Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection

Abstract

Background: The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi) pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.

Methodology/principal findings: We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2) or Argonaute-2 (AGO-2). We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.

Conclusions/significance: This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

Figure 1. Rearing at 18°C activates transgenic RNAi sensor strain mosquitoes.

(A) Schematic representation of the two transgenic constructs. 3×P3-DsRED (red block arrows), 3×P3-EGFP/3×P3-EGFPir (green block arrows), and Mos1 right and left arms (black block arrows) are indicated. (B) Photographs of typical individuals from Ae. aegypti 3×P3-sensor or 3×P3-RG transgenic strains following rearing at 18°C or 28°C using a Leica EGFP (top panel) or DsRED filter (bottom panel). (C) Real-time qPCR displaying changes in EGFP mRNA levels within each of four transgenic strains (two sensor strains, two 3×P3-RG strains) following rearing at 18°C (18) or 28°C (28). Error bars indicate the standard deviation among three biological replicates; *** indicates significance at the p<0.001 level as determined by two-tailed Student's t-test.

Figure 2. Loss of DCR-2 or AGO-2 transcripts does not affect EGFP expression in transgenic 3×P3-RG lines.

(A) Real-time qPCR displaying changes in EGFP mRNA levels following knock-down of ago-2 or dcr-2 transcripts in two 3×P3-RG transgenic lines and one 3×P3-sensor line. (B) Real-time PCR results confirming successful knockdown of ago-2 and dcr-2 transcripts. Error bars indicate one standard deviation corresponding to technical variation for a representative biological replicate.

Figure 3. Low-temperature activation of RNAi sensor mosquitoes is reversible and can be induced in adults.

Photographs taken at 7 or 14 days post emergence (7 d or 14 d) of typical individuals from Ae. aegypti RNAi sensor strain #2 following rearing at 18°C (A) or 28°C (B). Adult females were transferred to the indicated temperature at 1 day post-emergence; photographs are EGFP (top panel) or DsRED (bottom panel). Real-time qPCR of EGFP mRNA levels in 3×P3-sensor mosquito heads following rearing at 18°C (C) or 28°C (D), with newly emerged adults held at the alternate temperature for the indicated number of days. Error bars indicate one standard deviation corresponding to technical variation for a representative biological replicate. (E) Real-time qPCR of EGFP mRNA levels in transgenic RNAi sensor heads following rearing at 18°C or 28°C, with mosquitoes remaining at the same temperature as adults. Error bars indicate the standard deviation among three biological replicates; *** indicates significance at the p<0.001 level as determined by two-tailed Student's t-test.

Figure 4. Small RNA sequencing from 3×P3-sensor heads.

Histogram displaying the length distribution of EGFP-derived small RNAs from 3×P3-sensor mosquito heads following rearing at 18°C or 28°C. Numbers above bars indicates fold increase from 28°C to 18°C. Sense (solid bar) and antisense (cross-hatched) siRNA totals are displayed in the same stack for each sample.

Figure 5. CHIKV infection, RNA production and small RNA biogenesis following rearing at 18°C or 28°C.

(A) Infectivity of CHIKV for Ae. aegypti Liverpool strain following per os challenge. Each bar represents the average of three biological replicates of 27–50 mosquitoes each. Small RNA size distributions of CHIKV vsiRNAs and vpiRNAs (B) and absolute quantitation of CHIKV (+) strand RNA (C) at 8 hrs post injection into Ae. aegypti Liverpool strain females reared at 18°C or 28°C. Error bars indicate the standard deviation amongst three biological replicates. Significance was assessed via a two-tailed Student's t-test.

Figure 6. YFV infection of mosquitoes following rearing at 18°C or 28°C.

Infectivity of YFV for Ae. aegypti (Lvp strain) and Ae. albopictus (Wise County) reared at 18°C or 28°C. Each bar represents the average of three biological replicates of 39–50 mosquitoes each; error bars indicate one standard deviation. Significance was assessed via a two-tailed Student's t-test.