Is there a missing link? Effects of root herbivory on plant-pollinator interactions and reproductive output in a monocarpic species.

Herbivores can have a major influence on plant fitness. The direct impact of herbivory on plant reproductive output has long been studied, and recently also indirect effects of herbivory on plant traits and pollinator attraction have received increasing attention. However, the link between these direct and indirect effects has seldom been studied. In this study, we investigated effects of root herbivory on plant and floral traits, pollination success and reproductive outcome in the monocarpic perennial Cynoglossum officinale. We exposed 119 C. officinale plants to a range of root herbivore damage by its specialist herbivore Mogulones cruciger. We assessed the effect of herbivory on several plant traits, pollinator foraging behaviour and reproductive output, and to elucidate the link between these last two we also quantified pollen deposition and pollen tube growth and applied a pollination experiment to test whether seed set was pollen-limited. Larval root herbivory induced significant changes in plant traits and had a negative impact on pollinator visitation. Infested plants were reduced in size, had fewer flowers and received fewer pollinator visits at plant and flower level than non-infested plants. Also, seed set was negatively affected by root herbivory, but this could not be attributed to pollen limitation since neither stigmatic pollen loads and pollen tube growth nor the results of the hand-pollination experiment differed between infested and non-infested plants. Our observations demonstrate that although herbivory may induce significant changes in flowering behaviour and resulting plant-pollinator interactions, it does not necessarily translate into higher rates of pollen limitation. The observed reductions in reproductive output following infection can mainly be attributed to higher resource limitation compared to non-infested plants.

Severe outbreeding and inbreeding depression maintain mating system differentiation in Epipactis (Orchidaceae).

In hermaphroditic plants, theory for mating system evolution predicts that populations will evolve to either complete autonomous selfing (AS) or complete outcrossing, depending on the balance between automatic selection favouring self-fertilization and costs resulting from inbreeding depression (ID). Theory also predicts that selection for selfing can occur rapidly and is driven by purging of genetic load and the loss of ID. Therefore, selfing species are predicted to have low levels of ID or even to suffer from outbreeding depression (OD), whereas predominantly outcrossing species are expected to have high levels of ID. To test these predictions, we related the capacity of AS to the magnitude of early-acting inbreeding or OD in both allogamous and autogamous species of the orchid genus Epipactis. For each species, the level of AS was assessed under controlled greenhouse conditions, whereas hand-pollinations were performed to quantify early costs of inbreeding or OD acting at the level of fruit and seed production. In the autogamous species, the capacity of AS was high (> 0.72), whereas in the allogamous species AS was virtually absent (< 0.10). Consistent with our hypothesis, allogamous Epipactis species had significantly higher total ID (average: 0.46) than autogamous species, which showed severe costs of OD (average: -0.45). Overall, our findings indicate that strong early-acting ID represents an important mechanism that contributes to allogamy in Epipactis, whereas OD may maintain selfing in species that have evolved to complete selfing.

Secondary pollen presentation and the temporal dynamics of stylar hair retraction and style elongation in Campanula trachelium (Campanulaceae).

To increase the accuracy of pollen capture and transfer by pollinators some plant species have developed secondary pollen presentation structures. Because the presence of secondary pollen presentation structures at the pistil may reduce the spatial separation between the sexual functions and increase the risk of self-interference and selfing, temporal segregation of the sexual organs, triggered by visiting insects, can be expected to occur. We investigated secondary pollen presentation and the temporal dynamics of the sexual phases in combination with the physiological self-incompatibility system in Campanula trachelium, a protandrous insect-pollinated herb. Stylar hair retraction (male function) and curling of the stigmatic lobes (female function) were modelled using Gompertz growth functions. Finally, we performed pollination experiments in the lab and field to assess seed set and pollen limitation under natural conditions. About 68% of the total pollen load was captured by stylar hairs. Manual manipulation of the stylar hairs, mimicking pollinator visitation, significantly shortened the male phase and accelerated the female phase, resulting in a significant decline in temporal overlap between the two sexual functions. Conversely, when pollinators and/or manual manipulations were lacking, the male phase was substantially prolonged and sexual overlap was maximal. This suggests that spreading of the sexual phases and thus the risk of sexual interference are largely determined by the interaction between stylar hairs and visiting pollinators. Natural seed set was high and not pollinator limited. Overall, these results indicate that secondary pollen presentation and partial protandry resulted in efficient pollen capture, transfer and deposition.

Temporal and spatial variation in flower and fruit production in a food-deceptive orchid: a five-year study.

