Genetic constraints in genes exhibiting splicing plasticity in facultative diapause.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

Temperature dependence of gas exchange patterns shift as diapause progresses in the butterfly Pieris napi.

Insects have the capacity to significantly modify their metabolic rate according to environmental conditions and physiological requirement. Consequently, the respiratory patterns can range from continuous gas exchange (CGE) to discontinuous gas exchange (DGE). In the latter, spiracles are kept closed during much of the time, and gas exchange occurs only during short periods when spiracles are opened. While ultimate causes and benefits of DGE remain debated, it is often seen during insect diapause, a deep resting stage that insects induce to survive unfavourable environmental conditions, such as winter. The present study explores the shifts between CGE and DGE during diapause by performing long continuous respirometry measurements at multiple temperatures during key diapause stages in the green-veined white butterfly Pieris napi. The primary goal is to explore respiratory pattern as a non-invasive method to assess whether pupae are in diapause or have transitioned to post-diapause. Respiratory pattern can also provide insight into endogenous processes taking place during diapause, and the prolonged duration of diapause allows for the detailed study of the thermal dependence of the DGE pattern. Pupae change from CGE to DGE a few days after pupation, and this shift coincides with metabolic rate suppression during diapause initiation. Once in diapause, pupae maintain DGE even at elevated temperatures that significantly increase CO2 production. Instead of shifting respiratory pattern to CGE, pupae increase the frequency of DGE cycles. Since total CO2 released during a single open phase remains unchanged, our results suggest that P. napi pupae defend a maximum internal ρCO2 set point, even in their heavily suppressed diapause state. During post-diapause development, CO2 production increases as a function of development and changes to CGE during temperature conditions permissive for development. Taken together, the results show that respiratory patterns are highly regulated during diapause in P. napi and change predictably as diapause progresses.

RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathway.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

Low GSK3ß activity is required for insect diapause through responding to ROS/AKT signaling and down-regulation of Smad1/EcR/HR3 cascade.

Glycogen synthase kinase 3ß (GSK3ß) plays important roles in gene transcription, metabolism, apoptosis, development, and signal transduction. However, its role in the regulation of pupal diapause remains unclear. In this paper, we find that low GSK3ß activity in brains of diapause-destined pupae of Helicoverpa armigera is caused by elevated AKT activity. In response to ROS, AKT phosphorylates GSK3ß to decrease its activity. In developing pupal brains, GSK3ß can activate the transcription factor Smad1, which binds to the promoter region of the ecdysone receptor (EcR) gene and increases its expression. In the presence of 20-hydroxyecdysone (20E), EcR can bind to USP and increase the expression of 20E-response genes, including HR3, for pupal-adult development. In contrast, high levels of ROS in brains of diapause-destined pupae up-regulate p-AKT, which in turn decreases GSK3ß activity. Low GSK3ß activity causes low expression of EcR/HR3 via down-regulation of Smad1 activity, leading to diapause initiation. These results suggest that low GSK3ß activity plays a key role in pupal diapause via ROS/AKT/GSK3ß/Smad/EcR/HR3 signaling.

Fat enough for the winter? Does nutritional status affect diapause?

Many insects enter a dormant state termed diapause in anticipation of seasonal inhospitable conditions. Insects drastically reduce their feeding during diapause. Their reduced nutrient intake is paired with substantial nutrient costs: maintaining basal metabolism during diapause, repairing tissues damaged by adverse conditions, and resuming development after diapause. Many investigators have asked "Does nutrition affect diapause?" In this review, we survey the studies that have attempted to address this question. We propose the term nutritional status, a holistic view of nutrition that explicitly includes the perception, intake, and storage of the great breadth of nutrients. We examine the studies that have sought to test if nutrition affects diapause, trying to identify specific facets of nutritional status that affect diapause phenotypes. Curiously, low quality host plants during the diapause induction phase generally induce diapause, but food deprivation during the same phase generally averts diapause. Using the geometric framework of nutrition to identify specific dietary components that affect diapause may reconcile these contrasting findings. This framework can establish nutritionally permissive space, distinguishing nutrient changes that affect diapause from changes that induce other dormancies. Refeeding is another important experimental technique that distinguishes between diapause and quiescence, a non-diapause dormancy. We also find insufficient evidence for the hypothesis that nutrient stores regulate diapause length and suggest manipulations to investigate the role of nutrient stores in diapause termination. Finally, we propose mechanisms that could interface nutritional status with the diapause program, focusing on combined action of the nutritional axis between the gut, fat body, and brain.

