Deciphering the Survival Strategies of the Entomopathogenic Nematode Steinernema carpocapsae Using Rapid Desiccation Assisted by Nanoparticle-Based Emulsion

Deciphering the Survival Strategies of the Entomopathogenic Nematode Steinernema carpocapsae Using Rapid Desiccation Assisted by Nanoparticle-Based Emulsion | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Deciphering the Survival Strategies of the Entomopathogenic Nematode Steinernema carpocapsae Using Rapid Desiccation Assisted by Nanoparticle-Based Emulsion Jayashree Ramakrishnan , Satheeja santhi Velayudhan , Adi Faigenboim , Ahmed Nasser , Mohamed Samara , Eduard Belausov , Victoria Reingold , Amit Horn , Karthik Ananth Mani , Guy Mechrez , Itamar Glazer , David Shapiro-Ilan , Dana Ment doi: https://doi.org/10.1101/2025.07.14.664280 Jayashree Ramakrishnan a Department of Plant Pathology and Weed Research, Agricultural Research Organization (ARO), Volcani Institute , Rishon LeZion 7505101, Israel b Department of Agroecology and Plant Health, The Robert H. Smith Faculty of Agriculture, Food & Environment the Hebrew University of Jerusalem , Rehovot 7610001, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Satheeja santhi Velayudhan f Department of Entomology, Nematology and Chemistry Units, Agricultural Research Organization, Volcani Institute , 7505101, Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adi Faigenboim e Institute of Plant Science, ARO, The Volcani Institute , Rishon Le Zion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ahmed Nasser g Inter-Institutional Analytical Unit, Agricultural Research Organization, Volcani Institute , 7505101, Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mohamed Samara g Inter-Institutional Analytical Unit, Agricultural Research Organization, Volcani Institute , 7505101, Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eduard Belausov h Department of Ornamental Plants and Agricultural Biotechnology, Institute of Plant Science, Agricultural Research Organization, Volcani Institute , 7505101, Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Victoria Reingold a Department of Plant Pathology and Weed Research, Agricultural Research Organization (ARO), Volcani Institute , Rishon LeZion 7505101, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amit Horn a Department of Plant Pathology and Weed Research, Agricultural Research Organization (ARO), Volcani Institute , Rishon LeZion 7505101, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karthik Ananth Mani c Department of Food Science, Agricultural Research Organization (ARO), Volcani Institute , Rishon LeZion 7505101, Israel d Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem , Rehovot 7610001, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guy Mechrez c Department of Food Science, Agricultural Research Organization (ARO), Volcani Institute , Rishon LeZion 7505101, Israel d Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem , Rehovot 7610001, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Itamar Glazer f Department of Entomology, Nematology and Chemistry Units, Agricultural Research Organization, Volcani Institute , 7505101, Rishon LeZion, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site David Shapiro-Ilan i USDA-ARS Southeastern Fruit and Ttree Nut Research Station , Byron, GA 31008 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dana Ment a Department of Plant Pathology and Weed Research, Agricultural Research Organization (ARO), Volcani Institute , Rishon LeZion 7505101, Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: danam{at}volcani.agri.gov.il Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Water is essential for organisms’ survival. Entomopathogenic nematodes (EPNs), such as Steinernema carpocapsae , experience rapid desiccation (RD) upon application to foliar surfaces, significantly reducing their biocontrol efficacy. Protective strategies employed by the rapidly desiccating EPNs, and their potential improvement through formulations, have been largely unexplored. We determined the protective physiological and molecular mechanisms employed by S. carpocapsae in non-formulated (control) and formulated treatments to withstand RD under different humidity levels. Building on previous results, we hypothesized that titania Pickering emulsion (TPE) and silica Pickering emulsion gel (SPEG) will enhance EPN survival and efficacy through distinct changes in physiological and molecular mechanisms. We determined the effect of RD using gravimetric analysis, confocal microscopy, transcriptomics, ultra-high performance liquid chromatography and ultrastructural (scanning and transmission electron microscopy) assessments. Our results indicate that SPEG and TPE temper the impact of RD in EPNs through diverse physiological mechanisms. Formulated EPNs exhibited significantly delayed water loss and enhanced survival under low humidity compared to controls. Confocal microscopy indicated two distinct protective mechanisms: the primary mode of action in SPEG was retention of hydration on the nematode, whereas TPE effectively slowed water loss from the nematode body. These protective mechanisms correlated strongly with differential patterns of trehalose accumulation, providing biochemical confirmation of formulation efficacy. Transcriptome analysis and ultrastructural validation highlighted the adaptive mechanisms such as—extracellular matrix remodeling, including basement membrane integrity and cytoskeletal reorganization—as critical components of EPN’s adaptative response to RD. Introduction Water is essential for life’s fundamental biological processes. During desiccation, rapid water loss leads to significant reductions in cell volume associated with structural modifications and alterations in extracellular and intracellular solute concentrations ( Perry and Moens, 2011 ; Perry et al., 2012 ). These changes significantly impact the thermodynamics and kinetics of biomolecule interactions, promoting aggregation and precipitation, which critically influence biological function. The genetic, biochemical and molecular machineries that enable survival in extreme environments present a complex interplay of adaptation strategies that influence tolerance to water stress ( Romero-Perez et al ., 2023 ). Insights across groups (bacteria, yeast, plants, tardigrades, nematodes and insects) suggest presence of both conserved and diverse adaptation strategies to withstand water loss and desiccation ( Sogame and Kikawada, 2017 ; Koshland and Tapia, 2019 , 2019 ; Hibshman et al., 2020 ). Extremophilic organisms from varied environmental niches,, often tolerate rapid water loss ( Ricci and Caprioli, 2005 ; Wełnicz et al ., 2011 ; Wharton, 2011 ; Hand and Menze, 2015 ; Møbjerg and Neves, 2021 ). However, even these organisms favor a preconditioning period (slow water loss) for optimal desiccation tolerance, highlighting a broad spectrum of phenotypic and genetic plasticity ( Møbjerg and Neves, 2021 ; Vecchi et al ., 2024 ). Common survival strategies include metabolic rewiring of carbon flux ( Leprince and Buitink, 2015 ; Koshland and Tapia, 2019 ; Hibshman et al., 2020 ), activation of detoxification systems using glutathione and cytochrome P450 ( Fu et al., 2020 ), accumulation of bioprotectants such as antioxidants, polyamines, di/oligosaccharides ( Erkut et al ., 2013 ; Tapia and Koshland, 2014 ; Erkut and Kurzchalia, 2015 ), increased activity of effector enzymes (i.e. catalase and superoxide dismutase) ( Giovannini et al ., 2022 ; Rolland et al., 2024; Sadowska-Bartosz and Bartosz, 2024 ; Ujaoney et al ., 2024 ), and expression of stress proteins such as late embryogenesis abundant proteins (LEA), dehydrins and heat-shock proteins (HSPs) ( Browne et al., 2002 ; Pouchkina-Stantcheva et al ., 2007 ; Li et al ., 2024 ) enabling priming or adaptation to desiccation tolerance ( Gade et al ., 2020 ; Romero-Perez et al ., 2023 ). A universal strategy is developmental arrest or production of stress-resistant stages enabling survival ( Franchi et al ., 2011 ; Erkut et al ., 2013 ; Shapiro-Ilan et al., 2017 ; Sogame and Kikawada, 2017 ). However, in metazoans such as tardigrades and rotifers, as well as lichens and moss, desiccation tolerance spans all life stages. Entomopathogenic nematodes (EPNs) from Steinernematidae and Heterorhabditidae family are slow-dehydration strategists ( Piggott et al ., 2002 ; Gal et al., 2003a , 2005 ; Chen et al., 2006; Yaari et al ., 2016 ; Levy et al., 2020). Hence, the infective juvenile (IJ) stage of EPNs offers a valuable model to investigate desiccation tolerance due to their ability to survive prolonged dehydration through quiescence, characterized by significantly reduced metabolic activity until favorable conditions return ( Glazer, 2002 ; Shapiro-Ilan et al., 2019 ; Ramakrishnan et al., 2024). Several similar strategies of protection have been reported during preconditioning (72 h at high humidity) ( Ramakrishnan et al., 2024 , Gal et al., 2005 , Yaari et al., 2016 , Levy et al., 2020) ( O’Leary et al ., 2001 ; Erkut, 2011 ; Ramakrishnan, Ment and Glazer, 2024 , Solomon et al ., 2000 ; Gal et al., 2004 ) ( Erkut et al ., 2013 ; Yaari et al ., 2016 ; Levy, 2020 ), ( Chen, 2006 ; Tyson et al ., 2007 ; Yaari et al ., 2016 ). EPNs are used commercially to control arthropod pests ( Belien, 2019 ), but their effectiveness is inconsistent in foliar surfaces due to sensitivity to abiotic stresses ( Lacey and Georgis, 2012 ; Glazer, 2015 ; Glazer et al., 2023 ; Ramakrishnan et al., 2024), e.g., low tolerance to rapid desiccation (RD) ( Glazer 1992 ; Arthurs et al., 2004 ). RD, defined as the rapid water loss over minutes to hours, severely