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Scope: Munis Entomology & Zoology Publishes a Wide Variety of Papers Munis Entomology & Zoology Mun. Ent. Zool. 296 https://www.munisentzool.org/ (January, 2021) ISSN 1306-3022 © MRG ___________________________________________________________ INVESTIGATION OF LEPIDOPTERANS IN BANGLADESH BY DNA BARCODING OF MALAISE TRAP COLLECTION Santosh Mazumdar*, Paul D. N. Hebert** and Badrul Amin Bhuiya*** * Department of Zoology, University of Chittagong, BANGLADESH. E-mail: [email protected], ORCID ID: 0000-0001-6403-577X ** Centre for Biodiversity Genomics, University of Guelph, 50 Stone Road East, Guelph, Ontario, CANADA. ORCID ID: 0000-0002-3081-6700 *** Biodiversity Research for Environment & Ecosystem Protection (BREEP), Chattogram- 4325, BANGLADESH. [Mazumdar, S., Hebert, P. D. N. & Bhuiya, B. A. 2021. Investigation of Lepidopterans in Bangladesh by DNA barcoding of malaise trap collection. Munis Entomology & Zoology, 16 (1): 296-311] ABSTRACT: Lepidoptera form an essential part and one of the dominant groups of insects of natural terrestrial ecosystem, and they are widely distributed ranging from desert to rainforest, from lowland grasslands to mountain plateaus. The main aim of the present study is to identify the lepidopterans’ diversity, at a site in Bangladesh through DNA barcoding technique (658 bp sequence from the 5′-end of cytochromeoxidase I). Field collections were carried out by a Malaise trap at Chittagong university campus between April 2014 and March 2015. Including first country records of 52 species, 76 genera, 23 subfamilies and 19 families, a total of 87 lepidopteran species, 115 genera, 61 subfamilies and 38 families were confirmed. Most new records belong to moth group and they are associated with agricultural fields. All the specimen records, with the Barcode Index Numbers (BINs) (the species proxies), are available on the Barcode of Life Data System (BOLD). KEY WORDS: Lepidoptera, malaise trap, DNA barcode, Bangladesh Lepidoptera comprise the second most diverse insect order (Paniagua Voirol et al., 2018) with about 180,000 recognized species in 126 families (Ren et al., 2019). They are concentrated with human society through culture, agriculture, and natural resource conservation (Goldstein, 2017). Also, they are important bioindicators for monitoring changes in terrestrial habitat, biodiversity and environmental conditions (Munyuli, 2012; Bashar, 2015). Likewise, they represent one of the greatest radiations of herbivorous animals on the world (AUSDA, 2012). Previous studies on Order Lepidoptera were highlighted especially on morphological classification but since last decad DNA barcoding technique has been widely used in the phylogenetic and taxonomic studies of lepidopterans (Jiang, 2017). To confirm species delimitation for taxonomic, ecological and evolutionary studies, DNA barcoding technique was suggested by Hebert et al. (2003), and this modern taxonomic system has become a global research effort for identifying unknown specimens to species-level (Pentinsaari et al., 2019; Mazumdar et al., 2019). Though morphological studies may have been hampered from Malaise trap samples preserved in ethanol, extensive molecular research on lepidopterans has proven successful (Schmidt et al., 2019). Generally, micromoths can be challenging to identify based on morphlology so it is an Munis Entomology & Zoology Mun. Ent. Zool. https://www.munisentzool.org/ (January, 2021) 297 ISSN 1306-3022 © MRG ___________________________________________________________ effective technique to confirm suburban micromoth diversity using DNA barcoding of malaise trap samples (Aagaard et al., 2017). Based on morphological characters many new records of lepidopterons have been published every year from Bangladesh. Different categorical works have been done on Bangladeshi lepidopteran fauna such as Rahman et al. (1983) worked on lepidopteron pest diversity on soybean. Baksha (1990) reported lepidopteran forest pests. Gapud (1992) reviewed published articles on agricultural lepidopterons. Larsen (2004), Chowdhury & Hossain (2011) and Bashar (2015) studied on butterflies and skippers. In 2015, IUCN Bangladesh evaluated the IUCN red list status of Bangladeshi butterflies. In the line of university campus, restricted areas, the butterflies of the Rajshahi University campus have been identified by Mahdi et al. (2013). Akter et al. (2015) published a survey report on spatial and temporal dimensions of butterfly species diversity in Jahangirnagar University campus. Al Haidar et al. (2017) reported a field observation particular on Status, abundance and habitat preference of butterflies at Chittagong University Campus (CUC), Chittagong. Islam et al. (2013b) studied the pattern of butterfly abundance, their diversity with abiotic and biotic factors in the