16 research outputs found

    Rockefeller midguts have relatively lower bacterial 16S gene levels compared to Singapore midguts as measured by qPCR.

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    <p>We reared female Rockefeller and Singapore strain <i>A</i>. <i>aegypti</i> in parallel and mixed the breeding water between the strains three times during larval/pupal development. Adults were maintained on 3% sucrose upon eclosion. At 3–5 days post eclosion, females were either provided a sterile blood meal or sterile sucrose. Twenty-four hours post blood meal, we externally sterilized all females by washing with 70% EtOH and then dissected two pools of 8 midguts in sterile 1X PBS for each strain and feeding treatment (5–6 midguts were collected for 3 Singapore blood fed samples). Sugar fed individuals were collected from three independent replicate experiments and blood fed individuals were collected from two. We extracted DNA from each midgut pool and used qPCR to quantify levels of the bacterial16S rDNA gene and <i>A</i>. <i>aegypti</i> S7 reference gene. We averaged delta Ct values from pools from the same biological replicate before analysis to prevent pseudoreplication. Values in the figure were calculated using the delta delta Ct method, where Singapore is shown relative to Rockefeller within each feeding treatment. Error bars represent one standard error. Raw delta Ct values were analyzed in R by ANOVA followed by a Tukey’s test using the following model: Y<sub>ijk</sub> = μ + strain<sub>j</sub> + feeding status<sub>k</sub> + strain<sub>j</sub> * feeding status<sub>k</sub>. Both strain (p = 0.00064) and feeding status (p = 3.22 x 10<sup>−6</sup>) were highly significant and we failed to detect an interaction between strain and feeding status.</p

    Rockefeller female midguts have lower bacterial loads than Singapore females at multiple time points post blood feeding.

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    <p>We reared female <i>A</i>. <i>aegypti</i> from Rockefeller and Singapore strains in parallel and mixed the breeding water between strains three times during larval/pupal development. We maintained adults on 3% sucrose until 5 days post eclosion, at which point a subset of the females were given a sterile blood meal. At 24, 48 and 72 hours post blood feeding, we externally sterilized sugar and blood fed females from each strain with 70% EtOH, dissected midguts from each strain/feeding treatment, homogenized individual midguts in 1X PBS using a sterile pestle and diluted the homogenate 1:100 and 1:10<sup>4</sup> using additional 1X PBS. We spread 50ul of each dilution as well as undiluted homogenate on LB agar plates, allowed the plates to grow for 48 hours at room temperature and then counted the total CFUs on each plate. We performed a zero-inflated data analysis to test the effect of strain, feeding status and time post blood feeding, as well as all pairwise interactions and a three-way interaction between factors. We performed stepwise backwards selection of model terms using likelihood ratio tests to assess the significance of each dropped term. For count data, strain was a significant predictor of bacterial load (p = 0.0237) and we also detected a significant interaction between feeding status and time post blood feeding (p = 0.0060). For presence/absence data, we detected a significant interaction between strain and feeding status (p = 0.0390). Time post blood feeding was not a significant predictor for presence/absence data. Data were collected over three experimental replicates and total sample sizes are as follows: n<sub>rock.sug.24</sub> = 23; n<sub>rock.sug.48</sub> = 23; n<sub>rock.sug.72</sub> = 21; n<sub>sing.sug.24</sub> = 24; n<sub>sing.sug.48</sub> = 24; n<sub>sing.sug.72</sub> = 23; n<sub>rock.blood.24</sub> = 24; n<sub>rock.blood.48</sub> = 24; n<sub>rock.blood.72</sub> = 23; n<sub>sing.blood.24</sub> = 21; n<sub>sing.blood.48</sub> = 17; n<sub>sing.blood.72</sub> = 20.</p

    Biological process gene ontology terms enriched for genes showing transcript abundance changes in either Rockefeller or Singapore in response to blood feeding or bacterial reintroduction.