impacts EPN viability and hence biological efficacy. Therefore, studying the innate adaptation strategies employed by EPNs and enhancing their resistance to rapid water loss are critical to improving their persistence and biocontrol efficacy ( Navaneethan et al ., 2010 ; Dito et al ., 2016 ; Shapiro-Ilan et al., 2017 ). We recently characterized water-loss rates in EPNs at different relative humidities (RHs) under RD, demonstrating an inverse relationship between the two ( Ramakrishnan et al ., 2022 ). That study revealed distinct differences in membrane-packing abilities during the initial drying period, indicating strong water-binding properties of S. carpocapsae . Those observations led to questions regarding the underlying mechanisms enabling EPNs to withstand rapid water loss. We further found that formulations such as nanoparticle-stabilized Pickering emulsions (titania Pickering emulsion [TPE] and silica Pickering emulsion gel [SPEG]), tailored to reduce water loss, resulted in enhanced survival and efficacy of S. carpocapsae IJs ( Ramakrishnan et al ., 2023 ). Building on those findings, we hypothesized that EPN survival and efficacy under RD involves distinct changes in physiological, biochemical and molecular mechanisms. We therefore recruited various strategies to approach the broader question of what changes enable survival under RD. We established a systematic toolkit of techniques to characterize and elucidate the protective physiological and molecular mechanisms employed by S. carpocapsae in non-formulated (control) and formulated (TPE and SPEG) treatments to withstand RD under varying humidity levels. We examined water-loss rates and the formulations’ protective effects and conducted comprehensive transcriptomic analyses at different time points. Findings were validated by ultrastructural studies and biochemical assays (ultra-high performance liquid chromatography [UHPLC]). This information could provide deeper insights on the nuances of RD, aiding toward integrating intervention strategies that provide maximal protection while maintaining the organism functionality during desiccation stress. Results Physiologically, water loss-delaying strategies could moderate EPN IJ survival In-vitro survival of formulated and non-formulated S. carpocapsae IJs was assessed at 64% RH and 54% RH over 144 h ( Figure 1A ). Irrespective of humidity, survival of formulated IJs was significantly higher than that of the control, non-formulated IJs ( Figure 1B ). TPE- and SPEG-treated IJs maintained viability for up to 120 and 144 h, respectively, whereas non-formulated controls (nematodes in water) reached 0% survival at both 54% RH (F (14,349) = 48.0069, P < 0.0001) and 64% RH (F (14,317) = 39.6917, P 80% viability for up to 72 h. Comparing the formulated nematodes, survival rates for TPE and SPEG were not significantly different for the first 24 h (F (3,317) = 1.000, P > 0.05), but diverged significantly after 16 h at 54% RH (F (4,349) = 23.4109, P 72 h suggests destabilization of the emulsion system. Download figure Open in new tab Figure 1. Effect of low RH and formulations on survival and water-loss characteristics of S. carpocapsae infective juveniles (IJs) following rapid desiccation (RD). (A) Schematic illustration of the experimental design: non-formulated (nematode in water, NIW) and formulated (titania Pickering emulsion [TPE] and silica Pickering emulsion gel [SPEG]) IJs were incubated in desiccators at 64 ± 5% and 54 ± 5% RH (23°C) for RD, then transferred to 55-mm petri plates with 7–10 mL distilled water and incubated overnight for viability determination. (B) IJ survival was determined at 0–144 h post-RD at both RHs. Error bars represent standard error. (C1) Gravimetric estimation of water loss in IJs after RD exposure for 24 h at 64% RH or 54% RH. (C2) FTIR spectral fingerprint region of S. carpocapsae IJs post-RD for 72 h at 64% or 54% RH and after subtraction from respective negative emulsion-only controls. (D1) Confocal microscopy images of formulated and non-formulated IJs: NIW, TPE, SPEG drop-casted on slides and rapidly desiccated at 54% RH for 16 h. Oil phase of emulsion is labeled with Nile red, and nanoparticles are labeled with carboxyfluorescein (upper panel). Modified procedure with carboxyfluorescein labeling of water phase (lower panel). Visualization of oil phase and nanoparticle tracking (upper panel), and oil-phase and water-phase tracking (lower panel), highlighting localization and protective interactions in formulations. (D2) Fluorescence intensity of carboxyfluorescein, used as an indicator for hydration in Pickering emulsions after RD for 16 h at 54% RH. (D3) Ultrastructural validation of the protective mechanisms (upper panel) and glycocalyx enrichment in rapidly desiccated SPEG and NIW compared to controls maintained at 100% RH (lower panel). (E) Schematic representation summarizing the protective mechanisms conferred by SPEG (upper panel) and TPE (lower panel) against water loss under RD conditions. Effects of RH and formulation on water loss in IJs Gravimetric analysis was conducted on formulated vs. non-formulated IJs for 24 h ( Figure 1-C1 ). At the lower humidities (64% RH and 54% RH), the control IJs lost approximately 98% of their water content, compared to 50 and 70% water loss in SPEG and TPE IJs, respectively. Although no significant differences were observed in the rate of water loss between 54% RH and 64% RH (F (1,26) = 1.3032, P > 0.05), formulated IJs exhibited significantly reduced water loss compared to their non-formulated counterparts (F (2,26.1) = 19.3396, P < 0.001). These results demonstrate the formulations’ effective protection of IJs by significantly slowing their rate of water loss. Measurements beyond 24 h were not conducted due to negative values obtained upon subtraction from controls (emulsion only) (Supplementary data – confocal assays). The Fourier transform infrared (FTIR) spectral fingerprint regions for EPN IJs in water (control), TPE and SPEG varied in response to desiccation at the lower humidities (64% RH and 54% RH) for 72 h ( Figure 1-C2 ). As previously described, the entire FTIR spectrum is required to reliably predict IJ water content ( Ramakrishnan et al., 2022 ). However, Pickering emulsions complicate spectral interpretation due to the presence of overlapping regions of IR-active components/phases within the formulation. Therefore, in this study, we focused specifically on the 3000–3500 cm -1 region, corresponding primarily to OH bond vibrations, to reliably assess hydration retention in formulated EPNs. Accordingly, control IJ samples showed the lowest peak intensity compared to TPE and SPEG formulated IJs after 72 h of desiccation. SPEG-formulated IJs exhibited the highest peak intensity, indicating better hydroxyl bond (OH)-retention capacity under reduced humidity. At 54% RH, a noticeable decrease in OH bond peak intensity was observed in TPE-formulated IJs, corresponding to their significantly lower survival compared to SPEG-formulated IJs (F (1,349.1) = 65.8327; P < 0.0001). Download figure Open in new tab Figure 2: Quality assessment and preliminary screening of transcriptome data. (A) PCA depicting the clustering patterns of stressed, protected and control IJs. (B) Heatmap illustrating the differentially expressed genes (DEGs) in stressed vs. unstressed S. carpocapsae IJs. (C-C1, C2, C3). Hierarchical clustering and functional enrichment analysis using KOBAS databases groups DEGs into clusters based on correlations in expression trends over the experimental time course. Physiological protection mechanism imparted by emulsions for EPN survival To elucidate the protective mechanism and identify the roles of different components in the Pickering emulsions, we labeled the oil phase and titanium dioxide nanoparticles using Nile red and 5,6 carboxyfluorescein, respectively, by surface modification through amidation (EDC chemistry) ( Kotliarevski et al ., 2022 ). The SPEG silica (commercial hydrophobic particles AEROSIL® R 972) nanoparticles were not labeled due to the absence of functional groups required for surface modification. Microscopy images revealed titanium nanoparticles adhered to the EPN cuticle, presumably through physical adsorption or attachment ( Figure 1-D1 , upper panel). In contrast, control IJs suspended in water exhibited a shrunken and dried appearance at 16 h. A distinct thin red coating on formulated EPN cuticles indicated the presence of oil ( Figure 1D ). Similar behavior was noted with SPEG emulsions, where the hydrophobic nature of nanoparticles within the inverse W/O emulsion resulted in analogous red labeling patterns ( Yaakov et al ., 2022 ). Notably, this protective coating appeared only after RD, indicating its IJ protection specificity (Supplementary Figure 2). To determine if formulations retain hydration post-desiccation, the aqueous and oil phases were labeled with carboxyfluorescein and Nile red, respectively. In the control group, a layer of carboxyfluorescein was clearly deposited on the IJs ( Figure 1-D1 , bottom panel). As previously observed with TPE, IJs were coated with oil and titanium nanoparticles. In contrast, a distinct water/polymer layer surrounding the IJs was observed with SPEG ( Figure 1-D1 , bottom panel, right end). Fluorescence intensity measurements ( Figure 1-D2 ) confirmed higher carboxyfluorescein retention with SPEG (3 × 10 6 fluorescence intensity) compared to control (1 × 10 6 ) and TPE (1.2 × 10 6 ) IJs, further strengthening our findings. Moreover, IJs in SPEG retained motility after 16 h of RD at 55% RH (Supplementary video). Collectively, these findings indicate the distinct protective mechanisms