Small heat shock proteins with two alpha-crystallin domains: a new set of proteins in the earthworm Eisenia fetida with differential transcriptional responses to stressors

Small heat shock proteins with two alpha-crystallin domains: a new set of proteins in the earthworm Eisenia fetida with differential transcriptional responses to stressors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Small heat shock proteins with two alpha-crystallin domains: a new set of proteins in the earthworm Eisenia fetida with differential transcriptional responses to stressors NATASHA TILIKJ, Mercedes de la Fuente, Alejandro Martínez Navarro, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6718926/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Climate change and the growing environmental pollution are two of the main challenges that life faces today. Soil invertebrates such as earthworms are increasingly exposed to stress from climate change (e.g., rising temperatures and drought) and chemical pollution. The heat shock proteins are central to the stress response. Eisenia fetida, a widely used model for soil ecotoxicology, relies on molecular chaperones like small heat shock proteins (sHSPs) for stress tolerance. Furthermore, sHSPs generate interest for their potential as molecular indicators of soil pollution and for thermotolerance acquisition. Previously, we have described a set of genes coding for sHSPs with a single ACD. Here we report the first identification of sHSPs with multiple α-crystallin domains (ACDs) in an annelid. These genes were isolated from an E. fetida transcriptome, their domain architecture was defined, and their expression was analyzed under environmental stressors: heat shock, desiccation, and exposure to the pollutants bisphenol A (BPA) and endosulfan. Gene expression patterns were stimulus-specific. Prolonged sub-lethal heat and desiccation each induced distinct subsets of the multi-ACD sHSP genes, highlighting their tailored roles in abiotic stress. In contrast, exposure to BPA at optimal conditions did not produce a response, while endosulfan produced a minimal response. Combined exposure to endosulfan and elevated temperature triggered a significant upregulation of these chaperone genes indicating synergetic stress. This work relates the response of dimeric sHSPs to monomeric ones and provides perspective on the temporal changes of the small heat shock protein response and their contribution in earthworm adaptation in changing environments. Earthworms Environmental Stress Stress Biomarkers Heat Shock Response Climate Change Adaptation Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The Intergovernmental Panel on Climate Change (IPCC) indicates in its last report that the ongoing climate change poses a severe threat to living organisms (IPCC, 2022 ), which emphasizes the need to understand their ability to adapt and survive. Furthermore, the growing pollution from anthropogenic origins with different compositions and mechanisms of action bring additional challenges for organisms. However, living organisms are not defenseless, as they have endured exposure to natural toxins, chemicals, and changing environmental conditions for millions of years. Different molecular, cellular, and organismal mechanisms have evolved to ensure survival. The challenge lies in the growing diversity of chemicals and the pace of their release in nature, raising concerns about whether their adaptive mechanisms of organisms will be sufficient. To assess how organisms cope with environmental stressors, it is essential to first identify the mechanisms they employ in response. Among these, the stress response is a central pathway activated when environmental conditions change and disrupt cellular homeostasis. The heat shock proteins (HSPs), a set of proteins discovered in the sixties by Ritossa (Ritossa, 1962 , 1996 ), are principal components in this response. These proteins were originally believed to be triggered by heat, leading to their designation as heat shock proteins. However, later research showed they are involved in many cellular processes (Hu et al., 2022 ; Tutar & Tutar, 2010 ) and can be induced by different stressors (Jeyachandran et al., 2023 ; Kalmar & Greensmith, 2009 ; Mahmood et al., 2014 ). They are divided into different families depending on the size and the domains they present, primarily including large HSPs, HSP90, HSP70, HSP60, HSP40, and small HSP families (Hu et al., 2022 ). In this work, we have centered our attention on small HSPs, the family of proteins that show more diversity between species in number and sequence. They are characterized by a highly conserved α-crystallin domain (ACD), the hallmark of the family, and disordered N- and C-terminal regions (Gusev et al., 2002 ). In recent years, they have garnered attention because of their diversity and rapid evolution compared to the other HSPs, the different roles in cellular processes and disease, and their potential as biomarkers (Carra et al., 2019 ; Ecroyd et al., 2023 ; Gupta et al., 2010 ). Vertebrate sHSPs, primarily in humans, have been studied due to their medical relevance, there is less information on invertebrates, despite the extensive number of animal groups and diversity of physiologies adaptations they exhibit. While some of them have been described in rotifers (Park & Kwak, 2014 ), insects (Li et al., 2020 ; Ruan et al., 2022 ), crustaceans (Zhang et al., 2023 ), and mollusks (Lei et al., 2016 ; Sun & Hu, 2016 ), some groups remain poorly studied. Annelida is one of these groups, and although many putative sHSPs have recently been identified in silico (de la Fuente & Novo, 2022 ), experimental evidence on their roles is still lacking. The earthworm Eisenia fetida is a poikilotherm that depends on the environmental conditions. Humidity and temperature are two primary factors that can alter its metabolism (Diehl & Williams, 1992 ). Human activities are increasing soil chemical contamination, impacting earthworms in their natural habitat. With climate change and pollution altering their environment, it becomes critical to examine how these organisms respond to such stressors. This is essential for preserving critical ecosystem functions, including but not limited to agricultural productivity, carbon cycling, and the soil's capacity to recover from disturbances (Blouin et al., 2013 ; Fonte et al., 2023 ). By analyzing the transcriptome of E. fetida , model organism for ecotoxicity tests (OECD, 1984, 2016), several sHSPs proteins were identified. de la Fuente and Novo ( 2022 ) explored sHSPs at the evolutionary level, but the functional analysis was pending. The study identified different sets based on the α-crystallin domain (ACD) and the number of ACDs, differentiating three clusters of ACDs—identified as A, B, and C—through phylogenetic analyses (de la Fuente & Novo, 2022 ). Interestingly, certain sHSPs in E. fetida exhibit unique architectures, containing two or more ACDs. The first set of single-ACD sHSPs in E. fetida was recently characterized (Tilikj et al., 2025 ). The present study focuses on sHSPs with multiple ACDs, analyzing their functional response to temperature increase and desiccation as conditions intensified by climate change leading to extreme events. In addition, the response to two known pollutants, the plasticizer bisphenol A and the insecticide endosulfan, combined with temperature stress was analyzed to mimic the exposure in a polluted site impacted by climate change. The objectives of this work are twofold: First, an in silico characterization of the sequences and structures is performed to explore features that may influence their functionality. Second, their potential roles in the organism are investigated by analyzing their expression in response to temperature, desiccation, and chemical stress. Together, these aims could lay the groundwork for future studies on small heat shock proteins as biomarkers for ecotoxicity in annelids. 2. Materials and methods 2.1. Animals The earthworms used in the experiments belong to a unique genetic lineage of Eisenia fetida , provided by Dr. Domínguez from the University of Vigo and maintained by the Zoology Group at the Complutense University of Madrid. Culture conditions were 80% humidity and 21 ± 0.5 ºC. Earthworms were maintained in culture chambers in the dark using untreated and defaunated horse manure as the culture medium. Only adults (clitellated) were selected for the experiments. Prior to exposure, they were washed with distilled water, dried on filter paper and weighed. 2.2. Gene identification and characterization: Sequence analysis and structural predictions Nine Eisenia fetida transcripts from the work by de la Fuente and Novo ( 2022 ) were chosen based on the criteria that they encode two or more ACDs. To identify the most similar protein sequences from well-characterized reference species, SmartBLAST ( https://blast.ncbi.nlm.nih.gov/smartblast/smartBlast.cgi ) and standard BLAST searches in the NCBI databases ( https://blast.ncbi.nlm.nih.gov/ ) were conducted (Altschul et al., 1990 ; Sayers et al., 2022 ). Batch CD-Search tool was used to detect conserved domains in the protein sequences ( https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi ) (Marchler-Bauer et al., 2017 ; Wang et al., 2023 ). A range of protein parameters—including molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and GRAVY score—were calculated using the ProtParam tool from ExPASy ( http://web.expasy.org/protparam/ ; Gasteiger et al., 2003 ). The melting temperature of each protein was estimated with DeepStabP ( https://csb-deepstabp.bio.rptu.de/ ) (Jung et al., 2023 ). Predictions for subcellular localization were obtained using two web-based tools: 1) BUSCA ( http://busca.biocomp.unibo.it/ ), which integrates various predictors including DeepSig, TPpred3, PredGPI, BetAware, ENSEMBLE3.0, BaCelLo, MemLoci, and SChloro (Savojardo et al., 2018 ) and 2) DeepLoc-2.1 ( https://services.healthtech.dtu.dk/services/DeepLoc-2.1/ ) (Odum et al., 2024 ) Each protein’s 3D structure was modeled using AlphaFold 2.1 within ChimeraX version 1.8 (2024-06-10) (Goddard et al., 2018 ; Meng et al., 2023 ; Mirdita et al., 2022 ; Pettersen et al., 2021 ), with default settings while enabling PDB template use ( https://www.rbvi.ucsf.edu/chimerax ). Sequences of the separate ACDs in each protein were aligned, analyzed and then manually adjusted using, ClustalW within MEGA 11.0.13 program (Tamura et al., 2021 ), using default settings. The adjustment was performed taking into account the predicted structural information. The optimal amino acid substitution model was determined using Modeltest-NG (Darriba et al., 2020 ; Flouri et al., 2015 ), identifying LG + I + G4 as the best-fit model. Maximum likelihood (ML) phylogenetic analyses of protein sequences were performed with RAxML-HPC BlackBox 8.2.10 (Stamatakis, 2014 ), implemented via the CIPRES Science Gateway (Miller et al., 2010 ). Best-scoring tree was inferred under the selected model, and support values were estimated using 1000 replicates of the rapid bootstrapping algorithm. The phylogenetic tree was visualized and edited using iTOL v.6.1.1 (Letunic & Bork, 2007 , 2021 ) 2.3. Temperature and humidity stress treatments Two physical stressors, heat stress and desiccation stress were based on their relevance to predicted climate change impacts on earthworms, as outlined in the review by Singh et al. ( 2019 ), which highlights temperature and soil moisture as key factors affecting earthworm physiology and activity. To prevent lethal bacterial exposure, glass jars containing 5 g of untreated manure were incubated at 21°C for 24 h before introducing the earthworms. Following this, the stress treatments were applied to test the response of the selected genes. For heat stress experiments, the earthworms were placed in the prepared jars and exposed to 31 ± 0.5°C for up to 24 h as moderate heat stress. In a separate experiment, they were subjected to 35 ± 0.5°C for 2 h to simulate sublethal stress. Both exposures were at 80% humidity Desiccation stress, on the other hand, was induced by exposing the earthworms to two different humidity levels, 10 and 20%, at 21 ± 0.5 ºC. The exposure time was up to 24 hours, with sample collection at 2, 7, and 24 hours for both temperature and humidity. Ten animals were exposed per time and condition, while control was maintained at 21 ± 0.5 ºC and 80% humidity for the same duration. Eight individuals per condition were snap-frozen at -80 ºC for subsequent analysis. 