Impact of Chronic Infection on Resistance and Tolerance to Secondary Infection in Drosophila melanogaster.

Prior exposure to a pathogen can greatly influence the outcome of a secondary infection, and although invertebrates lack classically defined adaptive immunity, their immune response is still influenced by prior immune challenges. While the strength and specificity of such immune priming depends highly on the host organism and infecting microbe, chronic bacterial infection of the fruit fly Drosophila melanogaster with species isolated from wild-caught fruit flies provides broad nonspecific protection against a later secondary bacterial infection. To determine how chronic infection influences progression of secondary infection, we specifically tested how chronic infection with Serratia marcescens and Enterococcus faecalis impacted both resistance and tolerance to a secondary infection with an unrelated bacterium, Providencia rettgeri, by simultaneously tracking survival and bacterial load postinfection across a range of infectious doses. We found that these chronic infections increased both tolerance and resistance to P. rettgeri. Further investigation of S. marcescens chronic infection also revealed robust protection against the highly virulent Providencia sneebia, and that protection was dependent on the initial infectious dose for S. marcescens with protective doses corresponding with significantly increased diptericin expression. While the increased expression of this antimicrobial peptide gene likely explains the increased resistance, increased tolerance is likely due to other alterations in organismal physiology, such as increased negative regulation of immunity or tolerance of ER stress. These findings provide a foundation for future studies on how chronic infection influences tolerance to secondary infection.

Consequences of chronic bacterial infection in Drosophila melanogaster.

Even when successfully surviving an infection, a host often fails to eliminate a pathogen completely and may sustain substantial pathogen burden for the remainder of its life. Using systemic bacterial infection in Drosophila melanogaster, we characterize chronic infection by three bacterial species from different genera - Providencia rettgeri, Serratia marcescens, and Enterococcus faecalis-following inoculation with a range of doses. To assess the consequences of these chronic infections, we determined the expression of antimicrobial peptide genes, survival of secondary infection, and starvation resistance after one week of infection. While higher infectious doses unsurprisingly lead to higher risk of death, they also result in higher chronic bacterial loads among the survivors for all three infections. All three chronic infections caused significantly elevated expression of antimicrobial peptide genes at one week post-infection and provided generalized protection again secondary bacterial infection. Only P. rettgeri infection significantly influenced resistance to starvation, with persistently infected flies dying more quickly under starvation conditions relative to controls. These results suggest that there is potentially a generalized mechanism of protection against secondary infection, but that other impacts on host physiology may depend on the specific pathogen. We propose that chronic infections in D. melanogaster could be a valuable tool for studying tolerance of infection, including impacts on host physiology and behavior.

Sexual dimorphism in Drosophila melanogaster survival of Beauveria bassiana infection depends on core immune signaling.

In many animal species, females and males differ in physiology, lifespan, and immune function. The magnitude and direction of the sexual dimorphism in immune function varies greatly and the genetic and mechanistic bases for this dimorphism are often unknown. Here we show that Drosophila melanogaster females are more likely than males to die from infection with several strains of the fungal entomopathogen Beauveria bassiana. The sexual dimorphism is not exclusively due to barrier defenses and persists when flies are inoculated by injection as well as by surface exposure. Loss of function mutations of Toll pathway genes remove the dimorphism in survivorship. Surprisingly, loss of function mutation of relish, a gene in the Imd pathway, also removes the dimorphism, but the dimorphism persists in flies carrying other Imd pathway mutations. The robust sexual dimorphism in D. melanogaster survival to B. bassiana presents opportunities to further dissect its mechanistic details, with applications for biological control of insect vectors of human disease and insect crop pests.

Conditions Affecting Social Space in Drosophila melanogaster.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster - or other small insects - in a straightforward manner. As we previously demonstrated (1), in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing (1,2). Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior (1,3). Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans (4).

Systemic bacterial infection and immune defense phenotypes in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Thorax injury lowers resistance to infection in Drosophila melanogaster.

