Diapause Termination Research Articles

Cold‐adapted species in a warming world – an explorative study on the impact of high winter temperatures on a continental butterfly

AbstractIncreasing evidence suggests that global warming significantly alters the range and phenology of plants and animals. Whereas thermophilous species usually benefit from rising temperatures, the living conditions of taxa adapted to cooler or continental climates are deteriorating. The woodland ringlet butterfly, Erebia medusa Fabricius (Lepidoptera: Nymphalidae) is one of the continental species that are supposed to be adversely affected by climate change, especially by rising winter temperatures. Here, we conduct an explorative study on the effects of low, moderate, and high winter temperatures on the pre‐adult and adult stages of E. medusa in a laboratory experiment. Compared to the two other temperature regimes, the warm winter treatment led to an earlier termination of diapause and higher larval weights at the end of the winter, but significantly lower survival rates. The after‐effects of the warm treatment included lower weight of the pupae and adult females, shorter forewings of adult males, and earlier emergence of both adult males and females. In natural environments, which are characterized by a much greater thermal variability and a much higher frequency of soil freeze‐thaw events compared to our experiment, the effects of rising winter temperatures might be stronger than in this study. Thus, we conclude that warmer winters pose a non‐negligible long‐term threat to E. medusa.

N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16∶8 (LD) and LD12∶12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4°C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNANAT caused dysfunction of photoperiodism. dsRNAPER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNANAT decreased melatonin while dsRNAPER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNANAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

Ecdysteroid-Induced Programmed Cell Death Is Essential for Sex-Specific Wing Degeneration of the Wingless-Female Winter Moth

The winter moth, Nyssiodes lefuarius, has a unique life history in that adults appear during early spring after a long pupal diapause from summer to winter. The moth exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that cell death of the pupal epithelium of females appears to display condensed chromatin within phagocytes. To provide additional detailed data for interpreting the role of cell death, we performed light microscopy, transmission electron microscopy, and TUNEL assay. We consequently detected two modes of cell death, i.e., dying cells showed both DNA fragmentation derived from epithelial nuclei and autophagic vacuole formation. To elucidate the switching mechanism of sex-specific wing degeneration in females of N. lefuarius, we tested the effects of the steroid hormone 20-hydroxyecdysone (20E) on pupal diapause termination and wing morphogenesis in both sexes. When 20E (5.4 µg) was injected into both sexes within 2 days of pupation, wing degeneration started 4 days after 20E injection in females, whereas wing morphogenesis and scale formation started 6 days after 20E injection in males. We discuss two important findings: (1) degeneration of the pupal wing epithelium of females was not only due to apoptosis and phagocytotic activation but also to autophagy and epithelial cell shrinkage; and (2) 20E terminated the summer diapause of pupae, and triggered selective programmed cell death only of the female-pupal wing epithelium in the wingless female winter moth.

Expression and roles of As-NUPR1 protein from Artemia sinica during embryo development and in response to salinity stress

As-NUPR1, a stress-related protein, plays an important role in post-diapause during embryonic development in the brine shrimp Artemia sinica. In the present study, successful expression of As-NUPR1 from the cDNA sequence isolated from A. sinica was demonstrated using a prokaryotic expression system. The recombinant protein consisted of 132 amino acids with a molecular weight of 15kDa, and a predicted isoelectric point of 7.17. As-NUPR1 polyclonal antibodies were prepared by immunization of Balb/c mice with purified recombinant As-NUPR1 protein as an antigen, and immunological studies were carried out. Expression of As-NUPR1 during different developmental stages of the embryo and in response to salinity stress was analyzed in A. sinica using Western blots. The experimental results showed that the expression of As-NUPR1 is widely distributed at different developmental stages in A. sinica, and there was no tissue or organ specificity. Expression of As-NUPR1 decreased gradually during the diapause termination stage of embryo development, after which there was a general increase in expression after breaking the shell. In addition, As-NUPR1 expression was highly upregulated under conditions of high salinity. These results suggest that the As-NUPR1 protein is a stress-related protein that plays a role in protecting embryos from high salt damage in different embryonic developmental stages, especially during the post-diapause period.

