Age-related differences in the cloacal microbiota of a wild bird species

Background: Gastrointestinal bacteria play a central role in the health of animals. The bacteria that individuals acquire as they age may therefore have profound consequences for their future fitness. However, changes in microbial community structure with host age remain poorly understood. We characterised the cloacal bacteria assemblages of chicks and adults in a natural population of black-legged kittiwakes (Rissa tridactyla), using molecular methods. Results: We show that the kittiwake cloaca hosts a diverse assemblage of bacteria. A greater number of total bacterial OTUs (operational taxonomic units) were identified in chicks than adults, and chicks appeared to host a greater number of OTUs that were only isolated from single individuals. In contrast, the number of bacteria identified per individual was higher in adults than chicks, while older chicks hosted more OTUs than younger chicks. Finally, chicks and adults shared only seven OTUs, resulting in pronounced differences in microbial assemblages. This result is surprising given that adults regurgitate food to chicks and share the same nesting environment. Conclusions: Our findings suggest that chick gastrointestinal tracts are colonised by many transient species and that bacterial assemblages gradually transition to a more stable adult state. Phenotypic differences between chicks and adults may lead to these strong differences in bacterial communities. These data provide the framework for future studies targeting the causes and consequences of variation in bacterial assemblages in wild birds

Enhanced Zika virus susceptibility of globally invasive Aedes aegypti populations

The drivers and patterns of zoonotic virus emergence in the human population are poorly understood. The mosquito Aedes aegypti is a major arbovirus vector native to Africa that invaded most of the world’s tropical belt over the past four centuries, after the evolution of a “domestic” form that specialized in biting humans and breeding in water storage containers. Here, we show that human specialization and subsequent spread of A. aegypti out of Africa were accompanied by an increase in its intrinsic ability to acquire and transmit the emerging human pathogen Zika virus. Thus, the recent evolution and global expansion of A. aegypti promoted arbovirus emergence not solely through increased vector–host contact but also as a result of enhanced vector susceptibility

The histone methyltransferase G9a regulates tolerance to oxidative stress-induced energy consumption

Stress responses are crucial processes that require activation of genetic programs that protect from the stressor. Stress responses are also energy consuming and can thus be deleterious to the organism. The mechanisms coordinating energy consumption during stress response in multicellular organisms are not well understood. Here, we show that loss of the epigenetic regulator G9a in Drosophila causes a shift in the transcriptional and metabolic responses to oxidative stress (OS) that leads to decreased survival time upon feeding the xenobiotic paraquat. During OS exposure, G9a mutants show overactivation of stress response genes, rapid depletion of glycogen, and inability to access lipid energy stores. The OS survival deficiency of G9a mutants can be rescued by a high-sugar diet. Control flies also show improved OS survival when fed a high-sugar diet, suggesting that energy availability is generally a limiting factor for OS tolerance. Directly limiting access to glycogen stores by knocking down glycogen phosphorylase recapitulates the OS-induced survival defects of G9a mutants. We propose that G9a mutants are sensitive to stress because they experience a net reduction in available energy due to (1) rapid glycogen use, (2) an inability to access lipid energy stores, and (3) an overinduced transcriptional response to stress that further exacerbates energy demands. This suggests that G9a acts as a critical regulatory hub between the transcriptional and metabolic responses to OS. Our findings, together with recent studies that established a role for G9a in hypoxia resistance in cancer cell lines, suggest that G9a is of wide importance in controlling the cellular and organismal response to multiple types of stress.status: publishe

The Epigenetic Regulator G9a Mediates Tolerance to RNA Virus Infection in <i>Drosophila</i>

<div><p>Little is known about the tolerance mechanisms that reduce the negative effects of microbial infection on host fitness. Here, we demonstrate that the histone H3 lysine 9 methyltransferase <i>G9a</i> regulates tolerance to virus infection by shaping the response of the evolutionary conserved Jak-Stat pathway in <i>Drosophila</i>. <i>G9a</i>-deficient mutants are more sensitive to RNA virus infection and succumb faster to infection than wild-type controls, which was associated with strongly increased Jak-Stat dependent responses, but not with major differences in viral load. Genetic experiments indicate that hyperactivated Jak-Stat responses are associated with early lethality in virus-infected flies. Our results identify an essential epigenetic mechanism underlying tolerance to virus infection.</p></div

Hyperactivation of the Jak-Stat pathway renders flies hypersensitive to virus infection.

