Seasonality influences key physiological components contributing to Culex pipiens vector competence

Abstract

Mosquitoes are the most important animal vector of disease on the planet, transmitting a variety of pathogens of both medical and veterinary importance. Mosquito-borne diseases display distinct seasonal patterns driven by both environmental and biological variables. However, an important, yet unexplored component of these patterns is the potential for seasonal influences on mosquito physiology that may ultimately influence vector competence. To address this question, we selected Culex pipiens, a primary vector of the West Nile virus (WNV) in the temperate United States, to examine the seasonal impacts on mosquito physiology by examining known immune and bacterial components implicated in mosquito arbovirus infection. Semi-field experiments were performed under spring, summer, and late-summer conditions, corresponding to historically low-, medium-, and high-intensity periods of WNV transmission, respectively. Through these experiments, we observed differences in the expression of immune genes and RNA interference (RNAi) pathway components, as well as changes in the distribution and abundance of Wolbachia in the mosquitoes across seasonal cohorts. Together, these findings support the conclusion that seasonal changes significantly influence mosquito physiology and components of the mosquito microbiome, suggesting that seasonality may impact mosquito susceptibility to pathogen infection, which could account for the temporal patterns in mosquito-borne disease transmission.

Keywords: Culex pipiens; RNAi; Wolbachia; gene expression; mosquito; physiology; seasonality; semi-field.

Figure 1

Overview and temperature conditions of the semi-field experiments examining Culex pipiens seasonality. (A) Graphical overview of semi-field experiments where first-instar Culex pipiens larvae were reared outside at three time points (week 19, 30, and 35 of the year) to represent spring, summer, and late-summer conditions. Mosquitoes were reared outside for their entire development, first in metal trays for larval growth, then the pupae were collected and placed in eclosion chambers to allow for adult emergence. Adult female mosquitoes were collected 5–8 days post emergence for further molecular analysis. Mosquitoes were obtained from a laboratory colony of Cx. pipiens. (B) Conditions experienced by each study group, with temperatures reflecting the weekly average of the daily high, average, and low temperatures for Ames, IA, United States, in 2021 when the experiments were performed. (C) Photoperiod (daylight) averages are displayed by week, highlighting conditions for each study group. Graphics in (A) were created with BioRender.com.

Figure 2

Seasonality of Culex pipiens immune gene expression. Gene expression was examined using samples from our Cx. pipiens colony under standard rearing conditions (laboratory) and across seasonal conditions (spring, summer, and late summer) from semi-field experiments. Expression of (A) genes involved in the RNA interference (RNAi) pathway (AGO2 and DCR2), (B) the immune factor Vago, or (C) the antimicrobial gene CEC-A were evaluated by quantitative reverse transcription- polymerase chain reaction (qRT-PCR). Each replicate contained groups of four pooled mosquitoes, with three or more independent samples per experimental condition. Data are displayed as the mean ± SEM, with significance determined using a one-way ANOVA with a Tukey’s multiple comparison test. Significant differences between conditions are denoted by asterisks (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001); all other comparisons between conditions were not significant. SEM, standard error of the mean.

Figure 3

Seasonality of Wolbachia titers in Culex pipiens ovary and carcass samples. Wolbachia titers (16s RNA) were examined in individual Cx. pipiens ovary or carcass (remaining tissue following ovary dissection) samples reared in the laboratory or under spring, summer, and late-summer semi-field conditions by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) and normalized to Rpl32 expression. A one-way non-parametric ANOVA (Kruskal–Wallis) with a Dunn’s multiple comparison test was used to determine significance. Significant differences between conditions are denoted by asterisks (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001).