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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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.

Molecular characteristics of insect vitellogenins

Insect vitellogenin/lipophorin receptors: molecular structures, role in oogenesis, and regulatory mechanisms.

Vitellogenins (Vg) genes code for the major egg yolk protein precursor in insects and many other oviparous species. In insects, the Vg gene is expressed extra-ovarially in the fat body in sex-, tissue- and stage-specific manners. During the reproductive phase, the Vg mRNA is expressed in large quantities, which is then translated, secreted into hemolymph and ultimately taken up by the developing oocytes through receptor-mediated endocytosis. Once sequestered, the Vgs are stored as vitellin (Vn), the main nutritional reserve for the developing embryo. The regulation of Vg genes is directly under the control of hormones at the transcriptional level. Hormones involved in Vg gene transcription are juvenile hormone (JH), ecdysteroids and some neuropeptides. The overall understanding that has emerged is that the insects can be classified, based on the system of hormonal regulation of Vg gene transcription, into three groups: (i) insects (like most of hemipterans) that use only JH for Vg gene transcription; (ii) insects (like dipterans) that need both JH and ecdysteroids for Vg regulation; and (iii) insects like lepidopterans that require JH, ecdysteroids and additional hormones to regulate their reproductive biology. However, why insect species diverge in using different hormones to govern their reproductive physiology remains unclear. The present contribution focuses on the current status of knowledge regarding the regulation of Vg genes in insects. Besides a brief information on biochemical and molecular features, the role of Vg genes as a target of endocrine disruptors will be addressed. Also, the molecular mechanism of Vg gene regulation will be discussed.

Regulation of vitellogenin genes in insects

Structure and expressions of two circadian clock genes, period and timeless in the commercial silkmoth, Bombyx mori.

The life history characteristics and predation of the Japanese Neoseiulus californicus (McGregor) strain on the two-spotted spider mite, Tetranychus urticae Koch, were studied in the laboratory under 60‐70% RH and 16L: 8D conditions. Developmental time from egg to adult emergence decreased when temperature increased. Total development period of immature stages was longest at 15°C and shortest at 35°C for both male and female. Sex ratio favored females and temperature did not exert a critical effect on sex determination. The total degree-days required from egg to adult female were 71.43 degree-days with thermal constant of 10.64 °C. At 25°C, female laid a total of 34.73 eggs during 17.91 days of oviposition period. The net reproductive rate (R o ) was highest at 25°C (22.92 females/female) and lowest at 30°C (16.74 females/female). The mean generation time (T) decreased from 20.61 to 16.79 days with increasing temperature up to 30 °C. The intrinsic rate of natural increase (r m ) ranged from 0.162 to 0.285, and was maximal at 25 °C. A gravid N. californicus female consumed more eggs, larvae and nymphs than adult male or female of T. urticae. As T. urticae density increased, prey consumption likewise increased. However, increasing the number of adult male or female preys did not increase the number of eggs laid by a female predator. The results were used to assess the effectiveness of the Japanese N. californicus strain as an important biological control agent against T. urticae.

/pdf/biology-and-predation-of-the-japanese-strain-of-neoseiulus-5c2o5un57w.pdf

Makio Takeda

Papers

Molecular characteristics of insect vitellogenins

Insect vitellogenin/lipophorin receptors: molecular structures, role in oogenesis, and regulatory mechanisms.

Regulation of vitellogenin genes in insects

Structure and expressions of two circadian clock genes, period and timeless in the commercial silkmoth, Bombyx mori.

Biology and predation of the Japanese strain of Neoseiulus californicus (McGregor) (Acari: Phytoseiidae)