Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding. 2016

Lawrence E Reeves, and Chris J Holderman, and Jennifer L Gillett-Kaufman, and Akito Y Kawahara, and Phillip E Kaufman
Entomology and Nematology Department, University of Florida, PO Box 110620, 1881 Natural Area Drive, Gainesville, FL, 32611, USA. lereeves@ufl.edu.

Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program. We compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success. Preservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor. Our data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided.

UI MeSH Term Description Entries
D010209 Paper Thin sheets made from wood pulp and other fibrous substances, used for writing, drawing, printing, image duplication or wrapping. Papers
D011309 Preservation, Biological The process of protecting various samples of biological material. Biological Preservation,Preservation, Biologic,Biologic Preservation
D001769 Blood The body fluid that circulates in the vascular system (BLOOD VESSELS). Whole blood includes PLASMA and BLOOD CELLS.
D003080 Cold Temperature An absence of warmth or heat or a temperature notably below an accustomed norm. Cold,Cold Temperatures,Temperature, Cold,Temperatures, Cold
D003890 Desiccation Removal of moisture from a substance (chemical, food, tissue, etc.). Dessication
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D005247 Feeding Behavior Behavioral responses or sequences associated with eating including modes of feeding, rhythmic patterns of eating, and time intervals. Dietary Habits,Eating Behavior,Faith-based Dietary Restrictions,Feeding Patterns,Feeding-Related Behavior,Food Habits,Diet Habits,Eating Habits,Behavior, Eating,Behavior, Feeding,Behavior, Feeding-Related,Behaviors, Eating,Behaviors, Feeding,Behaviors, Feeding-Related,Diet Habit,Dietary Habit,Dietary Restriction, Faith-based,Dietary Restrictions, Faith-based,Eating Behaviors,Eating Habit,Faith based Dietary Restrictions,Faith-based Dietary Restriction,Feeding Behaviors,Feeding Pattern,Feeding Related Behavior,Feeding-Related Behaviors,Food Habit,Habit, Diet,Habit, Dietary,Habit, Eating,Habit, Food,Habits, Diet,Pattern, Feeding,Patterns, Feeding,Restrictions, Faith-based Dietary
D005374 Filtration A process of separating particulate matter from a fluid, such as air or a liquid, by passing the fluid carrier through a medium that will not pass the particulates. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Filtrations
D000072138 Mosquito Vectors Mosquitoes (members of the family CULICIDAE) that transmit pathogens or their intermediate forms from one host to another. Mosquito Vector,Vector, Mosquito,Vectors, Mosquito
D000330 Aedes A genus of mosquitoes (CULICIDAE) frequently found in tropical and subtropical regions. YELLOW FEVER and DENGUE are two of the diseases that can be transmitted by species of this genus. Aede

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