Molecular Characterization and Tissue Distribution of Calcineurin Regulatory B Subunit during the Prevention of Diapause in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed calcineurin regulatory B subunit (CNB) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca2+. The gene expression level of CNB was increased by HCl treatment and the changes of the gene expression were almost the same as that in the non-diapause eggs. As for diapause eggs, almost no gene expression of CNB was confirmed except just after oviposition. In the assay for phosphorylation by protein kinase CK2, recombinant CNB (rCNB) was phosphorylated in vitro. Additionally, a Ca2+ binding assay indicated that rCNB shows affinity for Ca2+. The distribution of CNB was investigated with an immunohistochemical technique using antiserum against rCNB in diapause eggs and HCl-treated diapause eggs. CNB was localized in serosa cells and yolk cells in both eggs. These data may suggest that CNB is activated by intracellular Ca2+ or efflux Ca2+ resulting from HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Transcriptomic analysis elucidates the molecular processes associated with hydrogen peroxide-induced diapause termination in Artemia-encysted embryos.

Treatment with hydrogen peroxide (H2O2) raises the hatching rate through the development and diapause termination of Artemia cysts. To comprehend the upstream genetic regulation of diapause termination activated by exterior H2O2 elements, an Illumina RNA-seq analysis was performed to recognize and assess comparative transcript amounts to explore the genetic regulation of H2O2 in starting the diapause termination of cysts in Artemia salina. We examined three groupings treated with no H2O2 (control), 180 µM H2O2 (low) and 1800 µM H2O2 (high). The results showed a total of 114,057 unigenes were identified, 41.22% of which were functionally annotated in at least one particular database. When compared to control group, 34 and 98 differentially expressed genes (DEGs) were upregulated in 180 µM and 1800 µM H2O2 treatments, respectively. On the other hand, 162 and 30 DEGs were downregulated in the 180 µM and 1800 µM H2O2 treatments, respectively. Cluster analysis of DEGs demonstrated significant patterns among these types of 3 groups. GO and KEGG enrichment analysis showed the DEGs involved in the regulation of blood coagulation (GO: 0030193; GO: 0050818), regulation of wound healing (GO:0061041), regulation of hemostasis (GO: 1900046), antigen processing and presentation (KO04612), the Hippo signaling pathway (KO04391), as well as the MAPK signaling pathway (KO04010). This research helped to define the diapause-related transcriptomes of Artemia cysts using RNA-seq technology, which might fill up a gap in the prevailing body of knowledge.

An Embryonic Diapause-like Adaptation with Suppressed Myc Activity Enables Tumor Treatment Persistence.

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generated models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts, and cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, attenuates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity or interfering with the diapause-like adaptation by inhibiting cyclin-dependent kinase 9 represent potential therapeutic strategies against chemotherapy-persistent tumor cells. Our study demonstrates that cancer co-opts a mechanism similar to diapause with adaptive inactivation of Myc to persist during treatment.

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause.

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate-limiting enzyme, ornithine decarboxylase-1, in both ovary and utero-embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero-embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3ß-hydroxysteroid dehydrogenase through extracellular signal-regulated kinase (ERK1/2)-mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B-cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero-embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2-difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero-embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero-embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero-embryonic unit thereby activating development in delayed embryo in C. sphinx.

Culture of Mink Preimplantation Embryos.

The reproduction of the mink, Neovison vison, has been extensively studied over the past 70 years. The endocrine control of pregnancy is reasonably well understood, but our understanding of early embryo development is limited. The mink is one of the best characterized mammals for the study of embryonic diapause, but in order to unravel the complex interactions that occur between the blastocyst and the uterus during diapause and reactivation, a defined culture media system that supports growth is essential. Until recently, culture of the mink blastocyst has been relatively unsuccessful. This chapter will describe a method for successfully obtaining and culturing mink blastocysts and will highlight some of the unique challenges in working with this species. Methods to age match prediapause embryos in a mammal that exhibits superfetation, and to synchronize collection of reactivation from diapause stages using prolactin will be discussed. Finally, a quantitative method to determine the extent of cell proliferation in the blastocyst, a hallmark of reactivation from diapause, will be detailed.

Distinct microRNA and mRNA responses elicited by ecdysone, diapause hormone and a diapause hormone analog at diapause termination in pupae of the corn earworm, Helicoverpa zea.

Ecdysone, diapause hormone and a diapause hormone analog are all capable of breaking pupal diapause and prompting initiation of adult development in the cotton earworm, Helicoverpa zea. In this study we asked whether these three chemically-distinct diapause terminators elicit the same effect on expression of a collection of microRNAs and transcripts encoding components of the ecdysone signaling pathway. Injection of all three endocrine agents resulted in downregulation of one miRNA, miR-277-3p, a miRNA previously linked to the insulin/FOXO signaling pathway, and all three agents promoted upregulation of spook, a member of the ecdysone biosynthesis pathway, and iswi, an ecdysone-responsive transcript. Other miRNA and mRNA responses varied depending on the agent used to terminate diapause, thus suggesting that different endocrine pathways and mechanisms can lead to the same final developmental response.

A glyphosate-based herbicide reduces fertility, embryonic upper thermal tolerance and alters embryonic diapause of the threatened annual fish Austrolebias nigrofasciatus.

