Plasmodium DNA in desiccated mosquitoes was consistently amplified from all storage treatment groups despite DNA losses, in detection or yields, observed in other studies. No differences in parasite DNA detection by real-time PCR were noted between treatment groups, with the exception of one temperature group, in which the ability to amplify parasite DNA improved. Therefore, for at least six months, cold chain maintenance of specimens (the gold standard) is not necessary for preserving field-collections for subsequent small base pair target PCR-detection of Plasmodium DNA in desiccated mosquitoes. As no negative relationship was demonstrated between the experimental conditions and the amount of detectable parasite target DNA, it is likely that Plasmodium parasites will remain detectable by PCR for much longer periods, even after exposure to relatively high ambient temperatures.
Despite the logical premise that low-temperature storage of desiccated mosquitoes would best facilitate the amplification of parasite DNA (yielding the lowest Ct values), the 28°C storage group retained nearly six times as much amplifiable template compared to samples stored at -80°C. Under sub-freezing conditions, biological decomposition processes and ultimately the DNA-cleaving enzymatic activity of nucleases were expected to be inhibited. It was, therefore, surprising to see such a positive effect on DNA amplification from specimens stored at a warmer temperature. It has previously been demonstrated that high temperatures increase the susceptibility of DNA to shearing ; subsequent DNA isolation by precipitation methods may result in diminished yields as small DNA fragments are more readily lost in this process. It is possible that the methodology adopted here circumvents losses in PCR detection due to two factors: 1) a DNA carrier was used which facilitates extraction yields during precipitation; and 2) a small base pair template sequence was targeted (111 base pairs) for amplification. Even significant amounts of shearing therefore, may not have affected a small template target (commonly used for real-time PCR studies) and loss of small fragments would have been minimized. Furthermore, though there is no evidence to suggest that the PCR-targeted region is more shear or decay-resistant than other similar-length regions within Plasmodium spp. genomes, one limitation of this study is that this factor cannot be ruled out.
As the effect of temperature was not consistent across experimental groups, differences may reflect some variability in sample desiccation. It was noted that mosquitoes stored at temperatures above -80°C appeared drier and more friable at the time of dissection. If prior to storage, the desiccation process was not entirely complete, mosquitoes held at warmer temperatures may continue to desiccate, facilitating DNA preservation and perhaps extraction of DNA from tissues. Samples stored at -80°C may have experienced greater exposure to active nucleases while cooling, but before enzymes became temperature inactivated, and then again upon thawing for laboratory processing. Ice-crystal formation during the freeze-thaw cycle may also have damaged the integrity of DNA, although no definitive evidence exists to confirm this in this study. Though the -20°C and 37°C experimental groups were not statistically different, all groups stored above -80°C experienced on average less DNA degradation. This suggests that desiccation is critical to DNA preservation, and is perhaps more important than storage temperature.
The statistical analyses suggest no consistent association exists between the variables studied and the ability to detect parasites. Furthermore, little of the variation observed statistically was explained by the effects of time or temperature. Other factors that may influence parasite detection may be related to minor handling differences associated with DNA extractions, and dissections conducted at different time points. Though perhaps less significant in laboratory work, storage differences are likely magnified in field settings and in temporary lab facilities where sterile processing techniques and ease of sample manipulation are not easily attained. Therefore, though parasite DNA persisted in the lab irrespective of time and temperature treatment, the importance of proper handing techniques should not be underestimated in the field, and will likely require additional (and more rigorous) investigations.
In addition to its an application in malaria research, PCR-based assays are commonly used to detect other vector-borne parasites in arthropod hosts. Different insects, however, require different preservation techniques [15–17, 23–26]. Mosquitoes and most dipterans, for example are quite small and desiccate rapidly, but for larger insects this medium is less effective with regard to successful PCR amplification . Presumably, DNA-destructive nucleases remain active for longer periods when desiccation occurs slowly.
This research and other studies  suggest that desiccation of small dipterans, like mosquitoes, is an effective means of preserving field collections and the DNA of their endoparasites. Combined with efficient DNA extraction methods and small-target PCR detection, the results suggest that the methods described herein are appropriate for the detection of malaria parasites in field-collected, desiccated vectors. Furthermore, desiccation storage is highly amenable to field work. Drierite®, the desiccant used in this study costs just under $19 USD for 5 pounds, enough for over 6,000 sample preparations, a little less than a third of a penny per sample tube . Furthermore, it can be “refreshed” in conventional ovens and reused. It is not toxic to skin, and not combustible, making it easy to travel with, and it can be used correctly by anyone without need for detailed training. Taking costs, logistics and effectiveness into consideration, desiccants combined with detection methods such as those described above should be standard for operational vector work in malaria studies.