Abstract
The general association between longevity and energy metabolism has
been well-documented for some time, yet the specific metabolic
processes that regulate longevity remain largely unexplored. In
contrast to the common active swimming daphnids (e.g., Daphnia
sinensis), Simocephalus vetulus is notable for being sedentary and
having a lower metabolic rate, yet it has a longer lifespan than D.
sinensis. In this study, metabolomic analysis and drug validation
experiments are employed to demonstrate that the lower pyruvate
dehydrogenase (PDH) activity reduces the locomotor performance of S.
vetulus and to identify PDH activity as a regulator of the lifespan of
daphnids. Inhibition of PDH activity in daphnids by CPI-613 attenuates
its ATP supply and locomotor performance but significantly induces
longevity. The study also determines that the invertebrate
neurotransmitter octopamine and temperature have a significant impact
on PDH activity and modulate daphnids lifespan. And when the effects of
temperature and octopamine on PDH activity are counteracted by
inhibitors or agonists, the impact on lifespan becomes ineffective.
These results support an important role for PDH in lifespan regulation
and locomotor performance in daphnids and provide insights into the
metabolic regulation of lifespan.
Subject terms: Animal physiology, Metabolism, Energy metabolism
__________________________________________________________________
Metabolism is known to be linked to lifespan in some species, though it
remains unclear how this effect is mediated. Here they show that
reduced pyruvate dehydrogenase (PDH) activity lowers locomotion but
extends lifespan in daphnids and show that inhibition of PDH can
increase lifespan in these species.
Introduction
Water flea, the most commonly used model organism in ecological and
toxicological studies, is emerging as a model organism for lifespan
studies due to its short life cycle, easily controlled growth
conditions, solitary reproduction, clear genetic background
(multi-genome sequencing), and abundant genetic resources^[40]1–[41]5.
Almost all daphnids live a planktonic life throughout their lives,
which largely depends on their grazing patterns and avoidance of
enemies. However, Simocephalus vetulus exhibits a markedly different
behavioral pattern in this regard. For the majority of its life
history, S. vetulus was essentially attached to water plants. It even
evolved a unique limb feature in which the outermost swimming setae of
its second antennae were curved in the form of hooks to facilitate
stable hooking to water plants. Daphnia sinensis, which inhabited the
same habitat as S. vetulus, were selected as a representative species
of common daphnids for comparison with S. vetulus. A noteworthy
observation was that the sedentary S. vetulus exhibited a lower
metabolic rate than the active D. sinensis, and also demonstrated a
greater longevity at a similar body size. The relationship between
energy metabolism, exercise and longevity has long been of
interest^[42]6. Differences in locomotor habits, metabolism, and
longevity between S. vetulus and D. sinensis have led to their
consideration as good material for studying the relationship between
locomotion, metabolism, and longevity.
The influence of metabolism on lifespan is a topic that has been
extensively studied in the field of lifespan research. A substantial
body of research has demonstrated a strong correlation between lifespan
and metabolic rate. In Caenorhabditis elegans, mutants with increased
longevity exhibit a reduction in metabolic rate. Once these mutants are
returned to their original lifespan using genetic inhibitors, their
metabolic rates also return to normal levels^[43]7. Furthermore, it has
been demonstrated that environmental conditions which reduce the
metabolic rate of C. elegans also result in an extension of their
lifespan. Thus, Van and Ward concluded that the extension of C. elegans
lifespan may not be a consequence of altered genetic pathways, but
rather a consequence of their reduced metabolic rate. Furthermore, the
effect of glucose metabolism on longevity seems to require more
attention in exploring the relationship between longevity and energy
metabolism^[44]8–[45]12. An increase in glucose intake is associated
with a reduction in lifespan, whereas impaired glucose metabolism is
linked to an extension of lifespans^[46]8,[47]9. It has been
demonstrated that sustained glycolysis tends to accelerate the ageing
process, and that lifespan interventions such as calorie restriction
can extend lifespan by reducing the metabolic flux of
glycolysis^[48]13. This result has also been validated in yeast
experiments^[49]14. This hypothesis is further supported by the results
of RNAi studies of two glycolytic genes in C. elegans^[50]10,[51]11.
Lionaki et al. demonstrated that reduced mitochondrial protein inputs
extend lifespan in C. elegans by facilitating metabolic shifts in the
conversion of glucose to serine, and that the essence of this action
lies in the fact that mitochondrial dysfunction limits the oxidative
phosphorylation process^[52]15. When mitochondrial dysfunction occurs,
glycolysis increases to compensate for the decrease in the amount of
aerobically produced ATP, but does not counteract the lifespan
extension for C. elegans. This suggests that the role of glycolysis on
lifespan lies not at its own level, but more likely in a limiting
effect on downstream oxidative phosphorylation processes. It has long
been reported that mild inhibition of mitochondrial respiration extends
the lifespan of many organisms^[53]16. Metabolic data from a number of
small rodent long-lived species supports this view. Both the naked mole
rat (Heterocephalus glaber) and the Damaraland mole rat (Fukomys
damarensis), which exhibit extraordinary longevity compared to their
counterparts, have low basal metabolic rates and both rely more on
glycolysis than oxidative phosphorylation^[54]17. Energy metabolism not
only has a major impact on the lifespan of an organism, but also has a
profound effect on its motility and athletic performance. Given the
observed differences in locomotor behavior and longevity between S.
vetulus and D. sinensis, it is plausible to suggest that energy
metabolism plays a significant role in the poor locomotor performance
and longevity of S. vetulus.
The present study has demonstrated an inverse correlation between
pyruvate dehydrogenase complex (PDH) activity and lifespan. The use of
metabolomic, behavioral and lifespan assays in S. vetulus and D.
sinensis revealed that decreased PDH activity not only limits Daphnids
locomotor performance by regulating glucose metabolism for energy
supply, but also regulates energy metabolism rates and extends Daphnids
lifespan. This provides insights into the intrinsic link between
metabolism and lifespan, helps us to understand the mechanism of life
aging and longevity more deeply, and also provides multiple
perspectives and ideas for human anti-aging research.
Results
Sedentary S. vetulus lives longer than active D. sinensis
Both S. vetulus and D. sinensis were obtained and established as clone
lines from the South Lake in Wuhan. The initial observation that led to
the study of these two species was the observation of divergent
locomotor habits, thus prompting an examination of the morphological
differences in their swimming limbs. The outermost swimming setae of
the second appendage of sedentary S. vetulus are curved and hooked at
the end. These setae are hooked at the distal end, which anchors S.
vetulus to the surface of the water plants (Fig. [55]1a). In contrast,
the outermost swimming setae of the second appendage of active D.
sinensis are covered with fine hairs, a feature that enhances its
swimming ability (Fig. [56]1a). The genome sequence and assembly of D.
sinensis was previously completed^[57]5. In order to achieve a more
profound comprehension of the evolutionary relationships and divergence
times of S. vetulus and D. sinensis, we undertook the sequencing and
assembly of the genome of S. vetulus. Third-generation genome
sequencing of S. vetulus yielded a 144.98 Mb genome with an N50 of
1.34 M and a GC content of approximately 39.50% (Fig. [58]1b). The
genomes of S. vetulus and D. sinensis were compared at the chromosomal
level using Minimap2 (version 2-2.26, parameters: -x asm5), and
covariance circles were plotted using Circos (version 0.69-6)
(Fig. [59]1c). The genomic chromosomes of S. vetulus and D. sinensis
display a significant number of co-linear segments, thereby confirming
the close affinity and high chromosomal synteny of the two species.
With regard to the divergence time, S. vetulus was separated from other
cladocerans as early as approximately 237.9 (17.2-308.6) million years
ago (Supplementary Fig. [60]1).
Fig. 1. Comparison of the genomic, life history, and behavioral
characteristics of S. vetulus and D. sinensis.
