Molecular and Biochemical Studies on Some Marine Shrimp Species
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Middle East Journal of Applied Volume : 06 | Issue :04 | Oct.-Dec. 2016 Sciences Pages: 646-652 ISSN 2077-4613 Molecular and Biochemical Studies on some Marine Shrimp Species 1Mohsen S. H., 1 Abdelghany M. F. and 2Ahmed M. Darwish 1Animal Production Department, Agriculture faculty, Al-Azhr University, Cairo, Egypt. 2Cell Biology Department, Genetic engineering Division, National Research Centre, Giza, Egypt. Received: 20 August 2016 / Accepted: 19 Sept. 2016 / Publication date: 05 Oct. 2016 ABSTRACT Three shrimps population (Penaeus semesulacatus, Penaeus japonicus,Meta Penaeus monoceros) were investigated for determination of the genetic structure by using (PCR-RAPD) and SDS-PAGE. The results obtained from RAPD technique showed the same percentages of similarity (100%) within the three studied population. On the other hand, the degrees of similarity detected by SDS- PAGE were 90%, 87% and 85% in Penaeus japonicus, Penaeus semesulacatus, and Metapenaeus monoceros, respectively. The present study revealed that the RAPD Technique is the powerful tool in detecting genetic variations. Key words: Metapenaeus monoceros, Penaeus japonicas, Penaeus semesulacatus, RAPD - SDS -PAGE - Similarity. Introduction Penaeid shrimp belong to the largest phylum in the Animal Kingdom, the Arthropoda, which is characterized by the presence of jointed appendages and an exoskeleton orcuticle that is periodically molted. The more highly evolved crustaceans (Class: Malacostraca) include the penaeid shrimp (Order Decapoda), Suborder: Natantia, Superfamily: Penaeoidea, Family: Penaeidae, Genus: Penaeus, Species: monodon, japonicus, indicus, merguiensis, vannamei (Bailey and Moss., 1992). Members of genus Penaeus are the most economically important around the world. Shrimps production (Penaeus monodon and Litopenaeus vannamei) has rapidly increased since the last two decades. It was estimated that approximately 341000 metric tons (MT) of farmed shrimps were produced by the shrimp industry in 1986 and this has more than doubled (638000-855500 MT) since 1993 (Rosenberry, 2001). Shrimps are an important part of the marine food web. Some species of shrimps are also cultivated in aquaculture in tropical countries (Gopala et al., 2009). Shrimps contribute about 20% by volume of the world seafood market (Bhavan et al., 2010 and Abdel-Salam 2013). The total amount of shrimp caught in Turkey was 6,339 tons in 2005 and 960 tons of this amount were from the Mediterranean Sea (Mustafa, 2010). Phylogenetic information can be used to guide captive breeding programs that are intended to produce superior captive strains through hybridization of closely related species. Unfortunately, recent studies indicate that some taxonomic relationships based solely on morphology, to some extent, cannot reflect the real evolutionary relationship. (Palumbi and Benzie, 1991) found surprisingly high genetic differentiation (10%) between two morphologically and ecologically similar Penaeus species. Anatomical features are particularly difficult to be used in penaeid shrimp species differentiation due to their phenotypic similarities and to removal of the external carapace, which is very frequent during the manufacturing process. Therefore, it is highly recommendable to develop the analytical tools necessary to make possible the distinction between these closely related species, preventing the inadvertent or deliberate mislabeling and adulteration of these products (Ignacio et al., 2009). Molecular phylogeny of penaeid shrimps has been reported based on nucleotide sequences of COI (Baldwin et al., 1998), 16S rDNA and COI (Lavery et al., 2004) and AFLP (Wang et al., 2004). Phylogenetic trees revealed close genetic relationships between Penaeus monodon and Penaeus semisulcatus (subgenera Penaeus) but distant relationships were observed among economically important shrimps from different genera (Penaeus monodon, Penaeus semisulcatus, F. merguiensis, L. vannamei and Marsupenaeus japonicus). Nevertheless, simple molecular markers for differentiation of these shrimps have not been reported. The use of polymerase chain reaction (PCR) in combination with restriction enzymes digestion,( RFLP) (Thaewnon et al., 2004) or single-stranded conformation polymorphism (SSCP) (Weder et al., 2001) are favored for identifying species origins of shrimp products due to their Corresponding Author: Abdelghany M. F., Animal Production Department, Agriculture faculty, Al-Azhr University, Cairo, Egypt. E-mail: [email protected] 646 Middle East J. Appl. Sci., 6(4): 646-652, 2016 ISSN 2077-4613 convenient and cost-effective. Some molecular techniques such as microsatellite DNA (Liu et al., 2004; Meng et al., 2009) RAPD