RESUMEN
Theory predicts unified sex ratios for most organisms, yet biases may be engendered by selfish genetic elements such as endosymbionts that kill or feminize individuals with male genotypes. Although rare, feminization is established for Wolbachia-infected Eurema butterflies. This paradigm is presently confined to islands in the southern Japanese archipelago, where feminized phenotypes produce viable all-daughter broods. Here, we characterize sex bias for E. hecabe in continental Australia. Starting with 186 wild-caught females, we reared >6000 F1-F3 progeny in pedigree designs that incorporated selective antibiotic treatments. F1 generations expressed a consistent bias across 2 years and populations that was driven by an ~5% incidence of broods comprising ⩾80% daughters. Females from biased lineages continued to overproduce daughters over two generations of outcrossing to wild males. Treatment with antibiotics of differential strength influenced sex ratio only in biased lineages by inducing an equivalent incomplete degree of son overproduction. Brood sex ratios were nevertheless highly variable within lineages and across generations. Intriguingly, the cytogenetic signature of female karyotype was uniformly absent, even among phenotypic females in unbiased lineages. Molecular evidence supported the existence of a single Wolbachia strain at high prevalence, yet this was not clearly linked to brood sex bias. In sum, we establish an inherited, experimentally reversible tendency for incomplete offspring bias. Key features of our findings clearly depart from the Japanese feminization paradigm and highlight the potential for more subtle degrees of sex distortion in arthropods.
Asunto(s)
Mariposas Diurnas/genética , Razón de Masculinidad , Animales , Australia , Mariposas Diurnas/microbiología , Femenino , Masculino , Linaje , WolbachiaRESUMEN
Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.
Asunto(s)
Aedes/microbiología , Aedes/virología , Virus del Dengue/fisiología , Dengue/prevención & control , Control Biológico de Vectores/métodos , Wolbachia/clasificación , Wolbachia/fisiología , Aedes/fisiología , Animales , Dengue/transmisión , Dengue/virología , Virus del Dengue/clasificación , Virus del Dengue/aislamiento & purificación , Femenino , Aptitud Genética , Humanos , Insectos Vectores/microbiología , Insectos Vectores/fisiología , Insectos Vectores/virología , Masculino , Reproducción/fisiología , Saliva/virologíaRESUMEN
Genetic manipulations of insect populations for pest control have been advocated for some time, but there are few cases where manipulated individuals have been released in the field and no cases where they have successfully invaded target populations. Population transformation using the intracellular bacterium Wolbachia is particularly attractive because this maternally-inherited agent provides a powerful mechanism to invade natural populations through cytoplasmic incompatibility. When Wolbachia are introduced into mosquitoes, they interfere with pathogen transmission and influence key life history traits such as lifespan. Here we describe how the wMel Wolbachia infection, introduced into the dengue vector Aedes aegypti from Drosophila melanogaster, successfully invaded two natural A. aegypti populations in Australia, reaching near-fixation in a few months following releases of wMel-infected A. aegypti adults. Models with plausible parameter values indicate that Wolbachia-infected mosquitoes suffered relatively small fitness costs, leading to an unstable equilibrium frequency <30% that must be exceeded for invasion. These findings demonstrate that Wolbachia-based strategies can be deployed as a practical approach to dengue suppression with potential for area-wide implementation.
Asunto(s)
Aedes/microbiología , Aedes/virología , Virus del Dengue/fisiología , Dengue/prevención & control , Dengue/transmisión , Control Biológico de Vectores/métodos , Wolbachia/fisiología , Aedes/fisiología , Animales , Dengue/microbiología , Dengue/virología , Virus del Dengue/aislamiento & purificación , Drosophila melanogaster/microbiología , Femenino , Humanos , Insectos Vectores/microbiología , Insectos Vectores/fisiología , Insectos Vectores/virología , Masculino , Queensland , Factores de Tiempo , Wolbachia/aislamiento & purificaciónRESUMEN
Wolbachia are inherited intracellular bacteria that infect a broad range of invertebrate hosts. They commonly manipulate host reproduction in a variety of ways and thereby favour their invasion into host populations. While the biology of Wolbachia has been extensively studied at the ecological and phenotypic level, little is known about the molecular mechanisms underlying the interaction between Wolbachia and their hosts. Recent comparative genomics studies of Wolbachia strains have revealed putative candidate genes involved in the expression of cytoplasmic incompatibility (CI) in insects. However the functional testing of these genes is hindered by the lack of available genetic tools in Wolbachia. To circumvent this problem we generated transgenic Drosophila lines expressing various Wolbachia CI candidate genes under the control of the GAL4/UAS system in order to evaluate their possible role in Wolbachia-related phenotypes in Drosophila. The expression of a number of these genes in Drosophila melanogaster failed to mimic or alter CI phenotypes across a range of Wolbachia backgrounds or in the absence of Wolbachia.