<p>(<b>A</b>) Experimental set-up. Expression of the <i>Upd</i> transgene was induced specifically in adult flies using the <i>Gal4/Gal80ts</i> system. <i>Gal80ts</i> is a temperature-sensitive allele of the Gal80 inhibitor that binds Gal4 to prevent activation of gene expression at 20°C. At 29°C, Gal80ts is degraded, allowing Gal4 to bind to the Upstream Activating Sequences (UAS) to induce gene expression. Flies were reared at 20°C, and 0 to 3-day-old adults were conditioned at 29°C for 3 days prior to viral challenge. (<b>B</b>) Expression levels by RT-qPCR of <i>Upd</i> and <i>TotA</i> in flies carrying the temperature-dependent <i>Upd</i> overexpression system (<i>UAS-Upd; tubulin-Gal4/Gal80ts</i>) after 3 days conditioning at 29°C. The <i>Gal4</i> and <i>Gal80ts</i> transgenes were combined with the <i>UAS-Upd</i> by standard genetic crosses at 20°C and 0 to 3-day-old adult offspring was cultured for 3 days at 20°C or at 29°C before RNA levels were analyzed by RT-qPCR. Transcript levels of <i>Upd</i> and <i>TotA</i> were normalized to RNA levels of the housekeeping gene <i>Ribosomal Protein 49</i>, and expressed as fold change relative to control flies carrying only the <i>UAS-Upd</i> transgene. (<b>C</b>) Survival of flies carrying the temperature-dependent <i>Upd</i> overexpression system (<i>UAS-Upd; tubulin-Gal4/Gal80ts</i>) and genetic control flies upon DCV infection (1,000 TCID₅₀ = units) at 29°C. Data are means and s.d. of three independent pools of at least 10 male flies for each genotype. Data in (<b>C</b>) are from one experiment representative of 2 independent experiments. Differences in expression of <i>Upd</i> and <i>TotA</i> were evaluated with a Student’s t-test (*<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

Loss of <i>G9a</i> does not affect viral loads upon DCV infection.

<p>(<b>A,B</b>) Wild-type or <i>G9a</i> mutant flies were inoculated with DCV and viral titers were determined over time in (<b>A</b>) whole flies, and (<b>B</b>) dissected fat bodies. Data represent means and s.d of three independent experiments. Each experiment contained three biological replicates of 5 female flies (<b>A</b>), or 10 fat bodies (<b>B</b>) per replicate for each genotype. (<b>C</b>,<b>D</b>) DCV RNA levels over the course of 3 days post-infection analyzed by RT-qPCR in (<b>C</b>) whole flies or (<b>D</b>) fat bodies of wild-type and <i>G9a</i> mutant flies. DCV RNA levels were normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and are calculated relative to the viral RNA levels in flies harvested immediately after inoculation (t₀). Data represent means and s.d. of three biological replicates of 5 female flies (<b>C</b>) or 10 fat bodies (<b>D</b>) per replicate for each genotype. Data in panel <b>C</b> and <b>D</b> are from one experiment representative of 2 independent experiments. *<i>P</i> < 0.05 (Student’s t-test).</p

Genetic interaction between <i>G9a</i> and the Jak-Stat pathway.

<p>(<b>A,B</b>) Survival upon DCV infection (1,000 TCID₅₀ units) of wild-type or <i>G9a</i> mutant and wild-type flies overexpressing (<b>A</b>) a dominant negative version of the <i>domeless</i> receptor (dome^ΔCyt), or (<b>B</b>) the negative regulator of Jak-Stat signaling <i>Socs36E</i>. The UAS/Gal4 system was used to drive transgene expression. Gal4 is expressed under control of the actin promoter (<i>Act-Gal4</i>) to drive ubiquitous expression of the <i>UAS-dome</i>^ΔCyt and <i>UAS-Socs36E</i> transgenes. Control flies expressing only the <i>Act-Gal4</i>, the <i>UAS-dome</i>^ΔCyt, or the <i>UAS-Socs36E</i> transgenes were included as controls (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s012" target="_blank">S5A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s012" target="_blank">S5B</a> Dataset). Mock infections where performed along the experiments and are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s006" target="_blank">S6A and S6B Fig</a>. (<b>C,D</b>) Expression of <i>TotA</i> and <i>vir-1</i> upon DCV infection of wild-type or <i>G9a</i> mutant flies, expressing (<b>C</b>) dome^ΔCyt, or (<b>D</b>) <i>Socs36E</i>. Expression of the gene of interest (by RT-qPCR) was normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and expressed as fold change relative to mock infection (Tris buffer). Data are means and s.d. of three independent pools of at least 15 male flies for each genotype. (<b>A</b>,<b>B</b>) A representative experiment of two independent experiments is shown. Differences in expression of <i>TotA</i> and <i>vir-1</i> were evaluated with a Student’s t-test (*<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

<i>G9a</i> targets genes of the Jak-Stat pathway.