or random amplification of polymorphic DNA (Meng et al., 2004) and analysis of some mitochondrial genes (Cui et al., 2007) have been used to investigate the genetic variability among many shrimp species. For wild populations development and application of molecular markers provides new possibilities for establishing kinship and reconstructing pedigrees in species where such information cannot be obtained from field observations alone (Tanya, 2007). Material and Methods Sample collection: Three shrimp populations (Penaeus semesulacatus, Penaeus japonicus and Metapenaeus monoceros) were used in the present study. Individual of shrimp samples were collected from Mediterranean Sea. Each sample comprises at least five individuals. Table 1: Morphological differentiation between the three studied shrimps population. Latin name Penaeus semesulacatus Penaeus japonicus Metapenaeus monoceros Local name Suez japonicus Read shrimp Caught mainly with otter trawls and Caught mainly with otter trawls Caught mainly with gill nets Method of drift nets also taken with boat and drift nets and set gill nets also and stake Catchment shore seines beam trawls, stake nets taken by stake traps and traps Body pale yellow to pink with Color Body pale brown sometimes red-brown to dark brown Body pink green transverse bands Rostrum Rostrum armed with 5 to 8 Rostrum armed with 9 to 11 Rostrum armed with 9 to 12 and teeth on dorsal and 2 to 4 teeth on dorsal and single tooth teeth on dorsal ventral teeth on ventral margin on ventral margin Maximum total length males,18cm; Maximum total length Size Maximum total length fmales, 23 cm males,15cm; males, 20cm Telson not armed with movable Telson armed with 3 pairs Telson not armed with Telson spines of movable spines movable spines Genomic DNA Extraction: The DNA extraction was performed using DNeasy Mini Kit (QIAGEN).according to the manual instruction.Three population (5 penaeus japonicus, 5 penaeus semesulacatus and 5 Metapenaeus monoceros) were examined by RAPD-DNA techniques using all primers (A09, A10, A18, B01, B06, B07, B09, B11, and C09) to investigate the variations between and within the three examined populations. Protein electrophoresis (SDS-PAGE): The protein was extracted according to Gorinstein, (1999) and run into polyaclaramid gel electrophoresis according to Laemmli, (1970). Statistical Analysis This data was introduced to SPSS software in order to analysis genetic distances for intra and inter population relationships. Within population similarities were calculated as average of SXY across all possible comparison between individuals within such population (Bardakci and Skibinski, 1994). Results and Discussion Analysis of RAPD markers: The results of RAPD-DNA patterns showed that 9 primers(A09, A10, A18, B01, B06, B07, B09, B11, and C09) amplified genomic of the examined three population and the other three primers (A05,B08,and C05) didn't succeeded in matching and amplifying regions of the genomic DNA of the populations Presented in figure (1). 647 Middle East J. Appl. Sci., 6(4): 646-652, 2016 ISSN 2077-4613 Fig. 1: Electrophrotic patterns of DNA amplified by 9 RAPD primers for the three shrimps studied populations. A total number of RAPD-DNA amplifications generated by all primers were 122 in all performed PCRs Table (1) 76 out of the 122 amplifications were polymorphic (DNA markers), and from seventeen six polymorphic amplifications 21, 32 and 23 bands for Penaeus semesulacatus, Penaeus japonicus and Metapenaeus monoceros, the highest number of specific DNA marker was 6 in Penaeus japonicus populations which generated by B11 primer. The highest number of Metapenaeus monoceros specific DNA marker generated by primer A10 was 5. The results obtained from RAPD technique showed the same percentages of similarity (100%) within the three studied population. Table 1: Number of amplicans and the number of polymorphic amplicans related to each population and generated by each primer for the three shrimps population : Total number of Number of polymorphic amplicons related to each population Primers code amplicons per Primers Penaeus semesulacatus Penaeus japonicus Metapenaeus monoceros A9 14 2 2 1 A10 16 2 4 5 A18 5 1 2 1 B1 10 3 3 1 B6 15 2 2 2 B7 17 4 5 5 B9 14 4 5 2 B11 13 3 6 4 C19 18 0 3 2 122 21 32 23 648 Middle East J. Appl. Sci., 6(4): 646-652, 2016 ISSN 2077-4613 The present study provides evidence that the RAPD markers can be effectively used to descriminate among shrimps populations. This result agreed with that obtained by Khamnamtong et al. (2005) who identified five penaeid shrimps (Penaeus monodon, Penaeus semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) and discriminated between them using single- stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Also Niamaimandi et al. (2010) investigated