Antibody responses of domestic animals to salivary antigens of Triatomainfestans as biomarkers for low-level infestation of triatomines

Hematophagous arthropods such as Triatomainfestans, the vector of Trypanosomacruzi, elicit host-immune responses during feeding. Characterization of antibody responses to salivary antigens offers the potential to develop immunologically based monitoring techniques for exposure to re-emergent triatomine bug populations in peridomestic animals. IgG-antibody responses to the salivary antigens of T.infestans have been detected in chickens as soon as 2 days after the first exposure to five adult bugs. Chickens and guinea pigs regularly exposed to this number of triatomines showed a significantly lower anti-saliva antibody titre than animals exposed to 25 adults and fifth instars of four different T.infestans strains originating from Bolivia and from Northern Chile. Highly immunogenic salivary antigens of 14 and 21 kDa were recognised by all chicken sera and of 79 kDa by all guinea pig sera. Cross-reactivity studies using saliva or salivary gland extracts from different hematophagous species, e.g. different triatomines, bed bugs, mosquitoes, sand flies and ticks, as well as chicken sera exposed to triatomines and mosquitoes, demonstrated that the 14 and 21 kDa salivary antigens were only found in triatomines. Sera from peridomestic chickens and guinea pigs in sites of known T.infestans challenge in Bolivia also recognised the 14 and 21 kDa antigens. These represent promising epidemiological markers for the detection of small numbers of feeding bugs and hence may be a new tool for vector surveillance in Chagas disease control programs.     

Spatial variation in abiotic and biotic factors in a floodplain determine anuran body size and growth rate at metamorphosis

Body size at metamorphosis is a critical trait in the life history of amphibians. Despite the wide-spread use of amphibians as experimental model organisms, there is a limited understanding of how multiple abiotic and biotic factors affect the variation in metamorphic traits under natural conditions. The aim of our study was to quantify the effects of abiotic and biotic factors on spatial variation in the body size of tadpoles and size at metamorphosis of the European common toad (Bufo b. spinosus). Our study population was distributed over the riverbed (active tract) and the fringing riparian forest of a natural floodplain. The riverbed had warm ponds with variable hydroperiod and few predators, whereas the forest had ponds with the opposite characteristics. Spatial variation in body size at metamorphosis was governed by the interactive effects of abiotic and biotic factors. The particular form of the interaction between water temperature and intraspecific tadpole density suggests that abiotic factors laid the foundation for biotic factors: intraspecific density decreased growth only at high temperature. Predation and intraspecific density jointly reduced metamorphic size. Interspecific density had a negligible affect on body size at metamorphosis, suggesting weak inter-anuran interactions in the larval stage. Population density at metamorphosis was about one to two orders of magnitudes higher in the riverbed ponds than in the forest ponds, mainly because of lower tadpole mortality. Based on our results, we conclude that ponds in the riverbed appear to play a pivotal role for the population because tadpole growth and survival is best in this habitat.     

Hemifusion arrest by complexin is relieved by Ca2+-synaptotagmin I

Synaptic transmission relies on an exquisitely orchestrated series of protein-protein interactions. Here we show that fusion driven by neuronal SNAREs is inhibited by the regulatory protein complexin. Furthermore, inner-leaflet mixing is strongly impaired relative to total lipid mixing, indicating that inhibition by complexin arrests fusion at hemifusion. When the calcium sensor synaptotagmin is added in the presence of calcium, inhibition by complexin is relieved and full fusion rapidly proceeds.     

Transition of galactosyltransferase 1 from trans-Golgi cisterna to the trans-Golgi network is signal mediated

The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate beta1,4-galactosyltransferase 1 (galT) and alpha2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT-siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.     

Integrated sampling procedure for metabolome analysis

Metabolome analysis, the analysis of large sets of intracellular metabolites, has become an important systems analysis method in biotechnological and pharmaceutical research. In metabolic engineering, the integration of metabolome data with fluxome and proteome data into large-scale mathematical models promises to foster rational strategies for strain and cell line improvement. However, the development of reproducible sampling procedures for quantitative analysis of intracellular metabolite concentrations represents a major challenge, accomplishing (i) fast transfer of sample, (ii) efficient quenching of metabolism, (iii) quantitative metabolite extraction, and (iv) optimum sample conditioning for subsequent quantitative analysis. In addressing these requirements, we propose an integrated sampling procedure. Simultaneous quenching and quantitative extraction of intracellular metabolites were realized by short-time exposure of cells to temperatures < or =95 degrees C, where intracellular metabolites are released quantitatively. Based on these findings, we combined principles of heat transfer with knowledge on physiology, for example, turnover rates of energy metabolites, to develop an optimized sampling procedure based on a coiled single tube heat exchanger. As a result, this sampling procedure enables reliable and reproducible measurements through (i) the integration of three unit operations into a one unit operation, (ii) the avoidance of any alteration of the sample due to chemical reagents in quenching and extraction, and (iii) automation. A sampling frequency of 5 s(-)(1) and an overall individual sample processing time faster than 30 s allow observing responses of intracellular metabolite concentrations to extracellular stimuli on a subsecond time scale. Recovery and reliability of the unit operations were analyzed. Impact of sample conditioning on subsequent IC-MS analysis of metabolites was examined as well. The integrated sampling procedure was validated through consistent results from steady-state metabolite analysis of Escherichia coli cultivated in a chemostat at D = 0.1 h(-)(1).     

