per 2 mL FSW, with mild aeration. After 24 h the conditioned seawater was filtered using a 0.45 μm MF-millipore membrane filter and transferred to the experimental well.
Settlement in response to Adult Conditioned Seawater (AD-SW): Five adult specimens of Strombus canarium were taken from the field. The conchs were rinsed with FSW and their shells were scraped to remove epibionts and fouling and were kept in 2 L FSW with moderate aeration. After 24 h, the seawater was filtered using a 0.45 μm MF-Millipore membrane filter and introduced to the experimental well.
Settlement in response to elevated ion concentrations (K+) and
neuroactive compound (GABA): There are many ions and neuroactive compounds
that have been found to affect larva settlement in marine mollusks, however,
only Potassium chloride (KCl) and γ-aminobutyric acid (GABA) were tested.
KCl was selected as it is cheap and economical for commercial application (Yu et al., 2008), whilst GABA has increasing been used by commercial hatcheries
because of its higher efficiency compared with other settlement methods (Bernal
and Beltran, 1998). The KCl stock solution of 1 M was prepared by dissolving
the chemicals in distilled water. Serial dilutions with FSW were made immediately
before commencing the experiments. Test concentrations were 5, 10 and 15 mM
K+. Stock solution of GABA was prepared by dissolving the chemicals
in FSW immediately prior to the experiments. The stock solutions of 10-2
M were diluted to prepare solutions of 10-7, 10-6 and
Data analysis: There were three replicates of each treatment. The percentage
of metamorphosis in each experimental treatment was transformed (arcsine) before
the statistical analysis was conducted. To improve the arcsine-transformation,
those replicates with zero were given the value of 1/(4n), where n is the number
of larvae in a single replicate (Yu et al., 2008). Treatments in which
percent metamorphosis was equal to zero for all replicates were not included
in the statistical analyses. Normality and homogeneity of variance were tested
using Anderson-Darling and Bartletts test respectively. Data complied
with the requirement for parametrical test were analyzed with a one-way ANOVA
utilizing a post-hoc Tukeys test to compare difference between treatments
and control (Yu et al., 2008). Otherwise, the Kruskal-Wallis test followed
by Dunns method was applied (Gebauer et al.,
1998). All statistical analyses were conducted using the MINITAB®
Statistical Software (Minitab Inc., State College, USA).
The cumulative percentage of metamorphosis after 24, 48, 72 and 96 h are presented
in Table 1. Analysis of variance showed a highly significant
different between treatments (oneway-ANOVA, F = 40.39, df = 35, p<0.05).
Post-hoc analysis (Table 2) showed three different groups
that significantly differed from one another. Higher percentages of metamorphosis
were observed in treatments using sediment taken from nursery habitat (SD-NU),
Thalassia detritus leachate (T-LC) and 5 mM KCl (p<0.05), followed
by a slightly lower metamorphosis in treatments using sediment conditioned seawater
(SD-SW), Thalassia detritus (T-DET), Halophila detritus (H-DET),
10 and 15 mM KCl (p<0.05).
|| Metamorphosis response of Strombus canarium larvae
exposed to different inducers
|Values are cumulative percentage of metamorphosis (Mean±SE,
N = 3). FSW = 0.22 μm filtered seawater. Assays with negative metamorphosis
response were not presented
|| Tukeys pairwise comparisons between all treatments
after 24 h exposure to the inducers
|*Denotes significantly different and zero (o) denotes not
significantly different, at 0.05 probability levels. SD-NU: Sediment from
nursery habitat, SD-SW: Sediment conditioned seawater, T-DET : Thalassia
detritus, T-LC: Thalassia detritus leachate, H-DET: Halophila
detritus, H-LC: Halophila detritus leachate , K-5: KCl 5 mM, K-10:
KCl 10 mM, G-7: GABA 10-7 M, G-6: GABA 10-6 M, G-5:
GABA 10-5 M
Lowest metamorphic responses were observed in treatments using GABA (10-7,
10-6 and 10-5 M) and Halophila detritus leachate
(H-LC) (p<0.05). There was no metamorphosis occurred in treatments using
fresh macrophyte blades (HA, TH, EA and UL), adult conditioned seawater (SD-SW),
sterile nursery habitat sediment (SD-ST) and sediment taken from outside conch
nursery habitat (SD-OT). In addition, there was no spontaneous metamorphosis
as none of the larvae in the control treatments has metamorphosed.
