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Predatoryp Erformance of Coccinella Transversalis and Its Molecular Identification

Susmita Sarker
Department of Zoology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Abdul Jabber Howlader
Department of Zoology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Faria Farhana Rain
Department of Zoology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Abu Faiz Md. Aslam
Department of Zoology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Abstract/ Executive Summary:

Predatory efficacy of Coccinella transversalis (Coleoptera: Coccinellidae) on three detrimental agriculturally important aphids (Aphis craccivora, A. fabaeand A.gossypii)was studied under laboratory condition. The 4thinstar larvae of C. transversalis consumed highest (21.56±1.72) number of A. craccivora aphids followed by A. fabae (12.33±1.74) and A. gossypii (13.99±0.77). Life cycle studies of C. transversalis on the above three aphid species revealed that it took maximum (27.66 ± 3.06) days to complete life cycle while reared on A. craccivora followed by A. fabae (25.66 ± 0.58days) and A. gossypii (22.33 ± 1.52 days) respectively. As C. transversalisis a potential predator, an attempt was taken to identify this biological control agent based on mitochondrial cytochrome c oxidase (COI) gene sequence. Sequenced gene was submitted to the NCBI GenBank database (Accession NO. MG 587947.1) followed by proper procedure. Phylogenetic relationship of the beetle was constructed based on mitochondrial COIgene. The nucleotide composition analysis revealed that the value of A+T (69.3%) was greater than G+C (30.7%). Such study of C. transversalis would be helpful in biological control programme of aphid pest.

Keywords: Coccinella transversalis, Predatory efficacy, DNA barcode

Location: Department of Zoology, Jahangirnagar University.

Start Date:

End Date:

Program Area: Pest Management

Non-commodity: Performance

Objectives:

Therefore, an initiative was undertaken for the molecular identification and characterization of C. transversalis based on COIgene sequences and the sequences will be submitted to the NCBI GenBank. The overall goal is to avail tools that will contribute to timely and accurate identification of these biological control agents, which should in turn facilitate quicker and effective implementation of pest management and strengthening of the IPM systems in countries affected by the aphid species.

Methodology:

Assessment of predatory efficacy: The predatory efficacy of larval stages of Coccinella transversalis were tested on three agriculturally important aphid species i.e. Aphis craccivora, A. fabaeand A. gossypii separately under controlled conditions (26±2ºC and 65±5% RH). To maintain a regular supply of the aphid and beetle population, a garden of bean, Lablab purpureus and brinjal plants, Solanum melongena were maintained at the Insect Rearing and Experimental Station, Department of Zoology, Jahangirnagar University. To investigate the predatory efficacy of larvae of the predator against aphids, newly hatched larvae of the predator were placed singly in six Petri dishes having a Whatman filter paper. A counted number of aphids (30) were provided in each Petri dish daily. Upper collar portion of the Petri dishes was treated with vaseline and covered with muslin cloth to avoid larval escape. Everyday, old leaves were substituted with new ones and unconsumed numbers of aphids were noted. The predation potential of larvae of the predator was investigated by feeding the grubs on aphids. The number of aphids consumed per day, during the period of study was recorded in each treatment by counting the number of remaining aphids and subtracting them from the total number of aphids provided. The larvae of the predator were also checked daily for their moulting to calculate the duration of each larval instar. This practice was continued until pupation. Molecular identification DNA isolation: Coccinella transversalis was collected from the stock culture of Molecular Entomology Laboratory of Jahangirnagar University. The genomic DNA was extracted from somatic tissue rich in mitochondria, e.g., leg or elytra (Levenbook and Williams 1956) using Wizard®Genomic DNA Purification Kit, USA, following the manufacturer’s protocol with slight modification as mentioned in Aslam et al. 2019. The remaining parts of insects and respective individuals were kept as voucher specimens. Processed DNA was stored at 4?C or −20?C.DNA quantification and quality measurement:The quantity and purity of DNA was measured by using Nano drop™ 2000 spectrophotometer (Thermo Fisher Scientific, USA). 1μlof nucleic acid was used to quantify nucleic acid. 260/280 Ratio, the ratio of absorbance was used to assess the purity of DNA. A ratio of ~1.8 was generally accepted as “pure” for DNA.Polymerase chain reaction (PCR): The extracted DNA was subjected to PCR amplification through Applied Biosystems® Veriti® 96-Well Thermal Cycler following standard protocols. Primers used were forward primer: (LCO 1490 5′-GGTCAACAAATCATAAAGATATTG G-3′) and reverse primer: (HCO 2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′). The PromegaGotaq® G2 Green Master Mix (Promega Corporation) was used that contained GoTaq® G2 DNA polymerase, dNTPs, MgCl2and reaction buffers at optimal concentrations for efficient amplification of a wide range of DNA templates by PCR. The PCR reaction mixtureconsisted of total volume 20 μl among that master mix -10 μl, forward primer -1μl (10 picomolar), reverse primer -1μl (10 picomolar), template DNA (50 mg), and adjustable nuclease free water. Thermocycling consisted of an initial denaturation of 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 49°C for 30 sec, extension at 72°C for 1 min, final extension: 72⁰C for 10 min and hold: 4⁰C. Data analysis: The chromatograms were converted to FASTA format using Finch TV chromatogram viewer software. The DNA sequences in ABI file were manually edited using BioEdit v.7.0.5. Results of sequence editing were analyzed using BLAST (Basic Local Alignment Search Tool) NCBI to indicate the homology from closest species. Phylogenetic tree was constructed using maximum likelihood method, calculation using Bootstrap with 1000 times of repetition in MEGA (Molecular Evolutionary Genetic Analysis) software program v.10.0.(Tamura et al. 2013). COIgene sequence of five coccinellid beetle (Coccinella transversalis) was also collected from National Centre for Biotechnology Information (NCBI) to compare with the research sample for their phylogenetic analysis.

Source of Information: Bangladesh J. Zool. 47(2): 229-241, 2019 ISSN: 0304-9027 (print) 2408-8455 (online)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This study is the first attempt of construction of a DNA reference dataset using the mitochondrial COIgene along with molecular characterization and other related bioinformatics analysis from Bangladesh. Though there are sequences of C. transversalis in NCBI GenBank, any published paper on molecular characterization of this important biological control agent was not available. This dataset can be effectively used to identify this biological control agent. DNA barcoding can help in identifying such agents in any stage of life. Such identification would help to use this predator as a biological control agent more effectively in nature. Thus, this system would help farmers to save cost of billion dollars from pest damage (Kaur 2015, Sarvananda 2018). Moreover, our results showed the potential role of C. transversalis in bio control of three detrimental agricultural aphids. However, further field based studies are needed to confirm this hypothesis.

Knowledge Management: Journal