A field-friendly, non-toxic fixative for integrated morphological and molecular research in non-model invertebrates

A field-friendly, non-toxic fixative for integrated morphological and molecular research in non-model invertebrates | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 18 September 2025 V1 Latest version Share on A field-friendly, non-toxic fixative for integrated morphological and molecular research in non-model invertebrates Authors : Olmo Irene del , Moreno Paula , Patricia Alvarez-campos , and Aida Verdes 0000-0002-9193-9253 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175822784.46833410/v1 235 views 56 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Formalin, ethanol, and RNAlater are the most commonly used fixatives for morphological and molecular studies. Formalin is preferred for preserving tissue morphology, whereas ethanol and RNAlater are used to obtain high-quality nucleic acids for molecular analyses, including emerging -omics techniques. Over the past few years, the study of non-model organisms has gained attention, but the lack of laboratory cultures for many species requires collecting and fixing the animals directly in the field. Often, few specimens are secured, limiting the possibility of using multiple fixatives for parallel analyses. A single fixative that preserves both morphology and molecules while being easy to handle in the field would therefore be highly valuable. KINFix, a non-toxic alcohol-based fixative, was developed to preserve histology, proteins, and nucleic acids simultaneously, enabling both morphological and molecular analyses with the same sample. Here, we evaluate the suitability of KINFix for electron microscopy, bulk RNA sequencing, and single-cell RNA sequencing (scRNA-seq) using three invertebrate species from distinct spiralian phyla. Our results demonstrate that KINFix preserves both morphology and RNA integrity better than other standard fixatives, maintaining RNA quality for over three months. Additionally, we show the potential of KINFix to preserve cellular integrity for scRNA-seq applications. While fixation conditions may require optimization for different species, our findings highlight KINFix as a versatile and valuable fixative that enables a wide range of morphological and molecular studies in non-model invertebrates. KINFix is particularly useful for field-based research where sample availability and preservation logistics are challenging. A field-friendly, non-toxic fixative for integrated morphological and molecular research in non-model invertebrates IRENE DEL OLMO 1 † , PAULA MORENO 1 † , PATRICIA ÁLVAREZ-CAMPOS 1* , AIDA VERDES 2 * 1 Departamento de Biología, Universidad Autónoma de Madrid 2 Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales (MNCN-CSIC) † These authors contributed equally * Corresponding authors: [email protected] ; [email protected] ABSTRACT Formalin, ethanol, and RNAlater are the most commonly used fixatives for morphological and molecular studies. Formalin is preferred for preserving tissue morphology, whereas ethanol and RNAlater are used to obtain high-quality nucleic acids for molecular analyses, including emerging -omics techniques. Over the past few years, the study of non-model organisms has gained attention, but the lack of laboratory cultures for many species requires collecting and fixing the animals directly in the field. Often, few specimens are secured, limiting the possibility of using multiple fixatives for parallel analyses. A single fixative that preserves both morphology and molecules while being easy to handle in the field would therefore be highly valuable. KINFix, a non-toxic alcohol-based fixative, was developed to preserve histology, proteins, and nucleic acids simultaneously, enabling both morphological and molecular analyses with the same sample. Here, we evaluate the suitability of KINFix for electron microscopy, bulk RNA sequencing, and single-cell RNA sequencing (scRNA-seq) using three invertebrate species from distinct spiralian phyla. Our results demonstrate that KINFix preserves both morphology and RNA integrity better than other standard fixatives, maintaining RNA quality for over three months. Additionally, we show the potential of KINFix to preserve cellular integrity for scRNA-seq applications. While fixation conditions may require optimization for different species, our findings highlight KINFix as a versatile and valuable fixative that enables a wide range of morphological and molecular studies in non-model invertebrates. KINFix is particularly useful for field-based research where sample availability and preservation logistics are challenging. Keywords: fieldwork, KINFix, non-model organisms, RNA integrity, RNAlater, sample fixation, sample preservation, SEM, single-cell RNA sequencing INTRODUCTION One of the main challenges