Induction and regulation of reversible suspended animation in C. elegans

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
Suspended animation, a state of profound metabolic, behavioral and developmental
quiescence, is a remarkable yet poorly understood stress resilience strategy in animals.
Here, we describe a previously uncharacterized form of suspended animation inducible
by high-population density in isosmotic liquids in C. elegans throughout larval
development and adulthood. Transcriptomic, metabolomic and live-cell activity reporter
imaging analyses reveal striking molecular and cellular landscape changes by such
liquid-induced suspended animation (LISA), including remodeling of gene expression
programs, energy metabolites, lysosomal and mitochondrial morphology. Genetic
screens identify mutants with altered stress responses and survival against LISA. While
key endo-lysosomal regulators promote survival during LISA, organelle remodeling and
a neuronal axis via downstream neuropeptide and cAMP/PKA signaling orchestrate
behavioral awakening from LISA. Our findings define a facile paradigm for reversible
SA, providing a powerful model system to uncover key molecular and cellular
mechanisms governing an extreme case of reversible life arrest and dormancy.

Introduction
Animals across diverse phyla have evolved remarkable strategies to endure extreme
environmental stress by entering reversible states of dormancy or metabolic
suppression1. Among these, suspended animation (SA) is one of the most profound,
characterized by a nearly complete cessation of development and microscopically
visible behavioral movement while preserving organismal viability2–11. Such states are
not only biologically intriguing but also of growing interest for their potential applications
in medicine, biotechnology, and space biology, where controlled metabolic arrest and
reversible hibernation-like states could extend viability under limiting conditions.

Studies in the multicellular animal model organism Caenorhabditis elegans have
provided a mechanistic foundation for understanding SA as an active, regulated
response to extreme environmental stress. In seminal work by Padilla, Roth, and
colleagues, complete oxygen deprivation (anoxia) was shown to induce a reversible SA
state in C. elegans, characterized by a coordinated arrest of development, cell division,
motility, and other energy-intensive processes until normoxic conditions are restored7,12.
In embryos, anoxia triggers hypometabolism marked by reduced ATP levels and
dephosphorylation of cell cycle-regulated proteins, with blastomeres arresting at specific
cell cycle phases in a manner that preserves viability and structural integrity. Genetic
dissection revealed that components of the spindle assembly checkpoint (e.g., san-1,
mdf-2) and nucleoporin-dependent regulation of CDK-1 activity are essential for proper
anoxia-induced arrest, underscoring that entry into and exit from SA require specific
molecular regulators rather than passive metabolic failure4,13,14. Furthermore, C.
elegans demonstrates differential responses to graded oxygen stress: while anoxia
elicits behavioral responses and SA independently of the hypoxia-induced transcription
factor HIF-1, milder hypoxic conditions engage HIF-1–dependent pathways15–17.
Collectively, this literature establishes C. elegans as a tractable model for dissecting the
cellular and genetic regulation of SA and situates this state within a broader spectrum of
environmentally induced dormancy phenomena.

In C. elegans, the dauer diapause has been extensively studied as an another
well-characterized form of developmental arrest or dormancy accompanied by
distinctive morphological and behavioral adaptations18–20. However, dauer occurs only
during a specific, programmatically defined alternative larval stage and reflects a
specialized developmental pathway rather than an arrest inducible across life stages.
By contrast, SA describes a more profound and inducible state of reversible arrest in
development and motility. Although SA has been documented in various systems,
including in nematodes and even select vertebrates2–11, fundamental questions remain
regarding molecular, cellular, and physiological regulatory mechanisms. In particular,
how animals orchestrate cell quiescence, metabolic reprogramming, and neural activity
to achieve a globally suspended yet fully reversible state is not well understood. The
diversity of SA induction and mechanisms remain underexplored. Addressing these
questions will provide critical insight into core principles of stress resilience and may
enable strategies for inducing protective stasis across diverse biological contexts.

Here, we identify static liquid incubation as a previously unrecognized and robust
trigger that induces suspended animation in C. elegans. Under this condition, animals
rapidly enter a reversible state characterized by coordinated arrest of developmental
progression and motility, satisfying established operational criteria for SA. We term this
induction paradigm liquid-induced suspended animation (LISA). We systematically
define the physiological and temporal features of LISA and uncover key molecular
mechanisms governing both survival during arrest and efficient reactivation upon return
to favorable conditions. Notably, the entry pathway for LISA appears mechanistically
distinct from previously characterized forms of SA or dauer formation, suggesting that
multiple upstream cues can converge on a shared reversible arrest program. By
leveraging this experimentally tractable system, we provide insights into how animals
dynamically suppress and subsequently restore core life processes, revealing principles
underlying reversible metabolic and developmental quiescence.

Results
Physiological specificity and reversibility of suspended animation

In exploring various stress conditions capable of robustly inducing SA in C. elegans, we
found that a remarkably simple “standing” procedure, i.e. allowing animals to settle at
high population density in isosmotic M9 buffer, independent of food supplementation,
was sufficient to trigger this profound quiescent state (Fig. 1a). Within hours of static
incubation, larvae exhibited a striking cessation of developmental progression and
visible motility, hallmarks of SA, while counterpart control animals maintained in low-
density (LD, n = 60 worms) or dispersed (in a roller) conditions continued to develop
normally (Fig. 1b). We therefore identify such static liquid incubation as a robust inducer
of SA and refer to this induction paradigm as liquid-induced SA (LISA) for clarity. This
phenomenon was sharply dependent on population density, with near-complete
penetrance observed above a defined threshold (Fig. 1c). Arrested animals retained
gross morphology but did not apparently proceed through expected larval
developmental transitions, including intestinal endomitosis, seam cell terminal
differentiation, or dendrite branching of PVD neurons, as visualized using cell or tissue-
specific reporters (Fig. 1d–e and Supplementary Fig. 1a). Notably, LISA was inducible
across a wide developmental range, from early larvae to adults, in wild type and
mutants for known stress-responding transcription factors, including the hypoxia-
inducible factor HIF-1 and heat-inducible HSF-1, suggesting a robust and broad
permissive window for entry into LISA (Fig. 1f and Supplementary Fig. 1b). Temporal
analyses revealed that LISA onset was progressive and standing duration–dependent,
whereas survival remained high even after extended time in LISA (Fig. 1g, h).

Importantly, we found this arrest response required isosmotic conditions: animals
exposed to hypotonic media such as water displayed reduced viability and inconsistent
arrest phenotypes (Fig. 1i). Medium supplementation with various amounts of bacterial
food, amino acids, or vitamins did not abrogate LISA, while canonical stressors such as
starvation, heat shock, or hypoxia alone were insufficient to induce it (Fig. 1j). Dauer-
defective mutants, including daf-16 and daf-22 that disrupt dauer stress responses and
pheromone pathways21–24, respectively, entered LISA normally, indicating that this
phenomenon operates independently of dauer regulatory programs (Supplementary Fig.
1b). Additionally, animals subjected to LISA during larval development exhibited normal
post-recovery lifespans and can re-enter LISA upon repeated exposure (Supplementary
Fig. 1c, d). After LISA and reintroduction into normal nutrient-rich media, animals exited
LISA in a coordinated and timely fashion, resuming development and locomotion with
temporal synchrony (Fig. 1k–l). Unlike anoxia-induced embryonic SA, LISA at multiple
larval and adult stages in our study is strictly population-density dependent and SAN-1
independent (Fig. 1c, Supplementary Fig. 1g); our method is also easier to set up
without using environmental anoxia chamber. In addition, LISA is robust across
common cultivation temperatures, genetic backgrounds, buffer compositions, and food
availability (Supplementary Fig. 1e-h). Together, these findings define a facile, tunable
and robust system for inducing and reversing LISA in C. elegans, establishing a
tractable platform for dissecting mechanisms of reversible metabolic suppression.