Deceptive orchids are generally characterized by low levels of fruit set; however, there may be substantial variations in fruit set between sites and years. Within a single population, individual plants may also differ greatly in their reproductive output as a result of differences in inflorescence size or local density. In this study, we determined flower and fruit production over 5 years in two populations of the food-deceptive orchid, Orchis purpurea. All plants were monitored annually for survival and flowering at each site to determine whether flowering and fruiting induced costs. The number of flowers per inflorescence varied considerably from year to year (min: 36.6, max: 49.5). Average fruit set was low (7%) and varied considerably among years and populations. A considerable proportion of plants also failed to set any fruit. However, the probability of producing at least one fruit was not affected by inflorescence size or local density. The number of fruits was significantly related to inflorescence size, but proportional fruit set was not. Local density also did not affect the number of fruits, nor proportional fruit set. There was also no evidence that plants with large inflorescence size or high fruiting success had a larger probability of remaining vegetative the year after flowering than plants with small inflorescence size or low fruiting success. Our results suggest that pollinator-mediated selective forces on inflorescence size through female reproductive success alone are weak, most likely because of the low overall level of visitation and the resulting uncertainty of pollination at the individual level. Our results further demonstrate that investigation of patterns of fruit set over several years is needed to better understand the variability in female reproductive success that is typical of most plant-pollinator interactions.

Floral display size and spatial distribution of potential mates affect pollen deposition and female reproductive success in distylous Pulmonaria officinalis (Boraginaceae).

In animal-pollinated plants, both the spatial distribution of flowering individuals and the number of flowers that an individual displays affect pollen deposition rates and female reproductive success. Heterostylous species are likely to be particularly sensitive to the contingencies of spatial distribution, as they are reproductively subdivided into distinct mating groups, which usually exhibit self- and intra-morph incompatibility and differ in floral morphology. In this paper, we explore the joint effects of both spatial distribution of potential mates and floral display size on morph-specific pollen deposition rates and seed set patterns in two natural populations of Pulmonaria officinalis, a distylous species with a weak self-incompatibility system. Both total stigmatic pollen load and the proportion of legitimate pollen decreased with increasing spatial isolation. Legitimate (intermorph) pollen transfer was, however, asymmetric and decreased more rapidly with decreasing proximity to a compatible legitimate mating partner in the S-morph than in the L-morph. Total stigmatic pollen loads per flower increased with increasing floral display size, indicating that large plants are disproportionately more visited than smaller individuals. However, because legitimate pollen deposition decreased with increasing floral display size, these results also suggest that larger numbers of flowers increase the degree of geitonogamous pollination. In both the L- and S-morph, seed set significantly decreased with increasing isolation from a legitimate mating partner, but in the L-morph seed set was less dependent on the spatial distribution of the S-morph. In addition, seed set significantly increased with floral display size in the L-morph, but not in the S-morph. These findings indicate that the spatial distribution of potential mates and variation in floral display size may cause morph-specific differences in pollen deposition rates and female reproductive success.

Morph-ratio variation, population size and female reproductive success in distylous Pulmonaria officinalis (Boraginaceae).

Theory predicts that morph ratios in heterostylous populations are governed by negative frequency-dependent selection typically resulting in equal morph ratios at equilibrium. Previous work on the distylous perennial herb Pulmonaria officinalis, however, showed asymmetric mating between floral morphs and a weak self-incompatibility system, with the long-styled morph (L-morph) producing significantly higher seed set following intramorph crosses and even selfing than the short-styled morph (S-morph), two aspects thought to affect female fecundity and morph-ratio variation. Here, we evaluated morph ratios and population size of all known P. officinalis populations in the northern part of Belgium. Morph ratios deviated significantly from 1:1 (range 0.09-1 L-morph frequency, mean = 0.58). Relative fecundity of the S-morph (i.e. mean seed set of the S-morph/mean seed set of the L-morph) was on average 0.73, was positively related to the frequency of the L-morph, and reached 1 (similar levels of female fecundity) at an average L-morph frequency of 0.66 in the population. As some small populations had the S-morph in majority, our results suggest that local morph ratios are influenced both by the relative fecundity of L- and S-morph individuals and by stochastic processes in small populations.

Some tips and tricks in reading cervical spine radiographs in trauma patients.

Despite the fact that we are living in the era of spiral CT and multidetector spiral CT allowing us to scan the whole spine in less than a minute and to make high quality multiplanar reformatted images, plain films remain important in clearing the spine of polytraumatised patients. Particularly hemodynamic unstable patients that must be urgently transferred to the operation room need to be rapidly cleared for (cervical) spine lesions, without being transported to the CT unit which is--in most hospitals--located relatively far away from the emergency room and the operation rooms. In these patients, for the time being, spiral CT cannot replace conventional radiographs for the detection of sometimes subtle lesions. Knowledge of the direct but also the indirect signs of fractures remains important in the evaluation of these plain films. This article demonstrates some of these sometimes subtle signs that can help in making the diagnosis of these lesions.

Benign bone tumours of the spine.

A wide variety of primary bone tumours can involve the spine. The imaging features of these lesions are often characteristic. We present an overview of the primary benign bone tumours of the spine. The role of plain film, CT and MRI is discussed.

Reconstitution of the human 5-HT(1D) receptor-G-protein coupling: evidence for constitutive activity and multiple receptor conformations.

