Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Molecular Identification of Parasitoid, Encarsia Formosa Gahan in Bemisia Tabaci (gennadius) and Determination of Its Secondary Endosymbionts

JAHAN, S. M. H
Associate Professor
Department of Entomology, Patuakhali Science and Technology University, Dumki, Patuakhali

LEE, K-Y
Department of Agricultural Biology, Kyungpook National University, Daegu 702-701, Korea

HOWLADER, M. I. A
Senior Scientific Officer
On-Farm Research Division, Bangladesh Agricultural Research Institute (BARI), Patuakhali

BASHAR H. M
Scientific Officer
On-Farm Research Division, BARI, Patuakhali

HASAN G. N
Scientific Officer, On-Farm Research Division, BARI, Patuakhali

Abstract/ Executive Summary:

In this study two pairs of primers based on mitochondrial cytochrome oxidase subunit (mtCOI) region and 28S ribosomal RNA (rRNA) gene region were used for identifying very tiny and morphologically indistinguishable parasitoid Encarsia formosa (Gahan) which are specific to this insect. The fragment amplified by these primer pairs were 860 and 650 bp in length. Species specificity test showed that all E. formosa specimens were detected with no cross reactions with other aphelinid species, including E. sophia (Girault & Dodd), E. luteola, E. Inaron and E. nigricephala. Using phylogenetic cladogram by the sequences analysis of both mtCOI and 28S rRNA genes could be detected in E. formosa accurately in all replicates. Cardinium and Wolbachia secondary endosymbiont were also detected in E. Formosa used by PCR amplification as well as sequence analysis of 16S-23S rDNA gene region. The molecular technique developed here would be useful for rapid and precise species identification, determination of the host spectrum and more effective utilization of E. formosa. This research work has been performed from January 2011 to June 2012 at the insect molecular physiology lab in the Republic of Korea.

Keywords: Molecular identification, Encarsia formosa, Secondary endosymbiont, Bemisia tabaci.

Location: Greenhouses of Kyungpook National University, Korea

Start Date:

End Date:

Program Area: Pest Management

Commodity:

Objectives:

The morphological characters have been reviewed that can be used to distinguish E. formosa and then to characterize the amount of genetic variation within and between the species, and to present a molecular assay that rapidly distinguishes E. formosa.

Methodology:

Adult individuals of E. formosa and parasitized nymphs of B. tabaci were collected from tomato and cucumber host plants in greenhouses of Kyungpook National University, Daegu, Andong, Weiseong and Sangju in Korea. The adult E. formosa and it’s immature stages in the nymphs of B. tabaci were collected with tomato and cucumber leaves in 2012, immediately preserved a part of samples in 99% ethanol after collection, and stored them at -20⁰C for further molecular analysis. A rest of immature stages as well as adults with cucumber and tomato leaf were kept on wet cotton for observing its lifecycle under the microscope Total genomic DNA was extracted from individual E. formosa according to Dellaporta et al. (1983) and Palmer et al. (1998) using Invitrogen Purelink Genomic DNA mini kit for further use. After removing the sample from ethanol had been washed with double-distilled water to remove alcohol, the extracted DNA template were directly used for PCR amplification or were kept at -20°C for later use (Jahan et al., 2011). E. formosa and its’ harbored secondary endosymbionts were determined using the genomic DNA which was extracted from collected parasitoid of whitefly from different places in Korea with the primers listed in Tables 1 and 2, using Polymerase Chain Reaction (PCR). All PCR reaction mixture performed in 20 μl volume that included 1 μl of each primer (Forward and Reverse), 1 μl of DNA template and 17 μl smart buffer which were supplied by the manufacturer(Smart taq pre-mix). All PCR reactions were carried out on the PTC-200 DNA engine thermal cycler (MJ Research PTC-200 DNA Engine Thermal Cycler PCR). Sequences of the PCR products were determined using a Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) and analyzed using a 3730XL DNA Sequencer (Applied Biosystems, Foster City, USA). Databases were searched using the BLAST algorithm (Altschul et al., 1997; Schäffer et al., 2001) in NCBI, and sequences were aligned using the MUSCLE program (Edgar, 2004). Mitochondrial COI and 28S ribosomal RNA sequences of E. formosa were analyzed using Bayesian MrBayes 3.0 software. Four Metropolises-coupled Markov Chain Monte Carlo (MCMC) chains were run until standard divergence of the split frequencies become lower than 0.01 (Ronquist and Huelsenbeck, 2003). All sequences were analyzed over 10 million generations, and four sequences were sampled every 100 generations. The first 25% of burn-in (SUMP and SUMT) cycles were discarded prior to the construction of consensus tree, which were visualized by MEGA 4.0 (Tamura et al., 2007).

Source of Information: Bangladesh J. Agril. Res. 39(4): 563-578, December 2014 ISSN 0258-7122

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The molecular techniques used in this study could easily be identified from the test groups of individuals for potential cross contamination in insectary and other colonies. This application may prove useful in quality control programs of large rearing facilities; particularly those in which E. formosa are being produced for biological control worldwide. In this study, we used two pairs of primers based on mtCOI region and 28S rRNA gene region for identifying E. formosa which are specific to E. formosa.

Knowledge Management: Journal