[61]Fig. 1
[62]Open in a new tab
a Diagrams of parthenogenic female S. vetulus and D. sinensis. b Genome
circle diagram of S. vetulus. (A, Genomic information; B, GC content
distribution; C, Depth distribution of second-generation reads; D,
Depth distribution of third-generation reads; E, Outer circle for pure
SNP distribution, inner circle for heterozygous SNP distribution; F,
Pure InDel distribution in the outer circle, heterozygous InDel
distribution in the inner circle; G, Distribution of genes on the
genome for complete comparison BUSCO, single-copy BUSCO in blue,
duplicated BUSCO in red). c Genomic covariance analysis of S. vetulus
and D. sinensis. The letters “chr” and “LG” represent the chromosomes
of S. vetulus and D. sinensis, respectively. d The lifespans of S.
vetulus and D. sinensis (n = 16). e Age of S. vetulus and D. sinensis
at sexual maturity (n = 15). f Body length of S. vetulus and D.
sinensis at sexual maturity (n = 10). g The inter-clutch period of S.
vetulus and D. sinensis (N = 15). h Brood size, average number of
offspring per litter of S. vetulus and D. sinensis (n = 15). i Total
number of offspring produced throughout the life history (n = 17). j
The frequency of hops for swimming of S. vetulus and D. sinensis
(N = 6). k Locomotion per unit time in S. vetulus and D. sinensis
(N = 3). l Duration of quiescence of S. vetulus and D. sinensis
(N = 5). m Feeding rate of S. vetulus and D. sinensis (N = 6). n Oxygen
consumption in S. vetulus and D. sinensis (N = 3). N, number of
replicates; n, number of samples per replicate. d–n The raw data are
presented as means ± SEM and were analyzed by unpaired two-tailed
Student’s t-test after normality and homogeneity of variance was
confirmed. Source data are provided as a Source Data file.
Further, the life-history characteristics of S. vetulus and D. sinensis
were compared. The life history observations of the two cladocerans
revealed that the average lifespan of S. vetulus is 53.4 days, while
the average lifespan of D. sinensis is only 44.4 days (Fig. [63]1d).
Moreover, S. vetulus exhibited a younger age at first reproduction and
a smaller body length at first spawning compared to D. sinensis
(Fig. [64]1e and f). S. vetulus exhibited a longer inter-clutch period
than D. sinensis, while also having smaller brood size (litters) and
total reproductive capacity (lifetime litters) compared to D. sinensis
(Fig. [65]1g, h and i). In general, fast development, early sexual
maturation leading to early reproductive effort, as well as production
of many offspring, have been linked to shorter lifespans^[66]18. D.
sinensis with greater total reproductive capacity and shorter
inter-clutch period had significantly lower longevity than S. vetulus.
In order to visually compare the differences in locomotor habits
between S. vetulus and D. sinensis, an investigation was conducted into
the swimming behavior and locomotion-related physiological indicators.
Behaviorally, the frequency of hopping and locomotor activity were
found to be significantly lower in S. vetulus compared to D. sinensis
(Fig. [67]1j and k). The three-minute video observation revealed that
S. vetulus exhibited a high degree of immobility, remaining stationary
for an average of 92.7% of the observation period. In contrast, D.
sinensis swims at almost all the time, averaging only about 2.3% of the
observation period without swimming (Fig. [68]1l). A high level of
exercise is often accompanied by a high metabolic rate, which is
reflected in the demand for food and oxygen. Research has demonstrated
that D. sinensis exhibits a markedly higher feeding rate and oxygen
consumption than S. vetulus (Fig. [69]1m and n). Overall, the
behavioral and physiological characteristics observed in both species
of daphnids were consistent with their respective locomotor habits.
Ability rather than willingness to exercise leads to differences in locomotor
choice
To further understand the reasons for the different locomotor choices
of the two species, forced exercise experiments were conducted on both
S. vetulus and D. sinensis. Petri dishes containing all the daphnids
were placed on a fixed-rail shaker. The fluctuations in the water
column and the smooth surface of the petri dishes forced S. vetulus and
D. sinensis to swim. Although the natural water body in which S.
vetulus lives is also non-stationary, a blade of watercress was placed
in a petri dish with S. vetulus to eliminate the influence of stress on
the results. The good condition of S. vetulus hooked itself on the
surface of the watercress leaf in the same fluctuating water body
indicates that water fluctuations in the forced-motion experiment do
not cause stress to S. vetulus. During the sixth hour of the
experiment, over 50% of swimming S. vetulus exhibited difficulty
swimming in the water and sank to the bottom of the petri dish
(Fig. [70]2a and b). In contrast, D. sinensis continued to swim
normally. After cessation of disturbance to the water column, S.
vetulus showed gradual recovery. ATP levels and lactate levels were
measured in both S. vetulus and D. sinensis after 6 hours of forced
exercise. ATP concentrations decreased significantly in both S. vetulus
and D. sinensis, but the decrease was greater in S. vetulus (56.9% in
S. vetulus, 22.9% in D. sinensis) (Fig. [71]2c). Lactate levels were
also significantly increased in S. vetulus but not in D. sinensis
(Fig. [72]2d). These results demonstrate the inferior ATP supply and
aerobic metabolism of S. vetulus compared to D. sinensis.
Fig. 2. Locomotor ability, but not locomotor willingness, influences
locomotor choice in S. vetulus and D. sinensis.
[73]Fig. 2
[74]Open in a new tab
a The horizontal and vertical distribution of S. vetulus and D.
sinensis in petri dishes following six hours of the forced exercise
experiment (N = 3). b The proportion of falls over time in the forced
exercise experiments with S. vetulus and D. sinensis (N = 3, n = 30).
Statistical significance was determined by two-way ANOVA with Tukey’s
multiple-comparisons test. c The ATP content of S. vetulus and D.
sinensis after 6 hours of forced exercise (N = 3). Statistical
significance was determined by two-way ANOVA with Sidak’s
multiple-comparisons test. d The lactate concentration of S. vetulus
and D. sinensis after 6 hours of forced exercise (N = 5). Statistical
significance was determined by two-way ANOVA with Sidak’s
multiple-comparisons test. All data are presented as means ± SEM.
Source data are provided as a Source Data file.
Lower PDH activity limits locomotor activity and prolongs lifespan in S.
vetulus
Forced exercise experiments demonstrated the inferior aerobic
metabolism of S. vetulus relative to D. sinensis, so the two species
were analyzed metabolically, and key metabolites of energy metabolism
were compared. The metabolomic analysis of S. vetulus and D. sinensis
revealed 153 differential metabolites (DMs) that were significantly
enriched in the pathways related to glucose metabolism (Fig. [75]3a).
The levels of pyruvate and lactate were found to be significantly
higher in S. vetulus than in D. sinensis, while the levels of acetyl
coenzyme A and ATP were found to be significantly lower in S. vetulus
than in D. sinensis (Fig. [76]3b). Normally, pyruvate, the end product
of glycolysis, is generated by pyruvate dehydrogenase (PDH) to produce
acetyl coenzyme A, which enters the tricarboxylic acid cycle and
ultimately generates ATP by oxidative phosphorylation to supply energy
to the body. Once the pyruvate oxidative decarboxylation process is
blocked, pyruvate accumulates in large quantities and is reversibly
converted to lactate. Furthermore, the production of pyruvate oxidative
decarboxylation products, acetyl coenzyme A and ATP, is also reduced.
The pyruvate dehydrogenase complex (PDC) is responsible for the
oxidative decarboxylation of pyruvate. The complex comprises three
components: pyruvate dehydrogenase (E1), dihydrolipoamide
acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3)^[77]19.
The nucleic acid sequences of the pyruvate dehydrogenase complex E1,
E2, and E3 from S. vetulus and D. sinensis were extracted from their
respective genomes. Sequence analysis software and SWISS-MODEL were
employed to facilitate the comparison of amino acid sequence and
protein structures (Supplementary Fig. [78]2 and [79]3). The structure
of the PDH α-subunit differs significantly between S. vetulus and D.
sinensis (Supplementary Fig. [80]4). Furthermore, prediction of the
interaction between pyruvate dehydrogenase and thiamine pyrophosphate
using AlphaFold 3. The interaction prediction between PDH from D.
sinensis and thiamine pyrophosphate yielded a higher ranking score
compared to that from S. vetulus. (Fig. [81]3c). The results of
metabolomics analysis and PDH sequence and structure analysis suggest
that the pyruvate oxidative decarboxylation process in S. vetulus may
be impaired (Fig. [82]3d).