Enhanced germline stem cell longevity in Drosophila diapause.

In many species including humans, aging reduces female fertility. Intriguingly, some animals preserve fertility longer under specific environmental conditions. For example, at low temperature and short day-length, Drosophila melanogaster enters a state called adult reproductive diapause. As in other stressful conditions, ovarian development arrests at the yolk uptake checkpoint; however, mechanisms underlying fertility preservation and post-diapause recovery are largely unknown. Here, we report that diapause causes more complete arrest than other stresses yet preserves greater recovery potential. During dormancy, germline stem cells (GSCs) incur DNA damage, activate p53 and Chk2, and divide less. Despite reduced niche signaling, germline precursor cells do not differentiate. GSCs adopt an atypical, suspended state connected to their daughters. Post-diapause recovery of niche signaling and resumption of division contribute to restoring GSCs. Mimicking one feature of quiescence, reduced juvenile hormone production, enhanced GSC longevity in non-diapausing flies. Thus, diapause mechanisms provide approaches to GSC longevity enhancement.

Changes in metabolism and antioxidant systems during tropical diapause in the sunflower caterpillar Chlosyne lacinia (Lepidoptera: Nymphalidae).

Insect diapause shares many biochemical features with other states of metabolic depression, including the suppression of global metabolism, reorganization of metabolic pathways and improved stress resistance. However, little is known about the biochemical changes associated with the diapause phenotype in tropical insects. To investigate biochemical adaptations associated with tropical diapause, we measured the activities of metabolic and antioxidant enzymes, as well as glutathione levels, in the sunflower caterpillar Chlosyne lacinia at different times after initiation of diapause (<1, 20, 40, 60, and 120 days) and after arousal from diapause. Biochemical changes occurred early in diapausing animals, between the first 24 h and 20 days of diapause. Diapausing animals had reduced oxidative capacity associated with a decrease in the activities of peroxide-decomposing antioxidant enzymes. There was no sign of redox imbalance either during diapause or after recovery from diapause. Noteworthy, glutathione transferase and isocitrate dehydrogenase-NADP+ activities sharply increased in diapausing animals and stand out as diapause-associated proteins. The upregulation of these two enzymes ultimately indicate the occurrence of Preparation for Oxidative Stress in the tropical diapause of C. lacinia.

Dormancy in laboratory-reared Asian longhorned beetles, Anoplophora glabripennis.

An insect's capacity to survive winter is critical for range expansion in temperate regions. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-boring insect native to China and the Korean peninsula and poses a high risk of invasion in North America and Europe. It is unclear whether A. glabripennis enters diapause, which means that diapause cannot be included in assessments of the risk of this species invading forests in temperate regions. Using a laboratory colony, we examine larval developmental arrest, metabolic rates, gas exchange patterns, thermal sensitivity, and body composition to characterize larval dormancy. Chilled larvae entered a temperature-independent developmental arrest which usually required more than four weeks of chilling to break, decreased their metabolic rate by as much as 63%, and maintained energy stores throughout the chilling period - results consistent with an obligate diapause. We also observed a switch to discontinuous gas exchange at low temperatures. Thermal sensitivity of metabolic rate did not differ between chilled and non-chilled larvae. Taken together, we conclude that A. glabripennis enters a larval diapause during chilling and terminates diapause after a requisite chilling period. These results will enhance our ability to predict phenology and potential distribution of current and future invasions of A. glabripennis.

Cold tolerance, water balance, energetics, gas exchange, and diapause in overwintering brown marmorated stink bugs.

Halyomorpha halys (Hemiptera: Pentatomidae) is an emerging pest which established in Ontario, Canada, in 2012. Halyomporpha halys overwinters in anthropogenic structures as an adult. We investigated seasonal variation in the cold tolerance, water balance, and energetics of H. halys in southwestern Ontario. We also induced diapause in laboratory-reared animals with short daylength at permissive temperatures and compared cold tolerance, water balance, energetics, and metabolism and gas exchange between diapausing and non-diapausing individuals. Halyomorpha halys that overwintered outside in Ontario all died, but most of those that overwintered in sheltered habitats survived. We confirm that overwintering H. halys are chill-susceptible. Over winter, Ontario H. halys depressed their supercooling point to c. -15.4 °C, and 50% survived a 1 h exposure to -17.5 °C. They reduce water loss rates over winter, and do not appear to significantly consume lipid or carbohydrate reserves to a level that might cause starvation. Overall, it appears that H. halys is dependent on built structures and other buffered microhabitats to successfully overwinter in Ontario. Laboratory-reared diapausing H. halys have lower supercooling points than their non-diapausing counterparts, but LT50 is not enhanced by diapause induction. Diapausing H. halys survive desiccating conditions for 3-4 times longer than those not in diapause, through decreases in both respiratory and cuticular water loss. Diapausing H. halys do not appear to accumulate any more lipid or carbohydrate than those not in diapause, but do have lower metabolic rates, and are slightly more likely to exhibit discontinuous gas exchange.

