WNV Diversity is Maintained in Mosquitoes Figure 5. Bayesian trees with the most parsimonious reconstruction of tissue character states for three mosquitoes. Blue = input (i), pink = midgut (m), gold = legs (l), black = salivary secretions (s), dotted = multiple tissues, with specific tissues indicated by abbreviations. Hatched branches indicate equivocal reconstruction of character states. Numbers above nodes are the posterior probabilities inferred for each clade. A) Hypothetical tree with predicted outcome assuming the presence of genetic bottlenecks. The most parsimonious reconstruction of ordered character steps for each tree for three representative mosquitoes is as follows, B) 7 dpi mosquito 2 (22 steps, p,0.0001), C) 14 dpi mosquito 1 (30 steps, p,0.0001), D) 21 dpi mosquito 3 (23 steps, p = 0.0001). doi:10.1371/journal.pone.0024466.g005 whether arboviruses experience genetic bottlenecks during infec- tion of the mosquito vector. Therefore we tracked the composition of WNV populations during mosquito infection to quantify genetic bottlenecks associated with infection of these hosts. Cx. p. quinquefasciatus mosquitoes were exposed to a bloodmeal containing a highly fit, genetically diverse WNV population (M24) that has been described in detail elsewhere [19]. Most mosquitoes exposed to M24 had WNV RNA in midgut tissues after either 7, 14 or 21 days EI. Although fewer mosquitoes had WNV in hemolymph and salivary secretions, at all timepoints at least three individual mosquitoes had WNV in midgut, hemolymph and salivary secretions. WNV from these mosquitoes was used to assess population bottlenecks associated with mosquito transmission. Extreme care was taken to minimize the possibility that WNV RNA from one tissue would contaminate other tissues. Hemo- lymph was first sampled from newly anesthetized mosquitoes by gently removing their legs. Second, the mosquito mouthparts were inserted into a pulled capillary tube charged with buffer and the mosquito was allowed to salivate for approximately 30 minutes. Finally, the midgut was removed from the mosquito and washed three times in PBS to remove hemolymph-associated WNV. This approach was validated by intrathoracically inoculating adult female mosquitoes with 26104 pfu/ml of WNV. Subsequently, five whole mosquitoes, five unwashed midguts and three times washed midguts were collected 45 minutes post inoculation. The presence of WNV RNA was determined by one-step RT-PCR. Expectedly, all five whole body mosquitoes were positive for WNV RNA along with two of five unwashed midguts. All five of the washed midguts were negative for WNV RNA (data not shown). Since the greatest concentration of WNV RNA tended to be in the mosquito midguts, handling this tissue last minimized the possibility of contaminating samples from the same mosquito. Several mosquitoes were detected that had midgut-limited infections, or WNV in hemolymph but not salivary secretions (data not shown). These results indicate that our efforts to minimize contamination were effective and that the samples selected for this study were not compromized by contaminating WNV RNA. Quantitative analysis of viral genetic diversity in mosquito midguts compared with the highly genetically diverse ‘input’ WNV M24 clearly demonstrated that virus population diversity is restricted in this tissue. Both the percent nucleotide diversity and PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24466