2.4. Chemical stress by contact test Chemical stress was induced by using two well-known toxicants: the organochlorine insecticide Endosulfan (END, CAS 115-29-7, Fluka Analytical) and the plasticizer Bisphenol A (BPA, CAS 80-05-7, purity > 99% Sigma Aldrich). Endosulfan (250 g/L in acetone) and Bisphenol A (2 g/L in ethanol) stock solutions were diluted to achieve the desired final concentrations. The contact exposure followed the method described by Novo et al. ( 2018 ). The concentrations tested were selected following the preliminary experiments conducted by Tilikj et al. ( 2024 ). Two filter papers soaked with 1 mL of 0.2 g/L BPA or 0.1 g/L END were placed on a Petri dish, until complete evaporation (ethanol or acetone). Later, 2 mL of dH 2 O and one earthworm per Petri dish were added. The exposure time was 2 and 7 hours at 21 ± 0.5°C or 26 ± 0.5°C and 80% humidity. Six animals per time and condition were collected and snap-frozen. The samples were maintained at -80 ºC until processing. 2.5. RNA extraction and retrotranscription For RNA extraction, the whole earthworm was homogenized to fine powder using a mortar and a pestle on dry ice. Subsequently, the powder was added to the TRIzol reagent (Invitrogen), and the RNA was extracted following the manufacturer's instructions. An RNAse-free DNAse I (Roche) treatment was carried out to prevent DNA contamination. The enzyme was removed by a phenol-chloroform-isoamyl alcohol extraction using Phase Lock Gel Light tubes (5PRIME). Quantification was done with a NanoDrop One Spectrophotometer (Thermo Scientific), and the samples were diluted to the adequate concentration for retrotranscription. For retrotranscription, 1 µg of RNA, 200 units of the M-MLV enzyme (Invitrogen), 0.5 µg oligo dT20 primer (Biotools), 0.5 µg random hexamers (Biotools), and 10 mM dNTPs (Biotools) for a final volume of 40 µl. After 50 minutes at 37 ºC, the reaction was stopped by heating at 70 ºC for 15 minutes. The cDNA samples were stored at -20 ºC until use. 2.6. Gene expression Gene expression was analyzed by Real-Time PCR. The primers were designed with the identified sequences using the Primer-Blast tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi ) for amplification at the same temperature. The primer data are shown in Table S1 of the Supplementary File S2. The specificity of amplification and the efficiency calculations were done the same way as in Tilikj et al. ( 2024 ). Briefly, efficiency was obtained by a five serial 1:2 dilution from a cDNA mix of the previously obtained cDNAs. The Real-Time was performed in a final volume of 10 µl. The master mix was prepared with 10X buffer (Biotools) and 1 µl of 10 mg/mL bovine serum albumin (BSA, Sigma). The primers were used at a final concentration of 550 nM each, and 500 ng of cDNA was amplified. The Taq polymerase was from Biotools. PCR program was 95 ºC for 5 seconds and 35 cycles of 95 ºC for 5', 62 ºC for 10 seconds, and 65 ºC for 15 seconds. A final melting curve from 65 ºC to 95 ºC was performed to ensure the specificity of the amplicons. As reference genes were used Actin-β ( Act ), ribosomal protein L11 ( rpL11 ), and the TATA-binding protein ( TBP ) genes (primers are described in Tilikj et al., 2024 ). The gene expression was determined by the 2 −ΔΔCt (Pfaffl, 2001). 2.7. Statistical analysis The data were analyzed for normality using the Shapiro-Wilk test, but they did not follow it. Consequently, the differences between the treatments and the control were analyzed by a nonparametric Kruskal-Wallis test for non-normally distributed data, performed with the SPSS 24 software (IBM, USA). Kruskal-Wallis tests were followed by pairwise post hoc comparisons using Dunn’s test with Bonferroni correction for multiple testing. In all cases, p-value < 0.05 was considered significant. 3. Results and discussion 3.1. Double ACD sHSPs in E. fetida : Sequence and structural analysis To determine the potential functions of the proteins encoded by the identified genes, we performed the following analyses: (1) identification of homologous sequences; (2) detection of conserved domains within the protein sequences; (3) calculation of physicochemical properties and (4) prediction and visualization of AlphaFold 3D structures. These analyses provide initial evidence for functional classification, particularly as small heat shock proteins. The results are summarized in Table 1 and 2, while 3D structures can be found in the Supplementary file Dataset S1. Functional predictions based on these analyses were subsequently supported by qPCR experiments assessing gene expression under stress conditions. BLAST finds similar sequences to infer possible function. A standard nucleotide BLAST search of the nine EfsHSP transcripts, purported to contain a double ACD region, was conducted using the Transcriptome Shotgun Assembly database (accessed on 10/10/2024). The analysis revealed that four of the previously identified transcripts were incomplete. The results of the BLAST search are summarized in Table 1, with the top BLAST hits determined based on query coverage and percentage identity. CD-search revealed that seven of the sequences contained two conserved regions with at least one of them being a metazoan ACD region (Supplementary File S2). However, two notable exceptions were observed: EfsHSP83.doubleACD, for which the CD-search analysis revealed only a single identifiable ACD region, likely due to lower sequence conservation of the second ACD region, and EfsHSP59.doubleACD, which exhibited three conserved regions, including the DNA-binding THAP domain. Physicochemical analysis of the sHSPs with two ACD domains are summarized in Table 2. Following the nomenclature in Tilikj et al. (2025), molecular weight (MW) was used to assign new identifiers for the proteins used in this study. Theoretical isoelectric points (pI) may be indicative of subcellular localization, potential interactions with other molecules, and, ultimately, the functional roles of the proteins. In the case of the sHSPs with multiple ACD domains analyzed in this study, all appear to be cytoplasmic or nuclear proteins, with EfsHSP59.doubleACD standing out as a protein for which the predictions clearly suggest a nuclear localization. Monomeric sHSPs share subcellular localization with dimeric sHSPs and are primarily found in the cytoplasm and nucleus (Tilikj et al., 2025). Cytoplasmic and nuclear sHSPs have also been described in the cardiac tissues of rats and humans, as well as in muscle tissues of humans (Mymrikov et al., 2011; Pipkin et al., 2003). Most of the proteins exhibit a slightly acidic pI, ranging from 4.9 to 6.4, while three, EfsHSP84.doubleACD, EfsHSP59.doubleACD, EfsHSP52.doubleACD are basic, which may affect their interactions or stability in different cellular environments (Tokmakov et al., 2021). Negative GRAVY scores reflect the hydrophilic nature of the sHSPs with two ACD domains, which indicates they are soluble proteins (Lanneau et al., 2008) In relation to the predicted melting temperatures (Tm), most of the proteins exhibit Tm values ranging between 40 and 45°C. Higher Tm can be indicative of functional stability under elevated temperatures, although it is not the sole determinant of gene expression or stress responsiveness (Gehring & Wehner, 1995; Ku et al., 2009) .Notably, EfsHSP55.doubleACD stands out with a higher Tm, ranging from 52.8 to 53.5°C, while EfsHSP33.doubleACD shows the lowest Tm at 38°C. This lower Tm for EfsHSP33.doubleACD is inconsistent with a supposed role as a thermal stress protein, as it suggests a reduced stability under heat stress conditions. To better understand the evolutionary origin and potential functional conservation of the double ACD sHSPs, we investigated sequence similarity and phylogenetic relationships with homologous proteins within metazoa. The search of highly similar proteins with SmartBLAST uncovers homology to the equivalent proteins in Caenorhabditis elegans , Mus musculus and Homo sapiens , however due to the protein length (aa) of double ACD sSHPs values like query cover and identity are all below 50% (Supplementary File S2). A phylogenetic analysis was performed, including the proteins analyzed in this study alongside these homologous proteins found in the SmartBLAST analysis, as well as the previously identified and studied monomeric sHSPs with a single ACD from E. fetida (de la Fuente & Novo, 2022; Tilikj et al., 2025). The results of this analysis allow for establishing a phylogenetic relationship between the proteins under study and these homologous proteins, as shown in Figures 1 and S2 in DatasetS1. It is important to note that all the first ACD domains of the double ACD proteins cluster together, which would suggest a common origin for all these domains prior to the differentiation of the various proteins. The only exception within the proteins with multiple ACDs studied is sHSP68, initially considered a protein with a single ACD (de la Fuente & Novo, 2022), but a more detailed previous study revealed it to be incomplete (Tilikj et al., 2025), uncovering the presence of three ACDs. These three ACDs group together, which could indicate a more recent triplication event and a different origin. The predicted structural models for double ACD sHSPs exhibit two ACD domains similar to those previously described for monomeric sHSPs in Tilikj et al. (2025). The high degree of conservation in these domains—both at the sequence level and in their characteristic secondary structure elements—is evident in Figure 1. Oligomerization is a defining characteristic of sHSPs, and the predicted structure of the sHSPs in this study closely resembles the well-characterized homo- and heterodimer interfaces, which are formed through interactions between β7-strands (Supplementary file Dataset S1; Haslbeck et al., 2019; Janowska et al., 2019). A notable feature of the sHSPs containing two ACD domains analyzed in this study, except for EfsHSP59.doubleACD, is an α-helix region of approximately 35 amino acids located at the N-terminal. In EfsHSP59.doubleACD this α-helix region is replaced by the THAP domain (Supplementary File Dataset S1). Lastly, EfsHSP59.doubleACD is the only double ACD sHSP analyzed that contains cysteine residues in the β7 strand (Figure S2 and Supplementary file Dataset S1). In human sHSPs, particularly in the oxidized form of HSPB1, these cysteine residues facilitate the formation of disulfide bonds across the dimer interface, enhancing chaperone activity under oxidative stress (Rajagopal et al., 2015). 