of working with non-model organisms in evolutionary and developmental research, is to obtain enough number of specimens to resolve a given biological question. Different experimental approaches can require important amounts of tissue or the preservation of multiple specimens in different fixatives (i.e. for morphological vs. molecular studies), posing a significant challenge when laboratory cultures are not available for the focal taxa. For these non-model organisms, the only practical option is often to collect and fix samples directly in the field. This requires a fixative that preserves both morphology and molecules, while also being easy to handle and store under field conditions. Over the years, different fixatives have been developed to preserve biological samples for a variety of purposes, often tailored to the specific organisms or tissues being studied. Formalin has long been considered the gold-standard in histology and clinical pathology due to its high efficiency preserving tissue morphology, its affordability, wide availability and ease of use. As a result, it has remained the fixative of choice for decades in a range of applications, including histopathological analyses, cellular biology and even taxonomic studies (e.g., Groelz et al., 2013; Stefanits et al., 2016). However, numerous studies have demonstrated that formaldehyde is a human carcinogen, linked to different pulmonary and neural diseases (Kilburn et al., 1985; Cogliano et al., 2005). In addition to its high toxicity, formalin fixation compromises the quality of nucleic acids, leading to DNA and RNA fragmentation and cross-linking with proteins, hampering downstream molecular analyses (Campos and Gilbert, 2012; Salehi and Najafi, 2014). Therefore, in recent years there has been a growing interest in safer, more effective and better suited fixatives to preserve not only tissue morphology and cellular architecture, but also high-quality nucleic acids and proteins for molecular applications (e.g., Moelans et al., 2011a, b; Stefanits et al., 2016). One promising alternative is the non-crosslinking fixative RCL2 (ALPHELYS, Plaisir, France), which has been successfully applied in immunohistochemistry (Bellet et al., 2008; Preusser et al., 2010), clinical pathology (Delfour et al., 2006; Moelans et al., 2011b; Masir et al., 2018), and proteomics studies (Undheim et al., 2014, 2015; Bellet et al., 2008), showing comparable results to formalin fixation. As a result, many laboratories modified their routine protocols to incorporate RCL2. However, RCL2 is a commercial product that has been occasionally discontinued, prompting Stefanits et al. (2016) to develop KINFix (Klinisches Institut für Neurologie Fixative), a cost-effective, easy-to-prepare alternative based on the same core components of RCL2. KINFix preserves tissue morphology as effectively as RCL2 and formalin (Stefanits et al., 2016), while also maintaining the integrity of proteins and nucleic acids. Since its publication, this innovative, and affordable fixative has been used in a growing number of studies (Stefanits et al., 2016; Chen et al., 2019; Tanaka et al., 2020; Verdes et al., 2022), demostrating its value for both morphological and molecular research. While DNA and RNA have been successfully extracted from KINFix-fixed paraffin-embedded tissues for PCR applications (Stefanits et al., 2016), its performance has not been yet directly compared to RNAlater (Invitrogen) — the current gold standard for nucleic acid preservation (Mutter et al., 2004). KINFix has proven compatible with some advanced –omics techniques such as MALDI mass spectrometry imaging (MALDI-IMS) (Hempel et al., 2022), its suitability for other cutting-edge approaches, such as single cell RNA sequencing (scRNA-seq), remains unexplored. For many small-sized organisms, scRNA-seq requires tissue dissociation of many individuals to provide the cellular resolution required for constructing comprehensive cell atlases (e.g., Sebé-Pedrós et al., 2018; Massri et al., 2021; Álvarez-Campos et al., 2024). This poses a significant challenge when working with non-model organisms that are difficult to culture in the lab. Therefore, a fixative that allows temporary preservation of specimens until additional individuals of the same species and developmental stage can be collected would greatly expand the applicability of these advanced molecular techniques. In this study, we evaluated the efficiency of KINFix as an alternative to ethanol and formalin in morphological studies requiring scanning electron microscopy (SEM). To evaluate its suitability for molecular applications, we compared RNA yield and quality from freshwater and marine invertebrate tissues fixed with KINFix versus RNAlater . We also tested the stability of KINFix working solution after storage over 120 days—the limit tested by Stefanits