Stress tolerance and proteins involved in damage prevention

Our results suggest that diapause embryos could respond by significantly altering their metabolic profile due to the dehydration stress imposed by the environment. Embryos subjected to dehydration also have greater oxygen availability, and in this sense, the dehydration stress would increase reactive oxygen species (ROS) formation that can lead to lipid peroxidation, denaturation of proteins, and DNA damage [32]. It is known that organisms regulate their defenses against oxidative stress by increasing the production of antioxidant enzymes when dehydrated [33].

Taking this into account, the embryo should trigger a response consecutive to changes in environmental conditions, which could involve biosynthesis of a number of new molecules, particularly proteins involved in the scavenging of ROS. Among the proteins known for their protective function against oxidative damage, the most notable change found in DIA concerns two superoxide dismutases (SOD), Cu/Zn SOD and Mn SOD. These are metalloenzymes with a primary role of catalyzing the conversion of superoxide radicals to molecular oxygen and hydrogen peroxide, which are then enzymatically converted by catalase into molecular oxygen and H2O. Cu/Zn SOD is usually the most abundant SOD and can be found in the cytosol and in the extracellular space, while Mn SOD is a mitochondrial matrix enzyme that scavenges oxygen radicals produced by the oxidation–reduction and electron transport reactions that occur in the mitochondria [34]. Although catalase was present in our study, no change was detected under the conditions tested.

Wagner et al. [35] showed that tolerance to H2O2 reaches a peak during Austrofundulus limnaeus DII, while DIII embryos exhibit the lowest tolerance of all stages studied. However, these authors saw that protection against oxidative damage relies on small-molecule antioxidants during early development and on enzymatic systems towards the end of embryonic development, although most enzymatic antioxidant activity did not seem to depend on SOD [35]. Nevertheless, strategic antioxidant protection associated with increased SOD expression was described for other diapausing organisms [36]. Interestingly, it has been suggested that FoxO plays a conserved role in setting diapauses [37].

The targets of the FoxO transcription factor include genes that encode antioxidant proteins whose function is to eliminate ROS. Among these targets are human Mn-SOD and murine Cu/Zn-SOD. Catalase is also regulated by FoxO [38]. Although FoxO was not among the proteins identified in A. charrua, as a transcription factor its expression level can be low, and it is worth looking specifically to see if it also exhibits differential expression during DIA.

It is also striking that NAD/NADP-dependent betaine aldehyde dehydrogenase (ALDH) was expressed exclusively in DIA. ALDH enzymes metabolize aldehydes derived from lipid peroxidation during oxidative stress, many of which are extremely reactive and toxic. Upregulation of ALDHs is a practically universal response against a variety of environmental stressors, such as dehydration and ultraviolet radiation. In C. elegans during the dauer life stage, the signaling pathways that modulate antioxidant skills induce the expression of ALDHs [39]. In addition, in plants, the toxic aldehyde intermediate betaine aldehyde is transformed into glycine betaine by the same ALDH isoform that is exclusively expressed in DIA. Glycine betaine is an osmolyte that protects plants from dehydration and other unfavorable conditions, such salinity, cold and oxidative stress [40].

