Acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio species that secrete the toxin PirAB, resulting in significant shrimp mortality by degenerating the epithelial cells of the hepatopancreas. Various mitigation strategies have been explored, but their implementation has often been inadequate or ineffective. In this study, we developed an in vitro probiotic selection pipeline to identify Bacillus strains that could potentially mitigate AHPND. Bacillus strains were tested for in vitro inhibition of an AHPND strain, quorum sensing (QS) disrupting abilities, secretome profiles, lectin binding profile and PirAB toxin degradation. Bacillus strains could be divided into four groups. Group 1 showed a distinct secretome profile 3, strong N-acylhomoserine lactone (AHL, a quorum sensing molecule) and PirB degrading capacity, wheat germ agglutinin (WGA) lectin binding preference, and low glycan diversity. Group 2 was characterized by secretome profile 1, strong AHL degrading capacity, no PirB degrading capacity, WGA lectin binding preference and high glycan diversity. Group 3 showed secretome profile 1, no AHL or PirB degrading capacity, concanavalin A (ConA) lectin binding preference and a high glycan diversity. Group 4 was characterized by secretome profile 2, no AHL or PirB degrading capacity, ConA lectin binding preference and low glycan diversity. Next, B. megaterium LMG9300 of group 1, the only PirB degrading strain, was used at a 5 x 107 CFUmL-1 during a Penaeus vannamei immersion challenge test with Vibrio parahaemolyticus strain TW01, enhancing shrimp survival by 15 %, though not significantly compared to the challenge control. A probiotic mixture, including B. megaterium LMG9300 (group 1), B. megaterium DSM1668 (group 2), and B. licheniformis DSM12370 (Group 4), each at 5 x 106 CFUmL-1, was also tested and significantly increased survival by 40 % compared to the challenge control, suggesting that composing mixtures with diverse in vitro characteristics might be a promising strategy.

IntroductionBacteria belonging to the Harveyi clade of vibrios, including Vibrio campbellii infect many wild and cultured aquatic organisms and cause major losses in global aquaculture. Although not a primary human pathogen, V. campbellii can act opportunistically, particularly following exposure to marine environments or seafood, and in immunocompromised individuals, highlighting the links between aquatic ecosystems, food production systems, and human health. Carvacrol, a phenolic monoterpenoid found in oregano and thyme essential oils, is approved for use in human and animal food. Beyond its safety profile, this compound has also been reported to possess diverse pharmacological effects, including anticarcinogenic, anti-inflammatory and antimicrobial.MethodsUsing gnotobiotic brine shrimp Artemia as an in vivo model, we examined whether carvacrol can provide dual protection against V. campbellii by inhibiting the production of bacterial virulence and modulating host immune responses.Results and discussionCarvacrol significantly improved the survival of Artemia during V. campbellii challenge while showing low toxicity at effective concentrations. The protection was associated with reduced bacterial virulence, including decreased biofilm formation and lower hemolytic and caseinase activities. Additionally, carvacrol modulated the expression of defence-related genes (hsp70, prophenoloxidase, transglutaminase, and ferritin) in time-dependent and stochastic patterns, rather than sustained upregulation. Overall, these findings suggest that carvacrol enhances disease resistance through both pathogen- and host-directed mechanisms. Given its food-grade safety status, carvacrol holds strong translational potential as a functional antimicrobial strategy to support disease control and health management in aquaculture, warranting further evaluation under realistic farming conditions.

Recent ecological changes in Urmia Lake, marked by a dramatic transition from rainy to drought conditions, have altered key environmental parameters and may affect the immune system of local organisms, including Artemia urmiana. Among the environmental factors associated with these changes, temperature and salinity are the primary and critical factors directly influencing survival, growth, and immune function in aquatic organisms. Accordingly, this study aimed to evaluate the effects of temperature and salinity changes on the expression of prophenoloxidase (proPO), a key component of innate immunity, as well as non-lethal heat shock (NLHS)–induced proPO expression in Artemia urmiana. qPCR assay was developed to evaluate the influence of three-decade ecological crisis, focusing on temperature and salinity, on basal and NLHS-induced proPO expression of nauplii of Artemia urmiana, originating from cysts collected from 1994 (rainy period) to 2020 (drought period). To obtain partial cds of proPO, four regions of this cDNA were sequenced using Sanger method. Student’s t-test was conducted for statistical comparisons between two groups, whereas one-way ANOVA was utilized for the analysis of more than two groups. Before expression analysis, four regions of proPO cDNA were sequenced (the accession numbers: OQ784234, OQ784235, OQ784236, OQ784237) and then assembled into a larger partial cds (the accession numbers: OQ784174). qPCR results demonstrated that ecological changes caused proPO expression shifting, which was highest in 2005 (95