Fig. 3. Impaired energy metabolism limits locomotion but extends longevity in
S. vetulus.
[83]Fig. 3
[84]Open in a new tab
a KEGG pathway enrichment analysis of differential metabolites of S.
vetulus and D. sinensis. b Differential metabolites related to glucose
metabolism. c Binding site prediction and conformation simulation of
pyruvate dehydrogenase (PDH) and thiamine pyrophosphate by AlphaFold 3.
SVPDH, S. vetulus pyruvate dehydrogenase; DSPDH, D. sinensis pyruvate
dehydrogenase; The two α subunits are labelled green and blue and the
two β subunits are labelled red and yellow. d Schematic representation
of glucose metabolism and oxidative phosphorylation. e The mortality of
S. vetulus treated with gradient concentrations of CPI-613 (N = 3). f
The mortality of D. sinensis treated with gradient concentrations of
CPI-613 (N = 3). g The PDH activities of S. vetulus and D. sinensis
treated with gradient concentrations of DCA (N = 3). h The PDH
activities of S. vetulus and D. sinensis treated with gradient
concentrations of CPI-613 (N = 3). i The exercise trajectories and
locomotor activity of 10 μM CPI-613-treated D. sinensis and 10 μM
DCA-treated S. vetulus (N = 3). j The ATP content of S. vetulus and D.
sinensis treated with gradient concentrations of CPI-613 (N = 6). k The
ATP content of S. vetulus and D. sinensis treated with gradient
concentrations of DCA (N = 6). l The metabolic rate of 10 μM
DCA-treated S. vetulus and 1 μM CPI-613-treated D. sinensis (N = 3). m
The lifespan of CPI-613-treated S. vetulus and D. sinensis (n = 30). n
The lifespan of DCA-treated S. vetulus and D. sinensis (n = 30). SV, S.
vetulus. DS, D. sinensis. For (e–i), all data are presented as
means ± SEM. Statistical significance was determined by two-way ANOVA
with Dunnett’s multiple-comparisons test (e, f, j and k), two-way ANOVA
with Sidak’s multiple-comparisons test (g and h), unpaired two-tailed
Student’s t-test (i) or Survival curve comparison with
Gehan-Breslow-Wilcoxon test (m and n). Source data are provided as a
Source Data file.
Furthermore, CPI-613 (Devimistat, a PDH indirect inhibitor) and DCA
(sodium dichloroacetate, a PDH indirect agonist) were utilized in the
treatment of S. vetulus and D. sinensis^[85]20–[86]22. Higher
concentrations (≥10 μM) of the PDH inhibitor CPI-613 resulted in
increased mortality rates at 24 h, 96 h, and 240 h in both species
(Fig. [87]3e and f), and all concentrations of DCA used in the
experiments did not result in the death of daphnids. DCA
dose-dependently promoted PDH activities, and CPI-613 dose-dependently
inhibited PDH activities over the concentration range tested
(Fig. [88]3g and h). Subsequently, locomotor trajectories were
recorded, and locomotor vigor was calculated for 10 μM DCA-treated S.
vetulus for 24 h and 10 μM CPI-613-treated D. sinensis for 24 h. The
results showed that 10 μM DCA significantly enhanced PDH activities in
S. vetulus. 10 μM DCA significantly enhanced the locomotor vigor of S.
vetulus, and 10 μM CPI-613 also promoted the locomotion of D. sinensis
(Fig. [89]3i), and acetyl coenzyme A supplementation improves
indirectly exercise performance and ATP production (Supplementary
Fig. [90]4). The effects of DCA and CPI-613 on PDH activities were
ultimately reflected in the ATP content (Fig. [91]3j and k).
In aquatic animal studies, standard metabolic rate (SMR) is usually
measured using respirometry (measurement of oxygen consumption), and
thus oxygen consumption is used as a reflection of the overall
metabolic rate of daphnids^[92]23,[93]24. The oxygen consumption of D.
sinensis was found to be higher than that of S. vetulus under normal
conditions. The agonistic and inhibitory effects of DCA and CPI-613 on
PDH activities elevated the oxygen consumption of S. vetulus and
decreased the oxygen consumption of D. sinensis, respectively
(Fig. [94]3l). The relationship between metabolic rate and lifespan was
further examined by quantifying the lifespan of S. vetulus and D.
sinensis treated with DCA and CPI-613, respectively. The lifespan of D.
sinensis, which originally exhibited a high metabolic rate, was found
to be significantly extended after its metabolism was reduced by
CPI-613 (Fig. [95]3m). In contrast, S. vetulus, which initially
exhibited a lower metabolic rate, demonstrated an elevated metabolic
rate and a significantly diminished lifespan following DCA treatment
(Fig. [96]3n). Diminished reproductive capacity and decreased heart
rate are considered important phenotypes of senescence in
daphnids^[97]2,[98]25. Statistics on reproductive capacity and heart
rate on days 20 and 40 of both species showed that CPI-613 and DCA
slowed and accelerated senescence in daphnids, respectively
(Supplementary Fig. [99]5). To further validate the role of PDH in
determining lifespan in daphnids, the pdha gene was knocked out in D.
sinensis (Supplemental Video [100]1). Unfortunately, all pdha mutants
died before 30 hours (Supplementary Fig. [101]6). None of the pdha of
the individuals that continued to develop were mutated, and therefore
adult homozygous mutants could not be obtained for lifespan studies.
PDH activity not only appears to affect daphnids motility, a key valve
for energy supply, but is likely to be involved in lifespan regulation
by affecting metabolic rates.
Octopamine enhances motility and shortens lifespan of daphnids by enhancing
PDH activity
Octopamine is responsible for coordinating the transition from a
resting state to a more active state in insects, and plays a
significant role in the induction and maintenance of invertebrate
locomotion^[102]26. Octopamine-deficient Drosophila have reduced
physical activity and lower resting metabolic rate^[103]26,[104]27.
Octopamine levels were assayed in S. vetulus and D. sinensis due to the
important role of octopamine in the regulation of locomotion and
organismal metabolic rates in invertebrates. The enzyme-linked
immunosorbent assay demonstrated that the octopamine levels in S.
vetulus were markedly lower than those observed in D. sinensis
(Fig. [105]4a). To ascertain whether octopamine plays a role in the
regulation of locomotion in daphnids, the locomotor activity and
trajectory in octopamine-treated S. vetulus and D. sinensis were
measured. The experimental results demonstrated that octopamine
treatment could significantly induce locomotion in S. vetulus and D.
sinensis in a dose-dependent manner (Fig. [106]4b). Furthermore, 1 μM
octopamine has been demonstrated to stimulate PDH activity
(Fig. [107]4c), increase ATP content (Fig. [108]4d) and enhance oxygen
consumption (Fig. [109]4e) in both S. vetulus and D. sinensis. Given
the enhancement of PDH activity by octopamine, we observed the lifespan
of S. vetulus and D. sinensis under long-term treatment with 1 μM
octopamine. As anticipated, 1 μM octopamine markedly reduced the
lifespan of S. vetulus and D. sinensis (Fig. [110]4f). To determine
whether octopamine affects locomotor performance and longevity through
PDH rather than other pathways, octopamine and the PDH inhibitor
CPI-613 were co-treated in both species. 1 μM CPI-613 was used to
counteract the enhancing effect of octopamine on PDH activity
(Fig. [111]4g), and octopamine was no longer able to shorten lifespan
in both species (Fig. [112]4h). These results provide further support
for the involvement of octopamine in the regulation of life span by
influencing PDH activity.
Fig. 4. Octopamine could enhance exercise and metabolism, but impairs
longevity.