The proteins contained in the yolk are the source of amino acids that can have a direct role as building blocks for new protein synthesis or can sustain the generation of energy in the embryo. Among exclusive DIA proteins, the one with the highest representation was glutamate dehydrogenase (GDH), an enzyme that catalyzes oxidative deamination of the amino acid glutamate, generating α-ketoglutarate and ammonia while reducing NAD+ or NADP+. The obtained α-ketoglutarate is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, GDH function is linked to the maintenance of cellular redox homeostasis. GDH controls the intracellular levels of its product α- ketoglutarate and controls the next TCA metabolite, fumarate, which binds to and activates a reactive oxygen species that can be scavenged by the enzyme glutathione peroxidase [41]. Although this may be of great importance for diapause embryos since they must overcome stressors, another factor must be considered. The alpha-ketoglutarate obtained by GDH could produce2-hydroxyglutarate in a reaction catalyzed by 3-phosphoglycerate dehydrogenase (PHGDH) or by the action of malate dehydrogenase [42, 43] Of the two enzymes, the only one present in the current work was the malate dehydrogenase (but without reaching the required levels to be considered differentially expressed). Interestingly, a metabolomic analysis carried out in Austrofundulus limnaeus embryos suffering from dehydration stress during their diapause detected the metabolite 2-hydroxyglutarate [44]. The 2-hydroxyglutarate competitively inhibits α-ketoglutarate-dependent dioxygenases, and among its targets are prolyl hydroxylase domain (PHD) proteins, which are critical elements in stabilizing the hypoxia inducible factor HIF1α and hence the regulation of the transcription factor HIF-1. This factor plays a prominent role in the metabolic adjustments that drive cellular adaptation to low oxygen availability [45]. However, since DIII embryos have abundant availability of oxygen, the responsibility of arranging metabolic changes would not fall on HIF-1 coordination. Similarly, if there were changes in the epigenetic scenery of A. charrua DIII as suggested by changes that we will discuss later, they most likely would not adhere to the inhibition of DNA demethylases and histone demethylases by 2-hydroxyglutarate.

Heat shock proteins (HSPs) are damage prevention proteins that rapidly change their expression patterns in response to environmental shock. It is well established that HSP expression can change on several diapauses; however, an increase does not always occur. During diapause of insects, HSPs may increase, decrease, or remain unchanged [46]. For example, it is argued that HSPs would not play any role during Bombyx mori diapause because its expression does not change between diapause and nondiapause eggs [47, 48].

In Sarcophaga crassipalpis, HSP90 expression is high in nondiapausing and early diapause-destined individuals but drops significantly thereafter [49], and Nasonia presents this same expression pattern [48]. Meanwhile, our results indicate that HSPs were present in PRE and DIA, with only the 63 kDa chaperonin overrepresented in DIA. HSP90 and cognate HSP70 and HSP71 were overrepresented in PRE, while the protein that interacts with Hsc70 was exclusive to this stage. This scenario could be a sign that changes in HSPs occur prior to rather than in reaction to diapause, suggesting that HSPs would be ready to protect proteins from irreversible denaturation imposed by the stress associated with diapause. A similar method was previously proposed by King and MacRae [50]. The only available "omic" study of DIII is a transcriptomic approach in the annual killifish Nematolebias whitei [20]. This study showed high differential expression of HSP cognate 71 kDa protein-like between DIA and free-swimming larvae. In our study, this protein appeared to be overrepresented in PRE, but given that the present proteomic study was performed in a different species, we do not know what happens to the expression of messenger RNA in DIA embryos of A. charrua. In addition, we cannot ignore the fact that these results reflect what occurs in a time window; therefore, kinetic studies should be carried out to obtain a complete picture of A. charrua embryo HSPs expression.

Although increased expression of proteins with alpha-crystallin domains with chaperone or heat-shock functions during dormancy has been identified in different organisms, particularly invertebrates [46, 51], the six diapause increased proteins identified as crystalline in the present work were from the beta and gamma families. This suggests that the observed overrepresentation could be due to the lens growth of the embryo between the prehatching stage (ST 41) and DIII (ST 43).

Energy metabolism

It is usually assumed that during diapauses, metabolism is depressed; however, our results suggest metabolic changes that appear to indicate energy metabolism enhancement compared to the prehatching stage. DIA proteome profiling would indicate increased function of glycolysis that would not generate biomass if the diapause embryo does not grow or proliferate [2, 7, 18]. (we must also note that the cell cycle could be arrested by DIA periplakin and vimentin exclusivity as we will discuss later).

