Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictus

Abstract

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

Fig 1. Extrinsic incubation temperature alters ZIKV transmission efficiency in Aedes mosquitoes.

Percent of Ae. aegypti (average number per EIT group = 86, S3 Table) (A & B) and Ae. albopictus (average number per EIT group = 80, S3 Table) (C & D) with ZIKV in midgut, legs, and saliva at 7 (A & C) and 14 (B&D) days post feeding. The bar represents the mean, and the open circles represent the value of each experiment with SEM shown with error bars.

Fig 2. Extrinsic incubation temperature alters virus diversification, selection and divergence during mosquito infection.

ZIKV population diversity at varying EITs was determined using measures of nucleotide diversity (A) and natural selection (d_N/d_S) (B). d_N/d_S was also examined by virus coding region for virus that replicated in Ae. aegypti (C), and Ae. albopictus (D). Measured results for E, NS1, NS3, and NS5 protein coding regions are shown. d_N/d_S was near 1 for E, prM NS2A, NS2B, NS4A, NS4B (not shown). Nucleotide diversity (E) and divergence (F-G) were determined for ZIKV in different mosquito compartments. Divergence from input population (y-axis) and cumulative divergence between tissues (x-axis) (F-G) is presented for Midguts (M), legs (L), and Saliva (S). Consensus change counts also are presented (H) by EIT and mosquito species, alongside majority variants accumulated (H), as markers of population diversity. Significance was tested using the Kruskal-Wallis test with Dunn’s correction (B-D, p < 0.05). Figures present the mean and SEM.

Fig 3. Fluctuating extrinsic incubation temperatures impose purifying selection on ZIKV during systemic mosquito infection.

d_N/d_S (mean with SEM) for ZIKV CDS (closed circles), structural sequence (Boxes), and non-structural sequence (open circles), at indicated EITs including fluctuating EITs, in Ae. aegypti (A-E) and Ae. albopictus (F-J).

Fig 4. EIT and species control ZIKV variant frequency during systemic infection.

Frequencies of L330V E (A & E), W98G NS1 (B & F), M220T NS1 (C & G), and G83 NS5 (D & H) in input, midgut, legs and saliva shown at 25, 35 and fluctuating 25–35°C in Ae. aegypti (A-D) and Ae. albopictus (E-H). Mean and SEM of competitions from three biological replicates shown.

Fig 5. High extrinsic incubation temperature increases variant fixation in Aedes aegypti.

Indicated mutations were engineered into a ZIKV-PR-IC and mixed with a ZIKV-REF virus. The proportion of each competitor (grey, mean with 95% CI, *p-value < 0.05 compared with ZIKV-PR-IC, Kruskal-Wallis and Dunn’s) and rate of fixation (*p-value < 0.05 compared with ZIKV-PR-IC, Two-tailed Fisher’s exact test) was determined from mosquito bodies at 14-dpi for Ae. aegypti mosquitoes held at constant EIT’s 25°C (A) & 35°C (B). Fixation indicates that 100% of the sequenced nucleotides were from the competitor virus. Initial viral inoculum (ratio of competitor virus to reference) is shown in red symbols.