Ryuichi Yamada

Distinct Shifts in Microbiota Composition during Drosophila Aging Impair Intestinal Function and Drive Mortality

Alterations in the composition of the intestinal microbiota have been correlated with aging and measures of frailty in the elderly. However, the relationships between microbial dynamics, age-related changes in intestinal physiology, and organismal health remain poorly understood. Here, we show that dysbiosis of the intestinal microbiota, characterized by an expansion of the Gammaproteobacteria, is tightly linked to age-onset intestinal barrier dysfunction in Drosophila. Indeed, alterations in the microbiota precede and predict the onset of intestinal barrier dysfunction in aged flies. Changes in microbial composition occurring prior to intestinal barrier dysfunction contribute to changes in excretory function and immune gene activation in the aging intestine. In addition, we show that a distinct shift in microbiota composition follows intestinal barrier dysfunction, leading to systemic immune activation and organismal death. Our results indicate that alterations in microbiota dynamics could contribute to and also predict varying rates of health decline during aging in mammals.

Host Adaptation of a Wolbachia Strain after Long-Term Serial Passage in Mosquito Cell Lines

ABSTRACT The horizontal transfer of the bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. The experimental transfer of Wolbachia between distantly related host species often results in the loss of infection, presumably due to an inability of Wolbachia to adapt quickly to the new host. To examine the process of adaptation to a novel host, we transferred a life-shortening Wolbachia strain, wMelPop, from the fruit fly Drosophila melanogaster into a cell line derived from the mosquito Aedes albopictus. After long-term serial passage in this cell line, we transferred the mosquito-adapted wMelPop into cell lines derived from two other mosquito species, Aedes aegypti and Anopheles gambiae. After a prolonged period of serial passage in mosquito cell lines, wMelPop was reintroduced into its native host, D. melanogaster, by embryonic microinjection. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into the original host, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility compared to the original strain. We interpret these shifts in phenotype as evidence for genetic adaptation to the mosquito intracellular environment. The use of cell lines to preadapt Wolbachia to novel hosts is suggested as a possible strategy to improve the success of transinfection in novel target insect species.

High Anti-Viral Protection without Immune Upregulation after Interspecies Wolbachia Transfer

Wolbachia, endosymbionts that reside naturally in up to 40–70% of all insect species, are some of the most prevalent intracellular bacteria. Both Wolbachia wAu, naturally associated with Drosophila simulans, and wMel, native to Drosophila melanogaster, have been previously described to protect their hosts against viral infections. wMel transferred to D. simulans was also shown to have a strong antiviral effect. Here we directly compare one of the most protective wMel variants and wAu in D. melanogaster in the same host genetic background. We conclude that wAu protects better against viral infections, it grows exponentially and significantly shortens the lifespan of D. melanogaster. However, there is no difference between wMel and wAu in the expression of selected antimicrobial peptides. Therefore, neither the difference in anti-viral effect nor the life-shortening could be attributed to the immune stimulation by exogenous Wolbachia. Overall, we prove that stable transinfection with a highly protective Wolbachia is not necessarily associated with general immune activation.

Male development time influences the strength of Wolbachia-induced cytoplasmic incompatibility expression in Drosophila melanogaster.

Cytoplasmic incompatibility (CI) is the most widespread reproductive modification induced in insects by the maternally inherited intracellular bacteria, Wolbachia. Expression of CI in Drosophila melanogaster is quite variable. Published papers typically show that CI expression is weak and often varies between different Drosophila lines and different labs reporting the results. The basis for this variability is not well understood but is often considered to be due to unspecified host genotype interactions with Wolbachia. Here, we show that male development time can greatly influence CI expression in D. melanogaster. In a given family, males that develop fastest express very strong CI. The “younger brothers” of these males (males that take longer to undergo larval development) quickly lose their ability to express the CI phenotype as a function of development time. This effect is independent of male age effects and is enhanced when flies are reared under crowded conditions. No correlation is seen between this effect and Wolbachia densities in testes, suggesting that a more subtle interaction between host and symbiont is responsible. The observed younger brother effect may explain much of the reported variability in CI expression in this species. When male development time is controlled, it is possible to obtain consistently high levels of CI expression, which will benefit future studies that wish to use D. melanogaster as a model host to unravel CI mechanisms.

High carbohydrate–low protein consumption maximizes Drosophila lifespan

Dietary restriction extends lifespan in a variety of organisms, but the key nutritional components driving this process and how they interact remain uncertain. In Drosophila, while a substantial body of research suggests that protein is the major dietary component affecting longevity, recent studies claim that carbohydrates also play a central role. To clarify how nutritional factors influence longevity, nutrient consumption and lifespan were measured on a series of diets with varying yeast and sugar content. We show that optimal lifespan requires both high carbohydrate and low protein consumption, but neither nutrient by itself entirely predicts lifespan. Increased dietary carbohydrate or protein concentration does not always result in reduced feeding-the regulation of food consumption is best described by a constant daily caloric intake target. Moreover, due to differences in food intake, increased concentration of a nutrient within the diet does not necessarily result in increased consumption of that particular nutrient. Our results shed light on the issue of dietary effects on lifespan and highlight the need for accurate measures of nutrient intake in dietary manipulation studies.

