THE APPLICATION OF DNA SEQUENCING IN IDENTIFYING THE
BIODIVERSITY OF CERITHIDEA OBTUSA ALONG THE MANGROVES OF PARIT KEROMA, MUAR,
JOHOR
by
ZOEY ZEFFREY AZMAN, 2K3
This proposal is submitted to Pn.
Syakina, Research Instructor of Kolej PERMATApintar Negara, UKM, in fulfillment
of the Requirement for the Research and Methodology subject for Foundation 2
November 2015
********************************************
ABSTRACT
This
study is on the application of DNA sequencing in identifying the biodiversity
of Cerithidea obtusa along the mangroves of Parit Keroma, Muar, Johor. Cerithidea
obtusa is a species of sea snail, a marine gastropod mollusk in the family
Potamididae. It is used as food in Malaysia where it is known by the name
"Siput Sedut". This research is concentrated only on Cerithidea
obtusa. This study will be conducted in the mangrove areas along
the coastal side of Parit Keroma, Muar, Johor, facing the Strait of Malacca; at
about 10 locations from latitude 2.005010, 102.572937 to longitude 2.001547,
102.574021. Visits to the sampling area at the end of each month for 5
consecutive months and collecting Cerithidea obtusa sp. in 10 different
locations. Each location will be marked on the nearest mangrove tree. The
samples of Cerithidea obtusa sp. will be collected during low tides via hand,
and will be placed into containers with 95% alcohol, with it being labeled
after (date collected, place collected). The process being carried out is DNA
extraction, Polymerase Chain Reaction (PCR), gel electrophoresis and cycle
sequencing.
**************************************************
CHAPTER 1
INTRODUCTION
1.1 Background
of Study
Marine
molluscs are seafood commodities. They inhabit their natural habitat, the
intertidal area, which are usually close to estuaries. In Malaysia, the mangrove
ecosystem has been an important resource for the coastal, estuarine and to a
certain extent, the riverine communities. Mangrove trees are found along the
west coast of Peninsular Malaysia, generally associated with mudflats and clay
swamps, which provide good environments for living organisms including
gastropods. According to the Department
of Fisheries, the Malaysia Fisheries Directory documented 27 bivalves and 18
species of gastropods in Malaysia's coastal areas.
Cerithidea
obtusa is a species of sea snail, a marine gastropod mollusk in the family
Potamididae. The Obtuse Horn Shell also known as "Mud Creeper" is a
relatively common snail found in muddy coastal areas. It grows to around 5-6
cm. It is used as food in Southeast Asia where it is known by the name
"Siput Sedut" or "Belitung". This research is concentrated
only on Cerithidea obtusa along the coasts of Parit Keroma, Muar, Johor, facing
the Strait of Malacca. The objective of this research is to determine the
actual species of the Cerithidea obtusa along the coasts of Parit Keroma; to
understand the lifestyles of Cerithidea obtusa along the coasts of Parit
Keroma; and for profiling in the GenBank and BOLD.
1.2 Statement
of the Problem
Is
the species of Cerithidea obtusa located in mangrove areas along the coast of
Parit Keroma, Muar, Johor unique in a way that it has been profiled into
GenBank, or is it similar to the other species all across Malaysia which have
already been profiled?
1.3 Objective
of Study
1.
To
determine the actual species of the Cerithidea obtusa along the coasts of Parit
Keroma;
2.
For
profiling in the GenBank and BOLD; and
3.
To
understand the lifestyles of Cerithidea obtusa along the coasts of Parit Keroma
1.4 Limitations
of the Study
The
equipment in the prepared lab of Kolej PERMATApintar Negara, the Biology lab,
may not be enough for the DNA extraction and sequencing.
1.5 Basic
Assumptions
1.
The
species of Cerithidea obtusa found in the mangroves of Parit Keroma is
different from the species found in the other parts of Malaysia, making it a
unique species of Cerithidea obtusa, which has not been found or documented.
2.
The
species of Cerithidea obtusa found in the mangroves of Parit Keroma is the same
as the species found in the other parts of Malaysia.
