Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology, and effects of Bacillus thuringiensis endotoxin

Analysis of larval Aedes aegypti midgut using scanning electron microscopy, nuclear and mitochondrial dyes, response to Bacillus thuringiensis israelensis CryIVB toxin, and electrophysiology is described. The anterior ventriculus ("stomach") region is found to have much lower mitochondrial densities than other midgut regions. The transitional region is distinguished by apical surface architecture, and by region-specific effects of CryIVB endotoxin. In this region CryIVB causes holes ranging from 1.0 to 7.0 microm in diameter (mean 3.3+/-0.53 microm, N=12), blisters 16.9+/-1.54 microm in diameter (N=10), and separation of adjacent cells. The holes are not consistent with damage due to the colloid osmotic lysis model of delta-endotoxin activity. The posterior ventriculus possesses a distinctive cellular architecture consisting of hemispherical, domed apical membranes surrounded by deep clefts. Functional and morphological heterogeneity is revealed within the posterior ventriculus, with the anterior end dominating the electrical profile of isolated, perfused preparations and showing the greatest response to serotonin. Hyperpolarization of the transepithelial potential by serotonin occurred in conjunction with a decrease in the space constant lambda, ruling out closure of ion channels as the mechanism of action of serotonin.     

Distinct domains in the ARC region of the potato resistance protein Rx mediate LRR binding and inhibition of activation

Plant nucleotide binding and leucine-rich repeat (NB-LRR) proteins contain a region of homology known as the ARC domain located between the NB and LRR domains. Structural modeling suggests that the ARC region can be subdivided into ARC1 and ARC2 domains. We have used the potato (Solanum tuberosum) Rx protein, which confers resistance to Potato virus X (PVX), to investigate the function of the ARC region. We demonstrate that the ARC1 domain is required for binding of the Rx N terminus to the LRR domain. Domain-swap experiments with Rx and a homologous disease resistance gene, Gpa2, showed that PVX recognition localized to the C-terminal half of the LRR domain. However, inappropriate pairings of LRR and ARC2 domains resulted in autoactive molecules. Thus, the ARC2 domain is required to condition an autoinhibited state in the absence of elicitor as well as for the subsequent elicitor-induced activation. Our data suggest that the ARC region, through its interaction with the LRR, translates elicitor-induced modulations of the C terminus into a signal initiation event. Furthermore, we demonstrate that physical disruption of the LRR-ARC interaction is not required for signal initiation. We propose instead that this activity can lead to multiple rounds of elicitor recognition, providing a means of signal amplification.     

The isolated anterior stomach of larval mosquitoes (Aedes aegypti): voltage-clamp measurements with a tubular epithelium

The anterior stomach of larval Aedes aegypti was isolated and perfused via two pipettes. For transepithelial voltage (V(te)) measurement, the inflow pipette and the bath were connected via agar bridges to calomel electrodes. For voltage-clamping, the lumen of the tissue contained an Ag/AgCl wire held by the outflow pipette, and the preparation was placed in a bath within a spiral of Ag/AgCl wire. After equilibrating the tissue in mosquito saline on both sides, a V(te) of -8+/-1 mV was measured (+/-S.E.M., N=32). Current-voltage curves (+/-100 mV) demonstrated ohmic behaviour of the epithelium. Short-circuiting resulted in a current (I(sc)) of 103+/-16 microA cm(-2) and a mean transepithelial conductance (G(te)) of 11.8+/-1.3 mS cm(-2) (+/-S.E.M., N=32). A Yonath-Civan plot of G(te) of individual preparations over the corresponding I(sc) resulted in a straight line (r(2)=0.8422), indicating that the difference in I(sc) of individual preparations is mainly based on different transcellular conductances (G(c)). This analysis allowed to estimate the mean leak conductance (G(l) approximately 3.9 mS cm(-2)) and the mean transcellular electromotive force (E(c) approximately 13 mV). After administering 0.2 micromol L(-1) serotonin, I(sc) and G(te) significantly increased, to 457+/-49 microA cm(-2) and to 21.3+/-2.3 mS cm(-2) (+/-S.E.M., N=31, P<0.05), respectively. The Yonath-Civan plot after serotonin resulted again in a straight line (r(2)=0.8219), indicating a mean G(l) of about 1 mS cm(-2) and a mean E(c) of about 22 mV. Dinitrophenol (2.5 mmol L(-1)) almost abolished I(sc) and significantly reduced G(te) (N=6). Concanamycin A (100 micromol L(-1)) reduced I(sc) by more than 90% without significantly affecting G(te).     

Immuno-electron microscopy reveals that the excitotoxin quinolinate is associated with the plasma membrane in human peripheral blood monocytes/macrophages

Quinolinate (QUIN), a tryptophan-derived excitotoxin, was localized ultrastructurally in human peripheral blood monocytes/macrophages (M?) by immuno-electron microscopy. A combined carbodiimide/glutaraldehyde/paraformaldehyde-based fixation procedure was developed for optimal retention of QUIN in the cell as well as minimal loss of ultrastructure; a silver-enhanced colloidal gold detection system was used for electron-microscopic analysis. Gold particles representing QUIN immunoreactivity were associated with the inner side of the plasma membrane in normal M?. The number of gold particles increased significantly when QUIN levels were elevated by treatment with its precursor kynurenine, but location of the gold particles remained essentially the same under this condition. Treatment with interferon-γ increased the number of Golgi bodies, vacuoles and pseudopodia, reflecting the activated state of the cell. Significantly increased numbers of gold particles representing QUIN were detectable in approximately the same location as in the case of kynurenine treatment. Combined treatment with kynurenine and interferon-γ maximally increased the number of gold particles at the periphery of the cell. The pseudopodia were intensely stained with gold particles, while they were not detectable in the inner part of the cytoplasm or in any other organelle even under this activated condition. The significance of the specific location of QUIN revealed in the present study and its relation to the release and subsequent actions of QUIN are discussed. Received: 30 May 1996 / Accepted: 22 May 1997     

How shrub encroachment under climate change could threaten pollination services for alpine wildflowers: A case study using the alpine skypilot,Polemonium viscosum

Under climate change, shrubs encroaching into high altitude plant communities disrupt ecosystem processes. Yet effects of encroachment on pollination mutualisms are poorly understood. Here, we probe potential fitness impacts of interference from encroaching Salix (willows) on pollination quality of the alpine skypilot, Polemonium viscosum. Overlap in flowering time of Salix and Polemonium is a precondition for interference and was surveyed in four extant and 25 historic contact zones. Pollinator sharing was ascertained from observations of willow pollen on bumble bees visiting Polemonium flowers and on Polemonium pistils. We probed fitness effects of pollinator sharing by measuring the correlation between Salix pollen contamination and seed set in naturally pollinated Polemonium. To ascertain whether Salix interference occurred during or after pollination, we compared seed set under natural pollination, conspecific pollen addition, and Salix pollen addition. In current and past contact zones Polemonium and Salix overlapped in flowering time. After accounting for variance in flowering date due to latitude, Salix and Polemonium showed similar advances in flowering under warmer summers. This trend supports the idea that sensitivity to temperature promotes reproductive synchrony in both species. Salix pollen is carried by bumble bees when visiting Polemonium flowers and accounts for up to 25% of the grains on Polemonium pistils. Salix contamination correlates with reduced seed set in nature and when applied experimentally. Postpollination processes likely mediate these deleterious effects as seed set in nature was not limited by pollen delivery. Synthesis: As willows move higher with climate change, we predict that they will drive postpollination interference, reducing the fitness benefits of pollinator visitation for Polemonium and selecting for traits that reduce pollinator sharing.