The Glacier Ice Worm, Mesenchytraeus solifugus, Elevates Mitochondrial Inorganic Polyphosphate (PolyP) Levels in Response to Stress

Simple Summary Energy maintenance in living organisms is crucial for survival. The ice-obligate worm, Mesenchytraeus solifugus, displays an unusual bioenergetic pattern, namely that intracellular ATP levels increase with declining temperature. In this study, we address the effects of stress on mitochondrial inorganic polyphosphate (polyP) and its relationship with ATP. Mitochondrial inorganic polyphosphate is a ubiquitous polymer whose role in the maintenance of prokaryotic and mammalian bioenergetics has been broadly demonstrated. We show here that polyP levels in ice worms increase with thermal stress, in contrast with those observed in other annelid worms. Thus, polyP may function as an energetic buffer in ice worms, effectively storing phosphate groups under stress and replenishing ATP under normal physiological conditions. Abstract The inorganic polymer, polyphosphate (polyP), is present in all organisms examined to date with putative functions ranging from the maintenance of bioenergetics to stress resilience and protein homeostasis. Bioenergetics in the glacier-obligate, segmented worm, Mesenchytraeus solifugus, is characterized by a paradoxical increase in intracellular ATP levels as temperatures decline. We show here that steady-state, mitochondrial polyP levels vary among species of Annelida, but were elevated only in M. solifugus in response to thermal stress. In contrast, polyP levels decreased with temperature in the mesophilic worm, Enchytraeus crypticus. These results identify fundamentally different bioenergetic strategies between closely related annelid worms, and suggest that I worm mitochondria maintain ATP and polyP in a dynamic equilibrium.


Introduction
Primary energy generation pathways are well conserved across domains of life, and bioenergetic regulation is fundamental to the survival of all organisms [1]. Inorganic polyphosphate (polyP)-a negatively charged polymer associated with bioenergeticscomprises monomers of orthophosphate linked by high-energy phosphoanhydride bonds, which are isoenergetic to those present in ATP [2]. A rapid inter-conversion of phosphoryl groups between ATP and polyP has been observed in yeast [3]. Accordingly, the regulatory role of polyP in cellular bioenergetics has been broadly demonstrated [4][5][6][7][8][9][10][11][12][13][14][15][16][17] For example, polyP facilitates the enzymatic synthesis of ADP in Escherichia coli [13] and mediates primary metabolism in yeast [11]. In mammalian cells, we and others have demonstrated the functional role of mitochondrial polyP in (i) the regulation of mitochondrial free calcium crucial for ATP production [18], (ii) preserving the equilibrium between oxidative phosphorylation (OXPHOS) and glycolysis [15,16], and (iii) maintenance of the appropriate oxidative status of cells [17,19].
Mitochondrial inorganic polyphosphate is present in all studied organisms, from prokaryotes to mammals [20], showing a mostly ubiquitous cellular distribution. In eukaryotes, polyP can be found in various subcellular locations including the cytoplasm, nucleus and mitochondria, and can be associated with membrane proteins as well as the extracellular space [8]. In mammalian cells, a co-localization between polyP and mitochondria has been shown [5,12]. Moreover, polyP levels are highly dynamic and closely coupled with the metabolic state of mitochondria [15][16][17]21]. Recent evidence suggests that the mitochondrial F 0 F 1 ATP synthase is linked with the metabolism of mammalian polyP [22].
Glacier ice worms, Mesenchytraeus solifugus, display an atypical bioenergetic property, namely that ATP levels paradoxically increase with declining temperatures [23]. This has been interpreted as a compensatory mechanism to offset reductions in molecular motion and enzyme kinetics at cold physiological temperatures [23,24]. Ice worms complete their life cycle in hydrated, maritime glacier ice found throughout the Pacific northwestern region of North America [25,26], and thus are challenged with permanently cold temperatures hovering near 0 • C. To explore the putative role of polyP in the maintenance of mitochondrial bioenergetics in these worms, we tested their response to thermal stress and hypoxia in comparisons with mesophilic, aquatic (Helobdella austinensis and Lumbriculus variegatus) and terrestrial (Eiseniella andrei and Enchytraeus crypticus) worms. Our data show that steady-state mitochondrial polyP levels vary significantly between worms occurring at different habitats, and that M. solifugus uniquely elevates polyP as a function of increasing temperature.