Treatments using sediment from nursery habitat (SD-NU), KCl (5, 10 and 15 mM) and Thalassia detritus leachate (T-LC) showed the best result where all larvae in these treatments successfully metamorphosed after 48 h. Treatment using sediment conditioned seawater (SD-SW) also showed good metamorphic response where, 80% metamorphoses recorded after 96 h exposure. In treatment using Thalassia detritus (T-DET), up to 50% metamorphoses were recorded after 48 h, but no further metamorphosis recorded thereafter. For the treatments using Halophila detritus (H-DET) and Halophila detritus leachate (H-LC), metamorphoses were only observed during the first 24 h (at 20 and 3.33%, respectively).
The neurotransmitter GABA was less efficient as inducers for the species as only 3 to 7% of metamorphoses were recorded. The juveniles from these treatments were relatively in poor shape compared to the other treatments. They were inactive and showed very slow respond when disturbed and the animal remained partially retracted into the shell with their columella opening facing upwards. All juveniles exposed to GABA were subsequently died by day 3 (72 h).
Recruitment is an important process that plays a critical role in the population
dynamics and community structures of benthic marine organisms, especially those
that develop from an initial planktonic larval phase. Successful recruitment
can be influenced by both settlement success and early post-settlement mortality
and how well they adapt to their new environment after metamorphosis (Stoner
et al., 1996; Pinedo et al., 2000;
Roberts, 2001). Thus, to some extend there might be
some relationship between settlement and benthic invertebrates habitat
preferences. Previous studies showed that S. canarium is highly associated
with seagrass bed ecosystem and there is specific nursery habitat present where
small juveniles can be found in large numbers (Cob et
al., 2008, 2009a). However, there was no metamorphosis
response when the S. canarium larvae were exposed to fresh macrophyte
blades (fragments of Halophila ovalis, Thalassia hemprichii and
Enhalus acoroides), indicating that S. canarium settlement in
seagrass beds may be related to features other than the seagrass itself. The
seagrass ecosystem formed an important habitat and refuge for the species, but
there might be other factors more important for survivorship of the newly settled
Strombus canarium larvae on the other hand showed strong metamorphosis
responses when sediment and detrital substrata from their nursery habitat were
used, which is in agreement with previous studies on other Strombus species
(Davis and Stoner, 1994; Boettcher
and Targett, 1996; Stoner et al., 1996).
Many other invertebrate larvae also showed the same characteristic where positive
responses to associative cues from their favorable feeding habitat were reported
by Williams et al. (2007), Xing
et al. (2008) and Hayakawa et al. (2009).
This behavior might be important to ensure settlement in an environment most
appropriate for their survival, as juvenile survivorship differed greatly among
habitats with different physical and biological environments (Naylor
and McShane, 1997; Herbert and Hawkins, 2006; Walker,
Highest percentage of metamorphosis was observed when, larvae were exposed
to sediment taken from their natural nursery habitat. However, no metamorphosis
was observed in treatment using the same sediment but sterilized, indicating
that factors other than the physical characteristics of the substrate itself
are more important for S. canarium larvae. Benthic sediment has in many
instances been reported to emanate important metamorphic inducer for gastropods
(Pederson and Page, 2000; Smith
et al., 2005; Walker, 2007). The upper layer
of seagrass bed sediment is very rich in detritus and other associated epibionts
(Kharlamenko et al., 2001) that highly preferred
by juvenile Strombus. The present study indicated that these associated
epibionts within the nursery habitat might have emanated the most important
cues for S. canarium larvae. However, the types of epibionts and their
chemical properties however, need further investigation. According to Boettcher
and Targett (1998), the types of chemical cues involved in Strombus
metamorphosis and the mechanisms that controlling it share general features
with chemoreception in adult aquatic invertebrates. They normally consists of
chemicals with low molecular weight and soluble in water. Autoclaved sterilization
might have destroyed the epibionts as well as other important metabolites (cues)
thus, resulted in negative metamorphic response by the larvae.
