Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects

Abstract

Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.

Keywords: Aedes aegypti; Drosophila melanogaster; RNA viruses; adaptive immunity; antiviral memory; chromatin modifications; immune response; insects; transgenerational immune priming.

Graphical abstract

Figure 1

Drosophila melanogaster Flies Primed with SINV Transmit Antiviral Immunity to Their Progeny (A) Schematic of the protocol. w¹¹¹⁸ virgin female flies were infected with 100 PFU of SINV or mock infected and then crossed with non-infected males 2 days after priming. The F1 embryos were collected and treated with 50% bleach to eliminate the virus coming from the mother that could be present on the surface of the embryo. The F1 adult flies were recovered 3 to 4 days after emergence and challenged with 3,000 PFU of SINV-R. (B–D) Viral replication in F1 flies produced by primed (SINV/SINV) or unprimed (Mock inf/SINV) mothers was quantified 2 days after infection as Renilla luciferase counts (B) and by plaque assays (C and D). (E–H) F1 flies whose mothers were primed with 100 PFU of SINV or mock infected were challenged with a set of different viruses: 3,000 PFU of SINV-R (E), 5 TCID₅₀ of DCV (F), 5 TCID₅₀ of CrPV (G), or 5 TCID₅₀ of FHV (H). For (A), two independent experiments with three biological replicates consisting of n = 3 pools of three flies each per condition were analyzed. For (B), three independent experiments with three biological replicates consisting of n = 3 pools of three flies each per condition were analyzed. For (E)–(H), two independent experiments with at least three biological replicates of n = 3 pools of three flies each per condition were analyzed. Asterisks indicate statistical significance of pairwise comparisons by Mann-Whitney test (^∗∗p ≤ 0.01). The absence of asterisks indicates lack of statistical significance (p > 0.05). Error bars indicate SEM.

Figure 2

Inherited Antiviral Immunity Persists Across Generations and Is Long Lasting (A) To obtain the F2 and the subsequent generations, F1 females (produced by SINV-primed F0 mothers) were crossed with their male siblings. One half of the F2 flies were used for virus challenge experiments and viral titer quantification. The other half was used to obtain the F3 and so on. Antiviral immunity was inherited for 5 generations (F5). (B) Virgin flies of different ages (0, 1, 2, or 3 days after emergence) were primed with SINV and treated with the TGIP protocol depicted in Figure 1A. F0 mothers transmit antiviral immunity to their progeny when they are primed at 3 days old, suggesting that the maturity of the F0 immune response is relevant for TGIP. (C) F1 flies produced by primed or unprimed mothers were challenged with SINV (SINV/SINV and Mock inf/SINV, respectively), and viral load was measured at different time points (1, 2, 3, 4, 5, and 10 dpi). For (A) and (B), three independent experiments with three biological replicates of n = 3 pools of three flies each per condition were analyzed. For (C), filled black circles (SINV/SINV) and open gray squares (Mock inf/SINV) represent individual flies. Six flies per condition were analyzed. Asterisks indicate statistical significance of pairwise comparisons by Mann-Whitney test (^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001). The absence of asterisks indicates lack of statistical significance (p > 0.05). Error bars indicate SEM.

Figure 3

Antiviral Immunity Is Inherited Following Priming With Different Positive-Sense Single-Stranded RNA Viruses w¹¹¹⁸ virgin female flies were primed or mock infected with different RNA viruses. The F1 embryos were collected and treated with 50% bleach to eliminate the viruses coming from the mother that could be present on the surface of the embryo. (A) F0 flies were primed with 1 TCID₅₀/fly or mock infected, and the F1 flies were challenged with 5 TCID₅₀/fly (DCV/DCV and Mock inf/DCV, respectively). Three independent experiments with three biological replicates of n = 3 pools of three flies each per condition were analyzed. (B) F0 flies were primed with 1 TCID₅₀/fly or mock infected, and the F1 flies were challenged with 5 TCID₅₀/fly (CrPV/CrPV and Mock inf/CrPV, respectively). Four independent experiments with three biological replicates of n = 3 pools of three flies each per condition were analyzed. (C) F0 flies were primed with 1 TCID₅₀/fly or mock infected, and the F1 flies were challenged with 5 TCID₅₀/fly (FHV/FHV and Mock inf/FHV, respectively). Two independent experiments with three biological replicates of n = 3 pools of three flies each per condition were analyzed. (D) F0 flies were primed with 200 PFU/fly or mock infected, and the F1 flies were challenged with 200 PFU/fly (DXV/DXV and Mock inf/DXV, respectively). Three independent experiments with three biological replicates of n = 3 pools of three flies each per condition were analyzed. (E) F0 flies were primed with 100 PFU/fly or mock infected, and the F1 flies were challenged with 100 PFU /fly (VSV/VSV and Mock inf/VSV, respectively). Three independent experiments with three biological replicates of n = 3 pools of three flies each per condition were analyzed. Asterisks indicate statistical significance of pairwise comparisons by Mann-Whitney test (^∗∗p ≤ 0.01). The absence of an asterisk indicates lack of statistical significance (p > 0.05). Error bars indicate SEM.