Molecular and cellular landscape profiling of suspended animation

To elucidate the molecular landscape associated with LISA in C. elegans, we performed
transcriptomic profiling of animals subjected to high-density isosmotic LISA. RNA-seq
analysis revealed extensive transcriptional reprogramming, with a substantial number of
genes either up- or down-regulated in response to LISA (Fig. 2a and Supplementary
Data 1). Intriguingly, among the most robustly LISA-induced transcripts were members
of the small heat shock protein family, particularly the hsp-16 cluster, whose
upregulation was sharply localized to their genomic cluster locus without affecting
adjacent genes (Fig. 2b). This transcriptional induction was corroborated by hsp-
16p::GFP transcriptional and hsp-16p::hsp-16::mStayGold translational reporter
imaging, which showed minimal GFP or mStayGold abundance during LISA but a
dramatic increase following recovery, suggestive of a delayed protein translation, rather
than contemporaneous, stress response (Fig. 2c and Supplementary Fig. 2a–j).
Consistently, this induction was abrogated by hsf-1 RNAi, implicating the canonical heat
shock transcription factor HSF-1 in this response25–27 (Supplementary Fig. 2d). The
degree of hsp-16 activation was modulated by population density, echoing the
physiological parameters that define LISA itself (Supplementary Fig. 2e). RNAi
knockdown of the genes encoding the C. elegans homologs of known thermogenic
regulators, including UCP-4 or SCA-1 did not affect hsp-16 activation, whereas lower
environmental temperature progressively decreased it (Supplementary Fig. 2f, g). In
addition, the supernatant from LISA animals at high density was insufficient to induce
LISA or hsp-16 reporters of animals at low density (Supplementary Fig. 2k-m), arguing
against a stable diffusible pheromone that transduced the population density signal.
Together, these findings delineate a distinct LISA-induced transcriptional state, marked
by stress-responsive gene expression changes, likely reflecting the organism’s dormant
and physiologically primed state.

To further characterize the physiological and metabolic states underpinning LISA, we
employed a panel of fluorescent reporters and metabolomic analyses. Systematic
imaging of organelle-specific live reporters revealed that LISA triggers pronounced
remodeling of the mitochondrial network, favoring vesicular over fused forms, as
visualized by mitochondrial matrix-targeted MAI-2::GFP (Fig. 2d, e). This architectural
shift was accompanied by a notable reduction in steady-state mitochondrial calcium
levels, indicated by GCaMP-based sensors in body wall muscle cells, consistent with a
hypometabolic mitochondrial state. We used liquid chromatography-mass spectrometry
(LC-MS) based metabolomic profiling to further identify major energy metabolites
differentially regulated by LISA (Supplementary Data 2; see Methods). Principal
component analysis (PCA) of metabolites, including ATP, ADP, AMP, acetyl-CoA,
acetyl-carnitine, and various redox intermediates, showed clear segregation between
LISA and control animals, indicating a comprehensive rewiring of cellular bioenergetics
(Fig. 2f). We identified differential abundance of multiple metabolites critical to energy
and redox homeostasis, including LISA-downregulated acetyl-CoA and ratio of
NADH/NAD+, reinforcing the notion of metabolic suppression and adaptation (Fig. 2g
and Supplementary Data 2). Additionally, steady-state ATP and its dephosphorylated
form AMP/adenosine were slightly up- and downregulated, respectively, reflecting
overall reduced bioenergetic demands under LISA (Fig. S2g). Complementary gas
chromatography-mass spectrometry (GC-MS) metabolomic profiling of major glycolytic
and TCA cycle metabolites and amino acids further supported a stress-adaptive state
and hypometabolic signature characterized by elevated succinate and lactate levels
(Supplementary Fig. 3 and Supplementary Data 3). Collectively, these data reveal a
coordinated cellular and physiological program during LISA that conserves energy,
dampens mitochondrial activity and metabolism, and primes the organism for
reactivation upon stress release.

Stress-responsive transcriptional and auto-lysosomal programs facilitate survival
from suspended animation

To uncover genetic regulators that modulate LISA and organismal survival during this
profound quiescent state, we conducted forward genetic screens using ethyl
methanesulfonate (EMS) mutagenesis to isolate mutants that either fail to undergo LISA
by progression from L4 to adults or undergo LISA but with aberrant activation of the
hsp-16p::GFP reporter and increased viability under prolonged LISA. Though we did not
obtain fertile mutants that completely failed to undergo LISA so far, we isolated multiple
mutants with constitutive hsp-16p::GFP activation and enhanced viability after
prolonged LISA (Fig. 3a). Genetic mapping, complementation test and whole genome
sequencing identified causal mutant alleles in daf-21, encoding the Hsp90 ortholog28,
and lin-61, a chromatin regulator29 (Fig. 3a and Method). We confirmed that
backcrossed daf-21 mutants exhibited hsp-16 expression under baseline conditions and
showed further exaggerated induction after LISA, coinciding with enhanced survival
following extended periods of arrest (Fig. 3b, c). These phenotypes required the heat
shock transcription factor HSF-1, as RNAi-mediated knockdown suppressed hsp-
16p::GFP up-regulation in both daf-21 and lin-61 backgrounds (Fig. 3d). HSF-1 and the
FOXO transcription factor DAF-16 can cooperatively mediate stress-induced gene
expression to promote resilience25,30,31. Although hsf-1(sy441) single reduction-of-
function mutants32 showed largely normal LISA survival, double gene manipulations
revealed synergistic roles for HSF-1 and DAF-16 in LISA survival (Fig. 3e), suggesting
they act in coordination and convergent pathways to promote LISA resilience.

Beyond transcriptional regulation, cellular organelle remodeling processes appeared
integral to LISA survival. HSF-1 and DAF-16 can form a co-regulator complex to
promote stress resilience and longevity through auto-lysosomal pathways33–35. We next
used live imaging of an intestinal lysosomal reporter (nuc-1::mCherry) to monitor LISA-
induced lysosomal dynamics and found that LISA triggered marked tubulation of the
lysosomal network (Fig. 3f), a morphological signature often associated with stress
responses and increased degradative capacity33–35. Genetic disruption of key auto-
lysosomal pathway components36–38, including vps-15 (autophagosome formation), vps-
45 (autolysosome fusion), and hpo-27 (lysosomal fission), strongly compromised
survival, highlighting the functional importance of auto-lysosomal dynamics in sustaining
viability during LISA (Fig. 3g). In contrast, despite robust LISA-induced mitochondrial
remodeling, including altered morphology across multiple tissues (Supplementary Fig.
4a–f), mutants defective in key mitochondrial fusion and fission genes (fzo-1, mtp-18,
drp-1)39–41 displayed little or slightly increased LISA survival (Supplementary Fig. 4g),
suggesting that mitochondrial morphologic remodeling may be a correlative rather than
crucial feature of LISA resilience. Collectively, these findings reveal a critical axis of
stress-responsive transcriptional programs and autophagic-lysosomal remodeling that
jointly enable C. elegans to withstand prolonged metabolic stasis during LISA (Fig. 3h).

Neural circuitry orchestrates behavioral awakening from suspended animation

To delineate the neural substrates governing the transition from LISA to behavioral
reactivation, we conducted a comprehensive screen across strains perturbed in key
neurotransmitter systems, neuropeptides, and defined neuronal subtypes implicated in
sensory perception and sleep regulation42–46 (Fig. 4a and Supplementary Fig. 5a). This
analysis revealed that the AFD sensory neurons and their postsynaptic partners AIY
interneurons are critical for promoting timely awakening (defined as conspicuous
locomotion from quiescent behavioral state in LISA upon transfer to normal plate culture
conditions, see Methods), whereas the sleep-active RIS interneuron likely exerts an
antagonistic role by suppressing arousal. In parallel, we found that neuropeptide
signaling via PDF and its receptor PDFR-1 were indispensable for coordinating
behavioral recovery from LISA, supporting their previously established roles in
promoting behavioral “roaming”47–51. Notably, single-cell RNA-seq datasets52–54
revealed enriched expression of gcy-8, a guanylyl cyclase, in AFD neurons, and pdf-1 in
AIY and a few other neurons, supporting their respective roles in a molecularly defined
circuit for awakening regulation (Fig. 4b). Indeed, time-lapse imaging of behavioral
awakening showed a time-dependent delay of recovery in AFD-ablated or PDF-deficient
animals, while ablation of RIS resulted in premature awakening (Fig. 4c and
Supplementary Fig. 5b). These behavioral phenotypes suggest a neural antagonism
between arousal-promoting and sleep-maintaining pathways in modulating the exit from
LISA.

To monitor neural activity dynamics during the transition out of LISA, we employed pan-
neuronal and cell-specific calcium imaging. While global neuronal activity remained
largely unchanged during LISA and recovery, we found that both AFD and AIY neurons
exhibited marked silencing during LISA, followed by a progressive rise in calcium levels
shortly after reintroduction to normal feeding conditions. This coordinated reactivation
indicates early engagement of these neurons during awakening (Fig. 4d and
Supplementary Fig. 5c, d). cAMP and PKA signaling mediates PDF/PDFR-1 signaling
and constitute evolutionarily ancient regulators of awakening in animals43,55–57. To test
whether intracellular cAMP/PKA signaling mechanisms were sufficient to modulate
awakening, we manipulated cAMP levels via optogenetic activation55 and genetic gain-
of-function of adenylyl cyclase (acy-1)58. Both approaches led to precocious behavioral
reactivation from LISA (Fig. 5). However, LISA-induced silencing and reactivation of
AFD GCaMP activities were unaltered in PDF-1/PDFR-1 signaling-deficient or cAMP-
activated pde-4 mutant animals (Supplementary Fig. 5e), placing peptide/cAMP action
downstream or in parallel to AFD. In addition, we found that loss of PDE-4 or the RIS
neuron accelerated awakening even in the absence of AFD or AIY neurons
(Supplementary Fig. 6), arguing against the AFD-AIY neuronal axis acting downstream
of cAMP or the RIS neuron. These results converge on a model in which AFD/AIY
neurons act with downstream PDF/PDFR-1 neuropeptide signaling and subsequent
cAMP/PKA activation to drive awakening via the motor circuit, while RIS neurons likely
provide inhibition to maintain behavioral quiescence. Together, these findings uncover a
modular neural circuit and intracellular signaling logic that governs the coordinated
resumption of behavioral locomotion following LISA via cAMP/PKA signaling conserved
in animals.