Asunto(s)
Citoplasma/genética , Drosophila melanogaster/genética , Drosophila melanogaster/microbiología , Técnicas Genéticas , Wolbachia/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Animales Modificados Genéticamente/microbiología , Citoplasma/microbiología , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Femenino , Regulación de la Expresión Génica , Genes de Insecto , Masculino , Datos de Secuencia Molecular , Wolbachia/fisiologíaRESUMEN
alpha-DsbA1 is one of two DsbA homologues encoded by the Gram-negative alpha-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The alpha-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet alpha-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, b = 49.5, c = 69.3 A, beta = 107.0 degrees and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 A using a copper anode and data from SeMet alpha-DsbA1 crystals were recorded to 2.45 A resolution using a chromium anode.
Asunto(s)
Proteína Disulfuro Isomerasas/química , Wolbachia/química , Cristalización , Cristalografía por Rayos X , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/químicaRESUMEN
The mitochondria of legume root nodules are critical to sustain the energy-intensive process of nitrogen fixation. They also generate reactive oxygen species at high rates and thus require the protection of antioxidant enzymes and metabolites. We show here that highly purified mitochondria from bean nodules (Phaseolus vulgaris L. cv. Contender x Rhizobium leguminosarum bv. phaseoli strain 3622) contain ascorbate peroxidase primarily in the inner membrane (with lesser amounts detected occasionally in the matrix), guaiacol peroxidases in the outer membrane and matrix, and manganese superoxide dismutase (MnSOD) and an ascorbate-regenerating system in the matrix. This regenerating system relies on homoglutathione (instead of glutathione) and pyridine nucleotides as electron donors and involves the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and homoglutathione reductase. Homoglutathione is synthesized in the cytosol and taken up by the mitochondria and bacteroids. Although bacteroids synthesize glutathione, it is not exported to the plant in significant amounts. We propose a model for the detoxification of peroxides in nodule mitochondria in which membrane-bound ascorbate peroxidase scavenges the peroxide formed by the electron transport chain using ascorbate provided by L-galactono-1,4-lactone dehydrogenase in the inner membrane. The resulting monodehydroascorbate and dehydroascorbate can be recycled in the matrix or cytosol. In the matrix, the peroxides formed by oxidative reactions and by MnSOD may be scavenged by specific isozymes of guaiacol peroxidase, ascorbate peroxidase, and catalase.
Asunto(s)
Antioxidantes/metabolismo , Fabaceae/metabolismo , Glutatión/análogos & derivados , Mitocondrias/metabolismo , Raíces de Plantas/metabolismo , Ascorbato Peroxidasas , Catalasa/metabolismo , Citosol/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Fabaceae/enzimología , Glutatión/biosíntesis , Isoenzimas/metabolismo , Malato Deshidrogenasa/metabolismo , Proteínas de la Membrana/metabolismo , NADH Deshidrogenasa/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fijación del Nitrógeno/fisiología , Oxidorreductasas/metabolismo , Péptido Sintasas/metabolismo , Peroxidasa/metabolismo , Peroxidasas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/microbiología , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , SimbiosisRESUMEN
The thiol tripeptides glutathione (GSH) and homoglutathione (hGSH) are very abundant in legume root nodules and their synthesis is catalyzed by the enzymes gamma-glutamylcysteine synthetase (gammaECS), GSH synthetase (GSHS), and hGSH synthetase (hGSHS). As an essential step to elucidate the role of thiols in N(2) fixation we have isolated cDNAs encoding the three enzymes and have quantified the transcripts in nodules. Assay of enzyme activities in highly purified nodule organelles revealed that gammaECS is localized in the plastids, hGSHS in the cytosol, and GSHS in the cytosol and mitochondria. These results are consistent with sequence analyses. Subcellular fractionation of nodules also showed that bacteroids contain high thiol concentrations and high specific gammaECS and GSHS activities. Results emphasize the role of nodule plastids in antioxidant protection and in control of thiol synthesis, and suggest that plastids may be important in the stress response of nodules. Overall, our results provide further evidence that thiol synthesis is critical for nodule functioning.
Asunto(s)
Fabaceae/genética , Glutatión Sintasa/genética , Fijación del Nitrógeno , Péptido Sintasas/genética , Plantas Medicinales , Fracciones Subcelulares/enzimología , Secuencia de Aminoácidos , Bacteroides/enzimología , Northern Blotting , Compartimento Celular , Clonación Molecular , Citosol/enzimología , ADN Complementario/aislamiento & purificación , Fabaceae/enzimología , Fabaceae/microbiología , Glutatión Sintasa/metabolismo , Datos de Secuencia Molecular , Péptido Sintasas/metabolismo , Filogenia , Raíces de Plantas/enzimología , Raíces de Plantas/ultraestructura , Plastidios/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , SimbiosisRESUMEN
High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and gamma-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for gamma-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants.
Asunto(s)
Fabaceae/metabolismo , Glutatión Sintasa/metabolismo , Glutatión/análogos & derivados , Glutatión/biosíntesis , Péptido Sintasas/metabolismo , Plantas Medicinales , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , ADN Complementario , Fabaceae/genética , Glutatión Sintasa/química , Glutatión Sintasa/genética , Datos de Secuencia Molecular , Péptido Sintasas/química , Péptido Sintasas/genética , Raíces de Plantas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O(2) diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O(2) limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2-4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by gamma-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.
