Metabolic reserves associated with pupal diapause in the flesh fly, Sarcophaga crassipalpis

Larvae of Sarcophaga crassipalpis destined for pupal diapause (light:dark 12:12, 20°C) contain nearly twice as much lipid and twice the haemolymph protein concentration as larvae that will not enter diapause (light:dark 15:9, 20°C). This conspicuous difference in metabolic reserves provides the earliest indication of the developmental fate of the larva. Lipid reserves are utilized rapidly during the first half of diapause and then remain stable until adult eclosion. In contrast, residual dry weight changes very little early in diapause but drops sharply late in diapause, thus implying a transition from lipid utilization to protein or carbohydrate utilization in mid-diapause. We suggest that this metabolic transition marks the end of the “fixed latency period”: pupae readily respond to environmental or hormonal stimulation after this point. Diapause-destined larvae did not accumulate more glycogen than nondiapause-destined larvae, but an 80% decrease in glycogen at the onset of diapause and its elevation at the end of diapause suggests the utilization of glycerol or related compounds as cryoprotectants during diapause. Profiles of water content are very similar in short-day and long-day flies, thus suggesting that dehydration is not a mechanism exploited by the flesh fly to achieve cold hardiness. Adult flies that have experienced pupal diapause emerge from the puparium with lipid, glycogen, and water content nearly identical to flies that have not experienced diapause, but the residual dry weight is much lower. The severe depletion of protein may account for the reduced fecundity of flies that have experienced diapause.     

A novel trade-off of insect diapause affecting a sequestered chemical defense

Diapause allows insects to temporally avoid conditions that are unfavorable for development and reproduction. However, diapause may incur a cost in the form of reduced metabolic energy reserves, reduced potential fecundity, and missed reproductive opportunities. This study investigated a hitherto ignored consequence of diapause: trade-offs involving sequestered chemical defense. We examined the aristolochic acid defenses of diapausing and non-diapausing pipevine swallowtail butterflies, Battus philenor. Pipevine swallowtail larvae acquire these chemical defenses from their host plants. Butterflies that emerge following pupal diapause have significantly less fat, a female fitness correlate, compared to those that do not diapause. However, butterflies emerging from diapaused pupae are more chemically defended compared to those that have not undergone diapause. Furthermore, non-diapausing butterflies are confronted with older, lower quality host plants on which to oviposit. Thus, a trade-off exists where butterflies may have greater energy reserves at the cost of less chemical defense and sub-optimal food resources for their larvae, or have substantially less energetic reserves with the benefit of greater chemical defense and plentiful larval food resources.     

Differences in the pre‐diapause and pre‐oviposition accumulation of critical nutrients in adult females of the beetle Colaphellus bowringi

In many insect species, the differentiation of development between diapause and reproduction first becomes obvious during the diapause preparation (pre‐diapause) and pre‐oviposition phases. However, the differentiation of nutrient accumulation between these two phases remains unclear. We compared the weights of pre‐diapause and reproductive adult female Colaphellus bowringi Baly (Coleoptera: Chrysomelidae), and measured their triacylglycerol (TAG), protein, and carbohydrate content from emergence until they had fed for 4 days post‐eclosion. We also compared the ovarian development and accumulation of lipid droplets between pre‐diapause and reproductive adult females in order to determine whether we could visually detect differences in nutrient allocation. The weights of both pre‐diapause and reproductive females increased with duration of feeding. The fresh weight and water content of pre‐diapause females was significantly lower than that of reproductive females after feeding for 3 days post‐eclosion. Pre‐diapause females channeled their reserves into TAG in the fat body, whereas reproductive females converted nutrients into proteins and carbohydrates for egg development. These results quantify differences in nutrient accumulation between pre‐diapause and reproductive adult female C. bowringi, and provide clues for understanding the molecular mechanisms underlying differences in the allocation of nutrients between diapause and reproduction in insects.     

Insect photoperiodic calendar and circadian clock: independence, cooperation, or unity?

The photoperiodic calendar is a seasonal time measurement system which allows insects to cope with annual cycles of environmental conditions. Seasonal timing of entry into diapause is the most often studied photoperiodic response of insects. Research on insect photoperiodism has an approximately 80-year-old tradition. Despite that long history, the physiological mechanisms underlying functionality of the photoperiodic calendar remain poorly understood. Thus far, a consensus has not been reached on the role of another time measurement system, the biological circadian clock, in the photoperiodic calendar. Are the two systems physically separated and functionally independent, or do they cooperate, or is it a single system with dual output? The relationship between calendar and clock functions are the focus of this review, with particular emphasis on the potential roles of circadian clock genes, and the circadian clock system as a whole, in the transduction pathway for photoperiodic token stimulus to the overt expression of facultative diapause.     

