Efficiency of Three Mitochondrial Genes in Molecular Identification
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Research Journal of Biotechnology Vol. 14 (10) October (2019) Res. J. Biotech Efficiency of three mitochondrial genes in molecular identification and phylogenetic analysis of termites Reem Alajmi1, Ashraf Mashaly2*, Noura Al-Otaibi1,3, Ahmed Mahmoud1 and Tahany Ayaad4 1. Department of Zoology, College of Sciences, King Saud University, P.O. Box. 2455, Riyadh 11451, SAUDI ARABIA 2. Department of Zoology, Faculty of Science, Minia University, El Minia 61519, EGYPT 3. Department of Biology, Faculty of Science, Taif University, Taif 26571, SAUDI ARABIA 4. Department of Entomology, Faculty of Science, Cairo University, Giza 12613, EGYPT *[email protected] Abstract made structures, termites cause a huge damage to wood- Termites are eusocial insects with an economic impact made and valued commodities such as wooden structures, where they cause a great loss in crops and man-made warehouses, wooden buildings, constructional timber 12,28 valued commodities and buildings. Rapid and accurate etc. identification techniques of termite species are Termites belong to the order isoptera with more than 2800 important step for management approaches. Termite species.11 These species are grouped within different identification based on morphological features is not families based on their biological and ecological features reliable and could be misleading in many cases. In the such as the size of the colony, nesting, feeding habits and current study, we planned to investigate the efficiency reproduction.1 Of these families, the family Hodotermitidae of three mitochondrial genes; mt12S rRNA, mtCOI and includes members that have a great avidity to grasslands, mtCOII in molecular identification and phylogenetic crops and structural timber.46 Species belonging to the analysis of termites from Saudi Arabia. Partial family Kalotermitidae are characterized by feeding on dry sequences of mt12s rRNA and mtCOII were obtained wood and form small colonies without constructing definite 16 from 10 populations of Anacanthotermes ochraceus, 10 nests. The family Rhinotermitidae involves termites that populations of Psammotermes voeltzkowi and 10 are mainly subterranean and feed mainly on wood. Up to 600 species of termites that cause great losses in agriculture are populations of Microcerotermes arboreus. For the grouped into the family Termitidae.22 Species of the family mtCOI, partial sequences were obtained from 10 Termopsidae are known to form their nests within damp and populations of Anacanthotermes ochraceus and 10 decaying wood.22 populations of Microcerotermes arboreus. Proper identification techniques of termite species are Our sequence analysis revealed that mtCOII is essential to choose the suitable management strategy.35 evolving rapidly recording a large number of variable Identification of termite species is not an easy task due to: i) sites while mt12s rRNA is the most conserved. Intra- the significant overlapping of the morphological features, ii) and interspecific variation varied between population the difficulty in sampling morphologically distinguished 23,50 of the same genus and among the three genera. castes and iii) the scarcity of systematic studies. Morphology-based identification is not reliable and could be Phylogenetic analysis showed the distribution of misleading in many cases.17,26,36 In addition, the available termite species based on their geographic region. morphological keys are not valid to identify all the relevant Termite genera from Riyadh formed distinct clades. termite species.20 Molecular-based techniques offer rapid Populations of each genus formed different groups and reliable approaches for definite identification of termite based on their intraspecific variations. The topology of species. These techniques overcome the limitations of 12s rRNA tree was more informative than those of morphologically based tools and have been applied in mtCOI and mtCOII. several studies to identify termite species, population genetics and evolutionary relationships between Keywords: Isoptera, Saudi Arabia, 12s rRNA, mtCOI, species.3,4,29,49 mtCOII. The mitochondrial genome (mtDNA) was used in species Introduction identification of several invertebrate groups including 8,19,20,30,39 Termites are ecologically and economically important termites. The use of mitochondrial genes has eusocial insects.33 They are efficient lignocellulose advantages over nuclear ones such as stability, haploid decomposers and hence play a vital role in recycling of wood uniparental inheritance, lack of recombination and rapid 5,10,40 materials and sustaining of various ecosystems.7,31,43 On the evolutionary rates. The most commonly used mtDNA other hand, they represent serious pests to