Academic Journal of Entomology 8 (3): 104-109, 2015 ISSN 1995-8994 © IDOSI Publications, 2015 DOI: 10.5829/idosi.aje.2015.8.3.9623

DNA Fingerprinting of Some Major Genera of Subterranean (Isoptera) (, and Microtermes) from Western Saudi Arabia

1Abdel Rahman A. Faragalla, 2 Mohamed H. Alqhtani, 1 Isam Abu Zeid and 1,3 Mohamed M.M. Ahmed

1Department of Biological Sciences, Faculty of Science, P. O. Box 80203, King Abdulaziz University, Jeddah, 21589, Saudi Arabia 2College Applied Medical Sciences, Center of Excellence, King Fahad Medical Research Center (KFMRC), King Abdulaziz University, Jeddah, 21589, Saudi Arabia 3Department of Nucleic Acid Research, Genetic Engineering and Biotechnology Research Institute, City for Scientific Researches and Technology Application, Borg EL-Arab, P.O.box. 21934, Alexandria, Egypt

Abstract: RAPD fingerprinting technology was used to provide the basic information on the genetic similarity between12 members of three families (, RhinotermitidaeandTermitidae) containing five species of subterranean termites belonging to three genera (Anacanthotermes, PsammotermesandMicrotermes)through the use of RAPD-PCR technology. Three ten-mere primers were used in fingerprinting the 12 germplasms under studyused two of them have a 60-80% G+C contentand one primer has 40% G+C content. A phylogenetic analysis for the different species using a statistical computer programon the basis ofthe RAPD-PCR results showed a dendrogram of 2 groups depending on the average of band sharing ratio between different species under investigation.

Key words: RAPD PCR Subterranean Termites

INTRODUCTION recent surveys has shown that the infestation is on the rise. This is due to the wide extensive The subterranean termites (Isoptera) was well-known sprawling of major cities due to the conversion of date to contain various destructive and harmful termite palms grooves and traditional marginal land to housing species, with respect to their own preferences of eating projects and urban settlements. This has turned a wide wood, timber, wood works and a wide range of secondary range of desert lands of many oases to agricultural products. In tropical and subtropical regions of the world, production and increase irrigation that has created a they are specifically described as the most destructive favorable utopia for termite infestation by these cryptic, pests on various commodities and human valued insidious creatures. properties. Moreover, the overall annual cost of treatment Most contemporary ecological and biological studies control around the world was estimated to reach 20 billion are confronted with problems of determining correct dollars [1] with the risk of the total loss of structural taxonomic identification of recovered species because of timber. the apparent lack of renowned experts in many genera and The ubiquitous distribution of a rich fauna of species worldwide, especially when dealing with newly subterranean termite species manifested as losses discovered species or their siblings. Therefore modern and damages worth staggering sums of money from technologies have been used have been used in the a human perspective in valued properties including documentation of a variety of insect pests, mainly (screw houses, wood works, storage facilities and many worm (Infante [2]; Hematobiairritans [3,4] sarcophagid agricultural crops and commodities. The regions of flies [5]; Mosquito Culexquiniquifaciatus and Saudi Arabia, western and south western regions; Dengue mosquitoes [6, 7] and Eutropichthysvacha [8].

Corresponding Author: M.M.M. Ahmed, Department of Nucleic Acid Research, Genetic Engineering and Biotechnology Research Institute, City for Scientific Researches and Technology Application, Borg EL-Arab, P.O. box. 21934, Alexandria, Egypt. E-mail: [email protected]. 104 Acad. J. Entomol., 8 (3): 104-109, 2015

