Molecular Identification and Sequence Analysis of Dusky Cotton Bug, Oxycarenus hyalinipennis (Hemiptera:Lygaiedae) Infesting Cotton Field in Pakistan

Hand picking of dusky cotton bug (DCB) was done during 2017-2018 in cotton at Post Graduate Agriculture Research Centre, Faisalabad, Department of Horticulture, University of Agriculture Faisalabad as well as AARI Faisalabad and CCRI Multan. Adult specimens of were collected from infested opened bolls of cotton. Before proceeding molecular identification, morphological study of the species was carried out under laboratory using light microscope. Some of the collected specimens were preserved in 96% alcohol or either stored at −20 °C in a freezer or prior to analysis.

For molecular study, total genomic DNA from whole body of insect or parts of adult body (legs) of DCB was extracted using CTAB method with slight modifications to increase DNA yield (Ahmad et al., 2017; 2018). Mitochondrial cytochrome oxidase I (mtCOI) based primers were used for PCR. PCR amplifications were performed in PCR machine (Peqstar, Germany) for primers (LCO-1490 (5´-GGTCAACAAATCATAAAGATATTGG-3´) and H C O - 2 1 9 8 ( 5 ´ -TAAACTTCAGGGTGACCAAAAAATCA-3´) conditions as described by Folmer et al. (1994). Simply, an initial denaturation at 95 °C for 5 min, 40 cycles at 94 °C for 40 sec, 47 °C for 40 sec, extension at 72 °C for 45 min and final extension at 72 °C for 15 min. The amplified PCR products were tested for the confirmation of genomic DNA presence using gel electrophoresis on 2.0% agarose gel. The required size of DNA fragment from fruit fly samples was estimated by comparing with 1 kb DNA ladder markers (GeneMark). The amplified band corresponding to the target PCR product was documented using SYNGENE Gel documentation system under UV light. The amplified PCR products (710 bp) were sequenced directly in both directions using services of M/S Macrogen (Korea) with primers (LCO-1490/HCO-2198) after purification with a QIAquick PCR Purification Kit (QIAGEN, Valencia, CA). The obtained sequences were analyzed using Lasergene v. 7.1 software package (DNASTAR, USA) and were further aligned using CLUSTAL W method of Bio-Edit software. Comparison of obtained sequences with sequences available in GenBank was accomplished using BLAST (Basic Local Alignment Search Tool)service available at http://www.ncbi.nlm.nih.gov:80/BLAST. Pairwise alignment tree for our samples sequence query (dendrogram) was constructed with NCBI data base available. The studies were performed with software BLASTN 2.8.0+ employing a methodology for pairwise alignment for the construction of phylogenetic tree. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).

The nucleotide sequencing of DNA fragments were analyzed and aligned through BLASTN with sequence data of DCB previously reported in NCBI site (Fig. 1). The reference accession numbers of sequences used for alignment were DCB (HQ908084.1, JQ342988.1), (KM022344.1, KM023038.1, KJ541660.1, HQ105989, KM021657.1, KR918399.1 and KX053389.1. In base pair sequence alignment, homology of DCB spp. was compared with previously reported NCBI database through BLAST option. Pairwise alignment of dendrogram tree indicated that our dusky cotton bug Seq (>180220-034-O01-8-DCB-HCO-2198.ab1) or Seq (>180220-034-O01-8-DCB-LCO-1490.ab1) share same cluster with Oxycarenus

hyalinipennis OH-1 (JQ342987.1), Oxycarenus hyalinipennis OH2 (JQ342988.1) isolates and Oxycarenus laetus (HQ908084.1) while other species (Oxycarenus lavaterae and Oxycarenus modestus, Oxycarenus pallens) of same genus have separate clusters (Figs. 2, 3). In homology sequence study, DCB showed 100% similarity with themselves and 100% and 99% similarity with NCBI GenBank database, Oxycarenus hyalinipennis OH-1 (JQ342987.1), Oxycarenus hyalinipennis OH2 (JQ342988.1), respectively with 97-99 % good query coverage while 99 % similarity was observed for Oxycarenus laetus (HQ908084.1). The pairwise alignment also showed 87 to 94 % homology with other species of same genus (Accession numbers: KM022344.1, KM023038.1, KJ541660.1, HQ105989, KM021657.1) and 86% similarity for other Hemiprerans (KR918399.1, KX053389.1) (Table I).

The pairwise alignment of nucleotides indicated a very high and significant match confirming our sequence to be a part of Cytochrome C Oxidase subunit1 family with good query coverage (Fig. 1). The amplified sequenced segment of COI was closely related to insect species from the Order Hemiptera by using BLAST search (Figs. 2, 3). The pairwise phylolenetic tree (Fig. 2) indicated that all the species belonging to Oxycarenus being clustered together with high query coverage score 97-99. As we expected, our entry Oxycarenus hyalinipennis (605bp) was found to be clustered with the same species of members of the genus Oxycarenus showing 99-100% sequence similarity with

Table I.- Comparison and identity of fragment of the cytochrome oxidase I (COI) gene of Oxycarenus hyalinipennis of major cotton growing districts of Punjab with that of referred gene bank available in NCBI.

Oxycarenus hyalinipennis reported from USA (Nagoshi et al., 2012). Oxycarenus laetus has been reported and identified from India using (COI) based primers showing 99% similarity (Habeeb and Sanjayan, 2011). Currently morphological based identification methods for DCB complex are time consuming and technically difficult for closely related species. Further, for most of the adult and immature stages, diagnostic morphological characters are not completely suitable (White and Elson-Harris, 1992).

Our sequence will serve as an exclusive DNA barcode for this species. This is probably the first report of molecular identification of this insect from Pakistan.

Authors are thankful to HEC (Pakistan) and FICP (Norway) for providing funds for this work.

Authors have declared no conflict of interest.