3.2. Changes in double ACD sHSP gene expression in response to stress The small heat shock proteins (sHSPs) play a crucial role in cellular stress responses due to their chaperone-like activity and structural flexibility (Haslbeck et al., 2005). Previous studies on Eisenia fetida have explored the expression of various HSPs, hypoxia-related proteins, oxidative stress enzymes, and DNA repair proteins under heat, desiccation, and chemical stress, highlighting a coordinated multistress response and the role of sHSPs in adaptation (Tilikj et al., 2024). Using similar stress conditions, a more recent study focused specifically on monomeric sHSPs in E. fetida and their responses to environmental challenges (Tilikj et al., 2025). However, the role of sHSPs containing two ACD domains in this model soil organism remains largely unexplored. This study addresses this gap by examining the expression dynamics of nine double ACD sHSP genes in E. fetida under heat, desiccation, and chemical stress across multiple time points. Response to heat stress To investigate the response of the identified small heat shock proteins (sHSPs), their transcriptional activity was analyzed at two temperatures: 31 ºC and 35 ºC. The results are summarized in Table S2 in Supplementary File S2. At 31 ºC, three time points were examined: 2, 7, and 24 hours. No significant changes in gene expression were observed at 2 and 7 hours (Figure 2). These findings contrast with the expression patterns observed in monomeric sHSPs in E. fetida , where specific monomeric sHSPs, such as EfsHSP19 and EfsHSP21, displayed significant and consistent upregulation at earlier time points (Tilikj et al., 2024). At 24 hours, eight genes for dimeric sHSPs demonstrated increased transcriptional activity (Figure 2), indicating a delayed response to heat stress at this temperature. It is particularly striking that even double ACD sHSPs which belong in this same phylogenetic cluster as these two monomers have no significant activity at earlier time points. This is further supported by the observation that continuous 24-hour heat exposure elicited a two-phase transcriptional response of small heat shock proteins (sHSPs) in E. fetida This temporal separation suggests a potential sequential or synergistic role in thermotolerance: monomeric sHSPs may act as early responders, rapidly engaging in protective functions during the acute phase of heat stress, while double-ACD sHSPs contribute to sustained protection as stress persists. Although based solely on transcriptional profiles, this pattern may reflect a coordinated strategy in which different sHSP subtypes are mobilized at distinct stages of the cellular stress response to enhance resilience to prolonged thermal exposure. Synergistic models of thermotolerance via synergy between HSPs has been suggested in mouse models (Huang et al., 2007). Earlier transcriptional activity of triple-ACD and septuple-ACD containing genes has been reported in the whiteleg shrimp, with expression observed as early as 2 hours and peaking at 12 hours (Zhang et al., 2023). Tilikj et al. (2025) also reported on a sHSPs with three ACDs (EfsHSP68) which was only found to be upregulated only at 2h of moderate heat stress. At the lethal temperature of 35 °C, no induction was observed; moreover, two sHSPs containing two ACD domains, EfsHSP55.doubleACD and EfsHSP33.doubleACD, were downregulated (Figure 2). Interestingly, these proteins had the highest and lowest Tm, respectively, suggesting they may have distinct roles in cellular processes beyond thermal protection. Once again, clear differences emerge, as previous studies reported upregulation of two monomeric sHSPs, EfsHSP19 and EfsHSP21, at 2 hours under the same temperature. In the case of the whiteleg shrimp, Zhang et al. (2023) demonstrated that triple-ACD and septuple-ACD containing genes were involved in heat-resistance above 36 °C. The results suggest that the sHSPs with two ACDs have a later role in the response to mild temperature stress, probably related to the long-term response more than the acute response. Response to desiccation Desiccation poses a significant challenge for earthworms, as they rely on humid environments for survival (Singh et al., 2019 and references therein). To simulate desiccation stress, two different humidity levels of 10% and 20% were employed. The exposure durations were set at 2, 7, and 24 hours, consistent with previous experiments. A summary of the results is shown in Table S2 in Supplementary File S2. The response of double ACD sHSPs to desiccation stress differs markedly from that of single ACD sHSPs in E. fetida , reflecting distinct roles in managing abiotic stress. Under 20% humidity, monomeric sHSPs showed some upregulation at earlier time points (Tilikj et al., 2025). In contrast, sHSPs with two ACD domains exhibited delayed transcriptional modulation, with significant upregulation observed only at 24 hours (Figure 3). An exception was EfsHSP33.doubleACD, which was downregulated at 7 hours. At 10% humidity, a similar delayed upregulation was observed, with two double ACD sHSPs exhibiting a staggered transcriptional pattern. EfsHSP38.7.doubleACD and EfsHSP55.doubleACD showed early induction at 2 hours, followed by a reduction in activity at 7 hours (Figure 3). Notably, EfsHSP38.7.doubleACD was reactivated at 24 hours (Figure 3). For EfsHSP68, which contains three ACD domains, upregulation was observed only at 2 hours and 24 hours under 10% desiccation stress, suggesting that its transcriptional response is influenced by the severity of the stress rather than the duration (Tilikj et al., 2025). Overall, the transcriptional patterns of sHSPs with two and three ACD regions show little similarity, indicating they may play fundamentally different roles in the environmental stress response. Response to chemical stress The response of double ACD sHSPs to two well-known contaminants—the plasticizer bisphenol A and the insecticide endosulfan—was analyzed to assess their reaction to chemical stressors (Table S2 in Supplementary File S2). These exposures were conducted at the standard growth temperature (21 ºC) and an elevated temperature (26 ºC), simulating the worst-case climate change scenario as outlined in the 2022 IPCC report. This report predicts temperature increases ranging from 1.5 ºC by 2050 (scenario 1, most optimistic) to 4.4 ºC by 2100 (scenario 5, most severe) (IPCC, 2022). In E. fetida , sHSPs with single or double ACDs exhibit distinct responses to individual chemical stressors, yet under the combined stress of BPA exposure and elevated temperatures, both types show limited transcriptional modulation. Among the monomeric sHSPs, only EfsHSP21 showed significant upregulation under these conditions (Tilikj et al., 2025), while none of the double ACD sHSPs were upregulated at either temperature. This limited transcriptional activity underlines the need for further investigation into whether sHSPs contribute to long-term tolerance mechanisms under such combined stressors. Under END exposure, combined stress led to significant upregulation of sHSPs with two ACDs, paralleling the response observed in monomeric sHSPs. Notably, two double ACD sHSPs, EfsHSP38.7.doubleACD and EfsHSP59.doubleACD, were highly activated after 7 hours at the optimal temperature (Figure 4). SmartBLAST analysis revealed that these two dimers share similarity with heat shock protein beta proteins (HSPBs), with EfsHSP38.7.doubleACD exhibiting 47.95% identity to HSPB2 (Supplementary File S2) while the later exhibiting 34.83% identity to HSPB3 HSPB2 is known to form homo- and hetero-oligomers, displaying robust chaperone activity as a homo-oligomer (Boelens, 2020). Indeed Joosten et al. (2023) discussed the importance of regulating the HSPB2:HSPB3 balance for the purposes of subcellular distribution and chaperone activity However, oligomerization of sHSPs with multiple ACDs has not yet been studied. EfsHSP59.doubleACD contains a THAP domain, a feature linked to pro-apoptotic regulation in cells under abiotic stress (Balakrishnan et al., 2011). This characteristic may account for its delayed upregulation in response to the environmental stressors examined in this study. Interestingly, EfsHSP68, which contains three ACD domains, was the only sHSP upregulated under these conditions (Tilikj et al., 2025) suggesting that sHSPs with multiple ACDs may exhibit enhanced sensitivity to some types of chemical stress. This unique response highlights the potential for multi-ACD sHSPs to play specialized roles in chemical stress adaptation. Oligomerization and Its Role in Stress-Induced Chaperone Activity Many metazoan sHSPs with monomeric architecture are known for their ability to dimerize and oligomerize, forming large homo- or hetero-complexes (Tedesco et al., 2022). Under normal conditions, these sHSP oligomers remain stable; however, stress triggers rapid subunit exchange, leading to alterations in oligomeric states and unmasking new substrate interaction modes, thereby expanding the range of substrate binding (Boelens, 2020). Two distinct mechanisms have been proposed to explain how sHSP oligomers behave during heat stress. Eyles and Gierasch (2010) suggest that large oligomers undergo subunit exchange, forming even larger assemblies that incorporate both sHSPs and trapped unfolded proteins. In contrast, Haslbeck et al. (2005) propose that oligomers dissociate into smaller units, such as dimers or tetramers, which then bind unfolded proteins. Despite these differences, both models emphasize the dynamic nature and structural flexibility of sHSPs, key features that underpin their chaperone activity and ability to interact with diverse substrates. The delayed transcriptional activity of double ACD sHSPs under heat, desiccation, and chemical stress, compared to the earlier activity of monomeric sHSPs (Tilikj et al., 2024), may be related to the structural differences between these two groups. The early and robust response of monomeric sHSPs may be attributed to their inherent flexibility, which allows them to rapidly oligomerize as homo- or heterodimers under stress. In contrast, the presence of two ACD regions in may introduce structural rigidity, potentially making the oligomeric states of these proteins less adaptable. Thus, this dual ACD architecture could limit their capacity for rapid oligomerization or dynamic subunit exchange and could be indicative of an alternative mechanism of action. Another factor to consider is the strong preference for forming heterooligomeric complexes, extensively studied in the HSPB family and summarized by Boelens (2020). The dual ACD domain region in sHSPs might add structural complexity, which could hinder efficient heterooligomerization. Additionally, changes in oligomerization are influenced by posttranslational modifications, including phosphorylation, which can regulate sHSP binding affinity and structural dynamics (Carra et al., 2017). The number of ACD regions also appears to influence transcriptional dynamics. Studies on genes with one, three, and seven ACDs report earlier peaks for mono-ACD genes (6 hours) compared to those with three or seven ACDs (12 hours; Zhang et al., 2023). In contrast, sHSPs with two ACDs in this study showed significant upregulation only at 24 hours, suggesting that the even number of ACD regions may impose additional structural constraints that imply a different functional role in the stress response. While mono-ACD sHSPs act as the fastest responders and multi-ACD proteins (with three or more ACDs) activate at intermediate times, the double-ACD sHSPs appear to be the slowest to respond. This potential disadvantage associated with the even ACD configuration indicates that the double-ACD sHSPs might fulfill a complementary protective role during the later stages of stress exposure. Thus, the late-stage upregulation of double-ACD sHSPs likely serves to reinforce cellular protection after the initial surge of monomeric sHSP activity. By complementing the rapid, early action of single-ACD sHSPs, these dimeric sHSPs help establish a sustained chaperone network that is vital for survival under prolonged or severe stress conditionsFuture studies on the oligomerization and solubility of sHSPs with two ACDs are necessary to further elucidate a more specific contributions to stress tolerance. 