of each formulation: SPEG promotes survival through hydration retention, whereas TPE primarily reduces the rate of water loss in IJs during RD. Our TEM results consistently demonstrated glycocalyx enrichment in SPEG-treated samples ( Figure 1-D3 ). Interestingly, we observed a very dark epicuticle layer in rapidly desiccated IJs compared to controls (100% RH). However, whether this is due to increased epicuticle thickness or glycoprotein accumulation is unclear. In addition, glycocalyx enrichment was apparent in TPE-treated rapidly desiccated IJs, though visualization was inconsistent and unclear due to the coating of titanium nanoparticles. Download figure Open in new tab Figure 3. Venn diagram illustrating unique and shared enriched GO processes and KEGG pathways for the differentially expressed transcripts (upregulated and downregulated) of IJs that underwent RD, and preconditioned IJs subjected to osmotic desiccation or evaporative desiccation. Differential gene expression during RD An average of 23,654,640 reads were generated using the Illumina Novaseq 6000. The reads were assembled using Bowtie software and mapped to S. carpocapsae as the reference genome from WormBase ParaSite (Bioproject ID: PRJNA202318). In unstressed IJs, a total of 92.76% reads were mapped to the reference genome compared to an average of 95.48% reads in stressed, formulated and filtered IJs, indicating the quality of the obtained RNA. A total of 30,405 transcripts were identified based on FPKM. PCA showed distinct clustering of unstressed IJs (100% RH) as compared to desiccated, formulated and filtered IJs ( Figure 3A ). Contrary to our initial hypothesis, SPEG IJs (64% RH, 24 h) clustered together with desiccated IJs, indicating their ability to sense and respond similarly to different environments (i.e., both formulation and lower humidity). This clustering suggested activation of protection mechanisms in response to stress conditions. Moreover, notable variations were observed between biological repeats, where samples from one experiment did not cluster together. Hence, an initial screening was conducted using hierarchical clustering and functional enrichment analysis compared to controls. Differentially expressed transcripts were identified using DESeq software. Desiccated, formulated and filtered IJs were each compared separately to unstressed IJs to identify the DEGs. Functional enrichment of DEGS with outliers DEGs in each treatment group were identified in comparison to unstressed nematodes (log fold change (logFC) ≥ |1.50|, P ≤ 0.05, Supplementary Table 1). Approximately 3084 genes were differentially expressed, with 51–78% of them downregulated at high humidity (85% RH) with time. The most pronounced downregulation occurred at 6 h of desiccation, consistent with Yaari et al . (2016) . In contrast, among the differentially expressed at lower humidity (64% RH), 76% of the upregulated transcripts demonstrated an inverse relationship with desiccation tolerance, as previously observed by Somvanshi et. al. (2008) . Formulated IJs exhibited significant downregulation of ∼84% of the differentially expressed transcripts, indicating adaptation similar to that observed at high humidity. To gain an overall understanding of treatment differences without compromising the statistical robustness of the data, time series clustering of FPKM data was performed using mfuzzy soft clustering tool from SR plots ( https://www.bioinformatics.com.cn/en ). The DEGs were uniquely clustered into 9 groups (cluster 1 to cluster 9) based on similarity of FPKM expression data (Supplementary figure 3). Biologically interesting patterns, such as contrasting expression trends observed at different humidities or for formulated IJs and correlation among clusters were used as indicators to combine individual clusters: clusters 1 and 5, clusters 2 and 7, and clusters 3 and 6 were combined. The combined clusters yielded 1120 DEGs, presented in a heatmap to visualize the differences in expression patterns ( Figure 2B ). Expression trends in SPEG-treated samples showed significantly higher fold changes compared to different humidity treatments ( Figure 2B ), providing direct evidence for environmental buffering as a protective mechanism in IJs. In addition, clear distinctions in expression patterns between high and low humidity were observed. At high humidity, downregulated transcripts displayed modification over 24 h with peak downregulation at 6 h, indicating saturation of specific biological processes that were “switched off” during desiccation. Conversely, at low humidity, these processes continued to function, suggesting either a delayed response mechanism or different adaptative strategies during RD. Functional enrichment reveals core and novel pathways involved in RD tolerance Functional enrichment analysis revealed an overrepresentation of distinct biological processes and pathways within the combined clusters ( Figure 2C ). Cluster 1 and 5 encompassed 488 genes that were downregulated compared to controls yet maintained relatively higher expression levels under lower humidity conditions compared to higher humidity ( Figure 2-C1 ). Those enriched at lower humidity included HSP binding (GO:0031072), endopeptidase activity, G-protein-coupled receptor activity (GO:0004930), membrane components (GO:0016020), structural constituent of cuticle (GO:0042302), extracellular region (GO:0005576) and processes related to signal transduction (GO:0007165). Functional enrichment of Cluster 2 and 7 indicates membrane organization and sarcomere assembly as critical for desiccation tolerance Cluster 2 and 7 consisted of 316 genes that were significantly upregulated compared to the control group ( Figure 2-C2 ). Their expression trends varied across different time points and treatments, with the highest expression observed in formulated treatments (SPEG) and at higher vs. lower humidity levels. This cluster likely reflects adaptive changes promoting survival during RD over time. Notably, enriched GO categories related to cellular components and biological processes, such as structural constituent of muscle (GO:0008307), A band (GO:0031672) and M band (GO:0031430), cytoplasm (GO:0005737), and sarcomere organization (GO:0060298). Previous studies have suggested cytoskeletal reorganization as essential during desiccation and anhydrobiosis ( Erkut et al., 2016 ). Thus, our findings suggest that membrane organization and sarcomere assembly represent a critical remodeling process underlying desiccation tolerance. KEGG pathway analysis further identified significant upregulation of autophagy-related processes, suggesting that clearance of damaged or aggregated cellular components is critical for desiccation survival. Functional enrichment of Cluster 3 and 6 consists of core pathways and processes involved in desiccation tolerance Cluster 3 and 6 comprised 416 genes that were significantly upregulated compared to the control group ( Figure 2-C3 ). A consistent expression trend across humidity levels indicated the fundamental role of these genes in desiccation tolerance. Enriched biological processes within these clusters included transmembrane transport (GO:0055085), cell volume homeostasis (GO:0006884), UDP-glycosyltransferase activity (GO:0008194), hydrolase (GO:0016787) and oxidoreductase (GO:0016491). In addition, membrane-, cytoplasm- and proteolysis-related processes (GO:0016020, GO:0005737 and GO:0006508, respectively) were significantly enriched. KEGG pathway analysis confirmed the involvement of established pathways associated with desiccation or anhydrobiosis, including carbon metabolism, fatty acid metabolism and carboxylate/dicarboxylate metabolism, reinforcing their critical role in desiccation adaptation. Transcriptomic evidence suggests that SPEG formulation protects IJs via environmental buffering A transcriptomic comparison of upregulated genes between desiccated IJs at high humidity (85% RH, all time points) vs. formulated IJs (at low humidity) indicated 2024 (51.23%) genes shared among all samples (Supplementary Figure 4, Table 3). The next largest shared cluster included genes that were common to SPEG and the 6 h treatment at 85% RH (721 genes, 18.00%). Differentially expressed genes unique to SPEG (361 genes, 9.14%) were significantly upregulated with enriched pathways including mucin-O-type glycan biosynthesis (Cel00512, enrichment ratio [ER]: 0.27, log P = 2.68) and ECM–receptor interaction (Cel004512, ER: 0.375, log P = 2.93). The significantly enriched cellular component GO terms included transferase activity of glycosyl groups (GO:0016757, log P = 4.84), basement membrane (BM) (GO:0005604, log P = 6.02), extracellular matrix organization (GO:0030198, log P = 4.34), ecdysis, collagen and cuticulin-based cuticle (GO:0042395, log P = 3.31), peptidyl-serine phosphorylation (GO:0018105, log P = 12.47), chromosome (GO:0005694, log P = 10.83) and protein folding (GO:0006457, log P = 5.27). Conversely, downregulated processes uniquely associated with SPEG compared to high-humidity treatments were G protein-coupled receptor signaling pathway (GO:0007186; log P = 3.00), cytoplasm (GO:0005737; log P = 6.22), membrane (GO:0016020, log P = 18.60) and integral component of membrane (GO:0016021, log P = 16.19). RD mirrors osmotic rather than evaporative desiccation processes To determine fundamental differences between rapidly desiccated and slow-desiccated/preconditioned IJs, we utilized our previous data from preconditioned Heterorhabditis spp. that were subjected to osmotic or evaporative desiccation. The comparison between osmotic and evaporative desiccation of H. bacteriophora was conducted to