A maternal effect that eliminates pupal diapause in progeny of the flesh fly, Sarcophaga bullata

Flesh flies that have experienced pupal diapause produce progeny that will not enter diapause even when reared in a strongly diapause-inducing environment. The effect is determined, not by diapause itself, but by the short days previously received by the larvae during the programming of pupal diapause. Reciprocal cross matings indicate that the effect is transmitted solely by the female parent. Though the embryos develop within the uterus of the female, the maternal effect is transmitted prior to the onset of embryogenesis, probably during oögenesis. Only by rearing a generation in long-day (nondiapausing) conditions can the capacity for pupal diapause be restored in the progeny. The effect is likely to provide an adaptive mechanism for preventing an untimely diapause response among the progeny of overwintering females that emerge early in the spring.     

Trade-offs between storage and survival affect diapause timing in capital breeders

Many organisms spend the unfavourable part of the year, such as the winter season, in diapause or dormancy and reproduce in spring shortly after emergence. Reserves are acquired prior to diapause to cover metabolic costs and in some species also reproduction (capital breeding) directly after diapause. Storage is then a component of future reproduction, and capital breeders consequently pay a pre-breeding cost of reproduction as they risk dying while obtaining and carrying the reserves. How large should the reserves be, and to what extent should optimal storage, and thereby timing of diapause, depend on predation risk and reproductive strategy? We present a general and simplistic life history model of an arthropod (e.g. crustaceans or insects) that is exposed to background mortality risk when it accumulates reserves before diapause. The model optimizes diapause timing and resultant reserves for income, mixed and capital breeders, and predicts how mortality risk affects the degree of capital breeding. For income breeders, timing of diapause is insensitive to the risk while obtaining reserves as they, regardless of risk, acquire the minimum amount needed to survive the winter. For capital breeders, the higher the risk the earlier the diapause and less is consequently stored. Mixed breeders diapause late and store as much as pure capital breeders when exposed to low risk, but behave as income breeders and diapause early when mortality is high. Our model shows that the degree of capital breeding impacts phenology of diapause in a risk-dependent manner. This prediction should impact how diapause timing is thought of across a wide range of taxa, including the much studied marine copepods. Timing of diapause, including triggers and cues, can only be understood when the diversity of reproductive strategies and the adaptive value of storage is taken into account.     

Pre‐wintering conditions and post‐winter performance in a solitary bee: does diapause impose an energetic cost on reproductive success?

1. Diapause is a dynamic process of low metabolic activity that allows insects to survive periods of harsh conditions. Notwithstanding the lowered metabolism, and because diapausing insects have no access to food, diapause has an energetic cost that may affect post‐diapause performance. 2. Previous studies on the solitary bee Osmia lignaria have shown that prolonged pre‐wintering periods (the time during which individuals already in diapause remain at warm temperatures) are associated with elevated lipid consumption, fat body depletion, and body weight loss. The present study investigated whether prolonged pre‐wintering also affects reproduction, i.e. whether the costs associated with diapause could have an effect on post‐diapause performance in this species. 3. Females were exposed to a range of pre‐wintering conditions, and ovary development and individual post‐wintering performance were monitored throughout their adult life span. 4. No evidence of an effect of pre‐wintering duration on post‐diapause reproductive success was found. Expected differences in the timing of establishment were not observed because ovary maturation was, surprisingly, not arrested during pre‐wintering. Prolonged pre‐wintering duration did not result in decreased life span, probably because emerging females could rapidly replenish their metabolic reserves through feeding. However, there was a very strong effect of the duration of the pre‐emergence period on the likelihood of nest establishment. 5. Longevity, the main factor determining fecundity in Osmia, is subjected to high levels of intrinsic variability, even among females of similar size exposed to identical conditions during development and nesting.     

Diapause-specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization

In this study we probe the molecular events underpinning diapause observed in overwintering females of Culex pipiens. Using suppressive subtractive hybridization (SSH) we have identified 40 genes that are either upregulated or downregulated during this seasonal period of dormancy. Northern blot hybridizations have confirmed the expression of 32 of our SSH clones, including six genes that are upregulated specifically in early diapause, 17 that are upregulated in late diapause, and two upregulated throughout diapause. In addition, two genes are diapause downregulated and five remain unchanged during diapause. These genes can be categorized into eight functional groups: genes with regulatory functions, metabolically-related genes, those involved in food utilization, stress response genes, cytoskeletal genes, ribosomal genes, transposable elements, and genes with unknown functions.     