Glycolysis is the front door to obtain metabolic energy from carbohydrates, while gluconeogenesis works in the opposite direction, although it shares several steps with the glycolytic process. However, the glycolysis rate-limiting enzyme hexokinase exclusively found in DIA suggests that the glycolysis pathway was increased under diapausing conditions, utilizing glucose derived from the yolk’s glycogen [52]. Interestingly, concerning the DIA hexoquinase isotype (hexoquinase I), different experimental approaches concluded that the enzyme is bound to actively phosphorylating mitochondria facilitating the access to intramitochondrial ATP as substrate. This would further coordinate the first glycolysis reaction in the cytosol with the final oxidative stages of glucose metabolism in the mitochondria, thus ensuring an overall rate of glucose metabolism in line with cellular energy demands [53]. Furthermore, binding of hexoquinase I to mitochondria prevents Bax-induced cytochrome c release and therefore this binding inhibits apoptosis [54–56]. The hexokinase I-mitochondria interaction controlling both bioenergetics and cell death, aspects that would be relevant in diapause III, undoubtedly presents great interest and deserves to be explored. In addition, several enzymes involved in glycolytic metabolism were significantly more abundant in DIA, such as phosphoglucomutase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase and enolase, some of which are known to be upregulated during insect diapauses [48]. Similarly, Hussein et al [57] showed that glycolysis was the most significantly upregulated metabolic pathway from the preimplantation to the diapause stage based on RNA-seq analysis conducted on mouse blastocysts. Since mouse blastocyst diapause is a cell cycle arrest and growth dormancy stage, these authors pose an open question: why does diapause require such a high rate of glycolysis? It is important to bear in mind that glycolysis is among the biochemical changes created by the stress response that accompanies diapauses. In some cases, its activation is oriented towards glycerol biosynthesis, as seen in overwintering insects and in hibernating black bears [58, 59]. Under normal conditions, glycerol is mainly used to make triacylglycerols and phospholipids. However, some organisms exposed to cold environments use glycerol as a cryoprotectant for their macromolecules, and others accumulate glycerol during diapause and use it as an energy resource when diapause ends [60]. Glycerol-3-phosphate dehydrogenase (GDH) catalyzes the redox conversion of dihydroxyacetone phosphate to glycerol-3-phosphate, which can be used to generate glycerol. In A. charrua, the GDH protein was found specifically, in DIA. If glycerol synthesis would be increased, there could be fewer precursors available for glycolysis. However, GAPDH was also increased in DIA. Hence, it could be necessary for the embryo to alternate between these options, emphasizing GDH or GAPDH. Based on their own results in diapausing Nasonia and on older data from Bombyx mori diapause, Wolschin and Gadau [48] proposed a pathway in which switching between glycerol synthesis and glycolysis would limit glycerol gain to a restricted time window to ensure sufficient glycolysis. Recently, it was found that GDH is expressed specifically in the dormant brain tumor stem cell (BTSC) population. In a metabolomic assay, it was shown that the most notably enriched pathway in these cells is the taurine and hypotaurine pathway, which is significantly downregulated in GDH KO BTSCs. This pathway is essential for the cell stress response, as taurine is responsible for protection during osmotic stress and hypotaurine is protective during oxidative stress [61]. In this sense, glycerol-3-phosphate dehydrogenase is essential for Saccharomyces cerevisiae growth under osmotic stress, and significant induction of this enzyme was observed under abiotic stresses in maize [62, 63]. The increased level of DIA pyruvate dehydrogenase would provide acetyl-CoA to the Krebs cycle. Likewise, we found that the DIA citric acid cycle (TCA) could exhibit greater speed given the higher succinyl-CoA synthetase level. This enzyme functions in the TCA, coupling the hydrolysis of succinyl-CoA to the synthesis of either ATP or GTP, and thus represents the only substrate-level phosphorylation step in the cycle. In diapausing embryos, TCA could also gain acetyl-CoA derived from the enrichment of enoyl-CoA hydratase, which is essential to metabolize fatty acids via beta oxidation. The metabolic needs of lipids during killifish DIII are also covered by the yolk, which would fulfill a role similar to that of the insect fat deposits. These are the most important reserves used by insects to satisfy their energy demand during diapause, and it has been proposed that interactions between nutrient storage and metabolism can influence this diapause by utilizing signaling pathways [64]. Interestingly, our results showed that a member of the cyclic AMP signaling pathway, cAMP-dependent protein kinase, was increased in DIA. PKA-mediated protein phosphorylation is considered a major factor in the mobilization of triglycerides in insects, but this is an evolutionarily conserved mechanism that is also present in vertebrates [65]. Furthermore, PKA pathway-mediated fat mobilization is required for other stress situations in Caenorhabditis elegans [66]. In this transduction pathway, arrestins play an important role in regulating the function of G protein-coupled receptors, including receptor desensitization and internalization. Arrestin-3 (also called c) is phosphorylated by casein kinase II, and it is the phosphorylation state of arrestin-3 that promotes internalization of the phosphorylated receptor [67]. Importantly, while arrestin was expressed in DIA, casein kinase II was only expressed in PRE. This profile, which suggests that arrestin would remain inoperative during diapause, deserves further investigation. The role played by the DIA exclusive guanine nucleotide-binding protein (i) subunit alpha-1, which can act as an inhibitor of adenylate cyclase, thus modulating the signal, or in another pathway as a stimulator of K+ channels, also deserves attention [68]. For mitochondrial energy production, the reducing equivalents generated by the TCA cycle are transferred to the electron transport chain (ETC). Subunits of complex I, the core protein of complex III and electron transfer flavoprotein (ETF), were found exclusively in DIA. While ETF transfers electrons to ETC via ubiquinone, both complexes are proton pumps that collaborate in gradient formation and drive the synthesis of ATP by ATP synthase.