4. Conclusions This study highlights the complexity and diversity of small heat shock proteins (sHSPs) with two alpha-crystallin domains (ACDs) in Eisenia fetida. These double ACD sHSPs exhibit stimulus-specific responses under thermal, desiccation, and chemical stress conditions. The delayed transcriptional activation, compared to their monomeric counterparts, suggests a complementary role in stress adaptation, likely in long-term cellular protection or through an alternative mechanism of action. Future work should aim to elucidate the oligomerization mechanisms and substrate interactions of these proteins to better understand their contribution to stress tolerance. These findings not only expand our knowledge of sHSP diversity in invertebrates but also provide a foundation for further exploration of their ecological and functional roles in soil ecosystems under environmental stressors. Statements & Declarations Acknowledgements We would like to thank the members of the Soil Zoology Group from Complutense University of Madrid for laboratory support. as well as the Biology and Environmental Toxicology Group from UNED. Author Contributions Marta Novo and José-Luis Martínez-Guitarte conceived the project. Marta Novo, Natasha Tilikj, and Alejandro Martínez Navarro designed the experiments. Natasha Tilikj performed the Real-Time PCR analysis, and Mercedes de la Fuente carried out the gene characterization. Natasha Tilikj and Alejandro Martínez Navarro conducted the experiments and collected the data. Marta Novo, José-Luis Martínez-Guitarte, and Mercedes de la Fuente provided the necessary resources. Natasha Tilikj wrote the original draft. Marta Novo, José-Luis Martínez-Guitarte, Mercedes de la Fuente, and Alejandro Martínez Navarro reviewed and edited the manuscript. Natasha Tilikj and Mercedes de la Fuente prepared all tables and figures. Marta Novo supervised the project, coordinated the work and responsibilities, and acquired funding from the Spanish Government. Funding Marta Novo was supported by Ramón y Cajal Fellowship (RYC2018-024654-I) and this study was funded by Grants PGC2018‐ 094112‐A‐I00 and PID2021-122243NB-I00, from MCIN/AEI/10.13039/501100011033 and by “ESF: Investing in your future” and “ERDF: A way of making Europe”. Data availability Data available on request from the authors. Ethical approval This is not applicable. Consent to participate This is not applicable. Consent to publish All authors have given their permission for publishing this work. Competing interests The authors declare no competing interests. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., & Lipman, D.J. (1990). Basic local alignment search tool. Journal of molecular biology , 215(3), 403-410. doi:https://doi.org/10.1016/S0022-2836(05)80360-2 Balakrishnan, M. P., Cilenti, L., Ambivero, C., Goto, Y., Takata, M., Turkson, J., Li, X. S., & Zervos, A. S. (2011). 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Science of the Total Environment , 880, 163239. doi:https://doi.org/10.1016/j.scitotenv.2023.163239 Tables Tables 1 and 2 are available in the Supplementary Files section Supplementary Files DatasetS1.docx FileS2.xlsx Table1Table2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 07 Jan, 2026 Reviewers agreed at journal 18 Sep, 2025 Reviewers invited by journal 29 May, 2025 Editor invited by journal 27 May, 2025 Editor assigned by journal 27 May, 2025 First submitted to journal 23 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6718926","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463784900,"identity":"3a2df9df-4880-41eb-a410-3f50c646be7d","order_by":0,"name":"NATASHA TILIKJ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYFACxsYDCQfY5EjQwcbYANJiTIoWBoYDQJjYQLQOc/nmhgMPzvClbzh+OoHhwx8itFi2gRx2gy13w5ncDYwz24jQYnAMpOUDUMsN3g3MvMQ4D6Yl3QCk5Q8xDoNoucGWANYCCgwitCQCtZxhM5wJ9MvBXqL8cvj4w4c/jh2T5zt+duODH8Q4DAqOgckDxGtgYKghRfEoGAWjYBSMNAAAN6ZBeFeJXV8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7868-6936","institution":"Biodiversity, Ecology and Evolution Department, Faculty of Biology, Complutense University of Madrid","correspondingAuthor":true,"prefix":"","firstName":"NATASHA","middleName":"","lastName":"TILIKJ","suffix":""},{"id":463784901,"identity":"36b68b56-e03a-44ab-98c5-2ac256cf451e","order_by":1,"name":"Mercedes de la Fuente","email":"","orcid":"","institution":"UNED: Universidad Nacional de Educacion a Distancia","correspondingAuthor":false,"prefix":"","firstName":"Mercedes","middleName":"de la","lastName":"Fuente","suffix":""},{"id":463784902,"identity":"ecadcfea-afe6-4ab7-83a9-1091035f4df7","order_by":2,"name":"Alejandro Martínez Navarro","email":"","orcid":"","institution":"Complutense University of Madrid: Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"Martínez","lastName":"Navarro","suffix":""},{"id":463784903,"identity":"15e37428-5352-45d9-b4ed-10b42da69fee","order_by":3,"name":"Jose-Luis Martínez-Guitarte","email":"","orcid":"","institution":"UNED: Universidad Nacional de Educacion a Distancia","correspondingAuthor":false,"prefix":"","firstName":"Jose-Luis","middleName":"","lastName":"Martínez-Guitarte","suffix":""},{"id":463784904,"identity":"c06ba2db-b14d-45b8-8357-1f6c9607e601","order_by":4,"name":"Marta Novo","email":"","orcid":"","institution":"UNED: Universidad Nacional de Educacion a Distancia","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Novo","suffix":""}],"badges":[],"createdAt":"2025-05-21 18:24:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6718926/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6718926/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84033137,"identity":"a0375b4d-a205-4aee-8057-789dc5a67ce5","added_by":"auto","created_at":"2025-06-06 03:07:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2996212,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment of the alpha-crystallin domain (ACD) regions from small heat shock proteins (sHSPs) involved in the study. The clustering of the first ACD regions (blue letters) suggests a shared evolutionary origin. The second ACD regions (red letters) in sHSPs with a dual architecture group with monomeric sHSPs from \u003cem\u003eE. fetida\u003c/em\u003e (shown with a black background) and the triple ACD sHSPs (shown in bold letters), alongside other well-characterized metazoan sHSPs.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/799b917ec4e43ab7b2cce12a.png"},{"id":84032999,"identity":"98f1db65-79e9-4b27-8a20-035436498ee4","added_by":"auto","created_at":"2025-06-06 02:59:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1363770,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of \u003cem\u003eEfsHSP\u003c/em\u003e genes with dual ACD domain architecture under heat stress treatments of 31 °C and 35 °C across time points (2 h, 7 h, and 24 h). Missing data in panels (h), and (i) for the control samples at 2 h and 7 h are due to the RT-PCR amplification not reaching the threshold for detection, resulting in undetermined Ct values (NaN). This reflects the absence of detectable expression under these specific conditions. Asterisks represent significant differences (p \u0026lt; 0.05) between control and exposure. Numbers on y axis are arbitrary.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/dedb4bd41f6297ba8a9aa7a0.png"},{"id":84033005,"identity":"3a84ed73-f60a-41ae-a353-15c3c22ab81b","added_by":"auto","created_at":"2025-06-06 02:59:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1588514,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of \u003cem\u003eEfsHSP\u003c/em\u003e genes with dual ACD domain architecture under desiccation stress of H 20% and H 10% across time points (2 h, 7 h, and 24 h). Missing data in panels (h), and (i) for the control samples at 2 h and 7 h are due to the RT-PCR amplification not reaching the threshold for detection, resulting in undetermined Ct values (NaN). This reflects the absence of detectable expression under these specific conditions. Asterisks represent significant differences (p \u0026lt; 0.05) between control and exposure. Numbers on y axis are arbitrary.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/157f7e87ab26e1330a5349b8.png"},{"id":84033003,"identity":"dc298323-9e40-4392-893d-d084b7c557a0","added_by":"auto","created_at":"2025-06-06 02:59:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1409591,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of \u003cem\u003eEfsHSP\u003c/em\u003e genes with dual ACD domain architecture under chemical stress from endosulfan at two temperature conditions: 21 °C (optimal) and 26 °C (elevated, combined stress). The expression levels were measured at different time points (2 h and 7 h). Asterisks represent significant differences (p \u0026lt; 0.05) between control and exposure. Numbers on y axis are arbitrary.