The Potential Role of As-sumo-1 in the Embryonic Diapause Process and Early Embryo Development of Artemia sinica

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica.

Description of Rhagoletis cerasi (Diptera: Tephritidae) pupal developmental stages: indications of prolonged diapause.

The European cherry fruit fly, Rhagoletis cerasi (L.) (Diptera: Tephritidae), is the key pest of sweet and sour cherries in many European countries and west Asia. It is a univoltine species of the west Palaearctic zone that undergoes obligatory pupal diapause. In this study, the development of R. cerasi pupae that were brought to an optimum temperature for postdiapause development following a long chilling period is described. The six most representative developmental stages within the puparium are illustrated, and the developmental progression among the stages after the end of the chilling period is quantified. Within 20 d postchilling, there was a gradual progress from stage I to pharate adult. However, ∼30% of the pupae remained at the transitional stage II, after 20 d at 25°C (optimum temperature for development). This suggests that a proportion of pupae remain at an intermediate developmental stage for an extended period of time that goes beyond 20 d postchilling. The pupal stage II might be related to diapause termination and responsiveness to environmental cues. It may also define the time before developmental progress to pharate adult. This finding agrees with previous studies proposing that a number of R. cerasi pupae undergo prolonged diapause, though the morphological characteristics of these pupae have never been described before.

Identification of the glycerol kinase gene and its role in diapause embryo restart and early embryo development of Artemia sinica

Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization by transferring a phosphate from ATP to glycerol, yielding glycerol 3-phosphate, which is an important intermediate for both energy metabolism and glycerolipid production. Artemia sinica has an unusual diapause process under stress conditions of high salinity, low temperature and lack of food. In the process, diapause embryos of A. sinica (brine shrimp) accumulate high concentrations of glycerol as a cryoprotectant to prevent low temperature damage to embryos. Upon embryo restart, glycerol is converted into glucose and other carbohydrates. Therefore, GK plays an important role in the diapause embryo restart process. However, the role of GK in diapause termination of embryo development in A. sinica remains unknown.In the present study, a 2096bp full-length cDNA of gk from A. sinica (As-gk) was obtained, encoding putative 551 amino acids, 60.6kDa protein. As a crucial enzyme in glycerol uptake and metabolism, GK has been conserved structurally and functionally during evolution. The expression pattern of As-gk was investigated by quantitative real-time PCR and Western blotting. Expression locations of As-gk were analyzed using in situ hybridization. As-gk was widely distributed in the early embryo and several main parts of Artemia after differentiation. The expression of As-GK was also induced by stresses such as cold exposure and high salinity. This initial research into the expression pattern and stress response of GK in Artemia provides a sound basis for further understanding of the function and regulation of genes in early embryonic development in A. sinica and the stress response.

Both the anterior and posterior eyes function as photoreceptors for photoperiodic termination of diapause in the two-spotted spider mite

Photoreceptors involved in photoperiodism in insects and mites can be either the retinal photoreceptors in the visual system or nonvisual extraretinal photoreceptors. Mites with no eyes have a clear photoperiodic response, suggesting the involvement of extraretinal photoreceptors in mite photoperiodism. In mites equipped with eyes, however, it is not known whether the retinal or extraretinal photoreceptors are involved in photoperiodism. The two-spotted spider mite Tetranychus urticae possesses two pairs of eyes. Adult females of this species terminate diapause in response to long days. To investigate whether the eyes function as photoperiodic photoreceptors in T. urticae, their eyes were ablated using a laser ablation system. Mites with their eyes intact terminated diapause under long days after low temperature exposure, whereas they remained in diapause under short days. Under constant darkness, they did not terminate diapause. When all eyes were removed, the mites remained in diapause even when they were maintained under long days. In contrast, the mites showed clear photoperiodic response when only the anterior or posterior eyes were removed. These results indicate that both the anterior and posterior eyes function as photoreceptors in photoperiodic termination of diapause in T. urticae.

