Transcriptomic data support phylogenetic congruence and reveal genomic changes associated with the repeated evolution of annualism in aplocheiloid killifishes (Cyprinodontiformes).

Repeated evolution of novel life histories that are correlated with ecological variables offers opportunities to study convergence in genetic, developmental, and metabolic features. Nearly half of the 800 species of Aplocheiloid killifishes, a clade of teleost fishes with a circumtropical distribution, are "annual" or seasonal species that survive in ephemeral bodies of water that desiccate and are unfeasible for growth, reproduction, or survival for weeks to months every year. But the repeated evolution of adaptations that are key features of the annual life history among these fishes remains poorly known without a robust phylogenetic framework. We present a large-scale phylogenomic reconstruction of aplocheiloid killifishes evolution using newly sequenced transcriptomes obtained from a diversity of killifish lineages representing putative independent origins of annualism. Ancestral state estimation shows that developmental dormancy (diapause), a key trait of the killifish annual life cycle, may have originated up to seven times independently among African and South American lineages. To further explore the genetic basis of this unique trait, we measure changes in evolutionary rates among orthologous genes across the killifish tree of life by quantifying codon evolution using dN/dS ratios. We show that some genes have higher dN/dS ratios in lineages leading to species with annual life history. Many of them constitute key developmental genes or nuclear-encoded metabolic genes that control oxidative phosphorylation. Lastly, we compare these genes with higher ω to genes previously associated to developmental dormancy and metabolic shifts in killifishes and other vertebrates, and thereby identify molecular evolutionary signatures of repeated transitions to extreme environments.

Relationship of PSC to embryos: Extending and refining capture of PSC lines from mammalian embryos.

Pluripotent stem cell lines derived from preimplantation mouse embryos have opened opportunities for the study of early mammalian development and generation of genetically uncompromised material for differentiation into specific cell types. Murine embryonic stem cells are highly versatile and can be engineered and introduced into host embryos, transferred to recipient females, and gestated to investigate gene function at multiple levels as well as developmental mechanisms, including lineage segregation and cell competition. In this review, we summarize the biomedical motivation driving the incremental modification to culture regimes and analyses that have advanced stem cell research to its current state. Ongoing investigation into divergent mechanisms of early developmental processes adopted by other species, such as agriculturally beneficial mammals and birds, will continue to enrich knowledge and inform strategies for future in vitro models.

Advances in understanding Lepidoptera cold tolerance.

Ambient thermal conditions mediate insect growth, development, reproduction, survival, and distribution. With increasingly frequent and severe cold spells, it is critical to determine low-temperature performance and cold tolerances of ecologically and economically essential insect groups to predict their responses to global environmental change. This review covers the cold tolerance strategies of 49 species of Lepidoptera (moths and butterflies), focusing on species that are known as crop pests and crop storage facilities. We synthesize cold tolerance strategies of well-studied species within this order, finding that diapause is a distinctive mechanism that has independently evolved in different genera and families of Lepidoptera. However, the occurrence of diapause in each life stage is specific to the species, and in most studied lepidopteran species, the feeding stage (as larva) is the predominant overwintering stage. We also found that the onset of diapause and the improvement of cold tolerance are interdependent phenomena that typically occur together. Moreover, adopting a cold tolerance strategy is not an inherent, fixed trait and is greatly influenced by a species' geographic distribution and rearing conditions. This review further finds that freeze avoidance rather than freeze tolerance or chill susceptibility is the primary cold tolerance strategy among lepidopteran species. The cold hardiness of lepidopteran insects primarily depends on the accumulation of cryoprotectants and the depression of the supercooling point. We highlight variations in cold tolerance strategies and mechanisms among a subset of Lepidoptera, however, further work is needed to elucidate these strategies for the vast numbers of neglected species and populations to understand broad-scale responses to global change.

Phenotypical aspects of Culex pipiens biotype pipiens during diapause: Lipid utilization, body size, insemination, and parity.