To determine whether the mitochondrial and lysosomal dynamics observed during LISA
are functionally linked to recovery from LISA, we examined behavioral awakening in
mitochondrial and lysosomal mutants. Mutations in the genes eat-3 and fzo-1 essential
for mitochondrial fusion caused striking defects in the LISA-induced change of
mitochondrial morphology (Supplementary Fig. 4). Correspondingly, we found that eat-3
and fzo-1 mutants also exhibited markedly slower awakening from LISA
(Supplementary Fig. 7). Similarly, endo-lysosomal gene mutations in vps-15, vps-45,
rabs-5 and hpo-27 led to slower awakening phenotypes (Supplementary Fig. 7). By
contrast, mutations in the genes key for mitochondrial fission (mtp-18 LOF, drp-1 LOF)
or mitophagy (dct-1 LOF, dct-1 GOF, pdr-1 LOF)59,60 did not apparently affect
behavioral awakening post-LISA (Supplementary Fig. 7). Collectively, these results
indicate that endo-lysosomal dynamics is functionally important for not only surviving
but also awakening from LISA, whereas mitochondrial fusion is selectively required for
efficient behavioral reactivation from LISA, potentially by sustaining systemic energy
homeostasis needed for the timely engagement of neuro-sensory and arousal-
promoting circuits.

Methods
C. elegans strains

C. elegans strains were grown on nematode growth media (NGM) plates seeded with
Escherichia coli OP50 at 20 °C with laboratory standard procedures unless otherwise
specified. The N2 Bristol strain was used as the reference wild type, and the
polymorphic Hawaiian strain CB4856 was used for genetic linkage mapping and SNP
analysis70,71. Forward genetic screens after ethyl methanesulfonate (EMS)-induced
random mutagenesis were performed as described previously72,73. Approximately
15,000 haploid genomes were screened for dmaIs8 activators and LISA survivors,
yielding at least 12 independent mutants. Mutations were identified by whole-genome
sequencing and complementation was tested by crossing EMS mutants with daf-21 or
lin-61 heterozygous males. Feeding RNAi was performed as previously described74.
Transgenic strains were generated by germline transformation as described75.
Transgenic constructs were co-injected (at 10 - 50 ng/μl) with dominant unc-
54p::mCherry or GFP, and stable extrachromosomal lines of fluorescent animals were
established. Genotypes of mutant or transgenic strains used are as follows: RB1391
san-1(ok1580) I, XW9558 hpo-27(tm5336) I, VC1669 aptf-1(gk794) II, CU5991 fzo-
1(tm1133) II, EU2959 vps-15(or1235ts) II; unc-119(ed3) III; orIs21, KG518 acy-
1(ce2dm) III, LSC27 pdf-1(tm1996) III, LSC39 pdfr-1(lst34) III, PY1589 cmk-1(oy21) IV,
IK800 gcy-8(oy44) IV, PR767 ttx-1(p767) V, MT150 egl-3(n150ts) V, DR47 daf-
11(m47ts) V, MT14680 cat-2(n4547) II, MT18877 flp-11(n4765) X, OH15422 ceh-
14(ot900) X, VC4506 mtp-18(gk5577[loxPI + myo-2p::GFP::unc-54 3' UTR + rps-
27p::neoR::unc-54 3' UTR + loxP]) X, QJ4134 vps-45(tm246) X; arIs37; jqEx611,
KG744 pde-4(ce268) II, AML70 lite-1(ce314) X; wtfIs5, GN112 pgIs2 [gcy-8p::TU#813 +
gcy-8p::TU#814 + unc-122p::GFP + gcy-8p::mCherry + gcy-8p::GFP + ttx-3p::GFP],
JN579 peIs579 [ttx-3p::casp1 + ttx-3p::Venus + lin-44p::GFP], DMS63 dmaIs8 [hsp-
16p::GFP; unc-54p::mCherry] IV, DMS2974 dmaEx814 [hsp-16::hsp-16::mStayGold;
unc-54p::mCherry], DMS124 dmaIs8 IV; lin-61(dma19), DMS125 dmaIs8 IV; lin-
61(dma20), DMS407 dmaIs8 IV; lin-61(dma114), DMS2958 hsf-1(sy441) I; dmaIs8 IV,
KAB122 louIs8 [ges-1p::nuc-1::mCherry::unc-54 3'UTR], RN80 xmSi01 [mai-2p::mai-
2::GFP], SJU39 stjEx24[snb-1p:IlaC22/SL2/dsRed; myo-2p:gfp], DCR3055 wyls629
[gcy-8p::GCaMP6s; gcy-8p::mCherry; unc-122p::gfp], DMS3123 pde-4(ce268) II; pgIs2
[gcy-8p::TU#813 + gcy-8p::TU#814 + unc-122p::GFP + gcy-8p::mCherry + gcy-8p::GFP
+ ttx-3p::GFP], DMS3117 aptf-1(gk794) II; peIs579 [ttx-3p::casp1 + ttx-3p::Venus + lin-
44p::GFP], DMS3118 aptf-1(gk794) II, pgIs2 [gcy-8p::TU#813 + gcy-8p::TU#814 + unc-
122p::GFP + gcy-8p::mCherry + gcy-8p::GFP + ttx-3p::GFP].

Plasmid construction and germline transformation

The Phsp-16.48::hsp-16.48::mStayGold::unc-54 3′ UTR construct was generated by
PCR amplification. The hsp-16.48 promoter and coding sequence were amplified from
genomic DNA of wild-type strain N2 using the primer pair 5′-ATCTTCTGGCTTGA
ACTGCG-3′ and 5′-
GAAAAGTTCTTCTCCCGTTGAAACGGCTCCATGTTTTGCAACAAAATTAATGGGAAT
AG-3′. The mStayGold::unc-54 3′ UTR fragment was amplified from a plasmid (gift from
Dr. Mizumoto). The resulting fragments were fused by PCR to generate the full-length
Phsp-16.48::hsp-16.48::mStayGold::unc-54 3′ UTR expression construct.

For transgenic strain generation, the fused PCR product was microinjected at 15 ng μl⁻¹
together with unc-54p::mCherry (20 ng μl⁻¹) as a co-injection marker. A 1 kb DNA ladder
was added as carrier DNA to a final concentration of 100 ng μl⁻¹. Integrated transgenic
lines were generated by UV irradiation (UV Stratalinker 2400, Stratagene) at 600 Lux.

RNA interference (RNAi)

RNA interference (RNAi) was performed by feeding worms with E. coli strain HT115
(DE3) expressing double-stranded RNA (dsRNA) targeting endogenous genes. RNAi
clones were obtained from the Ahringer RNAi library. Bacterial clones were streaked
onto LB agar plates containing 100 μg ml⁻¹ ampicillin and grown at 37 °C for 16 h. A
single colony was inoculated into LB broth containing 100 μg ml⁻¹ ampicillin and
cultured overnight at 37 °C. dsRNA expression was induced with 1 mM isopropyl 1-thio-
β-D-galactopyranoside (IPTG), and bacteria were seeded onto NGM plates. Plates were
incubated at RT for 24 h before use. Developmentally synchronized embryos, obtained
by bleaching gravid adults, were placed onto RNAi plates and grown at 20 °C to the
indicated developmental stage. Animals were used for imaging, behavioral, or survival
assays as indicated.