[113]Fig. 4
[114]Open in a new tab
a The octopamine content of S. vetulus and D. sinensis(N = 3). b The
exercise trajectories of D. sinensis and S. vetulus treated with 1 μM,
10 μM and 100 μM octopamine (N = 3); The locomotor activity of D.
sinensis and S. vetulus was measured in a 10 μM octopamine treatment. c
PDH activity of D. sinensis and S. vetulus treated with 10 μM
octopamine (N = 3). d ATP content of D. sinensis and S. vetulus treated
with 10 μM octopamine (N = 4). e Metabolic rate of D. sinensis and S.
vetulus treated with 10 μM octopamine (N = 5). f The lifespan of 10 μM
octopamine-treated S. vetulus and D. sinensis (n = 30). g The PDH
activity in S. vetulus and D. sinensis co-treated with 10 μM CPI-613
and 10 μM octopamine (N = 3). h The lifespan of 10 μM CPI-613 and 10 μM
octopamine-treated S. vetulus and D. sinensis (n = 30). For (a–e and
g), all data are presented as means ± SEM. Statistical significance was
determined by unpaired two-tailed Student’s t-test (a), two-way ANOVA
with Sidak’s multiple-comparisons test (b, c, d, e and g) or Survival
curve comparison with Gehan-Breslow-Wilcoxon test (f and h). Source
data are provided as a Source Data file.
PDH activity is also involved in the regulation of daphnids lifespan by
temperature
It is evident that temperature is a crucial factor that influences the
lifespan of daphnids^[115]28. Lifespan analysis experiments have
corroborated the correlation between lifespan and temperature in S.
vetulus and D. sinensis. Both S. vetulus (Fig. [116]5a) and D. sinensis
(Fig. [117]5 b) exhibited a significantly shorter lifespan in
high-temperature (35 °C) environments and a prolonged lifespan in
low-temperature (15 °C) environments. Furthermore, Octopamine levels in
S. vetulus and D. sinensis exhibited a positive correlation with
increasing temperature (Fig. [118]5c), which is in line with the trend
of octopamine and temperature effects on lifespan. It is likely that
these potential lifespan regulators act synergistically in organisms,
particularly in their effects on metabolism. The detection of PDH
activity at varying temperatures in both S. vetulus (Fig. [119]5d) and
D. sinensis (Fig. [120]5e) revealed an increase of PDH activity with
rising temperature. The same applies to the ATP content (Fig. [121]5f)
and their locomotion (Fig. [122]5g). Similar to octopamine, the
longevity effect was also no longer significant after the inhibition of
PDH activity by low temperature was rescued by a PDH agonist (10 μM
DCA) (Fig. [123]5 h and i).The results of this study support the idea
that PDH activity plays an important role in the regulation of
locomotor performance and lifespan in S. vetulus and D. sinensis, and
that PDH inhibitors and agonists, temperature, and octopamine all
affect locomotor performance and lifespan in both S. vetulus and D.
sinensis through modulation of PDH activity (Fig. [124]5j).
Fig. 5. Low temperature extends Daphnids lifespan by inhibiting PDH activity.
[125]Fig. 5
[126]Open in a new tab
a The lifespan of S. vetulus at 15 °C, 25 °C and 35 °C (n = 30). b The
lifespan of D. sinensis at 15 °C, 25 °C and 35 °C (n = 30). c The
octopamine content of S. vetulus and D. sinensis at 15 °C, 25 °C and
35 °C (N = 5). d The PDH activity of S. vetulus at 15 °C, 25 °C and
35 °C (N = 3). e The PDH activity of D. sinensis at 15 °C, 25 °C and
35 °C (N = 3). f The ATP content of S. vetulus and D. sinensis at
15 °C, 25 °C and 35 °C (N = 6). g The exercise trajectories and
locomotor activity of S. vetulus and D. sinensis at 15 °C, 25 °C and
35 °C (N = 3). h The PDH activity of CPI-613-treated S. vetulus and D.
sinensis at 15 °C (N = 3). i The lifespan of CPI-613-treated S. vetulus
and D. sinensis at 15 °C (n = 30). j PDH is involved in regulating
motor performance and longevity in Daphnia. For (c–h), all data are
presented as means ± SEM. Statistical significance was determined by
Survival curve comparison with Gehan-Breslow-Wilcoxon test (a, b and
i), two-way ANOVA with Sidak’s multiple-comparisons test (c, f, g and
h) or one-way ANOVA conducted with Tukey’s multiple comparisons test (d
and e). Source data are provided as a Source Data file.
Discussion
The present study found that the lower PDH activity lowered the
locomotor ability and athletic performance of S. vetulus, while
elevated PDH activity was able to confer an enhancement of daphnids
locomotor ability at the cost of a shorter lifespan. Previous studies
on PDH have focused on its role in areas such as glucose-lipid
metabolism, cancer therapy and insulin resistance/diabetes
treatment^[127]29–[128]32. Although the pivotal role of PDH in energy
metabolism is well established, few studies have investigated the
effects of PDH on energy metabolism with the aim of intervening in
lifespan. The present study demonstrates that elevated PDH activity
promotes oxidative phosphorylation (ATP synthesis) and oxygen
consumption, yet it also shortens lifespan and enhances ATP
availability for daphnids motility. This not only implies the possible
discovery of a potential lifespan-modulating target but also suggests
that drugs that target or indirectly modulate PDH in the clinic pose a
pro-aging or short-life risk to patients.
The PDC is present in all organisms except viruses and is a fundamental
component of energy metabolism in both prokaryotes and
eukaryotes^[129]33. A reduction in PDH activity results in a
physiological disorder known as pyruvate dehydrogenase complex
deficiency (PDCD)^[130]34,[131]35. Patients with PDCD exhibit reduced
PDH activity in their cells, and typical symptoms include lactic
acidosis, elevated levels of pyruvate, paroxysmal exercise-induced
dyskinesia, and metabolic ataxia^[132]34. The elevated lactate and
pyruvate levels observed in S. vetulus in this study are consistent
with the typical symptoms of PDCD as described in the literature. The
results of subsequent enzyme activity assay experiments confirmed the
lower PDH activity of S. vetulus compared to D. sinensis. In human
patients, PDCD is frequently attributed to mutations in the components
that comprise the PDC^[133]36. PDH is the rate-limiting enzyme of the
pyruvate dehydrogenase complex (PDHC), a tetramer composed of two PDH α
subunits and two PDH β subunits. One of the most prevalent mutation
types is mutations in the PDHα subunit, which are predominantly
missense and code-shift mutations^[134]37. These mutations affect the
binding of E1α to thiamin pyrophosphate cofactors, the formation of
heterotetramers, and the ability to correctly target and translocate
into mitochondria^[135]34. Thanks to the rapid development of
artificial intelligence in structural biology, AlphaFold 3 was
developed and proved to be accurate in predicting protein-small
molecule interactions^[136]38. We predicted the interaction between PDH
proteins and thiamine pyrophosphate in both species. The lower docking
scores for the PDH protein-thiamine pyrophosphate interactions of S.
vetulus support that changes in the amino acid sequence and protein
structure of the S. vetulus PDH α-subunit may be the cause of the
reduced activity of S. vetulus PDH. Thus, when S. vetulus was forced to
exercise, the lower PDH activity resulted in a large accumulation of
lactate and a significant deficit in ATP supply. However, further
experimental validation is needed to confirm these predictions and
fully understand the functional consequences. S. vetulus might be
considered as a PDCD taxon in the daphnids. However, in contrast to the
high mortality rate observed among human patients^[137]34, S. vetulus
is able to survive in nature as an intact population. This is likely to
be related to the complex genesis of PDCD and the unique lifestyle of
S. vetulus. Changes in PDH activity in human patients with PDCD caused
by E1 mutations tend to be variably significant, ranging from
approximately 10% to 60% of those in healthy individuals^[138]34. S.
vetulus has only 30% to 40% of the PDH activity of D. sinensis, but is
still partially functional. Apparently, the lower PDH activity is not
sufficient to allow S. vetulus to exhibit the same level of
free-swimming behavior as D. sinensis. However, the persistence of
residual PDH activity ensures that glycolytic and oxidative
phosphorylation processes are still functioning at low levels in
resting S. vetulus to sustain life^[139]36.