agriculture and genes are rRNA (12S and 16S), transfer RNA (tRNA) and 20,38,47 household settings leading to more than $20 billion annually cytochrome oxidase genes (mtCOI and mtCOII). The worldwide.42 They were reported to cause a considerable 12S rRNA gene is highly conserved (i.e. low evolutionary loss of several important crops such as sugarcane, maize, rate) and was adopted in phylogenetic analysis of higher 41 rice and others.21,25 For household settings and other man- taxonomical levels. The sequence of mtCOI gene has been 30 Research Journal of Biotechnology Vol. 14 (10) October (2019) Res. J. Biotech used for accurate identification of termite species.18,34 The were assigned to test the validity of the three mtDNA genes mtCOII gene contains both highly conserved and variable in molecular identification and phylogenetic analysis. regions and have been used in accurate identification of Before starting the DNA isolation process, termites were Reticulitermes termites,15 Reticulitermes termites, washed with distilled water and air dried on filter paper to Macrotermes and Microtermes.27 get rid of ethanol. After that, the abdomens of the workers were removed and discarded and only the head of each In Saudi Arabia, 19 subterranean termite species were worker termite was ground in liquid nitrogen using a Pestle identified and grouped within 4 families: Kalotermitidae and Mortar. Genomic DNA was then isolated using QIAamp Hodotermitidae, Rhinotermitidae and Termitidae. Human DNA Mini Kit (Qiagen, Germany) according to the expansion activities such as city sprawl and the relocation of manufacturer’s instructions. Extracted DNA was Bedouins from rural areas and launching new settlements in resuspended in 60 µl of TE buffer. The quality and the marginal land resulted in increasing infestation by concentration of DNA were then measured using the termites.12,13 Therefore, establishing a rapid and reliable NanoDrop ND-1000 Spectrophotometer (Thermo Fisher identification technique will help to avoid the destructive Scientific Inc, USA) and DNA vials were stored at -20oC for effect of subterranean termites. further uses. In the current study, our aims were to: i) obtain the sequences PCR amplification of mitochondrial genes: Extracted of three mtDNA genes; mt12S rRNA, mtCOI and mtCOII DNA genomes from each family members were used as from termite workers collected from Riyadh, Saudi Arabia, templates to amplify the mt12S rRNA, mtCOI and mtCOII ii) the efficiency of these genes in molecular identification genes by FideliTaqTM PCR Master Mix Plus (GE and phylogenetic analyses and iii) compare the results of Healthcare, Buckinghamshire, UK) using the primer sets molecular identification with those of morphological listed in table 1. The PCR was carried out in a thermal cycler assessments. (Applied Biosystems; Thermo Fisher Scientific, USA) using the following cycling conditions; initial denaturation at 94°C Material and Methods for 15 min followed by 35 cycles of denaturation at 94°C for Study area, termite collection and storage: Riyadh city is 45 s, annealing (Table 1) for 45 s and extension at 68°C for located in the center of Saudi Arabia at latitude 38°–34°N 1 min and a final extension at 68°C for 10 min. and longitude 43°–46°E and about 600 m above the sea level. The city is characterized by a hot desert climate during The amplified products were visualized in 1.5 % agarose gel the summer with average high temperature 43.6 °C in stained with ethidium bromide and compared to a 100-bp August. The overall climate is arid with little rainfall during DNA ladder (Solis Biodyne). Amplified PCR products the summer and a fair amount of rain in March and April. corresponding to mt12S rRNA, mtCOI and mtCOII genes Riyadh is also frequently hit by dust storms.24 The termite were purified using IllustraTM GFX PCR DNA and a Gel samples used in this study were collected from different Band Purification Kit (GE Healthcare). locations in Riyadh city during the spring of 2016. Samples were collected twice a day: early morning (5–8 am) and DNA Sequencing and phylogenetic analysis: DNA before sunset (4–6 pm). sequencing of the amplified PCR products of mt12S rRNA, mtCOI and mtCOII was performed at Central Laboratory, The termites were collected from wood buried in soil by King Saud University, Riyadh. The purified PCR products shaking the wood into a large container. A total of 1577 were sequenced on both strands using a 3130xl Genetic worker termites were collected and transferred into labeled Analyzer (Biosystems; Thermo Fisher Scientific, USA). The vials containing 100% undenatured ethanol and stored at sequence chromatograms of each gene were individually 4°C for morphological and molecular analysis. The details assembled using Bioedit software, version 7.2.5 (Ibis of sampling such as the location