Metabolite extraction and GC-MS analysis

C. elegans L4 larvae from suspended animation and control conditions were harvested
and extracted in cold 90% methanol to achieve a final concentration of 80% methanol
per pellet. Samples were incubated at −20 °C for 1 hour, centrifuged at 20,000 × g for
10 minutes at 4 °C, and the resulting supernatants were collected. Quality control (QC)
samples were generated by pooling equal volumes of extract from all samples; process
blanks contained extraction solvent only. All extracts were dried en vacuo and stored
until derivatization. Dried extracts were resuspended in 40 µL of 40 mg/mL O-
methoxylamine hydrochloride (MOX) in anhydrous pyridine and incubated at 37 °C for 1
hour. Derivatization was completed by automatic addition of 60 µL N-methyl-N-
(trimethylsilyl)trifluoroacetamide (MSTFA + 1% TMCS) and further incubation at 37 °C
for 30 minutes. One microliter of the derivatized sample was injected into an Agilent
5977B GC-MS system in split mode (5:1 or 50:1, depending on metabolite abundance).
Chromatographic separation was performed on a 30 m Zorbax DB-5MS capillary
column (Agilent) with helium as the carrier gas at 1 mL/min. The GC oven temperature
was held at 60 °C for 1 minute, ramped at 10 °C/min to 325 °C, and held for 10 minutes.
Mass spectra were acquired using Agilent MassHunter and processed with MassHunter
Quant. Metabolites were identified using in-house, NIST, and Fiehn libraries, and peak
areas were exported for further analysis. Data were filtered based on %CV (<30% in QC
samples), QC-to-blank signal ratio (>1.5), and minimal signal intensity (>1000 counts).
Statistical analysis was conducted using MetaboAnalystR following log transformation,
sum normalization, and Pareto scaling. Principal component analysis (PCA), heatmaps
of the top 25 changing metabolites, and volcano plots were used to compare suspended
animation versus control groups. Metabolites with fold change >1.5 and raw P value
<0.05 were considered significantly altered.

Metabolite extraction for LC-MS analysis

C. elegans L4 larvae subjected to suspended animation or maintained under control
conditions were collected, flash frozen, and extracted using a chilled solution of
acetonitrile, water, and methanol (14:4:1 v/v) containing 0.1% ammonium hydroxide,
0.3 µM D9-carnitine, 8 µM D4-succinate, and 2.5 µM uniformly labeled amino acids
(Cambridge Isotope Laboratories). Samples were vortexed, homogenized in bead mill
tubes, chilled at −20 °C, centrifuged (20,000 × g, 10 min, 4 °C), and supernatants were
dried under nitrogen. Extracts were reconstituted in 80% acetonitrile with 0.1%
ammonium hydroxide and transferred to autosampler vials for LC-MS/MS analysis.
Metabolites were separated on a Waters BEH zHILIC column using a SCIEX 7600
Zeno-ToF mass spectrometer coupled to an Agilent 1290 Infinity II HPLC system in
positive ionization mode. Chromatographic separation employed a gradient of buffer A
(25 mM ammonium carbonate in water) and buffer B (99% acetonitrile with 5% water).
Data were acquired using high-resolution multiple reaction monitoring (MRM^HR), and
chromatograms were integrated using SCIEX Analytics software.

Raw data were drift-corrected using the SERRF algorithm, normalized to the internal
standard D9-carnitine, log-transformed, and Pareto-scaled for statistical analysis using
MetaboAnalystR. Principal component analysis (PCA) was used to assess overall
metabolic differences. Differential metabolite abundance was evaluated using two-sided
unpaired Student’s t-tests with Benjamini–Hochberg false discovery rate correction.
Metabolites with adjusted P < 0.05 and fold change > 1.5 were considered significantly
altered. Metabolite identities were confirmed by MS/MS and validated against in-house
standards and the Human Metabolome Database (HMDB). Only Level 1 confidence
metabolites (confirmed by retention time and reference standard) were included in the
final analysis.

Fluorescence microscopy and imaging

SPE confocal (Leica) and epifluorescence compound microscopes were used to capture
fluorescence images. Animals were randomly picked at the same stage and treated with
1 mM levamisole in M9 solution (31742-250MG, Sigma-Aldrich), aligned on a 2% agar
pad on a slide for imaging. Identical setting and conditions were used to compare
experimental groups with control. For quantification of GFP fluorescence, animals were
outlined and quantified by measuring gray values using the ImageJ software. The data
were plotted and analyzed by using GraphPad Prism.

RNA sequencing

C. elegans N2 animals were maintained at 20 °C. For liquid-induced suspended
animation (LISA) treatment, synchronized L4 larvae were either subjected to LISA 12
hours or maintained under control conditions prior to collection. Upon sample collection,
animals were washed off NGM plates using M9 buffer, collected into 1.5 ml
microcentrifuge tubes, and homogenized using a tissue disruptor. Total RNA was
extracted using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (RC112-01,
Vazyme) according to the manufacturer’s instructions. RNA quantity and purity were
assessed using a NanoDrop spectrophotometer, and RNA integrity was evaluated by
agarose gel electrophoresis. For each sample, 1 µg of total RNA was used for
sequencing library construction. Four biological replicates were included for each
treatment. Sequencing libraries were prepared and sequenced (paired-end, 150 bp) on
the DNBseq platform (Innomics). Differential gene expression analysis was performed
using the DESeq2 package. Adjusted p-value≤0.05 was used as the threshold to
identify the differentially expressed genes.

C. elegans LISA induction, survival and behavioral awakening assays

For the LISA assay, synchronized-stage worms at specific stages were washed off the
plate with OP50 as food and collected into a 1.5 mL microcentrifuge tube. For RNAi
strains, Day 1 adults (16-24 hours past the L4 stage when RNAi efficiency is sufficient)
were used. The worms were washed once with 1 mL of M9 buffer, then the tube was
placed on a rack and left to stand at room temperature for the specified duration.
Induction of LISA requires high animal density and was less efficient at LD (low density),
defined as fewer than 500 animals per 1 mL isotonic liquid buffer. Unless otherwise
specified, animal density for most experiments were ≥500 worms/mL. For determining
the percentage of LISA, after standing for the designated duration, the M9 supernatant
was removed and worms were transferred to a fresh NGM plate using pipette tips pre-
treated with 5% NP-40. After the residual M9 had evaporated, worms were immediately
examined under a standard optical microscope for phenotypes including body length
and staging with L4 characterized by the vulva crescent structure. For assessing
survival, after a specific duration of LISA, worms were transferred to a fresh NGM plate
and scored for survival after 16 h of recovery. Worms exhibiting a rigid, transparent body
and an absence of visible pharyngeal pumping were scored as dead. For assaying
awakening, approximately 100–150 worms after LISA were transferred using a 20 µl tip
(pre-washed with 5% NP-40) and placed at the center of a 35 mm NGM full-lawn plate.
The awakening process was recorded using the WormLab imaging system at room
temperature. For acute elevation of cAMP via optogenetic activation, worms were
transferred to the center of a 35 mm NGM plate immediately after LISA and exposed to
red LED at 2100 lux to induce cAMP production. WormLab recordings were taken every
10 min to monitor the awakening process. All awakening videos were analyzed to
determine the percentage of awakened worms, defined as the proportion of animals
located outside the original population cluster at 0-60 minutes after transfer.

Western blot analysis

L4-stage worms were subjected to one of three conditions: normal growth for 12 h,
liquid-induced suspended animation (LISA) for 12 h with OP50, or recovery for 12 h
following 12 h of LISA. The indicated genotypic worms were then washed off the plates
and collected using M9 solution. Worm samples (approximately 30 μl animals) were
lysed in 1× Laemmli sample buffer (Bio-Rad, #1616737) supplemented with DTT (50
mM; RPI, #D11000-10.0). The samples were mixed thoroughly, boiled at 98 °C for 10
min, and vortexed twice during the incubation. GFP and β-tubulin levels were detected
by western blotting using a rabbit anti-GFP monoclonal antibody (MP Biomedicals,
#L100036) at a 1:10,000 dilution or a rabbit anti-β-tubulin polyclonal antibody
(Proteintech, #10068-1-AP) at a 1:1,000 dilution. Blotting membranes (Millipore) were
imaged using a ChemiDoc™ Imaging System (Bio-Rad). Band intensities were
quantified using ImageJ (NIH). All western blotting experiments were independently
repeated at least three times.

Statistics

Numerical data were analyzed using GraphPad Prism 10 Software (Graphpad, San
Diego, CA) and presented as means ± s.e.m. unless otherwise specified, with P values
calculated by unpaired two-tailed t-tests (comparisons between two groups), one-way
ANOVA (comparisons across more than two groups) and two-way ANOVA (interaction
between genotype and treatment), with post-hoc Tukey and Bonferroni’s corrections.
The lifespan assay was plotted and quantified using Kaplan–Meier lifespan analysis,
and P values were calculated using the log-rank test.