To date, there has been substantial experimental evidence to suggest
that energy metabolism has a positive regulatory effect on
lifespan^[140]39–[141]42. Nevertheless, the molecular mechanisms by
which energy metabolism regulates lifespan remain poorly understood.
For a considerable period, the prevailing view has been that higher
energy metabolism rates are associated with shorter lifespans, and vice
versa. The findings of the present study indicate that an increasing in
the metabolic rate of daphnids will result in a significantly reduction
in lifespan. It is evident that this pattern is not exclusive to model
organisms employed in longevity studies. A recent sex-specific
Mendelian randomization survey in human subjects further supports this
view and suggests that higher basal metabolic rates may shorten
lifespan^[142]39. Furthermore, our findings indicate that the metabolic
rate of daphnids is significantly influenced by glucose metabolic
processes. A glucose-rich diet has been demonstrated to reduce lifespan
in C. elegans by influencing the activity of longevity-promoting
proteins, suggesting that glucose toxicity may be a contributing
factor. This is often associated with increased mitochondrial
respiration and oxidative stress^[143]43. The free radical theory of
ageing postulates that reactive oxygen species (ROS) are responsible
for the oxidative damage that occurs in organisms, which in turn leads
to the process of ageing. Mitochondria are the main ROS producers in
organisms, with the respiratory chain serving as the site of electron
transfer, resulting in the generation of superoxide anion^[144]44. In
their study, Lionaki et al. demonstrated that the effects of a
shortened lifespan resulting from diets high in glucose could be
mitigated by inhibiting mitochondrial protein input^[145]15. Inhibition
of mitochondrial protein input result in mitochondrial dysfunction,
which in turn limits the capacity for mitochondrial oxidative
phosphorylation, thereby prolonging the lifespan of C. elegans^[146]15.
In the present study, we found that inhibition of PDH activity
similarly also extends lifespan. Inhibition of PDH activity would be
directly reflected in the downstream mitochondrial respiratory process,
with reduced oxygen consumption and ATP production in daphnids. And the
life-extending effect of mild inhibition of mitochondrial respiration
has been demonstrated in a variety of organisms, including yeast,
worms, flies, and mice^[147]16. Overall, this study provides a
potential target for lifespan regulation, where PDH activity will
influence lifespan through its effects on metabolic rate and downstream
mitochondrial respiration.
In addition to the lifespan-regulating role of PDH activity, this study
identified several modulators of PDH activity, including octopamine and
temperature. Octopamine is a tyrosine-derived biogenic amine that has
been reported to be involved in the regulation of energy homeostasis
and the initiation of locomotion in invertebrates^[148]26. In
Drosophila, octopamine regulates flight behavior (the most
energy-intensive form of locomotion in insects) at several levels, in
particular playing an important role in the initiation and duration of
flight^[149]45. In this study, we found that octopamine levels were
more than four times higher in D. sinensis than in S. vetulus.
Exogenous octopamine was able to enhance locomotion in S. vetulus, and
this enhancement was based on the mobilization of total metabolic
levels in S. vetulus. Octopamine increased PDH activity, metabolic rate
(oxygen consumption rate) and ATP production in S. vetulus. The
enhancement of S. vetulus exercise performance by octopamine was based
not only on increased willingness to exercise, but also on increased
energy availability. In addition to enhancing glucose metabolism,
octopamine is also thought to promote fat utilization, with both
fasting and starvation capable of inducing octopamine
release^[150]46,[151]47. Octopamine-deficient Drosophila melanogaster
exhibit an obese phenotype, and studies in grasshoppers and crickets
have demonstrated the direct activation of fatty acid oxidation and
carbohydrate release by octopamine^[152]26. Long-term treatment with
octopamine also significantly shortened the lifespan of daphnids, which
may be attributed to its PDH activating effects and acceleration of
energy metabolism rates.
Temperature, another factor affecting locomotor performance and
lifespan in S. vetulus, was also found to correlate with octopamine
release and PDH activity. The traditional view of the effects of
temperature on enzyme activity, metabolism and lifespan of organisms is
based on purely thermodynamic effects, but recent studies have
increasingly emphasized the important role of genetic regulation in
that^[153]48,[154]49. In the present study, we found that the effects
of temperature on PDH activity and lifespan are not based on purely
thermodynamic effects, and that octopamine is significantly increased
in synthesis and release by temperature, and that this effect is
realized in both S. vetulus and D. sinensis. And the inhibition of low
temperature on PDH activity in both species was resecured by DCA
treatment, and the extension of their lifespan by low temperature was
counteracted. These results support a critical role for PDH activity in
temperature-to-lifetime modulation, rather than a simple thermodynamic
effect. Octopamine is a neuroactive substance that is the invertebrate
equivalent of adrenergic signaling compounds^[155]27. One possible
regulatory pathway is that stimulation of cladocerans by the external
environment first elicits a response in octopamine levels, which
mobilizes the body’s glycolipid metabolism and provides energy security
for the organism. Indeed, our results are biased in favor of octopamine
as a stress-responsive compound in dendrobatids.
The present study demonstrates that proper inhibition (not deletion) of
PDH in S. vetulus and D. sinensis reduces metabolism (level of oxygen
consumption) and contributes to longevity. As an additional consequence
of PDH inhibition, locomotor performance and exercise performance
subsequently deteriorated. However, there is a consensus in the human
world is that life is all about exercise. The longevity benefits of
exercise have been widely reported. Among other mechanisms mediating
protective effects of exercise, periodic exercise, while increasing
heart rate during exercise, appears to reduce resting heart
rate^[156]50, which in some respects may be a proxy for metabolic rate
and which also inversely correlates with lifespan in terrestrial
mammals. Although metabolic rate (e.g., as indicated by heart rate)
increases during exercise, the net effect of exercise is to reduce
overall heart rate (after cessation of exercise) and thus, plausibly,
total net metabolism. Athletes tend to have lower resting heart rates
and lower resting metabolic rates as well as longer life expectancies
compared to the general population. And a statistically significant
association between increased resting heart rate and mortality risk has
been reported^[157]51. Among daphnids, more physically active D.
sinensis had shorter lifespans, which seems to contradict the common
understanding that exercise is beneficial for lowering total net
metabolism and longevity. But the effects of exercise on metabolic rate
must be considered separately as the immediate effects (metabolic rate
tends to increase during exercise) and the long-term effects (total net
metabolism decreases due to the exercise of the body’s aerobic
respiratory capacity). In fact, it should not be overlooked that as a
zooplankton, D. sinensis, swims throughout its life, which is very
different from the periodic exercise performed by athletes. Exercise in
D. sinensis corresponds to a non-stop immediate effect resulting in a
persistent high metabolic rate, and therefore D. sinensis as an
“exercise enthusiast” does not enjoy the life-extending benefits of
exercise.
Methods
Animal culture and sample collection
The clones of S. vetulus (LN2017 strain) and D. sinensis (WSL strain)
were originally isolated from the South Lake (Wuhan, Hubei, China), and
the clone lines were initiated from a single parthenogenetic female and
cultured for years in the laboratory. The daphnids (Daphnia sinensis
and Simocephalus vetulus) used in this study were subjected to multiple
passages (> three generations) under standard laboratory conditions to
ensure that maternal effects were excluded. Daphnids are cultured in a
1 L conical flask in ADaM (Aachener Daphnien Medium) at 25 ± 1°C, a
16 h photoperiod and pH between 7-7.5^[158]5. The medium was changed
weekly to prevent poor medium quality, and the animals were fed once
daily with Chlorella pyrenoidosa at a final concentration of 3 × 10^5
algal cells/ml^[159]52 (Supplemental Video [160]2 and [161]3). With the
exception of the life history observation experiment and the longevity
experiment, all experimental daphnids in this study were 10-day-old
(adults) parthenogenetic individuals without eggs to ensure that the
daphnids were at the same developmental stage in each experiment. All
samples were rinsed 3-4 times with sterile ddH[2]O to remove
contaminating bacteria and daphnids samples for genome sequencing and
the LC-MS/MS experiment are placed in clean media a day in advance to
empty the bacteria and algae in the gut. All samples were snap frozen
in liquid nitrogen and then stored at -80 °C for using.