identify adaptive mechanisms enabling improved desiccation tolerance, ultimately enhancing storage capabilities during transport. To understand the fundamental differences between these regimes, we compared H. bacteriophora to S. carpocapsae . Specifically, IJs desiccated hygroscopically/osmotically (20% PEG, 36 h) and evaporatively (97% RH, 24 h, Satheeja et al ., unpublished results) were compared to rapidly desiccated S. carpocapsae IJs. Functional enrichment analysis was conducted, and significantly enriched GO processes and KEGG pathways (both upregulated and downregulated) were compared via Venn diagram differentiation among osmotic, evaporative and RD treatments ( Figure 3 ). Among the upregulated process, 58% (749 processes) were unique to RD, while 25% (337 processes) overlapped between osmotic desiccation and RD. Notably, only 1.4% of the processes were present in all stress conditions. These findings suggest that IJs encounter significant osmotic stress early during RD, potentially due to rapidly changing solute concentrations driven by the paracellular movement of water. Unique and shared terms for the RD, osmotic desiccation (PEG) and evaporative desiccation groups are presented in rectangular boxes in Figure 3 . Enriched terms encompassed BM (GO:0005604), extracellular matrix (GO:0031012), adherens junction (GO:0005912), hemidesmosome (GO:0030056), sarcomere (GO:0030017) and cytoskeleton (GO:0005856) organization, regulation of cell size (GO: 0008361) and integrin-mediated signaling pathways (GO:0007229). Although BM enrichment was observed during osmotic desiccation, it was not significant. Comparisons of pathways across the three desiccation conditions indicated that 34 (40.5%) of them were unique to RD, while 38 (44.18%) were shared between the osmotic and RD groups. Purine-metabolism and protein-export pathways were shared between evaporative desiccation and RD groups. Unique pathways identified in the RD group compared to previously published Steinernema data (Yaari dataset, 2016) included ECM–receptor interaction and DNA damage-response pathways such as the Fanconi anemia pathway, nucleotide excision repair and mismatch repair. Analysis of downregulated processes revealed suppression of water channel activity, proteolysis and oxidative phosphorylation (cytochrome I to IV) during RD. Interestingly, osmotic desiccation showed upregulation of water transport, water channel activity and cytochrome I, whereas cytochrome V (ATP synthase) and water transport were downregulated specifically during RD. These observations suggest that RD imposes significant metabolic and energetic demands on EPN IJs. Commonly enriched terms among all desiccation treatments included the unfolded protein response (UPR) mediated by the endoplasmic reticulum and structural components such as the striated muscle dense body (Z-disc). This emphasizes the critical roles of protein refolding and stress-signaling hubs in initiating downstream activation of stress effector proteins. Pathways common to rapidly desiccated IJs at different humidity levels Core challenges in desiccation involve the ability to perceive stress ( Erkut et al ., 2013 ), translate the perceived signals to downstream tissues, cells and organelles via signaling cascades ( Yaari et al ., 2016 ; Ma et al ., 2020 ; Wan et al ., 2021b ; Balakumaran et al ., 2022 ), neutralize reactive oxygen species (ROS) ( Erkut et al ., 2013 ) and toxins generated during desiccation, manage bioenergetics by optimizing shortcut pathways ( Penkov et al ., 2015 ; Erkut et al ., 2016 ), produce stress-protectant proteins and preserve the membrane via cytoskeleton remodeling. To identify specific pathways critical for desiccation tolerance, we screened several core stress-related pathways (Supplementary Figure 3). Carbon metabolism emerged as a central pathway for the control and bifurcation of the energy demands arising from desiccation (and other) stresses, a role consistently observed across various anhydrobiotic organisms, including plants, yeasts and nematodes ( Erkut et al ., 2016 ). Pathways such as trehalose metabolism and cytoskeletal reorganization were also identified. We further investigated well-established pathways known to be essential for desiccation tolerance, including antioxidant defense systems, detoxification pathways and stress proteins such as IDPs and HSPs. Functionally enriched KEGG pathways are provided in Supplementary Table 3. Guided by previous studies, we focused on the expression patterns of several key genes to clarify their roles in tolerance to RD as summarized in supplementary table 4. Gluconeogenesis and the Trehalose-Biosynthesis Pathway Rapidly desiccated nematodes showed enrichment of metabolic pathways previously reported in anhydrobiotic adaptation, including gluconeogenesis (ER: 0.16, log P = 2.2748), pentose phosphate pathway (non-oxidative phase), TCA cycle (ER: 0.48, log P = 9.76), glyoxylate and dicarboxylate cycle (ER: 0.41, log P = 8.45) and glyoxylate shunt. Additional enriched pathways included amino acid biosynthesis (ER: 0.30, log P = 10.26), unsaturated fatty acid biosynthesis (ER: 0.18, log P = 1.55), calcium signaling system (ER: 0.46, log P = 17.34) and ribosome biogenesis (ER: 0.34, log P = 14.05). These results indicate conserved core survival strategies regardless of environmental conditions during RD. Trehalose accumulation in formulated and non-formulated IJs Many anhydrobiotic organisms biosynthesize and accumulate trehalose during preconditioning ( Boothby et al ., 2017 ; Ma et al ., 2020 ; Wan et al ., 2021a ). We thus analyzed gene expression in the trehalose-biosynthesis pathway. The glyoxylate shunt pathway was significantly upregulated (ER: 0.42, log P = −8.457, Figure 4A ), consistent with trehalose accumulation ( Erkut and Kurzchalia, 2015 ; Erkut et al ., 2016 ). Gene-expression analyses showed significant differential expression patterns for trehalose biosynthesis genes ( tps-1, tps-2, icl-1 ) under varying humidities and in formulated treatments compared to controls at 100% RH ( Figure 4B ). Notably, ICL-1, associated with isocitrate breakdown to L-malate via glyoxylate, and referred to as glyoxylate shunt, was highly upregulated (logFC: 6.9) under low humidity compared to SPEG (logFC: 5.3). TPS-1 and TPS-2, responsible for trehalose synthesis, exhibited higher expression (logFC: 3.0) at low humidity compared to formulated SPEG IJs (logFC: 1.8), suggesting trehalose’s central role in protecting membranes and managing bioenergetics during dehydration and rehydration in RD. Download figure Open in new tab Figure 4. Mechanism of trehalose accumulation in S. carpocapsae IJs under RD. (A) Trehalose biosynthesis initiated through glyoxylate shunt metabolism highlighting key genes involved. (B) Heatmap of genes expressed in the pathway and their expression patterns under different RH conditions and time points. (C) Experimental design for validating trehalose accumulation: S. carpocapsae IJs at 85% and 64% RH and formulated IJs (64% and 54% RH) were rapidly desiccated for 0, 2, 6, 24 and 72 h (C1). Comparison of trehalose accumulation between formulated and non-formulated IJs, measured on a dry weight basis after 72 h of RD at different humidity levels (C2). (D) Schematic representation of trehalose accumulation across treatments, environmental conditions and time points. Bubble size and color indicate intensity of accumulation. (E) Proposed mechanism of trehalose accumulation in IJs during desiccation based on water-replacement hypothesis. To validate gene-expression findings, trehalose levels were quantified by UHPLC with a carbohydrate-specific column (Figure 5C-1). Trehalose accumulation varied significantly among treatments and conditions ( Figure 4C-2 ). Initially, filtered S. carpocapsae IJs had significantly higher trehalose levels than non-formulated controls (F (2,14) = 7.28732, P = 0.0068), aligning with previous studies indicating constitutive trehalose accumulation in Steinernema species under stress ( O’Leary et al ., 2001 ; Yaari et al ., 2016 ). Although trehalose levels increased over time at higher humidity, this was not statistically significant (F (3,19) = 2.2363, P > 0.05). Conversely, under low humidity, trehalose accumulation increased significantly with time (F (3,17.2) = 8.6373, P = 0.0010). At 64% RH, trehalose levels were threefold higher within 2 h than in controls (F (1,17) = 24.46, P < 0.0001) and 85% RH (F (1,37) = 15.7582, P < 0.0003). However, after 2 h, accumulation levels stabilized with no significant differences among humidity conditions. Comparisons between formulated and non-formulated IJs showed significant differences in trehalose accumulation across humidity conditions (F (4,70) = 5.532, P = 0.0006, Figure 4C-2 ). Control (100% RH) trehalose levels at 0 h were not significantly different from those of formulated samples (F (2,70) = 0.216, P > 0.05). Overall, irrespective of humidity, trehalose accumulation was highest in desiccated controls, intermediate in TPE, and lowest in SPEG (F (2,70) = 13.362, P < 0.0001). Unlike previous findings, trehalose accumulation in desiccated controls was significantly lower under harsher humidity compared to 64% RH (F (1,70) = 4.168, P = 0.0451). Membrane organization and ECM remodeling via BM assembly as critical factors for desiccation tolerance Enriched GO biological processes identified during RD included cytoskeletal reorganization, actin dynamics, cell volume homeostasis and KEGG pathways such as ECM–receptor interaction. Comprehensive data mining revealed differences in gene expression related to cytoskeleton, sarcomere and ECM organization which were visualized using