Egyptian Journal of Zoology (EJZ), Vol. 75: 1-13 (June, 2021) p-ISSN: 1110-6344 Publisher: The Zoological Society of A. R. Egypt e-ISSN: 2682-3160 DOI: 10.12816/ejz.2020.44427.1041

RESEARCH ARTICLE

GENETIC RELATIONSHIPS AMONG FOUR MUTELID SPECIES (: ) IN EGYPT REVEALED BY RAPD-PCR TECHNIQUE

Mona F. Fol1; Mostafa Y. Morad2; Irene S. Gamil2; Salwa F. Sabet1; Reda M. Mansour2* 1Zoology Department, Faculty of Sciences, Cairo University, Giza, Egypt 2Zoology and Entomology Department, Faculty of Science, Helwan University, Cairo, Egypt

ABSTRACT Article History: Random amplified polymorphic deoxyribonucleic acid- Received: 28 September 2020 polymerase chain reaction (RAPD-PCR) is considered as one Revised: 27 November 2020 Accepted: 30 November 2020 of the simple and quick methods that are used to resolve the genetic relationship among species of taxa that are Published Online: difficult to identify on the basis of morphological characters. 1 December 2020 Bivalves show great morphological variations that couple Keywords: with relatively few constant characters rendering them Chambardia letourneuxi a systematically difficult group, and many of them have Chambardia rubens numerous subspecies and several synonyms. Due to the Mutela dubia high degree of morphological variability within Order Mutela rostrata RAPD-PCR Unionoida, four species: Mutela dubia, M. rostrata, Chambardia rubens, and C. letourneuxi were collected from *Correspondence: Benha region (Qaluobiya Governorate, Egypt) to resolve Reda Mansour their degree of genetic similarity depending on RAPD-PCR Zoology and Entomology technique. Out of six primers used, only five primers Department, Faculty of Science Helwan University “UBC476, UBC477, UBC478, UBC483, and UBC486” Cairo, Egypt worked successfully. The study revealed that C. rubens and E-mail: C. letourneuxi are very closely related species, as they [email protected]. showed closely similar bands on using the primers edu.eg UBC477, UBC483, and UBC486.

INTRODUCTION affect these characters[2]. In Egypt, the Recent molecular approaches in species of family Mutelidae have showed have led to a steady increase in the so variable shell morphology due to identification of cryptic species. Prior to environmental changes and geographical molecular techniques, evolutionary relation- distribution leading to confusion in their ships among bivalves were determined nomenclature and classification[3-5]. Ibrahim using morphological species concepts[1]. et al.[3] reported that within Mutelidae there The advent of molecular techniques has are only two genera found in Egypt revealed that some morphological characters, namely, Mutela and Chambardia. Mutela such as shell structures in bivalves, are was reported to have three species; Mutela highly polymorphic and any change due to dubia, M. rostrata, and M. singularis; environmental and population density can while genus Chambardia has two species,