et al. (2016) — to determine whether crystalized sugar precipitation affects its effectiveness. Lastly, we examined whether KINFix preserves cellular integrity well enough to allow effective tissue dissociation and cell permeabilization, for its application in scRNA-seq studies. Our results are particularly relevant for research on non-model invertebrates, where specimens are often rare or available in limited numbers, making it difficult to conduct multiple types of analyses with the same samples. MATERIAL AND METHODS Sample preparation for Scanning Electron Microscopy imaging Specimens for SEM were fixed following four different protocols: (1) 10% formalin for 10 minutes, (2) 3% glutaraldehyde buffered with cacodylate for 30 minutes, (3) KINFix for 30 minutes, and (4) 5% EtOH for 10 min and then 30% EtOH for another 10 minutes. Then, all samples were dehydrated through an increasing ethanol series of 50, 70, 90, and 100%, for 10 minutes each grade. Fixed and dehydrated worms were stored in 100% EtOH at 4 ºC and then prepared on an Emitech K850 Critical Point Dryer, mounted with adhesive tabs on aluminum stubs, gold-coated with a Q150T-S Turbo-Pumper Sputter Coater, and examined with a Hitachi S-3000N SEM at the Servicio Interdepartamental de Investigación (SIdI) of the Universidad Autónoma de Madrid (UAM). Preservation of specimens for RNA extraction We analyzed a total of 62 samples representing three spiralian phyla (i.e. Nemertea, Platyhelminthes and Annelida) (Figure 1) for which we have laboratory cultures: the planarian Dugesia subtentaculata , (24 samples; Figure 1b ), the annelid Pristina leidyi (17 samples; Figure 1c ) and the nemertean Lineus lacteus (21 samples; Figure 1d ). Between 50 and 100 mg of tissue were used for each sample, which corresponds approximately to one adult planarian, 20 specimens of P. leidyi , and a 1 cm body fragment of L. lacteus . Samples were preserved using three different treatments ( Figure 1e ): (1) RNAlater solution (Invitrogen TM ) used as control, (2) KINFix solution within three months from preparation (KINFix “new”, KFNew), and (3) KINFix solution over three months from preparation (KINFix “old”, KFOld) to evaluate its stability over time, since Stefanitis et al., 2016 only tested the solution up to 3 months after preparation. After fixation with RNAlater or KINFix, all samples were incubated overnight at 4 °C and then stored at –20 °C until RNA extraction was performed. The stock solutions of KFNew and KFOld were stored at 4 ºC as recommended by Stefanitis et al., 2016 . Extraction and quality assessment of total RNA Fixed tissues were introduced in PowerBead Tubes with 1.4 mm ceramic beads (Qiagen) and 1 mL of TRIzol Reagent (Invitrogen TM ) and homogenised using the soft mode (2 cycles of 15 s with 30 s pause at 5,800 rpm) in a Precellys Evolution Touch Homogenizer (Bertin Technologies). Total RNA was extracted on ice to avoid RNA degradation, using the phenol-chloroform method with TRIzol, following the manufacturer’s instructions. The quantity and quality of the total RNA extracted was assessed with a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). We measured RNA concentration (ng/µl) and integrity based on absorbance values at 230, 260 and 280 nm. A sample is considered of good quality ( i.e . pure) when the A260/280 ratio is ∼2.0 and the A260/230 value is between 2.0 and 2.2 according to the NanoDrop™ Spectrophotometer manufacturer’s instructions. In addition, RNA integrity of three samples per treatment was assessed using the High Sensitivity RNA ScreenTape Analysis in the 4150 TapeStation System (Agilent). RNA quality is indicated by the RNA integrity number equivalent (RIN e ), with values that range from 1 when RNA is degraded, to 10 when RNA is intact. RIN e values over 7 are generally considered acceptable for RNA sequencing experiments (Padmanaban et al., 2012). Statistical analyses Differences in RNA concentrations (ng/µl), absorbance ratios (A260/230 and A260/280) and RIN e values were analyzed following the same statical methods. We first applied the ROUT test to identify and eliminate outliers (Q = 1%). Then we checked whether the data followed a normal distribution according to the Shapiro-Wilk test (P < 0.05). When the data did not follow a normal distribution, a logarithmic transformation was applied, and the Shapiro-Wilk normality test was repeated. Finally, to test for significative differences when the data followed a normal distribution, we performed a one-way ANOVA using Turkey’s multiple comparison test (P < 0.05). When the data did not follow a normal distribution even after logarithmic transformation, we applied the Kruskal-Wallis non-parametric tests using Dunn’s multiple comparison test (P < 0.05). This non-parametric test was also applied to TapeStation RIN values, as only three measurements