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Bacteria belonging to the genus Wolbachia are obligatory microendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by multiple-displacement amplification from limited infected host tissue (0.2 g or 2 x 10(7) cells). We obtained sufficient amounts (8 to 10 microg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.

Objective: To enable serodiscordant couples to reproduce while minimizing the risk of infecting the female partner and to demonstrate the feasibility of including these couples in an insemination program., Design: Prospective follow-up study., Setting: Ambulatory IUIs., Patient(s): Fifty-six HIV-1-serodiscordant couples with a male infected partner., Intervention(s): Sperm cell separation by the gradient density method followed by the swim-up method, virological detection, and IUI after ovarian stimulation., Main Outcome Measure(s): Pregnancy rate, HIV-1 test, and p24 antigenemia in women., Result(s): Two hundred thirteen IUIs were performed, and 37 pregnancies occurred, resulting in 33 births and 2 ongoing pregnancies. Fifty percent of couples have a child. No women were infected., Conclusion(s): Our study demonstrates the efficiency of an IUI program with prepared and virologically tested spermatozoa in serodiscordant couples with an HIV-1-infected male partner, allowing the couples to have children without transmission of the virus to the female partner.

Herbivores that feed on toxic plants must overcome plant defenses and occasionally may even benefit from them. The current challenge is to understand how herbivores evolve the necessary physiological adaptations and which changes at the molecular level are involved. In this context we studied the leaf beetles genus Chrysochus (Coleoptera, Chrysomelidae). Two species of this genus, C. auratus and C. cobaltinus, feed on plants that contain toxic cardenolides. These beetles not only avoid poisoning by the toxin but also use it for their own defense against predators. All other Chrysochus species feed on plants that are devoid of cardenolides. The most important active principle of cardenolides is their capacity to bind to and thereby block the ubiquitous Na(+)/K(+)-ATPase responsible for maintaining cellular potentials. By analyzing the DNA sequence of the putative ouabain-binding site of the alpha-subunit of the Na(+)/K(+)-ATPase gene of Chrysochus and its close relatives feeding on plants with or without cardenolides, we here trace the evolution of cardenolide insensitivity in this group of beetles. The most interesting difference among the sequences involves the amino acid at position 122. Whereas all species that do not encounter cardenolides have an asparagine in this position, both Chrysochus species that feed on cardenolide plants have a histidine instead. This single amino acid substitution has already been shown to confer cardenolide insensitivity in the monarch butterfly. A mtDNA-based phylogeny corroborates the hypothesis that the asparagine at position 122 of the alpha-subunit of the Na(+)/K(+)-ATPase gene as observed in Drosophila and other insects is the plesiomorphic condition in this group of leaf beetles. The later host-plant switch to cardenolide-containing plants in the common ancestor of C. auratus and C. cobaltinus coincides with the exchange of the asparagine for a histidine in the ouabain binding site.

Evidence for the use of defensive compounds for sexual purposes is scarce, even though sexual selection might have some importance for the evolution of defensive traits. This study investigates the effect of defense-related traits and body size on mating success in two sister species of leaf beetle differing in their type of chemical defense. Oreina gloriosa produces autogenous cardenolides, whereas O. cacaliae sequesters pyrrolizidine alkaloids from its food plant. Larger O. gloriosa males with more toxin or higher toxin concentration had a mating advantage, likely due to direct or indirect female choice. In the laboratory, particular pairings recurred repeatedly in this species, indicating mate fidelity. O. gloriosa females were also subject to sexual selection, possibly by male choice, because larger females and those with higher toxin concentration mated more readily and more often. In O. cacaliae, in contrast, sexual selection for toxicity and body size was not detected, or at best was much weaker. Because toxicity is heritable in O. gloriosa but environment-dependent in O. cacaliae, individuals of the former species could be choosing well-defended partners with "good genes." Our study suggests that sexual selection may contribute to the maintenance of heritable defensive traits.