Antibiotic-resistant Vibrio species pose a threat in aquaculture. This study evaluates the potential of phage vB_VibS_KarBoss under climate-influenced water conditions such as temperature, pH, and salinity. vB_VibS_KarBoss, isolated from fish market wastes, inhibited 48 of 89 nonredundant autochthonous Vibrio isolates. With a 10-min latent period and a burst size of 3 phages per cell, vB_VibS_KarBoss inhibited the growth of pathogenic species including Vibrio campbellii and Vibrio parahaemolyticus. With only 16.4% intergenomic similarity to related phages, we propose a new phage genus, KarBossvirus, for consideration by the International Committee on Taxonomy of Viruses. Considering the robustness of Artemia and their sensitivity to changes in environmental conditions, the virulence of the Vibrio isolates resulting in Artemia mortality and the efficacy of vB_VibS_KarBoss in these altered water conditions were studied. Results support the potential importance of vB_VibS_KarBoss in biocontrol in its ability to rescue >50% Artemia (p < 0.01) from mortality due to vibriosis.

The increasing resistance of aquatic pathogens to antibiotics demands alternative therapeutic strategies. Plant essential oils (EOs) and their active components offer promising antibacterial and antifungal properties. This study evaluated the growth-inhibitory effects of cinnamaldehyde (CN), citral, limonene, and pinene at four concentrations (0.1 %, 0.01 %, 0.001 %, and 0.0001 %) against Vibrio coralliilyticus, Vibrio splendidus, and Vibrio tubiashii. The tolerance of Magallana gigas juveniles to CN was assessed over a 7-day exposure, and the protective effects of 0.001 % CN against V. coralliilyticus infection were evaluated, including its impact on immune-related gene expression. CN demonstrated strong growth inhibition against all Vibrio species at 0.1 % and 0.01 %, while citral demonstrated efficacy at 0.1 %. Limonene and pinene partially inhibited V. splendidus and V. tubiashii growth at 0.1 %. At 0.001 %, CN partially inhibited V. coralliilyticus growth and was non-toxic to oysters, maintaining 100 % survival. In the challenge assay, oysters treated with 0.001 % CN exhibited significantly higher survival (44 %) compared to the untreated control (3 %). Gene expression analysis revealed that CN upregulated genes associated with immune signaling (MyD88), antioxidant defense (catalase), and ion channel regulation (the transient receptor potentials TRPV4.2, TRPM1.4, and TRPM2.4), supporting its role in enhancing immune and stress responses, while downregulating NF-kappa B, suggesting anti-inflammatory effects. These findings indicate that CN, at non-toxic concentrations, can enhance M. gigas survival against bacterial infection by modulating immune responses. This highlights CN's potential as a natural therapeutic alternative to antibiotics in oyster aquaculture. Further studies should explore synergistic effects with other EOs and assess long-term impacts in production systems.

We review the literature on microbial management in larval rearing and give recommendations for future research. Based on a range of different approaches, we show that, as for many farmed animals, detrimental host/microbe interactions are a main reason for low viability of young stages of farmed fish, crustacea and shellfish. The composition of the microbiota of larval stages is determined by both selection and stochastic processes, which makes it possible to steer the colonization of larval fish by controlling the abundance and the inventory of species of the microbes present in aquaculture facilities. The microbiota evolves rapidly during larval development, leading to need for a continuous management. It is important to emphasize that it is not which species, but which functions that the microbiota provide that is important for the viability of the larvae. These functions are difficult to quantify, and we have limited knowledge. We discuss a range of microbial management methods that have been proposed and evaluate current knowledge and use in the industry. We group these methods as 1) targeted and non-targeted decimation, 2) targeted enhancement, and 3) stimulation of the immune system. Many different methods within these three groups are discussed. There has been considerable progress with some methods, but few are well studied. Only a few methods are implemented in the industry. To develop a research program that aims to establish the scientific knowledge-based needed for development of microbial management methods we call for a concerted action with participants from the industry, the research community, and national and international funding agencies. The research should be based on a consensus understanding of the current problems and be more diverse than now, as complex problems need complex solutions.