Changes in microRNA abundance may regulate diapause in the flesh fly,Sarcophaga bullata

Diapause, an alternative developmental pathway characterized by changes in developmental timing and metabolism, is coordinated by molecular mechanisms that are not completely understood. MicroRNA (miRNA) mediated gene silencing is emerging as a key component of animal development and may have a significant role in initiating, maintaining, and terminating insect diapause. In the present study, we test this possibility by using high-throughput sequencing and qRT-PCR to discover diapause-related shifts in miRNA abundance in the flesh fly, Sarcophaga bullata. We identified ten evolutionarily conserved miRNAs that were differentially expressed in diapausing pupae compared to their nondiapausing counterparts. miR-289-5p and miR-1-3p were overexpressed in diapausing pupae and may be responsible for silencing expression of candidate genes during diapause. miR-9c-5p, miR-13b-3p, miR-31a-5p, miR-92b-3p, miR-275-3p, miR-276a-3p, miR-277-3p, and miR-305-5p were underexpressed in diapausing pupae and may contribute to increased expression of heat shock proteins and other factors necessary for the enhanced environmental stress-response that is a feature of diapause. In S. bullata, a maternal effect blocks the programming of diapause in progeny of females that have experienced pupal diapause, and in this study we report that several miRNAs, including miR-263a-5p, miR-100-5p, miR-125-5p, and let-7-5p were significantly overexpressed in such nondiapausing flies and may prevent entry into diapause. Together these miRNAs appear to be integral to the molecular processes that mediate entry into diapause.     

滞育昆虫营养物质的积累、转化与调控

滞育是昆虫度过不良环境的一种生存适应策略,营养物质的利用、积累和转化影响昆虫的滞育。滞育期间,昆虫体内发生一系列生理生化变化,包括脂类、碳水化合物和氨基酸等内源营养物质呈特异性地积累或转化,保障了滞育个体在逆境中存活及滞育解除后发育的能量需求。外源营养物质对昆虫滞育的影响较复杂,其种类、丰度、质量能通过"食料-寄主-天敌"营养关系进行传递,影响昆虫体内营养物质的积累和转化,改变昆虫的耐寒性、滞育率等,制约昆虫的滞育深度、滞育存活率。滞育昆虫营养物质积累、利用机制复杂多样,胰岛素信号通路-叉头转录因子以及激脂激素的调控效应在滞育诱导及营养物质转化中起到了关键的调控作用,但滞育的系统性调控仍需进一步深入研究。     

Analysis of cDNA transcripts from Coniothyrium minitans reveals a diverse array of genes involved in key processes during sclerotial mycoparasitism

Coniothyrium minitans colonises and destroys the sclerotia of Sclerotinia sclerotiorum in nature exhibiting ecologically obligate mycoparasitism as its spores remain dormant in soil and only grow actively in the presence of the sclerotia. Molecular mechanisms underlying sclerotial mycoparasitism are poorly defined. We identified 251 unisequences representing genes preferentially expressed by C. minitans during sclerotial mycoparasitism, substantially increasing the molecular knowledge of this commercially important biocontrol agent. Genes associated with signalling and cellular communication, degradation of host cell walls and energy reserves, nutrient utilisation, detoxification and stress response were identified suggesting that C. minitans employs a number of key processes during host colonisation. Several of these genes are novel to fungal-fungal interactions (e.g. PTH11-like GPCR and the ETP gene cluster). Secretin receptor-like GPCR and the TGF-beta signalling system have not yet been characterised in filamentous fungi. This study provides the basis for in-depth gene function analysis in sclerotial mycoparasitism.     

C. elegans DAF-18/PTEN mediates nutrient-dependent arrest of cell cycle and growth in the germline

The molecular pathways that link nutritional cues to developmental programs are poorly understood. Caenorhabditis elegans hatchlings arrest in a dormant state termed "L1 diapause" until food is supplied. However, little is known about what signal transduction pathways mediate nutritional status to control arrest and initiation of postembryonic development. We report that C. elegans embryonic germline precursors undergo G2 arrest with condensed chromosomes and remain arrested throughout L1 diapause. Loss of the DAF-18/PTEN tumor suppressor bypasses this arrest, resulting in inappropriate germline growth dependent on the AGE-1/PI-3 and AKT-1/PKB kinases. DAF-18 also regulates an insulin/IGF-like pathway essential for longevity and dauer larva formation. However, DAF-16/FoxO, which is repressed by this pathway, is not required for germline arrest in L1 diapause. Thus, these findings indicate that quiescence of germline development during L1 diapause is not a passive consequence of nutrient deprivation, but rather is actively maintained by DAF-18 through a pathway distinct from that which regulates longevity and dauer formation.     

