Identification of Stomatopod Larvae In Banten Bay, Indonesia Using DNA Barcoding
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1 Identification of Stomatopod Larvae In Banten Bay, Indonesia Using DNA Barcoding Mariana D. Bayu Intan, M.Si. Dr. Abinawanto Drs. Wisnu Wardhana, M.Si. International Seminar on Marine and Fisheries Product Processing and Biotechnology September 26, 2014
2 Introduction Praying mantis-like raptorial claws Lobster-like crustaceans
3 Introduction Distribution Map of Stomatopod 115 species (23,95%)
4 Introduction Economic Value Oratosquilla oratoria Hong kong Kuala Tungkal, Jambi Harpiosquilla raphidea Harpiosquilla harpax Taiwan Tang 2009; Wardiatno & Mashar 2011; Mulyono dkk. 2013
5 Introduction Ecological Value One of meroplankton community Indicator of climate-induced changes in temperature Kirby dkk Abundance of pelagic fish (Skipjack tuna, grouper, and snapper fish) Bullard dkk. 1999; Ahyong & Harling 2000; Kirby dkk. 2008; Tang dkk. 2010
6 Introduction Indonesia P. Jawa Diversity of meroplankton Incl. Stomatopod larvae 2% 4% 2% 91% Ikan Demersal karang and dan pelagic pelagisfish Udang Shrimp Kepiting Crap Kerang Shellfish Cumi-cumi Squid Maintaining the sum of pelagic fish
7 Introduction 115 species of stomatopod Hutomo & Moosa spesies 72,17% 32 spesies 27,82% Spesies sequences found in NCBI Alima pacifica Gonodactylus smithii Chorisquilla excavata Haptosquilla glyptocercus Chorisquilla hystrix Haptosquilla pulchella Echinosquilla guerinii Haptosquilla stoliura Erugosquilla woodmasoni Haptosquilla trispinosa Fallosquilla fallax Harpiosquilla harpax Gonodactylaceus falcatus Harpiosquilla raphidea Gonodactylaceus glabrous Hoplosquilla said Gonodactylellus affinis Lysiosquillina maculata Gonodactylellus annularis Miyakea nepa Gonodactylellus caldwelli Odontodactylus latirostris Gonodactylellus erdmanni Odontodactylus scyllarus Gonodactylellus espinosus Pseudosquilla ciliata Gonodactylinus viridis Pseudosquillana richeri Gonodactylus childi Raoulserenea oxyrhyncha Gonodactylus platysoma Taku spinosocarinatus Source: National Center for Biotechnology Information (NCBI) 2013
8 Introduction DNA barcoding New method in taxonomic research which aimed to identify organisms in species-level by comparing the sequences divergence within taxa. Hajibabaei dkk. 2007; Tang dkk DNA barcoding Cytochrome c oxidase subunit 1 (COI) Animals spesies Plants 520 spesies Protist spesies Fungy 410 spesies Research of DNA barcoding in Stomatopod larvae Barber & Boyce 2006 Stomatopod larvae Lui dkk Oratosquilla oratoria adults Tang dkk Stomatopod larvae
9 Aim Identify and give more understanding about the diversity of stomatopod larvae in Banten Bay, Indonesia using DNA barcoding. Assess the effectiveness of DNA barcoding application in identifying stomatopod larvae in Banten Bay. Complete database of COI sequence in Gene Bank.
