Munis Entomology & Zoology Mun. Ent. Zool. https://www.munisentzool.org/ (January, 2021) 457 ISSN 1306-3022 © MRG ___________________________________________________________ EXPLORING THE ACULEATA HYMENOPTERA OF 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. Exploring the Aculeata Hymenoptera of Bangladesh by DNA barcoding of Malaise trap collection. Munis Entomology & Zoology, 16 (1): 457-464] ABSTRACT: Aculeata hymenopterans play a crucial role in ecology and economics. Diversity analysis of 901 Aculeata wasps at Chittagong University Campus a site in Bangladesh was performed by sequencing DNA barcodes (658 bp sequence from the 5′-end of cytochromeoxidase I). Specimens were collected by a Malaise trap from April 2014 to March 2015. The results revealed 22 species and 42 genera from 20 families in three superfamilies namely Apoidea, Chrysidoidea and Vespoidea. Among them 15 species, 22 genera, two subfamilies (Dolichoderinae and Ponerinae), and one family named Dryinidae are the first county records in Bangladesh. All the specimen records, with the Barcode Index Numbers (BINs) are available on the Barcode of Life Data System (BOLD). KEY WORDS: Aculeata Hymenoptera, Malaise trap, DNA barcode, Bangladesh Aculeata hymenopterans or stinging wasps (Hymenoptera: Aculeata) such as bees, ants, and many wasps play vital ecological roles as predators and pollinators and some have medicinal value as well (Zimmermann & Vilhelmsen, 2016). In addition, aculeate hymenopterans assist in biodiversity conservation and the management of forest reserves (Ribeiro et al., 2019). Reliance on only morphological identification for biological classification has posed many challenges for taxonomists (Ojha et al., 2014). DNA barcoding technique has emerged as a trustworthy way for rapid and accurate species identification and biological diversity assessment (Hebert et al., 2003; deWaard et al., 2019). A partial fragment of cytochrome c oxidase I gene (COI) of Mitochondrial DNA (mtDNA) is used in DNA barcoding (Hebert et al., 2004; Mazumdar et al., 2015, 2019). Malaise trap is an effective insect collection apparatus commonly used for monitoring ecosystem compositions particularly in arthropod biodiversity assessment (Morinière et al., 2016; Ashfaq et al., 2018). Several studies have been carried out on Aculeata in Bangladesh by Bhuiya & Miah (1990), Gapud (1992), Hannan (2007 & 2008), Ahmed (2009), Mazumdar et al. (2010), Mazumdar et al. (2014), and they authors provided useful information on the aculeate hymenopterans fauna of Bangladesh. However, prior studies have mainly used morphological identification. The current study aimed to explore the aculeate hymenopterans through Malaise trap, and confirmed by coupling DNA Munis Entomology & Zoology Mun. Ent. Zool. 458 https://www.munisentzool.org/ (January, 2021) ISSN 1306-3022 © MRG ___________________________________________________________ barcoding and morphological appearances. The data generated in this study will provide an important reference for further work on stinging wasps in Bangladesh. MATERIALS AND METHODS Specimen sampling, processing, identification and specimens’ deposition By following the Standard Operating Protocol for the Global Malaise Trap Program (www.dnabarcoding.ca.), a Townes-style Malaise trap (BioQuip Inc. USA) was deployed at the Chittagong University Campus (CUC) (Lat. 22.46359°N; Long. 91.7808°E) in Bangladesh. In short, CUC consists of about 1753.88 acres of land, of which about 72% land consists of hills and hillocks ranging from 10-90m, and remaining are either plains or valleys. Climate of Campus is typically tropical monsoon, characterized by hot humid summer and dry chilled water. Region has a mean annual rainfall of about 275 cm. Temperature varies from 46°F in January to 95°F in May. Ecologically, CUC falls under tropical evergreen forest. Plains and valleys are now under cultivation of rice and vegetables. About 10% of land is now occupied by roads, building, inhabitants and gardens (CU, 2016; Shuvo, 2015; WOCU, 2018). Insect specimens were collected during March 2014 to February 2015. The samples were collected 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/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 DNA was extracted from a single leg of larger specimen and from the whole body of smaller taxa. 