1 Genetic relationships of four mutelid species by RAPD-PCR

Chambardia rubens and C. letourneuxi. Extraction of DNA and RAPD-PCR On the other hand, Harper et al.[4] and technique El-Assal et al.[5] have separated each of The four samples under investigation were Mutela and Chambardia into only two dissected and preservation of their soft species. Based on the morphological parts was carried out in absolute ethanol characters, shells of Mutela spp. devoid at 20°C until utilization. The extraction of the umbonal sculpture, while Chambardia total genomic DNA from frozen alcohol- spp. have shells with umbonal sculpture. preserved foot was achieved using Qiagen Shells of M. dubia and C. rubens are ovate- DNeasy tissue kit (Valencia, Santa Clarita, elongate, while those of M. rostrata and CA, USA) following the manufacturer’s C. letourneuxi are elongate. In addition, shell instruction. Six primers were used in the of M. dubia has posterior dorsal margin present investigation in RAPD-PCR[8-11]: showing the largest height, while M. rostrata 476: 5`-TTGAGGCCCT-3`, 477: 5`-TGTT has dorsal and ventral margins that are GTGCCC-3`, 478: 5`-CGAGCTGGTC-3`, almost parallel. On the other hand, C. rubens 479: 5`-CTCATACGCG-3`, 483: 5`-GCA has shell length/height (L/H) ratio equals CTAAGAC-3`, 486: 5`-CCAGCATCAG-3` to 1.6, while that of C. letourneuxi has L/H (Midland Certified Reagent Company, ratio measuring 1.8-2.0[3]. Midland, TX, USA). One of the DNA features that were used In the preliminary experiments, only to analyse the population genetic diversity five primers worked successfully “UBC476, is random amplified polymorphic DNA- UBC477, UBC478, UBC483, and UBC486”. polymerase chain reaction (RAPD-PCR) Amplifications were practiced following technique. RAPD-PCR standard method Williams et al.[12] with some modifications. uses short-single oligonucleotide (10-12 PCR mixture (50 µL) contained 2.0 µL bases) with the random order as a primer sample DNA, 1.0 µL primer, 25 µL master to amplify DNA genes in nanograms in mix and 22 µL distilled water (PCR low annealing temperature[6]. Therefore, the MyTaq™ HS Red Mix, Bioline, Memphis, present study aimed to use RAPD-PCR TN, USA). By utilization of each primer, method to determine the degree of each amplification reaction was repeated genetic similarity and evaluate the genetic three times to verify band autosimilarity[13]. relationship among the four mutelid species, Amplifications were produced in T-personal M. dubia, M. rostrata, C. rubens, and thermal cycler (TC-3000G, Techne Inc., C. letourneuxi from the River Nile in Egypt. Burlington, NJ, USA), programmed for 45 cycles of 94°C for 1.0 minute, 35°C MATERIAL AND METHODS for 1.0 minute, and 72°C for 1.0 minute. Collection of samples An initial denaturation step for 3.0 minutes Samples of the four mutelid species, at 94°C and a final extension holding for M. dubia, M. rostrata, C. rubens, and 10 minutes at 72°C were included in C. letourneuxi (n = 5, for each species) the first and last cycles, respectively. were collected from Benha (Qaluobiya The reaction products (10 µL) were resolved Governorate) along River Nile in Egypt by 2.0% agarose gel electrophoresis at 85 V (Figure 1) using a special net made of in 1.0 Tris-acetate-EDTA (TAE) buffer. hard metallic frame. Mussels were collected The gel was visualized using ethidium during spring 2017 and transferred to bromide stain and then photographed the laboratory of Invertebrates. Sorting with gel documentation system (SynGene, and maintenance of samples were done GeneTools - File version: 4.02.03, France). under the standard conditions of food Species -specific fragments were revealed and temperature. Identification of mussels using GeneRuler 1.0 kb Plus DNA Ladder followed the keys of Ibrahim et al.[3] and (Fisher Scientific, Toronto, Canada) to Graf and Cummings[7]. compare the amplified products.

2 Fol, M. F. et al.

Figure 1: Photographs showing the morphology of external shell surface of the four mutelid species: (a) Mutela dubia shell; (b) M. rostrata shell; (c) Chambardia rubens shell; (d) C. letourneuxi shell. GA: growth annuli, PS: posterior slope, U: umbo. Scale bar = 2 cm.

Molecular data analysis and estimation of similarity index (D-value) Molecular analysis was executed using gel of each primer among the studied mutelid documentation system for the dendogram species. RAPD-PCR amplification products

3 Genetic relationships of four mutelid species by RAPD-PCR took the score 1/0 for presence/absence as the mean and overall value for all of homologous bands[14] and analyses species and each primer. were accomplished using the NTSYSpc 2.2 software[15]. For RAPD markers, RESULTS similarity coefficient matrix was calculated DNA was successfully extracted from using Jaccard similarity algorithm[16]. the foot of the four studied species; Construction of dendograms was done Mutela dubia, M. rostrata, Chambardia using the unweighed pair-group method rubens, and C. letourneuxi. RAPD-PCR was with arithmetical algorithms averages carried out using six primers of which, (UPGMA)[17]. Genetic diversity was also primer (UBC 479) gave no reproducibility revealed as the percentage of polymorphic for all studied species, while the other bands. The percentage of polymorphism five primers provided strongly amplified was calculated for each species, as well fragments (Figures 2-6).