Butterfly Research Park at Bhawal National Park, Gazipur. Feeroz (2014) assessed lepidopteran biodiversity of Chunati Wildlife Sanctuary. Afterwards, Feeroz (2016) evaluated in the area of Inani Protected Forest. In 2016, Feeroz et al., worked on Village Common Forest lepidopterans. Shihan (2016) provided a photographic guide to the Butterflies of Bangladesh. Also, Islam et al. (2013a) reported moth’s fauna in the campus of Atomic Energy Research Establishment (AERE), Savar, Dhaka. In molecular taxonomy, very few attempts were made to produce DNA barcode data of lepidopterons like Ghosh et al. (2019) applied Cytochrome c oxidase subunit I gene for identification and phylogenetic relationships of seven Satyrinae butterflies collected by sweep net. Therefore, the present work was carried out to reveal lepidopteran diversity by applying DNA barcoding technique of Malaise trap collections as a first report from Bangladesh. The results of the present study will provide a significant reference for further work on lepidopteran’ fauna particularly in pest selection, and in effective conservation management of beneficial lelidopterans in Bangladesh. MATERIAL AND METHODS Specimen Collection, Processing, Identification and Specimen Deposition Collections were made by a Townes-style Malaise trap (BioQuip Inc. USA) installed in perceived flight paths at Chittagong University Campus (Lat. 22.46359°N; Long. 91.7808°E) in Bangladesh by following the Standard Operating Protocol for the Global Malaise Trap Program (www.dnabarcoding.ca.). Between March 2014 and February 2015, the samples were harvested weekly in a 500 mL plastic Nalgene bottle that was filled with 375 mL of 95% ethanol and placed in 500 mL of fresh ethanol before storage at -20°C until analysis. Collected insects were analyzed, following standard barcoding protocols (http://ccdb.ca/resources.php), at the Canadian Centre for DNA Barcoding within the Centre for Biodiversity Genomics, University of Guelph, Canada. Collection data, voucher information and taxonomy for each specimen are available in the Barcode of Life Data Systems (http://v3.boldsystems.org/ Munis Entomology & Zoology Mun. Ent. Zool. 298 https://www.munisentzool.org/ (January, 2021) ISSN 1306-3022 © MRG ___________________________________________________________ index.php/TaxBrowser_Taxonpage ?taxid=125). All the specimens analyzed in this study have been curated at the Centre for Biodiversity Genomics, University of Guelph, Guelph, Ontario, Canada. Molecular Analysis and Data Analysis A small portion of each specimen (usually 1-3 legs) was removed and used for DNA extraction and from the whole body of smaller taxa, and vouchers were recovered after DNA extraction for imaging and curation. Tissue lysis, DNA extraction, PCR amplification, cycle sequencing and sequence analysis were performed at the Canadian Centre for DNA Barcoding following the standard protocols (CCDB). PCR amplification of COI-5′ was performed with primers C_LepFolF and C_LepFolR (http://www.ccdb.ca/docs/CCDB_PrimerSets). following PCR conditions; 94°C (1 min), 5 cycles at 94°C (40 s), 45°C (40 s), 72°C (1 min); 35 cycles at 94°C (40 s), 51°C (40 s), 72°C (1 min) and a final extension at 72°C (5 min) and amplicons were sequenced using BigDye v3.1 (Applied Biosystems) on an ABI 3730XL. Sequences were assembled, aligned, and edited using CodonCode Aligner (CodonCode Corporation, USA) and submitted to Barcode of Life Data Systems (BOLD) (www.boldsystems.org). RESULT AND DISCUSSION During the study period a total of 1163 sequences of specimens were analyzed, and confirmed 87 species and 115 genera in 61 sub-families under 38 families. Of these, 52 species, 76 genera, 23 subfamilies and 19 families are newly recorded from the country. In the present study, Crambidae (subfamilies: 6, genera: 20, species: 16), Noctuidae (subfamilies: 6, genera: 13, species: 14) and Lycaenidae (subfamilies: 3, genera: 8, species: 8) are ranked first, second and third in position respectively for their all over diversification (Table 1). In eighteen families, no species were ensured by using DNA barcoding technique. In Bangladesh, by following morphological characters Shahjahan (1974) identified Sitotroga cerealella (Gelechiidae) and mentioned its damaging habit to unhusked stored rice. Baksha (1990) reported Tonica niviferana (Depressariidae) and Hypsipyla robusta (Pyralidae) as lepidopteran forest insect pest. EPPO (2011) noted Diaphania indica (Carambidae) as a pest of Momordica sp. Islam et al. (2013a) reported a total of 153 moth species belonging to 113 genera, 25 sub- families under 14 families were recorded. Including specis Thereta clotho clotho, Thereta pinastrina pinastrina, Rhyncholaba acteus (Sphingidae), Zaranga permagna (Notodontidae) and Meganola pseudohypena (Nolidae), the
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