*********************************************
CHAPTER 2
LITERATURE REVIEW
2.1 Studies
on mangrove biodiversity:
Mangroves
are found globally in tropical ad sub-tropical regions at the confluence of
marine and terrestrial environments and support a unique ecosystem of
considerable importance (Brandea Hookham, Aileen Tan Shau-Hwai, Benoit Dayrat
and William Hintz, 1-12, 2014). In Malaysia, the mangrove ecosystem has been an
important resource for the coastal, estuarine and to a certain extent, the riverine
communities. Mangrove trees are found along the west coast of Peninsular
Malaysia, generally associated with mudflats and clay swamps, which provide
good environments for living organisms including gastropods (Franklin Berandah
anak Edward Thomas, FS 2009 16).
According to the Department of Fisheries, Malaysia (2005), in 2005 - 2006,
the Malaysia Fisheries Directory documented 27 bivalves and 18 species of gastropods
in Malaysia's coastal areas.
Johor
has a total of 20533 hectares of mangrove forests which are mostly found in
Sungai Pulau Forest Reserve, Sungai Johor Forest Reserve, Sungai Santi Forest
Reserve and Sungai Lebam Forest Reserve (I. Faridah-Hanum, A. Latiff, Khalid
Rehman Hakeem, Munir Ozturk, 2012). The mangrove coastal area from Muar to Batu
Pahat has not been categorized as a mangrove forest reserve. Therefore, this
area slowly has been cleared and converted to small coconut plantations, palm
oil cultivations and aqua cultural activities (I. Faridah-Hanum, A. Latiff,
Khalid Rehman Hakeem, Munir Ozturk, 2012). This research is confined to the coastal
areas of Muar; specifically along the mangroves of Parit Keroma, Muar, Johor.
The encroachment to this mangrove area particularly at Parit Keroma has given
impact to the biodiversity, especially a certain species of gastropods, Cerithidea obtusa.
Research
has been conducted on the components of the composition of Cerithidea obtusa in
Indonesian islands and discovered that there are antioxidant compounds. Antioxidant
compounds are frequently produced by natural foods. Cerithidea obtusa is one of
fishery commodities which is commonly consumed by society and traditionally
used for therapeutic purposes. The aim of this research was to investigate the
nutrient composition and antioxidant activity of C. obtusa. The nutrient
compounds were analyzed using proximate contents according to AOAC method,
mineral contents using APHA method, and amino acid contents using AOAC method.
The results showed that Cerithidea obtusa contained 77.5% of moisture, 13.8% of
protein, 2.8% of fat, and 4.5% of ash. Analyses of macro- and micro-minerals
revealed that the highest level of macro-minerals was sodium 283.45 mg/100 g
and the lowest one was calcium 39.78 mg/100 g. The highest level of
micro-minerals was selenium 39.25 mg/100 g and the lowest one was copper 0.29
mg/100 g. The highest content of essential amino acids was isoleusin 4.82% and
the lowest one was arginine 0.95%. The highest content of non-essential amino
acids was glutamic acid 12.08% and the lowest one was cysteine 0.84%. Methanol
extract of C. obtusa displayed potential antioxidant activities with IC50 value
of 58,19 ppm, with IC50 of vitamin C as positive control was 3,555 ppm (Sri
Purwaningsih, 2012).
2.2 Study
of species of gastropods in Malaysia:
There
are quite a significant amount of researches on the gastropods' biodiversity in
mangrove areas in Malaysia. The recent studies were done in Langkawi and Sungai
Merbok, and they have reported that the gastropod species were rarer at the
sampling site, especially species of Cerithidea
obtusa (Brandea Hookham, Aileen Tan Shau-Hwai, Benoit Dayrat and William
Hintz, 1-12, 2014). The study on biodiversity and distributions of marine
invertebrate fauna was done at the areas of Mersing (Cob Z.C., Samat A., Muda
W.M.L.W., Mazlan A.G, 2012: 1-14) of which field sampling was carried out using
a transact line method, during low tide periods. The transact line was laid
down along the tidal height, perpendicular to the shoreline. All marine
invertebrates encountered within 1 meter areas of the line were recorded. Species were taken and preserved in 5% formalin
for analysis and identification.