Traditionally most identifications was based bio- from Hodotermitidae Anacanthotermesochraceus morphological characteristics but currently the state of (Burmeister) and Anacanthotermesvagans (Hagan). One the art innovative technologies (DNA, chromosomes, species from , Psammotermeshybostoma, Karyotyping, Microarray, PCR) and genetic markers has (Desneex) and two species from Termitidae been adopted on a large scale to document the (Macrotermitinae, Microtermesnajdensis (Harris) and phylogenetic relationship and accelerate the rate of proper Microtermesyemenensis (Wood)). They were assigned by identification of species [9] This will automatically lead to the following designations: rational application of remedial, alternative strategies to control these pests. Anacanthotermesochraceus (small size workers) The recent studies focused on the phylogeny of Anacanthotermesochraceus (large size workers) termites using both morphological characters[10] and Anacanthotermesvagans (large size soldiers) molecular characters [11] Some studies have focused on Anacanthotermesvagans (small size workers) specific species based on the genetic differences between Anacanthotermesvagans (large size workers) species, particularly within the family Rhinotermitidae Microtermesnajdensis(small size workers) especially with genus Reticulotermes [12]. Microtermesnajdensis (small size soldiers) The recovered information is divided on the genetic Psammotermeshybostoma(Large size soldiers) variation between populations and species of termites Psammotermeshybostoma(small size soldiers) that can be useful in determining the extent of gene flow Microtermesyemenensis (Wood)(small size workers) and for the development of molecular diagnostics for Microtermesyemenensis(Wood)(large size workers) species identification[13, 14]. The usefulness of the Microtermesyemenensis(Wood) (large size soldiers) cytochrome oxidase II (COll) region of the mitochondrial DNA (mt DNA) is well-demonstrated in genome and in the According to the kit, a large volume of salting study of phylogenetic relationships of termites [15, 16]. It methods of [19] are made with some modifications. The is known that mitochondrial genes evolve more rapidly adults of were crushed with mortar and the than nuclear genes and fingerprinting genomes [17] and resultant material was kept in a 1.5 ml eppendorf tube. therefore they make good markers for the analysis of Three hundred microliter TENS buffer (50 mMTris base relatively close ties within the genus/species. Data pH 7.5, 400 mMNaCl, 20 mM EDTA, 5% SDS) and 5 µl of generated from DNA sequences has been used to deduce proteinase K (10 mg/ml) were added and incubated at 55°C the origin of other Formosan subterranean termites for 20 min. Then, 85 µl of 5M NaCl were added and vortex (Coptotermesformosanusshiruki) infestation where the for 15 sec. Centrifugation was carried out at 13,000 rpm for genetic sequence of cytochrome oxidase II was used to 5 min. Then, carefully the upper phase was placed in a determine the eight lineages of certain species from 14 new tube and an equal volume of cold 100% ethanol was geographic locations across the southern U.S. or for added. Then a second centrifugation was carried out for colony demarcation [18]. 5 minutes. The liquid phase was removed and the pellet In the present work, RAPD fingerprinting technology was washed using 500 µl of 70% ethanol. The DNA was used for providing the information about the genetic pellets were dissolved in 30 µl 0.1X TE buffer and stored similarity among individuals of five major species at –20 °C. belonging to three subterranean termite genera including Anacanthotermes,Psammotermes and Microtermes; for PCR Primers: The three primers were randomly selected conservation, improvement and the use of marker-assisted and used in PCR amplifications. The nucleotide selection in identification programs. sequences, GC content and annealing temperatures for all primers are given in (Table 1). MATERIALS AND METHODS PCR Amplification and Agarose Gel Electrophoresis: DNA Isolation: DNA was extracted from members of three PCR amplificationswere conducted following the families of subterranean termites (Isoptera) including (The procedure of Wiliams et al., [20] using 3 arbitrary harvester termite Hodotermitidae, the old world termite, primers.These primers were ten to twenty nucleotides in Rhinotermitidae and the advanced family, Termitidae). A length and possessed 60-80% G + C content and only one total of 5 major species of the above mentioned families in primer (primer No. 16) possessed 40% G + C content. three genera have been recovered (Anacanthotermes, We obtained a series of several DNA fragments (figures Psammotermes and Microtermes) including two species 1,2 and 3) of the samples under investigation.