Asunto(s)
Oscuridad , Fabaceae/enzimología , Fabaceae/fisiología , Estrés Oxidativo , Raíces de Plantas/enzimología , Raíces de Plantas/fisiología , Plantas Medicinales , Antioxidantes/metabolismo , Ascorbato Peroxidasas , Metabolismo de los Hidratos de Carbono , Respiración de la Célula , Fabaceae/ultraestructura , Ferritinas/análisis , Glutatión/análogos & derivados , Glutatión/metabolismo , Nitratos/metabolismo , Fijación del Nitrógeno/fisiología , Nitrogenasa/metabolismo , Nucleótidos/metabolismo , Oxidantes/metabolismo , Oxígeno/metabolismo , Pisum sativum/enzimología , Pisum sativum/fisiología , Pisum sativum/ultraestructura , Peroxidasas/metabolismo , Raíces de Plantas/ultraestructura , Compuestos de Sulfhidrilo/análisis , Factores de TiempoRESUMEN
Ascorbate peroxidase (AP) is a key enzyme that scavenges potentially harmful H2O2 and thus prevents oxidative damage in plants, especially in N2-fixing legume root nodules. The present study demonstrates that the nodule endodermis of alfalfa (Medicago sativa) root nodules contains elevated levels of AP protein, as well as the corresponding mRNA transcript and substrate (ascorbate). Enhanced AP protein levels were also found in cells immediately peripheral to the infected region of soybean (Glycine max), pea (Pisum sativum), clover (Trifolium pratense), and common bean (Phaseolus vulgaris) nodules. Regeneration of ascorbate was achieved by (homo)glutathione and associated enzymes of the ascorbate-glutathione pathway, which were present at high levels. The presence of high levels of antioxidants suggests that respiratory consumption of O2 in the endodermis or nodule parenchyma may be an essential component of the O2-diffusion barrier that regulates the entry of O2 into the central region of nodules and ensures optimal functioning of nitrogenase.
Asunto(s)
Antioxidantes/metabolismo , Fabaceae/enzimología , Regulación de la Expresión Génica de las Plantas , Peroxidasas/biosíntesis , Plantas Medicinales , Ascorbato Peroxidasas , Ácido Ascórbico/metabolismo , Difusión , Fabaceae/fisiología , Regulación Enzimológica de la Expresión Génica , Medicago sativa , Oxígeno/metabolismo , Consumo de Oxígeno , Pisum sativum , Raíces de Plantas , Poaceae , Glycine maxRESUMEN
The effect of short-term nitrate application (10 mM, 0-4 d) on nitrogenase (N2ase) activity, antioxidant defenses, and related parameters was investigated in pea (Pisum sativum L. cv Frilene) nodules. The response of nodules to nitrate comprised two stages. In the first stage (0-2 d), there were major decreases in N2ase activity and N2ase-linked respiration and concomitant increases in carbon cost of N2ase and oxygen diffusion resistance of nodules. There was no apparent oxidative damage, and the decline in N2ase activity was, to a certain extent, reversible. The second stage (>2 d) was typical of a senescent, essentially irreversible process. It was characterized by moderate increases in oxidized proteins and catalytic Fe and by major decreases in antioxidant enzymes and metabolites. The restriction in oxygen supply to bacteroids may explain the initial decline in N2ase activity. The decrease in antioxidant protection is not involved in this process and is not specifically caused by nitrate, since it also occurs with drought stress. However, comparison of nitrate- and drought-induced senescence shows an important difference: there is no lipid degradation or lipid peroxide accumulation with nitrate, indicating that lipid peroxidation is not necessarily involved in nodule senescence.
RESUMEN
The involvement of activated oxygen in the drought-induced damage of pea (Pisum sativum L. cv Frilene) nodules was examined. To this purpose, various pro-oxidant factors, antioxidant enzymes and related metabolites, and markers of oxidative damage were determined in nodules of well-watered (nodule water potential approximately -0.29 MPa) and water-stressed (nodule water potential approximately -2.03 MPa) plants. Water-stressed nodules entered senescence as evidenced by the 30% decrease in leghemoglobin and total soluble protein. Drought also caused a decrease in the activities of catalase (25%), ascorbate peroxidase (18%), dehydroascorbate reductase (15%), glutathione reductase (31%), and superoxide dismutase (30%), and in the contents of ascorbate (59%), reduced (57%) and oxidized (38%) glutathione, NAD+ and NADH (43%), NADP+ (31%), and NADPH (17%). The decline in the antioxidant capacity of nodules may result from a restricted supply of NAD(P)H in vivo for the ascorbate-glutathione pathway and from the Fe-catalyzed Fenton reactions of ascorbate and glutathione with activated oxygen. The 2-fold increase in the content of "catalytic Fe" would also explain the augmented levels of lipid peroxides (2.4-fold) and oxidatively modified proteins (1.4-fold) found in water-stressed nodules because of the known requirement of lipid and protein oxidation for a transition catalytic metal.