17634 K. Sivasankaran et al./ Elixir Appl. Zoology 62 (2013) 17634-17639 Available online at www.elixirpublishers.com (Elixir International Journal) Applied Zoology Elixir Appl. Zoology 62 (2013) 17634-17639 Molecular studies of family Erebidae moths (Lepidoptera: Noctuoidea) using RAPD-PCR technique K. Sivasankaran 1, M. Ramakrishnan 2, D. Parandhaman 1 and S. Ignacimuthu 3,* 1Division of Taxonomy and Diversity, Entomology Research Institute, Loyola College, Chennai- 600 034, Tamil Nadu, India. 2Division of Plant Biotechnology, Entomology Research Institute, Loyola College, Chennai- 600 034, Tamil Nadu, India. 3Entomology Research Institute, Loyola College, Chennai- 600 034, Tamil Nadu, India. ARTICLE INFO ABSTRACT Article history: Experiments were conducted to assess the relatedness of different subfamilies of Erebidae Received: 28 July 2013; family using RAPD-PCR technique. The genomic DNA of thirteen different species of Received in revised form: Erebidae moths belonging to different subfamilies of Erebinae, Calpinae and Hypocalinae 20 August 2013; were subjected to RAPD-PCR analysis. A total of 300 bands were scored with five RAPD Accepted: 3 September 2013; primers, of which 291 were polymorphic bands while 9 were monomorphic bands; the percentage of polymorphism was 97%. Dendrogram revealed that the thirteen species were Keywords grouped into three major clusters and four subclusters. Lepidoptera, © 2013 Elixir All rights reserved. Erebidae, Molecular data, Population variation, RAPD-PCR. Introduction (Mitchell et al ., 2006; Zahiri et al ., 2010) to find distinct Molecular studies provide discrete data for phylogenetic characters. analysis of highly diverse groups of organisms. Insects presently In the present study, 13 species belonging to three constitute half the known species on the planet, with an subfamilies of Calpinae Hypocalinae and Erebinae of family estimated number of 5 million living species (Grimaldi & Engel, Erebidae i.e. Thyas coronata , Hypocala violacea , Oxyodes 2005). scrobiculata , Eudocima phalonia , Hypocala deflorata , Catocala Insects comprise the largest group in the animal kingdom macula , Pindara illibata , Serrodes campana , Eudocima and possess a vast undiscovered genetic diversity and gene pool salaminia , Sphingomorpha chlorea , Achaea serva and Erebus that can be better explored using molecular marker techniques. macrops were assessed for their relatedness using RAPD-PCR Polymerase chain reaction (PCR) technique is used for the technique. No such work has been carried out on Indian species phylogenetic analysis of many insects. This method employs till now. This molecular information would further facilitate the single random primers and results are used for the differentiation rapid identification of species of Erebidae moths. of species and the reconstruction of phylogeny. Materials and methods Noctuoid species are placed in the order Lepidoptera. Isolation of Genomic DNA Numerous classifications of the superfamily groups of Thirteen species of moths belonging to family Erebidae Noctuoidea have been proposed. Fibiger and Lafontaine (2005) were collected from various locations from Tamil Nadu part of proposed a new classification with ten families: Oenosandridae, Western Ghats, India. The species are listed in Table (1). The Doidae, Notodotidae, Strepsimanidae, Nolidae, Lymantridae, DNA was isolated using the method of Margam et al. (2010) Arctidae, Erebidae, Micronoctuidae and Noctuidae. with minor modifications. Insect tissue (from the head, thoracic The family Erebidae comes under superfamily Noctuoidea, and legs) of approximately 10–20 mg was homogenized. 500 µl most of which was previously classified in the family of CTAB buffer (100 mM Tris (P H: 9.5); 200 mM EDTA; 1.4 M Noctuidae. The family Erebidae is a predominantly tropical NaCl; 2% CTAB; 1% PEG 8000) was added to the homogenate subfamily and is widely distributed. The Erebidae is of and thoroughly mixed; then 4 µl of Proteinase–K (20 mg / µl) considerable economic importance as it contains many was added and incubated at 55º C for 4 hours. Thereafter 500 µl agricultural pests. Many of the genera included in Erebidae have of Phenol: Chloroform (1:1) was added; the tubes were gently been noted to feed as adults on fruits. inverted for a few seconds. The suspension was centrifuged for Several molecular studies have examined higher level 10 min at 12000 rpm at 4º C. The supernatant was taken and relationship within the Noctuidae. Weller et al. (1994), used equal volume of Chloroform: Isoamylalcohol (24:1) was added partial sequences of nuclear 28S rRNA (300 bp) and and centrifuged for 10 min. Aqueous phase was removed; 2.5 mitochondrial NDI (320 bp) from 26 Noctuoid species, volume of ice-cold absolute alcohol