Molecular identification of Hymenopteran insects collected by using Malaise traps from Hazarganji Chiltan National Park Quetta, Pakistan

In the order Hymenoptera, DNA barcoding evaluations have reported minor variation at the species level and demonstrated its ability to accurately identify different species. This study serves to bridge a current knowledge gap by performing the first-ever molecular identification of Hymenopteran insects that have been reported from the protected area (HCNP) of Balochistan. This process has the potential to assist taxonomists to accurately classifying these Hymenoptera insects.

The present study was carried out in Hazarganji-Chiltan National Park (HCNP), located 20 kilometers Southwest of Quetta city, in the Balochistan province, Pakistan. This national park is renowned for its diverse fauna and flora that has been officially designated as the 25th national park in Pakistan. Malaise traps made up of mash material were used for the collection of insects. These traps were provided by the International Barcode of Life (IBOL) and designed especially for the capturing of Hymenopteran insects. The geographic coordinates of each collection site were recorded using a global positioning system and data obtained were processed in Microsoft Excel 2013 (Microsoft 365) to create a study map using ArcGIS v 10.3.1. Five significant locations within the study area were selected for the placement of Malaise traps to collect insect specimens. These locations include the sub-campus of Balochistan University of Information Technology and Management Sciences (BUITM), Hazarganji Nullah, Kangari, Wadd and Garak. Insects were collected using Malaise traps from April 2019 to November 2019 (a total of eight months of sampling). Specimen collection was performed on weekends (i.e., Saturday and Sunday). The dates of collection were marked on bags and these specimens were brought to the Entomology Laboratory at the University of Balochistan, Quetta for further molecular analyses. The collected specimens underwent a cleaning process using 70% ethanol and rinsed with distilled water to remove any external impurities. A stereomicroscope (Olympus SZ61) was used to identify the specimens by observing diagnostic features such as color pattern, wing venation, body shape, antennae and head. These characters were examined using established reference materials including standard published keys, catalogs and electronic keys. In the present study, we follow the family and subfamily classifications with additional resolution from the published articles. The extracted genomic DNA from each morphologically identified specimen was used for conventional PCR to amplify the universal genetic marker, partial fragments of cytochrome c oxidase subunit 1 (cox1) gene. The PCR cycling conditions were as follows: initial denaturation at 98 °C for 30s, followed by 40 cycles of denaturation at 98 °C for 10s, annealing at 63 °C for 20s, elongation at 72 °C for 25s and a final extension at 72 °C for 5 minutes. The obtained amplified products were run