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/6f125046e85a1fe7d616fb67.png"},{"id":84033624,"identity":"13c1da5e-f5f8-46ba-a974-c45f3eb48952","added_by":"auto","created_at":"2025-06-06 03:16:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8709677,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/11ce2718-8bd6-4fb5-9549-0dbca24a5bad.pdf"},{"id":84033000,"identity":"05d43d96-2b0f-4aa8-af8f-fd055294296b","added_by":"auto","created_at":"2025-06-06 02:59:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7613725,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/f855fe7b65ef7dff1e3f7599.docx"},{"id":84032997,"identity":"4267bf0d-2a93-47d9-81f8-7fdb2d92b2f6","added_by":"auto","created_at":"2025-06-06 02:59:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":245311,"visible":true,"origin":"","legend":"","description":"","filename":"FileS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/5bb46ace95bd8516a2cd1824.xlsx"},{"id":84033138,"identity":"671489be-64b0-40fc-8225-7e7fb053ad25","added_by":"auto","created_at":"2025-06-06 03:07:59","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":31804,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6718926/v1/89c8a3bed4af921e8846d767.docx"}],"financialInterests":"","formattedTitle":"Small heat shock proteins with two alpha-crystallin domains: a new set of proteins in the earthworm Eisenia fetida with differential transcriptional responses to stressors","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe Intergovernmental Panel on Climate Change (IPCC) indicates in its last report that the ongoing climate change poses a severe threat to living organisms (IPCC, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which emphasizes the need to understand their ability to adapt and survive. Furthermore, the growing pollution from anthropogenic origins with different compositions and mechanisms of action bring additional challenges for organisms. However, living organisms are not defenseless, as they have endured exposure to natural toxins, chemicals, and changing environmental conditions for millions of years. Different molecular, cellular, and organismal mechanisms have evolved to ensure survival. The challenge lies in the growing diversity of chemicals and the pace of their release in nature, raising concerns about whether their adaptive mechanisms of organisms will be sufficient.\u003c/p\u003e \u003cp\u003eTo assess how organisms cope with environmental stressors, it is essential to first identify the mechanisms they employ in response. Among these, the stress response is a central pathway activated when environmental conditions change and disrupt cellular homeostasis. The heat shock proteins (HSPs), a set of proteins discovered in the sixties by Ritossa (Ritossa, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1962\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), are principal components in this response. These proteins were originally believed to be triggered by heat, leading to their designation as heat shock proteins. However, later research showed they are involved in many cellular processes (Hu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tutar \u0026amp; Tutar, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and can be induced by different stressors (Jeyachandran et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kalmar \u0026amp; Greensmith, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mahmood et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). They are divided into different families depending on the size and the domains they present, primarily including large HSPs, HSP90, HSP70, HSP60, HSP40, and small HSP families (Hu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this work, we have centered our attention on small HSPs, the family of proteins that show more diversity between species in number and sequence.\u003c/p\u003e \u003cp\u003eThey are characterized by a highly conserved α-crystallin domain (ACD), the hallmark of the family, and disordered N- and C-terminal regions (Gusev et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In recent years, they have garnered attention because of their diversity and rapid evolution compared to the other HSPs, the different roles in cellular processes and disease, and their potential as biomarkers (Carra et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ecroyd et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gupta et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Vertebrate sHSPs, primarily in humans, have been studied due to their medical relevance, there is less information on invertebrates, despite the extensive number of animal groups and diversity of physiologies adaptations they exhibit. While some of them have been described in rotifers (Park \u0026amp; Kwak, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), insects (Li et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ruan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), crustaceans (Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and mollusks (Lei et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sun \u0026amp; Hu, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), some groups remain poorly studied. Annelida is one of these groups, and although many putative sHSPs have recently been identified \u003cem\u003ein silico\u003c/em\u003e (de la Fuente \u0026amp; Novo, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), experimental evidence on their roles is still lacking.\u003c/p\u003e \u003cp\u003eThe earthworm \u003cem\u003eEisenia fetida\u003c/em\u003e is a poikilotherm that depends on the environmental conditions. Humidity and temperature are two primary factors that can alter its metabolism (Diehl \u0026amp; Williams, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Human activities are increasing soil chemical contamination, impacting earthworms in their natural habitat. With climate change and pollution altering their environment, it becomes critical to examine how these organisms respond to such stressors. This is essential for preserving critical ecosystem functions, including but not limited to agricultural productivity, carbon cycling, and the soil's capacity to recover from disturbances (Blouin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fonte et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). By analyzing the transcriptome of \u003cem\u003eE. fetida\u003c/em\u003e, model organism for ecotoxicity tests (OECD, 1984, 2016), several sHSPs proteins were identified. de la Fuente and Novo (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) explored sHSPs at the evolutionary level, but the functional analysis was pending. The study identified different sets based on the α-crystallin domain (ACD) and the number of ACDs, differentiating three clusters of ACDs\u0026mdash;identified as A, B, and C\u0026mdash;through phylogenetic analyses (de la Fuente \u0026amp; Novo, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, certain sHSPs in \u003cem\u003eE. fetida\u003c/em\u003e exhibit unique architectures, containing two or more ACDs. The first set of single-ACD sHSPs in \u003cem\u003eE. fetida\u003c/em\u003e was recently characterized (Tilikj et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The present study focuses on sHSPs with multiple ACDs, analyzing their functional response to temperature increase and desiccation as conditions intensified by climate change leading to extreme events. In addition, the response to two known pollutants, the plasticizer bisphenol A and the insecticide endosulfan, combined with temperature stress was analyzed to mimic the exposure in a polluted site impacted by climate change.\u003c/p\u003e \u003cp\u003eThe objectives of this work are twofold: First, an \u003cem\u003ein silico\u003c/em\u003e characterization of the sequences and structures is performed to explore features that may influence their functionality. Second, their potential roles in the organism are investigated by analyzing their expression in response to temperature, desiccation, and chemical stress. Together, these aims could lay the groundwork for future studies on small heat shock proteins as biomarkers for ecotoxicity in annelids.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eThe earthworms used in the experiments belong to a unique genetic lineage of \u003cem\u003eEisenia fetida\u003c/em\u003e, provided by Dr. Dom\u0026iacute;nguez from the University of Vigo and maintained by the Zoology Group at the Complutense University of Madrid. Culture conditions were 80% humidity and 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C. Earthworms were maintained in culture chambers in the dark using untreated and defaunated horse manure as the culture medium. Only adults (clitellated) were selected for the experiments. Prior to exposure, they were washed with distilled water, dried on filter paper and weighed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Gene identification and characterization: Sequence analysis and structural predictions\u003c/h2\u003e \u003cp\u003eNine \u003cem\u003eEisenia fetida\u003c/em\u003e transcripts from the work by de la Fuente and Novo (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) were chosen based on the criteria that they encode two or more ACDs. To identify the most similar protein sequences from well-characterized reference species, SmartBLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/smartblast/smartBlast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/smartblast/smartBlast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and standard BLAST searches in the NCBI databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were conducted (Altschul et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Sayers et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Batch CD-Search tool was used to detect conserved domains in the protein sequences (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Marchler-Bauer et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA range of protein parameters\u0026mdash;including molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and GRAVY score\u0026mdash;were calculated using the ProtParam tool from ExPASy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Gasteiger et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The melting temperature of each protein was estimated with DeepStabP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://csb-deepstabp.bio.rptu.de/\u003c/span\u003e\u003cspan address=\"https://csb-deepstabp.bio.rptu.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Jung et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Predictions for subcellular localization were obtained using two web-based tools: 1) BUSCA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://busca.biocomp.unibo.it/\u003c/span\u003e\u003cspan address=\"http://busca.biocomp.unibo.it/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which integrates various predictors including DeepSig, TPpred3, PredGPI, BetAware, ENSEMBLE3.0, BaCelLo, MemLoci, and SChloro (Savojardo et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and 2) DeepLoc-2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/DeepLoc-2.1/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/DeepLoc-2.