Location: Vegetable ResearchTitle: Method for DNA isolation from sweetpotato weevil collected in pheromone-baited traps Author
Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/19/2018
Publication Date: 12/26/2018
Citation: Wadl, P.A., Williams III, L.H., Harris-Shultz, K.R., McQuate, G.T. 2019. Method for DNA isolation from sweetpotato weevil collected in pheromone-baited traps. Journal of Economic Entomology. 112:1001-1003. https://doi.org/10.1093/jee/toy390.
DOI: https://doi.org/10.1093/jee/toy390 Interpretive Summary: Molecular techniques developed over the past two decades have revolutionized studies on insects. Logistics of sample collection is a limiting factor for studies of natural populations, particularly for studies where preservation and recovery of nucleic acids (DNA or RNA) is critical. This can pose challenges when dealing with insects because they are often small, cryptic, and difficult to capture. In addition, live material is often unavailable, or they may be serious agricultural pests that have been quarantined with restricted movement of living specimens. Genomic DNA can be isolated readily from samples frozen and stored at -80°C, preserved in ethanol, or dried in silica gel. However, these preservation methods may not be practical when insects are collected under field conditions. The sweetpotato weevil is distributed throughout tropical and subtropical regions of the world and is a serious pest of sweetpotato in the field and in storage facilities. The first report of sweetpotato weevil in the United States was near New Orleans in 1875 from insects presumed to have come from the West Indies. Now the sweetpotato weevil is found throughout the coastal plain of the southeastern United States. There is a lack of information regarding the genetic diversity of sweetpotato weevil in the U.S. An important first step toward assessing this diversity is the development of robust methods for isolation of genomic DNA from trapped specimens. During ongoing research by USDA scientists at Charleston, South Carolina working with sweetpotato weevil, it was discovered that it is difficult to isolate non-degraded genomic DNA from specimens collected biweekly. We discovered that under summer conditions typical of sweetpotato production, it is critical to collect specimens from traps at an optimum interval of one week. Furthermore, our results indicate that either 70% ethanol or storage at -20°C may be used to preserve samples that are no older than 7 days, depending on the resource limitations of the particular research project, without loss in the quality of genetic material. Our findings allow for scheduling of sampling at reasonable times without the need for any special materials. This has the added benefit of allowing individuals without special training to collect sweetpotato weevil specimens for genetic studies.
Technical Abstract: This study provides a protocol for isolation of high quality DNA from sweetpotato weevils [Cylas formicarius elegantulus (Summers)] collected from pheromone-baited aerial funnel traps. This study was based on our discovery that a two week collection interval of sweetpotato weevils from pheromone traps did not permit isolation of intact high quality genomic DNA. To test the effect of collection methods, i.e., sample collection interval and preservation method, on quality of isolated DNA, we placed freshly-killed male sweetpotato weevils into aerial funnel traps in the field and removed subsamples at several times thereafter. DNA yield from freshly isolated (day=0) samples was significantly greater than samples preserved in 70% ethanol or at -20°C, whereas there was no difference between 70% ethanol and -20°C storage. Likewise, DNA yield from freshly isolated (day=0) samples was significantly greater than for later sampling times. Quality assessment of genomic DNA through gel electrophoresis and PCR indicated isolation of high molecular weight DNA for all samples collected at t = 7d, but that DNA quality was degraded by 14d. Our goal was to develop a reliable method for isolation of genomic DNA from field-collected sweetpotato weevil suitable for direct use in PCR. We discovered that it is critical to collect specimens from traps at an interval of one week or less. Our findings allow for scheduling of sampling at reasonable intervals without the need for special materials. This has the added benefit of allowing individuals without special training to collect and prepare sweetpotato weevil specimens for genetic studies.