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    <p>Biological process gene ontology terms enriched for genes showing transcript abundance changes in either Rockefeller or Singapore in response to blood feeding or bacterial reintroduction.</p

    Amino acid metabolic signaling influences <i>Aedes aegypti</i> midgut microbiome variability

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    <div><p>The mosquito midgut microbiota has been shown to influence vector competence for multiple human pathogens. The microbiota is highly variable in the field, and the sources of this variability are not well understood, which limits our ability to understand or predict its effects on pathogen transmission. In this work, we report significant variation in female adult midgut bacterial load between strains of <i>A</i>. <i>aegypti</i> which vary in their susceptibility to dengue virus. Composition of the midgut microbiome was similar overall between the strains, with 81–92% of reads coming from the same five bacterial families, though we did detect differences in the presence of some bacterial families including <i>Flavobacteriaceae</i> and <i>Entobacteriaceae</i>. We conducted transcriptomic analysis on the two mosquito strains that showed the greatest difference in bacterial load, and found that they differ in transcript abundance of many genes implicated in amino acid metabolism, in particular the branched chain amino acid degradation pathway. We then silenced this pathway by targeting multiple genes using RNA interference, which resulted in strain-specific bacterial proliferation, thereby eliminating the difference in midgut bacterial load between the strains. This suggests that the branched chain amino acid (BCAA) degradation pathway controls midgut bacterial load, though the mechanism underlying this remains unclear. Overall, our results indicate that amino acid metabolism can act to influence the midgut microbiota. Moreover, they suggest that genetic or physiological variation in BCAA degradation pathway activity may in part explain midgut microbiota variation in the field.</p></div

    <i>A</i>. <i>aegypti</i> strains vary significantly in their LB-cultivable gut microbial load.

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    <p>We reared female <i>A</i>. <i>aegypti</i> from five strains in parallel and mixed the breeding water between strains three times during larval/pupal development. We maintained adults on 3% sucrose until 5 days post eclosion, at which point a subset of the females were given a sterile blood meal. At 48 hours post blood feeding, we externally sterilized all adults with 70% EtOH, dissected midguts from each strain/feeding treatment, homogenized individual midguts in 1X PBS using a sterile pestle and diluted the homogenate 1:100 using additional 1X PBS. We spread 80ul of undiluted and 1:100 diluted homogenate on LB agar plates and allowed the plates to grow for 48 hours at room temperature. We combined counts from all bacterial genera found in each individual to determine total CFUs per individual female midgut. We collected data over two experimental replicates; sample sizes are as follows: Bkk<sub>sucrose</sub>: n = 13, Bkk<sub>blood</sub>: n = 11, Orl<sub>sucrose</sub>: n = 18, Orl<sub>blood</sub>: n = 24, Rock<sub>sucrose</sub>: n = 18, Rock<sub>blood</sub>: n = 25, Sing<sub>sucrose</sub>: n = 13, Sing<sub>blood</sub>: n = 11, Waco<sub>sucrose</sub>: n = 7, Waco<sub>blood</sub>: n = 8. For many individuals, zero bacterial colonies grew on the LB plates, which resulted in this dataset being zero-inflated. We therefore performed a zero-inflated data analysis to test for the effect of strain, feeding status and a strain × feeding status interaction on the presence/absence of bacteria or total bacterial load. We then performed stepwise backwards selection of model terms using likelihood ratio tests to assess the significance of each dropped term. For count data (<i>i</i>.<i>e</i>. the part of the model analyzing individuals with at least one CFU), strain was a significant predictor of bacterial load (p = 6.19 x 10<sup>−14</sup>) and we detected no interaction between strain and feeding status. For presence/absence data (<i>i</i>.<i>e</i>. the part of the model analyzing the presence or absence of bacteria), we detected a significant interaction (p = 0.01739) between strain and feeding status.</p

    RNAi silencing of genes involved in Val-Leu-Ile degradation causes Rockefeller-specific increases in relative bacterial 16S rDNA.

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    <p>We reared Rockefeller and Singapore mosquitoes in parallel and injected them at 3–5 days post-eclosion with 200ng of dsRNA targeting one of the three experimental genes or eGFP as a control. At two days post injection, we dissected two pools of eight midguts for each strain/treatment combination. This entire experiment was repeated three independent times. We extracted DNA from each midgut pool and performed qPCR to quantify levels of the bacterial16S rDNA gene and <i>A</i>. <i>aegypti</i> S7 reference gene. We averaged delta Ct values from pools from the same biological replicate before analysis to prevent pseudoreplication, and Y-axis values are average inverse delta CT values, i.e. -1*(CT<sub>16S</sub> –CT<sub>S7</sub>) for each treatment. Because CT values are Log<sub>2</sub>, a difference of 1 on the y-axis corresponds to a 2-fold change in 16S DNA abundance. Error bars represent one standard error. Raw delta CT values were analyzed in R by ANOVA followed by a Dunnett’s test using the following model: Y<sub>ijk</sub> = μ + strain<sub>j</sub> + treatment<sub>k</sub> + strain<sub>j</sub> * treatment<sub>k</sub>. We detected a significant interaction between strain and treatment (p = 0.0044), and using a Dunnett’s test found that silencing all three genes caused a significant increase in 16S rRNA gene levels relative to Rock GFP-injected controls. No significant effects of silencing were detected in Sing females. DLD = dihydrolipoamide dehydrogenase, ACAD = acyl-CoA dehydrogenase, IVD = isovaleryl-CoA dehydrogenase.</p

