ELBA BIOFLUX

Extreme Life, Biospeology & Astrobiology

International Journal of the Bioflux Society

Influence of photoperiod on digestive enzyme

activities of the Angelwing clam (Pholas
orientalis)
Ruby U. Tizon, Augusto E. Serrano Jr., Rex F. M. Traifalgar
Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the
Philippines Visayas, Miagao, Iloilo, Philippines. Corresponding author: A. E. Serrano Jr.,
serrano.gus@gmail.com

Abstract. Thirty adult Angelwing clams, Pholas orientalis, (89.78 6.60 g wet weight; 118.13 2.92
mm standard length) were acclimated in the laboratory for 7 d, randomly segregated into 3 groups which
were exposed to different photoperiod regimes (24 h light, 12 h light - 12 h dark, 24 h dark). After 7 d,
the clams were sacrificed and their crystalline styles excised and the digestive enzymes -amylase, CMcellulase, agarase, laminarinase and protease were assayed. All the digestive enzymes, amylase,
cellulase, agarase, laminarinase and protease exhibited significantly the highest activities when the
clams were exposed to 24 h darkness for 7 d and those exposed to continuous artificial light or to equal
light and dark hours exhibited lower activities but were not significantly different from each other.
Key Words: Photoperiod, Pholas orientalis, digestive enzyme activities, alpha amylase, cellulase,
agarase, laminarinase, protease.

Introduction. Differences in day lengths or photoperiods have been used by many

marine invertebrates as an external factor to start biological and physiological processes
(DeCoursey 1983). Photoperiod can regulate reproduction and growth of marine species.
Manipulation of light-dark cycles has been shown to increase the growth of fish
(Stefansson et al 1989) and shrimp (Withyachumnarnkul et al 1990).
In fish, photoperiod is a factor triggering melatonin production that regulates
secretions of releasing hormones in the hypothalamus, which in turn control the
production of hormones in the pituitary, eventually leading to gonadal maturation
(Devauchelle & Mingant 1991; Maitra et al 2006). Melatonin, the endocrine signal of
photoperiod, is produced by the pineal gland and retina of the eye at significantly higher
amounts during the night than at daytime (Maitra et al 2006). Its presence has been
detected in marine invertebrates like freshwater prawn Macrobrachium rosenbergii
(Withyachumnarnkul
et
al
1992),
giant
tiger
shrimp
Penaeus
monodon
(Withyachumnarnkul et al 1995), sea hare Aplysia californica (Abran et al 1994), fiddler
crab Uca pugilator (Tilden et al 2001), snail Helix aspersa (Blanc et al 2003), and rotifer
Philodina sp. (Hardeland & Poeggeler 2003). Melatonin is reported to translate
environmental signals like photoperiod into rhythmic messages in fish (Maitra et al
2006). Although the chemical signal regulating enzyme activity has not been elucidated
yet in bivalves, melatonin has been shown to modulate the circadian rhythm in
invertebrates like crayfish Procambarus clarkii (Solis-Chagoyan et al 2008).
There is scarcity of reports elucidating the influence of photoperiod on the
digestive capacity of bivalve species. Expression of photoperiod phenomena is possible in
animals possessing some sort of a photoreceptor (Cronin 1986). Hecht (1927) has
reported that the photoreceptors in Pholas are located in the siphon and exposed parts of
the mantle. Bivalves respond to light stimulation. The shells of Mytilus edulis grown in
darkness are thin and brittle (Stromgren 1976b), a characteristic of Pholas orientalis. The
same author has demonstrated that when M. edulis are grown in the dark, the clam has
lighter pigmentation, again similar to Angelwing clams. Furthermore, Hecht (1927) has