Figure 2: RAPD-PCR profile of the mutelid species using UBC476 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC476 primer demonstrating the relationships of the species under study.

Figure 3: RAPD-PCR profile of the mutelid species using UBC477 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC477 primer demonstrating the relationships of the species under study.

4 Fol, M. F. et al.

Figure 4: RAPD-PCR profile of the mutelid species using UBC478 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC478 primer demonstrating the relationships of the species under study.

Figure 5: RAPD-PCR profile of the mutelid species using UBC483 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC483 primer demonstrating the relationships of the species under study.

Figure 6: RAPD-PCR profile of the mutelid species using UBC486 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC486 primer demonstrating the relationships of the species under study.

5 Genetic relationships of four mutelid species by RAPD-PCR

Each primer produced 3-6 polymorphic (Table 1). Some RAPD-PCR fragments were bands (Table 1). The number of amplified found to be unique; four unique bands in M. bands (monomorphic, polymorphic, and dubia, one unique band in M. rostrata, one unique) of the DNA of each species with unique band in C. rubens, and six unique different primers is given, and the genetic bands in C. letourneuxi, while the total diversity is also given as the percentage of polymorphic bands were found to be 90% the polymorphic bands for each primer among the four studied species (Table 2).

Table 1: Total number of bands (monomorphic and polymorphic) and the percentage of polymorphism of each primer in samples under investigation.

Primers Total bands number Monomorphic Polymorphic Polymorphism (%) UBC476 6 0 6 100 % UBC477 3 0 3 100 % UBC478 5 0 5 100 % UBC483 5 0 5 100 % UBC486 4 0 4 100 %

Table 2: Total number of bands for all studied primers (monomorphic, polymorphic, and unique) and the percentage of polymorphism revealed by RAPD markers among the four mutelid species.

Bands M. dubia M. rostrata C. rubens C. letourneuxi Total Amplified 6 6 11 16 39 Monomorphic 1 1 1 1 4 Polymorphic 5 5 10 15 35 Unique 4 1 1 6 12 Polymorphism (%) 83% 83% 90% 94% 90%

By running PCR products of the studied 6a). No differences between the four studied samples on agarose gel, the genetic species were shown using the primers variability was observed among the studied UBC476 and UBC478 (Figures 2a and 4a). Mutela and Chambardia species. The In addition, the dendrogram analysis using RAPD-PCR analysis was based on the the primers UBC477, UBC483, and number of bands that were different between UBC486 (Figures 3b, 5b, and 6b) confirmed any given pair of species (Figures 2-6). the results obtained with the RAPD-PCR Analysis showed natural differences or profile and those of the D-values, where polymorphism among the studied Mutela C. rubens and C. letourneuxi are the closest and Chambardia species under investigation. species, while M. dubia and M. rostrata are Results of RAPD-PCR using UBC477, distinct ones. UBC483, and UBC486 primers indicated D-values of the studied species is that C. rubens and C. letourneuxi showed presented in Table “3”. Species are bands at about the similar base pairs on considered similar when the D-value using the same primers (Figures 3a, 5a, and between two species is equal or close to 1.0.

6 Fol, M. F. et al.

Table 3: Similarity indices (D-values) of the studied species using the current five primers.