This study aimed to isolate AHL-degrading bacteria from the intestine of Penaeus vannamei and evaluate their ability to control pathogenic Vibrio harveyi in P. vannamei larvae. Twenty-seven isolates were obtained from the digestive tract of healthy Pacific white shrimp juveniles (P. vannamei) after six cycles of pasteurization at 70 °C, but only three isolates (E1LP2, E2LP1, and E2LP2) could degrade AHL. The 16S sequence results gave a high identity (>95%) with Bacillus sp. The isolates exhibited quorum-quenching abilities by degrading AHLs, thereby disrupting Vibrio quorum sensing and virulence. In Zoea and Mysis, the challenged larvae plus the administration of E1LP2 resulted in the lowest survival compared to the other groups. Isolates degrading N-acyl homoserine lactone improved the survival of shrimp Zoea and Mysis larvae when challenged with pathogenic V. harveyi. This is the first report on the use of quorum-sensing disrupter bacteria in P. vannamei larval shrimp culture. Our findings suggest that these Bacillus spp. strains have potential as biocontrol agents for sustainable shrimp aquaculture, reducing the reliance on antibiotics while mitigating vibriosis outbreaks.

IntroductionThe intensification of aquaculture to meet the growing demand for aquatic animal protein by a global population approaching 10 billion by 2050 has raised concerns about the increased risk of disease outbreaks in farmed species. These diseases account for over 50% of economic losses in commercial aquaculture, largely due to the reliance on ineffective and harmful therapeutic options like antibiotics, which contribute to multidrug resistance and pose serious global health concerns. However, non-antibiotic alternatives such as probiotics have emerged promising choices to enhance growth performance, immunity, and disease resistance in aquaculture species.MethodsIn this study, we present a novel, non-invasive protocol for isolating indigenous bacteria from sediment. This method utilizes minimal media supplemented with N-hexanoyl homoserine lactone (HHL) to select strains with key probiotic attributes.Results and discussionOur study isolated 24 bacterial isolates, 11 demonstrating quorum quenching (QQ) activity by degrading HHL, indicating potential for antivirulence therapy. Among these, eight were non-hemolytic, suggesting safety in the presence of host wounds. Six non-hemolytic isolates exhibited proteolytic activity, which is essential for aiding protein digestion. Whole genome sequencing revealed their identity as Priestia megaterium PMUG01 and PMUG02, Lysinibacillus fusiformis LFUG, Micrococcus yunnanensis MYUG, and two novel species tentatively named Kocuria crassamentum species nova strain KSNUG, and Heyndrickxia crassamentum species nova strain HSNUG. Despite some virulence-associated genes, none of the strains demonstrated pathogenicity in Artemia nauplii. Apart from an lsaB gene in P. megaterium, which confers resistance to lincosamides, no antibiotic resistance genes were detected. Our findings highlight these strains’ biosafety and probiotic potential for aquaculture, offering promising candidates for sustainable disease management and improved feed utilization in farmed species. These results pave the way for developing indigenous and effective, non-antibiotic-based probiotic solutions to mitigate disease risks in aquaculture.

Heat shock proteins (HSPs), particularly HSP70, play a vital role in fish immune defense against pathogens. The administration of DnaK (bacterial homolog of HSP70) may be a strategy to potentiate the immune response and survival of aquatic organisms. This study evaluates the effect of cells overexpressing DnaK on mortality and immune-related gene expression in gnotobiotic sea bass larvae challenged with Vibrio anguillarum. Larvae were subjected to different treatments: NB (no bacteria), YS0 (E. coli with no plasmid), YS1 (E. coli expressing truncated DnaK), and YS2 (E. coli expressing DnaK), and then infected with V. anguillarum at 7 days post-hatching (dph). Mortality was monitored, and RT-qPCR was used to evaluate immune gene expression at 0, 18, 24, 36, and 120 hpc. While no significant variations were recorded in the non-challenged larvae, constant and sustained mortality was observed in challenged larvae from 60 to 120 hpc. However, lower mortality was observed in the larvae treated with DnaK. DnaK treatment promoted the expression of antimicrobial (hepcidin, transferrin) and chemotaxis genes (ccl4), which was further enhanced after a challenge with V. anguillarum, in conjunction with the modulation of il1β and il-8 at 120 hpc. These findings suggest that DnaK induces a potent innate immune response, improving survival against V. anguillarum and supporting its potential use as a disease-preventive strategy in aquaculture.

