Genetic Relatedness of Six South Indian Agriculturally Important Moth Species (Lepidoptera: Noctuoidea) Based on 28S Rrna-D2 Region Sequence Analysis
Total Page：16
File Type：pdf, Size：1020Kb
Indian Journal of Biotechnology Vol 12, July 2013, pp 364-371 Genetic relatedness of six South Indian agriculturally important moth species (Lepidoptera: Noctuoidea) based on 28S rRNA-D2 region sequence analysis V Veeramani, S Sakthivelkumar and S Janarthanan* Department of Zoology, University of Madras, Chennai 600 025, India Received 27 January 2012; revised 19 September 2012; accepted 14 October 2012 Molecular phylogenetic evidence from 28S rRNA-D2 expansion segment was used to facilitate differentiation of six different pest species of moths from the superfamily Noctuoidea. The expansion segment of the large ribosomal subunit of 28S rRNA-D2 region was amplified, sequenced and used to construct a phylogenetic tree on the basis of N-J phylogeny method. The topology on the basis of this phylogenetic tree supported a large monophyly for Helicoverpa armigera Hub. and Spodoptera litura Fab. in one cluster, Euproctis fraterna More, Amsacta albistriga Walker and Pericallia ricini Fab. in another cluster, and Earias vitella Fab. in third cluster. The rRNA sequences were also analyzed for the genetic variation using the predicted secondary structure models for all the six species. This analysis revealed variation in the structure of D2 expansion segments at its various arms. Though the multiple sequence alignment showed one large cluster with three sub- clusters and 96% homology among these pest species, the predicted secondary structure models did not show any such close relationships. These results reflect the functional diversity occurred due to the changes acquired slowly at the rRNA structure to perform their function during evolution rather than the homology in the nucleotide sequences or rRNA morphometry among different species of insects. Keywords: Leipdopteran insect pests; molecular phylogeny; noctuoidea; secondary structure; 28S rDNA-D2 region Introduction of ancestral characters to provide an idea on Butterflies and moths belong to the order divergence of insects and their homology5,6. Lepidoptera, one of the four mega-diverse insect Morphological characters have a mosaic 1 orders with over 165000 described species . They are distribution and it is extremely difficult to find out prominent organisms of terrestrial ecosystem. The phylogenetic indications from such variable data with larval forms are primarily herbivores and feed on a small number of functional characters7. In addition, plants, while the adults are pollinators and feed on the phylogenetic analysis of species on the basis of plant nectar. They also constitute one of the most morphological attributes may be faced with problems damaging groups of pests to agriculture. These insects because these features are variable with the have become vital for ecosystem assessment and environment and geographical locations. Molecular conservation planning to promote environmental markers are more reliable in terms of the number of 2 awareness . Besides, these lepidopteran insects available taxonomic characters displaying an serve as important model organisms for studies appropriate level of variability and thus they offer an of genetics, physiology, development, ecology, alternate tool. The molecular techniques based on 3 evolutionary biology and insect-plant co-evolution . RFLP, PCR-RFLP, PCR-RAPD, SSR-PCR, etc Insect phylogenetic studies normally include use of are very frequently used as tools to assess the morphological features as phenotypic characters and phylogenetic variations among organisms8. Though ontogenic stages, or pathways as developmental traits. these tools are considered to be good molecular In recent times, RNA, DNA, allozymes and amino techniques for the study of genetic analysis, different acid sequences have been used to infer evolutionary rRNA genes are very often used in recent times for 4 relationships among insects as molecular markers . inferring the phylogenetic relationships between Such studies are more important to rebuild the status distant species, because its evolutionary rate is —————— relatively low. Among them, 28S rRNA is a highly *Author for correspondence: Tel:+91-44-22202841; Fax: +91-44-22352494/22353309 conserved region used to distinguish divergence E-mail: [email protected] within and between species of insects. VEERAMANI et al: GENETIC RELATEDNESS IN AGRICULTURALLY IMPORTANT MOTH SPECIES 365 Among lepidopteran insects, many noctuoid genera 160A). Aliquots of the DNA (100 ng) were then used include armyworms, cutworms, bollworms and stem for polymerase chain reaction (PCR). borers, and they are some of the most economically important forest and agricultural pests causing PCR Amplification and Sequencing massive economic