Specimens
Glacier ice worms, Mesenchytraeus solifugus, were collected from surface snow above the equilibrium line altitude on The South Sister Glacier (OR, USA). Specimens were stored in thermally insulated containers with field snow/ice during transport to Rutgers University, upon which they were transferred into glass bowls and maintained at 4 • C. No additional water or supplements were added to ice worm cultures. Specimens of Lumbriculus variegatus were purchased from local pet stores and maintained in 0.03% Instant Ocean (Blacksburg, VA, USA) equilibrated to 19 • C in glass bowls. Terrestrial worms, Enchytraues crypticus, were maintained in a laboratory colony with rich topsoil supplemented weekly with oatmeal and water, housed in covered plastic containers. Red wiggler earthworms, Eiseniella andrei, were purchased locally and identified by PCR barcoding. Worms were maintained in the absence of food (e.g., ice worms are maintained for over two years without feeding [23]), except E. crypticus, which was equilibrated to experimental temperatures for at least 24 h without food prior to harvesting; thus, any contribution of gut contents to the analyses was minimized.

Polymerase Chain Reaction (PCR) Species Identification
DNA extractions were performed with a Qiagen Blood and Tissue kit (Germantown, MD, USA), according to the manufacturer's specifications. For PCR, 1 µL of template was added to 24 µL of premixed DreamTaq (FisherScientific, Waltham, MA, USA) solution supplemented with 4 µM each of cytochrome c oxidase subunit 1 (CO1) universal primers HCO and LCO [27]. Cycling conditions were 95 • C-2 min; 95 • C-20 s; 50 • C-40 s; 72 • C-40 s; 35X. Reactions were screened on a 1% agarose gel viewed under UV light and positives were run through a GenJet PCR Clean-up kit (SignaGen, Frederick, MD, USA). Sanger sequencing was performed by Azenta (South Plainfield, NJ, USA) on both strands using HCO and LCO primers, respectively.

Dry Weight Quantification
Samples were weighed using a laboratory analytical balance and incubated in a Lindberg Blue M oven (FisherScientific, Waltham, MA, USA), at 55 • C for 24 h. Weights were recorded again after 55 • C incubation to obtain wet:dry weight ratios.

PolyP Quantification
Samples were lysed as described above for mitochondrial isolation. PolyP-DAPI shifts the fluorescence emission towards higher wavelengths (λ excitation = 405 nM), compared to wavelengths needed to visualize DNA-DAPI [30]. Utilizing this property, standard curves with synthetic polyP (0-10 µM) were employed to quantitate mitochondrial polyP. Briefly, 5 µL of isolated mitochondria was loaded in triplicate into 96-well black plates (transparent bottom) and incubated in the presence of 20 µM DAPI. After 15 min in the dark and at room temperature, plates were read using a BioTek spectrophotometer (ThermoFisher Scientific) as described [15,17]. Mitochondrial inorganic polyphosphate concentrations were normalized to wet:dry weight ratios.

Temperature-Induced Stress
Specimens were equilibrated and incubated at stated temperatures for 24 h under aquatic or terrestrial conditions, accordingly. Temperatures were chosen based on worms' viable thermal range.

Hypoxia
Specimens were placed in 6-well plates in a hypoxia chamber (COY, Grass Lake, MI, USA) under 0.1% O 2 atmosphere at their preferred temperature for 24 h. Subsequently, samples were lysed, and DAPI-PolyP assays were conducted as described above.