Species M. dubia M. rostrata C. rubens C. letourneuxi UBC476 M. dubia 1 M. rostrata 0 1 C. rubens 0 0 1 C. letourneuxi 0 0 0 1 UBC477 M. dubia 1 M. rostrata 0 1 C. rubens 0 0 1 C. letourneuxi 0 0 1 1 UBC478 M. dubia 1 M. rostrata 0 1 C. rubens 0 0 1 C. letourneuxi 0.2 0 0 1 UBC483 M. dubia 1 M. rostrata 0 1 C. rubens 0 0.33 1 C. letourneuxi 0 0.33 1 1 UBC486 M. dubia 1 M. rostrata 0 1 C. rubens 0 0 1 C. letourneuxi 0 0 0.8 1

On the other hand, when D-value is distant closest species. The similarity coefficient from 1.0, the two species are considered matrix of all primers showed that the highest as separate species. D-values between D -value (0.75) was found between C. rubens C. rubens and C. letourneuxi using the and C. letourneuxi, while the lowest D-value primers UBC477, UBC483, and UBC486 (0.12) was obtained between M. dubia was found to be close to 1.0, while it was and C. letourneuxi (Table 4). Moreover, distant from 1.0 for these two species and the UPGMA dendrogram of all primers the other studied species using the primers (Figure 7) revealed that C. rubens and UBC476 and UBC478 indicating no close C. letourneuxi are the most closely relationship (Table 3). As a result, C. rubens associated species, while M. dubia and and C. letourneuxi were found to be the M. rostrata are distinct ones.

7 Genetic relationships of four mutelid species by RAPD-PCR

Table 4: Similarity coefficient matrix of all primers calculated by NTSys of the studied mutelid species.

Species M. dubia M. rostrata C. rubens C. letourneuxi M. dubia 1 M. rostrata 0.56 1 C. rubens 0.37 0.43 1 C. letourneuxi 0.12 0.31 0.75 1

Figure 7: Dendrogram of cluster analysis for the four studied species according to RAPD- PCR data obtained using Jaccard coefficient and unweighed pair-group method with arithmetical algorithms averages (UPGMA).

DISCUSSION studying species with variable phenotypic The importance of taxonomy by using plasticity[24,25]. DNA analyses contrasts with morphological In molluscs, many previous results were studies in biodiversity research[18]. The obtained by different research groups[26-29] exclusive reliance on one or other method indicated that RAPD-PCR was an adequate may fail to detect variations. Comprehensive technique and a good initial approach studies including morphometrics and for distinguishing between morphologically molecular analyses may provide a more close species and even among the different accurate approach to species discrimination. populations within the same species. The genetic analyses of bivalve species In addition, Bin Dajem et al.[30] and identifications have been used to correct Oliveira et al.[31] amplified the genomic mislabelling[19,20]. Species determination by DNA and the extracted PCR products from means of molecular techniques could bypass Biomphalaria spp. and reported a fingerprint the problems of interspecific convergence pattern unique to these snails. They analysed and intraspecific variability, often impeding RAPD-PCR products using agarose gel as accurate morphological identification of good, easy, rapid, and cheap method, freshwater mussels[21-23]. RAPD-PCRs are while other groups of researchers used commonly used as markers to discriminate the alternative polyacrylamide gel electro- between particular taxa by the presence or phoresis method[25,32-34]. absence of certain diagnostic RAPD-PCR RAPD-PCR method was successfully bands[11]. In addition, the molecular data have used to determine genetic relationships been confirmed as an effective tool for among molluscs. Adult specimens of the