per condition were available. All statistical analyses and graphs were performed with the software GraphPad Prism v.9.5 (Ivashchenko et al., 2017). Cell dissociation and flow cytometry analysis Since tissue architecture and cell structure are well preserved with KINFix, we also tested the suitability of KINFix-fixed samples for single-cell transcriptomics (scRNA-seq). We used the ACetic-MEthanol (ACME) dissociation protocol described in (García-Castro et al., 2021) with approximately100 adult individuals of P. leidyi previously fixed in KINFix and preserved at 4°C. As a control, live specimens were directly dissociated using the same ACME protocol. Following dissociation, cells were cryopreserved in DMSO and stored at –80 ºC for downstream analyses. Thawed samples were filtered through 50-μm CellTrics strainers (Sysmex) and stained for flow cytometry. Nuclei and cytoplasm were labelled with DRAQ5 and Concanavalin-A respectively and visualized with a CytoFlex S Flow Cytometer (Beckman Coulter) at the SIdI-UAM. Flow cytometry profiles of KINFix-fixed, ACME-dissociated samples were evaluated by identifying distinct gated populations, quantifying the proportion of singlet cells (i.e., DRAQ5-positive and Concanavalin-A-positive single cells) with the staining and gating parameters described in García-Castro et al. (2021). RESULTS KINFix effectively preserves external morphology for SEM imaging and detailed morphological assessment One of the key features of KINFix is its ability to maintain tissue structure, preventing shrinkage during fixation. To assess this, we compared the quality of external morphology preservation for SEM imaging in P. leidyi specimens fixed with three commonly used fixatives for morphological studies in annelids ( i.e ., formalin, glutaraldehyde and ethanol), and KINFix ( Figure 2 ). Samples fixed with formalin, glutaraldehyde and ethanol showed noticeable body shrinkage ( Figure 2c–e ), hindering the observation of external morphological details. In contrast, specimens fixed in KINFix retained their original body shape and tissue integrity ( Figure 2a, b ), showing better preservation for detailed morphological analysis with SEM. Quality and integrity of RNA extracted from KINFix-fixed samples are compatible with downstream molecular applications We successfully extracted RNA from all samples fixed with either KINFix or RNAlater ( Supplementary Table S1 ). However, RNA concentration values varied significantly among species, with L. lacteus showing the highest concentration, followed by D. subtentaculata , and the annelid P. leidyi showing significantly lower concentration values (Kruskal-Wallis, H = 83.62, P < 0.0001). s According to NanoDrop measurements, RNA concentrations were consistently higher in KFOld compared to KFNew and RNAlater . In L. lacteus mean values were 744,74 ng/µl for KFOld, 199,98 ng/µl for KFNew, and 396,11 ng/µl for RNAlater . In D. subtentaculata , values averaged 122,41 ng/µl (KFOld), 112,18 ng/µl (KFNew) and 37,75 ng/µl ( RNAlater ), while P. leidyi showed considerably lower mean concentrations 5,52 ng/µl (KFOld), 4,20 ng/µl (KFNew) and 4,60 ng/µl ( RNAlater ) ( Figure 3a – c and Supplementary Table S1 ) . Although L. lacteus samples yielded the highest RNA concentrations, ranging from 418.7 to 1014.7 ng/µl with KFOld, and from 114.7 ng/µl to 315.9 ng/µl with KFnew, differences between KFOld and KFNew were smaller in D. subtentaculata (43.5–230.9 ng/µl vs. 26.2–273.8 ng/µl) and in P. leidyi (2.5–9.6 ng/µl vs. 2.8–6.3 ng/µl). Concentration of samples fixed in RNAlater showed minimal variability in P. leidyi (2.9–6 ng/µl), and a broader range in L. lacteus (27.35–879.6 ng/µl) and D. subtentaculata (12–57.1 ng/µl) ( Supplementary Table S1 ). Consequently, statistical analysis revealed significative differences in RNA concentration between KFOld and RNAlater in the nemertean and planarian samples, but not in the annelid P. leidyi ( Figure 3a – c and Table 1 ) . No significative differences were found between KFNew and RNAlater in any of the three species analyzed ( Figure 3a – c and Table 1 ). In both planarian and nemertean samples, absorbance ratios (260/280 and 260/230) were comparable between KINFix- and RNAlater -fixed samples, with similar values across all three conditions ( Figure 3d, e, g, h and Supplementary Table S1 ). In contrast, P. leidyi samples showed a significantly higher 260/280 ratio in KFNew compared to RNAlater , indicating slightly better RNA purity, with values closer to 2, in KFNew samples ( Figure 3f, i, Table 1 and Supplementary Table S1 ). To validate these results, we further assessed RNA quality using the Agilent TapeStation ( Figure 4, Table 1 and Supplementary Table S1 ). In contrast to Nanodrop results, TapeStation analysis generally showed higher RNA quality of samples fixed in RNAlater , especially