Mitochondrial Membrane Potential Assay
Freshly prepared mitochondria were isolated from worm specimens as described above. Protein quantitation was performed using a nanodrop spectrophotometer (Nan-odrop2000, ThermoFisher Scientific). Subsequently, 40 µg of protein was loaded into 96-well plates and incubated in the presence of 200 nM TMRM for 20 min at 37 • C. After incubation, plates were read using a BioTek spectrophotometer (ThermoFisher Scientific). Lastly, 20 µM FCCP was loaded into each well, the samples were left for 10 min at room temperature, and fluorescence was measured again, using the same parameters.

Statistical Analysis
Experiments and analyses were conducted in triplicate. In each case, one independent worm was used per experiment. Triplicates were conducted with different worms. Data are expressed as the mean ± SEM. Statistical analyses were carried out using Origin Lab (Northampton, MA, USA). Data were analyzed using non-parametric tests. Specifically, Chi-squared test was used in all the figures comparing more than two groups of data and Mann-Whitney U test was used in remaining Figures. Values of p ≤ 0.05 were considered significant (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Results
To establish steady-state levels of mitochondrial polyP across a diverse group of annelids, we assayed DAPI-polyP fluorescence using specific wavelengths, as described [15]. Mitochondrial inorganic polyphosphate levels were monitored in mitochondria isolated from five species of annelid worms: Mesenchytraeus solifugus (glacial), Lumbriculus variegatus (aquatic), Helobdella austinensis (aquatic), Eiseniella andrei (terrestrial) and Enchytraeus crypticus (terrestrial). Since water content differed between worms, wet and dry weights were calculated and normalized for all comparisons (Figure 1). Our data show that polyP levels were significantly different between worms occurring at different habitats, with highest levels observed in terrestrial species (E. andrei and H. austinensis) and lowest in aquatic species (L. variegatus and H. austinensis) ( Figure 2). This prompted us to assay mitochondrial membrane potential (using TMRM fluorescence) among worm species to confirm that mitochondria were functionally equivalent; comparable values were observed in all worms examined, but decoupling appeared to be less efficient in M. solifugus (Figure 3). Subsequently, we measured mitochondrial polyP levels as a function of temperature within each worm's viable thermal range (i.e., deviating accordingly from its preferred temperature). Mitochondrial polyP levels increased significantly with temperature in M. solifugus, remained relatively constant in L. variegatus and decreased significantly in E. crypticus ( Figure 4). Since oxygen solubility decreases with temperature, we measured polyP levels in response to hypoxia (also an independent stressor), a condition in which all worms significantly increased polyP ( Figure 5).
cally, Chi-squared test was used in all the figures comparing more than t data and Mann-Whitney U test was used in remaining Figures. Values of considered significant (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Results
To establish steady-state levels of mitochondrial polyP across a divers nelids, we assayed DAPI-polyP fluorescence using specific wavelengths, as d Mitochondrial inorganic polyphosphate levels were monitored in mitocho from five species of annelid worms: Mesenchytraeus solifugus (glacial), Lum gatus (aquatic), Helobdella austinensis (aquatic), Eiseniella andrei (terrestrial) an crypticus (terrestrial). Since water content differed between worms, wet an were calculated and normalized for all comparisons (Figure 1). Our data sh levels were significantly different between worms occurring at different highest levels observed in terrestrial species (E. andrei and H. austinensis) aquatic species (L. variegatus and H. austinensis) (Figure 2). This prompted u tochondrial membrane potential (using TMRM fluorescence) among worm s firm that mitochondria were functionally equivalent; comparable values w in all worms examined, but decoupling appeared to be less efficient in M. so 3). Subsequently, we measured mitochondrial polyP levels as a function o within each worm's viable thermal range (i.e., deviating accordingly from temperature). Mitochondrial polyP levels increased significantly with temp solifugus, remained relatively constant in L. variegatus and decreased sign crypticus (Figure 4). Since oxygen solubility decreases with temperature, polyP levels in response to hypoxia (also an independent stressor), a cond all worms significantly increased polyP ( Figure 5).     Using the same methods to assay polyP as under control conditions, we observed a significant temperature-dependent increase in the levels of polyP in M. solifugus, which was not present in the other mesophilic counterparts. Underlined temperature is the optimal temperature for each of the annelids. Data are expressed as mean ± SEM of, at least, three independent experiments conducted using biological triplicates. Values of p ≤ 0.05 were considered significant (** p ≤ 0.01).    Using the same methods to assay polyP as under control conditions, we observed a significant temperature-dependent increase in the levels of polyP in M. solifugus, which was not present in the other mesophilic counterparts. Underlined temperature is the optimal temperature for each of the annelids. Data are expressed as mean ± SEM of, at least, three independent experiments conducted using biological triplicates. Values of p ≤ 0.05 were considered significant (** p ≤ 0.01).    Using the same methods to assay polyP as under control conditions, we observed a significant temperature-dependent increase in the levels of polyP in M. solifugus, which was not present in the other mesophilic counterparts. Underlined temperature is the optimal temperature for each of the annelids. Data are expressed as mean ± SEM of, at least, three independent experiments conducted using biological triplicates. Values of p ≤ 0.05 were considered significant (** p ≤ 0.01). Using the same methods to assay polyP as under control conditions, we observed a significant temperature-dependent increase in the levels of polyP in M. solifugus, which was not present in the other mesophilic counterparts. Underlined temperature is the optimal temperature for each of the annelids. Data are expressed as mean ± SEM of, at least, three independent experiments conducted using biological triplicates. Values of p ≤ 0.05 were considered significant (** p ≤ 0.01).
Biology 2022, 11, x FOR PEER REVIEW Figure 5. When stress is induced by hypoxia, all Annelida included in our study showed the levels of polyP. The effects of hypoxia in the levels of mitochondrial polyP were also in our samples. However, no major differences were found in M. solifugus and the other me counterparts. Data are expressed as mean ± SEM of, at least, three independent experime ducted using biological triplicates. Values of p ≤ 0.05 were considered significant (** p ≤ 0.01