8 Fol, M. F. et al. two common freshwater bivalves, Caelatura in Thailand. Similar observations on high companyoi and Caelatura prasidens genetic polymorphism were detected by belonging to family Unionidae were RAPD-PCR analysis across investigated described by some authors as one species[35], species in the mud crabs “Scylla serrata, while Ibrahim et al.[3] separated them as S. oceanic, and S. tranquebarica”[37] and two different species. RAPD-PCR markers the cupped oysters of genera Saccostrea were used and confirmed that the two “S. cucullata, and S. forskali” and Striostrea specimens should be actually separated “ Striostrea (Parastriostrea)” mytiloides[38]. as two different species[9]. RAPD-PCR Some authors used morphological carried out to solve the problem of characters based-taxonomy and reported confusion between Coelatura species and that genus Chambardia contained two showed that the five distinct morpho- species in Egypt; C. rubens and logically species of Coelatura, may be only C. letourneuxi[3,7]. However, the similarity three different species namely Coelatura coefficient matrix and the UPGMA aegyptiaca, C. canopicus, C. parreyssi[11]. dendrogram obtained in the present study Therefore, the determination of genetic of all primers could confirm that C. rubens similarity degree for some of the species is is clearly very close to C. letourneuxi. necessary, using technique like RAPD-PCR. Similarly, Yoon[39] succeeded to differentiate As far as is known, no previous RAPD- between Korean scallop populations and PCR analysis has been so far carried out on Chinese scallop populations from different the freshwater bivalves family localities by using RAPD-PCR. The genetic species found in Egypt. The current study distance between individuals approved the presents a degree of genetic convergence for existence of close relatedness in the Korean the mutelid species inhabiting the River scallop population. In addition, the hierarchical Nile in Egypt, facilitating quick, low-cost, dendrogram of Euclidean genetic distances accurate, and reliable determination of adult individuals of Korean scallop population were specimens. RAPD-PCR analysis showed fairly distantly related to that of Chinese natural differences or polymorphism among scallop populations. Thus, it was suggested mutelid species under investigation. In that the PCR fragments revealed might be addition, dendrograms and D-values showed valuable as a DNA marker to discriminate that there are differences among the four between the two geographical populations[39]. studied species, using the five primers: In conclusion, the current study revealed UBC476, UBC477, UBC478, UBC483, and that C. rubens and C. letourneuxi are very UBC486. Genetic diversity was measured as closely related species, as they showed the percentage of polymorphic bands, 90% similar bands on using the primers UBC477, of the bands were polymorphic among the UBC483, and UBC486. Using of low four studied species. Some RAPD-PCR number of specimens per each species may fragments were found to be unique to be attributed to current study limitations. a particular species; four unique bands were However, further studies are needed in the found in M. dubia, one unique band was future aiming to use larger number of each found in M. rostrata and C. rubens, and six species to verify inter- and intra-specific unique bands were found in C. letourneuxi. genetic variation within populations of These results were similar to the study of mutelid species. Thaewnon-ngiw[36] who found that all scored RAPD-PCR bands generated by different COMPLIANCE WITH ETHICAL primers across overall species of Pila apple STANDARDS snails in Thailand were polymorphic. So, All procedures contributing to this work this suggested the potential of RAPD-PCR comply with the ethical standards of the analysis for determination of inter- and intra- relevant national guides on the care and use specific genetic differences of apple snails of laboratory and have been