Importantly, mitochondrial ATP synthase subunit g, which enables the transfer of protons from one side of a membrane to the other, was also exclusive to DIA, suggesting that ATP synthesis might be increased.

Emphasizing the lack of confirmatory functional studies, previous proteomic data suggest that in diapause would be increased the glycolysis, leading to an enhance in aerobic metabolism, probably facilitating dehydration stress tolerance and the selective synthesis of protective molecules. In agreement with an increase in protein synthesis, high levels of molecules involved in the translational machinery were found only in diapausing embryos: ribosomal proteins, eukaryotic translation initiation factors, elongation factor 1-alpha 1 and nascent polypeptide-associated complex subunit beta. These proteins are crucial elements in translation and protein synthesis regulation, and its increase could be indicating that both processes would potentially be more active in DIA than in PRE embryos although these must be validated by biochemical approaches.

Additionally, the enrichment of nucleotide metabolism attracts our attention considering that the diapausing embryo could not proliferate as in the case of Austrofundulus limnaeus embryos in diapause II [13]. As it was already mentioned above, we must also note that the cell cycle could be arrested by DIA periplakin and vimentin exclusivity as we will discuss later.

The increase of nucleotide metabolism is mainly linked to the increase in proteins of the purine synthesis pathway. Related to this rise could be the striking fact that several proteins involved in GDP/GTP exchange reactions were exclusively expressed in A. charrua DIA (Rho GDP-dissociation inhibitor 1, Ras-related protein Rap-1, Ran-specific GTPase-activating protein, Ras-related protein Rab-5A, guanine nucleotide binding protein G(i) subunit alpha-1, Rab GDP dissociation inhibitor beta, guanine nucleotide-binding protein G(k) subunit alpha, and Ras-related protein Rab-8A).

According to this it was found that the induction of purine synthesis enzymes is associated with environmental changes in some diapause insects [69].

Cytoskeleton and cell signaling proteins

Intermediate filaments are structures that provide mechanical and stress coping resilience to cells. A universal response to stress is the reorganization of the intermediate filament network. This event was most comprehensively studied in the context of keratins [70]. It is interesting to note that K18 keratins were found in DIA and K8 in PRE in our analysis. Epithelial tissues contain different keratins expressed in a tissue-specific manner. Keratin pair 8/18 is found in the intestine, liver, and pancreas [71]. This pair have a major role in the mechanical stability and integrity of cells but also executes functions in different cellular processes, including protection from apoptosis [72, 73], cellular stress response [74, 75] and cell signaling [76, 77].