2.3 Study
on DNA Sequencing to identify species of gastropods; Cerithidea obtusa sp.
Till
this day, I have not found any researches in Malaysia which involve studies on
DNA sequencing to identify new species of Cerithidea
obtusa sp. The application of DNA sequencing to identify species of gastropods
has done previously by D.G. Reid, P. Dyal, P. Lozouet, M. Glaubrecht, S.T. Williams
(2008). DNA was extracted from mantles or foot tissues of ethanol-preserved
materials using DNeasy Blood and Tissue Kits (Qiagen) following the manufacturer’s
instruction. Sequences were edited using Sequencher (v4.5, Gene Codes
Corporation, Ann Arbor, Michigan). There is one research conducted on radula
morphology, in Cerithidea obtusa
(Lamarck, 1822) which was analyzed with Scanning Electron Microscopes (Hafizul
Haque and Amalesh Choudry, 2014).
Most
of the 29 living species of Potamididae show a close association with
mangroves. The trees provide the snails with shelter, protection from
predators, a solid substrate and sometimes food. Using sequences from three
genes (nuclear 18S rRNA and 28S rRNA, mitochondrial COI) we derive a molecular
phylogeny and recognize six living genera (Terebralia, Telescopium,
Tympanotonos, Cerithidea, Cerithideopsis, Cerithideopsilla). The oldest modern
genera (Terebralia, Cerithideopsis) appeared in the Tethyan realm in the Middle
Eocene, shortly after the origin of mangrove trees. Whereas most potamidid
genera are now restricted to either the Indo-West Pacific (IWP) or to the
eastern Pacific plus Atlantic (EPA), sister clades of Cerithideopsis survive in
both realms. Based on a reinterpretation of the fossil record (particularly of
the monotypic Tympanotonos and extinct Potamides), and parsimonious reconstruction
of ancestral habitats, we suggest that the living potamidids are an adaptive
radiation that has always been closely associated with mangroves. The
specialized tree-climbing groups Cerithidea and Cerithideopsis were
independently derived from mud-dwelling ancestors (Reid D. G., 2008)
**************************************************
CHAPTER 3
PROCEDURES / METHODOLOGY
3.1 Study
Area and Sampling Procedure:
· This
study will be conducted in the mangrove areas along the coastal side of Parit
Keroma, Muar, Johor, facing the Strait of Malacca; at about 10 locations from
latitude 2.005010, 102.572937 to longitude 2.001547, 102.574021.
· Visits
to the sampling area at the end of each month for 5 consecutive months and
collecting Cerithidea obtusa sp. in
10 different locations. Each location will be marked on the nearest mangrove
tree.
·
The
samples of Cerithidea obtusa sp. will
be collected during low tides via hand, and will be placed into containers with
95% alcohol, with it being labeled after (date collected, place collected)
3.2 Experimental
Procedure
·
DNA Isolation:
Reagents:
|
Supplies
and Equipment:
|
Qiagen®
DNeasy Blood & Tissue Kit, including:
Buffer
ATL (180 µL)
Buffer
AL (200 µL)
Proteinase
K (20 µL)
100%
Ethanol (200 µL)
Buffer
AW1 (500 µL)
Buffer
AW2 (500 µL)
Buffer
AE (100 µL)
1
DNeasy Mini spin column plus 2 additional collection tubes (2 mL)
Specimen
tissue sample(s)
|
Container
with cracked or crushed ice
Microcentrifuge
2
Microcentrifuge tubes (1.5 mL)
Micropipettes
and tips (2-1000 µL)
Permanent
marker
Tweezers
and scissors
Water
bath or heating blocks at 56 ºC
Vortexer
(optional)
Microcentrifuge
tube rack
|
Prior to beginning:
·
Buffer AL may form a precipitate upon storage. If
necessary, warm to 56°C until the precipitate has fully dissolved.
·
Buffer AW1 and Buffer AW2 are supplied as
concentrates. Before using for the first time, add the appropriate amount of
100% ethanol as indicated on the bottle to obtain a working solution.
i.