The 5-hydroxytryptamine (5-HT) 1D/1B receptors have gained particular interest as potential targets for treatment of migraine and depression. G-protein coupling and other intrinsic properties of the human 5-HT(1D) receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT(1D) receptor with Galpha(i1), alpha(i2), alpha(i3), or Galpha(o)-proteins and Gbeta(1)gamma(2)-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [(3)H]5-HT to this receptor, whereas the Galpha(q)beta(1)gamma(2) heterotrimer was ineffective in this respect. Competition of [(3)H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT(1D) receptor with Galpha(i/o)beta(1)gamma(2) reconstitutes the receptor in a high affinity agonist binding state, having the same pharmacological profile as the receptor expressed in mammalian cells. Binding of the antagonist ocaperidone to the human 5-HT(1D) receptor in coupled or noncoupled state was analyzed. This compound competed with [(3)H]5-HT binding more potently on the human 5-HT(1D) receptor in the noncoupled state, showing its inverse agonistic character. Ocaperidone acted as a competitive inhibitor of [(3)H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [(35)S]GTPgammaS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT(1D) receptor. Taken together, the reconstitution of the human 5-HT(1D) receptor-G-protein coupling using baculovirus-infected Sf9 cells made possible the assessment of coupling specificity and the detection of different binding states of the receptor induced by G-protein coupling or ligand binding.

A novel expression cloning method to isolate mammalian transcription factors in Schizosaccharomyces pombe.

We describe a novel expression cloning strategy in the fission yeast for the isolation of mammalian transcription factors using a mammalian promoter as target. This strategy is possible because of the conservation between mammalian cells and Schizosaccharomyces pombe of the mechanism that leads to the selection of the transcription start site. It also opens new perspectives to investigate the transcriptional regulation of genes for which detailed promoter analysis is difficult.

Oct-1 is involved in the transcriptional repression of the von willebrand factor gene promoter.

The negative regulation of transcription of the human von Willebrand factor (vWF) gene was investigated in human umbilical vein endothelial cells (HUVECs) and HeLa cells. A fragment spanning -89 to +244 nucleotides (nt), containing the first exon, is active in HUVECs only but not in HeLa cells. The activity of this promoter is sharply reduced by mutagenesis of the GATA binding site at +221. Extension of the upstream sequences from nt -89 to -142 and to -496 results in progressive reduction of the activity of the -89 to +244 promoter identifying a negative regulatory element between nt -142 and -89. A factor present in nuclear extracts from endothelial and nonendothelial cells binds to an AT-rich sequence located between nt -133 and -125. Mutagenesis of the AT-rich sequence interferes with nuclear protein binding and restores the activity of the -142 to +244 fragment to the level of the -89 to +244 promoter. Binding of the nuclear protein to the vWF AT-rich sequence in mobility shift assays is inhibited by competition with a consensus Oct-1 binding site and with a silencer octamer-like sequence from the vascular cell adhesion molecule-1 (VCAM-1) promoter. Subsequent supershift experiments identified Oct-1 as the transcription factor that binds to vWF and VCAM-1 silencer elements. These results indicate that Oct-1 acts as a transcriptional repressor of promoters of genes expressed in endothelial cells.

Identical cis-acting elements and related trans-acting factors control activity of nonviral promoter in Schizosaccharomyces pombe and mammalian cells.

We have analyzed the transcriptional activity of the human plasminogen activator inhibitor-1 promoter in the fission yeast Schizosaccharomyces pombe. This promoter is active in S. pombe, and the initiation site of transcription corresponds to the site identified previously in mammalian cells. Mutations in the AP-1-binding site (PAI-1 A box) or the HLTF-binding site (the B box), which reduced the basal and phorbol ester-induced levels of PAI-1 expression in human cells, also decreased the transcriptional activity in S. pombe. Gel retardation assays showed that an S. pombe protein binds specifically to this B box element and displays the same B box sequence requirement as HLTF. Furthermore, this yeast protein binds specifically to other HLTF-binding sites in the human immunodeficiency virus-1 long terminal repeat (LTR) and the simian virus 40 (SV40) enhancer. The B box (but not a mutated B box) strongly stimulated transcription when combined with adh downstream promoter elements, indicating that the S. pombe B box-binding protein, like HLTF, is a transcriptional activator. We conclude that the transcriptional activity of the nonviral PAI-1 promoter is controlled by the same promoter elements in S. pombe as in mammalian cells. In addition, mammalian trans-acting factors that bind to these promoter elements were shown to have counterparts with conserved DNA-binding activity in S. pombe. These results further illustrate the conservation of the mechanism of transcription between mammalian cells and fission yeast.

Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe.

Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.

Pancreas divisum: a teenager with calculi in the duct of Santorini. Report of a case treated by double drainage procedure.

Since the age of sixteen, a teenager suffers from acute recurrent pancreatitis with a weight loss of 17 kg, without evident etiologic factor. At laparotomy the duct of Wirsung is absent; calculi are found in a grossly dilated duct of Santorini and the minor papilla is stenosed with periductal fibrosis and a large retention pseudocyst. Recurrent pancreatitis is cured by sphincteroplasty of the minor papilla and side-to-side pancreatico-jejunostomy. After a follow-up of 17 months the recurrent pancreatitis seems cured by sphincteroplasty of the minor papilla and side-to-side pancreatico-jejunostomy.