In this context, it is likely that K18, which was found exclusively in DIA embryos, would be involved in protection from apoptosis and cellular stress responses during DIII in A. charrua. Moreover, it has been reported that K18 phosphorylation at Ser34 in the head domain promotes its solubility and binding to adaptor protein 14-3-3. Overexpression of K18-S34A, K8 and K18 filaments in mouse pancreatic acinar cells induces them to retract from the basal nuclear region and become apically concentrated, and nuclear retention of adaptor protein 14-3-3 leads to partial mitotic arrest in the liver [78]. It has been shown that the 14-3-3ζ (zeta subunit) protein functions as a central node to promote chemoresistance pathways in cancer [79]. The 14-3-3ζ (zeta subunit) was identified as exclusively expressed in A. charrua DIII embryos. Taken together, these data suggest that 14-3-3ζcould play a role in mitotic arrest and cell survival maintenance during DIII in A. charrua embryos.

Another exclusive cytoskeletal protein found under DIA conditions was vimentin. Vimentin is a main component of the cytoskeleton networks, although accumulating evidence places vimentin at the cell surface or in the bloodstream, either soluble or as a vesicle-transported protein, suggesting it has important roles in the extracellular environment. Cell activation, apoptosis, senescence, inflammation and stress promote vimentin secretion [80, 81]. This protein acts as an integrator of cellular mechanical functions, including cell migration, adhesion and division [82]. It is also involved in organelle positioning, homeostasis and aggresome formation and protects the nucleus during mechanical stress. Moreover, it plays important roles in cell signaling, lipid metabolism, and gene expression [83]. Recently, it was suggested that vimentin acts as an organizer of proteins that regulate the balance of protein synthesis, refolding, and degradation. Morrow et al. [84] demonstrated the role of vimentin as a critical regulator of proteostasis in neural stem cells (NSCs) and the role of aggresomes in NSCs, which clear proteins during quiescent activation. It is possible that vimentin may play a role in safeguarding proteins aligned with HSPs during DIII in A. charrua embryos.

Periplakin, also identified as an exclusive DIA protein, is a cytolinker from the plakin family [85]. This protein is an interaction partner of protein kinase B (PKB or Akt) and localizes to the cell membrane, nucleus, mitochondria and the intermediate filament network through direct binding to vimentin. Periplakin localization is in part influenced by the presence of vimentin, suggesting that periplakin may act as a scaffold and as a localization signal for PKB (Akt) signaling [86]. Interestingly, as stated by Heuvel [86], the interaction of periplakin with PKB (Akt) and vimentin can lead to the inhibition of PKB (Akt)-mediated FOXO phosphorylation and modulation of transcriptional activity. Sequestering PKB (Akt) in the cytosol through binding periplakin/vimentin enhances FOXO transcriptional activity and cell cycle arrest [86]. As stated by Hand et al [4], the transcription factor FOXO is a prime candidate for activating the diapause phenotype in insects and nematodes. It is possible that during DIA, the cell cycle could be arrested by this mechanism, as periplakin and vimentin were found to be exclusive DIA proteins.

A member of the Forkhead box (FOX) family of transcription factors, the Forkhead box protein K2, was also identified as an exclusive protein under DIA conditions. Members of this family are known to regulate a wide range of biological processes, including metabolism, cell cycle progression, proliferation, survival, differentiation and apoptosis [87]. It has been shown that together with Forkhead box protein K1/K2 acts as negative regulators of autophagy in skeletal muscle cells in response to starvation. Interestingly, Forkhead box protein K2 enters the nucleus, binds the promoters of autophagy genes and represses their expression, preventing proteolysis of skeletal muscle proteins [88]. Forkhead box protein K1 and Forkhead610 box protein K2 were identified using a proteomics approach as targets of insulin action [89]. These transcription factors, in contrast to FoxO1, are translocated from the cytoplasm to the nucleus after insulin stimulation, a pattern that is reciprocal to that of FoxO1 after insulin stimulation. Forkhead box protein K1 and Forkhead box protein K2 are critical components in IR and IGF1R-mediated signal transduction, controlling cell proliferation and metabolism [90]. These factors are present in the cytoplasm under basal conditions, and inside the nucleus, they can both activate and repress gene expression [90].

