Bacteriophage Migration via Nematode Vectors: Host-Parasite-Consumer Interactions in Laboratory Microcosms

Pathogens vectored by nematodes pose serious agricultural, economic, and health threats; however, little is known of the ecological and evolutionary aspects of pathogen transmission by nematodes. Here we describe a novel model system with two trophic levels, bacteriophages and nematodes, each of which competes for bacteria. We demonstrate for the first time that nematodes are capable of transmitting phages between spatially distinct patches of bacteria. This model system has considerable advantages, including the ease of maintenance and manipulation at the laboratory bench, the ability to observe many generations in short periods, and the capacity to freeze evolved strains for later comparison to their ancestors. More generally, experimental studies of complex multispecies interactions, host-pathogen coevolution, disease dynamics, and the evolution of virulence may benefit from this model system because current models (e.g., chickens, mosquitoes, and malaria parasites) are costly to maintain, are difficult to manipulate, and require considerable space. Our initial explorations centered on independently assessing the impacts of nematode, bacterium, and phage population densities on virus migration between host patches. Our results indicated that virus transmission increases with worm density and host bacterial abundance; however, transmission decreases with initial phage abundance, perhaps because viruses eliminate available hosts before migration can occur. We discuss the microbial growth dynamics that underlie these results, suggest mechanistic explanations for nematode transmission of phages, and propose intriguing possibilities for future research

<i>LncRNA-HIT</i> Functions as an Epigenetic Regulator of Chondrogenesis through Its Recruitment of p100/CBP Complexes

<div><p>Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), <i>LNCRNA-HIT</i> in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, <i>LncRNA-HIT</i> was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of <i>LncRNA-HIT</i>-p100/CBP complexes by ChIRP-seq revealed <i>LncRNA-HIT</i> associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by <i>LncRNA-HIT-</i>p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in <i>LncRNA-HIT</i> or p100 transcripts revealed a significant decrease in expression of many of the <i>LncRNA-HIT</i>-associated loci. <i>LncRNA-HIT</i> siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the <i>LncRNA-HIT</i> siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that <i>LncRNA-HIT</i> is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using <i>LncRNA-HIT</i> and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.</p></div

Co-recruitment of <i>LncRNA-HIT</i> and p100 is required to stimulate gene expression from a synthetic locus.

<p><b>(A)</b> Analysis of UAS-luciferase reporter activation after GAL4-λN-BoxB<i>LncRNA-HIT</i> and GAL4-p100 recruitment. <b>(B)</b> Analysis of UAS-luciferase reporter activation in the absence of GAL4-p100. <b>(C)</b> Model of UAS-luciferase reporter activation in response to the recruitment of <i>LncRNA-HIT</i> and p100. For panels A and B, increasing dosages (0, 50, 100, 250, ng) of the BoxB or BoxB-<i>LncRNA-HIT</i> transcripts are represented by the black triangle.</p

siRNA-mediated loss of <i>LncRNA-HIT</i> affects H3K27ac at its site of nascent transcription.

<p><b>(A)</b> Relative location of <i>LncRNA-HIT</i> and its nearest neighbors, <i>Hoxa11</i> and <i>Hoxa13</i>. <b>(B)</b> Visualization of the nascent <i>LncRNA-HIT</i> (green peaks) detected by ChIRP-seq using the USCS Genome Browser. Peaks identified by the even and odd <i>LncRNA-HIT</i> probe sets are indicated on the left axis using even and odd probe sets specific for the <i>LncRNA-HIT</i> lncRNA and displayed by the UCSC genome browser. The localization of the fourteen H3K27ac peaks (black peaks) proximal to the <i>LncRNA-HIT</i> associated peaks as determined by overlaying the H3K27ac limb bud ChIP-seq dataset, GSE3064 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005680#pgen.1005680.ref062" target="_blank">62</a>]. <b>(C)</b> qChIP Analysis of the fourteen H3K27ac regions proximal to the nascent <i>LncRNA-HIT</i> peaks revealed a loss in fragment enrichment in response to the <i>LncRNA-HIT</i> siRNA treatments in limb bud mesenchyme. Values represent the mean fold change H3K27ac fragment enrichment compared to parallel treatments using scrambled <i>LncRNA-HIT</i> siRNA controls for three independent assays. Error bars represent the standard deviation of the mean for the three independent assays.</p

Subcellular localization of <i>LncRNA-HIT</i> and <i>Gapdh</i> transcripts in limb mesenchyme using single molecule RNA fluorescent in situ hybridization (FISH).

<p><b>(A-D)</b><i>LncRNA-HIT</i> transcripts are detected in the nucleus of undifferentiated limb mesenchyme at E 11.0. Red signal = detection of <i>LncRNA-HIT</i> probe sets labeled with CalFluor610. Blue signal = DAPI staining of the nuclear DNA. <b>(E</b> and <b>F)</b> <i>Gapdh</i> transcripts are detected in the cytoplasm of undifferentiated limb mesenchyme. Green signal = detection of Gaph probe sets labeled with CalFluor610 and pseudo-colored green. Blue signal = DAPI staining of the nuclear DNA. <b>(G)</b> Negative control using RNase A prior to hybridization with the <i>LncRNA-HIT</i> probe sets reveals no detected <i>LncRNA-HIT</i> (red signal) in the nucleus confirming the detected signal in panels A-D represent hybridization with the <i>LncRNA-HIT</i> transcript. The nuclear DNA was unaffected by the RNase A treatment and stained positively with DAPI (blue signal). <b>(H)</b> Negative control using RNase A prior to hybridization with the <i>Gapdh</i> probe sets reveals no detected <i>Gapdh</i> transcript (green signal) in the cytoplasm confirming the detected signal in panels E and F represent hybridization with the <i>Gapdh transcript</i>. The nuclear DNA was unaffected by the RNase A treatment and stains positively with DAPI (blue signal). Bar = 10 μm.</p

Model of enhancer RNA function of <i>LncRNA-HIT</i> at the 5’ HoxA locus.

<p>Model of enhancer RNA function of <i>LncRNA-HIT</i> at the 5’ HoxA locus.</p