Molecular cloning and functional characterization of the diapause hormone receptor in the corn earworm Helicoverpa zea

The diapause hormone (DH) in the heliothine moth has shown its activity in termination of pupal diapause, while the orthology in the silkworm is known to induce embryonic diapause. In the current study, we cloned the diapause hormone receptor from the corn earworm Helicoverpa zea (HzDHr) and tested its ligand specificities in a heterologous reporter system. HzDHr was expressed in Chinese Hamster Ovary (CHO) cells, which were co-transfected with the aequorin reporter, and was used to measure the ligand activities. A total of 68 chemicals, including natural DH analogs and structurally similar peptide mimetics, were tested for agonistic and antagonistic activities. Several peptide mimetics with a 2-amino-7-bromofluorene-succinoyl (2Abf-Suc) N-terminal modification showed strong agonistic activities; these mimetics included 2Abf-Suc-F[dA]PRLamide, 2Abf-Suc-F[dR]PRLamide, 2Abf-Suc-FKPRLamide and 2Abf-Suc-FGPRLamide. Antagonistic activity was found in the ecdysis triggering hormone in Drosophila melanogaster (FFLKITKNVPRLamide). Interestingly, HzDHr does not discriminate between DH (WFGPRLamide C-terminal motif) and another closely related endogenous peptide, pyrokinin 1 (FXPRXamide; a C-terminal motif that is separate from WFGPRLamide). We provide large-scale in vitro data that serve as a reference for the development of agonists and antagonists to disrupt the DH signaling pathway.

Diapause, signal and molecular characteristics of overwintering Chilo suppressalis (Insecta: Lepidoptera: Pyralidae)

Diapause is a complex and dynamic process. Chilo suppressalis, an important rice pest in Asia enters facultative diapause as larvae. Our results demonstrated in Yangzhou, China, diapause was initiated between September 4 and 12, 2010. After diapause termination, C. suppressalis remained in quiescence in the field for as long as three months. The average time between collection of field larvae of C. suppressalis and their pupation decreased as the season progressed from fall to next spring. Unexpectedly, the pupated ratio of female to male in the initiation of diapause was 0.22. The abundance of hsp90, hsp70, hsp60 and CsAQP1 all peaked on January 8 or 15, 2011. Nitric oxide (NO) is a secondary messenger that is positively correlated with the diapause of C. suppressalis. Among several geographically separated populations of C. suppressalis, there are no significant differences in the mRNA levels of hsp70, hsp60 or CsAQP1.

Months of Asynchrony in Offspring Production but Synchronous Adult Emergence: The Role of Diapause in an Ectoparasite's Life Cycle

Off-host stages of temperate zone ectoparasites must overcome two challenges: coping with unfavorable seasons and synchronizing their life cycles with host availability. In general, little is known about the seasonal cycles of insect ectoparasites of warm-blooded animals. The current study investigates the unusual phenology of a viviparous hippoboscid fly, the deer ked (Lipoptena cervi L.), that parasitizes boreal cervids. Despite months of asynchrony in offspring production, the adults emerge synchronously in mid-August across the northern boreal zone. We examined the role of diapause variation in the synchronization of life cycles by testing adult emergence success and time in relation to offspring birth month (October to April) and with respect to chilling time and photoperiod. Unexpectedly, we found that photoperiod had no role in regulating the life cycle, but diapause was maintained as long as pupae were exposed to cold. Pupae born before February needed a slightly longer exposure to high temperatures to terminate diapause if the cold period was short. Despite the apparent importance of a long period of chilling for life cycle synchrony, it was not required to terminate diapause. This finding of cold mainly preventing, rather than promoting, diapause termination is not novel among temperate insects, but it is rare. Slow diapause termination as a response to exceptionally long exposure to high, not low, temperatures seems to be a cornerstone for synchronizing the life cycle in the deer ked.