In temperate regions, female Culex pipiens biotype pipiens mosquitoes undergo diapause in winter. Diapausing biotype pipiens mosquitoes are potentially important winter reservoirs for mosquito-borne viruses, such as West Nile virus (WNV), Usutu virus (USUV), and Sindbis virus (SINV). Mosquitoes in diapause have not taken a bloodmeal prior to winter. Therefore, they do not become infected by taking an infectious bloodmeal and as a consequence, vertical transmission is considered the primary mechanism of mosquito-borne virus overwintering. Prior to winter, biotype pipiens mosquitoes build up fat reserves, which they utilize throughout winter. Furthermore, earlier studies have indicated that larger body size is correlated with increased survival during winter. However, not much is known about lipid utilization and body size of wild biotype pipiens mosquitoes in diapause. Therefore, we performed monthly collections of diapausing biotype pipiens mosquitoes in two consecutive winters (2020/2021 and 2021/2022) in bunkers of the New Hollandic Waterline in the Netherlands. Every month, we checked the proportion of inseminated and parous females via microscopy. In addition, we measured wing length as proxy for body size, and assessed total lipid content. Furthermore, we monitored indoor temperature in the overwintering locations. We show that the overwintering sites in our study provide relatively stable environments, in which temperatures rarely drop below 0 °C. The vast majority of biotype pipiens females were inseminated (84.1 %) and nulliparous (97.5 %). We detected differences in body size between but not within the two years of sampling. Additionally, we detected a difference in lipid content between the sampling years. We confirm that the vast majority of diapausing biotype pipiens females are inseminated and nulliparous. This indicates that they did not blood feed prior to winter, which underscores the likeliness of vertical transmission being the primary mechanism behind virus overwintering. The detected difference in body size between years can most likely be attributed to differences in summer conditions the mosquitoes were exposed to as larvae, although this needs confirmation. The difference in lipid depletion could not be explained by differences in climatic conditions. To shed more light on the links between climatic conditions, body size, lipid depletion and the consequences for mosquito population dynamics and arbovirus transmission, future experimental work, for example by arbovirus exposure followed by artificially induced diapause, is desired.

Probing metabolism in mouse embryos using Raman spectroscopy and deuterium tags.

The use of deuterated compounds is an interesting opportunity to expand the capabilities of Raman spectroscopy to study metabolism in living cells. Different biological objects have different tolerances to different deuterated compounds, and their metabolic chains may differ. Here, we explore the potential of this approach to probe metabolism in early mouse embryos. We investigated the Raman spectra of mouse embryos at different developmental stages cultured with deuterated amino acids, phenylalanine-d8 and leucine-d10, glucose-d7, and D2O. Embryos after in vitro culture with 20 % v/v D2O demonstrate Raman peak at 2186 cm-1 corresponding to newly synthesized proteins. Deuterated amino acids can slow down the development rate in 4-8 cell stage embryos, and deuterated glucose can be used at 2 mM concentration. For blastocyst, it was possible to achieve 75 % fraction of deuterated phenylalanine, when cultured with glucose, the maximal intensity ratio between CD and CH bands was 13.7 %. To demonstrate the capabilities of Raman spectroscopy reinforced by deuterium labeling, we investigated the short-term effect of cryopreservation and revealed that cryopreservation decreases the amount of saccharides in embryos and does not affect the activity of protein de novo synthesis.

The small GTPase RAB-18 is involved in regulating development/diapause by recruiting the intestinal cholesterol transporter NCR-1 onto the apical side in Caenorhabditis elegans.

