Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

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.

For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

The fat body plays major roles in the life of insects. It is a dynamic tissue involved in multiple metabolic functions. One of these functions is to store and release energy in response to the energy demands of the insect. Insects store energy reserves in the form of glycogen and triglycerides in the adipocytes, the main fat body cell. Insect adipocytes can store a great amount of lipid reserves as cytoplasmic lipid droplets. Lipid metabolism is essential for growth and reproduction and provides energy needed during extended nonfeeding periods. This review focuses on energy storage and release and summarizes current understanding of the mechanisms underlying these processes in insects.

Insect Fat Body: Energy, Metabolism, and Regulation

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Environmental and hormonal regulators of diapause have been reasonably well defined, but our understanding of the molecular regulation of diapause remains in its infancy. Though many genes are shut down during diapause, others are specifically expressed at this time. Classes of diapause-upregulated genes can be distinguished based on their expression patterns: Some are upregulated throughout diapause, and others are expressed only in early diapause, late diapause, or intermittently throughout diapause. The termination of diapause is accompanied by a rapid decline in expression of the diapause-upregulated genes and, conversely, an elevation in expression of many genes that were downregulated during diapause. A comparison of insect diapause with other forms of dormancy in plants and animals suggests that upregulation of a subset of heat shock protein genes may be one feature common to different types of dormancies.

Regulation of diapause.

A Bombyx EST cDNA database was searched using the Drosophila takeout gene and nine cDNAs were obtained. The homology search suggested that these genes are widespread in insects and organize a large gene family, and that they have hydrophobic ligands. A phylogenetic tree indicated that the genes are first divided into two large groups, juvenile hormone binding protein and other protein genes, and the latter group diversified within a short time at an early stage. The expression study of five Bombyx genes indicated that they are expressed in various tissues and are regulated by development and feeding conditions. The Bombyx genes might have roles related to the regulation of metabolism, growth or development related to nutritional conditions.

Cloning and expression analysis of takeout/JHBP family genes of silkworm, Bombyx mori.

Starvation suppresses cell proliferation that rebounds after refeeding in the midgut of the American cockroach, Periplaneta americana.

. An in vitro culture method is described in which embryonic development in Bombyx mori is traced at various temperatures and treatments. The results show that the induction, intensification and termination of diapause are distinct processes. Prediapause embryos, explanted from 40-h-old diapause-destined eggs and cultured in Grace's medium, continue to develop to the appendage-formation stage without arrest, which indicates that the isolated embryos have not entered diapause, whereas the development of embryos from diapausing eggs (15 days after being laid) is significantly slower. The rate of development of embryos dissected from diapause eggs increases during chilling (5 °C) and incubation (at 25 °C) gradually during chilling and dramatically at 25 °C. The in vitro experiments also reveal that sorbitol directly inhibits the development of embryos explanted from diapausing eggs but has no affect on the development of embryos from prediapause eggs. Neither alanine nor diapause hormone prevent isolated embryos from developing.

Effects of temperature, sorbitol, alanine and diapause hormone on the embryonic development in Bombyx mori: in vitro tests of old hypotheses.

We exposed midgut cells from primary cultures of Heliothis virescens larvae to cell-free previously used medium, the Vaughn X and HyQ SFtrade mark media used for serum-free culture of insect cell lines which do not support H. virescens midgut cells, and to toxin from Bacillus thuringiensis. A statistically significant increase in the percent of dying cells was counted in cell populations in Vaughn X medium. Use of the TUNEL method to detect apoptosis indicated a low rate (7.2%) of apoptosis in control cultures grown in Heliothis medium, an increase to approximately 20% in previously used and HyQ SFtrade mark media, and to approximately 45% of cells remaining after exposure to and initial destruction by B. thuringiensis toxin. Apoptotic nuclei were predominant (approximately 6%) in mature columnar cells in control cultures. Approximately 1% of goblet, stem, and differentiating cells were apoptotic. However, apoptosis rose to 12% in stem and differentiating cells exposed to used and unsuitable medium. B. thuringiensis exposure to toxin for 2-3 days resulted in visible membrane damage and necrosis, causing the death of 84% of the cells as measured by both the TUNEL and Annexin methods. Some of the columnar cells and stem and differentiating cells that remained also contained apoptotic nuclei. Stem and differentiating cells normally replace dying mature cells in the midgut. Thus, exposure of cultures of H. virescens midgut cells to adverse environments such as unsuitable or poisonous media appeared to induce down-regulation of the cell populations by apoptosis.

Apoptosis in cultured midgut cells from heliothis virescens larvae exposed to various conditions.

Roles of fat body trophocytes, mycetocytes and urocytes in the American cockroach, Periplaneta americana under starvation conditions: an ultrastructural study.

Makio Takeda

Papers

Cloning and expression analysis of takeout/JHBP family genes of silkworm, Bombyx mori.

Starvation suppresses cell proliferation that rebounds after refeeding in the midgut of the American cockroach, Periplaneta americana.

Effects of temperature, sorbitol, alanine and diapause hormone on the embryonic development in Bombyx mori: in vitro tests of old hypotheses.

Apoptosis in cultured midgut cells from heliothis virescens larvae exposed to various conditions.

Roles of fat body trophocytes, mycetocytes and urocytes in the American cockroach, Periplaneta americana under starvation conditions: an ultrastructural study.