in L. lacteus , were both RNA concentration and RIN e values were significantly higher compared to KINFix-fixed samples ( Figure 4a, c ). Samples of both L. lacteus and D. subtentaculata showed comparable RNA concentrations across all conditions, while significant differences were observed in RIN e values only in L. lacteus ( Figure 4a, c, e, g ). RNAlater samples showed slightly higher RIN e values on average, than KFNew and KFOld samples ( Figure 4e ). Only in nemertean samples, significative differences were found between KFOld and RNAlater ( Figure 4c and Table 1 ); however, in planarian samples, RNA integrity was high in all three conditions, with most RIN e values above 8 and a mean of 8.5, except for a single outlier ( Figure 4g and Supplementary Table S1 ). Electrophenogram profiles confirmed the significative differences in the lower RIN e values in samples preserved with KFOld, showing highly degraded RNA profiles compared to KFNew and RNAlater, ( Figure 4b-d ). In contrast, the electrophenogram profiles revealed the absence of RNA degradation in all KINFix-preserved planarian samples, demonstrating the fixative’s potential to yield high-quality, non-degraded RNA ( Figure 4f, h ). For P. leidyi samples, all conditions resulted in very low RNA concentrations, with means of 2,2 for KFNew and 1,9 ng/µl for KFOld, and 3,3 ng/µl for RNAlater , with significant differences found only between KFOld and RNAlater . ( Figure 4i and Supplementary Table S1 ). Due to these low concentrations outside the quantitative detection range of the TapeStation, RIN e values could not be calculated for these samples ( Supplementary Table S1) . Nevertheless, the electrophenogram profiles and gel image show no sign of RNA degradation in any of the three conditions analyzed ( Figure 4j, k), confirming the integrity of the extracted RNA despite its low abundance. Combining KINFix with ACME dissociation preserves cell morphology for flow cytometry. We dissociated 100 P. leidyi specimens previously fixed in KINFix and stored at 4°C using the ACME protocol and compared results to those obtained from freshly dissociated live specimens ( Figure 5 ). Our flow cytometry analysis recovered a well-defined DNA-containing population of cells (DRAQ5-positive cells), corresponding to singlet-cells in both conditions ( Figure 5a, b ). These singlets represent two clearly distinguishable cell populations, corresponding to “G1 cells” containing 2c DNA content, and “G2 cells” with 4c DNA content. Although the pellet obtained after ACME dissociation was similar in both samples, the total number of cells recovered in KINFix-fixed samples was lower (533 cells) compared to the dissociations obtained from live animals (1664 cells) ( Figure 5a, b ). However, the relative proportion of all the gated populations –total, G1 and G2– were comparable or slightly higher in the KINFix-fixed samples ( Figure 5b ). Moreover, we did not find differences in the proportion of cellular debris or aggregates between KINFix-fixed and live dissociations, and therefore, in both conditions they could be easily excluded from the singlets ( Figure 5a, b ). DISCUSSION Imaging techniques such as light, confocal and electron microscopy, are essential tools for morphological studies, often necessary for species descriptions and taxonomic identifications. Both transmission and scanning electron imaging (TEM and SEM) are commonly used to describe diagnostic features either to identify species or to describe developmental processes in spiralian taxa, including those analyzed in the present study (e.g., Magarlamov et al., 2018; Salgado de Oliveira et al., 2018; Kawamoto et al. 2005). In nemerteans and planarians, external morphological characters are often important for taxonomy; however, histological observations of the internal anatomy usually provide more reliable information for species-level identifications (e.g., Sundberg et al., 2009; Bartolomaeus and Döhren, 2010; Brubacher et al., 2014; Strand et al., 2014; Winsor and Sluys, 2018; de Miguel Bonet and Hartenstein, 2024). In contrast, the identification of annelid species often depends on the examination of external traits, such chaetal morphology, ciliary structures, mating organs or developmental stages (e.g., Bergter et al. 2004; Yáñez et al. 2006; Caramelo and Anselmi,2012; Álvarez-Campos and Verdes, 2017; Moreno-Martín et al., 2023). Preparation of specimens for SEM imaging, requires specific fixation and dehydration protocols to ensure complete desiccation before critical point drying. Tissue shrinkage during this process poses a challenge in the preparation of soft-bodied organisms, particularly annelids. Fixatives like formalin or glutaraldehyde are still commonly used to preserve the morphological features required for taxonomic identifications (e.g., Kawamoto et al., 2005; Caramelo