Winter compensatory growth under field conditions partly offsets low energy reserves before winter in a damselfly

Despite the survival value of high energy reserves during winter, animals often face energy deficits when entering winter. Compensatory growth in energy reserves during the winter period to buffer such deficits may increase winter survival and alleviate the need for costly compensatory mechanisms before or after winter when predation risk is much higher. However, such compensatory responses in energy reserves during winter have not been demonstrated under field conditions. We explored if Lestes eurinus damselfly larvae can compensate for suboptimal energy reserves during winter at 4°C when their ponds are covered with ice. In a field enclosure experiment, we demonstrated compensatory growth in terms of body mass and energy reserves in larvae whose energy status was previously manipulated in the laboratory. These results were supported by patterns in body mass and energy reserves over winter in two natural unmanipulated populations. Winter survival was high overall and not affected by compensatory growth. We hypothesize that the observed compensatory growth in energy reserves during winter may shape life history decisions in autumn and spring, and may make resource availability during winter as or more important than energy reserves before winter.     

Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8 kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8 kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8 kDa proteins function to block permeability of O₂ during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8 kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.     

哺乳动物胚胎滞育及其调控机制研究进展

胚胎滞育(Embryonic diapause)是存在于部分哺乳动物中的一种生存策略和繁殖状态.其具体过程为:胚胎在着床前停止或减缓发育形成胚胎滞育期,滞育期结束后胚胎再次活化,且滞育过程不会对随后的胚胎发育产生任何不良影响.胚胎滞育主要分为两种类型:兼性滞育(Facultative diapause)和专性滞育(Ob...     

An ultrastructural analysis of the dispersed cell phase during development of the annual fish,Cynolebias

Cell ultrastructure was investigated during the dispersion phase of development in the annual fish Cynolebias. Three cellular populations encompass the yolk mass during dispersion, namely, 1) the yolk syncytial layer (YSL) or periblast, which lies directly over the surface of the yolk; 2) the deep blastomeres of the blastoderm, which engage in morphogenetic movements on the surface of the YSL and beneath the enveloping layer prior to forming the future embryo; and 3) the enveloping layer (EVL) of the blastoderm, which is a cohesive epithelium that forms the outermost cell layer of the blastoderm. Deep blastomeres contain numerous mitochondria and scattered glycogen rosettes that appear to function in the utilization of energy reserves. These cells also possess surface extensions such as filopodia and ruffles. Numerous microfilaments running parallel to the plasma membrane occur in cell extensions and in the cortical cytoplasm of neighboring blastomeres. In bleb-like extensions such as ruffles, microfilamentous stress fibers run parallel to the plane of the plasma membrane and prevent cellular organelles from entering the hyaline cap of the ruffle. Deep blastomeres also have basal projections that contain glycogen as well as pits in the basal membrane. Blastomeres move about using the YSL as a substrate. The YSL possesses specializations for nutrient uptake, storage, and transport such as numerous multivesicular bodies and large amounts of glycogen. Glycogen, in the rosette form, occurs in extraordinary amounts, virtually occluding the cytoplasm. Glycogen reserves are postulated to serve as an energy source during diapause. Glycogen is sometimes contained within villous projections that extend from the apical surface of the YSL. This configuration suggests the possibility of glycogen transport to the overlying deep blastomeres. Specializations of the EVL include apical tight junctions and basal lateral zonulae adherentes that interdigitate with those of adjacent EVL cells. The EVL serves as an impermeable membrane that protects the developing egg from the vicissitudes of its environment.     

Changes in dry weight, protein, and nucleic acid content during diapause and normal development of the blowfly, Lucilia sericata

Dry weight (D.W.), protein, RNA, and DNA have been determined in the blowfly, Lucilia sericata, throughout all stages of normal development in long and short photoperiod regimes and during larval diapause. During normal development protein/D.W. levels fluctuate markedly during the larval and puparial stages, increased levels being correlated with the synthesis of new cuticle, etc. prior to ecdysis and the histogenesis of adult tissues prior to emergence. Protein levels remain relatively high and constant during adult life to senescence. RNA/D.W. levels are highest in first instar larvae but decline rapidly during larval development until just before puparium formation. Sharp increases are found prior to pupation and then again prior to adult emergence. In the adult stage, the levels decline steadily throughout the life span. DNA/D.W. levels are very low in the egg but rach their highest levels in early first instar larvae. They then decline during larval development, with small increases being found prior to puparium formation and adult emergence. Adult levels remain relatively constant throughout the life span. The ratio has extremely high values in the egg, indicating the high degree of synthetic activity that takes place during embryogenesis. There is a steady decline in values during adult life to senescence in both sexes, suggesting that physiological ageing in L. sericata is accompanied by a decrease in protein synthesis potential.During larval diapause all parameters, with the exception of the ratio, are maintained at low and constant levels, reflecting the fact that diapause is a period when synthetic and mitotic activity are minimal. The great variations in levels, however, indicate that individuals within a group of larvae can terminate diapause spontaneously at 24°C and return to the normal processes of morphogenesis.