Obtain
a piece of Cerithidea obtusa sp.
tissue ~10-20 mg or 1/4 inch diameter from each sample collected.
ii.
Place
tissue into a clean 1.5 mL microcentrifuge tube labeled with sample
identification number.
iii.
Add
180 μL of buffer ATL to each tube. Use different pipette tip for each sample.
iv.
Add
20 μL Proteinase K (20 mg/mL) to each tube and mix thoroughly the tissue with
the solutions. Use different pipette tip for each sample.
v.
Vortex
tubes for 5 seconds. Use a vortex if available.
vi.
Incubate
at 56°C for at least 3 hours on a rocking platform (incubator) until the sample
is completely lysed. Samples may appear sticky.
vii.
Remove
from incubator and vortex by hand or machine (if available) for 5 seconds.
viii.
Add
200 μL of buffer AL and 200 μL 100% Ethanol to each tube, vortex by hand or
machine for 5 seconds.
ix.
From
the mixture, transfer ~600 μL to a spin DNeasy Mini spin column labeled with
sample identification number. Spin column should be placed in a 2-ml collection
tube.
x.
Place
your tubes in a balanced configuration in a microcentrifuge, with cap hinges
pointing outward. Centrifuge for 1 minute at ≥6000 x g (8000 rpm).
xi.
Dispose
of the collection tube containing the flow-through and put the column in a
clean 2-mL collection tube.
xii.
Add
500 μL of buffer AW1.
xiii.
Place
your tubes in a balanced configuration in a microcentrifuge, with cap hinges
pointing outward. Centrifuge for 1 minute at ≥6000 x g (8000 rpm).
xiv.
Dispose
of the collection tube containing the flow-through and put the column in a
clean 2-mL collection tube.
xv.
Add
500 μL of buffer AW2.
xvi.
Place
your tubes in a balanced configuration in a microcentrifuge, with cap hinges
pointing outward. Centrifuge 3 minutes at ≥20,000 x g (14000 rpm).
xvii.
Dispose
of the collection tube containing the flow-through.
xviii.
Place
spin column on a clean 1.5-mL microcentrifuge tube previously labeled with
sample identification number.
xix.
Add
100 μL of buffer AE directly to the membrane.
xx.
Incubate
samples for 5 minutes at room temperature.
xxi.
Place
your tubes in a balanced configuration in a microcentrifuge, with cap hinges
pointing outward. Centrifuge 1 minute at ≥6000 x g (8000 rpm).
xxii.
Discard
spin column but keep your 1.5-mL microcentrifuge containing the eluted DNA.
·
Polymerase Chain Reaction (PCR)
Reagents:
|
Shared
Supplies and Equipment:
|
Appropriate
primer/loading dye mix (25 µL)* per reaction
DNA
from specimen(s) (from part II)*
1
Ready-To-Go PCR Bead in 0.2- or 0.5-mL PCR tube per reaction or NEB Taq 2X
Master Mix (12.5 µL)* per reaction
*Store
on ice
|
Container
with cracked or crushed ice
Microcentrifuge
tube rack
Micropipettes
and tips (1-100 µL )
Permanent
marker
Thermal
cycler
|
i.
Obtain
PCR tube containing Ready-To-Go PCR Bead. Label the tube with your
identification number.
ii.
Use
a micropipette with a fresh tip to add 23 µL of one of the following
primer/loading dye mixes (for Ready-To-Go PCR Beads) to each tube. Allow the
beads to dissolve for 1 minute.
Ñ
Vertebrate
(non-fish): COI primers (VF1_t1/ VF1d_t1/ VF1i_t1 / VR1d_t1/ VR1_t1/ VR1i_t1)
Ñ
Invertebrate
cocktail: COI primers (LCO1490/ HC02198)
iii.
Use
a micropipette with fresh tip to add 2 µL of your DNA directly into the
appropriate primer/loading dye mix. Ensure that no DNA remains in the tip after
pipetting.
iv.
Place
the PCR tube in a thermal cycler that has been programmed with the appropriate
PCR protocol.