Two calcium-binding chaperones from the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause.

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin-type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N-globular domain, a P-arm domain, and a highly charged C-terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause-related expression analysis in the whole body revealed an upregulation of both genes by post-diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause-related expression pattern in the entire body for both genes. Fat body-specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post-diapause. This study suggests that calcium-binding chaperones play similar and possibly gender-specific roles during diapause.

To mate, or not to mate: The evolution of reproductive diapause facilitates insect radiation into African savannahs in the Late Miocene.

Many tropical environments experience cyclical seasonal changes, frequently with pronounced wet and dry seasons, leading to a highly uneven temporal distribution of resources. Short-lived animals inhabiting such environments often show season-specific adaptations to cope with alternating selection pressures. African Bicyclus butterflies show strong seasonal polyphenism in a suite of phenotypic and life-history traits, and their adults are thought to undergo reproductive diapause associated with the lack of available larval host plants during the dry season. Using 3 years of longitudinal field data for three species in Malawi, dissections demonstrated that one forest species reproduces continuously, whereas two savannah species undergo reproductive diapause in the dry season, either with or without pre-diapause mating. Using additional data from field-collected and museum samples, we then documented the same three mating strategies for a further 37 species. Phylogenetic analyses indicated that the ancestral state was a non-diapausing forest species, and that habitat preference and mating strategy evolved in a correlated fashion. Bicyclus butterflies underwent rapid diversification during the Late Miocene, coinciding with expansions into more open savannah habitat. We conclude that the ability to undergo reproductive diapause was a key trait that facilitated colonization and eventual radiation into savannahs in the Late Miocene.

Developmental plasticity in metabolism but not in energy reserve accumulation in a seasonally polyphenic butterfly.

The evolution of seasonal polyphenisms (discrete phenotypes in different annual generations) associated with alternative developmental pathways of diapause (overwintering) and direct development is favoured in temperate insects. Seasonal life history polyphenisms are common and include faster growth and development under direct development than in diapause. However, the physiological underpinnings of this difference remain poorly known despite its significance for understanding the evolution of polyphenisms. We measured respiration and metabolic rates through the penultimate and final larval instars in the butterfly Pieris napi and show that directly developing larvae grew and developed faster and had a higher metabolic rate than larvae entering pupal diapause. The metabolic divergence appeared only in the final instar, that is, after induction of the developmental pathway that takes place in the penultimate instar in P. napi. The accumulation of fat reserves during the final larval instar was similar under diapause and direct development, which was unexpected as diapause is predicted to select for exaggerated reserve accumulation. This suggests that overwinter survival in diapause does not require larger energy reserves than direct development, likely because of metabolic suppression in diapause pupae. The results, nevertheless, demonstrate that physiological changes coincide with the divergence of life histories between the alternative developmental pathways, thus elucidating the proximate basis of seasonal life history polyphenisms.

Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

Bombyxin/Akt signaling in relation to the embryonic diapause process of the silkworm, Bombyx mori.

Our previous study showed that phosphorylation of glycogen synthase kinase (GSK)-3ß is related to the embryonic diapause process in Bombyx. However, the upstream signaling pathway was not clearly understood. In the present study, we examined bombyxin/Akt signaling in relation to the embryonic diapause process of B. mori. Results showed that GSK-3ß phosphorylation stimulated by dechorionation was blocked by LY294002, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, indicating involvement of PI3K in GSK-3ß phosphorylation in dechorionated eggs. Direct determination of Akt phosphorylation showed that dechorionation stimulated Akt phosphorylation. The Akt phosphorylation was blocked by LY294002. Temporal changes in Akt phosphorylation showed that different changing patterns exist between diapause and developing eggs. Relatively higher phosphorylation levels of Akt were detected between days 3 and 5 after oviposition in non-diapause eggs compared to those at the same stages in diapause eggs. Upon treatment with HCl, which prevents diapause initiation, Akt phosphorylation levels exhibited a later and much broader peak compared to diapause eggs. Examination of expression levels of the bombyxin-Z1 gene showed that in diapause eggs, a major peak occurred 1 day after oviposition, and its level then sharply decreased on day 2. However, in both non-diapause and HCl-treated eggs, a major broad peak was detected between days 1 and 4 after oviposition. These temporal changes in bombyxin-Z1 gene expression levels during embryonic stages coincided with changes in Akt phosphorylation, indicating that bombyxin-Z1 is likely an upstream signaling component for Akt phosphorylation. Taken together, our results indicated that PI3K/Akt is an upstream signaling pathway for GSK-3ß phosphorylation and is associated with the diapause process of B. mori eggs. To our knowledge, this is the first study to demonstrate the potential correlation between bombyxin/Akt signaling and the embryonic diapause process.

