Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and Rickettsia typhi.

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.

Host-driven population dynamics in an herbivorous insect.

Understanding the nature and relative importance of endogenous (density-dependent) and exogenous (density-independent) effects on population dynamics remains a central problem in ecology. Evaluation of these forces has been constrained by the lack of long time series of population densities and largely limited to populations chosen for their unique dynamics (e.g., outbreak insects). Especially in herbivore populations, the relative contributions of bottom-up and top-down effects (resources and natural enemies, respectively) have been difficult to compare because population data have rarely been combined with resource measurements. The feeding scars of a wood-mining herbivorous insect (Phytobia betulae Kangas; Diptera: Agromyzidae) of birch trees (Betula pendula and Betula pubescens) provided long time series data (47 and 65 years) of absolute abundance (larvae/tree) in replicated trees within replicated stands. Measurements of tree annual rings provided matching time series of host age and physiological status. Analyses showed a powerful exogenous effect of stand age on temporal variation in insect abundance (58 and 32% of the variance in two populations, respectively). With the additional effects of variation among trees, 77 and 64% of the total variance in abundance was attributable to exogenous bottom-up effects of host plants. Potential endogenous effects were evident as immediate linear density dependence, but only accounted for approximately 10% of the total variance. Abundance of Phytobia is primarily a function of disturbance history, which produces a mosaic of different aged birch stands that harbor Phytobia populations of different sizes. Density-dependence tends to regulate local populations around levels determined by host suitability.

Characterization of CD4+ T cells in mouse bone marrow. I. Increased activated/memory phenotype and altered TCR Vbeta repertoire.

A significant proportion of memory B cells home to bone marrow (BM) which is a major site of anamnestic antibody responses in mice. We hypothesized that memory T cells likewise accumulate in BM perhaps to provide help for antibody production, and that the compartment of CD4+ T cells in BM of unimmunized mice would be enriched for memory phenotype cells that might have been activated by environmental antigens. The phenotype of activated/memory CD4+ lymphocytes has been defined as CD44hi CD45RBlo CD62L-. Conversely, the phenotype of immunologically naive cells is CD44lo CD45RBhi CD62L+. Flow cytrometric analysis of tissue from normal, adult C57BL/6 mice identified 1-2 % CD3+CD4+ cells in BM. Up to 40 % of CD3+CD4+ cells in the BM expressed the activated/memory phenotype compared with < or = 10% in the spleen and lymph nodes. Analysis of TCR Vbeta repertoire revealed that expression of Vbeta3 and Vbeta7 genes was increased as much as fourfold in BM compared to the periphery; most of this increase was within the CD44hi T cells. The accumulation of activated/memory T cells and clonotypic expansion(s) was not seen in the BM of germ-free mice, indicating that it reflects the history of the animal's exposure to antigens. Finally, immunization of mice which express a transgenic T cell receptor specific for ovalbumin peptide resulted in appearance of antigen-specific T cells with activated/memory phenotype in the BM.

Age-related changes in antibody repertoire: contribution from T cells.

The immune system of aged mice produces antibodies that are characterized by low affinity, diminished protection against infections and autoreactivity. It has been shown that these antibodies may be encoded by different immunoglobulin V genes and that the mechanism of somatic hypermutation in the V genes is inefficient. Studies on scid mice reconstituted with B and T cells from donors of different ages suggested that both lymphocyte subsets may contribute to the age-related changes in antibody repertoire. With help provided by T cells from young mice, the response to a hapten, nitrophenyl(acetyl), became gradually dominated by B-cell clones that rearranged a particular germline VH gene (V186.2). However, help from the aged T cells resulted in a heterogeneous response of B cells expressing many different V segments. Analysis of discrete foci of primary antibody-forming cells suggested that the aged T-helper cells are unable to govern the normally-occurring competition between the B-cell clones that have different affinities for the hapten. It is proposed that a signaling disequilibrium from the aged T cells, which provide less efficient help in quantitative terms, supports the growth of low-affinity B cells. This process may be exacerbated due to the apparent hyperactivity of aged B cells to CD40-mediated mitogenic signal.

Variability in grape phylloxera preference and performance on canyon grape (Vitis arizonica).

