In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The role of microbial load during aging of the adult fruit fly Drosophila melanogaster is incompletely understood. Here we show dramatic increases in aerobic and anaerobic bacterial load during aging, both inside the body and on the surface. Scanning electron microscopy and cell staining analyses of the surface of aged flies detected structures resembling abundant small bacteria and bacterial biofilms. Bacteria cultured from laboratory flies included aerobic species Acetobacter aceti, Acetobacter tropicalis, and Acetobacter pasteurianus and anaerobic species Lactobacillus plantarum and Lactobacillus sp. MR-2; Lactobacillus homohiochii, Lactobacillus fructivorans, and Lactobacillus brevis were identified by DNA sequencing. Reducing bacterial load and antimicrobial peptide gene expression by axenic culture or antibiotics had no effect on life span. We conclude that Drosophila can tolerate a significant bacterial load and mount a large innate immune response without a detectable trade-off with life span; furthermore, microbes do not seem to limit life span under optimized laboratory conditions.

Affymetrix GeneChips were used to measure RNA abundance for Ϸ13,500 Drosophila genes in young, old, and 100% oxygenstressed flies. Data were analyzed by using a recently developed background correction algorithm and a robust multichip modelbased statistical analysis that dramatically increased the ability to identify changes in gene expression. Aging and oxidative stress responses shared the up-regulation of purine biosynthesis, heat shock protein, antioxidant, and innate immune response genes. Results were confirmed by using Northerns and transgenic reporters. Immune response gene promoters linked to GFP allowed longitudinal assay of gene expression during aging in individual flies. Immune reporter expression in young flies was partially predictive of remaining life span, suggesting their potential as biomonitors of aging.

Yeast FLP recombinase was used in a binary transgenic system ("FLP-OUT") to allow induced overexpression of catalase and/or Cu/Zn-superoxide dismutase (Cu/ZnSOD) in adult Drosophila melanogaster. Expression of FLP recombinase was driven by the heat-inducible hsp70 promoter. Once expressed, FLP catalyzed the rearrangement and activation of a target construct in which expression of catalase or Cu/ZnSOD cDNAs was driven by the constitutive actin5C promoter. In this way a brief heat pulse (120 or 180 min, total) of young adult flies activated transgene expression for the rest of the life span. FLP-OUT allows the effects of induced transgene expression to be analyzed in control (no heat pulse) and experimental (heat pulse) populations with identical genetic backgrounds. Under the conditions used, the heat pulse itself always had neutral or slightly negative effects on the life span. Catalase overexpression significantly increased resistance to hydrogen peroxide but had neutral or slightly negative effects on the mean life span. Cu/ZnSOD overexpression extended the mean life span up to 48%. Simultaneous overexpression of catalase with Cu/ZnSOD had no added benefit, presumably due to a preexisting excess of catalase. The data suggest that oxidative damage is one rate-limiting factor for the life span of adult Drosophila. Finally, experimental manipulation of the genetic background demonstrated that the life span is affected by epistatic interactions between the transgene and allele(s) at other loci.

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