1. The dynamics of the ventilatory response to carbon dioxide inhalation were studied in ten healthy young men using four different inspired fractions of carbon dioxide (FI, CO2) in air (0.015, 0.030, 0.045 and 0.060) successively increasing and decreasing stepwise. 2. Seven such different progressions were performed for each subject and each of seven different durations of the steps (t) ranging between 0.1 (i.e. one ventilatory cycle) and 10 min ('steady-state' conditions). The overall duration of one test (T) was taken as the sum of the seven successive FI, CO2 steps (t) plus one step, t, of air breathing. Thus, the values of T ranged between 0.8 (i.e. eight ventilatory cycles) and 80 min. Three subjects were tested twice. 3. We measured, as a function of T, the magnitude of the loops formed by the curves PA, CO2-VE and the value of the highest ventilatory response (VE max) to each progression. For all ten subjects, both functions had two maxima, one for T values of 2.6 or 8.0 min and one for T values of 24 or 40 min, and one minimum at T equal to 12 min. 4. The same measurements were made on tidal volume-response curves (PA, CO2-VT) and ventilatory frequency-response curves (PA, CO2-f) and yielded the same results except for the ventilatory frequency-response curves, for which we only found a statistically insignificant single maximum for T values of 24 or 40 min. 5. The locations of the maxima in loop magnitude and VE max were similar in duplicate tests in three subjects, whereas the quantitative values of these variables showed wide differences. 6. We compared our results with what is expected from the current linear dynamic model of ventilatory control submitted to the same forcing function: the first maximum in the loop magnitude is predicted by the model, but the second is not. The model shows no peak in the evolution of VE max. 7. We conclude that controlled system dynamics, which are the only ones included in dynamic models of ventilatory control, cannot by themselves account for our observations, and that one should take into consideration the dynamics of the controlling neuronal network.

1. The dynamics of the ventilatory response to isocapnic hypoxia were studied in seven healthy subjects using four different levels of hypoxia, (inspired oxygen pressures, PI,O2 equal to 110, 100, 80 and 60 mmHg) successively increasing and decreasing stepwise. 2. Five such progressions were performed for each subject, corresponding to five different durations of the steps (t) ranging between 0.33 and 5.00 min. The overall duration of one test (T) was taken as the sum of the seven successive PI,O2 hypoxic steps (t) plus one step t of air breathing. Thus, the values of T ranged between 2.6 and 40.0 min. 3. End-tidal CO2 pressure was maintained constant (+/- 1 mmHg) throughout the test by manipulation of inspired CO2 pressure. 4. We measured, as a function of T, (i) the magnitude of the loops formed by the ventilatory response curves (PA,O2-VE) as measured by their surface area (S), (ii) the magnitude of ventilatory response to each rising hypoxic step, and (iii) the difference between resting VE and VE observed at PA,O2 equal to 50 mmHg (delta V50). On average, we found one maximum in absolute value of S at T = 8 min and one minimum at T = 12 min, along with two maxima of ventilatory response at T values of 8 and 24 min. 5. The same measurements were made on tidal volume response curves (PA,O2-VT) and ventilatory frequency response curves (PA,O2-f): on average we observed two non-significant peaks in the progression with T of VT and S(VT) and two significant peaks in that of delta VT,50 for T = 8 and T = 24 min. No significant peak was observed in the progression with T of f curve parameters. 6. These results are discussed together with the current dynamic model of the ventilatory control system, which includes a central neural controller with no dynamics of its own and a linear response to chemoreceptor inputs. We discuss the physiological meaning of a negative loop area in relation to the previously described depressant effect of hypoxia upon the brain stem. 7. We conclude that the dynamics of the controlling neuronal network are responsible for the observed singularities which result from differential sensitivity properties of the controller. We propose the existence of discrete excitatory states of the controller as a possible explanation of the shape of the steady-state response curve to hypoxia and of the loop variations.