Co-occurrences of parasite clones and altered host phenotype in a snail-trematode system

The frequent co-occurrence of two or more genotypes of the same parasite species in the same individual hosts has often been predicted to select for higher levels of virulence. Thus, if parasites can adjust their level of host exploitation in response to competition for resources, mixed-clone infections should have more profound impacts on the host. Trematode parasites are known to induce a wide range of modifications in the morphology (size, shell shape or ornamentation) of their snail intermediate host. Still, whether mixed-clone trematode infections have additive effects on the phenotypic alterations of the host remains to be tested. Here, we used the snail Potamopyrgus antipodarum-infected by the trematode Coitocaecum parvum to test for both the general effect of the parasite on host phenotype and possible increased host exploitation in multi-clone infections. Significant differences in size, shell shape and spinosity were found between infected and uninfected snails, and we determined that one quarter of naturally infected snails supported mixed-clone infections of C. parvum. From the parasite perspective, this meant that almost half of the clones identified in this study shared their snail host with at least one other clone. Intra-host competition may be intense, with each clone in a mixed-clone infection experiencing major reductions in volume and number of sporocysts (and consequently multiplication rate and cercarial production) compared with single-clone infections. However, there was no significant difference in the intensity of host phenotype modifications between single and multiple-clone infections. These results demonstrate that competition between parasite genotypes may be strong, and suggest that the frequency of mixed-clone infections in this system may have selected for an increased level of host exploitation in the parasite population, such that a single-clone is associated with a high degree of host phenotypic alteration.     

Ongoing hybridization obscures phylogenetic relationships in the Drosophila subquinaria species complex

Inferring evolutionary relationships among recently diverged lineages is necessary to understand how isolating barriers produce independent lineages. Here, we investigate the phylogenetic relationships between three incompletely isolated and closely related mushroom‐feeding Drosophila species. These species form the Drosophila subquinaria species complex and consist of one Eurasian species (D. transversa) and two widespread North American species (D. subquinaria and D. recens) that are sympatric in central Canada. Although patterns of pre‐ and post‐mating isolation among these species are well characterized, previous work on their phylogenetic relationships is limited and conflicting. In this study, we generated a multi‐locus data set of 29 loci from across the genome sequenced in a population sample from each species, and then, we inferred species relationships and patterns of introgression. We find strong statistical support that D. subquinaria is paraphyletic, showing that samples from the geographic region sympatric with D. recens are most closely related to D. recens, whereas samples from the geographic region allopatric with D. recens are most closely related to D. transversa. We present several lines of evidence that both incomplete lineage sorting and gene flow are causing phylogenetic discordance. We suggest that ongoing gene flow primarily from D. recens into D. subquinaria in the sympatric part of their ranges causes phylogenetic uncertainty in the evolutionary history of these species. Our results highlight how population genetic data can be used to disentangle the sources of phylogenetic discordance among closely related species.     

Remodeling of the endoplasmic reticulum in Caenorhabditis elegans oocytes is regulated by CGH‐1

A key aspect of development in all metazoans is remodeling at the cellular level. During the development of gametes, remodeling occurs throughout the germ line. When Caenorhabditis elegans hermaphrodites become depleted of sperm after 4 days of adulthood, significant cellular remodeling occurs within the meiotically‐arrested oocytes, including the formation of ribonucleoprotein granules. Since major remodeling of the endoplasmic reticulum (ER) occurs in early embryos, we investigated the extent of ER remodeling in meiotically‐arrested oocytes. We found, using a combination of fluorescence reporters and transmission electron microscopy, that the ER in arrested oocytes accumulates in patches and sheets that are enriched at the cortex. Our findings suggest this remodeling is not due to simple displacement by large amounts of yolk that accumulate in arrested oocytes, and instead may be genetically regulated. We further identified the Ddx6 RNA helicase, CGH‐1, as a key regulator of ER in the germ line. In cgh‐1(tn691) oocytes, we detected cortical ER patches as well as aberrant granules of the RNA‐binding proteins, PAB‐1, MEX‐3, and CGH‐1. Taken together, our results suggest the possibility that the spatial organization of RNA binding proteins may regulate the translation of mRNAs associated with the ER that in turn, controls the organization of the ER in the adult germ line.     

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.     