Forkhead box protein K1 and Forkhead box protein K2 also positively regulates WNT/beta-catenin signaling by translocating DVL proteins into the nucleus [91]. Interestingly, the N-Myc downstream-regulated gene 2 (NDRG2) protein was found in DIA A. charrua embryos. This protein contributes to the regulation of cell proliferation, apoptosis and metastasis by regulating various signaling pathways, including the WNT/β-catenin, mitogen-activated protein kinase, transforming growth factor β and Akt signaling pathways [92–94]. Insulin-like signaling pathways are considered to play a central role in diapause regulation in a variety of lineages [6]. Insulin-like growth factor 2 mRNA-binding protein (IGFBP2), a member of this pathway, was also found to be an exclusive DIA protein in embryos. This suggests that this pathway could also be involved in the generation of this diapause phenotype, representing a universal mechanism to control cellular physiology via metabolic modifications, as stated by Podrabsky and Hand [6]. IGFBP2 generally inhibits IGF activity, mainly IGF2 [95]. However, it is important to mention that IGFBP2 also shows IGF-independent activity, playing a role in the regulation of energy status and metabolism [96].

DNA repair protein

Remarkably, in our analysis a deoxyribodipyrimidine photolyase was exclusively found in DIA embryos. These enzymes are involved in the repair of UV radiation induced DNA damage. They catalyze the light-dependent reversal of cyclobutane pyrimidine dimers, which are formed between adjacent bases on the same DNA strand upon exposure to ultraviolet radiation [97]. Wagner and Podrabsky [98] reported that embryos at the disperse blastomere stage (the stage at which DI can occur) and DII embryos of A. limnaeus possess an impressive tolerance to UV‐induced DNA damage. Their repair capacity appears to be strongly dependent on photolyase activity to reverse DNA lesions, suggesting that either embryos at these stages constitutively express a photolyase or that the expression of this enzyme is rapidly inducible. Taken together, these results suggest that the repair capacity of embryos during diapauses appears to be strongly dependent on the photo repair pathway.

Protein related to epigenetic changes

Another protein found exclusively in DIA is S-methylmethionine homocysteine S-methyltransferase. This enzyme is involved in methionine metabolism, catalyzing the reaction between homocysteine and S-methylmethionine, to produce two molecules of methionine. Methionine, or its metabolites participate in protein synthesis but also in the transmethylation pathway, where methionine is converted to S-adenosyl-L-methionine (SAM), a compound that participates in most biological methylation reactions. SAM, which is involved in chromatin methylation events, is critical for the maintenance and transmission of epigenetic information [99]. Indeed, in a recent transcriptomic study conducted in the annual killifish Nothobranchius furzeri embryonic diapause, Hu et al [17] found upregulated genes that are implicated in chromatin regulation through their abilities to methylate histone H3. There is also evidence indicating that epigenetic silencing plays a regulatory role in insect dormancy and growth arrest in response to the environment in plants [100, 101]. Although we did not obtain differentially abundant proteins directly linked to chromatin modification, we found that the presence of S-methylmethionine homocysteine S-methyltransferase in A. charrua DIA embryos could generate methylation events during diapause. This is a topic that deserves further investigation.

Environmental sensing

KEGG pathway analysis of overrepresented proteins in PRE and DIA (Fig 6) showed that the proportion of proteins in the “environmental adaptation” and “sensory system” categories increased in DIA conditions in relation to PRE.

One possible explanation is related to the fact that embryos in diapause could be able to tolerate rather harsh environments while retaining the ability to sense and respond to critical environmental cues [11]. Likewise, although maternal influences may program the embryo for one trajectory, environmental cues experienced by the embryo can override those maternal influences [102, 103]. Moreover, it is possible that embryos in DIA could be coupled in an inactive state, ensuring a quick response to react to the output signal of diapause and eventual hatching. It is important to consider that DIII embryos, unlike DII embryos, are ready to hatch, swim, and feed.