RAB family proteins, which are small GTPases, are integral to the process of eukaryotic membrane trafficking. In the nematode, Caenorhabditis elegans, 31 RAB proteins have been identified through genome sequencing. Using an RNAi screen specifically targeting C. elegans rab genes, we identified multiple genes that are involved in the regulation of larval development, in particular, the rab-18 gene. Our molecular genetic studies resulted in several findings. First, RAB-18 predominantly functions in the intestine to regulate larval development by modulating steroid hormone signaling. Second, the C. elegans cholesterol transporter NCR-1 is a target of RAB-18 in the intestine. Third, the membrane trafficking of NCR-1 to the apical side in intestinal cells is particularly influenced by RAB-18. Finally, RAB-18 and NCR-1 possibly co-localize on membrane vesicles. Our study is the first to demonstrate the relationship between a RAB protein and a cholesterol transporter, in which the RAB protein probably drives the transporter to the apical membrane in the intestine to regulate cholesterol uptake. This study provides insight into the molecular mechanisms underlying human disease stemming from a transport defect of cholesterol and its derivative.

Genome-wide analysis reveals transcriptional and translational changes during diapause of the Asian corn borer (Ostrinia furnacalis).

BACKGROUND: Diapause, a pivotal phase in the insect life cycle, enables survival during harsh environmental conditions. Unraveling the gene expression profiles of the diapause process helps uncover the molecular mechanisms that underlying diapause, which is crucial for understanding physiological adaptations. In this study, we utilize RNA-seq and Ribo-seq data to examine differentially expressed genes (DEGs) and translational efficiency during diapause of Asian corn borer (Ostrinia furnacalis, ACB). RESULTS: Our results unveil genes classified as "forwarded", "exclusive", "intensified", or "buffered" during diapause, shedding light on their transcription and translation regulation patterns. Furthermore, we explore the landscape of lncRNAs (long non-coding RNAs) during diapause and identify differentially expressed lncRNAs, suggesting their roles in diapause regulation. Comparative analysis of different types of diapause in insects uncovers shared and unique KEGG pathways. While shared pathways highlight energy balance, exclusive pathways in the ACB larvae indicate insect-specific adaptations related to nutrient utilization and stress response. Interestingly, our study also reveals dynamic changes in the HSP70 gene family and proteasome pathway during diapause. Manipulating HSP protein levels and proteasome pathway by HSP activator or inhibitor and proteasome inhibitor affects diapause, indicating their vital role in the process. CONCLUSIONS: In summary, these findings enhance our knowledge of how insects navigate challenging conditions through intricate molecular mechanisms.

mTOR activity paces human blastocyst stage developmental progression.

Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.

Transcriptome-Wide Evaluation Characterization of microRNAs and Assessment of Their Functional Roles as Regulators of Diapause in Ostrinia furnacalis Larvae (Lepidoptera: Crambidae).

microRNAs (miRNAs) function as vital regulators of diapause in insects through their ability to post-transcriptionally suppress target gene expression. In this study, the miRNA of Ostrinia furnacalis, an economically important global crop pest species, was characterized. For the included analyses, 9 small RNA libraries were constructed using O. furnacalis larvae in different diapause states (non-diapause, ND; diapause, D; diapause-termination, DT). The results identified 583 total miRNAs, of which 256 had previously been identified, whereas 327 were novel. Furthermore, comparison analysis revealed that 119 and 27 miRNAs were differentially expressed in the D vs. ND and DT vs. D, respectively. Moreover, the expression patterns of their miRNAs were also analyzed. GO and KEGG analysis of the target genes of differentially expressed miRNAs highlighted the importance of these miRNAs as diapause regulators in O. furnacalis, especially through metabolic processes, endocrine processes, 20-hydroxyecdysone, and circadian clock signaling pathways. In summary, this study highlighted the involvement of specific miRNAs in the control of diapause in O. furnacalis. To the best of our knowledge, this is the first study to identify miRNA expression patterns in O. furnacalis, thereby providing reference and novel evidence enhancing our current understanding of how small RNAs influence insect diapause.

Reproductive Strategies and Embryonic Development of Autumn-Spawning Bitterling (Acheilognathus rhombeus) within the Mussel Host.