The rotifer Brachionus sp. is of great importance for aquaculture, as the reproduction cycle under rearing conditions of many economically important species larvae depends on the use of rotifers as first live feed. Establishing a protocol that results in an improved tolerance of rotifers to environmental stressors will allow for a more stable rotifer production. The exposure to non-lethal heat shocks (NLHS) already proved to enhance the tolerance, not only to heat stress, but also to other stressors in several aquatic species, by activating the heat shock response and epigenetic mechanisms. This study aimed to determine the potential of a single NLHS to induce tolerance to different abiotic stressors in two strains of B. koreanus (MRS10 and IBA3) and to evaluate possible molecular mechanisms involved in the achievement of increased tolerance to hydrogen peroxide induced by NLHS. Cross-tolerance was achieved for both strains, namely to high salinity, cadmium chloride, and hydrogen peroxide. Scale-up tests resulted in increased tolerance to hydrogen peroxide only for MRS10. During the exposure to this substance, heat-shocked MRS10 rotifers showed an up-regulation of genes related to oxidative stress response and histone modifications, increased production of HSP70, and higher levels of total acetylation of histone H3. A single NLHS proved to induce epigenetic effects when rotifers were exposed to other stressor later in life. However, further studies should elucidate if the NLHS conditions used in this study can yield a persistent outcome, allowing the establishment of tolerant rotifer strain lines and, consequently, a more stable production.

Temperature plays an important role in the occurrence and performance of organisms in aquatic ecosystems and is also one of the main environmental factors affecting species' ' survival and growth in aquaculture. As an important species for aquaculture sustainability, the rotifer Brachionus sp. benefits from inducible phenotypic traits that allow the organisms to cope with environmental stress. The exposure to high temperature has shown to increase production of heat shock proteins (HSPs) and histone modifications in several organisms, resulting in induced thermotolerance. This study aimed to evaluate the potential of non-lethal heat shock (NLHS) to induce thermotolerance in two strains of B. koreanus and pinpoint some of the molecular mechanisms involved in the process. Exposure of organisms to 42 degrees C for 30 min, with a subsequent recovery at 25 degrees C for 8 h demonstrated to increase thermotolerance in up to three-fold when organisms were, in a posterior phase, subjected to a lethal temperature. This study also showed that one of the strains is tendentially more thermotolerant than the other. Indeed, NLHS exposure resulted in increased mRNA expression of different Hsp genes and production of HSP70 in general, but different patterns of expression were observed between strains. However, a single NLHS showed to have no effects in epigenetic mechanisms, suggesting that the induced capacity to tolerate heat stress was transient and some more cycles of NLHS may be needed to promote a persistent effect.