9 Genetic relationships of four mutelid species by RAPD-PCR approved/authorized by Institutional [7] Graf, D. L. and Cummings, K. Care and Use Committee (IACUC) of S. (2007). Preliminary review of Faculty of Science, Cairo University, Egypt. the freshwater mussels (: Bivalvia: Unionoida) of northern FUNDING SOURCE DISCLOSURE Africa, with an emphasis on the Nile. This research received no specific grant J Egypt Ger Soc Zool (D. Invertebrate from any funding agency in the public, Zoology), 53: 89-118. commercial, or not-for-profit sectors. [8] Ibrahim, A. M.; Aly, R. H.; Kenchington, E. et al. (2008). Genetic CONFLICT OF INTEREST polymorphism among five populations The authors have no potential conflict of of Pinctada radiate from the interest regarding the content of this article. Mediterranean coast in Egypt indicated by RAPD-PCR technique. Egyptian REFERENCES Journal of Zoology, 50: 467-477. [1] Bickford, D.; Lohman, D. J.; Sodhi, [9] Sleem, S. H. and Ali, T. G. N. S. et al. (2007). Cryptic species (2008). Application of RAPD-PCR as a window on diversity and in taxonomy of certain freshwater conservation. Trends in Ecology and bivalves of genus Caelatura. Global J Evolution, 22(3): 148-155. Mol Sci, 3: 27-31. [2] Lydeard, C. and Lindberg, D. R., [10] Yousif, F.; Ibrahim, A.; Sleem, S. 1948- (2003). Molecular Systematics et al. (2009). Morphological and and Phylogeography of Mollusks. genetic analysis of Meanoides Smithsonian Institution, Washington, tuberculata populations in Egypt. DC, USA. Global J Mol Sci, 4(2): 112-117. [3] Ibrahim, A. M.; Bishai, H. M. [ 11 ] El-Assal, F. M.; Sabet, S. F.; and Khalil, M. T. (1999). Fresh- Varjabedian, K. G. et al. (2014). water Molluscs of Egypt. National Pollution of freshwater Coelatura Biodiversity Unit, Egyptian Environ- species (Mollusca: Bivalvia: mental Affairs Agency, Cairo, Egypt. Unionidae) with heavy metals and [4] Harper, E. M.; Hide, E. A. and its impact on the ecosystem of the Morton, B. (2000). Relationships River Nile in Egypt. Int J Waste between the extant Anomalodesmata: Resources, 4(4): 1000163 (DOI: a cladistic test. In: The Evolutionary 10.4172/2252-5211.1000163). Biology of the Bivalvia (Harper, E. M.; [12] Williams, J. G.; Kubelik, A. R.; Taylor, J. D. and Crame, J. A., eds). Livak, K. J. et al. (1990). DNA Geological Society Special Publication, polymorphisms amplified by arbitrary 177, pp. 129-143. The Geological primers are useful as genetic Society, London, UK. markers. Nucleic Acids Res, 18(22): [5] El-Assal F. M.; Varjabedian, K. 6531-6535. G.; Tawfik, A. R. et al. (2010). [13] Perez, T.; Alboronz, J. and Reproduction and genetic variation Dominguiez, A. (1998). An evaluation in two Corbicula species (Bivalvia: of RAPD fragment reproducibility Veneroida) from the River Nile in and nature. Molecular Ecology, 7(10): Egypt. Journal of the Pennsylvania 1347-1357. Academy of Science, 84: 31-37. [14] Abdellatif, K. F. and Khidr, Y. A. [6] Bardacki, F. and Skibinski D. O. F. (2010). Genetic diversity of new maize (1994). Application of the RAPD hybrids based on SSR markers as technique in Tilapia fish: species and compared with other molecular and subspecies identification. Heredity, 73: biochemical markers. J Crop Sci 117-123. Biotech, 13: 139-145.

