Research ArticleMICROBIAL ECOLOGY

Carryover effects of larval exposure to different environmental bacteria drive adult trait variation in a mosquito vector

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Science Advances  16 Aug 2017:
Vol. 3, no. 8, e1700585
DOI: 10.1126/sciadv.1700585
  • Fig. 1 Experimental workflow.

    (A) Water and A. aegypti pupae (for later midgut dissection from emerging adults) were collected from sylvatic larval sites in gallery forests along the rivers and streams of Lopé National Park in Gabon and from domestic larval sites in a nearby village. (B) At domestic sites (lower pictures), samples were collected from artificial containers such as discarded plastic containers, tires, and metal tins. At sylvatic sites, samples were collected from rock pools (upper picture). (C) At each collection site, both water and pupae were collected into a sterile tube using a sterile pipette. The samples were brought back to the field station, and an aliquot of water was removed next to a Bunsen burner flame and frozen until processing. Back in the laboratory, the water samples were thawed and centrifuged, and the bacteria pellet was resuspended in sterile water and spotted on Whatman FTA cards for later DNA extraction. The pupae were held in the same collection tube until adults emerged. Midguts from adults were dissected within 12 hours of emergence next to a Bunsen burner flame and preserved for later DNA extraction. Midguts were also dissected from wild adult females caught by HLC. Deep-sequencing libraries were made using the V5-V6 hypervariable region of the 16S bacterial ribosomal RNA gene. The sequences were clustered into OTUs and used for analysis of taxonomical abundance and community structure. At the same time that an aliquot of water was frozen, another aliquot was also removed to make a glycerol stock. Upon return to the laboratory, the glycerol stocks were streaked out onto different medium types and individual colonies isolated. For functional assays in vivo, gnotobiotic larvae were created by adding a single bacterial isolate to sterile flasks containing axenic larvae. Adult mosquitoes that had undergone different gnotobiotic treatments as larvae were used to test for variation in life-history and antimicrobial phenotypes.

  • Fig. 2 Limited overlap of bacterial communities between habitat and sample types.

    Venn diagrams show the overlap between OTUs identified in (A) samples from the sylvatic environment, (B) samples from the domestic environment, (C) mosquito midguts, or (D) water samples. OTU diversity indices, taxonomical composition, and relative abundance by sample type are shown in figs. S1, S2, and S3, respectively. The normalized OTU count table is provided in file S8.

  • Fig. 3 Bacterial community structures differ between domestic larval development sites, sylvatic larval development sites, and mosquito midguts.

    Structure of bacterial communities was determined by deep sequencing the V5-V6 region of the bacterial 16S gene among individual samples and sample types, including domestic and sylvatic water samples, domestic and sylvatic midguts, and midguts dissected following emergence and collected by HLC. Bacterial community structure is represented by (A) nonmetric multidimensional scaling (NMDS) of Bray-Curtis dissimilarity index based on OTU abundance and (B) heat map of Bray-Curtis dissimilarity index based on k-mer presence/absence and hierarchical clustering. In the NMDS plot, Spearman correlation (ρ) and stress values are indicated. The normalized OTU count table used to perform the NMDS analysis is provided in file S8. In the heat map, samples are labeled according to their type (M, midgut; W, water) and habitat of origin (sylvatic or domestic). Midguts dissected following emergence are labeled to match the corresponding water sample (for example, midgut a1 was dissected from a mosquito that emerged from water sample a). Midguts from the same breeding site are marked with matching symbols. Red color indicates high similarity, and green color indicates low similarity.

  • Fig. 4 Different larval gnotobiotic treatments result in variation in pupation rate and adult body size, but not in adult life span.

    (A) Pupation rate was determined by counting the number of pupae each day in three replicate flasks of gnotobiotic and nonaxenic larvae in three independent experiments. Axenic larvae (gray line) were included as negative controls. Statistical significance of pairwise differences in pupation rate between treatments was determined by using a three-parameter model to compare the slope of the exponential phase and the day when 50% of larvae pupated. Statistical significance for each pairwise comparison is indicated by a star in the inset table. The shaded ribbon around each curve represents SEM. (B) Adult female life span was determined by counting the number of dead females in triplicate cages. No statistical difference in life span was detected between the different treatments (P = 0.54). (C) Boxplots represent the wing length of adult females from different gnotobiotic treatments. Statistical significance of pairwise differences between treatments was determined by t test. Letters above the graph indicate statistical significance in which treatments with a letter in common are not significantly different from each other.

  • Fig. 5 Adult antimicrobial phenotypes vary following different larval gnotobiotic treatments.