We investigated the reproductive strategies and embryonic development of Acheilognathus rhombeus (a bitterling species that spawns in autumn) within its freshwater mussel host in the Bongseo Stream, South Korea. By focusing on survival mechanisms during critical stages of embryonic development, the selective use of mussel gill demibranchs by the bitterlings and associated adaptive traits were observed over 1 year. A significant diapause phase occurs at developmental stage D, which lasts for approximately 7 months, allowing embryos to survive winter. Development resumes when the temperature exceeds 10 °C. Minute tubercles on the embryos (crucial for anchoring within the host gill demibranchs and preventing premature ejection) exhibit the largest height during diapause, and the height decreases when developmental stage E is reached, when growth resumes. Acheilognathus rhombeus embryos were observed in 30.5% of the mussels, mostly within the inner gills, thereby maximizing spatial use and oxygen access to enhance survival. These results highlight the intricate relationship between A. rhombeus and its mussel hosts, demonstrating the evolutionary adaptations that enhance reproductive success and survival. This study provides valuable insights into the ecological dynamics and conservation requirements of such symbiotic relationships.

Sensory integration of food and population density during the diapause exit decision involves insulin-like signaling in Caenorhabditis elegans.

Decisions made over long time scales, such as life cycle decisions, require coordinated interplay between sensory perception and sustained gene expression. The Caenorhabditis elegans dauer (or diapause) exit developmental decision requires sensory integration of population density and food availability to induce an all-or-nothing organismal-wide response, but the mechanism by which this occurs remains unknown. Here, we demonstrate how the Amphid Single Cilium J (ASJ) chemosensory neurons, known to be critical for dauer exit, perform sensory integration at both the levels of gene expression and calcium activity. In response to favorable conditions, dauers rapidly produce and secrete the dauer exit-promoting insulin-like peptide INS-6. Expression of ins-6 in the ASJ neurons integrates population density and food level and can reflect decision commitment since dauers committed to exiting have higher ins-6 expression levels than those of noncommitted dauers. Calcium imaging in dauers reveals that the ASJ neurons are activated by food, and this activity is suppressed by pheromone, indicating that sensory integration also occurs at the level of calcium transients. We find that ins-6 expression in the ASJ neurons depends on neuronal activity in the ASJs, cGMP signaling, and the pheromone components ascr#8 and ascr#2. We propose a model in which decision commitment to exit the dauer state involves an autoregulatory feedback loop in the ASJ neurons that promotes high INS-6 production and secretion. These results collectively demonstrate how insulin-like peptide signaling helps animals compute long-term decisions by bridging sensory perception to decision execution.

RpedOBP1 plays key roles in aggregation pheromones reception of the Riptortus pedestris.

Riptortus pedestris (Hemiptera: Alydidae) is a notable soybean pest, with diapause and non-diapause individuals showing different sensitivities to aggregation pheromones. This study aimed to investigate how R. pedestris detects aggregation pheromones through electroantennogram (EAG) and behavioral experiments, transcriptome sequencing and qRT-PCR, as well as competitive fluorescence-binding assay. Results indicated that diapausing females and males of R. pedestris exhibited a heightened EAG response and were more attracted to the aggregation pheromone components compared to their non-diapause counterparts. Transcriptome sequencing and qRT-PCR analyses revealed significantly higher expression of RpedOBP1 in the antennae of diapause females and males compared to non-diapausing R. pedestris. The competitive fluorescence-binding assay demonstrated that RpedOBP1 displayed the strongest binding affinity to E2HE2H, suggesting its crucial role in recognizing the aggregation pheromone. These findings have the potential to inform the development of integrated pest management strategies utilizing behavioral approaches for bean bug control.

A quantitative model of temperature-dependent diapause progression.