Diapause termination of Rhagoletis cerasi pupae is regulated by local adaptation and phenotypic plasticity: escape in time through bet‐hedging strategies

Persistence and thriving of univoltine, herbivore insect species of the temperate zone rely on obligate diapause response that ensures winter survival and synchronization with host phenology. We used a stenophagous fruit fly (Rhagoletis cerasi) with obligate pupae diapause to determine genetic and environmental effects on diapause intensity of geographically isolated populations with habitat heterogeneity. Pupae from two Greek and one German populations with various gene flow rates were exposed at five constant chilling temperatures (0-12°C) for different durations and then incubated at a high temperature until all adults have emerged. Pupae diapause intensity differs among Greek and German populations, suggesting an adaptive response to habitat heterogeneity (mostly differences in phenology patterns of local host cultivars). Moderately warm winter temperatures, such as 8°C, promote diapause termination in all three populations. Insufficient chilling (short duration or warmer temperatures) regulates the expression of prolonged dormancy. Interestingly, extended chilling (longer than required for terminating diapause) 'return' pupae to another (facultative) cycle of dormancy enabling adults to emerge during the next appropriate 'window of time'; a strategy first time reported for univoltine insects. Consequently, diapause duration of R.cerasi is determined both by i) the adaptive response to local climatic conditions (annual dormancy) and ii) the plastic responses to interannual climatic variability resulting in two types of long life cycles within populations, prolonged and facultative dormancy as response to insufficient chilling and extended exposure to chilling, respectively. Long life cycles are expressed as a part of dormancy bet-hedging strategies of R.cerasi populations.

Serotonin receptor B may lock the gate of PTTH release/synthesis in the Chinese silk moth, Antheraea pernyi; a diapause initiation/maintenance mechanism?

The release of prothoracicotropic hormone, PTTH, or its blockade is the major endocrine switch regulating the developmental channel either to metamorphosis or to pupal diapause in the Chinese silk moth, Antheraea pernyi. We have cloned cDNAs encoding two types of serotonin receptors (5HTRA and B). 5HTRA-, and 5HTRB-like immunohistochemical reactivities (-ir) were colocalized with PTTH-ir in two pairs of neurosecretory cells at the dorsolateral region of the protocerebrum (DL). Therefore, the causal involvement of these receptors was suspected in PTTH release/synthesis. The level of mRNA5HTRB responded to 10 cycles of long-day activation, falling to 40% of the original level before activation, while that of 5HTRA was not affected by long-day activation. Under LD 16:8 and 12:12, the injection of dsRNA5HTRB resulted in early diapause termination, whereas that of dsRNA5HTRA did not affect the rate of diapause termination. The injection of dsRNA5HTRB induced PTTH accumulation, indicating that 5HTRB binding suppresses PTTH synthesis also. This conclusion was supported pharmacologically; the injection of luzindole, a melatonin receptor antagonist, plus 5th inhibited photoperiodic activation under LD 16:8, while that of 5,7-DHT, induced emergence in a dose dependent fashion under LD 12:12. The results suggest that 5HTRB may lock the PTTH release/synthesis, maintaining diapause. This could also work as diapause induction mechanism.

Development characteristics of the box‐tree moth Cydalima perspectalis and its potential distribution in Europe

AbstractThe box‐tree moth Cydalima perspectalis (Walker) is an invasive pest causing severe damage to box trees (Buxus spp.). It is native to Japan, Korea and China, but established populations have been recorded in a number of locations across Europe since 2007 and the spread of the insect continues. The developmental investigations suggest that larvae overwinter mainly in their 3rd instar in Europe and that diapause is induced by a day length of about 13.5 h. One and a half to 2 months in the cold are necessary to terminate diapause. Threshold temperatures for development and number of degree‐days to complete a generation are slightly different from those calculated in previous studies in Japan. A bioclimatic (CLIMEX®) model for C. perspectalis in Europe was developed, based on climate, ecological and developmental parameters from the literature and new field and laboratory studies on diapause termination, thermal requirements and phenology. The model was then validated with actual distribution records and phenology data. The current distribution and life history of C. perspectalis in Europe were consistent with the predicted distribution. The climate model suggests that C. perspectalis is likely to continue its spread across Europe, except for Northern Fenno‐Scandinavia, Northern Scotland and high mountain regions. The northern distribution of C. perspectalis is expected to be limited by a number of degree‐days above the temperature threshold insufficient to complete a generation, whereas its southern range is limited by the absence of a cold period necessary to resume diapause. The model predicts relatively high Ecoclimatic Indices throughout most of Europe, suggesting that the insect has the potential of becoming a pest in most of its predicted range. However, damage is likely to be higher in Southern and Central Europe where the moth is able to complete at least two generations per year.