We tested the deme-formation hypothesis experimentally with four populations of leaf-galling grape phylloxera, Daktulosphaira vitifoliae, and its host, canyon grape (Vitis arizonica). An experiment designed to examine preference and performance showed that phylloxera populations did not significantly prefer their original host relative to other hosts in the percent available leaves galled. There were significant herbivore population effects (P<0.01), host effects (P<0.001), and population x host interaction effects (P<0.001). Herbivore populations had different colonizing abilities (performance, as measured in the mean number of galls per leaf) on an individual host (P<0.001), but there was no host effect. Host genotype significantly affected phylloxera performance, measured as survivorship (P<0.01), but a phylloxera population did not necessarily have higher survivorship on its original host. Differences in fecundity, an-other measurement of performance, were due to intrinsic differences among herbivore populations (P<0.05), and not related to host genotype. There was no correlation between distance from a phylloxera population in the field and a host's susceptibility to attack. There was a significant positive relationship between levels of infestation on a clone in the field and its susceptibility to colonization experimentally (P<0.05), suggesting inherent differences in host resistance and susceptibility. These results did not support the deme-formation hypothesis. In a second experiment, host clone x water treatment interactions affected phylloxera survivorship (P<0.05) and fecundity (P<0.05). We conclude that host genotype x environment interactions may prevent sessile, parthenogenetic herbivores from locally adapting to individual host genotypes.

Parasitoid pressure and the radiation of a gallforming group (Cecidomyiidae: Asphondylia spp.) on creosote bush (Larrea tridentata).

We tested the Enemy Impact Hypothesis, which predicts that communities of one tropic level are organized by the tropic level above. In the case of gallforming insect communities, the hypothesis predicts that gall morphology will diverge, minimizing the number of parasitoids shared among species. We used the monophyletic group of gallforming cecidomyiids (Asphondylia spp.) on creosote bush (Larrea tridentata) to test this hypothesis, predicting that species with thicker gall walls should exclude species of parasitoids with shorter ovipositors and have lower levels of parasitism. Of 17 parasitoid species reared from Asphondylia galls on creosote bush, 9 accounted for over 98% of parasitism. Seven of these 9 species had ovipositors long enough to penetrate 10 of 13 gall morphs measured. There was no significant relationship between gall wall thickness and number of associated parasitoid species (r (2)=0.01, P>0.05, n=13). There was no relationship between gall wall thickness and types of parasitoid species colonizing galls: parasitoids with the shortest ovipositors colonized all types of gall morphs and were dominant members of the parasitoid assemblages in galls with the thickest walls. Ultimately, there were no significant differences in percent parasitism among Asphondylia species, regardless of gall wall thickness. We found no difference in numbers of associated parasitoids or percent parasitism in galls with different textures (e.g. hairy versus smooth), different locations on the plant or different phenologies. Our results suggest that enemy impact has not influenced the diversity of this gall community. Gall wall thickness, phenology, location on the plant and surface structure do not appear to influence the distribution of parasitoid species. Other explanations are offered to account for diversity in gall morphology among these species.

Carbon-nutrient balance hypothesis in within-species phytochemical variation ofSalix lasiolepis.

Predictions of the carbon-nutrient balance hypothesis were tested using a study of within-species phytochemical variation in the arroyo willow,Salix lasiolepis. The prediction that a balance between nutrients (total protein) and carbon-based secondary metabolites (total phenols) should exist was supported using water treatment and fertilizer experiments and wild willow clones. Leaf nitrogen content and net photosynthetic rates of plants potted in soil in which parental plants grew was low, indicating that wild plants exist under relatively low nutrient status-high carbon balance conditions. The hypothesis also correctly predicted positive relationships between shoot length and phenols in glasshouse plants, wild plants, and plants in the water treatment experiment and negative relationships between shoot length and phenols in the fertilizer treatment experiment. Total phenolic glycosides, fragilin, picein, salicortin, tremulacin, and tremuloidin all correlated positively with shoot length in glasshouse plants on a carbon-biased balance, and male willows had generally lower levels of phenolic glycosides than females. Salicortin and tremulacin showed the strongest positive relationships with shoot length.

Why does the bud-galling sawfly, Euura mucronata, attack long shoots?

