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Tropical Agricultural Science Pertanika J. Trop. Agric. Sci. 43 (4): 523 - 535 (2020) TROPICAL AGRICULTURAL SCIENCE Journal homepage: http://www.pertanika.upm.edu.my/ Species Richness of Leaf Roller and Stem Borers (Lepidoptera) Associated with Different Paddy Growth and First Documentation of Its DNA Barcode Salmah Yaakop1,2*, Angeline David-Dass1, Ummi Shuhaida Shaharuddin1, Suliza Sabri1, Aqilah Sakinah Badrulisham1 and Che Mohd Zain Che-Radziah2 1Centre for Insect Systematics, Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia 2Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia ABSTRACT Leaf folder and stem borer are pest moths (Lepidoptera) of paddy crop and caused serious damage and significant rice yield loss. The richness, abundance, and diversity of the pest moths were calculated in one paddy planting season and sampled from a model conventional paddy field, located on the west coast of Peninsular Malaysia (Sabak Bernam, Selangor). The adult and immature stages of moths associated with paddy plants have been sampled using active sampling namely sweep net and stem cross-cutting. A total of 189 individuals belonging to five species under two families (Crambidae and Noctuidae) were recorded. Overall, the richness (R’), diversity (H’), and evenness (E’) index of lepidopteran species ARTICLE INFO were 0.76, 1.51, and 0.90, respectively. The richness and species abundance throughout Article history: Received: 5 May 2020 the paddy stages were discussed. The DNA Accepted: 28 July 2020 Published: 27 November 2020 barcode of five collected species using cytochrome oxidase subunit 1 (CO1) viz. DOI: https://doi.org/10.47836/pjtas.43.4.08 Cnaphalocrocis medinalis (Guenée) (leaf E-mail addresses: folder), Scirpophaga incertulas (Walker), [email protected] (Salmah Yaakop) [email protected] (Angeline David-Dass) Chilo auricilius Dudgeon, Sesamia inferens [email protected] (Ummi Shuhaida Shaharuddin) [email protected] (Suliza Sabri) (Walker), and Parapoynx stagnalis (Zeller) [email protected] (Aqilah Sakinah Badrulisham) [email protected] (Che Mohd Zain Che-Radziah) (stem borers) were presented. This study’s *Corresponding author outcomes are very important as the initial ISSN: 1511-3701 e-ISSN: 2231-8542 © Universiti Putra Malaysia Press Salmah Yaakop, Angeline David-Dass, Ummi Shuhaida Shaharuddin, Suliza Sabri, Aqilah Sakinah Badrulisham and Che Mohd Zain Che-Radziah stage for conservation purposes, especially by lepidopteran species (Syarifah Zulaikha in managing the strategy in handling the et al., 2018). pest species populations in the paddy field. DNA barcoding is a novel approach used to precisely identify any species using Keywords: Agricultural ecosystem, CO1, genetic, the short and standard gene region, the grain insect pests, infestation, Lepidoptera, Malaysia cytochrome oxidase subunit 1 (CO1). This approach enables taxonomists to precisely identify species and helps stakeholders to INTRODUCTION combat pests and the invasive species so as Lepidoptera is the second-largest insect to ensure food safety and security (Hebert group comprising moths and butterflies. & Gregory, 2005). The latest findings reveal It plays a pivotal role as a food source for that identification using DNA barcoding birds and small mammals, apart from being helps to resolve problems related to species potential dispersal agents (Lee at al., 2002), diversity (Hebert et al., 2004a, 2004b). and nutrient recyclers (Mercks et al., 2013). This approach is found to be the most Lepidopteran is also very diverse in many effective technique to support and reconfirm agricultural ecosystems such as in paddy morphological identification (Halim et fields, oil palm plantations, orchards, and al., 2018; Nor Atikah et al., 2019). Moth secondary forest ecosystems. This group species are very difficult and complicated of species mostly acts as pests during their to accurately identify because of damaged larval stages such as the bagworm species, scale structures in the adult stage, as well Metisa plana (Halim et al., 2017), fruit as its very dull colouration. As leaf rollers bunch moth, Tirathaba sp. (Yaakop & Abdul and stem borers become pests during the Manaf, 2015), and other lepidopteran pests larval stage, this approach enables species (Ghazali et al., 2015). identification during their immature stages. Paddy fields are considered temporary Few researchers work on moths (Scoble, aquatic and terrestrial habitats for these 1992), hence many moths are still not species. The paddy ecosystem or area is yet identified. In addition, distributional flooded with water throughout the planting and host studies are also lacking (Janzen, season and is dried after the harvesting 1988). Several studies in Malaysia and season. Thus conditions become an ideal other Southeast Asian countries have been environment for lepidopteran species indirectly conducted on leaf folder and stem (Bahaar & Bhat, 2011). Paddy grains are borers in terms of diversity and abundance one of the most economically important and