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demonstrated that the siphon of Pholas dactylus is very sensitive to light. Similarly,
Corda et al (1998) have shown that direct light exposure causes shortening of P.
orientalis siphon, resulting to lower filtration rate. The decrease in the enzyme activity
observed in the present study among bivalves exposed to continuous 24 h light was in
agreement with these findings. The decrease may be related to the depressed ability of
the animal to feed and since enzyme activity is considered to be related to availability of
food substrate in the gut. Continued darkness results in a higher shell length growth in
mussel M. edulis which is correlated with higher defecation rate than in natural daylight
and continuous light (Nielsen & Stromgren 1985). Moreover, the growth rate of M. edulis
(Seed 1969; Stromgren 1976a, 1976c), Modiolus modiolus (Stromgren 1976b) and snail
Heliosoma duryi (Kunigelis & Saleuddin 1978) are significantly improved when the
animals are kept in dark environment. Nielsen & Stromgren (1985) have suggested that
light affects the bivalves filtering capacity, leading to reduced shell formation since
calcification is dependent upon filtration rate and food intake. Moreover, Nielsen &
Stromgren (1985) propose that the bivalves showing positive correlation with darkness
(M. edulis and M. modiolus) are pigmented and epifaunal while light-insensitive clams
like Cerastoderma edule are unpigmented and sand burrower. In contrast, several
authors have reported beneficial effects of lighted environment. Increasing light exposure
to 15 h d-1 resulted in a higher percentage of spawning in Pecten maximus scallop
(Devauchelle & Mingant 1991). Sick et al (1973) report that ingestion rate of pelleted
food by juvenile shrimp Penaeus setiferus is directly proportional with light intensity.
This study aimed to measure the influence of photoperiod on the Angelwing clams
in laboratory condition on the activities of digestive enzymes namely -amylase, CMcellulase, agarase, laminarinase and protease.
Material and method
Experimental set up. Thirty adult Angelwing clams, P. orientalis, (89.78 6.60 g wet
weight; 118.13 2.92 mm standard length) were acclimated in the laboratory for 7 d,
randomly segregated into 3 groups and each group exposed to different photoperiod
regimes (24h light, 12h light - 12h dark, 24h dark). Angelwing clams were placed in
aquaria and fed equal proportions of the four algal diets (Chaetoceros calcitrans,
Isochrysis galbana, Thalassiosira sp., and Tetraselmis tetrathele). Those exposed to 24
h-light treatment were provided with 40 W fluorescent lamp (5.7 Klux), while aquaria of
those in 24 h dark treatment was totally covered with black cloth. Those in the 12 h light
- 12h dark were exposed to 12 h with 40 W fluorescent lamp and 12 h totally covered
with black cloth. After 7 d treatment, the clams were excised, the crystalline styles
removed and stored in ultra low freezer (-85oC) until assay.
Preparation of enzyme extracts. Crystalline styles were excised, weighed and stored
in ultra low freezer (-85oC) until assay. All preparation procedures were done at 4oC
unless otherwise stated. During enzyme extraction, the crystalline styles were thawed,
weighed and washed with cold citrate phosphate buffer, pH 7.0. To the style was added
extraction buffer at 1:30 w/v, homogenized in an Ultraturrax homogenizer, and
centrifuged at 4000 rpm for 15 min. The supernatant was filtered and used as enzyme
extract for the assays.
Enzyme assays. The digestive enzymes -amylase, cellulase, agarase, laminarinase and
protease activities were assayed at 25 oC. All measurements were done in triplicates,
with corresponding blank and control samples.
Carbohydrases. Alpha amylase, CM-cellulase, agarase and laminarinase activities were
measured following the method of Areekijseree et al (2004) modified from Bernfield
(1955). For amylase, the reaction mixture consisted of 0.2 mL enzyme extract, 1.8 mL
phosphate and 1 mL of substrate (1.0 % soluble starch dissolved in buffer) in a final
volume of 2.0 mL. The reaction was stopped by adding 1.0 mL 3,5-dinitrosalicylic acid
(DNS) solution after 15 min of the reaction. The solution was placed in boiling water bath
for 10 min until the color of the solution turned from yellow to dark red and was allowed