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Odum et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eEach protein\u0026rsquo;s 3D structure was modeled using AlphaFold 2.1 within ChimeraX version 1.8 (2024-06-10) (Goddard et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Meng et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mirdita et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pettersen et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with default settings while enabling PDB template use (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rbvi.ucsf.edu/chimerax\u003c/span\u003e\u003cspan address=\"https://www.rbvi.ucsf.edu/chimerax\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSequences of the separate ACDs in each protein were aligned, analyzed and then manually adjusted using, ClustalW within MEGA 11.0.13 program (Tamura et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), using default settings. The adjustment was performed taking into account the predicted structural information. The optimal amino acid substitution model was determined using Modeltest-NG (Darriba et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Flouri et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), identifying LG\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 as the best-fit model. Maximum likelihood (ML) phylogenetic analyses of protein sequences were performed with RAxML-HPC BlackBox 8.2.10 (Stamatakis, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), implemented via the CIPRES Science Gateway (Miller et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Best-scoring tree was inferred under the selected model, and support values were estimated using 1000 replicates of the rapid bootstrapping algorithm. The phylogenetic tree was visualized and edited using iTOL v.6.1.1 (Letunic \u0026amp; Bork, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Temperature and humidity stress treatments\u003c/h2\u003e \u003cp\u003eTwo physical stressors, heat stress and desiccation stress were based on their relevance to predicted climate change impacts on earthworms, as outlined in the review by Singh et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which highlights temperature and soil moisture as key factors affecting earthworm physiology and activity. To prevent lethal bacterial exposure, glass jars containing 5 g of untreated manure were incubated at 21\u0026deg;C for 24 h before introducing the earthworms. Following this, the stress treatments were applied to test the response of the selected genes. For heat stress experiments, the earthworms were placed in the prepared jars and exposed to 31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C for up to 24 h as moderate heat stress. In a separate experiment, they were subjected to 35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C for 2 h to simulate sublethal stress. Both exposures were at 80% humidity\u003c/p\u003e \u003cp\u003eDesiccation stress, on the other hand, was induced by exposing the earthworms to two different humidity levels, 10 and 20%, at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C. The exposure time was up to 24 hours, with sample collection at 2, 7, and 24 hours for both temperature and humidity. Ten animals were exposed per time and condition, while control was maintained at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026ordm;C and 80% humidity for the same duration. Eight individuals per condition were snap-frozen at -80 \u0026ordm;C for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Chemical stress by contact test\u003c/h2\u003e \u003cp\u003eChemical stress was induced by using two well-known toxicants: the organochlorine insecticide Endosulfan (END, CAS 115-29-7, Fluka Analytical) and the plasticizer Bisphenol A (BPA, CAS 80-05-7, purity\u0026thinsp;\u0026gt;\u0026thinsp;99% Sigma Aldrich). Endosulfan (250 g/L in acetone) and Bisphenol A (2 g/L in ethanol) stock solutions were diluted to achieve the desired final concentrations. The contact exposure followed the method described by Novo et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The concentrations tested were selected following the preliminary experiments conducted by Tilikj et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Two filter papers soaked with 1 mL of 0.2 g/L BPA or 0.1 g/L END were placed on a Petri dish, until complete evaporation (ethanol or acetone). Later, 2 mL of dH\u003csub\u003e2\u003c/sub\u003eO and one earthworm per Petri dish were added. The exposure time was 2 and 7 hours at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C or 26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and 80% humidity. Six animals per time and condition were collected and snap-frozen. The samples were maintained at -80 \u0026ordm;C until processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. RNA extraction and retrotranscription\u003c/h2\u003e \u003cp\u003eFor RNA extraction, the whole earthworm was homogenized to fine powder using a mortar and a pestle on dry ice. Subsequently, the powder was added to the TRIzol reagent (Invitrogen), and the RNA was extracted following the manufacturer's instructions. An RNAse-free DNAse I (Roche) treatment was carried out to prevent DNA contamination. The enzyme was removed by a phenol-chloroform-isoamyl alcohol extraction using Phase Lock Gel Light tubes (5PRIME). Quantification was done with a NanoDrop One Spectrophotometer (Thermo Scientific), and the samples were diluted to the adequate concentration for retrotranscription. For retrotranscription, 1 \u0026micro;g of RNA, 200 units of the M-MLV enzyme (Invitrogen), 0.5 \u0026micro;g oligo dT20 primer (Biotools), 0.5 \u0026micro;g random hexamers (Biotools), and 10 mM dNTPs (Biotools) for a final volume of 40 \u0026micro;l. After 50 minutes at 37 \u0026ordm;C, the reaction was stopped by heating at 70 \u0026ordm;C for 15 minutes. The cDNA samples were stored at -20 \u0026ordm;C until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Gene expression\u003c/h2\u003e \u003cp\u003eGene expression was analyzed by Real-Time PCR. The primers were designed with the identified sequences using the Primer-Blast tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for amplification at the same temperature. The primer data are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e of the Supplementary File S2. The specificity of amplification and the efficiency calculations were done the same way as in Tilikj et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, efficiency was obtained by a five serial 1:2 dilution from a cDNA mix of the previously obtained cDNAs. The Real-Time was performed in a final volume of 10 \u0026micro;l. The master mix was prepared with 10X buffer (Biotools) and 1 \u0026micro;l of 10 mg/mL bovine serum albumin (BSA, Sigma). The primers were used at a final concentration of 550 nM each, and 500 ng of cDNA was amplified. The Taq polymerase was from Biotools. PCR program was 95 \u0026ordm;C for 5 seconds and 35 cycles of 95 \u0026ordm;C for 5', 62 \u0026ordm;C for 10 seconds, and 65 \u0026ordm;C for 15 seconds. A final melting curve from 65 \u0026ordm;C to 95 \u0026ordm;C was performed to ensure the specificity of the amplicons.\u003c/p\u003e \u003cp\u003eAs reference genes were used Actin-β (\u003cem\u003eAct\u003c/em\u003e), ribosomal protein L11 (\u003cem\u003erpL11\u003c/em\u003e), and the TATA-binding protein (\u003cem\u003eTBP\u003c/em\u003e) genes (primers are described in Tilikj et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The gene expression was determined by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e (Pfaffl, 2001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed for normality using the Shapiro-Wilk test, but they did not follow it. Consequently, the differences between the treatments and the control were analyzed by a nonparametric Kruskal-Wallis test for non-normally distributed data, performed with the SPSS 24 software (IBM, USA). Kruskal-Wallis tests were followed by pairwise post hoc comparisons using Dunn\u0026rsquo;s test with Bonferroni correction for multiple testing. In all cases, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. Double ACD sHSPs in \u003cem\u003eE. fetida\u003c/em\u003e: Sequence and structural analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the potential functions of the proteins encoded by the identified genes, we performed the following analyses: (1) identification of homologous sequences; (2) detection of conserved domains within the protein sequences; (3) calculation of physicochemical properties and (4) prediction and visualization of AlphaFold 3D structures. These analyses provide initial evidence for functional classification, particularly as small heat shock proteins. \u0026nbsp;The results are summarized in Table 1 and 2, while 3D structures can be found in the Supplementary file Dataset S1. Functional predictions based on these analyses were subsequently supported by qPCR experiments assessing gene expression under stress conditions.\u003c/p\u003e\n\u003cp\u003eBLAST finds similar sequences to infer possible function. A standard nucleotide BLAST search of the nine EfsHSP transcripts, purported to contain a double ACD region, was conducted using the Transcriptome Shotgun Assembly database (accessed on 10/10/2024). The analysis revealed that four of the previously identified transcripts were incomplete. The results of the BLAST search are summarized in Table 1, with the top BLAST hits determined based on query coverage and percentage identity. CD-search revealed that seven of the sequences contained two conserved regions with at least one of them being a metazoan ACD region (Supplementary File S2). However, two notable exceptions were observed: EfsHSP83.doubleACD, for\u0026nbsp;which the CD-search analysis revealed\u0026nbsp;only a single identifiable ACD region, likely due to lower sequence conservation of the second ACD region, and EfsHSP59.doubleACD, which exhibited three conserved regions, including the DNA-binding THAP domain.\u003c/p\u003e\n\u003cp\u003ePhysicochemical analysis of the sHSPs with two ACD domains are summarized in Table 2. Following the nomenclature in Tilikj et al. (2025), molecular weight (MW) was used to assign new identifiers for the proteins used in this study. Theoretical isoelectric points (pI) may be indicative of subcellular localization, potential interactions with other molecules, and, ultimately, the functional roles of the proteins. In the case of the sHSPs with multiple ACD domains analyzed in this study, all appear to be cytoplasmic or nuclear proteins, with\u0026nbsp;EfsHSP59.doubleACD standing out as a protein for which the predictions clearly suggest a nuclear localization. Monomeric sHSPs share subcellular localization with dimeric sHSPs and are primarily found in the cytoplasm and nucleus (Tilikj et al., 2025). Cytoplasmic and nuclear sHSPs have also been described in the cardiac tissues of rats and humans, as well as in muscle tissues of humans (Mymrikov et al., 2011; Pipkin et al., 2003). Most of the proteins exhibit a slightly acidic pI, ranging from 4.9 to 6.4, while three, EfsHSP84.doubleACD, EfsHSP59.doubleACD, EfsHSP52.doubleACD are basic, which may affect their interactions or stability in different cellular environments (Tokmakov et al., 2021).\u0026nbsp;Negative GRAVY scores reflect \u0026nbsp;the hydrophilic nature of the sHSPs with two ACD domains, which indicates they are soluble proteins (Lanneau et al., 2008) In relation to the predicted melting temperatures (Tm), most of the proteins exhibit Tm values ranging between 40 and 45°C. Higher Tm can be indicative of functional stability under elevated temperatures, although it is not the sole determinant of gene expression or stress responsiveness (Gehring \u0026amp; Wehner, 1995; Ku et al., 2009) .Notably, EfsHSP55.doubleACD stands out with a higher Tm, ranging from 52.8 to 53.5°C, while EfsHSP33.doubleACD shows the lowest Tm at 38°C. This lower Tm for EfsHSP33.doubleACD is inconsistent with a supposed role as a thermal stress protein, as it suggests a reduced stability under heat stress conditions.