Collection and maintenance of adults

The protocols used in this work were approved by the Animal Experimentation Committee from the Universidad de la República, CHEA (Comisión Honoraria de Experimentación Animal; protocol code 240011-002308-14).

Adult females and males of Austrolebias charrua were collected in ephemeral ponds from La Coronilla, Departamento de Rocha, Uruguay (latitude _33.92, longitude _53.52). Adults were kept in 20-L tanks filled with reverse osmosis filtered water reconstituted with marine salt (Tetra Marine Salt Pro, 300 mS, pH 7–7.5) with continuous air bubbling. Fish were exposed to natural light, and the water temperature was maintained at 19°C. Water was partially changed every 5 days. Specimens were fed once a day with live bloodworms. Spawning occurred naturally with fish groups consisting of one male/two females isolated in tanks using a container filled with glass spheres (500 mm diameter Thomas Scientific, Swedesboro, NJ) on the bottom.

Diapause III induction

Fertilized eggs were collected and kept in Yamamoto’s solution at 25°C [104] until the following features appeared: advanced organogenesis, pigmentation of the retina and skin and tip of the tail in contact with the head (stage 41, early prehatching [2, 105]). At this stage, the embryos were induced to DIII. To induce DIII, embryos were first rolled through filter paper to clean their surface. They were treated for 5 min with a 0.03% solution of sodium hypochlorite in Yamamoto’s solution to help prevent fungal and bacterial infections [18] and rinsed 2 times in Synergy water. Embryos were placed on wet filter paper with Yamamoto’s solution and kept in darkness in a plastic bag inside a container at room temperature. Humidity was checked periodically. Filter paper and bags were UV irradiated 45 min before use.

In terms of DIII embryos in natural environments, the only field data available come from the Mexican annual killifish Millerichthys robustus and Nothobranchius [29, 30]. Researchers found embryos arrested in DIII close to the end of the dry season before flood. Considering these data, to mimic these conditions, we placed the embryos in moistened paper in our experimental design.

After 3 months, embryos were observed by photonic microscopy with a blue filter (to minimize activation). DIII embryos were characterized by full development of the body with the tail extending over the head, most of the yolk consumed and cessation of mandibular and opercular contractions that mimic breathing in nondormant embryos (stage 43, late prehatching embryos [2, 105]).

We used three biological replicates per stage to compare differential protein expression profiles (Stage 41 early prehatching: 1 n = 20 embryos, 2n = 20 embryos, 3 n = 20 embryos; Stage 43 diapause III: 1 n = 17 embryos, n = 20 embryos, n = 8 embryos).

Sample preparation for LC-MS/MS analysis

Protein extraction was performed following the protocol described by Link et al. [106] to avoid vitellogenin sample contamination.

Protein quantification was carried out by densitometry analysis following SDS-PAGE. For that purpose, samples were run in precast gels (NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well, Invitrogen). The LMW-SDS Marker Kit (GE Healthcare) was used as a standard. After staining with colloidal Coomassie blue, gel images were digitized with a UMAX Power-Look 1120 scanner and LabScan 5.0 software (GE Healthcare). Quantification was performed using ImageQuant TL software (v8.1), a 1D analysis module.

Twenty micrograms of protein extracted from each sample was run on a 3-cm-long SDS-PAGE gel. Each lane was excised into 3 fragments, transferred to Eppendorf tubes and processed independently. Proteins were in-gel reduced and alkylated by incubation with 10 mM DTT for 1 h at 56°C followed by incubation with 50 mM iodoacetamide for 45 min at room temperature. In-gel protein digestion was performed by incubation with 2 μg of trypsin (sequence grade, Promega) in 50 mM ammonium bicarbonate overnight at 37°C. Peptides were extracted from gel pieces by the addition of 60% acetonitrile/0.1% formic acid with vigorous agitation followed by vacuum drying and resuspension in 0.1% formic acid. Samples were centrifuged at 16,000xg for 30 min at 4°C.