PTEN expression responds to transcription factor POU and regulates p-AKT levels during diapause initiation in the cotton bollworm, Helicoverpa armigera.

Diapause is a complex physiological response accompanied by many signaling pathways participating in the process. Previous studies have shown that p-AKT levels in brains of diapause-destined pupae are elevated by ROS, and the activated AKT promotes Glut expression for glucose uptake during diapause entry in Helicoverpa armigera. However, the mechanism by which ROS activate AKT is still unclear. Here, we show that PTEN, a PI3K/p-AKT signaling inhibitor, was significantly lower in the brains of diapause-destined pupae and that p-AKT levels were elevated by a lack of PTEN dephosphorylating PIP3. In addition, POU was identified as a transcription factor that binds to the PTEN promoter and regulates its expression. POU expression was enhanced by ecdysone but suppressed by ROS, suggesting that POU/PTEN plays a central role in responding to ROS signaling and regulating p-AKT levels. These results suggest that ecdysone and ROS participate together in the regulation of insect diapause through downregulation of POU/PTEN, which elevates p-AKT levels.

Diapause in a tropical oil-collecting bee: molecular basis unveiled by RNA-Seq.

BACKGROUND: Diapause is a natural phenomenon characterized by an arrest in development that ensures the survival of organisms under extreme environmental conditions. The process has been well documented in arthropods. However, its molecular basis has been mainly studied in species from temperate zones, leaving a knowledge gap of this phenomenon in tropical species. In the present study, the Neotropical and solitary bee Tetrapedia diversipes was employed as a model for investigating diapause in species from tropical zones. Being a bivoltine insect, Tetrapedia diversipes produce two generations of offspring per year. The first generation, normally born during the wet season, develops faster than individuals from the second generation, born after the dry season. Furthermore, it has been shown that the development of the progeny, of the second generation, is halted at the 5th larval instar, and remains in larval diapause during the dry season. Towards the goal of gaining a better understanding of the diapause phenomenon we compared the global gene expression pattern, in larvae, from both reproductive generations and during diapause. The results demonstrate that there are similarities in the observed gene expression patterns to those already described for temperate climate models, and also identify diapause-related genes that have not been previously reported in the literature. RESULTS: The RNA-Seq analysis identified 2275 differentially expressed transcripts, of which 1167 were annotated. Of these genes, during diapause, 352 were upregulated and 815 were downregulated. According to their biological functions, these genes were categorized into the following groups: cellular detoxification, cytoskeleton, cuticle, sterol and lipid metabolism, cell cycle, heat shock proteins, immune response, circadian clock, and epigenetic control. CONCLUSION: Many of the identified genes have already been described as being related to diapause; however, new genes were discovered, for the first time, in this study. Among those, we highlight: Niemann-Pick type C1, NPC2 and Acyl-CoA binding protein homolog (all involved in ecdysteroid synthesis); RhoBTB2 and SASH1 (associated with cell cycle regulation) and Histone acetyltransferase KAT7 (related to epigenetic transcriptional regulation). The results presented here add important findings to the understanding of diapause in tropical species, thus increasing the comprehension of diapause-related molecular mechanisms.

NMR-based untargeted metabolomic study of hydrogen peroxide-induced development and diapause termination in brine shrimp.