heatmaps ( Figure 6D ). Notably, BM-related genes exhibited differential expression across humidity conditions and in the SPEG formulation. Download figure Open in new tab Figure 6: Impact of membrane organization and ECM remodeling of S. carpocapsae IJs during rapid desiccation (RD) assessed by ultrastructural microscopy. (A) Transverse section of nematodes illustrating ECM layers (cuticle, hypodermis/hemidesmosome and basement membrane [BM]); image courtesy Wormbook. (B1 & 2) Rapid shrinking of S. carpocapsae IJs after 2.5 h exposure to 43% RH. (C) Effect of RD on cuticle thickness, especially the epicuticle (calculated from cryo-TEM images) and annulation spacing (calculated from SEM images). (D) Heatmap of enriched processes, such as BM, hemidesmosome, focal adhesion and ECM. (E) Cryo-TEM images of desiccated IJs after 24 h at 54% RH. Treatments include control (100% RH), rapidly desiccated IJs and formulated IJs (TPE and SPEG). Green arrowheads indicate BM; orange arrowheads indicate sarcomere assembly. (F) Network analysis of genes associated with BM, ECM and hemidesmosome (HD) positively correlated to RD. Different colors represent distinct GO terms. (G) Proposed protective mechanisms involved in control vs. formulated IJs. To validate these transcriptomic findings, cryo-TEM imaging was used to compare the membrane organization in rapidly desiccated IJs (formulated and non-formulated) at low humidity ( Figure 6E ). Sarcomere organization was severely disrupted in all RD treatments compared to controls (100% RH). Membrane organization was particularly disrupted in rapidly desiccated IJs treated with water and TPE, while notably preserved in SPEG-treated IJs. This suggests that membrane structural plasticity significantly influences nematode survival under abiotic stress conditions. In addition, we observed structural integrity differences in the BM. Rapidly desiccated IJs exhibited extensive damage below the dense Z-disc layer along the circumferential axis, a pattern that was absent in TPE- and SPEG-treated IJs, which retained intact ECM structures. This finding aligns with the survival data, reinforcing the importance of intact ECM for nematode survival, while tissue integrity and membrane organization are essential for IJ efficacy. Further analysis explored changes within ECM layers, specifically targeting the cuticle. Genes associated with structural components of the cuticle, their maintenance, turnover and synthesis were predominantly downregulated, as reported previously ( Erkut et al ., 2013 ). However, genes involved in cuticle crosslinking were upregulated (Supplementary Figure 5). We hypothesized that if collagen-crosslinking within the cuticle or ECM contributes to desiccation tolerance, evidence could be obtained through measurements of cuticle thickness. To validate this, cuticle thickness across layers (epicuticle, cortical and median layers combined, basal layer, hypodermis) was quantitatively measured via cryo-TEM ( Figure 6C-1 ). Treatments significantly influenced overall cuticle thickness (F (3,23) = 6.6008; P = 0.0028), which was significantly greater in rapidly desiccated IJs compared to TPE ( P = 0.00081; 95% CI: 284.45, 384.15), SPEG ( P = 0.0166;95% CI: 311.26, 375.98) and controls (100% RH, P = 0.0116; 95% CI: 246.79, 372.26). No significant differences were detected among TPE ( P = 0.9099), SPEG ( P = 0.7987) and control groups, suggesting a protective effect of the emulsions. Epicuticle thickness varied significantly among treatments (F (3,23) = 49.5867; P < 0.0001), with rapidly desiccated IJs showing a 154% increase compared to controls (100% RH, P < 0.0001; 95% CI: 3.907, 19.35), TPE ( P < 0.0001; 95% CI: 10.603, 15.287) and SPEG ( P 0.05), emphasizing epicuticle thickness modulation as critical for maintaining a physiological barrier against water loss, consistent with previous studies ( O’Leary et al., 1998 ; Patel and Wright, 1998 ). The hypodermis is the thin fiber sheet-like layer present just below the basal zone of the cuticle. Our data suggest that hypodermis thickness increased by approximately 65% (data not shown), indicating possible active collagen-crosslinking within the hypodermis and BM of ECM layers. Annulation spacing, measured by scanning electron microscopy (SEM), for IJs that were rapidly desiccated at 74% RH ( Figure 6C-2 ) demonstrated significant differences compared to controls (100% RH) (F (2, 29) = 74.63; P < 0.0001), similar to Wharton (1988, 2013). Both high and low IJ concentrations demonstrated significant spacing reductions (∼37% and 21%, respectively) compared to controls (100% RH, P < 0.0001). Furthermore, images ( Figure 6B1-B2 ) revealed pronounced volume loss within the IJ sheath after 2.5 h under RD conditions, a response expected to intensify with decreasing humidity. Discussion We investigated the physiological, molecular and biochemical strategies underlying S. carpocapsae survival during RD. We previously reported that S. carpocapsae IJ survival drastically declines below 64% RH, rendering them ineffective for infection (and therefore, biocontrol) ( Ramakrishnan et al., 2022 ). Here, we assessed physiological responses, including rates of survival and water loss, in formulated and non-formulated IJs under low humidities (64% and 54% RH), and the formulations’ protection mechanisms. We further investigated molecular strategies through transcriptomic analyses, comparing results to previous slow-desiccation studies ( Yaari et al., 2016 and unpublished Heterorhabditis data) to understand the fundamental differences between slow and rapid desiccation. We then validated our novel findings through biochemical (trehalose accumulation) and ultrastructural approaches. Physiological basis of survival and protection mechanisms provided by formulations We systematically characterized the physiological, biochemical, molecular and protective roles of two Pickering emulsions: TPE (O/W) and SPEG (W/O) during RD of IJs. In-vitro results demonstrated extended IJ survival through reduced water-loss rates, particularly at humidity below 64% RH, with survival prolonged to 144 h compared to 16 h in non-formulated controls. These findings, along with the variability observed in survival (post-72 h), highlight the protective efficacy of the formulations and indicate subtle yet significant temporal differences in their performance under RD stress. The observed survival differences correlated well with the gravimetric and FTIR data. Specifically, FTIR analysis revealed distinct hydration dynamics for TPE and SPEG formulations: TPE showed faster water loss compared to SPEG, yet still maintained IJ survival effectively until 120 h. These results were reinforced by a significant increase in efficacy with SPEG, as indicated in Ramakrishnan et al . (2023) . This raised the question of how TPE and SPEG formulations protect EPN IJs. Direct examination of the mechanisms underlying these protective differences by confocal microscopy showed that TPE delays water loss, whereas SPEG retains the water phase. TPE is an O/W emulsion, with water as the major phase. Its protective capability arises primarily from a coating of oil and nanoparticles on the IJs, which reduces water permeability— as supported by our epicuticle thickness results—and consequently slows water loss, similar to the mechanism observed in Ditylenchus dipsaci , a foliar plant-pathogenic nematode ( Wharton et al ., 2008 ). In contrast, SPEG is a W/O emulsion that protects IJs by maintaining hydration through a superabsorbent polymer layer, which may be further stabilized by an additional oil or nanoparticle layer, creating a layered protection system ( Figure 2-E ). Biochemically, trehalose accumulation in formulated IJs increased consistently until 72 h of RD but remained lower than in non-formulated rapidly desiccated IJs. TPE and SPEG also exhibited contrasting accumulation patterns, further validating their distinct protective mechanisms and corresponding gene-expression trends. The variation in trehalose accumulation between the two emulsions suggests that until 72 h, the protective effect of SPEG primarily stems from its ability to retain hydration, whereas the protection conferred by TPE is largely due to its surface coating, as supported by cytoskeleton integrity observations. Both formulations preserved ECM integrity, thereby enabling survival of the EPN IJs. Glycocalyx enrichment and involvement of Mucin-O-glycan pathway Transcriptome analysis revealed that SPEG activates pathways similar to those activated in desiccated IJs but physiologically resembles the response of IJs at 85% RH, notably involving oxidation–reduction processes and DNA-repair systems. Interestingly, mucin-O-glycan biosynthesis and glycosyltransferase were uniquely enriched in SPEG samples. Specifically, GalNac (N-acetylgalactosyltransferase; Tn antigen)—encoded by gly-7 and initiating the mucin-biosynthesis pathway by synthesizing core 1 O-glycans—showed significant enrichment (logFC: 6.2) (Supplementary Table 5). Moreover, several other genes related to this pathway exhibited FCs of at least 1.5 across all humidity conditions (Supplementary Table 4). Mucins are high-molecular-weight serine–threonine backbone-containing O-glycosylated proteins present in the cuticle and surface coat (glycocalyx) of many animal- and plant-parasitic nematodes ( Blaxter and Bird, 1997 , D. Bird and T.J Chen, unpublished data, Phani et al ., 2018 ). In nematodes, mucin biosynthesis is contested between the secretory–excretory system ( Khoo et al ., 1991 ), and hypodermis or seam cells ( Wright, 1987 ; Gravato-Nobre et al ., 2011 ). Several reports suggest their role in