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to cool to room temperature. The optical density (OD) of the clear solution was read at
546 nm. Mixtures with no substrate or no enzyme or both were used as blank samples
for correction of innate activity in the crude extract and for the exclusion of spontaneous
hydrolysis of the substrate, respectively.
Similar procedure was employed in the assays of CM-cellulase, agarase and
laminarinase varying only in the substrate used. CMC, agarose, and laminarin were
dissolved in corresponding buffers and used as substrate for CM-cellulase, agarase and
laminarinase assays, respectively. The reaction mixture for the CM-cellulase assay
consisted of 0.3 mL enzyme extract, 1.0 mL 0.25 % CMC and 1.7 mL citrate-phosphate
buffer, pH 6.0, in a final volume of 3.0 mL. That for agarase consisted of 0.1 mL enzyme
extract, 1.0 mL agarose substrate (0.2 %) and 1.9 mL citrate-phosphate buffer, pH 6.0,
in a final volume of 3.0 mL. The reaction mixture for the laminarinase was 0.3 mL
enzyme extract, 1.0 mL 0.1 % laminarin, 1.7 mL of citrate-phosphate buffer, pH 6.0, in a
final volume of 3.0 ml. The reactions were stopped after 15 and 30 min for CM-cellulase
and agarase, and 30 min for laminarinase, respectively.
All carbohydrase activities were quantified using glucose as standard except for
agarase activity in which galactose was used as standard. Protein was determined
following the procedure of Bradford (1976) using bovine serum albumin as standard.
Alpha amylase, CM-cellulase and laminarinase activities were expressed as mol glucose
liberated min-1 mg-1 protein while agarase activity was expressed as mol galactose
liberated min-1 mg-1 protein.
Protease. Proteolytic activity was measured following the method of Kunitz (1947) with
some modifications. The reaction mixture consisted of 1.0 mL 1.0 % casein dissolved in
0.01 N NaOH, 1.5 mL phosphate buffer, pH 7.0 and 0.5 mL enzyme extract, in a final
volume of 3.0 mL. After 60 min, the reaction was stopped by adding 1.0 mL ice-cold 5 %
trichloroacetic acid, allowed to stand for 15 min, centrifuged and filtered. The optical
density of the clear supernatant was read at 280 nm. Mixtures with no substrate or no
enzyme or both were used as blank samples. Tyrosine was used as standard for the
expression of enzyme activity as g tyrosine released hr-1 mg-1 protein.
Results and Discussion. All the digestive enzymes, amylase, cellulase, agarase,
laminarinase and protease exhibited significantly the highest activities when the clams
were exposed to 24 h darkness for 7 d and those exposed to continuous artificial light or
to equal light and dark hours exhibited lower activities but were not significantly different
from each other (Figures 1-5).
Although the Angelwing clam is a mud burrower and has minimal pigmentation,
digestive capacity was enhanced in dark environment probably because it resembled the
clams natural setting being found in subtidal areas of about 8 m from the water surface
(Laureta & Marasigan 2000). In addition, it burrows itself into muddy substrate at a
depth of 0.3-0.6 m with only its siphon sticking out of the substrate to feed on
suspended particles. According to Loosanoff & Nomejko (1946), natural oyster beds are
situated at a considerable depth (10-30 ft, a depth comparable to that of P. orientalis
bed) with suspended matter blocking the sun rays. Thus, oyster exists in near-darkness
even during very strong daylight, a condition not so dissimilar to the dark regime in the
present study, and the natural setting of P. orientalis beds. Another possible reason for
increased enzyme activity at dark environment is the nightly vertical migration of
phototactic algae in the natural environment. Phytoplankton are concentrated on the
water surface at daylight, but migrate to deeper areas at night time (Staker & Bruno
1980), making the algae available for the filter feeding bivalves. Thus, the bivalves might
have been accustomed to feeding at dark times, triggering the significant increase of
digestive enzyme activities in the dark photoperiod regime.

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-1

-1

mol glucose min mg protein

27.0
24.0

21.0
18.0
15.0
12.0
9.0
6.0
3.0
0.0
24h light

12h light-12h dark

24h dark

Photoperiod
Figure 1. Alpha amylase activities of clams exposed to different photoperiods. The assay
mixture containing 0.2 mL enzyme extract, 1.8 mL buffer (pH 7.0) and 1.0 mL 1.0 %
soluble starch (w/v) was incubated for 15 min. Bars represent mean standard
deviation; means with different letters indicate significant differences between
treatments (P < 0.05, Tukey Test).

Figure 2. CM-cellulase activities of clams exposed to different photoperiods. The assay

mixture containing 0.3 mL enzyme extract, 1.7 mL buffer (pH 6.0) and 1.0 mL 0.25 %
CMC (w/v) was incubated for 15 min. Bars represent mean standard deviation; means
with different letters indicate significant differences between treatments (P < 0.05, Tukey
Test).

