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

Toxic substances in the environment generate adverse effects at all levels of biological organization from the molecular level to community and ecosystem. Given this complexity, it is not surprising that ecotoxicologists have struggled to address the full consequences of toxic substance release at ecosystem level, due to the limits of observational and experimental tools to reveal the changes in deep structure at different levels of organization. -Omics technologies, consisting of genomics and ecogenomics, have the power to reveal, in unprecedented detail, the cellular processes of an individual or biodiversity of a community in response to environmental change with high sample/observation throughput. This represents a historic opportunity to transform the way we study toxic substances in ecosystems, through direct linkage of ecological effects with the systems biology of organisms. Three recent examples of -omics advance in the assessment of toxic substances are explored here: (1) the use of functional genomics in the discovery of novel molecular mechanisms of toxicity of chemicals in the environment; (2) the development of laboratory pipelines of dose-dependent, reduced transcriptomics to support high-throughput chemical testing at the biological pathway level; and (3) the use of eDNA metabarcoding approaches for assessing chemical effects on biological communities in mesocosm experiments and through direct observation in field monitoring. -Omics advances in ecotoxicological studies not only generate new knowledge regarding mechanisms of toxicity and environmental effect, improving the relevance and immediacy of laboratory toxicological assessment, but can provide a wholly new paradigm for ecotoxicology by linking ecological models to mechanism-based, systems biology approaches.

Omics approaches can monitor responses and alterations of biological pathways at genome-scale, which are useful to predict potential adverse effects by environmental toxicants. However, high throughput application of transcriptomics in chemical assessment is limited due to the high cost and lack of "standardized" toxicogenomic methods. Here, a reduced zebrafish transcriptome (RZT) approach was developed to represent the whole transcriptome and to profile bioactivity of chemical and environmental mixtures in zebrafish embryo. RZT gene set of 1637 zebrafish Entrez genes was designed to cover a wide range of biological processes, and to faithfully capture gene-level and pathway-level changes by toxicants compared with the whole transcriptome. Concentration-response modeling was used to calculate the effect concentrations (ECs) of DEGs and corresponding molecular pathways. To validate the RZT approach, quantitative analysis of gene expression by RNA-ampliseq technology was used to identify differentially expressed genes (DEGs) at 32 hpf following exposure to seven serial dilutions of reference chemical BPA (10-10EμM) or each of four water samples ranging from wastewater to drinking water (relative enrichment factors 10-6.4 × 10). The RZT-ampliseq-embryo approach was both sensitive and able to identify a wide spectrum of biological activities associated with BPA exposure. Water quality was benchmarked based on the sensitivity distribution curve of biological pathways detected using RZT-ampliseq-embryo. Finally, the most sensitive biological pathways were identified, including those linked with adverse reproductive outcomes, genotoxicity and development outcomes. RZT-ampliseq-embryo approach provides an efficient and cost-effective tool to prioritize toxicants based on responsiveness of biological pathways.