and Anselmi, 2012). Fixation in ethanol 96-98% is also common and can provide good quality SEM images for key diagnostic characters in many groups (e.g., Álvarez-Campos and Verdes, 2017; Tilic et al., 2022; Moreno-Martín et al., 2023). In P. leidyi , several fixation methods have been developed for the histological analysis of internal structures (Özpolat et al., 2016), but none are optimized for SEM imaging. Moreover, SEM preparation methods commonly used for other freshwater oligochaetes (Bouché et al., 1999; Yáñez et al., 2006), led to complete tissue disintegration or shrinkage in P. leidyi ( Figure 2 ). In contrast, our results show that KINFix not only preserves internal histology and morphology, as previously reported (Stefanitis et al., 2019) but also represents an effective alternative for high-quality SEM imaging of the external morphology, even in species with reduced body size like P. leidyi . Transcriptome analysis represents a powerful tool for modern biology by enabling precise quantification of gene expression and providing comprehensive sequence information. However, extracting high-quality RNA remains challenging due to its rapid degradation, mainly caused by cytoplasmic RNases, making RNA preservation methods essential. RNAlater has become a standard solution for preserving small tissue samples, as it stabilizes and protects cellular RNA integrity without the need to freeze the tissue (Mutter et al., 2004; Salehi and Najafi, 2014). While effective, RNAlater is expensive and does not preserve tissue morphology. Other alternatives, such as the nucleic acid preservation (NAP) buffer, can preserve DNA and RNA at a lower cost (Camacho-Sanchez et al., 2013), but also fail to maintain tissue morphology. Our results support the use of KINFix as a dual-purpose fixative to preserve both RNA integrity and tissue morphology. KINFix provides a more affordable solution than RNAlater (~7.5 € for 100 mL of KINFix compared to ~189 € for 100 mL of RNAlater) with the added benefit of preserving tissue morphology (Stefanitis et al., 2016). In addition, by comparing RNA quantity and quality across multiple parameters and methods, we also assessed whether long-term storage KINFix (>120 days) compromises RNA integrity, as precipitation of crystalized sugar may alter the concentration of the solution (Stefanitis et al., 2016). In general, RNA concentration and quality were not affected by the fixative used, showing no significant differences in most comparisons ( Table 1 ). Furthermore, when significant differences were detected, RNA extracted from KINFix samples was of sufficient quality for sequencing ( Figures 3, 4 and Table 1 ). Notably, NanoDrop measurements consistently showed higher RNA concentration in KINFix-fixed samples ( Figure 3a, b, f ). In P. leidyi , the A260/280 ratio—that indicates protein or phenol contamination—was considerably lower in RNAlater -fixed samples than in KINFix-fixed samples ( Figure 3f and Supplementary Table S1 ), with none reaching the minimum value of 1.85 expected for pure RNA (Gayral et al., 2011). Notably, absorbance ratios across all species—especially in P. leidyi —were generally lower than values indicative of high-quality RNA, likely due to the very low RNA concentrations (<10 ng/µl) which can compromise the accuracy of Nanodrop measurements ( Supplementary Table S1 ) . However, NanoDrop usually overestimates RNA concentration compared to more accurate methods like the TapeStation (Hussing et al., 2015, 2018). Accordingly, our TapeStation results recovered lower RNA concentrations—often at least half—across all samples. This difference was more pronounced in KINFix samples, probably due to ethanol carryover affecting NanoDrop spectrophotometric readings (Oxford Nanopore technologies documentation, https://nanoporetech.com/documentation). TapeStation results consistently showed higher RNA concentrations and RIN e values in RNAlater -fixed samples, except for D. subtentaculata ( Figure 4a, c, g, i ). Nevertheless, TapeStation measurements show RNA of sufficient quality for sequencing across all samples. Overall, our results indicate that KINFix is a valuable and cost-effective alternative for RNA preservation, providing comparable results to the gold standard RNAlater with the added advantage of preserving tissue morphology. Moreover, long-term storage of KINFix does not seem to greatly affect RNA quality, although RNA concentration was lower in samples fixed with KINFix stored for over 120 days, in both the planarian and annelid species ( Figure 4e, i ). In addition, RNA degradation was considerably higher in L. lacteus samples fixed with KFOld ( Figure 4d ), indicating storage time might impact some species more than others. Therefore, we recommend testing the optimal fixation conditions for each model species and proceeding with caution