<p>(<b>A</b>) Expression levels of <i>domeless</i>, <i>dPIAS</i>, and <i>Socs36E</i> at 24 hpi in fat bodies of 3 to 5-day-old female wild-type or <i>G9a</i> mutant flies challenged with DCV (10,000 TCID₅₀ units). Data are expressed as fold change relative to mock infection (Tris buffer). (<b>B</b>) Basal expression levels of Jak-Stat genes measured by RT-qPCR on fat bodies of 3 to 5-day-old unchallenged female wild-type and <i>G9a</i> mutant flies. Basal expression is presented as dCt (difference between Ct of the gene of interest and the Ct of <i>Ribosomal Protein 49)</i>. (<b>C</b>) Representative example of a <i>G9a</i> target locus within the <i>domeless</i> gene, defined as a genomic region in which the H3K9me2 mark is present in wild-type flies, but not in <i>G9a</i> mutants, in a previous study [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.ref020" target="_blank">20</a>]. Blue and red plots represent sequence reads in H3K9me2 ChIP-seq analyses of wild-type and <i>G9a</i> mutants, respectively [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.ref020" target="_blank">20</a>]. Gene structure is indicated with boxes for exons, lines for introns, and gray boxes for untranslated regions. The arrow represents the position of the amplicon generated by qPCR after Chromatin-Immunoprecipitation (ChIP-qPCR). (<b>D</b>) H3K9me2 ChIP-qPCR on fat bodies of wild-type or <i>G9a</i> mutant flies. Fold enrichment is the percentage of input of the gene of interest normalized to that of a reference gene with very low H3K9me2 marks (<i>moca</i>). Specificity control experiments for ChIP-qPCR experiments are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004692#ppat.1004692.s005" target="_blank">S5E–S5J Fig</a>. Data are means and s.d. of (<b>A,B</b>) three independent pools of at least 10 fat bodies, or (<b>D</b>) three independent pools of 80 female fat bodies, for each genotype. Data are from one experiment representative of 2 (<b>A</b>,<b>B</b>) or 6 (<b>D</b>) independent experiments. *<i>P</i> < 0.05 (Student’s t-test).</p

Hyperactivation of the Jak-Stat pathway by virus infection of <i>G9a</i> mutants.

<p>(<b>A</b>,<b>B</b>) Expression of inducible immune genes at 24 hours after DCV infection (TCID₅₀ = 10,000) determined by RT-qPCR in (<b>A</b>) whole flies, and (<b>B</b>) fat bodies of wild-type or <i>G9a</i> mutant flies. Expression of the gene of interest was normalized to transcript levels of the housekeeping gene <i>Ribosomal Protein 49</i> and expressed as fold change relative to mock infection (Tris buffer). Data are means and s.d. of three independent pools of (<b>A</b>) 30 female flies and (<b>B</b>) 30 fat bodies for each genotype. (<b>C,D</b>) Basal expression levels of the indicated genes measured by RT-qPCR on 3 to 5-day-old unchallenged wild-type and <i>G9a</i> mutant female flies (<b>C</b>) or fat bodies <b>(D</b>). Basal expression levels are expressed as dCt values (difference between Ct of the gene of interest and the Ct of <i>Ribosomal Protein 49)</i>. (<b>E</b>-<b>I</b>) Expression of inducible Jak-Stat dependent immune genes at (<b>E</b>-<b>H</b>) 24 hpi or (<b>I</b>) 7 dpi with 10,000 TCID₅₀ units of (<b>E</b>) CrPV, (<b>F</b>) DXV, (<b>G</b>) FHV or (<b>H</b>,<b>I</b>) 14,000 TCID₅₀ units of IIV-6. Data are means and s.d. of three independent pools of at least 15 female flies (<b>C,E-I</b>) or at least 10 fat bodies (<b>D</b>) per genotype. Data are from one experiment representative of 3 (<b>A,B,E</b>), and 2 (<b>C</b>,<b>D</b>) independent experiments. *<i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001 (Student’s t-test).</p