Discussion
Our results show that disparate annelid worms display highly variable mit drial polyP levels under basal conditions. Considering that wet:dry ratios were rel constant between these worms, it remains unclear why polyP levels were signif elevated in terrestrial species. Nonetheless, these differences are suggestive of b getic patterns that distinguish these species, particularly the glacier ice worm, chytraeus solifugus. For example, Lumbriculus variegatus (aquatic) and Enchytraeus cr (terrestrial) displayed similar membrane potential:decoupling ratios, but these ap to be reduced in M. solifugus, suggesting an enhanced ability of the latter to depola mitochondrial membrane (see Figure 3). Importantly, temperature is a well-know ulator of protein dyshomeostasis [31], and possibly the stenothermic environmen worms (i.e., ~0 °C over geological time) stabilizes machinery associated with mem potential; alternatively, the apparent requirement for elevated ATP in M. solifugus may have provided strong evolutionary pressure to select for robust OXPHOS co in these worms.
Under thermal stress, levels of mitochondrial polyP increased significantly in ifugus, in contrast to their mesophilic counterparts, where levels were either not (L. variegatus) or decreased (E. crypticus). These opposite responses have been ob elsewhere; specifically, bacterial polyP increases with stress [32,33], while mam polyP levels could decrease with stress. In fact, levels of mitochondrial polyP are d ent on the status of mitochondrial physiology, and they decrease as mitochond comes dysfunctional [21]. Mitochondrial dysfunction has been broadly described stress conditions [34]. These differences seem to reflect fundamentally different str for maintaining bioenergetic activity in the context of polyP. In bacteria and in vi tems, polyP has been proposed to function as a molecular chaperone [35][36][37]. Since chaperoning is a highly energy-dependent process [38], the roles of polyP as a re of bioenergetics and of protein homeostasis could be linked. In mammalian cells, p thought to regulate levels of mitochondrial free calcium, as well as opening the mit drial permeability transition pore [4,5,14]. Both are closely related to the genera