LC-MS/MS analysis

Samples were analyzed by nano-LC MS/MS by means of a shotgun strategy using a nano-HPLC (EASY-nLC 1000, Thermo Scientific) coupled online to an LTQ Velos mass spectrometer (Thermo Scientific). Tryptic peptides were injected into an Acclaim® PepMap 100 nanotrap column (75 μm x 2 cm, Thermo Scientific) and separated on a 50 μm x 15 cm C18 Easy Spray column (PepMap® RSLC, 2 μM, 100 Å, Thermo Scientific) at a constant flow rate of 250 nL/min at 45°C. Peptide elution was achieved with a 70 min gradient from 0% to 55% mobile phase B (A: 0.1% formic acid; B: 0.1% formic acid in acetonitrile). Online MS analysis was carried out in data-dependent acquisition mode in two steps: acquisition of full MS scans in positive ion mode with an m/z range from 400 to 1200 Da followed by CID fragmentation of the ten most intense ions using a dynamic exclusion list (exclusion duration 45 s). The following parameters were set: spray voltage, 2.3 kV; capillary temperature, 260°C; normalized collision energy, 35; activation Q energy, 0.25; and activation time, 15 ms.

Protein identification and analysis

PatternLab for Proteomics v4.0 software (http://www.patternlabforproteomics.org) was employed to generate a target-reverse database using sequences from the Austrolebias charrua genome [107]. Additionally, 127 common mass spectrometry contaminants were incorporated. Thermo raw files were searched against the database using the software integrated Comet search engine applying the following parameters: trypsin as proteolytic enzyme with full specificity and 2 missed cleavages allowed; oxidation of methionine as a variable modification, carbamido methylation of cysteines as a fixed modification; 800 ppm of tolerance from the measured precursor m/z. Each of the three fragmentation fractions belonging to the same sample were searched together, giving rise to a unique results file for each one. XCorr and Z-Score were used as the primary and secondary search engine scores, respectively. Peptide spectrum matches were filtered out using the Search Engine Processor (SEPro), and acceptable FDR criteria were set at 1% at the protein level. Comparison between proteins detected in each sample set was performed using PatternLab’s Approximately Area Proportional Venn Diagram module. Proteins uniquely detected in each stage were determined if they were found in at least two replicates of one condition and were absent from the other. PatternLab’s T-Fold module was used to detect proteins present in both conditions but at significantly different relative abundances by spectral count analysis.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [108] partner repository with the dataset identifier PXD025196.

Gene ontology and KEGG orthology analyses

GO terms were assigned using InterProScan v5.31–70.0 [109] with the option–goterms. GO enrichment was performed using the R package TopGO v2.34.0 [110], using all genes with an assigned GO (19.332 of 40.933 total proteins) as the universe and those genes either upregulated or exclusive to diapause III and prehatching as genes of interest. GO terms with a p-value <0.05 in the weighted Fisher test were considered significant. Enriched GO terms in each condition and their p-values were plotted in R (version 3.5.2, R Core Team, 2018 [111], using the ggplot2 (version 3.2.1, [112]) and cowplot (version 1.0.0, [113]) packages.

KEGG Orthology terms were assigned to all proteins using the KEGG Automatic Annotation Server [114] using GHOSTZ as the search algorithm and the SBH Method for assignment. The gene dataset included the default eukaryotic gene set plus the fish Paramormyrops kingsleyae, Kryptolebias marmoratus (mangrove rivulus), Takifugu rubripes (torafugu), Sinocyclocheilus rhinocerous and Xiphophorus maculatus (southern platyfish). KO terms were mapped to KEGG pathways using KEGG Mapper [115].

Protein sequences exclusively expressed or upregulated in each condition were analyzed for pathway enrichment using KOBAS3.0 [116] with default parameters (online version: http://kobas.cbi.pku.edu.cn/anno_iden.php), using D. rerio as a reference organism for analysis of KEGG pathways.