The difficulties and high costs of live microalgae production have driven the search for alternative diets in bivalve aquaculture. The baker's yeast (Saccharomyces cerevisiae) represents a promising, cost-effective, highquality protein for aquaculture. However, its application is restricted by poor digestibility due to its thick, rigid cell wall. Previously, we observed that the deletion of mnn9 gene in S. cerevisiae, leading to defective mannan synthesis and increased beta-glucan exposure, enhances growth and immune response in Magallana gigas oysters. Nevertheless, using Delta mnn9 in aquaculture is not feasible due to restrictions on genetically modified organisms (GMO) containing antibiotic-resistance genes. This study aimed to create a non-GMO cell-wall defective yeast mutant (JH40) via ethyl methanesulfonate (EMS) mutagenesis and evaluate its nutritional value for M. gigas juveniles. Phenotypical differences between wild-type S. cerevisiae (WT), Delta mnn9, and JH40 were characterized using fluorescein isothiocyanate (FITC)-labeled lectin analysis, microscopy, cell diameter measurements, growth curves, and osmosensitivity tests. The nutritional value of a microalgae-based diet (Chaetoceros muelleri: Tisochrysis lutea, 50:50 based on dry weight) and its 50% substitution with JH40 or WT was assessed over 21 days in M. gigas juveniles. A complementary test also explored higher substitution levels (63%, 75%, and 100%) of the based diet with JH40 and the addition of extra JH40 (+25 %) in the 63% and 75% replacement diets. Results indicated that JH40 forms clumps, grows slower than the WT, has a larger cell diameter, higher binding affinity to WGA and LEL lectins than the WT, and is highly sensitive to hypo-osmotic stress, which are characteristics of cell-wall defective yeasts. Oysters fed a diet with 50% JH40 substitution showed significantly higher growth compared to those fed the WT-containing diet. JH40 could replace 50% of the algal diet without significantly affecting oyster growth and survival. However, higher inclusion levels of JH40 (62 %, 75 %, and 100 %) did not achieve the oyster growth observed with the algae-based diet, even with additional yeast. These findings are attributed to the limited polyunsaturated fatty acid content in yeast cells. Further research on dosing and administration frequencies is needed to evaluate the immunostimulatory potential of JH40.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging threat causing high mortality in penaeid shrimp, triggered by deadly toxins secreted by a virulent strain of Vibrio parahaemolyticus. This strain acquires its pathogenicity by harboring a 63-70 kb AHPND-associated plasmid (pVA1), which encodes the binary PirAB<^>VP toxin, comprising the PirA<^>VP and PirB<^>VP subunits. Degrading these toxins presents a potential strategy for controlling AHPND. This study evaluated the ability of various Bacillus strains to degrade crude and pure AHPND toxins in vitro, as confirmed by SDS-PAGE analysis. Previous research has indicated that Bacillus-based treatments can enhance shrimp survival when exposed to AHPND pathogens. However, despite advancements in detection methods, the exact mode of action and effective therapies for AHPND remain unclear. In vivo challenge tests using gnotobiotic Artemia franciscana revealed that certain Bacillus strains improved larval survival against crude and pure AHPND toxins. Moreover, a mixed culture of Bacillus strains significantly increased Artemia survival compared to the positive control, whereas individual strains showed no significant difference when directly challenged with V. parahaemolyticus M0904. The Artemia model provides a controlled, ethical, and cost-efficient system for high-throughput screening of microbial interactions, especially for assessing cytotoxicity and evaluating the effectiveness of potential probiotics in mitigating toxin-induced mortality. These findings suggest that the protective effect of Bacillus strains likely stems from their ability to degrade toxins.

This study evaluated the efficacy of dietary supplementation with two indigenous probiotic strains, Lysinibacillus fusiformis LFUG and Priestia megaterium PMUG01 in improving growth, health, and disease resistance in Nile tilapia juveniles. Each strain was administered at two concentrations (106 and 108 CFU.g-1 of feed) during a 60-day feeding trial. Regardless of concentration, probiotic-supplemented diets significantly improved growth performance indicators, including weight gain, biomass gain, specific growth rate (SGR), and feed conversion ratio (FCR). This was likely due to enhanced nutrient bioavailability and feed utilization. There were no notable changes in fish proximate nutrient composition or condition indices (condition factor, VSI and HSI), signifying that the probiotics did not adversely affect the nutritional quality and welfare of the fish. After the feeding trial, a 14-day challenge with virulent clinical Providencia sp. (strain PSNUG) revealed significant protection in probiotic-fed groups. This protection was attributed to the modulation of gut microbiota, suppression of pathogenic taxa and attenuation of pathogen virulence factors. Histological analysis showed reduced hepatic and splenic inflammation in treated fish, while molecular assays demonstrated modulation of immune markers (IL1 beta, IL6, and CXCL8), and the complement component C3, indicating enhanced mucosal immunity and reduced systemic inflammation. Overall, both strains showed promise as safe and multifunctional probiotics capable of boosting growth, immune competence, and disease resistance in Nile tilapia juveniles. These results support their potential for improving fish health and aquaculture productivity. Exploration of their applicability across other aquaculture species and environments is recommended to promote sustainable fish farming practices.