Roundup is the most popular glyphosate-based herbicide (GBH) worldwide. These formulations kill a wide range of plants. Despite that, non-target species can be jeopardized by GBH, such as the annual fish Austrolebias nigrofasciatus. This species occurs in wetlands that dries annually. Key-adaptations permit them to live in such harsh habitats, e. i. Elevated fertility, drought-tolerant diapausing embryos and elevated thermal tolerance. We aimed to evaluate acute (96 h) effects of Roundup exposure (0.36 or 3.62 mg a. e./L) in reproduction, diapause pattern and embryonic upper thermal tolerance (EUTT) of A. nigrofasciatus. For such, we evaluated the number and diameter of embryos produced by exposed fish. Also, recently fertilized embryos were exposed and its diapause pattern was evaluated. Following 15 post exposure days (PED), we evaluated the number of somite pairs and following 30, 35 and 40 PED we evaluated the proportion of pigmented embryos (PPE). Finally, the critical thermal maximum (CTMax) of exposed embryos was assessed. Results demonstrated that couples exposed to 0.36 mg a. e./L Roundup produced less but larger embryos. Similarly, embryos exposed to 3.62 mg a. e./L Roundup had a reduced PPE following 30 PED. Finally, embryos exposed to 0.32 mg a. e./L Roundup had a CTMax reduction of 2.6 °C and were more sensitive to minor increases in heating rates. These results indicate that Roundup have negative outcomes in fish reproduction, embryonic development and EUTT. This information is of particular interest to the conservation of annual fish, considering that those are key-adaptations that permit these animals to survive the harsh impositions of ephemeral wetlands.

Pheromone modulates two phenotypically plastic traits - adult reproduction and larval diapause - in the nematode Caenorhabditis elegans.

BACKGROUND: Animals use information from their environment to make decisions, ultimately to maximize their fitness. The nematode C. elegans has a pheromone signalling system, which hitherto has principally been thought to be used by worms in deciding whether or not to arrest their development as larvae. Recent studies have suggested that this pheromone can have other roles in the C. elegans life cycle. RESULTS: Here we demonstrate a new role for the C. elegans pheromone, showing that it accelerates hermaphrodites' reproductive rate, a phenomenon which we call pheromone-dependent reproductive plasticity (PDRP). We also find that pheromone accelerates larval growth rates, but this depends on a live bacterial food source, while PDRP does not. Different C. elegans strains all show PDRP, though the magnitude of these effects differ among the strains, which is analogous to the diversity of arrested larval phenotypes that this pheromone also induces. Using a selection experiment we also show that selection for PDRP or for larval arrest affects both the target and the non-target trait, suggesting that there is cross-talk between these two pheromone-dependent traits. CONCLUSIONS: Together, these results show that C. elegans' pheromone is a signal that acts at two key life cycle points, controlling alternative larval fates and affecting adult hermaphrodites' reproduction. More broadly, these results suggest that to properly understand and interpret the biology of pheromone signalling in C. elegans and other nematodes, the life-history biology of these organisms in their natural environment needs to be considered.

Effects of juvenile hormone analogue (methoprene) and 20-hydroxyecdysone on reproduction in Polygonia c-aureum (Lepidoptera: Nymphalidae) in relation to adult diapause.

We investigated the effects of juvenile hormone analogue (methoprene) and 20-hydroxyecdysone on female and male reproduction in a nymphalid butterfly, Polygonia c-aureum. This butterfly has a facultative adult diapause controlled by the corpora allata and brain. Methoprene seems to terminate reproductive diapause, although transplantation experiments indicate that the activity of the corpora allata does not affect male mating behavior Endo (Dev Growth Differ 15:1-10, 1973a), suggesting that the brain may be involved in diapause. We found that exposure to methoprene promoted the development of ovaries and of the male accessory glands and simplex. On the other hand, exposure to 20-hydroxyecdysone did not promote the development of female and male reproductive organs and eupyrene sperm movement from the testis to the duplex in the adult stage. Ecdysteroid titer in both sexes was consistently low in adults. These results suggest that imaginal diapause is largely regulated by juvenile hormone in this butterfly.

Pupal diapause termination in Bactrocera minax: an insight on 20-hydroxyecdysone induced phenotypic and genotypic expressions.

The Chinese citrus fruit fly, Bactrocera minax, is an economically important pest of citrus. It exhibits pupal diapause from November to May to combat harsh environmental conditions. Such a long pupal diapause is a barrier for laboratory rearing and development of control strategies against this pest. In the present study, 20-hydroxyecdysone (20E) was used to break pupal diapause of B. minax by topical application. After diapause termination by 20E treated, the pupal ontogenetic processes were observed along the temporal trajectory. The pupal response time to 20E was estimated by detecting the relative expression of 20E responsive genes at different times after 20E-treatment. Results revealed that 20E could effectively terminate the pupal diapause in a dose-dependent manner and significantly shorten the time for 50% adult emergence (Et50). 20E response genes, including ecr, broad and foxo, were up-regulated within 72h, indicating these genes are involved in pupal metamorphosis and diapause termination processes. Morphological changes showed the pupal metamorphosis began ~7 days after 20E-treatment at 22 °C. This study does not only pave the way for artificial rearing in the laboratory through manipulating of pupal diapause termination, but also deepens our understanding of the underlying pupal diapause termination mechanism of B. minax.