Winter diapause in insects is commonly terminated through cold exposure, which, like vernalization in plants, prevents development before spring arrives. Currently, quantitative understanding of the temperature dependence of diapause termination is limited, likely because diapause phenotypes are generally cryptic to human eyes. We introduce a methodology to tackle this challenge. By consecutively moving butterfly pupae of the species Pieris napi from several different cold conditions to 20 °C, we show that diapause termination proceeds as a temperature-dependent rate process, with maximal rates at relatively cold temperatures and low rates at warm and extremely cold temperatures. Further, we show that the resulting thermal reaction norm can predict P. napi diapause termination timing under variable temperatures. Last, we show that once diapause is terminated in P. napi, subsequent development follows a typical thermal performance curve, with a maximal development rate at around 31 °C and a minimum at around 2 °C. The sequence of these thermally distinct processes (diapause termination and postdiapause development) facilitates synchronous spring eclosion in nature; cold microclimates where diapause progresses quickly do not promote fast postdiapause development, allowing individuals in warmer winter microclimates to catch up, and vice versa. The unveiling of diapause termination as one temperature-dependent rate process among others promotes a parsimonious, quantitative, and predictive model, wherein winter diapause functions both as an adaptation against premature development during fall and winter and for synchrony in spring.

Physiological and transcriptional changes associated with obligate aestivation in the cabbage stem flea beetle (Psylliodes chrysocephala).

Aestivation is a form of seasonal dormancy observed in various insect species, usually coinciding with the summer season. The cabbage stem flea beetle, Psylliodes chrysocephala (Coleoptera: Chrysomelidae), is a key pest of oilseed rape that obligatorily aestivates as adult in late summer. Since the physiological and transcriptional processes linked to aestivation in P. chrysocephala are still little understood, we analyzed relevant physiological parameters and performed RNA-seq analyses on laboratory-reared beetles in their pre-aestivation, aestivation, and post-aestivation stages. We found that the beetles reached aestivation at 15 days post-eclosion, showing strongly reduced metabolic activity, with less than 50% CO2 production, compared to pre-aestivating individuals. Under constant laboratory conditions, the beetles aestivated for about 25 days. Female beetles reached reproductive maturity at a median of 52 days post-eclosion. Furthermore, aestivating beetles had significantly reduced carbohydrate reserves and increased lipid reserves compared with pre-aestivating beetles, indicating that aestivation is associated with drastic changes in energy metabolism. Aestivating beetles contained 30% less water and their survival rates under high-temperature conditions (30 °C) were 40% higher compared to pre-aestivating beetles. RNA-seq studies showed that, in particular, gene ontology terms related to carbohydrate and lipid metabolism, digestion, and mitochondrial activity were enriched, with clear differences in transcript abundance between beetles in aestivation compared to pre- or post-aestivation. Specifically, mitochondrial transcripts, such as respiratory chain I subunits, and digestion-related transcripts, such as trypsin, were less abundant during aestivation, which supports the idea that aestivation is associated with decreased metabolic activity. This study represents the first exploration of the transcriptomic and physiological processes linked to aestivation in P. chrysocephala.

Developmental ecology in embryos of an estuarine pupfish endemic of the Yucatan peninsula: Survival out of water, metabolic depression, and asynchronous hatching.

BACKGROUND: Theory predicts that drought-resistant embryos with extended incubations are evolutionarily favored in environments with high mortality of larvae but safe for eggs. Here, we experimentally test, under common garden conditions, the effect of three incubation temperatures and media on embryonic developmental length, extended incubation out of the water, survival, metabolic rate, and hatching dynamics in the estuarine pupfish Garmanella pulchra. We also described the morphological changes of embryonic cortical structures related to air exposure. RESULTS: We found that embryos incubated out of water in low and medium temperatures present an extended incubation period beyond their hatching capability with a deep metabolic depression. Also, these embryos exhibited a hatching asynchrony not related to water availability. Embryos incubated at high temperatures did not show extended incubation, with decreased probability of survival out of water. Our morphological observations of the embryonic cortical structures reveal that the perivitelline space and hair-like filaments buffer the deleterious drought effects. CONCLUSIONS: Our results reveal that G. pulchra possesses life-history traits typical of two separate phenomena: delay hatching and diapause; supporting a true continuum between them, rather than a dichotomy. The evolution of these traits may respond to aerial exposure during low tides in the estuaries of Yucatán they inhabit.

Changes in bumblebee queen gut microbiotas during and after overwintering diapause.

