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Title: A Protocol to Preserve the Integrity of Stable Fly (Diptera: Muscidae) DNA for Long Distance Shipment

Author
item KNEELAND, K - University Of Nebraska
item Skoda, Steven
item CLAASSEN, ELIZABETH - University Of Nebraska
item FOSTER, JOHN - University Of Nebraska

Submitted to: PAKISTAN ENTOMOLOGIST
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/25/2015
Publication Date: 8/18/2015
Citation: Kneeland, K.M., Skoda, S.R., Claassen, E., Foster, J.E. 2015. A Protocol to preserve the integrity of stable fly (Diptera: Muscidae) DNA for Long distance shipment. PAKISTAN ENTOMOLOGIST. 37(1):1-4.

Interpretive Summary: Stable flies are an important pest of livestock in the US and worldwide. Because of this and their potential for developing resistance to control tactics there is increased interest in developing better knowledge of stable fly genetics. Following the protocol presented herein prevents the deterioration of preserved stable flies collected and shipped from distant locations while adhering to rules and regulations of the US Postal Service. Separate samples of adult stable flies were first soaked in 95% ethanol for 6, 12, 24 or 48 hours. Alcohol was then poured off, the samples were held for 2, 4, 6 and 8 days to simulate transit time, and then fresh replacement ethanol was added. DNA was then extracted from subsamples of the differently treated (hours of soaking and days held) stable flies. Stable flies soaked in ethanol for 6 hours yielded high quality and quantity DNA after 2 and 4 days but quantity of DNA declined abruptly when held without ethanol for 6 and 8 days. Samples soaked in ethanol for 24 hours consistently yielded high quality and quantity of DNA for all days held. When stable flies shipped for molecular genetic analyses will be in transit for less than 4 days soaking the samples in ethanol for 6 hours is adequate. But when transit times are expected to be greater than 4 days and up to 8 days the stable fly samples should be soaked in ethanol for 24 hours to ensure high quality and quantity of DNA for molecular genetic analyses. This protocol may also be applicable for shipping other species of insects from distant sample locations for use in molecular genetic analyses.

Technical Abstract: Population genetic studies on a global scale may be hampered by the ability to acquire quality samples from distant countries. Preservation methods must be adequate to prevent the samples from decay during shipping, so an adequate quantity of quality DNA can be extracted for analysis, and materials used must not be prohibited by the Postal Service. We tested the efficacy of a shipping protocol for stable flies which preserves the samples and adheres to Postal regulations, using a time-point experiment. Samples were soaked in 95% ethanol for 6, 12, 24 and 48 hours; the ethanol was then poured off. Samples remained without ethanol for 2, 4, 6 and 8 days to simulate time in transit; ethanol was replaced at the end of each time point. Soaking for 6 hours produced the most DNA when samples were only in transit for 2 or 4 days, but the quantity of DNA declined abruptly after 4 days. Soaking for 24 hours was more effective than soaking for 12 or 48 hours, and was the best protocol for 6 and 8 days in transit. These results show that samples collected from distant locations should be imbibed in 95% ethanol for 24 hours before shipping, but within the United States or other short distances samples imbibed for only 6 hours would produce a large quantity of quality DNA.