adhesion, hydrogel-forming abilities and protective properties ( Casaravilla et al., 2003 ), conferring a negative charge to the cuticle due to their polyanionic nature ( Zuckerman, et al ., 1979 ), while RNAi targeting of mucin genes leads to reduced adherence of endospores ( Phani et al ., 2018 ; Davies et al ., 2023 ). Although mucins have not previously been reported in Steinernematidae, wheat-germ agglutinin lectin-binding assays revealed carbohydrate moieties throughout the cuticle of S. carpocapsae , whereas they were restricted to the anterior tip in H. bacteriophora (Wray Carl Hansen (III) master’s thesis, Ment lab unpublished results; Glazer et al ., 2024 ), similar to observations from studies on Caenorhabditis elegans ( Laughlin and Bertozzi, 2009 , McClure and Zuckerman, 1982 ; Zuckerman and Kahane, 1983 ; Link et al., 1988 ; Link et al ., 1992 ) and Pristionchus pacificus ( Sun et al ., 2022 ). Proteins encoded by let-653 are structural constituents of the cuticle, and serine/threonine-rich proteins mimicking mammalian mucins cause larval lethality when mutated in C. elegans ( Jones and Baillie, 1995 ). These proteins were significantly upregulated in SPEG (logFC: 3.1) and at 85% RH (logFC: 3.0) compared to RD at lower humidity (logFC: 2.5). Moreover, mucin-related genes were similarly upregulated under desiccation stress in Ditylenchus destructor ( Ma et al ., 2020 ), reinforcing their critical role in nematode desiccation tolerance. Based on TEM-observed glycocalyx enrichment, validating the transcriptomic observations, we propose that the highly negatively charged glycoproteins present in the glycocalyx attract the positively charged polymer layers in the formulations via electrostatic interactions, resulting in the retention of a water-rich layer on the IJ cuticle. Supporting this hypothesis and observations by Phani et al . (2018) , we propose that electrostatic interactions between the cuticle and polymer hydroxyl groups are influenced by membrane-associated mucins and steric effects of surface-protruding long glycoprotein chains, as evidenced by changes in epicuticle thickness and sarcomere integrity. Rapidly desiccated vs. preconditioned IJs Comparative analysis of enriched pathways and processes in preconditioned and rapidly desiccated IJs revealed that RD requires ∼50% more metabolic processes and pathways for adaptation, indicating a markedly high bioenergetic cost for the rapid stress response. A noteworthy finding was the similarity of processes activated between RD and later time points (36 h) of osmotic desiccation with PEG, where shared processes were marked by the upregulation of water-transport mechanisms. This suggests that in hygroscopic desiccation, osmotic pressure equilibrates with the external medium. Conversely, in RD, the sudden changes in solute concentrations lead to severe osmotic imbalance, primarily driven by rapid paracellular water movement. This acute imbalance is correlated with the observed downregulation of water-channel transport, reflecting a severe metabolic and osmotic impact within a short period. Consequently, the protective mechanisms during RD are rapidly activated, underscoring heightened metabolic demand to counteract rapid water loss and ensure survival. The minimal overlap between evaporative desiccation and RD indicates that gradual physiological adaptations at higher humidity (97% RH) differ significantly from those elicited by rapid exposure to lower humidity (85% or 64% RH). This highlights the differences between various desiccation regimes and the corresponding variations in phenotypic tolerance observed among EPNs ( Ramakrishnan et al ., 2022 ). Interestingly, for the common pathways shared by all types of desiccation conditions, we observed significant enrichment of the IRE-1-mediated UPR in the endoplasmic reticulum and Z-discs. This indicates that protein misfolding and aggregation are common stress responses across various desiccation conditions. Furthermore, Z-discs, serving as structural anchors linking the cuticle to the cytoskeleton, may act as critical signaling hubs (Lecroisey et al., 2007, Brouilly et al ., 2015 ), potentially activating downstream protective effector proteins and pathways. It is also important to note that processes involving BM integrity and mechanosensory responses were enriched and upregulated during osmotic desiccation, albeit not significantly. During RD, both terms showed significant enrichment, highlighting a heightened physiological response to harsher conditions. Collectively, these findings suggest that RD represents a unique and highly demanding form of desiccation stress, characterized by significant bioenergetic costs and extensive damage to structural and biomechanical integrity, crucial aspects determining the organism’s survival capacity. Core pathways and strategies for survival under desiccation stress A major (desiccation-survival) strategy involves accumulation of hydrophilic proteins termed hydrophilins or IDPs ( Erkut et al ., 2013 ; Koshland and Tapia, 2019 ). During RD, we observed significant upregulation of LEA and DUR ( Table 1 ), indicating that IDPs uniquely prevent or stabilize biomolecular entropy changes induced by desiccation, acting as molecular shields that prevent interactions (Goyal et al., 2007, Gal et al., 2005 ). In contrast, tardigrades produce diverse groups of IDPs that stabilize cellular structures through gelation (Shraddha KC et al., 2024). Although significant gaps remain in our understanding of the physiological roles and cooperative mechanisms of these protective proteins, cytoskeletal reorganization and its potential role in preserving or stabilizing tissues, membranes and organelles during anhydrobiosis remain key areas for further investigation View this table: View inline View popup TABLE 1. Significantly upregulated and downregulated genes involved in detoxification, protein folding and heat-shock responses during rapid desiccation Trehalose accumulation does not guarantee survival Trehalose acts as a chemical chaperone and is known for its high thermodynamic stability ( Crowe, 2007 ) and protection of membranes and proteins via “water-replacement theory” ( Crowe et al ., 1989 ; Erkut, 2011 ; Erkut et al ., 2013 ) and/or water entrapment and vitrification (formation of bioglass) ( Crowe, Carpenter and Crowe, 1998 ; Boothby et al ., 2017 ; Nguyen et al ., 2022 ; Ramirez et al ., 2023 ). Intracellular accumulation of trehalose has been documented under various stresses. Alternatively, a diverse array of protectants have also been reported among metazoans ( Tunnacliffe et al., 2005 , Nguyen et al ., 2022 ; Sanchez-Martinez et al ., 2023 ; Barilla et al., 2024 ). Bioprotection strategies vary among EPN species, e.g., preference for glycerol in S. carpocapsae and S. feltiae to trehalose accumulation in H. bacteriophora ( O’Leary et al ., 2001 ; Gal, Glazer and Koltai, 2003b ). Erkut et al . (2013) suggested a minimum required threshold of trehalose accumulation during preconditioning in nematodes. Our findings suggest significant variations in trehalose accumulation over time. Correlating trehalose accumulation with prior results of survival and water-loss rates ( Ramakrishnan et al ., 2022 ), we concluded that the initial 6 h of RD are critical in delaying water loss, indicating that timing of accumulation is crucial. During this window, organisms enhance permeability barriers, lipid packing and membrane stability, providing additional time to activate protective mechanisms, including trehalose accumulation. Abusharkh et al . (2014) found that polar headgroups preferentially switch to phosphotidylethanolamine, facilitating trehalose insertion to replace hydrogen bonds. Our results show rapid trehalose accumulation at low humidity but also low survival, indicating a lower protective effect of trehalose insertion and suboptimal membrane stabilization. Conversely, at higher humidity, membrane fatty acids are surrounded by hydration shells providing optimal membrane adjustments for high survival, as depicted in Figure 5E. Does rapid trehalose accumulation negatively impact chaperone activity or indicate a shift in protection strategy? There are conflicting reports on trehalose accumulation and survival in yeast desiccation during the stationary phase ( Ratnakumar and Tunnacliffe, 2006 ). In Escherichia coli, higher trehalose concentration leads to overstabilization and rigid protein conformations, resulting in increased aggregation and precipitation, and reduced survival, attributed to the destabilization effects of these osmolytes on proteins ( Singh et al ., 2011 ; Łupkowska et al ., 2023 ; Kuczyńska-Wiśnik, et al., 2024 ). Recent evidence also suggests water entrapment as a viable protective strategy for osmolytes such as sucrose at physiologically low water contents ( Stachura et al., 2019 ). Moreover, even highly desiccation-tolerant organisms require mild adaptation periods ( Rappaport and Oliverio, 2024 ). Thus, under increasingly severe conditions, chemical chaperones may shift their protective mechanism toward water entrapment. Other novel mechanisms identified in this study BM assembly and ECM remodeling in desiccation tolerance BMs are thin, dense extracellular layers composed of collagen and laminin, critical for intracellular structural rigidity as they cover tissues and link the cuticle to cytoskeleton ( Jayadev and Sherwood, 2017 ; Jayadev et al ., 2022 ). BMs are poroelastic with ∼20 nm pore size and continuously remodeled throughout development and stress ( Elosegui-Artola, 2021 ; Khalilgharibi and Mao, 2021 ). Hence, BMs’ serve as sensitive indicators of mechanical stress that directly impacts ECM