Bumblebees are key pollinators with gut microbiotas that support host health. After bumblebee queens undergo winter diapause, which occurs before spring colony establishment, their gut microbiotas are disturbed, but little is known about community dynamics during diapause itself. Queen gut microbiotas also help seed worker microbiotas, so it is important that they recover post-diapause to a typical community structure, a process that may be impeded by pesticide exposure. We examined how bumblebee queen gut microbiota community structure and metabolic potential shift during and after winter diapause, and whether post-diapause recovery is affected by pesticide exposure. To do so, we placed commercial Bombus impatiens queens into diapause, euthanizing them at 0, 2 and 4 months of diapause. Additionally, we allowed some queens to recover from diapause for 1 week before euthanasia, exposing half to the common herbicide glyphosate. Using whole-community, shotgun metagenomic sequencing, we found that core bee gut phylotypes dominated queen gut microbiotas before, during and after diapause, but that two phylotypes, Schmidhempelia and Snodgrassella, ceased to be detected during late diapause and recovery. Despite fluctuations in taxonomic community structure, metabolic potential remained constant through diapause and recovery. Also, glyphosate exposure did not affect post-diapause microbiota recovery. However, metagenomic assembly quality and our ability to detect microbial taxa and metabolic pathways declined alongside microbial abundance, which was substantially reduced during diapause. Our study offers new insights into how bumblebee queen gut microbiotas change taxonomically and functionally during a key life stage and provides guidance for future microbiota studies in diapausing bumblebees.

Molecular identification of two thioredoxin genes and their function in antioxidant defense in Arma chinensis diapause.

Thioredoxin (Trx), an important part of thioredoxin systems, plays crucial role in maintaining the intracellular redox balance by scavenging reactive oxygen species (ROS). However, few Trxs have been functionally characterized in Arma chinensis, especially in diapause. In this study, diapause induction condition promoted hydrogen peroxide accumulation and increased CAT enzymatic activity and ascorbate content, suggesting that A. chinensis was exposed to high level of ROS. Therefore, we identified AcTrx2 and AcTrx-like, and investigated the relationship with antioxidant defense. It was found that AcTrx2 expression was significantly induced, whereas AcTrx-like expression was the highest on day 10 under diapause conditions. The expression of AcTrx2 and AcTrx-like in fat body, a central metabolic organ of resisting oxidative stress, was significantly increased under diapause conditions, and was significantly improved by 5/15°C (diapause temperature). We investigated the knockdown of AcTrx2 and AcTrx-like in A. chinensis and found that some selected antioxidant genes were upregulated, indicating that the upregulated genes may be functional compensation for AcTrx2 and AcTrx-like silencing. We also found that the enzymatic activities of SOD and CAT, and the metabolite contents of hydrogen peroxide, ascorbate increased after AcTrx2 and AcTrx-like knockdown. These results suggested the AcTrx2 and AcTrx-like may play critical roles in antioxidant defense of A. chinensis diapause.

Optimal phenology of life history events in Calanus finmarchicus: exit from diapause in relation to interannual variation in spring bloom timing and predation.

Respiration of lipids by copepods during diapause (overwintering dormancy) contributes to ocean carbon sequestration via the seasonal lipid pump (SLP). Parameterizing this flux in predictive models requires a mechanistic understanding of how life history adaptation in copepods shapes their timing of exit from diapause. We investigate the optimal phenology of Calanus finmarchicus in the Norwegian Sea using an individual-based model in which diapause exit is represented as a trait characterized by phenotypic mean and variance. Without interannual variability, optimal exit correlated with the onset of the spring phytoplankton bloom and phenotypic variance was of no benefit. In contrast, copepods endured reduced fitness and adopted bet-hedging strategies when exposed to interannual variability in bloom timing and predation: later exit from diapause and phenotypic variance maintained adult numbers in anomalous late-bloom years. Exit nevertheless remained well before the peak of the bloom which is a favorable strategy when low predation early in the year enhances survival of eggs and early developmental stages. Our work highlights the complex interactions between C. finmarchicus and its environment and the need for improved understanding of bet-hedging strategies and the cues of diapause exit to progress the representation of the SLP in global biogeochemical models.