Quorum-sensing (QS) interference is a promising antivirulence strategy in aquaculture. This study investigated six indigenous bacterial strains for their ability to degrade N-hexanoyl homoserine lactone, a key QS signaling molecule. All strains demonstrated significant QS interference, indicating potential as biocontrol agents. Furthermore, we assessed the key in vitro traits relevant to probiotic application. These included growth rate, pH tolerance and salinity resilience. MYUG presented the fastest growth, followed by KSNUG, PMUG01, LFUG, PMUG02, and HSNUG, representing promising establishment potential within production systems and in fish guts. All strains tolerated pH (3—9) and salinity (1

Aquaculture is the world’s fastest-growing food production sector, yet larviculture remains a problem due to inconsistent live feed quality. Although Artemia is the most used live feed due to its availability and broad species acceptance, its nutritional quality varies across strains and batches. Thus, optimizing Artemia nutritional profile is crucial. To enhance nutritional reliability, Artemia is commonly enriched. However, strain-dependent enrichment responses and FA degradation mechanisms remain unclear. This study aims to elucidate PUFA degradation dynamics in Artemia, evaluating the effects of enrichment, strain differences, and temperature regimes. Nauplii from three Artemia strains—Vinh Chau (VC), Great Salt Lake (GSL), and San Francisco Bay (SFB)—were hatched under controlled conditions. The treatment group was enriched, while the control remained unenriched. After gut evacuation, animals were stored at 27 ± 1 °C or 16 ± 1 °C. Fatty acid methyl ester (FAME) analysis was performed at multiple time points. Forty-two FAs were identified across strains, and degradation slopes were calculated. Comparisons were made within the same strain between enriched and unenriched nauplii and temperatures to assess enrichment and temperature effects, respectively, and across strains within the same temperature and enrichment conditions to determine strain-specific PUFA degradation patterns. Results indicate that both enrichment and strain significantly affect Artemia FA profiles, with n-3 PUFAs responding best to enrichment. VC and SFB showed greater adaptability in FA metabolism, particularly under enrichment and high temperatures. These findings underscore the complex interplay between enrichment, temperature, and genetic factors in determining Artemia’s nutritional quality, providing key insights for optimizing live feed strategies in aquaculture.

Mammalian mitochondria undergo Ca2+-induced and cyclosporinA (CsA)-regulated permeability transition (mPT) by activating the mitochondrial permeability transition pore (mPTP) situated in mitochondrial inner membranes. Ca2+-induced prolonged openings of mPTP under certain pathological conditions result in mitochondrial swelling and rupture of the outer membrane, leading to mitochondrial dysfunction and cell death. While the exact molecular composition and structure of mPTP remain unknown, mammalian ATP synthase was reported to form voltage and Ca2+-activated leak channels involved in mPT. Unlike in mammals, mitochondria of the crustacean Artemia franciscana have the ability to accumulate large amounts of Ca2+ without undergoing the mPT. Here, we performed structural and functional analysis of A. franciscana ATP synthase to study the molecular mechanism of mPTP inhibition in this organism. We found that the channel formed by the A. franciscana ATP synthase dwells predominantly in its inactive state and is insensitive to Ca2+, in contrast to porcine heart ATP synthase. Single-particle cryo-electron microscopy (cryo-EM) analysis revealed distinct structural features in A. franciscana ATP synthase compared with mammals. The stronger density of the e-subunit C-terminal region and its enhanced interaction with the c-ring were found in A. franciscana ATP synthase. These data suggest an inactivation mechanism of the ATP synthase leak channel and its possible contribution to the lack of mPT in this organism.

Extremophiles evolved capacities to survive extended exposure to harsh environmental conditions such as complete desiccation (anhydrobiosis) and freezing (cryobiosis). Accumulation of the three-carbon polyhydric alcohol glycerol is commonly observed in anhydrobiotic organisms, although it is considered to preferentially enhance cryobiosis rather than anhydrobiosis. Here, using dormant stages of the halophilic extremophile crustacean Artemia franciscana, we show that this role is reversed. We find that A. franciscana and related branchiopods evolved co-opted entomoglyceroporin (Eglp)-like aquaporin-type channels previously only characterized in hexapods. Phylogenomic and site-directed mutagenesis analyses indicate that EglpL orthologs likely evolved during the early Cambrian in the common ancestor of the Pancrustacea. RNAi-mediated knockdown experiments show that the A. franciscana EglpL glycerol transporter is subfunctionally co-regulated with canonical aquaglyceroporins (Glps) to mediate glycerol accumulation in the diapause cysts. Termination of diapause using either desiccation or hydrogen peroxide and further exposure of the cysts to freezing suggest that the acquired glycerol plays a more critical role in anhydrobiosis rather than cryobiosis. These findings uncover the essential role of evolutionary divergent aquaporin-type glycerol channels in the accrual of glycerol in an anhydrobiotic organism and reveal a previously overlooked function of this polyol for desiccation tolerance.

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