stiffness by altering its composition(i.e., collagen, laminin and glycoprotein/ proteoglycan) ( Khalilgharibi et al ., 2016 ; Guimarães et al ., 2020 )( Töpfer et al ., 2022 ). Hence under desiccation, the differential fluid pressure could result in rearrangement of water within the BM ( Fabris et al ., 2018 ) and critical for intracellular membrane organization. Specifically, this mechanical force transduction could be moderated during preconditioning where the rate of change in cell volume and tissue is slower. However, during RD, rapid (within 6 h) IJ shrinkage likely causes severe mechanical stress, surpassing the BM’s strain threshold below 64% RH, and potentially leading to structural damage and cell detachment. The altered ECM stiffness through its degradation, consequently impaired cell attachment and reduced survival ( Vafaie et al ., 2014 ). Our cryo-TEM results support this hypothesis, showing mechanical disruption in non-formulated IJs but intact BM structures in formulated IJs under RD, highlighting the critical importance of BM integrity for IJ survival. Transcriptomic analyses revealed downregulation of structural cuticle-related genes during RD, suggesting that remodeling existing structures may be more energy-efficient than synthesizing new materials under stress. Hence, collagen remodeling could be desiccation-adaptation strategy as previously identified ( Page and Winter, 2003 ; Adhikari, Wall and Adams, 2009 ; Reardon et al ., 2010 ; Page et al ., 2014 ), similar to our results in Heterorhabditis (data not shown). Notably, collagen genes col-77 (logFC: 2.5–3.2) and col-54 (logFC: 0.5–2.5), known structural constituents of the cuticle, were significantly upregulated. Collagen crosslinking in the ECM likely involves matrix metalloproteinases (MMPs), were upregulated in RD data. Among 50 identified MMP-related genes, 16 were significantly upregulated (logFC ≥ 1.5), while 9 were downregulated, suggesting a crucial role for MMP-mediated ECM remodeling as a desiccation-tolerance mechanism (Supplementary Figure 6) ( Park et al ., 2010 ; Gomis-Rüth, Trillo-Muyo and Stöcker, 2012 ; Marrone et al ., 2012 ; Page et al ., 2014 ). Specifically, astacin-related MMPs, evolutionarily conserved zinc metalloproteases were upregulated in our data and are involved in longevity and stress responses ( Park et al., 2010 ). S. carpocapsae contains 15 homologues of C. elegans astacin-related proteins (Supplementary Table 6). The top 3 expressed MMP with lfc-2.7-3.9 were nas-27 expressed in hypodermis, nas-13 expressed in muscle cells (supplementary results). Mechanotransduction could act as an indicator in desiccation-sensing Involvement of mechanosensory pathways in hydration-sensing was hypothesized by Thunberg in 1905 but only recently proven ( Russell et al ., 2014 ). Genes involved in osmolarity-sensing, such as osm-9 , ocr-2 and osm-11 , expressed in ASH head neurons, are also implicated in hydration-sensing, suggesting shared molecular mechanisms between the two functions ( Erkut et al ., 2013 , Erkut et al., 2016 ). Shrinkage of EPN IJs associated with cell volume and shape changes during RD has been reported previously ( Glazer, 1992 ; Tyson et al ., 2012 ). Behavioral adaptations such as coiling ( Solomon, Paperna and Glazer, 1999 ; Shannon et al ., 2005 ; Wharton and Aalders, 2013 ) and reduced annulation spacing ( Wharton et al ., 1988 ; Wharton, 1996 ) suggest that muscle contraction is a stress-induced response. Muscle contractions transmit mechanical forces intracellularly via dense bodies (Z-discs) and fibrous organelles connecting muscles to the cuticle ( Cox and Hardin, 2004 ; Teuscher et al ., 2019 , 2024 ; Ewald and Nyström, 2023 ). Our current dataset indicates significant enrichment of fibrous organelles, emphasizing the potential importance of mechanical stress and force transduction in enhancing desiccation responses. Recent research has suggested that C. elegans body-wall muscles act as mechanoreceptors for signal transduction independently of traditional degenerin ion channels ( Yan et al ., 2020 ). Our cryo-TEM analysis revealed substantial structural loss and reorganization of body-wall muscles and Z-discs under RD conditions. The Z-discs connect body-wall muscles to the ECM and exoskeleton (cuticle) and host essential cell-surface receptors for intracellular signaling ( Jayadev et al ., 2019 ; Teuscher et al ., 2024 ). The ECM–receptor interaction pathway (Cel: 04512) indicated enrichment of collagen–, laminin– and integrin–receptor interactions (Supplementary Figure 7). This pathway is recognized for transmitting mechanical stress signals and activating downstream signaling cascades, including PI3K-AKT and MAPK pathways, regulating cytoskeletal reorganization and cell survival ( Sun et al., 2016 ; Kanchanawong and Calderwood, 2023 )( Aikawa et al ., 2002 ). Thus, alongside head neuron-mediated hygrosensation, we propose mechanotransduction from muscle contractions or size reduction as a potential mechanism activating downstream signaling for desiccation tolerance. However, the specific molecular and genetic foundations of mechanotransduction in desiccation-sensing require further validation. Conclusion The last decade of EPN research has focused on improving application and formulation systems (Stock et al., 2025). In this study, we expanded upon this research by exploring EPN biology under RD conditions and examining the protective effects of IJ formulations. Our findings demonstrate that RD is a unique form of desiccation stress, characterized by significant bioenergetic demands and substantial disruptions to mechanical stability in IJs. Using physiological, biochemical, molecular and ultrastructural analyses, we showed that RD adversely affects ECM integrity by disrupting BM assembly and cytoskeletal organization. Utilizing protected IJs as “gain-of-function” models, we confirmed that preserving ECM and sarcomere integrity by reducing water-loss rates is crucial for IJ survival and efficacy under RD conditions. This study thus provides fundamental insights into the biological mechanisms underpinning EPN tolerance and survival strategies during RD. Materials and Methods EPNs The original EPN populations were provided by e-nema GmbH (Schwentinental, Germany). S. carpocapsae-All strain were cultivated in the last instar larva of Galleria mellonella at 23°C ( Ramakrishnan et al. 2022 ). The infected larvae were transferred to a modified White trap, and emerging IJs were collected and stored at 8°C until further use. Formulation treatments SPEG was prepared from commercial hydrophobic silica (Aerosil R972, Evonik, Germany, fumed silica treated with dimethyldichlorosilane, estimated primary particle size of 16 nm) in paraffin oil and water (analytical grade; Sigma-Aldrich, St. Louis, MO, USA). Silica nanoparticles were dispersed in paraffin oil by sonication for 5 min (Sonics Vibra-Cell 750 W, 25% amplitude) with a silica content of 0.5 wt%. A solution of 0.5% potassium polyacrylic acid (0.5 g completely dissolved in 99.5 mL water by stirring for 1–2 h; Ramakrishnan et al., 2023 ) was added at a water-in-oil (W/O) ratio of 40:60 by volume and the mixture was sonicated for 10 min (at 25% amplitude) for emulsification. TPE was derived from amine-functionalized titania in water and mineral oil. The titania-NH 2 nanoparticles were sonically dispersed in deionized water for 5 min at1 wt%. Next, mineral oil was added at oil-in-water (O/W) ratio of 4:6 by volume. The mixture was sonicated for 10 min at 25% amplitude to achieve emulsification ( Kotliarevski et al ., 2022 ). S. carpocapsae IJs were vacuum-filtered, suspended in distilled water and adjusted to a concentration of 1000 nematodes/mL. The suspension was centrifuged at 4000 g for 2 min and the pellet was vaccum filtered. An required volume of formulation—SPEG (W/O) or TPE (O/W) emulsion—was suspended with IJ pellet. This procedure was followed unless otherwise mentioned. Survival of formulated EPN in vitro Survival of S. carpocapsae that had been rapidly desiccated at 65% or 55% RH was estimated and compared as described previously ( Ramakrishnan et al., 2022 ) with some modifications. To establish stable 64% RH, saturated calcium chloride dihydroxide salt and sodium nitrite were kept at opposite ends of the humidity chamber. Approximately 5000 vacuum-filtered IJs were suspended in 100 µL of treatment solution (water, TPE or SPEG), spotted on Teflon discs and dried in a laminar airflow chamber for 20–45 min to remove excess water. Samples were transferred to a desiccator at 65% or 55% RH maintained at 23°C with continuous humidity monitoring by data loggers (LOG32TH and SSN23E). Samples were collected at 0 (immediately after drying), 24, 48, 72, 96, 120 and 144 h of RD. Samples were excised from the disc, suspended in distilled water and incubated overnight to assess nematode survival rate by randomly counting the number of live or dead IJs out of 200 IJs under a binocular (Olympus SZH10, 30× magnification). IJs were scored as live based on active movement in response to probing. Data were used to calculate survival rate as percentage of total number of nematodes. The experiment was repeated three times with five replicates per treatment. Evaluating EPN water content by gravimetric method Water content of EPNs during RD was estimated using the gravimetric method described by Ramakrishnan et al ., (2022) with slight modifications. Water or emulsions (120–150 µL) were drop-casted on slides with Teflon sheets, and IJs (25,000–50,000) were vacuum-filtered and gently transferred to the slides. This prevented any loading errors for IJs and emulsions. The samples were dried under