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Thereactions were performed in the total volume of in determining the relationship betweensubterranean 25 µl consisting of 0.8 units of Taq DNA polymerase termites and as marker-assisted selection. In taxonomic (enzyme), 25 pecomoldNTPs (Di-deoxy nucleotide and systematic relationships, molecular markers could be triphosphate) and 25 pecomol of random primer, 2.5 µl 10x used as an effective tool to check for the establishment reaction buffer and 40 ng of genomic DNA. The final for the status of an organism.Usually in taxonomic studies reaction mixture was placed finally in the DNA thermal and evolution as has been adopted previously in honey cycler (Eppendorf). The PCR program included an initial bees [24-26]. denaturation step at 94°C for 2 min, followed by 45 cycles Our results reflect the usefulness of such modern (94°C for 30 sec, annealing as mentioned with each primer, techniques and fine molecular methods. These results 72°C for 30 sec) and a final extension at 72 °C for 10 min. demonstrate the benefits of the RAPD approach for the The samples were kept at 4°C. The amplified DNA detection of DNA similarity or polymorphisms fragments were separated on 2.5% agarose gels and ofsubterranean termites and the establishment of stained with ethidium bromide. The amplified patterns relationships between and among different species [27] of were visualized on a UV trans-illuminator and the following different termite species:1. photographed using a gel documentation system (Image Anacanthotermesocharceus, 2. A. ochraceus, 3. A. Analyzer). vagans, 4. A. vagans, 5.A. vagans, 6. Microtermesnajdensis, 7. M. najdensis, 8. Scoring and Analysis of RAPDs: RAPD bands were Psammotermeshybostoma, 9. P. hybostoma, 10. M. scored as 1 for presence and (0) for absence, in the RAPD yemenensis (Wood), 11. M. yemenensis(Wood), 12. profile of the subterranean termites were as designated M.yemenensis(Wood). during DNA isolation. All of the amplification products were found to be A dendrogram was constructed using the Average reproducible when repeated under the same reaction Linkage Between Groups (ALBG) as described by Sneath conditions. We obtained a series of several DNA and Sokal [21] based on the resulting RAPD results. fragments (Fig. 1, 2 and 3) from the samples under investigation. The number of fragments generated per RESULTS AND DISCUSSION primer varied between one and seven fragments. The genetic variation among the species may be due to All the subterranean termites genera and species differences in sex, function and the evolution of each were individually tested. Each produced a RAPD detected caste and include the following: 1. fingerprint presented in Figures 1, 2 and 3. The fingerprint Anacanthotermesocharceus, 2. A. ochraceus, 3. A. species patterns 1-3 (Fig. 1) were as follows: vagans, 4. A. vagans, 5.A. vagans, 6. Microtermesnajdensis, 7. M.najdensis, 8. A. ochraceus (small workers) Psammotermeshybostoma, 9. P. hybostoma, 10. M. A. ochraceus (large workers) yemenensis(Wood), 11. M. yemenensis(Wood), 12. A.vagans (large soldiers) M.yemenensis(Wood). Data obtained showed the advantages of RAPD respectively using the arbitrarily primer 3 (Table 1). markers for detecting similarities between and among the 1. Anacanthotermesocharceus, 2. A. ochraceus, 3. A. A phylogenetic analysis using the statistical vagans, 4. A. vagans, 5.A. vagans, 6. computer program Statistic 5 was conducted for three Microtermesnajdensis, 7. M.najdensis, 8. different specieson the basis of 3 random amplified Psammotermeshybostoma, 9. P. hybostoma, 10. M. primers, polymorphic DNA. Which showed a dendrogram yemenensis (Wood), 11. M. yemenensis(Wood), 12. as in Figure 4 exhibited two groups based on the average M.yemenensis (Wood) of different individuals. rates of band sharing similarity percentages between the three genera of subterranean termites including Table 1: The nucleotide sequences of the primers used and their annealing Anacanthotermes,Psammotermes and Microtermes. temperatures The sensitivity of RAPD technique played a good Primer No. Sequences 5-3 [20] Annealing Tem. / Sec. role in the detection of the genetic differences as noted by 1 AGA CCC AGA G 42 Hawksworth [22] and Haig [23] Thus, the RAPD profile 2 CTG AGA CGG A 42 generated for each species can be effectively used as a 3 GGG CCA CTC A 42 support fora marker for taxonomic classification purposes,

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Fig. 1: RAPD-PCR results of Subterranean termites DNA, generated by primer 1, Lane M, molecular size marker Lanes 1.A. ocharceous2. A. ochraceus; 3 A. vagans 4., A. vagans 5.A. vagans; 6.Micotermesnajdensis; 7.M. najdensis; 8. P. hybostomma; 9 P. hybostoma;10.M. yemenensis11. M. yemenensisand 12.M.yemenensiswith primer 1.

Fig. 2: RAPD-PCR products of Subterranean termites DNA generated by primer 2, Lane M, molecular size marker Lanes 1.A. ocharceous2. A. ochraceus; 3 A. vagans 4., A. vagans 5.A. vagans; 6.Micotermesnajdensis; 7.M. najdensis; 8. P. hybostomma; 9 P. hybostoma;10.M. yemenensis11. M. yemenensisand 12.M.yemenensiswith primer 2.

Fig. 3: RAPD-PCR products of Subterranean termites DNA, Lane M, molecular size marker Lanes 1.A. ocharceous2. A. ochraceus; 3 A. vagans 4. A. vagans 5.A. vagans; 6.Micotermesnajdensis; 7.M. najdensis; 8. P. hybostomma; 9 P. hybostoma;10.M. yemenensis11. M. yemenensisand 12.M.yemenensiswith primer 3.

Fig. 4: Dendrogram using average of similarity percentages (between groups) by of Statistica Program based on the RAPD-PCR data produced from the three primers. 1.A. ocharceous2. A. ochraceus; 3.A. vagans 4. A. vagans 5.A. vagans; 6. Micotermesnajdensis; 7.M. najdensis; 8. P. hybostomma; 9.P. hybostoma;10.M. yemenensis (Wood) 11. M. yemenensis (Wood) and 12.M.yemenensis (Wood) based on 3 RAPD primers.

