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 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 Kingdom, the Arthropoda, which is characterized by the presence of jointed appendages and an exoskeleton orcuticle that is periodically molted. The more highly evolved (Class: ) include the penaeid shrimp (Order ), Suborder: Natantia, Superfamily: Penaeoidea, Family: , 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 genetic variation among population of the green tiger prawn, Penaeus semisulcatus, in Bushehr waters, Persian Gulf by RAPD technique. They found that the percentage of polymorphism was 14.8%. In another study, Kumar et al. (2011) examined genetic variation in Tiger prawn, Penaeus monodon Using Random amplified polymorphic DNA (RAPD) analysis. Out of the total number of 26 loci scored, 16 were polymorphic (61.5 %). Bardakci and skibinsai (1994), who found that the RAPD method has been successfully used to detect variation among populations, sub populations and species. They used a group of 13 random premiers to evaluate the similarity among three species of Tilapia genus Oreochromis and four subspecies of Oreochromis niloticus. Benzie et al. (2000) also found that the RAPD method is capable of revealing nuclear DNA variations as extremely conserved species. Barman et al. (2003) reported that the information on the genetic structure of cultivable fish species is essential for studying molecular systematic and optimizing fisheries management and fish farming.

Analysis of SDS-PAGE markers:

Three populations (8 Penaeus japonicus, 8 Penaeus semesulacatus and 8 Metapenaeus monoceros) were examined by SDS-PAGE techniques to investigate the variations between and within the three examined populations.

P. semesulacatus

Eight individual (four males and four female) of Penaeus semesulacatus from Mediterranean Sea were randomly sampled and the saline soluble muscle proteins were extracted and separated using SDS–PAGE Technique. The molecular weight, relative front and band frequencies in presented in table (2) A maximum of 12 banding table (2) were dispersed according to their relative fronts along the gel and the positioned in 15 loci. These bands were not all present in all individual samples. Out of these fifteen band loci, sex were monomorphic loci while the rest 9 band loci were polymorphic ones. The average similarity value with in Penaeus semesulacatus population was 87%.

Table 2: Polymorphism within Penaeus semesulacatus of Shrimp population from the analysis of SDS_PAGE pattern of saline muscle proteins. BN RF MW BF 1 0.118 130.267 0.125 2 0.199 105.255 0.75 3 0.248 89.172 1 4 0.251 75.802 1 5 0.278 66.125 1 6 0.302 49.824 0.875 7 0.326 38.265 1 8 0.438 30.502 1 9 0.514 27.279 1 10 0.526 21.745 0.875 11 0.565 17.749 0.625 12 0.583 15.125 0.25 13 0.628 11.202 0.125 14 0.870 10.540 0.5 15 0.912 9.237 0.25 BN : Band number RF: Relative front MW: Molecular weight BF: Band frequencies

Penaeus japonicas

Eight individual (four males and four female) of Penaeus japonicus from Mediterranean Sea were randomly sampled and the saline soluble muscle proteins were extracted and separated using SDS –PAGE Technique. The molecular weight, relative front and band frequencies in presented in table (3) A maximum

649 Middle East J. Appl. Sci., 6(4): 646-652, 2016 ISSN 2077-4613 of 10 banding table (3) were dispersed according to their relative fronts along the gel and the positioned in 10 loci .These bands were not all present in all individual samples. Out of these ten band loci, sex were monomorphic loci while the rest 4 band loci were polymorphic ones. The average similarity value with in Penaeus japonicus population was 90%.

Table 3: Polymorphism within Penaeus japonicus of Shrimp population from the analysis of SDS_ PAGE pattern of saline muscle proteins BN RF MW BF 1 0.241 552.004 1 2 0.342 402.646 0.125 3 0.441 234.620 0.625 4 0.544 138.182 1 5 0.611 79.662 0.75 6 0.680 55.674 1 7 0.734 38.495 1 8 0.776 28.840 0.5 9 0.837 23.039 1 10 0.914 16.626 1 BN : Band number RF: Relative front MW: Molecular weight BF: Band frequencies

Metapenaeus monoceros

Eight individual (four males and four female) of Metapenaeus monoceros from Mediterranean Sea were randomly sampled and the saline soluble muscle proteins were extracted and separated using SDS – PAGE Technique. The molecular weight ,relative front and the band frequencies in presented in table ( 4) A maximum of 11 banding table (4 ) were dispersed according to their relative fronts along the gel and the positioned in 16 loci .These bands were not all present in all individual samples. Out of these sixteen band loci, 4 were monomorphic loci while the rest 11 band loci were polymorphic ones. The average similarity value with in Metapenaeus monoceros population was 85%.

Table 4: Polymorphism within Metapenaeus monoceros of Shrimp population from the analysis of SDS_ PAGE pattern of saline muscle proteins BN RF MW BF 1 0.158 190.320 0.25 2 0.169 187.214 0.25 3 0.224 160.354 1 4 0.323 140.365 1 5 0.364 136.253 0.125 6 0.429 130.254 0.25 7 0.572 126.821 0.75 8 0.645 93.580 1 9 0.716 70.845 0.25 10 0.731 61.868 0.75 11 0.750 45.988 1 12 0.778 31.537 0.75 13 0.787 20.622 0.125 14 0.833 11.863 0.875 15 0.868 10.359 0.875 16 0.915 8.316 0.5 BN : Band number RF: Relative front MW: Molecular weight BF: Band frequencies

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. Many electrophoretic studies have been conducted to identify the differences among fish species and populations within species over the entire world. In this regard, different protein sources were examined and included. As proved by different authors, protein electrophoresis was successfully employed to study the variations among and within fish populations (Skorkowski et al., 1995; Saad et al., 2002; Ibrahim et al., 2005 and Rashed et al., 2007).

650 Middle East J. Appl. Sci., 6(4): 646-652, 2016 ISSN 2077-4613

Table 5: The average percentages of similarity values within the three studied populations by SDS-PAGE and RAPD techniques. Test type Penaeus semisulcatus Penaeus japonicas Metapenaeus monoceros SDS-PAGE 87 90 85 RAPD 100 100 100

Rashed et al., (2000) studied some African catfish (Clarias gariepinus) populations collected from six different locations in Egypt. Yilmaz et al. (2007) used Native-PAGE and SDS-PAGE for serum proteins of Leuciscus cephalus, Acanthobrama marmid and Chondrostoma regium (Cyprinidae) taken from the Karakaya Dam (Malatya, Turkey) to study the population structure of these fish species and the differences among them. The results showed that there were differences in the molecular weight (MW) of the serum proteins among the three studied fish species. Studies in the wide world have been directed at the level of proteins, Electrophoretic polymorphism at 30 protein loci in 27 wild and cultered populathons in three shrimp species Penaeus japonicus, P. semesulacatus and Metapenaeus monoceros were investigated (Rognon et al.,1996) plasma and muscle protein patterns were used to assess the intra and the inter population variation and to infer the phylogenetic relationships in catfish (Rashed et al., 2000). Alcohol and saline soluble shecleted muscle proteins were involved the study the population structure in same Tilapia species . The study revealed tow T. zilli species protein markers (14.3 and 16.9 KDa). Also detected a high level of were genetic within studied T. zilli and O. aureus population based on saline and alcohol soluble proteins 0.98, 0.89, 0.95 and 0.98 respectively (Saad et al., 2002).

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