10 Fol, M. F. et al.

[15] Rohlf, F. J. (2009). NTSYSpc key to the North and Central European Numerical Taxonomy and Multivariate species. Ecology and Evolution, 2(4): Analysis, version 2.2, Getting Started 740-750. Guide. Applied Biostatistics Inc., Port [24] Lobato Paraense, W.; Pointier, J. P.; Jefferson, NY, USA. Delay, B. et al. (1992). Biomphalaria [16] Real, R. and Vargas, J. M. (1996). The prona (Gastropoda: planorpidae): probabilistic basis of Jaccard's index a morphological and biochemical of similarity. Systematic Biology, study. Mem Inst Oswaldo Cruz, Rio de 45(3): 380-385. Janeiro, 87(2): 171-179. [17] Sneath, P. H. A. and Sokal, R. R. [25] Spatz, L.; Vidigal, T. H. D. A.; (1973). Numerical Taxonomy: The Silva, M. C. A. et al. (2000). Principles and Practice of Numerical Characterization of Biomphalaria Classification. Freeman, San Francisco, orbignyi, Biomphalaria peregrina and CA, USA. Biomphalaria oligoza by polymerase [18] Herrera, N. D.; ter-Poorten, J. J.; chain reaction and restriction enzyme Bieler, R. et al. (2015). Molecular digestion of the internal transcribed phylogenetics and historical bio- spacer region of the RNA ribosomal geography amid shifting continents gene. Mem Inst Oswaldo Cruz, Rio de in the cockles and giant clams Janeiro, 95(6): 807-814. (Bivalvia: Cardiidae). Molecular [26] Larson, S. E.; Anderson, P. L.; Miller, Phylogenetics and Evolution, 93: 94- A. N. et al. (1996). Use of RAPD- 106. PCR to differentiate genetically [19] Espiñeira, M.; Gonzalez-Lavin, defined lines of an intermediate N.; Vieites, J. M. et al. (2009). host of Schistosoma mansoni, Development of a method for the Biomphalaria glabrata. J Parasitol, genetic identification of commercial 82(2): 237-244. bivalve species based on mitochondrial [27] Abdel-Hamid, A. Z.; de Molfetta, J. B.; 18S rRNA sequences. J Agric Food Fernandez, V. et al. (1999). Genetic Chem, 57(2): 495-502. variation between susceptible and non- [20] Cartaxana, A. (2015). Morphometric susceptible snails to Schistosoma and molecular analyses for infection using random amplified populations of Palaemonlon girostris polymorphic DNA analysis (RAPDs). and Palaemon garciacidi (Crustacea, Rev Inst Med Trop S Paulo, 41(5): Palaemonidae): Evidence for a single 291-295. species. Estuarine, Coastal and Shelf [28] Spada, R. G. M.; Da Silva, D.; Abdel- Science, 154(5): 194-204. Hamid, A. Z. et al. (2002). Genetic [21] Watters, G. T. (1994). Form and markers between Biomphalaria function of unionoidean shell sculpture glabrata snails susceptible and and shape (Bivalvia). American resistant to Schistosoma mansoni Malacological Bulletin, 11: 1-20. infection. Mem Inst Oswaldo Cruz, [22] Zieritz, A.; Hoffman, J. I.; Amos, W. Rio de Janeiro 97(1): 53-58. et al. (2010). Phenotypic plasticity and [29] Da Silva, D.; Spada, R. G.; Sobral- genetic isolation-by-distance in the Hamaguchi, S. S. et al. (2004). freshwater mussel Unio pictorum Biomphalaria tenagophila: genetic (Mollusca: Unionoida). Evolutionary variability within intermediate snail Ecology, 24: 923-938. hosts susceptible and resistant to [23] Zieritz, A.; Gum, B.; Kuehn, R. et al. Schistosoma mansoni infection. (2012). Identifying freshwater mussels Parasite, 11: 43-49. (Unionoida) and parasitic glochidia [30] Bin Dajem, S. M.; Ibrahim, E. H.; larvae from host fish gills: a molecular Al-Quraishy S. A. et al. (2011).

11 Genetic relationships of four mutelid species by RAPD-PCR

Fingerprint of Biomphalaria arabica, [35] Mandahl-Barth, G. (1988). Studies the intermediate host of Schistosoma on African Freshwater Bivalves. mansoni in Saudi Arabia, using Danish Bilharziasis Laboratory, RAPD-PCR. Gene, 485(2): 69-72. Charlottenlund, Denmark. [31] Oliveira, A. L. D.; Da Silva, D.; [36] Thaewnon-ngiw, B.; Klinbunga, S.; Manzano, B. C. et al. (2010). Genetic Phanwichien, K. et al. (2004). Genetic differences between strains of diversity and molecular markers in Biomphalaria glabrata (Planorbidae) introduced and Thai native apple snails that are susceptible and unsusceptible (Pomacea and Pila). Journal of to schistosomiasis. Genet Mol Res, Biochemistry and Molecular Biology, 9(3): 1450-1459. 37(4): 493-502. [32] Abdel-Hamid, A. Z.; Rawi, S. M. and [37] Klinbunga, S.; Ampayup, P. and Arafa, A. F. (2006). Identification of Tassanakajon, A. (2000). Development a genetic marker associated with the of species-specific markers of the resistance to Schistosoma mansoni tropical oyster (Crassostrea belcheri) infection using random amplified in Thailand. Mar Biotechnol, 2: 476- polymorphic DNA analysis. Mem Inst 484. Oswaldo Cruz, Rio de Janeiro, 101(8): [38] Klinbunga, S.; Ampayup, P. and 863-868. Tassanakajon, A. (2001). Genetic [33] Oliveira, A. L. D.; Da Silva, D.; diversity and molecular markers of Zanotti-Magalhaes, E. M. et al. cupped oysters (genera Crassostrea, (2008). Schistosome/mollusk: genetic Saccostrea, and Striostrea) in Thailand compatibility. Genet Mol Res, 7(2): revealed by randomly amplified 518-526. polymorphic DNA analysis. Mar [34] Teixeira, K. N.; Souza, K. N.; Biotechnol, 3: 133-144. Vidigal, T. H. D. A. et al. (2010). [39] Yoon, J.-M. (2017). Genetic distances Size polymorphism in alleles of of scallop (Chlamys farreri) the myoglobin gene from populations investigated by PCR Biomphalaria mollusks. Genes, 1(3): procedure. Dev Reprod, 21(4): 435- 357-370. 440.