Vertebrate
(non-fish) cocktail
(VF1_t1/ VF1d_t1/ VF1i_t1/
VR1d_t1/ VR1_t1/ VR1i_t1)
|
Initial step: 94°C 1 minute
35 cycles of the following profile:
o
Denaturing step: 94°C
15 seconds
o
Annealing step: 54°C
15 seconds
o
Extending step: 72°C
30 seconds
One final step to preserve the sample: 4°C ad
infinitum
|
Invertebrate cocktail
(LCO1490/ HC02198)
|
Initial step: 94°C 1 minute
35 cycles of the following profile:
o
Denaturing step: 95°C
30 seconds
o
Annealing step: 50°C
30 seconds
o
Extending step: 72°C
45 seconds
One final step to preserve the sample: 4°C ad
infinitum
|
·
Gel Electrophoresis:
Reagents:
|
Supplies
and Equipment:
|
2%
agarose in 1x TBE (hold at 60°C) (50 mL per gel)
pBR322/BstNI
marker (20 µL per gel)*
PCR
products
SYBR
Green DNA stain(6 µL per group)
1x
TBE buffer (300 mL per gel)
*Store
on ice
|
Container
with cracked or crushed ice
Digital
camera or photodocumentary system
Gel-casting
tray and comb
Gel
electrophoresis chamber and power supply
Latex
gloves
Masking
tape
Microcentrifuge
tube rack
3
Microcentrifuge tubes (1.5 mL)
Micropipette
and tips (1–100 µL)
Microwave
UV
transilluminator and eye protection
Water
bath for agarose solution (60°C)
|
i.
Seal
the ends of the gel-casting tray with masking tape, or other method appropriate
for the gel electrophoresis chamber used, and insert a well-forming comb.
ii.
Pour
the 2% agarose solution into the tray to a depth that covers about one-third
the height of the comb teeth.
iii.
Allow
the agarose gel to completely solidify; this takes approximately 20 minutes.
iv.
Place
the gel into the electrophoresis chamber and add enough 1x TBE buffer to cover
the surface of the gel.
v.
Carefully
remove the comb and add additional 1x TBE buffer to fill in the wells and just
cover the gel, creating a smooth buffer surface.
vi.
Use
a micropipette with a fresh tip to transfer 5 µL of each PCR product to a fresh
1.5mL microcentrifuge tube. Add 2 µL of SYBR Green DNA stain to tube.
vii.
Add
2 µL of SYBR Green DNA stain to 20 µL of pBR322/BstNI marker.
viii.
Orient
the gel, so that the wells are along the top of the gel. Use a micropipette
with a fresh tip to load 20 μL of pBR322/BstNI size marker into the far left
well.
ix.
Use
a micropipette with a fresh tip to load each sample from Step 6 in your
assigned wells.
x.
Run
the gel for approximately 30 minutes at 130V. Adequate separation will have
occurred when the cresol red dye front has moved at least 50 mm from the wells.
xi.
View
the gel using UV transillumination. Photograph the gel using a digital camera
or photodocumentary system.
·
Cycle Sequencing:
Fluorescent
dyes are added to the reactions, and a laser within an automated DNA sequencing
machine is used to analyze the DNA fragments produced.
****************************************
RESULTS:
The
samples will be collected at the sampling area, for a total of 5 times, from 10
different spots in the sampling area. From each sample collected, DNA
sequencing will be carried out, and will be profiled to see if there are any
matches in GenBank, and will be repeated for each sample in different months.
The purpose is to determine if all samples are of the same species, or if they
are different species. At the end of the research, we will have a complete
compilation of DNA sequences of Cerithidea obtusa along the mangrove areas of
Parit Keroma, Muar, Johor.
**************************************************
DISCUSSION
The
results of this study will be restated and evaluated in light of the initial
hypotheses. If the results are as predicted, there will be a unique species of
Cerithidea obtusa living in the coasts of Parit Keroma, Muar, Johor, profiled
in GenBank and BOLD. Limitations of the current research will be identified,
along with suggestions for how future research can build upon the findings of
the current study. Finally, the results and importance of this study will be
summarized.
*****************************************************
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12. Sukarno Wagiman. Imposeks
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