There was no metamorphosis response when cues associated with conspecific adult
conch were used. The result of this experiment suggest that the important cues
for S. canarium larvae settlement are not aggregative (cues associated
with conspecifics), but rather associative (cues associated with non-conspecific-hosts,
prey, or biofilm) in nature. Associative cues produced by marine algae, particularly
the rhodophytes, were important in the settlement and metamorphosis of many
invertebrate larvae (Morse, 1992; Huggett
et al., 2005; Hayakawa et al., 2009).
Although, juvenile Strombus lives in colony gregarious settlement has
never been reported within the genus. This behavior might be an evolutionary
selection to avoid being accidentally eaten up by the adults, which also engulf
sediment and other particulate matter during feeding.
In KCl exposure trial, the recovery after 24 h exposure was relatively high
and comparable with treatments using natural inducers (i.e., sediment from nursery
habitat and Thalassia detritus leachate). A similar study on S. gigas
reported between 52%-95% recoveries within 16 h exposure at 15 mM (Davis
et al., 1990). Another study by Boettcher and
Targett (1998) reported KCl concentration between 20 and 22 mM induced levels
of metamorphosis equivalent to those induced by the algal extract. Low concentrations
of KCl (between 5 and 15 mM) are therefore suggested for large scale metamorphosis
of the species studied, but test on commercial scale still yet to be conducted.
Elevated K+ concentration induced metamorphosis in marine invertebrates
presumably by depolarizing sensory cells (Boettcher and
Targett, 1998). Potassium chloride has considerable potential for S.
canarium aquaculture (spat production) mainly because it is readily available,
inexpensive, effective and non-toxic (Yu et al., 2008). It has been applied
as metamorphosis inducer in other mollusks hatcheries with consistent dosage
(Pechenik and Heyman, 1987; Davis
et al., 1990; Yu et al., 2008).
The GABA is one of neurotransmitters that had the ability to inhibit velar
cilia movement thus, induces the larvae metamorphosis behavior (Kaspar
and Mountfort, 1995). GABA has been used by an increasing number of commercial
hatcheries, because of it higher efficiency over other settlement methods (Bernal
and Beltran, 1998). It has been reported to effectively induced settlement
and metamorphosis in many mollusks larvae (Doroudi and Southgate,
2002; Zhao et al., 2003; Garcia-Lavandeira
et al., 2005). In contrast, GABA was less effective as metamorphic
inducer for S. canarium larvae studied. The percentage of metamorphosis
was very low in this treatment. Boettcher and Targett (1996)
reported no significant effect on larval metamorphosis when exposed to 1 and
100 μM GABA. The current study also showed evidence of toxic effects, which
has been reported in other settlement studies using GABA (e.g., Bryan
and Qian, 1998; Li et al., 2006; Yu
et al., 2008). The toxicity might be due to long exposure to the
chemical or through bacterial infestation as. GABA provide a suitable substrate
for bacterial growth.
The present study showed that S. canarium larvae demonstrate active habitat selection during metamorphosis. The metamorphosis responses were strongly influenced by the sediment and detrital substrata taken from their natural nursery habitat. Observations suggested that associated epibiont within the conch nursery habitat elicited the important cues for S. canarium larvae metamorphosis. As biological and chemical cues play great roles in the species metamorphosis, further investigations are therefore needed for extraction and characterization of the active compounds and the mechanism of the cues. The experiment also demonstrates that potassium chloride (KCl) has considerable potential in S. canarium aquaculture. It may provide a potentially effective and inexpensive method for enhancing large populations of synchronized spat production in S. canarium hatchery. As difficulties in the commercial culturing of mollusks mainly associated with larval metamorphosis, the establishment of metamorphosis cues for this species provide strong basis for advancement of the species into aquaculture.
The authors would like to thank the deanery and staffs of the Biology Department, Universiti Putra Malaysia and School of Environmental and Natural Resource Sciences, Universiti Kebangsaan Malaysia for technical supports, equipments and laboratory facilities provided. This study was funded by the Malaysian government through Universiti Kebangsaan Malaysia research grant No. UKM-ST-08-FRGS0001-2008.
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