10 Material and Methods
11 Material and Methods Research Location Banten Bay Samples collection October-November 2013 Morphotype identification and molecular analysis Lab. Genetic, Departement of Biologi, FMIPA, UI
12 Material and Methods Samples collection 10 minutes Kec. 1-2 knot (1,87-3,74 km/jam) ❷ ❶ Samples collection Formalin 4% (10 drops) ❶ ❷ Samples stored in cool box
13 Material and Methods Morphotype identification 40x magnification Carapace Median spine Ventrolateral spine
14 Material and Methods Molecular analysis Extracted with kit DNA extraction COI amplification Reagent Volume (µl) Profil PCR: Destiled water PCR Ready mix LCO 1490 HCO 2198 DNA template 18,5 25 1,25 1,25 8 Total C 95 C 03:00 00:35 45 C 00:30 35x 72 C 00:50 72 C 07:00 4 C Sequencing
15 Material and Methods Molecular analysis Software ❶ Chromas Lite & BioEdit v7.0.9 Evaluating and editing electropherogram ❷ MEGA 6 Multi-sequence alignments and construct phylogeny tree
16 Result and Discussion
17 Result and Discussion Microscopic morphotype identification Morphotype n Morphotype n Morphotype n Morphotype n Sto 1 2 Sto 4 3 Sto 7 4 Sto 10 7 Sto 2 3 Sto 5 1 Sto 8 2 Sto 3 3 Sto 6 8 Sto 9 4 n Total 37
18 Percentage (%) Result and Discussion Effectiveness of DNA barcoding COI Percentation of identified stomatpod larvae MOTU MOTU 1 MOTU 2 MOTU 3 MOTU 4 MOTU 5 *Molecular operational taxonomic units ,95% Species level Tingkat spesies 54,05 % Family level Tingkat famili Species Anchisquilla fasciata Famili Squillidae Stomatopoda sp. 1 RWKT-2009 Famili Squillidae Stomatopoda sp. PHB-2006 morphotype E
19 Protosquillidae Squillidae
20 Protosquillidae Squillidae
21 Result and Discussion Effectiveness of DNA barcoding COI Species range of minimum K2P variation among clades was less than 3% Barber & Boyce 2006 Morphotype MOTU Maximum divergence within clades (%) Minimum divergence among clades (%) Closest sister group Sto 1 Sto 2 MOTU 1 1,1 0,4 Anchisquilla fasciata Sto 3 Sto 4 MOTU 2 0,9 8,9 Clorida decorata Sto 5 MOTU 3 n.a 0,5 Stomatopoda sp. 1 RWKT-2009 Sto 6 Sto 7 Sto 8 Sto 9 Sto 10 MOTU 4 0,7 5,2 Stomatopoda sp. 2 RWKT-2009 MOTU 5 2,4 1,5 Stomatopoda sp. PHB-2006 morphotype E
22 Result and Discussion Effectiveness of DNA barcoding COI Relative percentage (%) Effectiveness of barcoding gap between K2P intra- and DNA barcoding interspesific variation COI Meyer & Paulay 2005 Effectiveness of DNA barcoding Cytochrome c oxidase sub-unit 1 (COI) barcoding gap Intraspesies Interspesies Sequence variation (%) Intraspecies Interspecies Frequency distributions of genetic variability (Kimura 2-Parameter distances) (37 individuals in 5 MOTU)
23 Result and Discussion Diversity of Stomatopod in Banten Bay No. Species Family 1. Anchisquilla fasciata Squillidae 2. Unidentified sp. 1 BTN-2013 Squillidae 3. Stomatopoda sp. 1 RWKT-2009 Squillidae 4. Unidentified sp. 2 BTN-2013 Squillidae 5. Stomatopoda sp. PHB-2006 morphotype E Squillidae Mulyono Harpiosquilla harpax Squillidae 2. Harpiosquilla raphidea Squillidae 3. Miyakea nepa Squillidae
24 Conclusion ❶ DNA barcoding COI effective to identified stomatopod larvae in Banten Bay with variation of variation of Kimura-2 parameter distance ranged between 0,7% to 2,4%. ❷ There were three species of stomatopod larvae (Anchisquilla fasciata, Stomatopoda sp. 1 RWKT-2009 and Stomatopoda sp. PHB-2006 morphotype E) and 2 unidentified Squillidea species of stomatopod larvae (MOTU 2 and 4) were found in this study.
25 Thank You
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