Sequence characterization and expression patterns of defensin and lysozyme encoding genes from the gut of the reduviid bug Triatoma brasiliensis

The cDNAs encoding an intestinal defensin (def1) and lysozyme (lys1) of the reduviid bug Triatoma brasiliensis have been amplified by PCR using specific oligonucleotide primers and 5'- and 3'-RACE, cloned and sequenced. The 576 bp clone has an open reading frame of 282 bp and encodes a pre-prodefensin with 94 amino acid residues, containing a putative signal and activation peptide cleavage site at Ser19 and Arg51, respectively. The genomic DNA contains a second defensin gene with similar characteristics, 88.3% identity and also one intron of 107 nucleotides. The 538 bp clone has an open reading frame of 417 bp, encoding a pre-lysozyme with 139 amino acid residues. The putative signal peptide is cleaved at alanine 18. Using whole mount in situ hybridization, high expression of both genes has been found, distributed uniformly throughout the entire cardia and the blood-storing stomach and to a much lower extent in the digesting small intestine. Using quantitative real-time PCR, the expression level of def1 was also shown to be very low in small intestine, rectum and salivary glands; in the stomach, expression was 500-2500 times higher than in the cardia and fat body. No expression of lys1 could be detected in the salivary glands and rarely a very low expression in the small intestine, rectum and fat body. Lys1 expression in the stomach was 60-300 times higher than in the cardia. Comparing the levels in unfed fifth instars and up to 15 days after feeding, a strong def1 induction was evident in the fat body at 15 days after feeding and in the stomach a maximum level of def1 and lys1 at 5 days after feeding.     

Modulation of the adrenocortical response to acute stress with respect to brood value, reproductive success and survival in the Eurasian hoopoe

Reproducing parents face the difficult challenge of trading-off investment in current reproduction against presumed future survival and reproduction. Glucocorticoids are supposed to mediate this trade-off because the adrenocortical response to stress disrupts normal reproductive behaviour in favour of self-maintenance and own survival. According to the brood-value hypothesis, individuals with a low survival probability until the next reproductive season have to invest in current reproduction, a process driven by a down-regulation of their adrenocortical response. If the adrenocortical response to stress effectively mediates the trade-off between current reproduction versus future survival and reproduction, we expect a negative relationship with reproductive success and a positive correlation of the adrenocortical stress response with survival. We studied the relationship between corticosterone secretion in parents and their current brood value, reproductive success and survival in a short-lived multi-brooded bird, the Eurasian hoopoe Upupa epops. The adrenocortical response to acute handling stress was correlated with the brood value within the individual (first and second broods of the year) and between individuals. Birds breeding late in the season mounted a lower total corticosterone response to acute stress than birds breeding earlier, while females showed lower levels than males. We observed a negative relationship between the adrenocortical stress response and rearing success or fledging success in females, as predicted by the brood-value hypothesis. However, we could not evidence a clear link between the adrenocortical stress response and survival. Future research testing the brood-value hypothesis and trade-offs between current reproduction and future survival should also measure free corticosterone and carefully differentiate between cross-sectional (i.e. between-individual) and individual-based experimental studies.     

Impact of density and environmental factors on population fluctuations in a migratory passerine

1. Populations of plants and animals typically fluctuate because of the combined effects of density-dependent and density-independent processes. The study of these processes is complicated by the fact that population sizes are typically not known exactly, because population counts are subject to sampling variance. Although the existence of sampling variance is broadly acknowledged, relatively few studies on time-series data have accounted for it, which can result in wrong inferences about population processes. 2. To increase our understanding of population dynamics, we analysed time series from six Central European populations of the migratory red-backed shrike Lanius collurio by simultaneously assessing the strength of density dependence, process and sampling variance. In addition, we evaluated hypotheses predicting effects of factors presumed to operate on the breeding grounds, at stopover sites in eastern Africa during fall and spring migration and in the wintering grounds in southern Africa. We used both simple and state-space formulations of the Gompertz equation to model population size. 3. Across populations and modelling approaches, we found consistent evidence for negative density-dependent population regulation. Further, process variance contributed substantially to variation in population size, while sampling variance did not. Environmental conditions in eastern and southern Africa appear to influence breeding population size, as rainfall in the Sahel during fall migration and in the south African wintering areas were positively related to population size in the following spring in four of six populations. In contrast, environmental conditions in the breeding grounds were not related to population size. 4. Our findings suggest negative density-dependent regulation of red-backed shrike breeding populations and are consistent with the long-standing hypothesis that conditions in the African staging and wintering areas influence population numbers of species breeding in Europe. 5. This study highlights the importance of jointly investigating density-dependent and density-independent processes to improve our understanding of factors influencing population fluctuations in space and time.