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Figure 3. Agarase activities of clams exposed to different photoperiods. The assay

mixture containing 0.1 mL enzyme extract, 1.9 mL buffer (pH 6) and 1.0 mL 0.2 %
agarose (w/v) was incubated for 15 min. Bars represent mean standard deviation.
Means with different letters indicate significant differences between treatments (P <
0.05, Tukey Test).

Figure 4. Laminarinase activities of clams exposed to different photoperiods. The assay

mixture containing 0.3 mL enzyme extract, 1.7 mL buffer (pH 6.0) and 1.0 mL 0.1 %
laminarin (w/v) was incubated for 30 min. Bars represent mean standard deviation.
Means with different letters indicate significant differences between treatments (P <
0.05, Tukey Test).

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240

-1

-1

g tyrosine h mg protein

ab
210

180
150
120
90
60
30
0
24h light

12h light-12h dark

24h dark

Photoperiod
Figure 5. Protease activities of clams exposed to different photoperiods. The assay
mixture containing 0.5 mL enzyme extract, 1.5 mL buffer (pH 7.0) and 1.0 mL 1.0 %
casein (w/v) was incubated for 1 h. Bars represent mean standard deviation. Means
with different letters indicate significant differences between treatments (P < 0.05, Tukey
Test).
The natural feeding activity of P. orientalis in the wild has not been elucidated to date.
The present results showed higher enzymatic activity at low light setting, indicating that
the clam exhibited a heightened feeding activity at low-light conditions. The present
findings agreed with previous works in other bivalve species, M. edulis (Stromgren
1976a, 1976c) and M. modiolus (Stromgren 1976c) elucidating that exposure to low light
levels enhances growth and metabolic activities. Although these earlier reports have
shown the positive effects of low light exposure to growth of bivalves, the physiological
mechanism involved in such effects remains unclear. Nielsen & Stromgren (1985) have
demonstrated that the improvement of M. edulis growth due to continuous low light
exposure is attributed to higher feed intake and to higher defecation rate, indicating a
heightened feeding activity. Similarly, Hecht (1927) has shown that that the siphon of
Pholas dactylus is extremely sensitive to light. In addition, Corda et al (1998) have
demonstrated that the filtration rate of P. orientalis is influenced by light exposure.
Angelwing clams exposed to continual light exhibited a decreased filtration rate. The
present study agreed with these earlier reports elucidating the enhanced digestive
activities of clams maintained in dark environment. The present work was the first
evidence indicating that digestive enzyme activity in a bivalve species can be influenced
by light exposure. Moreover, it could provide additional evidence on previous works,
showing faster growth rates in bivalves maintained in low lighted environment.
Conclusions. All the digestive enzymes, namely, -amylase, CM-cellulase, agarase,
laminarinase and protease exhibited significantly the highest activities when the clams
were exposed to 24 h darkness for 7 d and those exposed to continuous artificial light or
to equal light and dark hours exhibited lower activities and were not significantly different
from each other.
Acknowledgements. The authors wish to thank the Philippine Council for Aquatic and
Marine Research and Development (PCAMRD) of the Department of Science and

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Technology (DOST) for providing the research fund and for the scholarship (Accelerated
Science and Technology Human Resource Development Program).
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Received: 20 February 2013. Accepted: 21 March 2013. Published online: 01 May 2013.
Authors:
Ruby Ursula Tizon, Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the
Philippines Visayas, Philippines, Miagao, 5023 Iloilo, e-mail: ruby.tizon@yahoo.com
Augusto Erum Serrano Jr., Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the
Philippines Visayas, Philippines, Miagao, 5023 Iloilo, e-mail: serrano.gus@gmail.com
Rex Ferdinand Mallare Traifalgar, Institute of Aquaculture, College of Fisheries and Ocean Sciences, University
of the Philippines Visayas, Philippines, Miagao, 5023 Iloilo, e-mail: skerferd@yahoo.com
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.
How to cite this article:
Tizon R. U., Serrano Jr. A. E., Traifalgar R. F., 2013 Influence of photoperiod on digestive enzyme activities of
the Angelwing clam (Pholas orientalis). ELBA Bioflux 5(1):61-68.

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