Data availability

RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under
accession number GSE320035. All other data supporting the findings of this study, as
well as reagents generated and/or analyzed during the current study, are provided and
available from the corresponding author upon reasonable request.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Animals rely heavily on sensory and stress response pathways to regulate and adapt their metabolism to their environment. One such phenomena is the global, yet reversible, downregulation of metabolic activity termed suspended animation. Like sleep, suspended animation involves an arrest in most movement and consciousness. Unlike sleep, suspended animation is untethered to circadian rhythms while also arresting growth and developmental programming. And unlike sleep, we have a limited understanding of the signaling and molecular mechanisms that regulate and mediate suspended animation. Here, Liu et al use the C. elegans system to examine a form of suspended animation that occurs when these nematodes are incubated at high density in isotonic solution (in this case, the standard M9 phosphate saline buffer used for typical C. elegans studies). The authors analyze the various parameters required for this form of suspended animation, including time required in solution to induce suspended animation, time to recovery once out of solution, nematode density required for solution, et cetera. They test the stress conditions of starvation, hypoxia, and heat shock to show that these do not mimic this new form of suspended animation. They also test known genetic effectors for response to these stressors, including for dauer diapause arrest. They use RNA-seq and metabolomics approaches to examine gene expression and metabolic changes caused by suspended animation treatment, finding that heat shock chaperones are upregulated, combined with a shift in metabolism towards NAD+, ATP, and lactate accumulation. Using a combination of fluorescent reporters and survival assays following suspended animation treatment, the authors screen for mutants with either altered ability to enter suspended animation or altered kinetics of recovery (i.e., awakening) from it. Although they did not identify genes that mediate entry into suspended animation, they did find that DAF-21 and LIN-61 have an antagonistic role towards recovery and survival following suspended animation, as their loss of function mutants maintain Hsp16 gene expression longer and survive better than wild type. Finally, they explore whether key neural circuits and their related signaling molecules regulate exit from suspended animation, and they find that the AFD-AIY neural circuit promotes recovery, whereas the RIS circuit antagonizes recovery. Overall, the topic of how suspended animation works is quite interesting and of important biological and perhaps even biomedical relevance. The technical approaches employed by the authors are sound, reasonable, and largely well supported with data. However, three major issues limited my enthusiasm for the manuscript in its current form. First, suspended animation has been studied in C. elegans under many different contexts previously, and the background material and the discussion do not give much of the field the justice it deserves. Specifically, it is well known that anoxia (rather than hypoxia) induces suspended animation; indeed, I suspect that the new form of suspended animation that the authors describe is anoxia-induced suspended animation. From experiments using a Clark electrode approach to measure oxygen consumption in solution, it’s well established that nematodes in solution will rapidly deplete that solution of oxygen in the time span of minutes to hours. A critical mass of nematodes is required to deplete such oxygen levels in a test tube, which is consistent with the authors’ observation that their phenomena is dependent on a high density of animals. Unlike the adaptive changes observed under hypoxia, suspended animation does not require the HIF pathway, similar to the observations from the authors of worm suspended animation post-M9. Like under anoxia, the authors observe mitochondrial fragmentation during their suspended animation paradigm. In short, can the authors demonstrate that this is a novel form of suspended animation? Or have they just found an easier way to study anoxia-induced suspended animation? Either way, they should discuss this issue in the paper and cite the appropriate references. Second, the authors use a combination of RNA-seq and GFP-based transgenic reporters to show that various HSP-16 genes are regulated by their suspended animation paradigm. Interestingly, the GFP reporters for hsp-16 don’t become visible until after the recovery period – not at the end of the M9 incubation period. The authors thus state, “This activation occurs upon re-exposure to permissive environments, indicating that the stress response is not preemptively deployed during arrest but is part of a reawakening mechanism that buffers the transition from dormancy to active metabolism.” Yet hsp-16 mRNAs are elevated in worms exposed to the standing M9 incubation versus control in the RNA-seq data, which is done immediately at the end of the M9 incubation period, not after the recovery period, showing the transcriptional changes are happening during suspended animation even though those transcriptional changes are not being translated into protein abundance changes until the recovery period. Thus, I don’t agree with the statement that this Hsp16-based gene expression response is part of a reawakening response, as described in the Discussion. Rather, the delay in the appearance of GFP from the transgenic reporters likely either reflects suppressed mRNA translation in the absence of oxygen or perhaps reduced fluorophore maturation in the absence of oxygen (the latter being testable by Western blot) Third, the authors argue that the recovery from suspended animation is a process that is activity mediated by changes in neural signaling. The authors state, “Beyond cellular and molecular adaptation mechanisms, our work delineates a neural circuit that governs the behavioral reanimation or “awakening” from SA… These findings suggest that SA exit is not solely governed by passive metabolic recovery but involves a regulated neurobehavioral decision underpinned by antagonistic circuit motifs. The cAMP–PKA axis downstream of AFD-AIY appears to serve as a signaling switch, integrating environmental inputs with internal state to orchestrate behavioral recovery.” I’m not sure that I buy that AFD-AIY is acting as a switch in the sense that the circuit has sensed the exit from M9 liquid and now activates a behavioral program to awaken the worm. Rather, couldn’t it just be that the AFD-AIY circuit is robbed of oxygen during M9 incubation and now is simply coming back online with the same level of activity it had before the anoxic exposure? I suppose the authors would counter this point by stating that M9 incubation shuts down the level of AFD::GCaMP fluorescence relative to rab-3p::NLS::GCaMP fluorescence (Figure 4d), suggesting that AFD activity specifically, and no other neurons, is responding to M9 incubation (and subsequent recovery from suspended animation), but the pan-neuronal GCaMP used to measure the activity of the other neurons appears to have an NLS targeting it to the nucleus, which might cause it to experience a different calcium environment compared to the cytosolic AFD::GCaMP. No details were provided regarding the rab-3p::NLS::GCaMP pan-neuronal strain, making it difficult to verify just what this transgenic construct is. Regardless, a single neuron GCaMP control more analogous to AFD::GCaMP would have been more convincing. It would have also been interesting to observe calcium dynamics changes in AIY and RIS, too, given the importance of these neurons highlighted by the authors. Minor Issues that should be addressed: It’s unclear what the length of M9 incubation time was for the RNA-seq analysis. This needs to be indicated. Figure 1c. What does LD stand for? Presumably low density, but there is no discussion of what constitutes low density versus high density, including in the Materials and Methods. Figure 1g shows that just 3 hours of standing in M9 is enough to induce suspended animation, but the authors don’t try any shorter period to truly map the timing inflexion point. Fluorescent green text on a white background (e.g., Figure 2e,f) is not easy to read. Given that these are not dual color labels, the authors should change such text to high-contrast black. Figure 4a lists a short table of genetic strains tested based on their deficiency in various neurons. Unfortunately, strain names rather than genotypes are used to indicate the genetic manipulations, making the graph difficult to decipher, particularly for non-worm researchers not familiar with strain nomenclature. A column should be added that spells out the genotype. There is not a lot of discussion for the two genes that were isolated based on augmented Hsp16 expression and augmented survival during recovery from suspended animation. For example, there are published roles for DAF-21 and Hsp90 in general for clearing out histones during chromatin remodeling, thereby affecting gene expression. And LIN-61 binds to methylated histones (particularly H4K20me1), where it binds and represses transcriptional activity. Indeed, there is likely a build-up of methylated histones in their suspended animation worms due to the lack of oxygen needed for demethylases to remove those methyl groups. LIN-61 binding to chromatin is probably higher because of that, which likely mediates broad transcriptional repression of genes near such regions of methylated histones, contributing to general suspension of activity as well as the reporter genes for hsp-16. Some discussion of the relevant literature for these genes and how they might be operating would be welcome.", "reviewer_3": "In this study, the authors demonstrated that culturing nematodes at high density in isotonic M9 solution induces a state of dormancy, characterized by halted development and movement, termed Sustained Animation (SA). SA triggers the expression of heat shock proteins, such as HSP-16, which maintain survival during this state. Additionally, SA alters mitochondrial morphology and affects the accumulation of energy metabolites, which are essential for the resumption