when using older KINFix preparations. Single-cell transcriptomics (scRNA-seq) enables the analysis of thousands of mRNA molecules in large numbers of individual cells. This method has significantly advanced biological research by revealing cellular diversity and dynamic processes that were previously hidden in bulk RNA analyses. Despite its huge potential, several technical challenges remain unsolved, with recent studies focusing on preservation of dissociated cells and avoidance of compositional and gene expression changes during sample processing (e.g., Alles et al. 2017; García-Castro et al 2021; Phan et al. 2021; Burja et al. 2022; Gutiérrez-Franco et al. 2023). However, the need to preserve entire organisms in the field—critical when laboratory culturing is unfeasible or when individuals at the same developmental stage are hard to collect simultaneously—has not been addressed. Our results indicate that KINFix can be effectively combined with the ACME dissociation and fixation protocol to preserve whole organisms for downstream flow cytometry and scRNA-seq analyses ( Figure 5 ). KINFix-fixed samples required greater amounts of tissue to achieve a comparable number of recovered cells to those of fresh tissue ( Figure 5a, b ), but the possibility of preserving whole organisms eliminates the need of immediate processing allowing time to collect enough material. Thus, KINFix provides experimental flexibility facilitating the application of scRNA-seq when working with rare, field-collected, or specific developmental stages of non-model organisms. CONCLUSION Our results shows that KINFix provides a valuable and cost-effective fixative for studies with limited biological material and multiple downstream applications that might require different fixation techniques. This is particularly advantageous when working with non-model organisms, as the material obtained in the field is often limited, and keeping organisms alive or using different fixatives for multiple analyses may not be feasible. Unlike more expensive options such as RNAlater, KINFix provides a versatile, safe, and cost-efficient solution effective for high-quality preservation for both morphological and molecular analyses, offering a practical and unique solution for integrative research. ACKNOWLEDGEMENTS The authors want to thank all colleagues that helped with collection and maintaining of specimens used in this study, including Paloma Mas, Guillermo San Martín, and former and current members of P.Á-C lab at UAM. We are also thankful to Ana Riesgo and Jordi Solana for fruitful discussions about this work. We also appreciate the support provided by the staff at SIdI-UAM for their assistance with cytometry and SEM imaging. I.dO was funded by an FPI-UAM 2022 from the Universidad Autónoma de Madrid, and P.M-M by FPI-CAM grant from Comunidad de Madrid (PIPF-2022/SAL-GL-26101). P.Á-C was supported by the Madrid Government (Comunidad de Madrid, Spain) through the Multiannual Agreement with the Universidad Autónoma de Madrid to encourage young research doctors under the V Regional Programme of Research and Technological Innovation (PRICIT) (SI1/PJI/2019-00532), and by the MCIN/AEI/10.13039/501100011033 and the European Union “Next Generation EU”/PRTR (CNS2023-145193). A.V. was supported by the Spanish Ministry of Science MCIN/AEI/10.13039/501100011033, European Union Next Generation EU/PRTR (grant IJC2020-045256-I) and by a fellowship from the ‘la Caixa’ Banking Foundation (ID 100010434), with the code LCF/BQ/PR24/12050011. DATA AVAILABILITY STATEMENT All the required data are uploaded as supplementary material. REFERENCES Alles J, Karaiskos N, Praktiknjo SD, Grosswendt S, Wahle P, Ruffault PL, Ayoub S, Schreyer, Anastasiya Boltengagen L, Birchmeier C, Zinzen R, Kocks C, Rajewsky N.2017. Cell fixation and preservation for droplet-based single-cell transcriptomics. BMC Biol 15, 44. Álvarez-Campos P, García-Castro H, Emili E, Pérez-Posada A, del Olmo I, Peron S, Salamanca-Díaz DA, Mason V, Metzger B, Bely AE, Kenny NJ, Özpolat BD, Solana J. 2024. Annelid adult cell type diversity and their pluripotent cellular origins. Nat Commun 15:3194. Álvarez-Campos P, Verdes A. 2017. Syllids inhabiting holdfasts of Lessonia spicata in Central Chile: diversity, systematics, and description of three new species. Systematics and Biodiversity 15(6), 520–531. Bartolomaeus T, von Döhren J. 2010. Comparative morphology and evolution of the nephridia in Nemertea. Journal of Natural History 44(37–40), 2255–2286. Bellet V, Boissière F, Bibeau F, Desmetz C, Berthe ML, Rochaix P, Maudelonde T, Mangè A, Solassol J. 2008. Proteomic analysis of RCL2 paraffin-embedded tissues. Journal of Cellular and Molecular Medicine 12:2027–2036. Bergter A, Beck LA, Paululat A. 2004. Embryonic development of the oligochaete Enchytraeus coronatus : An SEM and histological