Microbes Promote Amino Acid Harvest to Rescue Undernutrition in Drosophila

Microbes play an important role in the pathogenesis of nutritional disorders such as protein-specific malnutrition. However, the precise contribution of microbes to host energy balance during undernutrition is unclear. Here, we show that Issatchenkia orientalis, a fungal microbe isolated from field-caught Drosophila melanogaster, promotes amino acid harvest to rescue the lifespan of undernourishedxa0flies. Using radioisotope-labeled dietary components (amino acids, nucleotides, and sucrose) to quantify nutrient transfer from food to microbe to fly, we demonstrate that I.xa0orientalis extracts amino acids directly from nutrient-poor diets and increases protein flux to the fly. This microbial association restores body mass, protein, glycerol, and ATP levels and phenocopies the metabolic profile of adequately fed flies. Our study uncovers amino acid harvest as a fundamental mechanism linking microbial and host metabolism, and highlights Drosophila as a platformxa0for quantitative studies of host-microbe relationships.

Functional test of the influence of Wolbachia genes on cytoplasmic incompatibility expression in Drosophila melanogaster.

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.

The Small interfering RNA pathway is not essential for Wolbachia-mediated antiviral protection in Drosophila melanogaster

ABSTRACT Wolbachia pipientis delays RNA virus-induced mortality in Drosophila spp. We investigated whether Wolbachia-mediated protection was dependent on the small interfering RNA (siRNA) pathway, a key antiviral defense. Compared to Wolbachia-free flies, virus-induced mortality was delayed in Wolbachia-infected flies with loss-of-function of siRNA pathway components, indicating that Wolbachia-mediated protection functions in the absence of the canonical siRNA pathway.

Identification of yeast associated with the planthopper, Perkinsiella saccharicida: Potential applications for Fiji leaf gall control

Yeasts associate with numerous insects, and they can assist the metabolic processes within their hosts. Two distinct yeasts were identified by PCR within the planthopper Perkinsiella saccharicida, the vector of Fiji disease virus to sugarcane. The utility of both microbes for potential paratransgenic approaches to control Fiji leaf gall (FLG) was assessed. Phylogenetic analysis showed one of the microbes is related to yeast-like symbionts from the planthoppers: Laodelphax striatellus, Nilaparvata lugens, and Sogetella furcifera. The second yeast was a member of the Candida genus, a group that has been identified in beetles and recently described in planthoppers. Microscopy revealed the presence of yeast in the fat body of P. saccharicida. The Candida yeast was cultured, and transformation was accomplished by electroporation of Candida albicans codon optimized plasmids, designed to integrate into the genome via homologous recombination. Transgenic lines conferred resistance to the antibiotic nourseothricin and expression of green fluorescent protein was observed in a proportion of the yeast cells. Stably transformed yeast lines could not be isolated as the integrative plasmids presumably replicated within the yeast without integration into the genome. If stable transformation can be achieved, then this yeast may be useful as an agent for a paratransgenic control of FLG.

Acidic Food pH Increases Palatability and Consumption and Extends Drosophila Lifespan

BACKGROUNDnDespite the prevalent use of Drosophila as a model in studies of nutrition, the effects of fundamental food properties, such as pH, on animal health and behavior are not well known.nnnOBJECTIVESnWe examined the effect of food pH on adult Drosophila lifespan, feeding behavior, and microbiota composition and tested the hypothesis that pH-mediated changes in palatability and total consumption are required for modulating longevity.nnnMETHODSnWe measured the effect of buffered food (pH 5, 7, or 9) on male gustatory responses (proboscis extension), total food intake, and male and female lifespan. The effect of food pH on germfree male lifespan was also assessed. Changes in fly-associated microbial composition as a result of food pH were determined by 16S ribosomal RNA gene sequencing. Male gustatory responses, total consumption, and male and female longevity were additionally measured in the taste-defective Pox neuro (Poxn) mutant and its transgenic rescue control.nnnRESULTSnAn acidic diet increased Drosophila gustatory responses (40-230%) and food intake (5-50%) and extended survival (10-160% longer median lifespan) compared with flies on either neutral or alkaline pH food. Alkaline food pH shifted the composition of fly-associated bacteria and resulted in greater lifespan extension (260% longer median survival) after microbes were eliminated compared with flies on an acidic (50%) or neutral (130%) diet. However, germfree flies lived longer on an acidic diet (5-20% longer median lifespan) compared with those on either neutral or alkaline pH food. Gustatory responses, total consumption, and longevity were unaffected by food pH in Poxn mutant flies.nnnCONCLUSIONSnFood pH can directly influence palatability and feeding behavior and affect parameters such as microbial growth to ultimately affect Drosophila lifespan. Fundamental food properties altered by dietary or drug interventions may therefore contribute to changes in animal physiology, metabolism, and survival.