pesticide applications, i.e. by Bhatnagar products because it is a food source for (2004), Faleiro et al. (2006), and Ooi et al. human beings (Sie et al., 2008), but the (2015). However, information is scarce grains have also been recorded to be infested and limited; none of the studies focussed 524 Pertanika J. Trop. Agric. Sci. 43 (4): 523 - 535 (2020) Species Richness of Pest Moth and Its DNA Barcode on molecular work such as DNA barcoding were also collected from a total of five analysis for Malaysian species. Hence, the paddy plants that were selected randomly abundance and richness of moth pests are from each subplot. Paddy stems were later still unclear, making conservation efforts cut longitudinally to examine the presence more difficult. This study is thus carried out of larvae and pupae. to provide information on paddy-associated moth species and to measure the abundance Laboratory Work and richness of paddy field moths, as well Field specimens were brought back to the as to provide the DNA barcode of obtained Entomology Laboratory for the sorting and moth species. identification process. The adult and larval stages were preserved in 100% alcohol for METHODS molecular work. Sampling Locations This study was conducted at a conventionally Species Identification cultivated paddy field in Parit 4 Timur, The identification of species was based on Sungai Panjang, Sabak Bernam, Selangor, external morphological characters (adult Malaysia (3°42’ 763” N 101°46’317”E), as only) and conducted up to species level a model sampling site. The plot size was using a stereo microscope Zeiss Stemi DV5 approximately 10250 hectares (102.5 m x by referring to a species key by Hampson 100 m). (1896) and Khan et al. (1988, 1991). Seven (7) individual representatives (different Insect Sampling morphology) were labelled with RC, The sampling area was divided into four LFC, LFM, LFG, KC, KK, and KH then plots and then each plot was divided into identified using the molecular approach four subplots. Two-line horizontal transects or DNA barcoding (Hebert et al., 2003). of 50 m each were selected randomly in The DNA barcoding procedure involves the subplot and the adult stage sampled DNA extraction, polymerase chain reaction by using the sweep net, then larval stage (PCR), DNA purification, DNA sequencing, specimens of leaf rollers were handpicked and DNA sequence analysis. from the subplots. Sampling was carried out from November 2017 till March 2018 (1 DNA Extraction and Polymerase Chain Reaction (PCR) season) according to paddy growth stages; vegetative, reproductive and mature stages The DNA of the moth specimens was at three time-zone replicates i.e. morning extracted using appropriate protocols by (1000-1100 hr), afternoon (1200-1300 hr), QIAGEN DNeasy® Blood and Tissue Kit and evening (1400-1500hr) for the netting (Germany). Extraction procedures were methodology. A total of 20 stems/ plants conducted using the manual provided by the Pertanika J. Trop. Agric. Sci. 43 (4): 523 - 535 (2020) 525 Salmah Yaakop, Angeline David-Dass, Ummi Shuhaida Shaharuddin, Suliza Sabri, Aqilah Sakinah Badrulisham and Che Mohd Zain Che-Radziah manufacturer. An Eppendorf machine was Alignment, Basic Local Alignment used to perform polymerase chain reaction Search Tool Analysis (BLAST) and (PCR) while cytochrome oxidase subunit 1 Phylogenetic Analysis (CO1) was amplified using a set of primers, The online software, Basic Local Alignment forward: LCO1490 5’ GGT CAA CAA Search Tool (BLAST) was used to ensure ATC ATA AAG ATA TTG G 3’ reverse: that the amplified sequence is the sequence HCO22198 5’ TAA ACT TCA GGG TGA of choice. BLAST showed a maximum hit CCA AAA AAT CA 3’ (Hebert et al., 2003). for the respective species only, as available The PCR condition is as follows; initial in GenBank (http://www.ncbi.nlm.nih.gov/ denaturation at 95°C for 3 min followed by genbank). PAUP* 4.0 was implemented to denaturation at 95°C for 30 s, annealing at construct the neighbour joining (NJ) tree 52.2°C for 1 min and extension at 72°C for using Kimura’s two parameter algorithm 30 s, and the final extension at 72°C for 10 model with bootstrap analysis (1,000 min in a total of 30 cycles. For PCR reagents, replications). 25 μL consisted of 12.5 μL of 1× GoTaq® Green Master Mix, 7.5 μL of ddH2O, 1-3 Data Analysis μL of 10 ng DNA template, 1.0 μL of 200 nm forward and reverse primers. The PCR Ecological indexes such as the Shannon product was later purified using the GF-1 diversity index (H’), evenness index (E’), PCR Clean-Up kit (Vivantis) to remove and Margalef’s richness index (R’) were dNTP, primers, and buffer. The end product evaluated using paleontological statistical was then used to perform electrophoresis gel software (PAST) version 2.17c. at 90V and 30 min using agarose gel 1.5%. RESULTS Sequencing Analysis, Editing, and Overall Composition of Lepidoptera Alignment of DNA A total of 189 individuals belonging to five The purified PCR product was sent to First species under two families were collected Base Sdn.
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