    Genes showing significant changes in transcript abundance in response to blood feeding or bacterial re-introduction in either Rockefeller or Singapore female midguts.

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    <p><b>(A) Experimental design to measure gene expression differences between Rockefeller and Singapore female midguts.</b> Rockefeller and Singapore females were reared in parallel and provided with 3% sucrose + antibiotics (pen/strep + gent) upon eclosion. At 5–7 days post eclosion, females were maintained on sucrose, given a sterile blood meal, or given a blood meal spiked with a mixed culture of seven common mosquito midgut bacteria (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005677#pntd.0005677.s011" target="_blank">S2 Table</a>). Midguts were dissected from each strain/feeding treatment 12 hours post blood feeding and genome wide gene expression was measured for each strain/feeding treatment relative to a pooled reference sample using custom Agilent gene expression microarrays. Comparisons of interest in this experiment (indicated by black arrows) are as follows: Rock sucrose vs. Rock blood fed, Sing sucrose vs. Sing blood fed, Rock blood fed vs. Rock bacteria fed, Sing blood fed vs. Sing bacteria fed. We were also interested in how response to treatment differed between the strains (yellow stars), <i>i</i>.<i>e</i>. transcript changes observed in one strain that are smaller or absent in the other strain. <b>(B) Strain-specific changes in transcript abundance in response to blood feeding.</b> Change in transcript abundance in response to blood feeding (<i>i</i>.<i>e</i>. sucrose fed vs. blood fed) was measured for both Rockefeller and Singapore females. Genes that responded to treatment in either or both strain(s) are shown in the Venn diagram. Genes that increased or decreased in transcript abundance in only Rockefeller or only Singapore (<i>i</i>.<i>e</i>. strain-specific responses) are shown in the bar graphs along with functional categorization data based on gene ontology. <b>(C) Strain-specific changes in transcript abundance in response to bacterial re-introduction.</b> Change in transcript abundance in response to bacterial re-introduction (<i>i</i>.<i>e</i>. blood fed vs. blood + bacteria fed) was measured for both Rockefeller and Singapore females. Genes that responded to treatment in either or both strain(s) are shown in the Venn diagram. Genes that increased or decreased in transcript abundance in only Rockefeller or only Singapore (<i>i</i>.<i>e</i>. strain-specific responses) are shown in the bar graphs along with functional categorization data based on gene ontology. UNK = unknown function, DIV = diverse functions, TRP = transport, RTT = replication, transcription, and translation, R/S/M = redox, stress and mitochondrion, PROT = proteolysis, MET = metabolism, IMM = immunity, DIG = blood and sugar digestion, CSR = chemosensory reception, CS = cytoskeletal and structural.</p

    Prostaglandins regulate humoral immune responses in Aedes aegypti.

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    Prostaglandins (PGs) are immuno-active lipids that mediate the immune response in invertebrates and vertebrates. In insects, PGs play a role on different physiological processes such as reproduction, ion transport and regulation of cellular immunity. However, it is unclear whether PGs play a role in invertebrate's humoral immunity, and, if so, which immune signaling pathways would be modulated by PGs. Here, we show that Aedes aegypti gut microbiota and Gram-negative bacteria challenge induces prostaglandin production sensitive to an irreversible inhibitor of the vertebrate cyclooxygenase, acetylsalicylic acid (ASA). ASA treatment reduced PG synthesis and is associated with decreased expression of components of the Toll and IMD immune pathways, thereby rendering mosquitoes more susceptible to both bacterial and viral infections. We also shown that a cytosolic phospholipase (PLAc), one of the upstream regulators of PG synthesis, is induced by the microbiota in the midgut after blood feeding. The knockdown of the PLAc decreased prostaglandin production and enhanced the replication of Dengue in the midgut. We conclude that in Ae. aegypti, PGs control the amplitude of the immune response to guarantee an efficient pathogen clearance
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