of movement post-SA. Changes in lysosomal morphology, which are crucial for survival during SA and the initiation of movement thereafter, were also observed. Furthermore, increased levels of the neuropeptide PDF-1 and its receptor PDFR-1, along with cAMP/PKA in AFD sensory neurons and AIY interneurons, facilitate the initiation of movement following SA. While the phenomenon is potentially intriguing, the lack of precision in the experimental conditions renders the discussion challenging to comprehend. The specific issues are detailed below. The presence or absence of food in the SA induction experiments should be described more precisely. Food availability is crucial not only for dauer but also for inducing other nematode diapause, such as L1 diapause and ARD (adult reproductive diapause). However, this experiment appears to have been conducted under conditions where OP50 was not sufficiently removed. Without accurately describing this point, it is impossible to discuss the differences between the other types of dormancy. Whether the worms are motile under conditions where OP50 is sufficiently washed off must be precisely determined. Similarly, the meaning of ST+OP50 in Fig. 1j changes entirely depending on whether OP50 was added to a food-free environment or an environment where OP50 was already present. Although it states that the strain is transferred to an NGM plate during recovery experiments, the presence or absence of OP50 is not clearly explained. The presence or absence of OP50 and any carryover of OP50 must be rigorously discussed in all experiments. Temperature conditions are also critical. While temperature conditions are important for inducing dauer and CID, the SA induction experiment was conducted at room temperature, making it impossible to discuss the effects of temperature. However, because the authors have demonstrated the involvement of temperature adaptation mechanisms such as hsf-1, the temperature-responsive neuron AFD, and temperature-responsive genes gcy-8, ttx-1, and ttx-3 in SA induction and recovery, the influence of temperature must be rigorously confirmed and described. Furthermore, the study lacked rigor in terms of worm density during SA induction. While Fig. 1c compares the SA induction efficiency at different densities, subsequent experiments failed to provide the specific worm density used. Worm density should be precisely documented as ≥500 worms/mL, not the total worm count used in the experiment. Line 56: What exactly are the “agitated (rolling) conditions”? Fig. 1c: The study indicates that nematode density is crucial for SA induction, but why is this the case? Is it because physical movement is restricted or because pheromones are secreted? How does this differ from dormancy induced by starvation or low temperature? Did all individuals that did not enter SA develop into young adults? Are phenotypes such as bagging (internal hatching) or individual death not induced? Information on individuals that did not undergo SA is also important. Fig. 1f: Starvation during the L1>L2 and L3>L4 stages induces dauer and adult reproductive diapause, respectively. How did you distinguish these from SA? Fig. 1i: It has been shown that SA is induced by isotonic M9, but is it also induced by isotonic solutions other than phosphate buffer? If only M9 can induce SA, what is the reason for this? Fig. 1j: The legend and methods are insufficiently explained. What does ST in the figure represent? How much OP50, etc., is being added? In addition, why does feeding not promote growth during SA induction? Ex Fig. 1c: The authors confirmed the effects of post-SA on lifespan but did not strictly describe the duration of SA. The growth stage that induces SA should be discussed, including information such as temperature and the presence or absence of food. Fig. 2f in the main text does not correspond to its position in that figure. Figures 2f-i in the figure panel are not referenced in the main text. Fig. 3g: The expression of lysosome-related genes vps-15, vps-45, rabs-5, and hpo-27 should be confirmed during SA induction and recovery to determine whether they are regulated by hsf-1, daf-21, and daf-16. Fluorescent observation using nematodes: Epistasis between daf-21 and hpo-7 should be confirmed by survival during the SA phase in double mutant strains or by double knockdown using RNAi. Furthermore, daf-21 regulates dauer formation via the TGF-β pathway together with daf-11/A transmembrane guanylyl cyclase, thus requiring discussion of both daf-11 and the TGF-β pathway. Heat shock factor-1 intertwines insulin/IGF-1, TGF-β, and cGMP signaling to control development and aging Janos Barna, Andrea Princz, Monika Kosztelnik, Balazs Hargitai, Krisztina Takacs-Vellai & Tibor Vellai BMC Developmental Biology volume 12, Article number: 32 (2012) The explanation for Fig. 3h is not included in the main text. Many aspects of the connection between these phenomena remain unclear. Does the increase in neuropeptide PDF-1 and its receptor PDFR-1, along with cAMP/PKA, in AFD sensory neurons and AIY interneurons alter HSP gene expression? Fig. 3h indicates that HSP expression affects lysosomal morphology; however, where is the data supporting this? Does the change in metabolite accumulation during SA affect mitochondrial or lysosomal morphology? Is autophagy induction enhanced in the lysosomes during SA? Fig. 4f, described in the text, is not included in the figures."}, "author_response": "Re: Thank you for your overall evaluation and supportive comments. Re: Thank you for these insightful and constructive questions. In the original version, we cited reviews and most of the early pioneering work on suspended animation but described the literature only briefly. In the revised version, we have substantially expanded the introduction and discussion on this topic, cited more references, and highlighted the novel aspects of suspended animation in our study in the context of the earlier foundational work by Padilla, Roth and colleagues. We posit that suspended animation in our study is indeed a novel form, which can be induced at multiple larval and adult stages (Fig. 1f) characterized by increased ATP/AMP ratio (Fig. 2i) whereas earlier work using anoxia showed decreased ATP/AMP ratio as expected for C. elegans embryo under suspended animation exposed to anoxia (PMID:12006646). Unlike anoxia-induced suspended animation, our scheme is strictly population density dependent yet SAN-1 independent (Fig. 1c, and new Extended Data Fig. 1g). In addition, suspended animation in our study is fundamentally different to the well-established dauer diapause since the latter occurs only during L2-L3 transition, shows distinctly specialized morphologic and physiological changes, and genes required for dauer formation are not required for suspended animation in our study (Extended Data Fig. 1b). Finally, as you also commented, our method of inducing SA is indeed also easier to set up since it does not require environmental anoxia condition, which is strictly required for anoxia-induced embryo suspended animation (PMID:12006646). Re: Thank you for pointing this out. We apologize for the inadequate description in our statement and fully agree that the delay in the appearance of GFP from the transgenic reporters likely reflects suppressed mRNA translation as both the hsp-16p::GFP transcriptional and newly generated hsp-16p::hsp-16::mStayGold translational reporters showed strongly up-regulated GFP and mStayGold signals only upon exit from suspended animation (new Fig. 2 and Extended Data Fig. 2h). We have revised the text to clarify accordingly. As suggested by this reviewer, we now also provide Western blot results (new Extended Data Fig. 2i-j) showing that HSP-16::mStayGold levels did not increase until recovery, consistent with the imaging data and your interpretation. Re: Thank you for these critical and insightful comments. We have included the detailed info about the GCaMP strains used in the Methods, including rab-3p::NLS::GCaMP6s (AML70, lite-1(ce314) X; wtfIs5.). Observation of suppressed activity in AFD rather than globally in all neurons upon SA suggests a specific effect on AFD instead of passive and global suppression of oxygen-related metabolism. Though pan neuronal nuclear GCaMP signals reflect cytosolic calcium and help to discern global C. elegans neuronal responses, we agree that they are not as ideal as single-neuron GCaMPs to reveal neuronal specificity in responses. Following your suggestions, we monitored the calcium dynamic changes in AIY and RIS in control and SA conditions using previously generated and currently available GCaMP3 strains (made by Bringmann and Bargmann labs). The data indicate that RIS::GCaMP3 did not show apparent changes by SA whereas AIY::GCaMP did show similar suppression of activity as AFD::GCaMP during SA and rebound during behavioral recovery (see below Reviewer Only Fig. 1). These results together with those in Fig. 4 support the notion that the neuronal AFD-AIY axis is not only specifically regulated but also essential for behavioral recovery after SA, whereas the RIS neuron might play a permissive role or show more subtle dynamics than AFD-AIY neurons. Given the caveats (response sensitivities and neuronal specificities) of such experiments using early-generation extrachromosomal arrays for RIS::GCaMP3 and AIY::GCaMP3, we remain cautious in interpreting the results and plan to generate and analyze integrated GCaMP6 reporters in follow-up studies. As such, we have also toned down the instructive roles of RIS and AFD-AIY neurons. Reviewer only Fig. 1 AIY::GCaMP3 but not RIS::GCaMP3 responds to SA. (a) Representative images and quantification of RIS::GCaMP3 responses to SA in L4-stage animals after standing in M9 for 12 hours, showing no significant change. (b) Representative images and quantification of AIY::GCaMP3 responses to SA in L4-stage animals after standing in M9 for 12 hours, showing suppressed and restored AIY::GCaMP3 fluorescence during and after SA, respectively. Re: 12 hours now included. Re: yes, LD stands for low density, defined as fewer than 500 animals per 1 mL isotonic liquid buffer (Fig. 1c), resulting in incomplete induction of SA, info now included in Method of the revised version. Re: Shorter than 3 hours makes it more practically difficult to assess developmental arrest in SA based on stage-specific markers and body lengths since unambiguous developmental stage transitions