Comparative Proteomics and Metabonomics Analysis of Different Diapause Stages Revealed a New Regulation Mechanism of Diapause in Loxostege sticticalis (Lepidoptera: Pyralidae).

Histone acetylation is an important epigenetic mechanism that has been shown to play a role in diapause regulation. To explore the physiological and molecular mechanisms of histone deacetylase in the diapause process, LC-MS/MS analysis was used to perform TMT proteomic and metabolomic analysis on non-diapause (ND), pre-diapause (PreD), diapause (D), cold treatment (CT), and post-diapause (RD) stages of the meadow moth. A total of 5367 proteins were identified by proteomics, including 1179 differentially expressed proteins. We found 975 (602 up-regulated and 373 down-regulated), 997 (608 up-regulated and 389 down-regulated), 1119 (726 up-regulated and 393 down-regulated), 1179 (630 up-regulated and 549 down-regulated), 94 (51 up-regulated and 43 down-regulated), 111 (63 up-regulated and 48 down-regulated), 533 (243 up-regulated and 290 down-regulated), 58 (31 up-regulated and 27 down-regulated), and 516 (228 up-regulated and 288 down-regulated) proteins in ND and PreD, ND and D, ND and CT, ND and RD, PreD and D, PreD and CT, PreD and RD, D and CT, D and RD, and CT and RD stages, respectively. A total of 1255 differentially expressed metabolites were annotated by metabolomics. Through KEGG analysis and time series analysis of differentially expressed metabolites, we found that phospholipids were annotated in significantly different modules, demonstrating their important role in the diapause process of the meadow moth. Using phospholipids as an indicator for weighted gene co-expression network analysis, we analyzed the most relevant differentially expressed proteins in the module and found that ribosomal 40s and 60s subunits were the most relevant proteins for diapause. Because there have been studies that have shown that histone deacetylase is associated with the diapause of meadow moths, we believe that histone deacetylase regulates the 40s and 60s subunits of ribosomes, which in turn affects the diapause of meadow moths. This finding expands our understanding of the regulation of meadow moth diapause and provides new insights into its control mechanism.

Integrating ATAC-Seq and RNA-Seq Reveals the Signal Regulation Involved in the Artemia Embryonic Reactivation Process.

Embryonic diapause is a common evolutionary adaptation observed across a wide range of organisms. Artemia is one of the classic animal models for diapause research. The current studies of Artemia diapause mainly focus on the induction and maintenance of the embryonic diapause, with little research on the molecular regulatory mechanism of Artemia embryonic reactivation. The first 5 h after embryonic diapause breaking has been proved to be most important for embryonic reactivation in Artemia. In this work, two high-throughput sequencing methods, ATAC-seq and RNA-seq, were integrated to study the signal regulation process in embryonic reactivation of Artemia at 5 h after diapause breaking. Through the GO and KEGG enrichment analysis of the high-throughput datasets, it was showed that after 5 h of diapause breaking, the metabolism and regulation of Artemia cyst were quite active. Several signal transduction pathways were identified in the embryonic reactivation process, such as G-protein-coupled receptor (GPCR) signaling pathway, cell surface receptor signaling pathway, hormone-mediated signaling pathway, Wnt, Notch, mTOR signaling pathways, etc. It indicates that embryonic reactivation is a complex process regulated by multiple signaling pathways. With the further protein structure analysis and RT-qPCR verification, 11 GPCR genes were identified, in which 5 genes function in the embryonic reactivation stage and the other 6 genes contribute to the diapause stage. The results of this work reveal the signal transduction pathways and GPCRs involved in the embryonic reactivation process of Artemia cysts. These findings offer significant clues for in-depth research on the signal regulatory mechanisms of the embryonic reactivation process and valuable insights into the mechanism of animal embryonic diapause.