Brazilian Journal of Biological Sciences, 2015, v. 2, n. 4, p. 295-302. ISSN 2358-2731 Identification and phylogenetic inference in different mollucs nudibranch species via mitochondrial 16S rDNA Ali Alqudah¹,*, Shahbudin Bin Saad¹, Noor Faizul Hadry² and Deny Susanti¹ ¹Kulliyyah of Science, International Islamic University Malaysia. Bandar Indera Mahkota, 25200 Kuantan Pahang, Malaysia. ²Kulliyyah of Engineering, International Islamic University Malaysia. Kuala Lumpur, Malaysia. Email: [email protected]. Abstract. The molecular analysis of marine life is an essential approach to discover new classes of natural products and to improve Received the management and sustainable utilization of these useful genetic July 30, 2015 resources. Mitochondrial DNA markers are frequently utilized to identify any organism up to the species level. This study provides Accepted insight into the significant role of species discrimination based on 16S August 17, 2015 rDNA, and this method could be a powerful tool for the identification Released of animal species. 16S rDNA sequences of each studied species December 31, 2015 (Phyllidia varicosa (Lamarck, 1801) and Phyllidiella pustulosa (Cuvier, 1804)) from two locations in the coastal waters of Balok in Open Acess Full Text Article Pahang State and Bidong Island in Terengganu State were aligned to identify the phylogenetic relationships among them. The phylogenetic tree produced in this study is consistent with those previously presented in the literature. The divergence of sequences was adequate to identify the samples at the species level with the assistance of the GenBank database and the BLAST tool. The results presented here provide useful information for a better understanding of this marine genetic resource. This genetic approach could simplify the identification of the species accurately for further analysis. Keywords: 16S rDNA, Phyllidia varicosa, Phyllidiella pustulosa, Bidong Island, Balok. Introduction astonishing creatures with distinctive biological peculiarities such as several Molluscs comprise a varied group developmental patterns, loss of the hard of animals with an estimated 200,000 protective shell, development of aposematic species, making it the second most diverse colourations, and acquisition of toxic phylum after Arthropoda. Molluscs present defences (Grande et al., 2002). In an extraordinary morphological array of gastropods, the mitochondrial genome is species diversity, including gastropods, approximately 14 kilobases (kb) long and bivalves, cephalopods, scaphopods, and circular. It is inherited in most cases chitons and the more obscure such as maternally, and there is no recombination tryblidia, solenogasters, tryblidia, and within it, although remarkable exceptions scutopods. Nudibranch molluscs are shell- exist among the bivalves. The less gastropods that are often considered as mitochondrial DNA (mtDNA) is a the main group of the Opisthobranchia meaningful marker due to distinctive including approximately 3,000 species, and characteristics such as rapid evolutionary they are widely distributed in all oceans and rate, maternal inheritance, lack of seas from the intertidal zone to the deep sea recombination, and the availability of (Wollscheid and Wägele, 1999). They are universal primers (Thollesson, 1999; ISSN 2358-2731/BJBS-2015-0128/2/4/12/295 Braz. J. Biol. Sci. http://revista.rebibio.net 296 Alqudah et al. Lydeard et al., 2000; Le et al., 2002; Li et was promoted by a series of observations al., 2015). A part of mitochondrial 16S assessing its morphological characteristics rDNA is easily amplified using universal in the east coast of peninsular Malaysia. primers, and this has been applied to Balok is a coastal area located near an species identification and phylogenetic industrial region that might be influenced studies of various groups and at different by various pollutants deposited into the levels (Han and McPheron, 1997; water. Anthropogenic activities have Wollscheid et al., 2001; Valdés, 2003; introduced various contaminants into the Wilson and Lee, 2005; Pola et al., 2007; De aquatic ecosystem of Balok, and this can Masi et al., 2015). deteriorate water quality that in turn directly The dorid nudibranchs Phyllidia or indirectly affects the ecological balance varicosa and Phyllidiella pustulosa of the environment. In order to recognize (Gastropoda, Opisthobranchia, the heterogeneity of habitats and the various Nudibranchia) are among the most levels of anthropogenic effects, Bidong widespread and abundant of intertidal Island was selected as a high water quality nudibranchs from tropical Indo-Pacific station compared to Balok. There are slight (Brunckhorst, 1993). The two species are differences in collected adult species in more or less