The bud-galling sawfly, Euura mucronata, attacked longer shoot length classes on its host, Salix cinerea, more frequently than shorter shoots. Shoot length accounted for 76 to 93 percent of the variance in number of galls per 100 shoots in three habitats: forest, watermeadow, and lakeside. The reasons for this pattern were addressed with studies on shoot length in relation to: 1. Number of resources (buds) per shoot; 2. Success in establishment of larvae in galls; 3. Gall size and resources per gall; and 4. Survival of larvae after establishment as influenced by plant resistance and natural enemy attack. The most important factors proved to be success in establishment of larvae, with percent of variance accounted for ranging from 57 to 77 percent in three of four sites where relationships were significant, and survival after establishment of larvae, with variance accounted for ranging from 40 to 54 percent in the same three sites. The pattern of survival was dictated by plant resistance and not by natural enemies. These two additive factors resulted in a general relationship across all sites of increasing emergence of fully developed larvae per cohort as shoot length increased, accounting for 78 percent of the variance. These adaptive advantages to attacking longer shoots are sufficient to account for the pattern of increased probability of shoots being attacked as they increase in length.

Plant age and attack by the bud galler, Euura mucronata.

As ramets of the willow, Salix cinerea L. (Salicaceae) aged shoot length decreased in the six populations studied in S.E. Finland. Many traits correlated positively with shoot length: basal diameter, number of internodes, internode length, leaf size, and length of growing period. The bud-galling sawfly, Euura mucronata (Hartig) Man. (Churchill) (Hymenoptera: Tenthredinidae), responded positively to shoot length or correlated traits, and negatively to ramet age in three forest populations. This herbivore attacked the most vigorous plants in a population, and numbers of attacks declined as ramets aged and senesced. The generality of this kind of herbivore response to plant quality is emphasized.

Patterns of intra- and interspecific association of gall-forming sawflies in relation to shoot size on their willow host plant.

Four species of gall-forming sawflies were each frequently found to have clumped distributions among shoots within their willow host plant across four sites and three years. When all species were considered together by clone, year, and site, species showed independence of distribution among shoots two thirds of the time and showed positive covariance one third of the time. When pairs of species were considered separately, but clones were combined within sites and years, 60% of the chi-square tests of association were significant. All but one of the significant tests showed positive associations between pairs of species. The stem galler was positively associated with the leaf folder at all sites in all years, and the petiole galler was positively associated with the stem galler and leaf folder for most year by site combinations. When species paris co-occurred on shoots they were usually found at the same or higher density as when found alone on shoots. Only 2 of 100 tests showed a depressed density of a species when co-occurring on shoots with heterospecifics.All sawfly species were found on shoots that were significantly larger (mean node number) than on shoots without sawflies, and species responded to shoot size variation similarly. Sizes of shoots occupied by heterospecific species combinations were usually significantly larger than shoots with only conspecifics, for all species. These data supported the hypothesis that similar species' responses to within-plant variation would lead to positive rather than negative or random species associations. The data do not support the hypothesis that interspecific competition was important in determining shoot choice or species density.

Variation in herbivore density among host plants and its consequences for community structure : Field studies on willow sawflies.

The densities of four species of gall-forming sawflies were found to vary significantly among willow host plant clones. Two of the speices varied among host plants at four sites in each of three years. The other two species varied in density among host plants at most of the sites in two of the three years. Total sawfly density also varied significantly among clones. Individual species densities on willow clones were significantly positively correlated between years when all sites were combined and frequently when sites were considered separately. Most pairwise species combinations were independent in density between years, but some negative correlations existed between the stem galler and the leaf galler. Gall-former densities also were largely independent among clones within years with all sites combined and with sites considered separately. The significant correlations were nearly all positive. At all four sites the combination of significant variation in sawfly densities among willow clones in the field and independence of species densities among clones resulted in significantly different communities (relative abundance of species) among willow clones in three years. Although sawfly abundances differed substantially among the four sites, this remained true. It is argued that the pattern of community structure among clones is the result of variation in host plant quality of clones. We propose an hypothesis to account for patterns of herbivore species associations based on intrapopulation host plant variation.

Trail odors: recognition by insects parasitic on cocoons.

Female parasitic insects in the genera Pleolophus, Endasys, and Mastrus (Hymenoptera: Ichneumonidae) search the ground cover for hosts and avoid areas they have already inspected. Females respond to their own trail odor, and recognition occurs also between conspecific, congeneric, and intergeneric individuals. This is the first direct evidence for recognition between parasitoids at sites other than on the host itself, and between females of different species.