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All the amplifications produced were found to be 4. Castiglioni, L. and H.E.M.C. Bicudo, 2005. reproducible when reactions were carried out under the Molecular characterization and relatedness of same conditions. These results reflected the relationship Haematobiairritans (hornfly) populations, between the RAPD patterns and quantitative trait position by RAPD-PCR. Genetica, 124: 11-21. (QTL) of each genotype. Our results are consistent with 5. Bajpai, N. and R.R. Tewari, 2010. the findings of several authors e.g. [28-30]. Genetic characterization of sarcophagid flies by RAPD analysis has been used to build a Random Amplified Polymorphic DNA- Polymerase phylogenetic tree (Fig. 4) using the average linkage Chain Reaction (RAPD-PCR), Natl. Acad. Sci. Lett., rescaled distance between cluster groups arranged after 33: 3-4. their combination [21]. This method has been used to 6. Santos, J.M.M., E.C. Fraga, J.F. Maia and W.P. Tadei, construct phylogenetic trees in other organisms such as 2011. Genetic Diversity in Dengue Mosquito, insects [31], in chicken [30], in farm [32] and in Aedesaegypti (Diptera: Culicidae) from Amazon tilapia fish [33-35]. Region: Comparative Analysis with Isozymes and In conclusion, it may be emphasized that most of the RAPD Loci, Open Trop. Med. J., 4: 11-20. RAPD primers used have amplified genomic DNA 7. Sharma, A.K., M.J. Mendki, S.N. Tikar, K. Chandel, successfully of the subterranean termites under study. All D. Sukumaran, Parashar B.D. Vijay Veer, comparative results gave the same specific bands O.P. Agarwal and Shri Prakash, 2009. between 3 and 5 and also 6 and 7. This similarity of DNA Genetic variability in the geographical populations of fingerprinting provides a rapid, effective method of Culexquinquifasciatus Say (Diptera: Culicidae) from detecting the genetic variation of different insect India based on random amplified polymorphic DNA analysis, Actatropica, 112: 71-76. genotypes and to be employed as a marker-assisted 8. Chandra, G., A. Saxena and A. Barat, 2010. selection during Subterranean termitesidentification Genetic diversity of two riverine populations of programs. Eutropiichthysvacha (Hamilton, 1822) using RAPD markers and implications for its Conservation, J. Cell ACKNOWLEDGEMENTS Mol. Biol., 8(2): 77-85. 9. Williams, K.P. and D.P. Bartel, 1995. PCR product This project was funded by the Deanship of Scientific with strands of unequal length, Nucleic Acids Res., Research (DSR), King Abdulaziz University, Jeddah, 23(20): 4220-4221. under grant no. (482/02/1429H). The authors, therefore, 10. Donovan, S.E., D.T. Jones, W.A. Sands and acknowledge with thanks DSR’s technical and financial P. Eggleton, 2000. Morphological phylogenetics of support. termites (Isoptera). Biol. J. Linneansoc, 70: 467-513. 11. Kambhampati, S., K.M. Kjer and B.L. Thorne, 1996. REFERENCES phylogenetic relationship among termite families based on DNA sequence of mito 16s ribosomal RNA 1. Su, N.Y., 2002. Novel technique for subterranean gene. Insect molec. Bio., 5: 229-238. termite control. Sociobiology, 40: 95-101. 12. Austin, J.W., A.L. Szalanski, P. Liva, A.G. Bagneres 2. Skoda, S.R., J.L. Figarola, S. Pornkulwat and and A. Kence, 2002. A comparative genetic J.E. Foster, 2013. Inter-and intra-specific analysis of the subterranean genus Reticulotermes. identification of the screwworm, Ann. Entomol. Soc. Am., 95: 753-760. Cochliomyiahominivorax, using random amplified 13. Szalansky, A.L., J.W. Austin and C.B. Owens, polymorphic DNA-polymerase chain reaction, 2003. Identification of Reticulotermes spp. J. Insect Sci., 13(76): 1-15. (Isoptera: Reticulotermitidae) from the South Central 3. Brito, L.G., L.C.A. Regitano, M.E.F. Huacca, United States by PCR-RFLP. J. Econ. Entomol. (in E. Carrilho, M.J. Paes and BG.E. orja, 2008. Press). Genotype characterization of the Haematobiairritans 14. Jenkins, B.N. Dean, B.E. and B.T. Forschler, 2002. (Diptera: Muscidae) from Brazil, Dominican Republic DNA Technology, Interstate Commerce and the likely and Colombia based on randomly amplified origin of the Formosan subterranean termites polymorphic DNA (RAPD) analysis, Revista (Isoptera: Rhinotermitidae) Infestation in Atlanta, Brasileira de ParasitologiaVeterinaria, 17(4): 179-184. Gerogia. J. Econ. Entomol., 95(2): 381-389.

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