Figure 4

Aedes aegypti Mosquitoes Infected With CHIKV Transmit Antiviral Protection to Their Progeny (A) Schematic of the protocol. Female Aedes aegypti mosquitoes were infected with 100 PFU of CHIKV or mock infected by injection. Three days later, mosquitoes were fed with a non-infectious blood meal to provide the nutrients for egg production. Eggs were hatched and F1 females were challenged with 100 PFU of CHIKV between 2 and 5 days after mosquito emergence. (B) Viral load was measured for individual mosquitoes by plaque assays 2 days after the viral challenge for mosquitoes produced by primed F0 mosquitoes (CHIKV/CHIKV) or by mock-infected F0 mosquitoes (Mock inf/CHIKV). (C) Schematic of the protocol. Female Aedes aegypti mosquitoes were infected with a blood meal containing 10⁶ PFU/mL of CHIKV or carrier buffer. Seven days after the infection, the mosquitoes were fed with a non-infectious blood meal to provide the nutrients for egg production. Eggs were hatched and F1 females were challenged with a blood meal containing 10⁶ PFU/mL of CHIKV. (D) Viral load was measured for individual mosquitoes by plaque assays 3 days after the viral challenge for mosquitoes produced by primed F0 mosquitoes (CHIKV/CHIKV) or by mock-infected F0 mosquitoes (Mock inf/CHIKV). For (B) and (D), filled black circles (CHIKV/CHIKV) and open black circles (Mock inf/CHIKV) represent individual mosquitoes. Between 18 and 22 mosquitoes per condition were analyzed. Asterisks indicate statistical significance of pairwise comparisons by Mann-Whitney test (^∗∗p ≤ 0.01, ^∗∗∗∗p ≤ 0.0001). Bars indicate the mean.

Figure 5

TG Antiviral Immunity Is Viral RNA Dependent and Sequence Specific (A and B) w¹¹¹⁸ virgin female flies were primed with 700 bp dsRNAs corresponding to two different regions of the SINV virus genome (dsSINV) or with control dsRNAs (dsCtrl). Flies were crossed with non-treated males, and the F1 flies were challenged with 3,000 PFU of SINV-R (dsSINV/SINV and dsCtrl/SINV, respectively). Viral replication was estimated at 2 dpi by measuring Renilla luciferase activity (A) or by plaque assays (B). (C) F1 females (produced by F0 dsSINV-primed flies) were crossed with their male siblings in order to obtain the F2 generation. One half of the F2 generation was used for virus challenge experiments and viral titer quantification. The other half was used to obtain the F3 and the following generations. Antiviral immunity was inherited by the first 5 generations (F5). See also Figure S3. Two independent experiments with three biological replicates of n = 3 pools of three flies each per condition were analyzed. Asterisks indicate statistical significance of pairwise comparisons by Mann-Whitney test (^∗p ≤ 0.05, ^∗∗p ≤ 0.01). The absence of an asterisk indicates lack of statistical significance (p > 0.05). Error bars indicate SEM.

Figure 6

A vDNA Form Is Transmitted From Infected Flies to Their Progeny (A) Schematic of the protocol. w¹¹¹⁸ virgin female flies were primed with 100 PFU of SINV or mock infected and crossed with non-infected males 2 days later. The F1 embryos were collected and treated with 50% bleach to eliminate the viruses coming from the mother that could be present on the surface of the embryo. (B and C) SINV-infected flies (B) and the F1 flies (C) were recovered, and vDNA (top) or viral RNA (bottom) was amplified by PCR or RT-PCR, respectively, from individual flies. (D) Schematic protocol. Embryos from w¹¹¹⁸ flies naturally harboring persistent DCV infection were treated with 50% bleach in order to produce DCV-free F1 and F2 generations. (E and F) F0 flies persistently infected with DCV (E) and F2 flies (F) were recovered, and vDNA (top) or viral RNA (bottom) was amplified by PCR or RT-PCR, respectively, from individual flies. To confirm the absence of viral replication in the F2 flies, two different pairs of primers were used for RT-PCR. Rp49 was used as housekeeping control.

Figure 7

The Transcriptome of the F2 Progeny Produced by Primed F0 Parents Differs from the Transcriptome of the F2 Progeny Produced by Unprimed F0 Parents F1 females (from SINV- or mock-infected F0 female flies) were crossed with their male siblings to obtain the F2 generation. The F2 flies were infected with SINV or mock infected. Two days later, RNA was extracted from three pools of four flies per condition to perform RNA-seq to produce a transcriptome for Gene Ontology enrichment analysis. Conditions: SM (F0 SINV/F2 mock infected), SS (F0 SINV/F2 SINV), MS (F0 mock infected/F2 SINV), MM (F0 mock infected/ F2 mock infected). In (A) and (B), the first two components of principal-component analysis (PCA) with percentages of variance associated with each axis are shown. (A) The PC1 separates infected from non-infected flies regardless of the priming status of their F0 mothers, while the PC2 separates the F2 progeny of primed F0 mothers (red circles) from the F2 progeny of unprimed F0 mothers (blue circles). (B) To avoid the effect of different levels of infection between the F2 flies produced by primed F0 mothers (red circles) and unprimed F0 mothers (blue circles), only the non-infected F2 flies were analyzed. The PC1 and PC2 are sufficient to differentiate the progeny produced by primed or non-primed F0 flies. (C) Gene Ontology enrichment analysis of upregulated genes in the SM progeny compared to the MM progeny shows an enrichment in the expression of genes related to chromatin and DNA binding.