losses. Earlier molecular studies The D2 region of 28S rRNA was amplified from the total genomic DNA by PCR using a universal on these genera have used only a small number of 11 markers, usually a few gene regions9. In the present primer . The forward and reverse primer sequences study, a new molecular marker, the large subunit used were 5′-CGTGTTGCTTGATAGTGCAGC-3′ and 5′-TTGGTCCGTGTTTCAAGACGGG-3′, (LSU) 28S rRNA-D2 region sequence data, is used to analyze the phylogeny of six most important pest respectively. PCR was carried out in 25 µL reaction species of Noctuoidea (moths), collected from volume in a Long Gene MG25 plus Thermal Cycler. agricultural fields in and around Chennai, Tamil It was mixed well and centrifuged briefly. A control Nadu, South India. reaction was prepared without template DNA (negative control) and without enzyme (positive Materials and Methods control). The PCR reaction conditions were 94°C for 1 min (denaturation), 56°C for 1 min (annealing) and Sample Collection 72°C for 90 sec (extension) for 30 cycles. To the The larvae and adults of the Helicoverpa armigera amplified product (5 µL), sample buffer (1 µL) was Hub. (Noctuidae) from Gossypium hirsutum, mixed and checked using 1.2% agarose gel, followed Spodoptera litura Fab. (Noctuidae) from Vigna by electrophoresis at 50 V for 3 h using 1× TAE unguiculata, Amsacta albistriga Walker (Arctiidae) buffer (Tris-acetate 40 mM, pH 8.0; EDTA 1 mM, from Arachis hypogaea, Pericallia ricini Fab. pH 8.0)10. The PCR products were gel purified (Arctiidae) from Moringa oleifera, Euproctis fraterna and sequenced using Ocimum Biosolutions DNA More (Lymantriidae) from Ricinus communis and Sequencing Services, Hyderabad, India. The GC Earias vitella Fab. (Nolidae) from Abelmoschus content was determined using GC calculator esculentus were collected from the agricultural fields (http://www.genomicsplace.com/gc_calc.html). at different locations in Chennai (Tamil Nadu), South India. Freshly collected larvae (matured 5th instar) Analysis of 28S rRNA Sequences were dissected, digestive tract was removed and The gene sequences were aligned in FASTA stored frozen at −20°C. format and then analysed using ClustalW, followed by 12 T-Coffee (version 8.06) . The program was then used to Extraction of Genomic DNA calculate the best match of similarities and differences Total DNA was extracted using phenol-chloroform for the selected sequences, through viewing the 10 method . The larval fat body tissue (100 mg) was phylograms based on N-J phylogeny. The 28S rRNA-D2 ground with pre-chilled mortar and pestle to fine region sequences were manually aligned in the powder using liquid nitrogen. It was then suspended FASTA format for secondary structure prediction. in 1 mL digestion buffer (100 mM Tris-HCl, pH 8.0; The individual sequences were analyzed using 10 mM EDTA pH 8.0; 1.4 M NaCl; 1% SDS; 0.2% RNAfold core programs. It was then used to predict mercaptoethanol with 100 µg/mL proteinase K). The the minimum free energy (MFE) for the secondary sample was incubated with occasional shaking in structures using dynamic programming algorithm13. In tightly capped micro-tubes for 30 to 60 min at 50°C. addition, minimum free energy folding and The sample was then extracted with equal volume of equilibrium base-paring probabilities were carried phenol-chloroform and was centrifuged for 10 min at out14. Mountain plot representation of the MFE 4000 rpm. The top aqueous layer was transferred to a structure and thermodynamic ensemble of RNA new tube. To the supernatant, 0.5 volume of 7.5 M structures were also carried out15-17. The centroid ammonium acetate and 2 volumes of 100% ice cold structures were done by using a method described ethanol were added to precipitate the DNA. It was earlier18. centrifuged for 2 min at 4000 rpm. The pellet was washed with 70% ethanol, dried and resuspended in Results TE buffer (pH 8.0). DNA concentration was measured In the present study, phylogenetic relationships at 260 nm in a spectrophotometer (Shimatzu UV of six Lepidopteran moth species, viz., H. armigera, 366 INDIAN J BIOTECHNOL, JULY 2013 S. litura, A. albistriga, P. ricini, E. fraterna and sequence alignment using Clustal W programme E. vitella , belonging to the superfamily Noctuoidae revealed three major clusters for the 6 different was traced on the basis of 28S rRNA-D2 region species of lepidopteran moths belonging to the specific gene sequence. PCR amplification of total family Noctuoidae. One cluster consisted of cotton genomic DNA of various lepidopteran larvae with bollworm, H. armigera and tobacco cutworm, universal 28S rRNA-D2 region specific primers S. litura. The second cluster contained with all yielded a fragment of DNA in all the samples, which hairy caterpillars including the red hairy caterpillar, varied in length from 581 to 849 bp. A. albistriga, hairy caterpillar,