Medicines and reagents
Sodium dichloroacetate (DCA) (purity > 98%) and octopamine
hydrochloride (OA) (purity ≥ 98%) were purchased from Shanghai Yuanye
Bio-Technology Co. (Shanghai, China). 6,8-Bis(benzylthio)octanoic acid
(CPI-613) (purity > 98%) was purchased from Tokyo Chemical Industry
Co., Ltd. All drugs were stored at −20°C, protected from light, and
dissolved in dimethyl sulfoxide (Nanjing Jiancheng Bioengineering
Institute, China) before use.
Genome sequencing, assembly, and annotation of the S. vetulus
Genomic DNA was extracted using the Qiagen kit (Qiagen, Germany)
according to the manufacturer’s instructions. Genomic DNA purity
(OD260/280 and OD260/230), concentration and integrity were determined
using Nanodrop 2000 (Thermo Fisher Scientific, USA), Qubit 3.0
(Invitrogen, USA) and 1% agarose gel electrophoresis (voltage: 100 V,
electrophoresis time: 40 min). Sequencing libraries for Illumina
NovaSeq and single-molecule real-time sequencing (SMRT) on the PacBio
Sequel platform were constructed from qualified DNA samples. Library
quality of the library was assessed using Qubit 3.0 and Agilent 2100
(Agilent, USA) to ensure that sequencing requirements were met.
Hi-C sequencing library construction. After formaldehyde crosslinking
and fixation of live S. vetulus isolates, the crosslinking products
were enzymatically cleaved using restriction endonucleases to produce
sticky ends. Biotin-labelled nucleotides were then added for end repair
to form flat ends, and ligase was used for the flat-end joining
reaction to form a molecular loop. Hi-C samples were then prepared by
DNA purification and extraction. After passing the test, the
end-labelled biotin was removed, cleaved by ultrasound, end repaired,
base A added, and sequencing junctions added to form the junction
product; then the PCR conditions were screened and amplified to obtain
the library product. After passing quality control, the constructed
library was sequenced on the Illumina HiSeq platform to provide raw
data for subsequent analysis.
The Pacbio sequencing data were corrected, trimmed and assembled using
Canu software to assemble the reads into contigs and generate consensus
sequences. The raw data were compared to the assembled genome using
blasr software, and then the completed bam files were compared using
samtools to sort by position and indexed using pbindex. The assembled
fasta files were also indexed using samtools, and then the next step of
error correction could be performed using arrow. In the
three-generation polish with arrow, the number of threads will be set
as large as possible to speed up the run, minCoverage is set to 15, the
result file of the blasr comparison and the assembled genome are input,
and error corrected genome is output. The results obtained will then be
genome survey of the second generation of data, through the BWA
software will be reads compared to the three generations of Polish
after the genome, using pilon software for error correction. The
genomes were de-redundant after preliminary assembly and error
correction using the software purge haplotigs.
Hi-C-assisted assembly: After the downstream Hi-C raw downstream data
were filtered and de-redundant, the resulting Hi-C reads of S. vetulus
were compared to the whole genome outline by BWA and then processed by
samtools to obtain high-quality comparison data. The filtered
comparison results were analyzed using Lachesis for clustering, sorting
and orientation of super-scaffolds, and finally, genomic overlapping
regions were identified based on sequence homology and the highly
phasic nature of distant interaction patterns, and scaffolds were
merged based on the overlapping regions and anchored to the chromosomes
to obtain the final chromosome-level scaffolds. The final
chromosome-level scaffolds were obtained using juicer software for
evaluation and correction of the assembly results and visualization in
the form of heat maps. Genomic circle maps were generated using the
Circos software and used to present the assessment results as a whole.
The repetitive sequences of the genome were masked, and then genomic
functional annotation and non-coding RNA annotation were performed. The
genome with blocked repetitive sequences was used for genomic
functional annotation, and all gene sequences were compared with
InterPro, GO, KEGG, Swissprot, TrEMBL, TF, Pfam, NR, and KOG protein
databases using BLASTX software with the parameter setting E-value of
BLASTX software: 1e-5.
Comparative genomics analysis
Fourteen species for which genomic data were available were downloaded
from the NCBI database: Acyrthosiphon pisum, Aphis gossypii, Myzus
persicae, Sipha flava, Cinara cedri, Armadillidium nasatum, Eumeta
japonica, Frankliniella occidentalis, Penaeus vannamei, and Tigriopus
californicus, coding sequence (CDS) sequences of D. sinensis, D. magna,
D. pulex, and Actinia tenebrosa. Blast comparison (e-value ≤ 1e-5) of
the CDS of 15 species was first performed, and then gene family
clustering analysis was performed using OrthoMCL (v.2.0.9) software.
Based on the results of the OrthoMCL run, single-copy homologous genes
were selected from all the homologous genes to construct the protein
sequences of the supergenes, and the sequences of the 15 species were
compared using MAFFT (v.7.294). The conserved sites in the multiple
sequence alignment results were then extracted using Gblocks software
(v.0.91b) software according to the default parameters. A. tenebrosa
was used as an outgroup to construct a maximum likelihood phylogenetic
tree using IQ-TREE (v.1.6.9) with a bootstrap value of 1000. Species
divergence time were estimated using MCMCTREE software, and the
divergence time pairs were obtained from the TimeTree website
([162]http://www.timetree.org) and the related literature. The
divergence times in the literature were used to correct the obtained
time correction points. The amplification and contraction analysis of
S. vetulus gene family was then performed using CAFE software
([163]http://sourceforge.net/projects/cafehahnlab/). The proteins of S.
vetulus and D. sinensis were compared using Blastp (v.2.6.0), and then
based on the comparison results, covariance analysis was performed
using MCScanX ([164]http://chibba.pgml.uga.edu/mcscan2/) to plot the
chromosome covariance scatterplots and circle plots of S. vetulus and
D. sinensis.
Life history observation experiment
A healthy parthenogenetic individual (F[0]) with eggs was selected and
its offspring were used to study the life history^[165]5. After being
released from the parthenogenetic mother (F[0]), more than 10 newborns
(F[1]) were cultured separately and observed until death. During the
experiment, S. vetulus were fed with S. obliquus every day, the medium
was changed every three days, F[2] were removed every day, and the body
length and reproduction of the newborns (F[1]) were measured and
recorded every single day. We then calculated the life span (in days),
body length at sexual maturity, age at sexual maturity, time gap
between each reproduction (the inter-clutch period), the number of
offspring per clutch (brood size) and total number of offspring for
each individual (total reproductive capacity). Maturity was considered
as the moment at which eggs can be observed in the female’s brood pouch
for the first time^[166]53. The rearing conditions were as previous
description.
Measurement of several physiological and behavioral parameters
Hopping frequency
Swimming by hopping is a characteristic movement of daphnids, caused by
rhythmic hopping of the second antennae. The hopping frequency is the
total number of hops in a given time period^[167]54.
Swimming trajectory and locomotor activity
The swimming trajectory and locomotor activity of daphnids were
measured using the method described by Bownik. The swimming behavior of
S. vetulus and D. sinensis in 6-well plates was videotaped for 1 min
using a fixed digital video camera. The swimming tracks were analyzed
on a frame-by-frame basis using the Tracker® 6.0, and the locomotor
activity is expressed as the distance swum per unit of time
(mm/min)^[168]55.
Duration of quiescence
the duration of quiescence was used in the study to characterize the
specific swimming habits of S. vetulus. This period of time when no
movement is observed is referred to as quiescence^[169]54. The
movements of the daphnids were recorded to count the time of immobility
and expressed as the ratio of the time of immobility to the total
duration.