Comparison of Diapause Termination in Tarnished Plant Bugs (Hemiptera: Miridae) from the Mississippi Delta and Springfield, Illinois

Abstract In a laboratory test, we compared diapausing adult tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), from Stoneville, MS, and Springfield, IL, for their ability to break diapause when kept at 25°C on a nutritious food source (broccoli, Brassica oleracea variety botrytis L.) under a diapause-maintaining photoperiod of 10:14 light:dark hours. The temperature, photoperiod, and food simulated environmental conditions in the mid-South region during December when overwintering tarnished plant bugs on blooming winter hosts break diapause. It was unknown if plant bugs from areas such as Springfield (upper Midwest) in which the winters are too cold for hosts to be available also had the ability to break diapause under a diapause-maintaining daylength. One-day-old diapausing adults from Stoneville and Springfield were kept in environmental cabinets for 5 weeks. Males and females from both locations were dissected at weekly intervals to determine their reproductive status. Diapausing adults from...

Frequency and Distribution of Extended Diapause in Nebraska Populations of <I>Diabrotica barberi</I> (Coleoptera: Chrysomelidae)

The frequency of extended diapause in populations of the northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), was measured in eastern Nebraska. Adult collections were made during late summer in 2008 and 2009 from eight sites each year (seven sites were consistent over years). Eggs were obtained from 12 to 20 females per site and were held on moist soil under appropriate temperature profiles to facilitate egg survival, diapause development, and diapause termination. Percentage egg hatch was recorded after the first and second year for the 2008 and 2009 collections. Additional extended diapause expression was estimated for the 2008 and 2009 collections by counting remaining live eggs after the second-year egg hatch was completed. These data also were used to estimate each site's maximum egg viability. Results collectively indicate that the extended diapause trait was present in all eastern Nebraska populations; however, significant variation in the frequency of extended diapause was observed within and among D. barberi populations (first-year mean percentage egg hatch range: 2008 collections: 4.5-38.6%; 2009 collections: 10.7-42.5%). Geographically, the highest incidence of extended diapause was found along a north-south transect in eastern counties of Nebraska that parallels the Missouri River. In general, the frequency of extended diapause was lower in sites found west of the north-south transect. This study contributes to the knowledge base needed to develop appropriate D. barberi management strategies in Nebraska. These data can be used to pinpoint areas of Nebraska that may have the greatest risk of economic injury in first-year corn (Zea mays L.) when D. barberi densities are high.

Overwintering Biology of <I>Culex</I> (Diptera: Culicidae) Mosquitoes in the Sacramento Valley of California

At temperate latitudes, Culex (Diptera: Culicidae) mosquitoes typically overwinter as adult females in reproductive arrest and also may serve as reservoir hosts for arboviruses when cold temperatures arrest viral replication. To evaluate their role in the persistence of West Nile virus (WNV) in the Sacramento Valley of California, the induction and termination of diapause were investigated for members of the Culex pipiens (L.) complex, Culex tarsalis Coquillett, and Culex stigmatosoma Dyar under field, seminatural, and experimental conditions. All Culex spp. remained vagile throughout winter, enabling the collection of 3,174 females and 1,706 males from diverse habitats during the winters of 2010-2012. Overwintering strategies included both quiescence and diapause. In addition, Cx. pipiens form molestus Forskäl females remained reproductively active in both underground and aboveground habitats. Some blood-fed, gravid, and parous Cx. tarsalis and Cx. pipiens complex females were collected throughout the winter period. Under both field and experimental conditions, Cx. tarsalis and Cx. stigmatosoma females exposed to autumnal conditions arrested primary follicular maturation at previtellogenic stage I, with primary to secondary follicular ratios <1.5 (indicative of a hormonally induced diapause). In contrast, most Cx. pipiens complex females did not enter reproductive diapause and ovarian follicles matured to >or=stage I-II (host-seeking arrest) or were found in various stages of degeneration. Diapause was initiated in the majority of Cx. tarsalis and Cx. stigmatosoma females by mid-late October and was terminated after the winter solstice, but host-seeking seemed limited by temperature. An accrual of 97.52 +/- 30.7 and 162.85 +/- 79.3 degree-days after the winter solstice was estimated to be necessary for diapause termination in Cx. tarsalis under field and seminatural conditions, respectively. An increase in the proportion of blood-fed Culex females in resting collections occurred concurrently with diapause termination in field populations based on ovarian morphometrics. WNV RNA was detected in one pool of 18 males and in a single blood-fed female Cx. tarsalis collected during winter. Therefore, both vertically and horizontally infected Culex females may persist through winter and possibly transmit WNV after diapause termination in late winter or early spring in the Sacramento Valley of California.

Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm,Bombyx mori

To explore whether glutathione regulates diapause determination and termination in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapause- and nondiapause-egg producers, as well as those in diapause eggs incubated at different temperatures. The activity of thioredoxin reductase (TrxR) was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was observed in the ovary of diapause-egg producers, due to weaker reduction of oxidized glutathione (GSSG) to the reduced glutathione (GSH) catalyzed by glutathione reductase (GR) and TrxR. This indicates an oxidative shift in the glutathione redox cycle during diapause determination. Compared with the 25°C-treated diapause eggs, the 5°C-treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm.

Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos

BackgroundAs a response to harsh environments, the crustacean artemia produces diapause gastrula embryos (cysts), in which cell division and embryonic development are totally arrested. This dormant state can last for very long periods but be terminated by specific environmental stimuli. Thus, artemia is an ideal model organism in which to study cell cycle arrest and embryonic development.Principal FindingOur study focuses on the roles of H3K56ac in the arrest of cell cycle and development during artemia diapause formation and termination. We found that the level of H3K56ac on chromatin increased during diapause formation, and decreased upon diapause termination, remaining basal level throughout subsequent embryonic development. In both HeLa cells and artemia, blocking the deacetylation with nicotinamide, a histone deacetylase inhibitor, increased the level of H3K56ac on chromatin and induced an artificial cell cycle arrest. Furthermore, we found that this arrest of the cell cycle and development was induced by H3K56ac and dephosphorylation of the checkpoint protein, retinoblastoma protein.Conclusions/SignificanceThese results have revealed the dynamic change in H3K56ac on chromatin during artemia diapause formation and termination. Thus, our findings provide insight into the regulation of cell division during arrest of artemia embryonic development and provide further insight into the functions of H3K56ac.

Geographic variation in diapause induction and termination of the cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

Overwintering diapause in Helicoverpa armigera, a multivoltine species, is controlled by response to photoperiod and temperature. Photoperiodic responses from 5 different geographical populations showed that the variation in critical photoperiod for diapause induction was positively related to the latitudinal origin of the populations at 20, 22 and 25°C. Diapause response to photoperiod and temperature was quite different between northern and southern populations, being highly sensitive to photoperiod in northern populations and temperature dependence in southern populations. Diapause pupae from southern population showed a significantly shorter diapause duration than from northern-most populations when they were cultured at 20, 22, 25, 28 and 31°C; by contrast, overwintering pupae from southern populations emerged significantly later than from northern populations when they were maintained in natural conditions, showing a clinal latitudinal variation in diapause termination. Diapause-inducing temperature had a significant effect on diapause duration, but with a significant difference between southern and northern populations. The higher rearing temperature of 22°C evoked a more intense diapause than did 20°C in northern populations; but a less intense diapause in southern population. Cold exposure (chilling) is not necessary to break the pupal diapause. The higher the temperature, the quicker the diapause terminated. Response of diapause termination to chilling showed that northern populations were more sensitive to chilling than southern population.All results demonstrate that H. armigera is not genetically homogeneous throughout its range, but rather is composed of distinct populations genetically adapted to local environmental conditions despite the potential for gene flow via seasonal migration of adults.