laminar airflow for 20 min, and set as the 0 h samples; these were weighed and transferred to desiccators maintained at the specified RHs (64 ± 5% or 54 ± 4%). Control treatments consisted of 120–150 µL of water or emulsions that were similarly processed. All samples and controls were weighed at 24 h. Gravimetric measurements post-24 h were not reliable due to negative values obtained for SPEG samples when the negative control (SPEG emulsion without IJs) was subtracted. Dry weight was estimated by oven-drying at 90 or 105°C for 12 h or 3 h, respectively. Water weight was calculated by subtracting the dry weight of the nematode samples from the total estimated weight at different time points. The weight of the control samples was also deducted to remove the effect of residual water or emulsions on water loss. Hence, the water content of nematodes estimated as weight (mg) was expressed as a percentage (%), i.e., normalized body water content, using the formula: where, weight 0 is the initial sample weight at 0 h and weight 24 is the sample weight at 24 h. Total RNA extraction Samples of rapidly desiccated IJs from the different treatments (nematodes in water [100% RH], filtered IJs, formulated IJs), RHs (85%, 64%), time points (0, 2, 6, 24 h) were collected in tubes with 500 µL TRI reagent (Sigma-Aldrich), flash-frozen in liquid nitrogen and kept at −80℃ pending extraction. Samples were homogenized in a Geno/Grinder at 1500 rpm for 1 min 30 s (repeated four times with 15-s intervals between pulses), and extracted following the TRI reagent protocol. Total RNA was further purified with a Zymo RNA Clean and Concentrator Kit as per the manufacturer’s instructions. RNA quality was checked in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and on a 1.5% agarose gel. Samples displaying clean 18S and 25S bands were selected and shipped for Illumina (San Diego, CA, USA) sequencing. Bioinformatics Analysis Approximately 496 million paired-end reads (average 18.4 million per sample) were mapped to the reference genome of S. carpocapsae (PRJNA202318; https://parasite.wormbase.org/ ) using STAR software (v2.7.10a) (Dobin et al., 2012). Gene abundance was estimated using Cufflinks (v. 2.2) (Trapnell et al., 2010) combined with gene annotations from the Wormbase database. Principal component analysis (PCA) and heatmap visualization were performed in R Bioconductor (Gentleman et al., 2004). Gene-expression values were computed as fragments per kilobase of transcript per million mapped reads (FPKM). Differential expression analysis was performed with the DESeq2 R package (Love et al., 2014). Genes with ≥2-fold differential expression and a false discovery-corrected statistical significance of at most 0.05 were considered differentially expressed (Benjamini and Hochberg, 1995). KOBAS 3.0 tool (Xi et al., 2011) ( http://kobas.cbi.pku.edu.cn/kobas3/?t=1 ) was applied to determine the statistical enrichment of differentially expressed genes (DEGs) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) processes. Venny tool was utilized to assess the unique and shared differentially expressed transcripts under different conditions. SR plots ( https://www.bioinformatics.com.cn/en ) were used to generate hierarchical clustering via the mfuzzy clustering tool with default settings, and heatmaps were generated from the SR plots. Network Analysis Cytoscape software and CoNet plugin were used to analyze interaction networks. Pearson correlation coefficient (r) was used to analyze interactions among the genes and was set to 0.8. Genes were represented as nodes, with color indicating the corresponding GO category. Other biochemical and microscopy bioassays are provided in the supplementary methods (See supplementary section) Statistical analysis Data from repeated experiments were combined to analyze the differences in formulated vs. control IJ survival. Survival data were arcsine-transformed and subjected to ANOVA, and significant differences among pairs were further elucidated through Tukey-HSD or Student’s t-test comparisons. Water-loss measurements were subjected to two-way ANOVA comparisons. Results for trehalose accumulation in rapidly desiccated IJs were subjected to ANOVA and test slices were carried out for post hoc comparison by humidity and time point. Trehalose accumulation at 0 h was analyzed by one-way ANOVA followed by Tukey multiple comparisons. Trehalose accumulation for formulated IJs were subjected to ANOVA and significant differences among each pair were further elucidated through Tukey-HSD or Student’s t-test comparisons. Data related to cuticle and epicuticle thickness and annulation spacing were tested by one-way ANOVA with means and compared using Tukey multiple comparison post-hoc. All comparisons were carried out with JMP statistical software. Functional enrichment of Cluster 2 and 7 indicates membrane organization and sarcomere assembly as critical for desiccation tolerance Cluster 2 and 7 consisted of 316 genes that were significantly upregulated compared to the control group ( Figure 3-C2 ). Their expression trends varied across different time points and treatments, with the highest expression observed in formulated treatments (SPEG) and at higher vs. lower humidity levels. This cluster likely reflects adaptive changes promoting survival during RD over time. Notably, enriched GO categories related to cellular components and biological processes, such as structural constituent of muscle (GO:0008307), A band (GO:0031672) and M band (GO:0031430), cytoplasm (GO:0005737), and sarcomere organization (GO:0060298). Download figure Open in new tab Figure 3. Quality assessment and preliminary screening of transcriptome data. (A) PCA depicting the clustering patterns of stressed, protected and control IJs. (B) Heatmap illustrating the differentially expressed genes (DEGs) in stressed vs. unstressed S. carpocapsae IJs. (C-C1, C2, C3). Hierarchical clustering and functional enrichment analysis using GO and KOBAS(KEGG?) databases groups DEGs into clusters based on correlations in expression trends over the experimental time course. Previous studies have suggested cytoskeletal reorganization as essential during desiccation and anhydrobiosis ( Erkut et al., 2016 ). Thus, our findings suggest that membrane organization and sarcomere assembly represent a critical remodeling process underlying desiccation tolerance. KEGG pathway analysis further identified significant upregulation of autophagy-related processes, suggesting that clearance of damaged or aggregated cellular components is critical for desiccation survival. Functional enrichment of Cluster 3 and 6 consists of core pathways and processes involved in desiccation tolerance Cluster 3 and 6 comprised 416 genes that were significantly upregulated compared to the control group ( Figure 3-C3 ). A consistent expression trend across humidity levels indicated the fundamental role of these genes in desiccation tolerance. Enriched biological processes within these clusters included transmembrane transport (GO:0055085), cell volume homeostasis (GO:0006884), UDP-glycosyltransferase activity (GO:0008194), hydrolase (GO:0016787) and oxidoreductase (GO:0016491). In addition, membrane-, cytoplasm- and proteolysis-related processes (GO:0016020, GO:0005737 and GO:0006508, respectively) were significantly enriched. KEGG pathway analysis confirmed the involvement of established pathways associated with desiccation or anhydrobiosis, including carbon metabolism, fatty acid metabolism and carboxylate/dicarboxylate metabolism, reinforcing their critical role in desiccation adaptation. Transcriptomic evidence suggests that SPEG formulation protects IJs via environmental buffering A transcriptomic comparison of upregulated genes between desiccated IJs at high humidity (85% RH, all time points) vs. formulated IJs (at low humidity) indicated 2024 (51.23%) genes shared among all samples (Supplementary Figure 4, Table 3). The next largest shared cluster included genes that were common to SPEG and the 6 h treatment at 85% RH (721 genes, 18.00%). Genes(DEGs?) unique to SPEG (361 genes, 9.14%) were significantly upregulated with enriched pathways including mucin-O-type glycan biosynthesis (Cel00512, enrichment ratio [ER]: 0.27, log P = 2.68) and ECM–receptor interaction (Cel004512, ER: 0.375, log P = 2.93). The significantly enriched cellular component GO terms included transferase activity of glycosyl groups (GO:0016757, log P = 4.84), basement membrane (BM) (GO:0005604, log P = 6.02), extracellular matrix organization (GO:0030198, log P = 4.34), ecdysis, collagen and cuticulin-based cuticle (GO:0042395, log P = 3.31), peptidyl-serine phosphorylation (GO:0018105, log P = 12.47), chromosome (GO:0005694, log P = 10.83) and protein folding (GO:0006457, log P = 5.27). Conversely, downregulated processes uniquely associated with SPEG compared to high-humidity treatments were G protein-coupled receptor signaling pathway (GO:0007186; log P = 3.00), cytoplasm (GO:0005737; log P = 6.22), membrane (GO:0016020, log P = 18.60) and integral component of membrane (GO:0016021, log P = 16.19). Acknowledgements We would like to acknowledge and thank Reut Amar Feldbaum and Michael Brichka for providing emulsions, Dr. Einat Zelinger, Dr. Tally Kossovsky for assistance in Cryo-TEM sample preparation and imaging. Our heartfelt gratitude and thanks to Dr. Hillary Voet for statistical consultations. This study was partially funded by the Chief Scientist of the Israeli Ministry of Agriculture (project number 20-06-0085) and US-Israel Binational Agricultural Research and Development Fund (BARD IS-5183-19). 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