Oxygen consumption
The respiration rate of daphnids was calculated by measuring dissolved
oxygen concentrations assay in the culture medium. Dissolved oxygen
measurements were performed using Hach Portable Water Quality
Instruments. The initial dissolved oxygen concentration in the medium
and the dissolved oxygen concentration after 6 h of sealing were
measured and the oxygen consumption rate was calculated (mg O[2]
ind.^−1 h^−1).
Feeding rate
Algae were sampled from each well of a six-well plate at 4 and 10 h
after feeding^[170]55. Algal densities were determined directly using a
light microscope (Nikon BM2000) at × 400 magnification. The feeding
rate (
[MATH: I :MATH]
, cells animal individuals^−1 h^−1, i.e., clearance rate) was
calculated as the difference in algal densities between the beginning
and the end of the experimental wells according to the following
equations, which are commonly used in plankton:
[MATH: FR=VIn(C0−ΔCb)−lnC1−ΔCbNt :MATH]
[MATH: I=FRC0+C12 :MATH]
Where
[MATH: FR :MATH]
is the filter rate (mL/ind./h);
[MATH: V :MATH]
is the volume of the culture (mL);
[MATH: C0
:MATH]
and
[MATH: C1
:MATH]
are the algal density at the beginning and the end of the experimental
wells (cells/mL), respectively;
[MATH: ΔCb :MATH]
is the difference in algal density in the blank wells (no grazers) at
6 hours;
[MATH: N :MATH]
is the number of daphnids and
[MATH: t :MATH]
is the duration of the experiment (h);
[MATH: I :MATH]
is the feeding rate (cells/ind./h).
Heart rate and thoracic limb activity
The number of heartbeats per minute is defined as the heart rate of D.
sinensis. The number of oscillations of thoracic limb per minute is
defined as thoracic limb activity of daphnids. Each water flea
individual was recorded for at least 2 min using a digital microscope
(DMS-2020A, MPI) with video recording and imaging capabilities. Video
editing software (Adobe premiere pro 2020) was used to slow down the
video by a factor of 0.2 to facilitate the counting of thoracic limb
activity and the heart rate.
LC-MS/MS data acquisition and analysis
Sample collection: S. vetulus and D. sinensis of the same age (10 days)
and feeding environment were selected for metabolomics analysis. Every
200 daphnids were considered as one sample, and three technical
replicates were set up for S. vetulus and D. sinensis, respectively,
for a total of six samples. Quality control was performed in this
experiment, and the researchers also prepared QC samples, which were
aliquots of all the samples mixed together. QC samples were used to
equilibrate the chromatography-mass spectrometry (CS-MS) system and to
determine the status of the instrument, and were used for the
evaluation of the system’s stability throughout the experiment.
Sample pre-treatment: Metabolites were extracted from homogenized
samples with 1 ml 70% methanol. Samples were first vortexed for 5 min
and placed on ice for 15 min. The samples were then centrifuged at
13523 × g for 10 min at 4 °C and 400 μL of the supernatant was stored
overnight at -20°C. Finally, the samples were centrifuged at 13523 × g
for 3 min at 4 °C and 200 μL of supernatant was used for metabolite
detection.
The instrumentation for data acquisition consisted mainly of
ultra-performance liquid chromatography (UPLC) (ExionLC AD, AB Sciex,
Framingham, MA, USA) and tandem mass spectrometry (MS/MS, QTRAP5500, AB
Sciex, Framingham, MA, USA).
Mainly liquid phase conditions: (1) Column: Waters ACQUITY UPLC HSS T3
C18 1.8 µm, 2.1 mm*100 mm; (2) mobile phase: ultrapure water (0.1%
formic acid) in phase A, and acetonitrile (0.1% formic acid) in phase
B. The mobile phase was 0.1% formic acid (0.1% formic acid); (3)
elution gradient: 0 min water/acetonitrile (95:5 V/V), 10.0 min
10:90 V/V, 11.0 min 10:90 V/V, 11.1 min 95:5 V/V, 14.0 min 95:5 V/V.
The elution rate was 0.4 ml/kg; (4) flow rate of 0.4 ml/min; column
temperature of 40 °C; injection volume of 2 μL.
The mass spectrometry conditions mainly included: Electrospray
ionization (ESI) temperature 500 °C, mass spectrometry voltage 5500 V
(positive), -4500 V (negative), ionization gas I (GS I) 55 psi, gas II
(GS II) 60 psi, curtain gas (CUR) 25 psi, and the collision-activated
dissociation (CAD) parameter was set to high. In a triple quadrupole
(Qtrap), each ion pair was detected by scanning based on optimized
declustering potential (DP) and collision energy (CE)^[171]56.
Data processing and quantification: Chromatographic peak areas and
retention times were extracted using MultiQuant 3.0.2. Retention times
were corrected using energy metabolite standards for metabolite
identification^[172]57. Ion peak areas of metabolites were normalized
by internal standards L-Glutamate-d5 and Succinate-d6. Standard curves
were plotted based on the intensity values of the standards. The
up-sampling concentrations of metabolites were calculated using the
standard curve. The concentration of metabolites in the sample was
calculated by substitution according to the pretreatment treatment
(Supplementary Data [173]1).
Amino acid sequence comparison and prediction of protein-ligand interactions
Multiple Sequence Alignment in DNAMAN (version 9.0) was used for amino
acid sequence alignment. Access the AlphaFold Server via a web browser,
upload sequence data including the respective PDH protein sequences of
D. sinensis and S. vetulus and their ligand TPP, and select the
corresponding sequence input format^[174]38. After completing the data
upload, the prediction results were viewed, and the 3D protein
structures were visualized using PyMOL (Version 3.1.3.). Based on the
results provided by AF3, the interactions between the respective PDH
proteins of D. sinensis and S. vetulus and TPP were analyzed, including
the predicted protein structures with docking scores (Fig. [175]1b).
Here, we assessed the quality of the predicted protein-ligand
interactions using the ranking score, which is a composite metric
including pLDDT, PAE, fraction disordered, and has_clash values
calculated as 0.8 × ipTM + 0.2 × pTM + 0.5 × disorder − 100 ×
has_clash.
Pyruvate dehydrogenase (PDH) activity assay
The PDH Activity Assay Kit (Beijing Solarbio Science & Technology Co.,
Ltd) was used to quantify PDH activity in daphnids. Three replicates of
100 daphnids per treatment were collected 24 hours after treatment,
washed three times in ultrapure water, and frozen at −80 °C. Daphnids
were homogenized in an ice bath according to the manufacturer’s
instructions, and the supernatant was centrifuged at 11,000 g for
10 min at 4 °C. 10 μL of supernatant and 180 μL of the working solution
were added to the assay plate, and the OD value at 605 nm was
determined. The same supernatant used to measure the total protein
concentration using a BCA protein assay kit (Jiancheng, Nanjing,
China). ATP levels were normalized to the protein content.
Forced exercise experiments
Based on observations of the perching and locomotion habits of S.
vetulus, there are two main conditions under which S. vetulus remains
stationary: i. climbing on the surface of aquatic grasses or other
substrates in water and, under laboratory conditions, on the walls of
culture vessels. Second, use the tension of the water surface when
suspended in water. The forced exercise experiment was designed
according to these two points. A clean and smooth glass petri dish was
chosen as the experimental vessel so that S. vetulus could not climb to
the surface of the vessel. The petri dishes containing S. vetulus and
D. sinensis were placed on a shaker and shaken slowly at a frequency of
40 Hz at a constant speed to disturb the water column so that the aged
low-fronted grebes could not use the water surface tension to stay
still on the water surface. The movements of S. vetulus and D. sinensis
were then observed and recorded hourly by counting the number of
daphnids individuals that remained suspended and swam and the number of
daphnids sinking at each time point. Samples of S. vetulus and D.
sinensis were collected after 6 hours of forced exercise, and ATP
content and lactate level were examined in S. vetulus and D. sinensis
before and after exercise.
