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.

Summary Two peptides, midgut differentiation factors 1 and 2 (MDF-l and 2), were previously isolated from conditioned medium in which Manduca sexta midgut cells were grown. They are identical to two portions of the terminal sequence of the protein, fetuin, a prenatal vertebrate liver product. In cultures of Lepidopteran midgut, the MDFs act as cytokines or growth factors by inducing midgut stem cells to differentiate to mature columnar, goblet, endocrine, or nerve-like cells. A polyclonal rabbit antiserum to one of these peptides, MDF-l, was used in this study to stain cultured cells from midguts of 4th instar larvae of the Lepidopteran, Heliothis virescens. Paraffin sections of midgut taken from newly molted, mid- 4th instars, and pre-molting 4th instar larvae were also stained with the antibody. Intense positive immunostain was observed in basal areas of the cultured columnar cells and in basal lateral areas of columnar cells in the sections of mid-instar and premolt 4th instar midgut. Other cell types ...

Immunohistochemical detection of a putative insect cytokine, midgut differentiation factor 1 (MDF-1) in midgut columnar cells of Heliothis virescens

DNA encoding a fragment of putative arylalkylamine N-acetyltransferase (NAT) of the American cockroach, Periplaneta americana, was amplified by PCR with degenerate primers based on the two peptides previously purified from the testicular system of this species. A full clone was obtained by RACE-PCR. The clone consisted of 89 bp 5'-UTR, 753-bp open reading frame and 712-bp 3'-UTR. The amino acid sequence of 251 residues deduced from this ORF corresponded to the predicted molecular mass of 28.5 kDa. The predicted amino acid sequence had an overall identity of 35% with NAT1 and 27% with NAT2 of Drosophila melanogaster, respectively. Structural analysis revealed that NAT from P. americana contained two motifs characteristic of the NAT superfamily and three conserved regions (C/c-1, D/c-1, D/c-2) distinguishing aaNAT subfamily. Northern blot analysis showed that the mRNA of approximately 1.5 kb was transcribed at a high level in the testicular system, and corresponded to the length of the cDNA, i.e., 1,554 bp. Significant levels of NAT transcript were also detected in the midgut, ovary and the accessory glands and at much lower levels in the fat body and brain. Southern blot analysis suggested the presence of a single copy of the cloned gene in the genome.

Molecular cloning of a cDNA encoding arylalkylamine N‐acetyltransferase from the testicular system of Periplaneta americana: Primary protein structure and expression analysis

The effects of climate change on the northern limit of the trivoltine area were clarified in the fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae). The Takaoka and Kanazawa populations used in the present study occur in locations to the north of Fukui (36.07°N), where H. cunea has recently shifted from a bivoltine to a predominantly trivoltine life cycle. The life-history traits of these populations were investigated and compared to those in the Fukui population. The lower threshold temperature for development and the thermal constant for one generation were 11.3°C and 674.5°d in the Takaoka population, and 11.2°C and 680.7°d in the Kanazawa population. The critical photoperiod for diapause induction at 25°C was 14 h 34 min in the Takaoka population and 14 h 28 min in the Kanazawa population. These critical photoperiods at 25°C were longer by 24 min and 18 min than the Fukui population, respectively. These results, together with climate data, suggest that the Takaoka population maintains a bivoltine life cycle, and a small part of the Kanazawa population has three generations per year, a predominantly bivoltine life cycle. Thus, the present northern limit of the trivoltine area lies around Fukui in districts along the Japan Sea.

Northerly shift in voltinism watershed in Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) along the Japan Sea coast: Evidence of global warming?

This study was aimed to elucidate the developmental and life history characteristics of Coboldia fuscipes (Diptera:Scatopsidae) which causes serious damage to the oyster mushroom, Pleurotus ostreatus, in Korea. The average number of eggs oviposited by a single female was 282. Eggs are usually laid in a mass on the spawn. The total life span was 44.8, 34.4, 24.5 and 18.9 days in males and 43.2, 33.3, 24.1 and 17.9 days in females at 18, 21, 25 and 28°C, respectively. The developmental zero temperatures (t0) for each stage were estimated from the regression lines as follows: 11.6°C for eggs, 11.5°C for larvae, 9.2°C in male pupae, and 8.8°C in female pupae. The required heat units were 38 degree-days for egg development, 180 for larval development, 66 for male pupal development, and 67 for female pupal development. The developmental threshold based on the total pre-emergence period was 11.6°C for males and 11.1°C for females, and the required heat units were 277 and 283 degree-days for males and females, respectively.

Makio Takeda

Papers

Immunohistochemical detection of a putative insect cytokine, midgut differentiation factor 1 (MDF-1) in midgut columnar cells of Heliothis virescens

Molecular cloning of a cDNA encoding arylalkylamine N‐acetyltransferase from the testicular system of Periplaneta americana: Primary protein structure and expression analysis

Northerly shift in voltinism watershed in Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) along the Japan Sea coast: Evidence of global warming?

Developmental and life history characteristics of the oyster mushroom fly, Coboldia fuscipes (Diptera:Scatopsidae)

Characterization of indolamine N-acetyltransferase activity from the head ganglia of the American cockroach, Periplaneta americana