and 1/10 th volume of 3M including 10 noctuid to differentiate each other. Two recent Sodium Acetate were added and mixed gently by inversion. The molecular studies on ditrysian Lepidoptera analyze members of sample was stored at -20º C for overnight and then spun (12000 controversial family Noctuidae and superfamily Noctuoidea rpm for 20 min). The supernatant was discarded; the DNA pellet was washed with 70 % Ethanol twice and spun down at 14000 Tele: E-mail addresses: [email protected] © 2013 Elixir All rights reserved 17635 K. Sivasankaran et al./ Elixir Appl. Zoology 62 (2013) 17634-17639 rpm for15 min. The pellet was air dried, dissolved in 1X TE band. RAPD amplification patterns are shown in figures 1, 2, Buffer and stored at -20ºC for immediate use at -80º C for long and 3. term storage. The DNA sample was quantified and its purity was Figure 1. M. Marker; 1. Thyas coronata ; 2. Hypocala determined using UV spectrophotometer. violacea ; 3. Oxyodes scrobiculata ; 4. Eudocima phalonia ; 5. RAPD Analysis Hypocala deflorata ; 6. Catocala macula ; 7. Pindara illibata ; 8. Random Amplification of DNA by PCR Serrodes campana ; 9. Eudocima salaminia ; 10. DNA was amplified by the RAPD-PCR technique using Sphingomorpha chlorea ; 11. Achaea serva ; 12. Sprima Random primers R2, R5, R7, R11 and R12. The details of retorta ; 13. Erebus macrops primers are given in table 2. PCRs were programmed as follows: an initial denaturation period of 95º C for 5 min and followed by denaturation at 95º C for 30seconds; primer annealing was done at 37º C for 1 min and extension at 72º C for 2 min. 35 cycles were run. An additional 7 min at 72º C was allowed for final extension. PCR products were stored at 4º C. Gel analysis The amplified PCR products were run on a 1.4 % agarose gel using 1x TBE buffer and stained with Ethidium bromide. The electrophoresis was performed until good separation of RAPD bands occurred; the gels were photographed under UV illumination. Dendrogram Plot - Dendrogram were constructed for the data obtained with each of the three primers, using nearest neighbour analysis of hierarchical clustering (UPGMA software package Figure 2. M. Marker; 1. Thyas coronata ; 2. Hypocala Version 4.0 genomes. urv.cat/UPGMA/ Garcia-Vallve et al. violacea ; 3. Oxyodes scrobiculata ; 4. Eudocima phalonia ; 5. (1999). A bootstrap process was used to assess the reliability of Hypocala deflorata ; 6. Catocala macula ; 7. Pindara illibata ; 8. the dendrogram using 1000. Polymorphic Informative Content Serrodes campana ; 9. Eudocima salaminia ; 10. (PIC) values were analyzed for each primer, and Cophenetic Sphingomorpha chlorea ; 11. Achaea serva ; 12. Sprima Correlation Coefficient (CP) calculation was using the following retorta ; 13. Erebus macrops . formula. Data Collection and Analysis The bands were keyed by scoring the presence (1) or absence (0) of the bands. The (AMOVA) procedure partitioned the total variation both among populations and within populations of Erebidae moths. The populations were grouped based on the subfamilies. The analysis of molecular variance (AMOVA) was executed the software ( GelAlEx 6.5 , Peakall & Smouse, 2012) using to access the variation among the population of family Erebidae moths. Results The concentration of DNA extracted from the thirteen species ranged between 1500-4430 ng/ µl as determined by Figure 3. M. Marker; 1. Thyas coronata ; 2. Hypocala spectrophotometric method. violacea ; 3. Oxyodes scrobiculata ; 4. Eudocima phalonia ; 5. RAPD-PCR analysis Hypocala deflorata ; 6. Catocala macula ; 7. Pindara illibata ; 8. The RAPD-PCR patterns were assessed using five random Serrodes campana ; 9. Eudocima salaminia ; 10. primers namely, R2, R5, R7, R11and R12 with the genomic Sphingomorpha chlorea ; 11. Achaea serva ; 12. Sprima DNA isolated from thirteen species of Erebidae moths. For each retorta ; 13. Erebus macrops primer, a multiple band profile comprising one to five major amplification products, a varying number of weak products and a faintly smeared region, were observed. The number and size of amplified products varied depending upon the sequence of random primers and genotype used. A total of 300 bands were produced (Table 3). The size of the amplified products ranged from 200bp to 3500bp having different intensities. Different primers produced different banding patterns. Only primers R5, R7 and R11produced distinct and highly reproducible bands; only those were selected for analysis and comparison. The total numbers of bands obtained from each selected primer were 107 (primer R5), 132 (R7) and 61 (R11). Primer R7 generated maximum 16 bands, while primer R11 generated minimum 1 17636 K. Sivasankaran et al./ Elixir Appl. Zoology 62 (2013) 17634-17639 Figure 4. Dendrogram of 13 Erebidae