\u003c/p\u003e\n\u003cp\u003eTo better understand the evolutionary origin and potential functional conservation of the double ACD sHSPs, we investigated sequence similarity and phylogenetic relationships with homologous proteins within metazoa. The search of highly similar proteins with SmartBLAST uncovers homology to the equivalent proteins in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, \u003cem\u003eMus musculus\u003c/em\u003e and \u003cem\u003eHomo sapiens\u003c/em\u003e, however due to the protein length (aa) of double ACD sSHPs values like query cover and identity are all below 50% (Supplementary File S2). A phylogenetic analysis was performed, including the proteins analyzed in this study alongside these homologous proteins found in the SmartBLAST analysis, as well as the previously identified and studied monomeric sHSPs with a single ACD from \u003cem\u003eE. fetida\u003c/em\u003e (de la Fuente \u0026amp; Novo, 2022; Tilikj et al., 2025). The results of this analysis allow for establishing a phylogenetic relationship between the proteins under study and these homologous proteins, as shown in Figures 1 and S2 in DatasetS1. It is important to note that all the first ACD domains of the double ACD proteins cluster together, which would suggest a common origin for all these domains prior to the differentiation of the various proteins. The only exception within the proteins with multiple ACDs studied is sHSP68, initially considered a protein with a single ACD (de la Fuente \u0026amp; Novo, 2022), but a more detailed previous study revealed it to be incomplete (Tilikj et al., 2025), uncovering the presence of three ACDs. These three ACDs group together, which could indicate a more recent triplication event and a different origin.\u003c/p\u003e\n\u003cp\u003eThe predicted structural models for double ACD sHSPs exhibit two ACD domains similar to those previously described for monomeric sHSPs in Tilikj et al. (2025). The high degree of conservation in these domains—both at the sequence level and in their characteristic secondary structure elements—is evident in Figure 1. Oligomerization is a defining characteristic of sHSPs, and the predicted structure of the sHSPs in this study closely resembles the well-characterized homo- and heterodimer interfaces, which are formed through interactions between β7-strands (Supplementary file Dataset S1; Haslbeck et al., 2019; Janowska et al., 2019). A notable feature of the sHSPs containing two ACD domains analyzed in this study, except for EfsHSP59.doubleACD, is an α-helix region of approximately 35 amino acids located at the N-terminal. In EfsHSP59.doubleACD this α-helix region is replaced by the THAP domain (Supplementary File Dataset S1). Lastly, EfsHSP59.doubleACD is the only double ACD sHSP analyzed that contains cysteine residues in the β7 strand (Figure S2 and Supplementary file Dataset S1). In human sHSPs, particularly in the oxidized form of HSPB1, these cysteine residues facilitate the formation of disulfide bonds across the dimer interface, enhancing chaperone activity under oxidative stress (Rajagopal et al., 2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Changes in double ACD sHSP gene expression in response to stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe small heat shock proteins (sHSPs) play a crucial role in cellular stress responses due to their chaperone-like activity and structural flexibility (Haslbeck et al., 2005). Previous studies on \u003cem\u003eEisenia fetida\u003c/em\u003e have explored the expression of various HSPs, hypoxia-related proteins, oxidative stress enzymes, and DNA repair proteins under heat, desiccation, and chemical stress, highlighting a coordinated multistress response and the role of sHSPs in adaptation (Tilikj et al., 2024). Using similar stress conditions, a more recent study focused specifically on monomeric sHSPs in \u003cem\u003eE. fetida\u003c/em\u003e and their responses to environmental challenges (Tilikj et al., 2025). However, the role of sHSPs containing two ACD domains in this model soil organism remains largely unexplored. This study addresses this gap by examining the expression dynamics of nine double ACD sHSP genes in \u003cem\u003eE. fetida\u003c/em\u003e under heat, desiccation, and chemical stress across multiple time points.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResponse to heat stress\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the response of the identified small heat shock proteins (sHSPs), their transcriptional activity was analyzed at two temperatures: 31 ºC and 35 ºC. The results are summarized in Table S2 in Supplementary File S2. At 31 ºC, three time points were examined: 2, 7, and 24 hours. No significant changes in gene expression were observed at 2 and 7 hours (Figure 2). These findings contrast with the expression patterns observed in monomeric sHSPs in \u003cem\u003eE. fetida\u003c/em\u003e, where specific monomeric sHSPs, such as EfsHSP19 and EfsHSP21, displayed significant and consistent upregulation at earlier time points (Tilikj et al., 2024). At 24 hours, eight genes for dimeric sHSPs demonstrated increased transcriptional activity (Figure 2), indicating a delayed response to heat stress at this temperature. It is particularly striking that even double ACD sHSPs which belong in this same phylogenetic cluster as these two monomers have no significant activity at earlier time points. This is further supported by the observation that continuous 24-hour heat exposure elicited a two-phase transcriptional response of small heat shock proteins (sHSPs) in \u003cem\u003eE. fetida\u003c/em\u003e This temporal separation suggests a potential sequential or synergistic role in thermotolerance: monomeric sHSPs may act as early responders, rapidly engaging in protective functions during the acute phase of heat stress, while double-ACD sHSPs contribute to sustained protection as stress persists. Although based solely on transcriptional profiles, this pattern may reflect a coordinated strategy in which different sHSP subtypes are mobilized at distinct stages of the cellular stress response to enhance resilience to prolonged thermal exposure. Synergistic models of thermotolerance via synergy between HSPs has been suggested in mouse models (Huang et al., 2007).\u003c/p\u003e\n\u003cp\u003eEarlier transcriptional activity of triple-ACD and septuple-ACD containing genes has been reported in the whiteleg shrimp, with expression observed as early as 2 hours and peaking at 12 hours (Zhang et al., 2023). Tilikj et al. (2025) also reported on a sHSPs with three ACDs (EfsHSP68) which was only found to be upregulated only at 2h of moderate heat stress.\u003c/p\u003e\n\u003cp\u003eAt the lethal temperature of 35\u0026nbsp;°C, no induction was observed; moreover, two sHSPs containing two ACD domains, EfsHSP55.doubleACD and EfsHSP33.doubleACD, were downregulated (Figure 2). Interestingly, these proteins had the highest and lowest Tm, respectively, suggesting they may have distinct roles in cellular processes beyond thermal protection. Once again, clear differences emerge, as previous studies reported upregulation of two monomeric sHSPs, EfsHSP19 and EfsHSP21, at 2 hours under the same temperature. In the case of the whiteleg shrimp, Zhang et al. (2023) demonstrated that triple-ACD and septuple-ACD containing genes were involved in heat-resistance above 36\u0026nbsp;°C. The results suggest that the sHSPs with two ACDs have a later role in the response to mild temperature stress, probably related to the long-term response more than the acute response.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResponse to desiccation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDesiccation poses a significant challenge for earthworms, as they rely on humid environments for survival (Singh et al., 2019 and references therein). To simulate desiccation stress, two different humidity levels of 10% and 20% were employed. The exposure durations were set at 2, 7, and 24 hours, consistent with previous experiments. A summary of the results is shown in Table S2 in Supplementary File S2. The response of double ACD sHSPs to desiccation stress differs markedly from that of single ACD sHSPs in \u003cem\u003eE. fetida\u003c/em\u003e, reflecting distinct roles in managing abiotic stress. Under 20% humidity, monomeric sHSPs showed some upregulation at earlier time points (Tilikj et al., 2025). In contrast, sHSPs with two ACD domains exhibited delayed transcriptional modulation, with significant upregulation observed only at 24 hours (Figure 3). An exception was EfsHSP33.doubleACD, which was downregulated at 7 hours. At 10% humidity, a similar delayed upregulation was observed, with two double ACD sHSPs exhibiting a staggered transcriptional pattern. EfsHSP38.7.doubleACD and EfsHSP55.doubleACD showed early induction at 2 hours, followed by a reduction in activity at 7 hours (Figure 3). Notably, EfsHSP38.7.doubleACD was reactivated at 24 hours (Figure 3). For EfsHSP68, which contains three ACD domains, upregulation was observed only at 2 hours and 24 hours under 10% desiccation stress, suggesting that its transcriptional response is influenced by the severity of the stress rather than the duration (Tilikj et al., 2025). Overall, the transcriptional patterns of sHSPs with two and three ACD regions show little similarity, indicating they may play fundamentally different roles in the environmental stress response.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResponse to chemical stress\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe response of double ACD sHSPs to two well-known contaminants—the plasticizer bisphenol A and the insecticide endosulfan—was analyzed to assess their reaction to chemical stressors (Table S2 in Supplementary File S2). These exposures were conducted at the standard growth temperature (21 ºC) and an elevated temperature (26 ºC), simulating the worst-case climate change scenario as outlined in the 2022 IPCC report. This report predicts temperature increases ranging from 1.5 ºC by 2050 (scenario 1, most optimistic) to 4.4 ºC by 2100 (scenario 5, most severe) (IPCC, 2022).\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eE. fetida\u003c/em\u003e, sHSPs with single or double ACDs exhibit distinct responses to individual chemical stressors, yet under the combined stress of BPA exposure and elevated temperatures, both types show limited transcriptional modulation. Among the monomeric sHSPs, only EfsHSP21 showed significant upregulation under these conditions (Tilikj et al., 2025), while none of the double ACD sHSPs were upregulated at either temperature. This limited transcriptional activity underlines the need for further investigation into whether sHSPs contribute to long-term tolerance mechanisms under such combined stressors.