The route of infection can profoundly affect both the progression and outcome of disease. We investigated differences in Drosophila melanogaster defense against infection after bacterial inoculation into two sites--the abdomen and the thorax. Thorax inoculation results in increased bacterial proliferation and causes high mortality within the first few days of infection. In contrast, abdomen inoculation results in minimal mortality and lower bacterial loads than thorax inoculation. Inoculation into either site causes systemic infection. Differences in mortality and bacterial load are due to injury of the thorax and can be recapitulated by abdominal inoculation coupled with aseptic wounding of the thorax. This altered resistance appears to be independent of classical immune pathways and opens new avenues of research on the role of injury during defense against infection.

How the fly balances its ability to combat different pathogens.

Health is a multidimensional landscape. If we just consider the host, there are many outputs that interest us: evolutionary fitness determining parameters like fecundity, survival and pathogen clearance as well as medically important health parameters like sleep, energy stores and appetite. Hosts use a variety of effector pathways to fight infections and these effectors are brought to bear differentially. Each pathogen causes a different disease as they have distinct virulence factors and niches; they each warp the health landscape in unique ways. Therefore, mutations affecting immunity can have complex phenotypes and distinct effects on each pathogen. Here we describe how two components of the fly's immune response, melanization and phagocytosis, contribute to the health landscape generated by the transcription factor ets21c (CG2914) and its putative effector, the signaling molecule wntD (CG8458). To probe the landscape, we infect with two pathogens: Listeria monocytogenes, which primarily lives intracellularly, and Streptococcus pneumoniae, which is an extracellular pathogen. Using the diversity of phenotypes generated by these mutants, we propose that survival during a L. monocytogenes infection is mediated by a combination of two host mechanisms: phagocytic activity and melanization; while survival during a S. pneumoniae infection is determined by phagocytic activity. In addition, increased phagocytic activity is beneficial during S. pneumoniae infection but detrimental during L. monocytogenes infection, demonstrating an inherent trade-off in the immune response.

Listeria monocytogenes infection causes metabolic shifts in Drosophila melanogaster.

Immunity and metabolism are intimately linked; manipulating metabolism, either through diet or genetics, has the power to alter survival during infection. However, despite metabolism's powerful ability to alter the course of infections, little is known about what being "sick" means metabolically. Here we describe the metabolic changes occurring in a model system when Listeria monocytogenes causes a lethal infection in Drosophila melanogaster. L. monocytogenes infection alters energy metabolism; the flies gradually lose both of their energy stores, triglycerides and glycogen, and show decreases in both intermediate metabolites and enzyme message for the two main energy pathways, beta-oxidation and glycolysis. L. monocytogenes infection also causes enzymatic reduction in the anti-oxidant uric acid, and knocking out the enzyme uric oxidase has a complicated effect on immunity. Free amino acid levels also change during infection, including a drop in tyrosine levels which may be due to robust L. monocytogenes induced melanization.

Infection-related declines in chill coma recovery and negative geotaxis in Drosophila melanogaster.

Studies of infection in Drosophila melanogaster provide insight into both mechanisms of host resistance and tolerance of pathogens. However, research into the pathways involved in these processes has been limited by the relatively few metrics that can be used to measure sickness and health throughout the course of infection. Here we report measurements of infection-related declines in flies' performance on two different behavioral assays. D. melanogaster are slower to recover from a chill-induced coma during infection with either Listeria monocytogenes or Streptococcus pneumoniae. L. monocytogenes infection also impacts flies' performance during a negative geotaxis assay, revealing a decline in their rate of climbing as part of their innate escape response after startle. In addition to providing new measures for assessing health, these assays also suggest pathological consequences of and metabolic shifts that may occur over the course of an infection.

Pioneering immunology: insect style.

Insects are a powerful tool for discovering and then dissecting interesting new immunology. Recent insect research has made productive forays into non-classical immune areas including tolerance, immune priming (trained immunity), and environmental effects on immunity. Environments which affect immunity not only include diet and metabolism, but also social interactions and the animal's microbiota. We argue that every process that affects immunity should be considered as part of the immune response and that it is the broad phenomena discovered in insects that will be translated to other organisms rather than fine mechanistic details.