sympatric and prevalent in total size and colour, and this incites selected study areas. Both species occupy questions towards the genetic structure and essentially similar feeding niches. diversity among the species. The aim of this Nudibranch species are apparently study was to identify two nudibranch vulnerable to predators because their shell species collected from the coastal waters of is completely absent, which leaves no Balok (Pahang) and Bidong Island obvious morphological defence structure (Terengganu) Malaysia. This study against predation. These creatures have investigated a PCR direct sequencing evolved chemical compounds for protection method for species identification by using from predator attacks. Nudibranchs have partial sequences of the mitochondrial 16S two methods to select bioactive molecules rDNA genes of selected nudibranch species either from retaining defensive chemicals or amplified with universal primers. The de novo biosynthesis, and this has resulted different 16S rDNA sequences of in an extraordinary library of active nudibranch species were used for compounds that are not present in their phylogenetic analysis, in order to clarify the terrestrial counterparts. Some of these relationships among species of the compounds are already employed in a broad Phyllidiidae Family. array of antibacterial, antifungal, anticancer, anti-inflammatory and Material and methods antifeedant activities (Miyamoto et al., 1996; Karuso and Scheuer, 2002; Gerwick Specimens and DNA extraction and Moore, 2012; Nuzzo et al., 2012). Samples of both species were Therefore, these creatures represent collected from the coastal waters of Balok valuable models to study their biological (Pahang) and Bidong Island (Terengganu), role in the marine environment and their Malaysia. Nudibranch species collected in secondary metabolites. Most of the this study were initially identified through identification approaches for the nudibranch the colouration pattern of their mantle and molluscs were based on morphological and external morphology. Specimens were kept anatomical characteristics due to some alive in the aquarium for several days to let difficulties in DNA isolation (Pereira et al., them empty their alimentary canals, after 2011). Due to the lack of genetic which they were transferred and preserved methodologies utilized for the at −20 °C until DNA extraction. The determination of species identity due to the Nudibranch genomic DNA was extracted intrinsic structure of mollusc tissue, we using Qiagen DNA mini kit according to propose mitochondrial 16S rDNA as a the manufacturer’s instructions, and the genetic marker for species identification. incubation time at 56 °C was adjusted to Initial interest in the nudibranch species run overnight instead of 3 h. Braz. J. Biol. Sci., 2015, v. 2, n. 4, p. 295-302. Identification and phylogenetic in molluscs nudibranch via mitochondrial 16S rDNA 297 Amplification, gel imaging and Bioinformatics sequencing Four rDNA sequences were Amplification of the target region generated in this study from Phyllidia of the mitochondrial large ribosomal DNA varicosa (Lamarck, 1801) and Phyllidiella gene (16S rRNA) was performed with pustulosa (Cuvier, 1804) genomic DNA primers developed by Palumbi et al. (1991) and were analysed using Sequence Scanner as universal 16S primers including forward v1.0. All the sequences of the studied 16Sar-L [5’-cgcctgtttatcaaaaacat-3’] and species were subjected to the BLAST tool reverse 16Sbr-H [5’- to identify registered sequences that ccggtctgaactcagatcacgt-3’]. Double- correspond to the sample sequences. The stranded DNA was amplified in a total 16S rRNA of Scaphander lignarius reaction volume of 50 µL containing 25 µL (Linnaeus, 1767) was chosen as the out of master mix, 15 µL sterile distilled water, group sequence. Mitochondrial 16S rDNA 2.5 µL of each primer and 5 µL of DNA sequence of selected species was aligned sample. The PCR program comprised an against corresponding sequences of initial denaturation step at 94 °C for 3 min. phyllidiid nudibranchs reported by The second denaturation step was 94 °C for Thollesson (1999) and Valdés (2003) and 1 min, followed by an annealing step at retrieved from the NCBI nucleotide 55 °C for 1 min and an extension step at database. The alignment file was edited to 72 °C for 1 min, and a final extension step remove gaps observed within the sequences at 72 °C for 5 min. The second, third and and then the bootstrap phylogenetic tree fourth steps were cycled 29 times. PCR was constructed using the Neighbor Joining products were visualized on 0.8% agarose (NJ) Method. All the sample sequences, gels and photographed using Gel imager GenBank sequences and out group (Alphaimager™ 2200, Germany) under UV sequences were aligned