\u003c/p\u003e\n\u003cp\u003eUnder END exposure, combined stress led to significant upregulation of sHSPs with two ACDs, paralleling the response observed in monomeric sHSPs. Notably, two double ACD sHSPs, EfsHSP38.7.doubleACD and EfsHSP59.doubleACD, were highly activated after 7 hours at the optimal temperature (Figure 4). SmartBLAST analysis revealed that these two dimers share similarity with heat shock protein beta proteins (HSPBs), with EfsHSP38.7.doubleACD exhibiting 47.95% identity to HSPB2 (Supplementary File S2) while the later exhibiting 34.83% identity to HSPB3 HSPB2 is known to form homo- and hetero-oligomers, displaying robust chaperone activity as a homo-oligomer (Boelens, 2020). Indeed Joosten et al. (2023) discussed the importance of regulating the HSPB2:HSPB3 balance for the purposes of subcellular distribution and chaperone activity However, oligomerization of sHSPs with multiple ACDs has not yet been studied. EfsHSP59.doubleACD contains a THAP domain, a feature linked to pro-apoptotic regulation in cells under abiotic stress (Balakrishnan et al., 2011). This characteristic may account for its delayed upregulation in response to the environmental stressors examined in this study.\u003c/p\u003e\n\u003cp\u003eInterestingly, EfsHSP68, which contains three ACD domains, was the only sHSP upregulated under these conditions (Tilikj et al., 2025) suggesting that sHSPs with multiple ACDs may exhibit enhanced sensitivity to some types of chemical stress. This unique response highlights the potential for multi-ACD sHSPs to play specialized roles in chemical stress adaptation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOligomerization and Its Role in Stress-Induced Chaperone Activity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMany metazoan sHSPs with monomeric architecture are known for their ability to dimerize and oligomerize, forming large homo- or hetero-complexes (Tedesco et al., 2022). Under normal conditions, these sHSP oligomers remain stable; however, stress triggers rapid subunit exchange, leading to alterations in oligomeric states and unmasking new substrate interaction modes, thereby expanding the range of substrate binding (Boelens, 2020).\u003c/p\u003e\n\u003cp\u003eTwo distinct mechanisms have been proposed to explain how sHSP oligomers behave during heat stress. Eyles and Gierasch (2010) suggest that large oligomers undergo subunit exchange, forming even larger assemblies that incorporate both sHSPs and trapped unfolded proteins. In contrast, \u0026nbsp;Haslbeck et al. (2005) propose that oligomers dissociate into smaller units, such as dimers or tetramers, which then bind unfolded proteins. Despite these differences, both models emphasize the dynamic nature and structural flexibility of sHSPs, key features that underpin their chaperone activity and ability to interact with diverse substrates.\u003c/p\u003e\n\u003cp\u003eThe delayed transcriptional activity of double ACD sHSPs under heat, desiccation, and chemical stress, compared to the earlier activity of monomeric sHSPs (Tilikj et al., 2024), may be related to the structural differences between these two groups. The early and robust response of monomeric sHSPs may be attributed to their inherent flexibility, which allows them to rapidly oligomerize as homo- or heterodimers under stress. In contrast, the presence of two ACD regions in may introduce structural rigidity, potentially making the oligomeric states of these\u0026nbsp;proteins less adaptable. Thus, this dual ACD architecture\u0026nbsp;could limit their capacity for rapid oligomerization or dynamic subunit exchange and could be indicative of an alternative mechanism of action. Another factor to consider is the strong preference for forming heterooligomeric complexes, extensively studied in the HSPB family and summarized by Boelens (2020). The dual ACD domain region in sHSPs might add structural complexity, which could hinder efficient heterooligomerization. Additionally, changes in oligomerization are influenced by posttranslational modifications, including phosphorylation, which can regulate sHSP binding affinity and structural dynamics (Carra et al., 2017).\u003c/p\u003e\n\u003cp\u003eThe number of ACD regions also appears to influence transcriptional dynamics. Studies on genes with one, three, and seven ACDs report earlier peaks for mono-ACD genes (6 hours) compared to those with three or seven ACDs (12 hours; Zhang et al., 2023). In contrast, sHSPs with two ACDs in this study showed significant upregulation only at 24 hours, suggesting that the even number of ACD regions may impose additional structural constraints that imply a different functional role in the stress response.\u0026nbsp;While mono-ACD sHSPs act as the fastest responders and multi-ACD proteins (with three or more ACDs) activate at intermediate times, the double-ACD sHSPs appear to be the slowest to respond. This potential disadvantage associated with the even ACD configuration indicates that the double-ACD sHSPs might fulfill a complementary protective role during the later stages of stress exposure. Thus, the late-stage upregulation of double-ACD sHSPs likely serves to reinforce cellular protection after the initial surge of monomeric sHSP activity. By complementing the rapid, early action of single-ACD sHSPs, these dimeric sHSPs help establish a sustained chaperone network that is vital for survival under prolonged or severe stress conditionsFuture studies on the oligomerization and solubility of sHSPs with two ACDs are necessary to further elucidate a more specific contributions to stress tolerance.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study highlights the complexity and diversity of small heat shock proteins (sHSPs) with two alpha-crystallin domains (ACDs) in \u003cem\u003eEisenia fetida.\u003c/em\u003e These double ACD sHSPs exhibit stimulus-specific responses under thermal, desiccation, and chemical stress conditions. The delayed transcriptional activation, compared to their monomeric counterparts, suggests a complementary role in stress adaptation, likely in long-term cellular protection or through an alternative mechanism of action. Future work should aim to elucidate the oligomerization mechanisms and substrate interactions of these proteins to better understand their contribution to stress tolerance. These findings not only expand our knowledge of sHSP diversity in invertebrates but also provide a foundation for further exploration of their ecological and functional roles in soil ecosystems under environmental stressors.\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the members of the Soil Zoology Group from Complutense University of Madrid for laboratory support. as well as the Biology and Environmental Toxicology Group from UNED.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e Marta Novo and Jos\u0026eacute;-Luis Mart\u0026iacute;nez-Guitarte conceived the project. Marta Novo, Natasha Tilikj, and Alejandro Mart\u0026iacute;nez Navarro designed the experiments. Natasha Tilikj performed the Real-Time PCR analysis, and Mercedes de la Fuente carried out the gene characterization. Natasha Tilikj and Alejandro Mart\u0026iacute;nez Navarro conducted the experiments and collected the data. Marta Novo, Jos\u0026eacute;-Luis Mart\u0026iacute;nez-Guitarte, and Mercedes de la Fuente provided the necessary resources. Natasha Tilikj wrote the original draft. Marta Novo, Jos\u0026eacute;-Luis Mart\u0026iacute;nez-Guitarte, Mercedes de la Fuente, and Alejandro Mart\u0026iacute;nez Navarro reviewed and edited the manuscript. Natasha Tilikj and Mercedes de la Fuente prepared all tables and figures. Marta Novo supervised the project, coordinated the work and responsibilities, and acquired funding from the Spanish Government.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e Marta Novo was supported by Ram\u0026oacute;n y Cajal Fellowship (RYC2018-024654-I) and this study was funded by Grants PGC2018‐ 094112‐A‐I00 and PID2021-122243NB-I00, from MCIN/AEI/10.13039/501100011033 and by \u0026ldquo;ESF: Investing in your future\u0026rdquo; and \u0026ldquo;ERDF: A way of making Europe\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Data available on request from the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e This is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e All authors have given their permission for publishing this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltschul, S.F., Gish, W., Miller, W., Myers, E.W., \u0026amp; Lipman, D.J. 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Science of the Total Environment\u003cem\u003e, \u003c/em\u003e880, 163239. doi:https://doi.org/10.1016/j.scitotenv.2023.163239\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Earthworms, Environmental Stress, Stress Biomarkers, Heat Shock Response, Climate Change, Adaptation","lastPublishedDoi":"10.21203/rs.3.rs-6718926/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6718926/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eClimate change and the growing environmental pollution are two of the main challenges that life faces today. Soil invertebrates such as earthworms are increasingly exposed to stress from climate change (e.g., rising temperatures and drought) and chemical pollution. The heat shock proteins are central to the stress response. Eisenia fetida, a widely used model for soil ecotoxicology, relies on molecular chaperones like small heat shock proteins (sHSPs) for stress tolerance. Furthermore, sHSPs generate interest for their potential as molecular indicators of soil pollution and for thermotolerance acquisition. Previously, we have described a set of genes coding for sHSPs with a single ACD. Here we report the first identification of sHSPs with multiple α-crystallin domains (ACDs) in an annelid. These genes were isolated from an E. fetida transcriptome, their domain architecture was defined, and their expression was analyzed under environmental stressors: heat shock, desiccation, and exposure to the pollutants bisphenol A (BPA) and endosulfan. Gene expression patterns were stimulus-specific. Prolonged sub-lethal heat and desiccation each induced distinct subsets of the multi-ACD sHSP genes, highlighting their tailored roles in abiotic stress. In contrast, exposure to BPA at optimal conditions did not produce a response, while endosulfan produced a minimal response. Combined exposure to endosulfan and elevated temperature triggered a significant upregulation of these chaperone genes indicating synergetic stress. This work relates the response of dimeric sHSPs to monomeric ones and provides perspective on the temporal changes of the small heat shock protein response and their contribution in earthworm adaptation in changing environments.\u003c/p\u003e","manuscriptTitle":"Small heat shock proteins with two alpha-crystallin domains: a new set of proteins in the earthworm Eisenia fetida with differential transcriptional responses to stressors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 02:59:54","doi":"10.21203/rs.3.rs-6718926/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2026-01-07T11:39:26+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-18T09:09:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-29T17:43:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2025-05-27T13:13:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-27T04:48:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2025-05-23T14:16:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d1ef1ca5-2e9f-4441-abc1-61e3482dc2df","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T09:49:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-06 02:59:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6718926","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6718926","identity":"rs-6718926","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}