Expression of the B7.1 costimulatory molecule on pancreatic beta cells abrogates the requirement for CD4 T cells in the development of type 1 diabetes

Although HLA-DQ8 has been implicated as a key determinant of genetic susceptibility to human type 1 diabetes, spontaneous diabetes has been observed in HLA-DQ8 transgenic mice that lack expression of murine MHC class II molecules (mII(-/-)) only when the potent costimulatory molecule, B7.1, is transgenically expressed on pancreatic beta cells. To study the contribution of HLA-DQ8 to the development of diabetes in this model, we crossed RIP-B7.1mII(-/-) mice with a set of transgenic mouse lines that differed in their HLA-DQ8 expression patterns on APC subpopulations, in particular dendritic cells and cortical thymic epithelial cells. Surprisingly, we found that even in the absence of HLA-DQ8 and CD4 T cells, a substantial fraction of the RIP-B7.1mII(-/-) mice developed diabetes. This disease process was remarkable for not only showing insulitis, but also inflammatory destruction of the exocrine pancreas with diffusely up-regulated expression of MHC class I and ICAM-1 molecules. Expression of HLA-DQ8 markedly increased the kinetics and frequency of diabetes, with the most severe disease in the lines with the highest levels of HLA-DQ8 on cortical thymic epithelial cells and the largest numbers of CD4 T cells. However, the adoptive transfer of diabetes was not HLA-DQ8-dependent and disease could be rapidly induced with purified CD8 T cells alone. Expression of B7.1 in the target tissue can thus dramatically alter the cellular and molecular requirements for the development of autoimmunity.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

Genetic diversity of dodder (<Emphasis Type="Italic">Cuscuta</Emphasis> spp.) collected from commercial cranberry production as revealed in the<Emphasis Type="Italic">trn</Emphasis>L (UAA) intron

Swamp dodder (Cuscuta gronovii) is a parasitic plant detrimental to cranberries. Observation of emergence of dodder seeds collected from a cultivated cranberry bog in Massachusetts revealed 2 or more peak emergence times during 4 consecutive growing seasons. Molecular methods were used to investigate genetic variation among the emerging dodder seedlings. On emergence, dodder seedlings were collected and analyzed for DNA sequence diversity in thetrnL (UAA) intron, a noncoding region of chloroplast DNA. DNA sequence analysis of 87 seedlings collected during the 1999 and 2000 growing seasons revealed the presence of 2 dodder ecotypes, designated A and B. Comparative DNA sequence analysis indicated that in thetrnL (UAA) intron, the sequence of ecotype A is identical to that ofCuscuta gronovii, whereas the sequence of ecotype B is closest to that ofCuscuta attenuata (99.3% sequence identity; 293 bases considered). ABg/II restriction enzyme cut site was identified that distinguished between thetrnL (UAA) introns of ecotypes A and B. Restriction fragment length polymorphism (RFLP) was used to analyze the sequences of 100 seedlings collected during the growing seasons of 2001 and 2002. Only 10 of the 187 samples were ecotype A, all of which emerged on or before May 7 in the growing seasons. Therefore, the predominant dodder haplotype found in this study may be a close relative ofC. attenuata and notC. gronovii, the common species found in cranberry bogs.     

Combined effect of CCR5-Delta32 heterozygosity and the CCR5 promoter polymorphism -2459 A/G on CCR5 expression and resistance to human immunodeficiency virus type 1 transmission

Exposed seronegative individuals (ES) with persistent high-risk sexual behavior may be less susceptible to human immunodeficiency virus type 1 (HIV-1) infection because they carry the chemokine receptor (CR) gene alleles CCR5 open reading frame (ORF) Delta32, CCR5 promoter -2459G, or CCR2 ORF 64I (CCR2-64I), all of which have been found to diminish HIV-1 infectivity and/or disease progression. To investigate this, we determined the haplotypes for these three genetic loci in 93 ES and 247 low-risk control individuals. To test if protective haplotypes exert their effect by modulating CR expression, we measured the protein expression of CCR5 and CXCR4 on circulating CD4+ T cells and CD14+ monocytes in 71 ES and 92 controls. To avoid investigator bias, the analysis was performed without knowledge of each subject's risk and genotype. The CCR5 -2459G allele was significantly enriched in ES Caucasian men, who constituted the majority (84%) of the ES cohort, compared to the control Caucasian men (P = 0.02). This increase was mostly attributable to a higher frequency of the -2459 A/G versus the -2459 A/A genotype in individuals heterozygous for the delta32 allele (P = 0.012). No protective influence of the CCR2-64I allele was observed. The haplotypes CCR5 ORF delta32/CCR5 -2459A (in complete linkage disequilibrium) and CCR5 ORF wt/CCR5 -2459G had a cumulative negative effect on the expression of CCR5, since we measured significantly reduced CCR5 densities on both T-helper cells and monocytes only when both haplotypes were present. Densities of CCR5 on lymphocytes and monocytes were correlated (r = 0.59; P < 0.0001), indicating concordance of CCR5 expression patterns across different cell types. We conclude that the CCR5 ORF delta32/wt-CCR5 -2459 A/G genotype combination offers an advantage in resisting sexual HIV-1 transmission and that this effect is mediated by a relative paucity of CCR5 on potential target cells of HIV-1.