Reactive oxygen species production and Brugia pahangi survivorship in Aedes polynesiensis with artificial Wolbachia infection types

Abstract

Heterologous transinfection with the endosymbiotic bacterium Wolbachia has been shown previously to induce pathogen interference phenotypes in mosquito hosts. Here we examine an artificially infected strain of Aedes polynesiensis, the primary vector of Wuchereria bancrofti, which is the causative agent of Lymphatic filariasis (LF) throughout much of the South Pacific. Embryonic microinjection was used to transfer the wAlbB infection from Aedes albopictus into an aposymbiotic strain of Ae. polynesiensis. The resulting strain (designated "MTB") experiences a stable artificial infection with high maternal inheritance. Reciprocal crosses of MTB with naturally infected wild-type Ae. polynesiensis demonstrate strong bidirectional incompatibility. Levels of reactive oxygen species (ROS) in the MTB strain differ significantly relative to that of the wild-type, indicating an impaired ability to regulate oxidative stress. Following a challenge with Brugia pahangi, the number of filarial worms achieving the infective stage is significantly reduced in MTB as compared to the naturally infected and aposymbiotic strains. Survivorship of MTB differed significantly from that of the wild-type, with an interactive effect between survivorship and blood feeding. The results demonstrate a direct correlation between decreased ROS levels and decreased survival of adult female Aedes polynesiensis. The results are discussed in relation to the interaction of Wolbachia with ROS production and antioxidant expression, iron homeostasis and the insect immune system. We discuss the potential applied use of the MTB strain for impacting Ae. polynesiensis populations and strategies for reducing LF incidence in the South Pacific.

Figure 1. ROS levels in Ae. polynesiensis strains.

The concentration of H₂O₂ was measured in APM, APMT and MTB after feeding on sucrose (black) and 24 hours after a blood meal (white). Sucrose fed MTB had significantly higher levels of ROS than APM (ANOVA, p<0.05). Blood feeding was associated with a significantly reduced ROS level in APMT and MTB (ANOVA, p<0.05) but not APM (p = 0.75). After blood feeding, APM maintained higher ROS levels than APMT and MTB (Tukey, p<0.05), which were equivalent (p = 0.9). The data shown are the means of five replicates.

Figure 2. Mean number of L3s within Ae. polynesiensis strains.

APM, APMT and MTB were given a Brugia pahangi infected blood meal. After ten days, mosquitoes were dissected and the number of infective stage L3s was counted. The MTB strain had a significantly lower worm load (p<0.0001) than APM and APMT, which were equivalent (p = 0.47). Despite significant variation between replicates (p<0.0001), the relationship between strains (APM = APMT>MTB) was consistent across the three replicates (p = 0.77). Each symbol represents a single mosquito. Lines represent mean values.

Figure 3. Survivorship of Ae. polynesiensis strains after a Brugia-infected blood meal.

Mean survivorship of APM (black), APMT (white) and MTB (grey) ten days after feeding on a Brugia pahangi-infected blood meal. Contrast comparisons demonstrate significant differences in survivorship for each pair-wise comparison between strains. A) The mean survival of the four replicates. Error bars represent standard error. B) Contrast comparisons. C) The relationship between strains (APM>APMT>MTB) was consistent across all four replicates (GLM: χ² = 6.27, df = 6, p = 0.39). For all tests, α = 0.05.

Figure 4. Survivorship of Ae. polynesiensis strains after feeding on Brugia-infected blood vs. uninfected blood.

A) Mean survivorship of APM, APMT and MTB ten days after feeding on Brugia pahangi-infected blood (black) vs. uninfected blood (white). The presence of Brugia pahangi in the blood meal was associated with decreased survivorship for APM (p<0.05) and MTB (p<0.05). APMT survivorship was independent of blood meal type (p = 0.89). Error bars represent standard error for three replicates. B) Contrast comparisons for strains fed Brugia-infected and uninfected blood. C) Contrast comparisons for strains fed Brugia-infected blood. D) Contrast comparisons for strains fed uninfected blood.

Figure 5. Survivorship of Ae. polynesiensis strains after blood feeding vs. sucrose only.

A) Mean survivorship of APM, APMT and MTB ten days after blood feeding (white) vs. sucrose only (black). Survival increased in APMT (p<0.05) and MTB (p<0.05) when fed sucrose only. However, decreased survival was observed in APM when not given a blood meal (p<0.05). Error bars represent standard error of two replicates. B) Contrast comparisons for strains fed blood or sucrose only. C) Contrast comparisons for strains fed blood. D) Contrast comparisons for strains fed sucrose only.