Pdha Knockout by CRISPR/Cas9
The pdhα knockout lines of D. sinensis were generated using
CRISPR/Cas9^[176]58. The cDNA and DNA sequences of pdhα were identified
from the D. sinensis genome in our laboratory, with clear delineation
of exon and intron regions. Utilizing the CRISPRscan online platform,
target sequences within the pdhα DNA sequence were identified. The
OligoEvaluator online tool was then employed to assess the quality of
these target sequences. The local BLAST component of NCBI (National
Center for Biotechnology Information) was employed to confirm that the
target sequences correspond to a unique locus within the D. sinensis
genome, thereby finalizing the target sequences for subsequent use. The
gRNA for the pdhα knockout lines of D. sinensis was synthesized using
the GeneArt™ Precision gRNA Synthesis Kit (Invitrogen, USA), following
the manufacturer’s protocol. The obtained gRNA product, after being
quantified for concentration, was diluted to a working solution of
1000 ng/μL and aliquoted for storage at -80°C. More details of the
experiment are in the Supplementary Material.
Before the injection procedure, the components (Table [177]1),
including TrueCut^TM Cas9 Protein v2 at a concentration of 5000 ng/μL
(Thermo Fisher Scientific Inc., USA), were mixed and then introduced
into capillary glass needles prepared using the NARISHIGE PC−100
programmable vertical micromanipulator (Micrology (Wuhan) Precision
Insuments, Ltd.). Using the two-stage pull method (NO. 1/NO. 2 heater
level: 80°C/75°C; weight: 2 type light and 0 type heavy), one glass
capillary (G−1) is pulled on the Micropipette Puller (PC−100) for
capillary pulling operation, carefully remove and mirror check the
pulled capillary (Supplemental Video [178]4). Then fix the capillary at
an inclination of 30 in the groove of the Micropipette Grinder
(KDG-01), rotate the knob so that capillary slowly down. Start the
timer when the tip of the capillary coincides with its shadow, after
about 10 s carefully remove and mirror check, if the capillary fails to
open successfully, repeat the above operation until you get the
capillary tip as seen in the video. The above instruments were
purchased from Micrology (Wuhan) Precision Induments, Ltd.
Microinjection was performed using Digital Pneumatic Microinjection
Pump (DMP-300, Micrology (Wuhan) Precision Insuments, Ltd.).
Table 1.
Reagent Ratios
Reagent Volume
pdhα gRNA (1000 ng/μL) 1 μL
TrueCut^TM Cas 9 Protein (5000 ng/μL) 1 μL
Phenol Red 0.5 μL
DNase/RNase-Free Water 2.5 μL
Total 5 μL
[179]Open in a new tab
After the embryos had developed for 30 hours post-injection, genomic
DNA from the injected embryos is extracted. Subsequently, the target
gene DNA segment, which includes the target site, was amplified using
PCR. Agarose gel electrophoresis was then employed to detect any
mutations in the targeted region. Table [180]2 delineates the pdhα gRNA
targets and their corresponding primers employed in this research.
Table 2.
The pdhα gRNA targets and their corresponding primers
For genotypin /qPCR analysis Forward primer (5′ - 3′) Reverse primer
(5′ - 3′)
pdhα-48 TAATACGACTCACTATAGGCTGCTTTGAAGGAAC
TTCTAGCTCTAAAACATCGGTTCCTTCAAAGCAG
pdhα-64 TAATACGACTCACTATAGAGCGGCCGGTAATTTA
TTCTAGCTCTAAAACTGTATAAATTACCGGCCGC
pdhα-76 TAATACGACTCACTATAGGAACTTGTCCCGAATA
TTCTAGCTCTAAAACCCTTTATTCGGGACAAGTT
pdhα-T48-10 GCCCAACAACCTTTGGGTG CGTAGAGCTGGCCTTGATTGG
pdhα-T64-2 CAGCCCTTCAAACTGCACAA AATCTAACGAACTTGTCCCGA
pdhα-T76-4 TCATCGAGGCTCTCATACCGA GAAGGATGGAAACAGCGGC
[181]Open in a new tab
Detection of ATP content and lactate levels
All experimental daphnids for the measurement of ATP content and
lactate level were 10-day-old parthenogenetic individuals (adult
individuals) without eggs. The ATP assay kit from Beyotime
Biotechnology was used to measure ATP levels. Daphnids were treated
with different drug concentrations or DMSO (control) in triplicate for
24 hours. Following the manufacturer’s instructions, the samples were
homogenized and centrifuged at 4°C for 5 min at 12,000 g. The
supernatant was mixed with the assay working solution, and luminescence
was measured using a SpectraMax i3x Multi-Mode Microplate Reader
(Molecular Devices). ATP levels were normalized to protein levels.
Lactate assay kits (Nanjing Jiancheng Bioengineering Institute, China.)
and β-hydroxybutyrate and acetylacetone assay kits (Beijing Solarbio
Science & Technology Co., Ltd.) were used to determine lactate and
β-hydroxybutyrate and acetylacetone levels. The samples were
homogenized and centrifuged according to the manufacturer’s
instructions to obtain the supernatant. The supernatants were mixed
with the appropriate working reagents. The absorbance of lactate was
measured at 530 nm and that of β-hydroxybutyrate and acetylacetone were
measured at 340 nm.
Assay of octopamine levels
Octopamine levels were quantified using a one-step double antibody
sandwich enzyme-linked immunosorbent assay (Octopamine ELISA Assay Kit,
Abmart (Shanghai) Co., Ltd.) in daphnids. Octopamine antibody-coated
wells were sequentially coated with sample, standard, and HRP-labeled
detection antibodies. The wells were then washed thoroughly and
incubated at a constant temperature. The color was generated using TMB
as the substrate, which was catalyzed to blue by peroxidase and later
converted to the final yellow color by the addition of acid. Absorbance
at 450 nm was measured using a SpectraMax i3x Multi-Mode Microplate
Reader (Molecular Devices). In addition, the protein concentration in
the samples was simultaneously determined using the BCA Protein Assay
Kit (Jiancheng, Nanjing, China), and octopamine levels were normalized
to protein levels.
Lifespan Analysis
Healthy female daphnids with eggs from the same batch were carefully
selected and newly hatched juveniles were collected for lifespan
analysis. The juveniles were reared under standardized conditions in a
sterile and constant temperature incubator (GZX-400BSH-III, CIMO,
Shanghai, China) with appropriate lighting, and survival rates were
recorded daily. At 3 days of age, the drug was added to the sterilized
medium. An equivalent concentration of DMSO, the drug solvent, was
added to the culture medium of pups in the control group. To ensure the
effective concentration of the drug, the culture media were changed
every other day.
Statistical Analysis
Data were analyzed and visualized using GraphPad Prism 8.02. Results
are expressed as the mean ± SEM. The Shapiro-Wilk test was used to test
for normal distribution. Unpaired two-tailed Student’s t-test, one-way
ANOVA and Tukey’s multiple range test, two-way ANOVA with Sidak’s
multiple-comparisons, two-way ANOVA with Dunnett’s multiple-comparisons
test and Survival curve comparison with Gehan-Breslow-Wilcoxon test
were used to assess the significance of differences between groups.
Reporting summary
Further information on research design is available in the [182]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[183]Supplementary information^ (2.6MB, pdf)
[184]Peer review file^ (2.5MB, pdf)
[185]41467_2025_58666_MOESM3_ESM.pdf^ (491.9KB, pdf)
Description of Additional Supplementary Files
[186]Supplementary Data 1^ (15KB, xlsx)
[187]Supplementary Movie 1^ (13.7MB, mov)
[188]Supplementary Movie 2^ (14.9MB, mov)
[189]Supplementary Movie 3^ (14MB, mov)
[190]Supplementary Movie 4^ (10.9MB, mov)
[191]Reporting Summary^ (93KB, pdf)
Source data
[192]Source Data^ (111.4KB, xlsx)
Acknowledgements