    (A) Antibacterial activity of the hemolymph. Bars show the percentage of individual adult mosquitoes whose hemolymph was able to inhibit M. luteus growth on an agar plate in two replicate experiments of 10 females each. Vertical bars indicate 95% confidence intervals of the percentages. Statistical significance of pairwise differences was determined with a χ2 test. (B) Infectious titer of dengue virus disseminated to the head tissues. The boxplot represents the concentration of infectious dengue virus particles expressed as the log10-transformed number of focus-forming units (FFU) in the head of adult females 14 days after oral exposure. Statistical significance of pairwise differences was determined with a t test. Prevalence of midgut infection and systemic viral dissemination in both experiments is shown in fig. S4. Letters above the graphs indicate statistical significance in which treatments with a letter in common are not significantly different from each other.

  • Table 1 Test statistics of wing length, hemolymph lysozyme-like activity, and titer of disseminated dengue virus.

    Wing length was compared with an analysis of variance. The proportion of hemolymph extracts with detectable antibacterial activity were analyzed with a logistic regression and analysis of deviance. FFU counts in head tissues were log10-transformed and compared with an analysis of variance. With the exception of wing length, the model includes the effect of the isolate (Ssp_ivi, Esp_ivi, Rsp_ivi, and nonaxenic), the experiment (two repetitions), and their interaction. Df, degrees of freedom; LR, likelihood ratio. *P < 0.05; **P < 0.01; ***P < 0.001.

    Wing length
    DfFP
    Isolate39.4158.635 × 10−6***
    Lysozyme-like activity in adult hemolymph
    DfLR χ2P
    Isolate39.3430.0251*
    Experiment12.1500.1426
    Isolate × experiment33.2720.3516
    Dengue virus FFU in adult head tissues
    DfFP
    Isolate32.7160.0470*
    Experiment10.2110.6471
    Isolate × experiment31.4340.2355

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700585/DC1

    fig. S1. Bacterial communities are richer (but not more diverse) in larval breeding site water than in mosquito midguts.

    fig. S2. Bacterial families differ between habitat and sample types.

    fig. S3. Dominant OTUs differ between habitat and sample types.

    fig. S4. No difference in the prevalence of midgut infection or systemic dissemination of dengue virus following different gnotobiotic treatments.

    file S1. Test statistics of richness and Shannon diversity index between sample and habitat type.

    file S2. Differentially abundant OTUs between sample types.

    file S3. Test statistics for larval growth rate and time to 50% pupation.

    file S4. Test statistics for the relationship between the amount of bacteria present in larval flasks and pupation rate.

    file S5. Habitat description of larval breeding sites sampled.

    file S6. Original oligonucleotide sequences used for 16S sequencing.

    file S7. Final oligonucleotide sequences used for 16S sequencing.

    file S8. Normalized OTU count table used for OTU-based analyses.

    file S9. Pairwise comparisons of growth rates (that is, slope of the exponential growth phase) among the 16 candidate bacterial isolates.

    file S10. Identity of cultivable bacteria present in midguts of adults exposed to different bacteria as larvae 4 to 6 days after emergence and maintained under standard insectary conditions as adults.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Bacterial communities are richer (but not more diverse) in larval breeding site water than in mosquito midguts.
    • fig. S2. Bacterial families differ between habitat and sample types.
    • fig. S3. Dominant OTUs differ between habitat and sample types.
    • fig. S4. No difference in the prevalence of midgut infection or systemic dissemination of dengue virus following different gnotobiotic treatments.
    • Legends for files S1 to S10

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    Other Supplementary Material for this manuscript includes the following:

    • file S1 (Microsoft Excel format). Test statistics of richness and Shannon diversity index between sample and habitat type.
    • file S2 (Microsoft Excel format). Differentially abundant OTUs between sample types.
    • file S3 (Microsoft Excel format). Test statistics for larval growth rate and time to 50% pupation.
    • file S4 (Microsoft Excel format). Test statistics for the relationship between the amount of bacteria present in larval flasks and pupation rate.
    • file S5 (Microsoft Excel format). Habitat description of larval breeding sites sampled.
    • file S6 (Microsoft Excel format). Original oligonucleotide sequences used for 16S sequencing.
    • file S7 (Microsoft Excel format). Final oligonucleotide sequences used for 16S sequencing.
    • file S8 (.txt format). Normalized OTU count table used for OTU-based analyses.
    • file S9 (Microsoft Excel format). Pairwise comparisons of growth rates (that is, slope of the exponential growth phase) among the 16 candidate bacterial isolates.
    • file S10 (Microsoft Excel format). Identity of cultivable bacteria present in midguts of adults exposed to different bacteria as larvae 4 to 6 days after emergence and maintained under standard insectary conditions as adults.

    Download Files S1 to S10

    Files in this Data Supplement:

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