Artemia diapause has been extensively studied in embryonic biology for a long time. It has been demonstrated that hydrogen peroxide (H2O2) can increase the hatching rate in the development and diapause termination of Artemia cysts. This study used an untargeted 1H NMR-based metabolomic approach to explore the physiological regulation of H2O2 in initiating the development and terminating the diapause of Artemia cysts. This experiment was divided into two parts. In the first part, we analyzed three groups with or without H2O2 as control-0h, control-5h and H2O2 (180µM)-5h; in the second part, after 7-d incubation, the non-hatching cysts were treated with different H2O2 concentrations as low as 180µM and as high 1800µM. The results showed that arginine and proline metabolism were up-regulated after 5h, and H2O2 up-regulated valine, leucine and isoleucine biosynthesis in the development of cysts. In the second part, low H2O2 (180µM) showed alanine, aspartate and glutamate metabolism, but high H2O2 (1800µM) also up-regulated arginine and proline metabolism, as in the control group without H2O2 stimulus. These results suggest that enough H2O2 can catalyze cell transcription and translation in Artemia cysts, and it improves the cell growth rate, thus allowing embryo cells to grow again.

TGF-ß signaling regulates p-Akt levels via PP2A during diapause entry in the cotton bollworm, Helicoverpa armigera.

Akt, which is a key kinase in the insulin signaling pathway, plays important roles in glucose metabolism, cell proliferation, transcription and cell migration. Our previous studies indicated that low insulin levels and high p-Akt levels are present in diapause-destined individuals. Here, we show that PI3K, which is upstream of Akt, is low in diapause-destined pupal brains but high in p-Akt levels, implying that p-Akt is modified by factors other than the insulin signaling pathway. Protein phosphatase 2A (PP2A), which is a key regulator in the TGF-ß signaling pathway, can directly bind to and dephosphorylate Akt. Low PP2A expression and activity in diapause-destined individuals suggest that a weak Akt dephosphorylation contributes to p-Akt accumulation. In addition, transforming growth factor-ß receptor I (TßRI), which is upstream of PP2A, increases the activity of PP2A and decreases the p-Akt levels. These results show that TGF-ß signaling decreases p-Akt levels by increasing the activity of PP2A. This is the first report showing that TGF-ß signaling negatively regulates the insulin pathway in insect development or diapause.

Molecular characterization and functional analyses of a diapause hormone receptor-like gene in parthenogenetic Artemia.

In arthropods, mature females under certain conditions produce and release encysted gastrula embryos that enter diapause, a state of obligate dormancy. The process is presumably regulated by diapause hormone (DH) and diapause hormone receptor (DHR) that were identified in the silkworm, Bombyx mori and other insects. However, the molecular structure and function of DHR in crustaceans remains unknown. Here, a DHR-like gene from parthenogenetic Artemia (Ar-DHR) was isolated and sequenced. The cDNA sequence consists of 1410bp with a 1260-bp open reading frame encoding a protein consisting of 420 amino acid residues. The results of real-time PCR (qRT-PCR) and Western blot analysis showed that the mRNA and protein of Ar-DHR were mainly expressed at the diapause stage. Furthermore, we found that Ar-DHR was located on the cell membrane of the pre-diapause cyst but in the cytoplasm of the diapause cyst by analysis of immunofluorescence. In vivo knockdown of Ar-DHR by RNA interference (RNAi) and antiserum neutralization consistently inhibited diapause cysts formation. The results indicated that Ar-DHR plays an important role in the induction and maintenance of embryonic diapause in Artemia. Thus, our findings provide an insight into the regulation of diapause formation in Artemia and the function of Ar-DHR.

SETD4 Regulates Cell Quiescence and Catalyzes the Trimethylation of H4K20 during Diapause Formation in Artemia.

As a prominent characteristic of cell life, the regulation of cell quiescence is important for proper development, regeneration, and stress resistance and may play a role in certain degenerative diseases. However, the mechanism underlying quiescence remains largely unknown. Encysted embryos of Artemia are useful for studying the regulation of this state because they remain quiescent for prolonged periods during diapause, a state of obligate dormancy. In the present study, SET domain-containing protein 4, a histone lysine methyltransferase from Artemia, was identified, characterized, and named Ar-SETD4. We found that Ar-SETD4 was expressed abundantly in Artemia diapause embryos, in which cells were in a quiescent state. Meanwhile, trimethylated histone H4K20 (H4K20me3) was enriched in diapause embryos. The knockdown of Ar-SETD4 reduced the level of H4K20me3 significantly and prevented the formation of diapause embryos in which neither the cell cycle nor embryogenesis ceased. The catalytic activity of Ar-SETD4 on H4K20me3 was confirmed by an in vitro histone methyltransferase (HMT) assay and overexpression in cell lines. This study provides insights into the function of SETD4 and the mechanism of cell quiescence regulation.