Discussion
Our results show that disparate annelid worms display highly variable mitochondrial polyP levels under basal conditions. Considering that wet:dry ratios were relatively constant between these worms, it remains unclear why polyP levels were significantly elevated in terrestrial species. Nonetheless, these differences are suggestive of bioenergetic patterns that distinguish these species, particularly the glacier ice worm, Mesenchytraeus solifugus. For example, Lumbriculus variegatus (aquatic) and Enchytraeus crypticus (terrestrial) displayed similar membrane potential:decoupling ratios, but these appeared to be reduced in M. solifugus, suggesting an enhanced ability of the latter to depolarize the mitochondrial membrane (see Figure 3). Importantly, temperature is a well-known modulator of protein dyshomeostasis [31], and possibly the stenothermic environment of ice worms (i.e.,~0 • C over geological time) stabilizes machinery associated with membrane potential; alternatively, the apparent requirement for elevated ATP in M. solifugus [23,24] may have provided strong evolutionary pressure to select for robust OXPHOS coupling in these worms.
Under thermal stress, levels of mitochondrial polyP increased significantly in M. solifugus, in contrast to their mesophilic counterparts, where levels were either not altered (L. variegatus) or decreased (E. crypticus). These opposite responses have been observed elsewhere; specifically, bacterial polyP increases with stress [32,33], while mammalian polyP levels could decrease with stress. In fact, levels of mitochondrial polyP are dependent on the status of mitochondrial physiology, and they decrease as mitochondria becomes dysfunctional [21]. Mitochondrial dysfunction has been broadly described under stress conditions [34]. These differences seem to reflect fundamentally different strategies for maintaining bioenergetic activity in the context of polyP. In bacteria and in vitro systems, polyP has been proposed to function as a molecular chaperone [35][36][37]. Since protein chaperoning is a highly energy-dependent process [38], the roles of polyP as a regulator of bioenergetics and of protein homeostasis could be linked. In mammalian cells, polyP is thought to regulate levels of mitochondrial free calcium, as well as opening the mitochondrial permeability transition pore [4,5,14]. Both are closely related to the generation of ATP via OXPHOS. The depletion of mitochondrial polyP in these organisms has a deleterious effect on OXPHOS function, entirely disrupting cellular bioenergetics [15][16][17].
The functional role of polyP as a molecule of energy storage has been demonstrated by us and others [21,39]. Thus, a kinetically favorable interconversion between the two highly energetic molecules, ATP and polyP, could buffer their behavior and place them in a dynamic equilibrium. The unique response of M. solifugus to thermal stress, namely an increase in mitochondrial polyP as a function of temperature (see Figure 4), is in direct contrast with a paradoxical decrease in ATP observed in these worms [23,24]. Taken together, it appears that mitochondrial polyP in M. solifugus is positioned to donate high energy phosphates to ADP in an energy deficit cellular environment, similar to a phosphagen [40], and may otherwise function to stabilize proteins critical to maintaining mitochondrial function (e.g., membrane potential), in a chaperone-like role [35,41,42].

Conclusions
Our data suggest that polyP and ATP are in a dynamic equilibrium within ice worm mitochondria, such that the paradoxical decline of ATP with temperature is coupled with a corresponding increase in polyP. These observations are consistent with a putative role of polyP as a stress-related chaperone, similar to what has been proposed in bacteria. Importantly, this response appears to be ice worm-specific amongst Annelida based on our analyses of disparate segmented worms, suggesting a novel bioenergetic function within this worm lineage.  Data Availability Statement: Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author.