take about 8-10 hours for each larval cycle. Re: Corrected. Re: Corrected. Re: Thank you for the suggestion. We have revised the Discussion accordingly. Re: Thank you for raising these important considerations. We described the food condition in the Method section and have now revised the text to clarify that OP50 is present in our initial setup for suspended animation (SA) and assays for survival post SA, though SA was robustly induced even after M9 washing, indicating food independence. We have performed new experiments to show that the absence of OP50, incubation temperature at 15, 20, 25C using precisely temperature-controlled incubators, or incubation in various isotonic buffers does not impact the entry into or exit from suspended animation (new Extended Data Figure 1e-h). We have also revised the text to document the worm density for most experiments as ≥500 worms/mL. Re: The same number of worms in the tube kept on a roller rather than standing still. We changed the word “agitated” to “dispersed” to avoid confusion. Re: We also find the density requirement intriguing (and fascinating). To address these questions more fully, we have performed experiments showing that the supernatant from suspended animation worms at high density is insufficient to induce suspended animation at low density (new Extended Data Fig. 2i). This would argue against a stable diffusible pheromone that transduced the density signal. We also found that most if not all individuals enter SA at high density while standing in the tube, and individuals that did not enter SA at low density or under rolling/dispersion condition developed into young adults in the presence of OP50 in the tube (Fig. 1j). Unlike starvation, our SA conditions (standing in M9 for 12-16 hrs, in the absence of presence of OP50) did not cause phenotypes such as bagging (internal hatching) or individual death. Re: Unlike dauer or adult reproductive diapause when cultured on plates with solid media, worms undergoing SA in our paradigm when incubated in isotonic liquid media at high population density do not apparently alter morphologic appearance (Fig. 1b, f). Molecular and metabolic signatures from our transcriptome and metabolomic profiling results (new Fig. 2, Fig. 3 and Supplementary tables), as well as genetic requirements (daf-16 and daf-22 dependency specifically for dauer) are also distinctly different. Re: Worms standing in S-basal buffer or PBS buffer showed also SA normally (new Extended Data Fig. 1f), indicating robustness of our method inducing SA. Re: Thank you for pointing this out. Detailed info has been included. ST represents tube “standing” still with animals inside the tube with isotonic buffer. OP50 was washed along with animals into the tube but washed once with M9 before typical SA assays. However, we found the washing step did not affect SA induction, which involves arrested motility and pharyngeal pumping thus feeding did not appear to promote growth. We have performed new experiments to show that the absence of OP50 during and after standing in isotonic liquid, incubation at 15, 20, 25C temperatures using precisely temperature-controlled incubators, or incubation in various isotonic buffers does not impact the entry into or exit from SA (new Extended Data Figure 1e-h). Re: Info now included: “L4-stage, room temperature and in the presence of OP50.” Re: Corrected. Re: We apologize for our inadequate description that led to possible misunderstanding by this reviewer. Mutants for the autolysosome-related genes vps-15, vps-45, rabs-5, and hpo-27 were used to assess the roles of autophagy and lysosomal fusion processes in promoting survival post SA, not to show that these genes are in the downstream and regulated by HSF-1, DAF-21 or DAF-16. Consistent with published studies, vps-15, vps-45, rabs-5, and hpo-27 are constitutively expressed and were not identified as direct targets of HSF-1 or DAF-16 (PMID: 27496166; PMID: 25156270). The direct transcriptional HSF-1/DAF-16 target genes responsible for modulating auto-lysosomal functions and lysosomal tubulation during SA in our paradigm remain to be identified and are among the foci of our follow-up studies. Re: We have attempted double RNAi but found efficiency of daf-21 RNAi with control vector only RNAi is quite limited, without even effectively activating hsp-16p::GFP. Thus, we tested whether RNAi against hpo-27 can suppress enhanced survival in daf-21 mutants. Percent survival rates of daf-21, WT+ hpo-27 RNAi and daf-21 + hpo-27 RNAi showed that hpo-27 RNAi can partially suppress effects of daf-21 mutants (Reviewer only Fig. 2), suggesting that DAF-21 recruits HPO-27 dependent and independent mechanisms. Given the caveats of double RNAi and single RNAi in knockdown efficiency and limitation of genetic epistasis in interpreting mechanistic relationship, we have toned down the conclusion on the mechanisms and would like to leave such results out in our manuscript and plan to investigate in depth by follow-up studies. For reviewer only Fig. 2 Testing genetic epistasis between daf-21 and hpo-27. Percent survival rates post-SA after standing 36 hrs in M9 showing partial suppression of daf-21 mutants by hpo-27 RNAi. Re: Thank you for raising this point. We have tested daf-11 mutants, which showed normal SA induction (new Extended Data Fig. 1g, h), suggesting the DAF-11/TGF-β pathway is indispensable for dauer formation but not for SA induction. We have included this result and added discussion on this point. Re: This info is now included. Re: We apologize for our inadequate description that led to possible misunderstanding by this reviewer. We used the PDF-1, PDFR-1, gcy-8, and cAMP/PKA mutants to study processes that regulate awakening post SA, whereas hsp-16 expression is up-regulated by HSF-1 activation during SA, not post SA. hsp-16 driven HSP-16 protein increases (new Extended Data Fig. 2) occur post SA as a consequence of restored protein translation. In addition, Fig. 3h shows HSP expression as an indicator of HSF-1 activation, which together with DAF-16 regulate lysosomal morphology (Fig. 3h). We do not claim that HSP expression affects lysosomal morphology given lack of evidence. Following the reviewer’s suggestion, we have also imaged mitochondrial or lysosomal markers upon exogenous metabolites including inositol, succinate and spermidine, the top 3 up-regulated metabolites by GC-MS. Under the conditions tested (1-10 mM supplementation), we did not observe apparent effects as SA had, perhaps due to absorption barriers or limitation of single metabolites at tested doses. We have also analyzed (by imaging and Western blot) the autophagy induction marker ATG-9::GFP and autophagosome marker LGG-1::GFP but didn’t observe striking signal changes, perhaps due to detection threshold/sensitivity of the reporters or alternative autophagy pathway activation. We agree that many aspects of the connection between these phenomena remain unclear and are under our ongoing and future studies. Meanwhile we have revised the manuscript to tone down the unsubstantiated mechanisms and sharpen our scope on the novelty and discovery of these fascinating phenomena. Re: This info is now corrected."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The key technical points I raised in my previous review have largely been addressed. However, I am still not convinced that this form of suspended animation isn't simply one previously described: anoxia-induced suspended animation (SA). Five hundred worms per mL of M9 rapidly depletes the liquid buffer of oxygen, exposing the worms to anoxia. The fact that regularly dispersing the animals by rolling the microfuge tube blocks this new form of SA induction would suggest that simply aerating the buffer by regularly mixing with the remaining air in the tube is sufficient to restore aerobic levels of oxygen. Indeed, if the model is that there is some sort of quorum-sensing signal released by worms, why would mixing the solution disrupt its function? Yes, the authors provide convincing data that their new SA phenomena is unlikely to be the kind of anoxia-induced SA described by Padilla, Roth, and colleagues, which only occurs in embryos and is SAN-1-dependent. However, there is another form of anoxia-induced suspended animation that occur to larvae and adults (e.g., PMID:24385935) that is dependent on EGL-9, SKN-1, and STL-1, but not HIF-1. This is likely the same phenomenon the authors are observing. I do not see any experiments in the paper that rule out this possibility. Either the authors need to do experiments to show that this new form of SA is not anoxia-induced SA, or they need to seriously discuss this caveat in the Discussion section, including highlighting that they cannot exclude the possibility that these two forms of SA are the same or rely on the same mechanism. Even if we accept that the form of SA described by the authors is not anoxia-induced SA, all these different forms of SA, each with their own different requirements, raises an important concern: the paper highlights findings as if this new form of SA were generalizable to ALL FORMS OF SA. Indeed, the title (Pausing and restarting life from reversible suspended animation) implies that the mechanism described within is likely broadly generalizable to suspended animation rather than being specific to this new form of suspended animation triggered by a combination of isotonic buffer and high animal density. Indeed, the authors’ own experiments demonstrating that this new form of suspended animation is distinct genetically, developmentally, environmentally, and physiologically from most previously described forms of SA clearly show that they are studying something that is fairly narrow and specific rather than generalizable to all forms of SA. To suggest otherwise is to misrepresent the findings and potential impact of the paper; thus, I can’t support its publication unless the changes are made to the text that tone down the generalizable nature of their findings: (1) The title needs to be changed. Perhaps something like: Isosmotic liquid exposure and population density induce a novel reversible form of suspended animation in C. elegans. (2) They need to replace the abbreviation “SA,” which suggests applicability to all forms of SA, with something that highlights this specific form of SA. Perhaps “IDSA” for isosmotic and density-induced suspended animation? Alternatively, “LISA” for liquid-induced SA? “SA” can be