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 the target gene region Cytochrome Oxidase 1 (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).The amplicons were sequenced using BigDye v3.1 (Applied Biosystems) on an ABI 3730XL sequencer. Sequences were assembled, aligned, and edited using CodonCode Aligner (CodonCode Corporation, USA) and submitted to the Barcode of Life Data Systems (BOLD) (www.boldsystems.org). RESULT AND DISCUSSION The present study barcoded 901 specimens of sting wasps that represented three superfamilies namely Apoidea, Chrysidoidea and Vespoidea. A total number Munis Entomology & Zoology Mun. Ent. Zool. https://www.munisentzool.org/ (January, 2021) 459 ISSN 1306-3022 © MRG ___________________________________________________________ of 872 of the 901 specimens were assigned to 258 BINs. DNA barcodes of 240 specimens of 10 families in the superfamily Apoidea were assigned to 97 BINs. In the superfamily Chrysidoidea, with 3 families, DNA barcoded 126 specimens and the sequences were assigned to 56. Finally, 525 specimens of the superfamily Vespoidea included 6 families, and were assigned to 105 BINs (Table 1). Table 2 shows the list of the confirmed species of aculeate hymenopterans in the present study. Table 1. Number of DNA barcodes and BINs recovered for aculeate Hymenoptera collected by a Malaise trap at Chittagong University Campus. A total of 22 species and 42 genera were confirmed through DNA barcoding which belonged to three superfamilies viz. Apoidea, Chrysidoidea and Vespoidea. Superfamily Apoidea consists of 10 families viz. Ampulicidae (no genus/ no species identified), Apidae (4 genera/2 species), Bembicidae (1 genus/ 1 species), Colletidae (1 genus/ no species identified), Crabronidae (9 genera/3 species), Halictidae (5 genera/2 species), Megachilidae (3 genera/no species identified), Pemphredonidae (1 genus/ no species identified), Philanthidae (2genera/ 1species), Psenidae (no genus/ no species identified) and Sphecidae (1 genus/1species). Superfamily Chrysidoidea consists of Bethylidae (no genus/ no species identified), Chrysididae (1 genus/ no species identified) and Dryinidae (no genus/ no species identified). Identified specimens of the superfamily Vespoidea belonged to the family Formicidae (13 genus/ 11 species), Mutillidae (no genus/no species identified), Pompilidae (no genus/ no species identified), Scoliidae (no genus/ no species identified), Tiphiidae (no genus/ no species identified) and Vespidae (2 genera/2 species). Munis Entomology & Zoology Mun. Ent. Zool. 460 https://www.munisentzool.org/ (January, 2021) ISSN 1306-3022 © MRG ___________________________________________________________ Table 2. List of DNA barcoded Aculeata Hymenoptera collected at the Chittagong University. Munis Entomology & Zoology Mun. Ent. Zool. https://www.munisentzool.org/ (January, 2021) 461 ISSN 1306-3022 © MRG ___________________________________________________________ Psenidae New Vespinae Vespa tropica (L., 1758) Aktar et al., 2020 Munis Entomology & Zoology Mun. Ent. Zool. 462 https://www.munisentzool.org/ (January, 2021) ISSN 1306-3022 © MRG ___________________________________________________________ Bhuiya & Miah (1990) have previously published a report on 18 bees from Chittagong, Bangladesh which included four apidid, seven xylocopid, four megachillid, two anthophorid, and one halictid bees. Tadauchi & Alam (1993) surveyed bees, and have recorded one megachilid, one andrenid, two anthophorid, and three halictid bee species from 33 mustard fields in Bangladesh. The prospects of beekeeping for increased production of honey from the Sundarban