The potential for flower nectar to allow mosquito to mosquito transmission of Francisella tularensis

Abstract

Francisella tularensis is disseminated in nature by biting arthropods such as mosquitoes. The relationship between mosquitoes and F. tularensis in nature is highly ambiguous, due in part to the fact that mosquitoes have caused significant tularemia outbreaks despite being classified as a mechanical vector of F. tularensis. One possible explanation for mosquitoes being a prominent, yet mechanical vector is that these insects feed on flower nectar between blood meals, allowing for transmission of F. tularensis between mosquitoes. Here, we aimed to assess whether F. tularensis could survive in flower nectar. Moreover, we examined if mosquitoes could interact with or ingest and transmit F. tularensis from one source of nectar to another. F. tularensis exhibited robust survivability in flower nectar with concentrations of viable bacteria remaining consistent with the rich growth medium. Furthermore, F. tularensis was able to survive (albeit to a lesser extent) in 30% sucrose (a nectar surrogate) over a period of time consistent with that of a typical flower bloom. Although we observed diminished bacterial survival in the nectar surrogate, mosquitoes that fed on this material became colonized with F. tularensis. Finally, colonized mosquitoes were capable of transferring F. tularensis to a sterile nectar surrogate. These data suggest that flower nectar may be capable of serving as a temporary source of F. tularensis that could contribute to the amplification of outbreaks. Mosquitoes that feed on an infected mammalian host and subsequently feed on flower nectar could deposit some F. tularensis bacteria into the nectar in the process. Mosquitoes subsequently feeding on this nectar source could potentially become colonized by F. tularensis. Thus, the possibility exists that flower nectar may allow for vector-vector transmission of F. tularensis.

Fig 1. F. tularensis survives in flower nectar.

F. tularensis bacteria were incubated in TSBc, yellow squash nectar, or water. Bacterial suspensions were incubated at 22°C, serial diluted and plated to determine CFU at the indicated time points. Graphed values represent mean CFU ± SE of three combined independent experiments. In some cases, error bars are smaller than the graph symbols. Differences in the average number of F. tularensis LVS recovered (CFU/mL) over the course of the assay were determined by repeated measures two-way ANOVA with Tukey’s multiple comparison test as a post hoc analysis comparing the change in bacterial burden between experimental, positive control, and negative control groups (ANOVA, P < 0.0001; post hoc, Nectar vs. TSBc not significantly different; Nectar vs Water, P < 0.001; TSBc vs. Water, P < 0.001).

Fig 2. F. tularensis exhibits limited survival in a sucrose solution, a nectar surrogate.

F. tularensis bacteria were incubated in TSBc, 30% sucrose, or water. Bacterial suspensions were incubated at ambient temperature, serial diluted and plated to determine CFU at the indicated time points. Graphed values represent mean CFU ± SE of four combined independent experiments. In some cases, error bars are smaller than the graph symbols. Differences in the average number of F. tularensis LVS recovered (CFU/mL) over the course of the assay were determined by repeated measures two-way ANOVA with Tukey’s multiple comparison test as a post hoc analysis comparing the change in bacterial burden between experimental, positive control, and negative control groups (ANOVA, P < 0.0001; post hoc, 30% Sucrose vs. TSBc, P < 0.001; 30% Sucrose vs. Water; P < 0.001; TSBc vs. Water, P<0.001).

Fig 3. Mosquitoes become colonized with F. tularensis after interacting with flower nectar surrogate.

A. Tubes holding a 30% sucrose solution inoculated with F. tularensis were placed within insect chambers for 6 days. As many as six mosquitoes per group were extracted daily, washed with gentamicin to kill surface bacteria, homogenized and plated on media selective for Francisella. Data shown represent the percentage of mosquitoes colonized with F. tularensis LVS (recovery of at least one CFU) on the days indicated and are a combination of three independent experiments. B. DNA was extracted from bacteria isolated from mosquito homogenates plated on media selective for F. tularensis. Only colonies that produced a similar morphology to F. tularensis were selected. The extracted DNA was subjected to PCR using primers specific for mglA of Francisella sp. Agarose gel electrophoresis was used to compare amplicons produced from bacteria isolated from mosquitoes to those generated from bona fide F. tularensis DNA. PCR from only one isolate is shown for simplicity, but all other colonies with similar morphologies produced a similar amplicon band (not shown). PCR reactions lacking Francisella template DNA did not produce amplicons (negative control). C. Cell extracts from isolates were separated by SDS-PAGE and electroblotted onto nitrocellulose. Following blocking, Western blots were probed with an anti-IglC monocolonal antibody. An alkaline-phosphatase anti mouse secondary antibody was used for detection.

Fig 4. Mosquitoes transfer F. tularensis from one nectar surrogate source to another.

DNA was extracted from bacteria isolated from media selective for F. tularensis. Only colonies that produced a similar morphology to F. tularensis were selected. The extracted DNA was subjected to PCR using primers specific for mglA of Francisella sp. Agarose gel electrophoresis was used to compare amplicons produced from bacteria isolated from nectar surrogate to those generated from bona fide F. tularensis DNA. PCR from only one isolate is shown for simplicity, but all other colonies with similar morphologies produced a similar amplicon band. PCR reactions lacking template DNA did not produce amplicons.