How to cite this article: Fol, M. F.; Morad, M. Y.; Gamil, I. S.; Sabet, S. F. and Mansour, R. M. (2021). Genetic relationships among four mutelid species (Bivalvia: Unionida) in Egypt revealed by RAPD-PCR technique. Egyptian Journal of Zoology, 75: 1-13 (DOI: 10.12816/ejz.2020.44427.1041).

12 المجلة المصرية لعلم الحيىان المجلد 57 )يىويى 0202(

العالقة الجيىية ألبرععة نوىا مه المحابريات الىهرية ”BIVALVIA: UNIONIDA“ في مصر عىاسطة جقىية ”RAPD-PCR“

مىً فححً فل2، مصطفً يحيً مراد0، ايريه سامح جميل0، سلىي فروق ثاعث2، برضا محمد مىصىبر0 2قسى ػهى انحيٕاٌ، كهيت انؼهٕو، جايؼت انقاْشة، جيزة، يصش 0قسى ػهى انحيٕاٌ ٔانحششاث، كهيت انؼهٕو، جايؼت حهٕاٌ، انقاْشة، يصش

يؼخبش حفاػم انبهًشة انًخسهسم ان ؼًُخًذ ػهٗ انخضاػف انؼشٕائٗ يخؼذد األشكال نهذَا )RAPD-PCR) يٍ انطشق انبسيطت ٔانسشيؼت ان ًُسخخذيت نخحذيذ انؼالقت انجيُيت بيٍ األَٕاع في انحاالث انخصُيفيت انخي يصؼب ححذيذْا ػهٗ أساس ان س ًاث انًٕسفٕنٕجيت. ح ُظ ٓش رٔاث انًصشاػيٍ حُٕع ظاْشٖ كبيش يصاحبّ انقهيم يٍ ان س ًاث انثابخت يًا يجؼهٓا يجػًٕت يصؼب حصُيفٓا، ٔانكثيش يُٓا نذيّ انؼذيذ يٍ األَٕاع انفشػيت ٔانًشادفاث انًخػُٕت. َٔخيجت نهذسجت انؼانيت يٍ انخُٕع انًٕسفٕنٕجي داخم سحبت "Unionoida"، فقذ حى حجًيغ أسبؼت إَٔاع ْي "M. rostrata ٔ Mutela dubia ٔ C. letourneuxi ٔ Chambardia rubens" يٍ يُطقت بُٓا )يحافظت انقهيٕبيت( فٗ يصش، ٔرنك نبياٌ دسجت انخشابّ انجيُٗ نٓزِ األَٕاع اػخًادا ػهٗ حقُيت "RAPD-PCR". فًٍ أصم سخت بٕادئ يسخخذيت ، ُٔ جذ أٌ خًست بٕادئ فقط ْي "UBC486 ٔ UBC483 ٔ UBC478 ٔ UBC477 ٔ UBC476" قذ ػًهج بُجاح. ٔقذ أظٓشث انذساست أٌ انػُٕيٍ "C. letourneuxi ٔ C. rubens" قشيبا انصهت حيث أظٓشا ششائط يخًاثهت نحذ كبيش ألجزاء يٍ انحًض انُٕٖٔ ٔرنك باسخخذاو انبٕادئ "UCB 486 ٔ UCB 483 ٔ UBC 477".

13