used in the first two sentences of the abstract where they refer to the general state of SA or other forms of SA induced by other stimuli (e.g., anoxia). It can also be used at lines 33, 40, 42, 53, 64, 69, 75, 80, 266, 273, 276, and 328 in the text for the same reason. But all other mention of SA should indicate that the SA described is specific to this novel form of SA, whether that be with IDSA, LISA, or equivalent abbreviation. Finally, the authors argue that this new form of SA triggers a drop in neural activity in the AFD neurons (Figure 4d) monitored by an AFD::GCaMP6 transgene, but no such change in other neurons monitored by a Prab-3::NLS::GCaMP6 transgene. I had previously raised the concern that the NLS::GCaMP6 was not the appropriate control for the AFD::GCaMP6 given the former’s subcellular localization to the nucleus. The authors responded with a figure meant only for reviewers (Reviewer only Fig. 1) showing no decrease in an RIS::GCaMP reporter, and a decrease in AIY::GCaMP upon SA, similar to that observed in AFD. It's great that they now see a drop in AIY activity. But the AIY::GCaMP pictures in Reviewer only Fig. 1b clearly show that many neurons besides AIY (presumably indicated by arrows, although this was never stated in the description) drop in fluorescence intensity upon SA induction. Thus, it’s not just AFD and AIY that show a drop in neural activity under these conditions. Why not just fess up to that point and include this figure in the paper? Indeed, the drop in AIY activity correlates nicely with their demonstrated requirement of AIY in the AIY-ablation experiments (e.g., ttx-3 mutants). Minor Issues: I appreciate that the authors have now defined the difference between high density and low density (LD) in the Methods, but why must the reader dig through the methods for something so crucial? The paper would be easier to follow if this was spelled out in the Results (around lines 75-83) and in the legend for Figure 1a-c. For example, exactly how many worms were monitored in the “LD” column of the graph in Figure 1c? What is the control column in this panel? Presumably no liquid immersion at all, but it’s not spelled out. Please spell these out, at least in the figure legends. Lines 245-247. This sentence (To further determine whether SA-induced mitochondrial and lysosomal dynamics we observed might link to awakening from SA, we examined behavioral recovery from SA in mitochondrial and lysosomal mutants) is rather confusing. Did you mean, “To further determine whether THE SA-induced mitochondrial and lysosomal dynamics THAT we observed might BE linkED to awakening from SA?” It needs to be rewritten to make it more clear.", "reviewer_3": "The authors have adequately addressed the issues I raised."}, "author_response": "Response: We thank the reviewer for raising this important point. We were aware of the suspended animation phenotype described in PMID:24385935 and have cited this study in the Introduction. We agree that anoxia is likely present in our liquid-induced SA paradigm. However, anoxia alone cannot account for several key features of our observations, including SAN-1 independence and stage independence. To directly address the reviewer’s concern, we examined our liquid-induced suspended animation (LISA, see below) by mutants in the EGL-9 pathway previously implicated in anoxia-induced SA. Specifically, we tested egl-9, skn-1, and hif-1 mutants (unfortunately, stl-1 mutants were not available in our laboratory or at the CGC and therefore could not be analyzed). We found that all three mutants entered LISA with penetrance comparable to wild type, with no significant differences in the percentage of animals undergoing developmental and behavioral arrest (Reviewer Figure 1a). Thus, entry into LISA in our paradigm does not require EGL-9, SKN-1, or HIF-1. Recovery kinetics in egl-9, hif-1, and skn-1 mutants were also comparable to wild type, with no significant differences in recovery timing (Reviewer Figure 1b, c). Survival following LISA was comparable between wild type, egl-9, and hif-1 mutants. In contrast, skn-1 mutant exhibited mildly improved 24-hour survival (Reviewer Figure 1d). Together, these results indicate that the canonical EGL-9/HIF oxygen-sensing pathway is dispensable for both induction and recovery of LISA under our experimental conditions. Reviewer Figure 1. a, Oxygen-sensing pathways are dispensable for LISA induction and recovery. Percentage of animals entering liquid-induced suspended animation (LISA) under high-density liquid conditions. WT, egl-9(sa307), hif-1(ia4), and skn-1(zj15) mutants exhibited comparable LISA penetrance (no significant differences). (n > 500 animals per condition, N > 3 trials). b-c, Time-serial brightfield images and population quantification showing locomotion behavioral recovery post-LISA in wild type and various strains (egl-9, hif-1, and skn-1 mutants), indicating recovery timing in egl-9, hif-1, and skn-1 mutants was comparable to wild type (no significant differences; p values shown). (n = 100-150 worms in each of 3 independent experiments). d, Percent survival 24 hours after LISA. Survival rates were comparable between wild type, egl-9, and hif-1 mutants, whereas skn-1 mutants exhibited significantly improved survival relative to wild type. (n > 500 animals per condition, N > 3 trials). Data are presented as mean ± SEM. Statistical analyses were performed using student’s two-tailed unpaired t-test (a,d) or Two-way ANOVA with Bonferroni post-test was used to compare mutant data with WT (c). Together, these data argue that the LISA phenotype described here is mechanistically distinct from the EGL-9–dependent anoxia-induced LISA reported previously. While anoxia may contribute to the physiological context of our assay, LISA does not depend on the canonical EGL-9/HIF pathway. Instead, our findings suggest that LISA engages multiple stress-responsive pathways, including HSF-1 and DAF-16 as described earlier for survival under LISA. We are actively pursuing the regulatory pathway for entry into LISA and mechanistic relationships underlying these observations; however, a detailed dissection of these pathways is beyond the scope of the present study. Response: We thank the reviewer for this thoughtful critique. We agree that our previous title could be interpreted as implying broad generalizability across all forms of suspended animation, which was not our intention. Our goal is to characterize a highly experimentally tractable, completely penetrant, and mechanistically distinct form of reversible SA induced by isosmotic liquid exposure at high population density. We acknowledge that multiple forms of SA exist in C. elegans, each with distinct genetic and environmental requirements. Our own data demonstrate that the LISA described here is genetically, developmentally, and environmentally distinguishable from previously reported paradigms. We agree that it would be inappropriate to imply that the mechanisms identified in this study universally apply to all forms of SA. To address this concern, we revised the title to: “Induction and regulation of a reversible form of suspended animation in C. elegans.” This revised title more accurately reflects the scope of our findings without overgeneralization. We have also modified the Abstract and Discussion to clearly state that our conclusions pertain specifically to this liquid- and density-induced SA paradigm. At the same time, we maintain that dissecting the molecular and physiological mechanisms underlying any experimentally tractable form of suspended animation provides important conceptual insight into how animals reversibly pause and resume biological activity. While our findings should not be generalized to all forms of SA, they offer a defined framework for understanding reversible dormancy within this specific context and may inform comparative analyses across different paradigms. We appreciate the reviewer’s guidance in ensuring that the scope and impact of the manuscript are represented accurately and precisely. Response: We thank the reviewer for this helpful suggestion. We agree that using “SA” throughout may imply broad applicability. To ensure precision, we adopted LISA (liquid-induced suspended animation) to refer specifically to the form characterized in this study. The term “SA” is retained only when referring to suspended animation in general or to previously described forms (including in the locations noted by the reviewer). All other instances have been revised to “LISA” throughout the manuscript to clearly define the scope of our findings. Response: We thank the reviewer for this careful evaluation. The original AIY::GCaMP reporter used in Reviewer-only Fig. 1 was an extrachromosomal array, which exhibited mosaic and variable expression between animals and trials. As a result, signal changes in non-AIY neurons were inconsistently labeled and could be misinterpreted. To address this concern more rigorously, we have repeated the experiments and generated new datasets with carefully controlled imaging and analysis. The new results confirm that AIY neuronal activity is significantly reduced during LISA induction and recovers upon exit. These revised data are now included in the manuscript (New Extended Data Figure 5c, d). Importantly, the reduction in AIY activity is consistent with our functional data demonstrating a requirement for AIY (e.g., ttx-3 mutants) in this process. We agree that these findings strengthen the mechanistic link between neural activity changes and LISA induction, and we have incorporated the revised data accordingly. Response: Corrected. Thanks for the suggestion. Response: Yes. Corrected."}, {"round": 3, "reviewer_comments": {"reviewer_1": "My concerns have been nicely addressed. I highly recommend that Reviewer Figure 1 showing that LISA is independent of EGL-9, SKN-1, and HIF-1 being included in the paper (supplement or main figure - either would be fine), as I'm sure I'm not the only one in the field who would have this question. A sentence or two in the Results and Discussion highlighting this point is appropriate, as it further strengthens the finding that LISA is distinct (genetically) from anoxia-induced SA (which should maybe now be called AISA?)."}, "author_response": "Thank you. We agree and have included the EGL-9/HIF-1 results in the Supplementary Figure 1 with appropriate discussion of the mechanisms for LISA distinct to AISA."}]

Decision letter