THE APPLICATION OF DNA SEQUENCING IN IDENTIFYING THE BIODIVERSITY OF CERITHIDEA OBTUSA ALONG THE MANGROVES OF PARIT KEROMA, MUAR, JOHOR

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

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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.

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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.

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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)

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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.

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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. 

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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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REFERENCES:

1.    Brenda Hookham, Aileen Tan Shau-Hwai, Benoit Dayrat & William Hintz (2014). A baseline measure of tree and gastropod biodiversity in replanted and natural mangrove stands in Malaysia: Langkawi Island and Sungai Merbok. Tropical Life Sciences Research, 25 (1), 1-12.

2.    Cheng W. H. & Yap C. K. (2015). Potential human health risks from toxic metals via mangrove snail consumption and their ecological risk assessments in the habitat sediment from Peninsular Malaysia. Chemosphere. 2015 Sep;135:156-65. doi: 10.1016/j.chemosphere.2015.04.013. Epub 2015 May 15.

3.    Cob Z. C., Samat A., Muda W. M. L. W., & Mazlan A. G. (2012). Preliminary checklist of marine invertebrate fauna within the intertidal of Teluk Penyabong and Teluk Gorek, Mersing, Johor, Malaysia. Journal of Tropical Marine Ecosystem 1, 1-14.

4.    David A. Micklos, Bruce Nash & Uwe Hilgert. Genome Science: A Practical and Conceptual Introduction to Molecular Genetic Analysis in Eukaryotes. Cold Spring Harbor Laboratory Press.

5.    Franklin Berandah anak Edward Thomas (2009). An Assessment of Metal Distribution and Metal Soluble Fractions in the Edible Molluscs from Malaysia.

6.    Hafizul Haque & Amalesh Choudhury (2014). Radula Morphology In Cerithidea Obtusa (Lamarck, 1822) (Gastropoda: Potamiddiae) From Sundarbans. International Journal of Engineering Science Invention. ISSN (Online): 2319-6734. ISSN (Print): 2319-6726.

7.    Harinder Rai Singh (2012). Biodiversity - mangroves & gastropods. Marine Biodiversity Expedition 2012.

8.    I. Faridah Hanum, A. Latiff, Khalid Rehman Hakeem & Munir Ozturk (2012). Mangrove Ecosystems of Asia. Status, Challenges and Management Strategies

.

9.    Kumar Krishnan, Elias Saion, Halima M. K., Yap C. K. & Muhd Suhaimi Hamzah (2014). Distributions of heavy metal in the surface sediments of mangrove from west coast of Peninsular Malaysia. International Journal of Application or Innovation in Engineering & Management. Volume 3, Issue 7. ISSN 2319-4847.

10. Reid D. G., Dyal P., Lozouet P., Glaubrecht M., Williams S. T. (2008) Mudwhelks and mangroves: The evolutionary history o an ecological association (Gastropoda: Potamididae). Molecular Phylogenetics and Evolution 47: 680-699.

11. Sri Purwaningsih (2012). Antioxidant Activity and Nutrient Composition of Matah Merah Snail (Cerithidea Obtusa). Ilmu Kelantan Maret 2012. Vol. 17 (1) 39-48. ISSN 0853-7291.

12. Sukarno Wagiman. Imposeks dalam Siput Haliah, Thais sp. sebagai penunjuk biologi pencemaran tributil tanah di Perairan Semenanjung Malaysia.

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