study of embryogenesis from one‐cell stage to hatching. Journal of Morphology 261(1), 26–42. Bouché MLL, Biagianti-Risbourg S, Vernet G. 1999. A light and scanning electron microscope study of the morphology of the chaetae of Tubifex tubifex in a non-polluted medium. Hydrobiologia 411:39–44. Brubacher JL, Vieira AP, Newmark PA. 2014. Preparation of the planarian Schmidtea mediterranea for high-resolution histology and transmission electron microscopy. Nat Protoc 9:661–673. Burja B, Paul D, Tastanova A, Edalat SG, Gerber R, Houtman M, Elhai M, Bürki K, Staeger R, Restivo G, Lang R, Sodin-Semrl S, Lakota K, Tomšič M, Levesque MP, Distler O, Rotar Z, Robinson MD, Frank-Bertoncelj M. 2022. An optimized tissue dissociation protocol for single-cell RNA sequencing analysis of fresh and cultured human skin biopsies. Frontiers in Cell and Dev Biol 10:872688. Camacho-Sanchez M, Burraco P, Gomez-Mestre I, Leonard JA. 2013. Preservation of RNA and DNA from mammal samples under field conditions. Mol Ecol Resour 13:663–673. Campos PF, Gilbert TM. 2012. DNA extraction from formalin-fixed material. Methods Mol Biol 840:81–5. Caramelo C, Ansemil E. 2012. Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy. Turkish Journal of Zoology 36(1), 1-14. Chen L, Lu Y, Li W, Ren Y, Yu M, Jiang S, Fu Y, Wang J, Peng S, Bilyk KT, Murphy KR, Zhuang X, Hune M, Zhai W, Wang W, Xu Q, Cheng C-HC. 2019. The genomic basis for colonizing the freezing Southern Ocean revealed by Antarctic toothfish and Patagonian robalo genomes. GigaScience 8:giz016. Cogliano VJ, Grosse Y, Baan RA, Straif K, Secretan MB, Ghissassi FE. 2005. Meeting Report: Summary of IARC Monographs on Formaldehyde, 2-Butoxyethanol, and 1-tert-Butoxy-2-Propanol. Environ Health Perspect 113:1205–1208. Delfour C, Roger P, Bret C, Berthe M-L, Rochaix P, Kalfa N, Raynaud P, Bibeau F, Maudelonde T, Boulle N. 2006. RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells. The Journal of Molecular Diagnostics 8:157–169. García-Castro H, Kenny NJ, Iglesias M, Álvarez-Campos P, Mason V, Elek A, Schönauer A, Sleight VA, Neiro J, Aboobaker A, Permanyer J, Irimia M, Sebé-Pedrós A, Solana J. 2021. ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics. Genome Biology 22:89. Groelz D, Sobin L, Branton P, Compton C, Wyrich R, Rainen L. 2013. Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality. Exp Mol Pathol 94:188–194. Gutiérrez-Franco A, Ake F, Hassan MN, Chaves Cayuela N, Mularoni L, Plass M, 2023. Methanol fixation is the method of choice for droplet-based single-cell transcriptomics of neural cells. Commun Biol 6, 522 Hempel B-F, Damm M, Petras D, Kazandjian TD, Szentiks CA, Fritsch G, Nebrich G, Casewell NR, Klein O, Süssmuth RD. 2022. Spatial Venomics─Cobra Venom System Reveals Spatial Differentiation of Snake Toxins by Mass Spectrometry Imaging. Journal of Proteome Res 22(1):26-35. Hussing C, Kampmann ML, Mogensen HS, Børsting C, Morling N. 2015. Comparison of techniques for quantification of next-generation sequencing libraries. Forensic Science International: Genetics Supplement Series 5:e276–e278. Hussing C, Kampmann M-L, Mogensen HS, Børsting C, Morling N. 2018. Quantification of massively parallel sequencing libraries – a comparative study of eight methods. Sci Rep 8:1110. Kawamoto S, Yoshida‐Noro C, Tochinai S. 2005. Bipolar head regeneration induced by artificial amputation in Enchytraeus japonensis (Annelida, Oligochaeta). Journal of Experimental Zoology Part A: Comparative Experimental Biology 303(8), 615-627. Kilburn KH, Warshaw R, Boylen CT, Johnson S-JS, Seidman B, Sinclair R, Takaro T. 1985. Pulmonary and Neurobehavioral Effects of Formaldehyde Exposure. Archives of Environmental Health: An International Journal 40:254–260. Magarlamov TY, Chernyshev AV, Turbeville JM. 2018. Pseudocnidae of archinemerteans (Nemertea, Palaeonemertea) and their implications for nemertean systematics. Journal of Morphology 279: 1444–1454. Masir N, Ghoddoosi M, Mansor S, Abdul-Rahman F, Florence CS, Mohamed-Ismail NA, Tamby M-R, Md-Latar NH. 2012. RCL2, a potential formalin substitute for tissue fixation in routine pathological specimens. Histopathology 60:804–815. Massri AJ, Greenstreet L, Afanassiev A, Berrio A, Wray GA, Schiebinger G, McClay DR. 2021. Developmental single-cell transcriptomics in the Lytechinus variegatus sea urchin embryo. Development 148:dev198614. de Miguel Bonet M del M, Hartenstein V. 2024. Ultrastructural analysis and 3D reconstruction of the frontal sensory-glandular complex and its neural projections in the platyhelminth Macrostomum lignano . Cell Tissue Res 397:147–177. Moelans CB, ter Hoeve N, van Ginkel J-W, ten Kate FJ, van Diest PJ. 2011a. Formaldehyde Substitute Fixatives: Analysis of Macroscopy, Morphologic Analysis, and Immunohistochemical Analysis. American Journal of Clinical Pathology 136:548–556. Moelans CB, Oostenrijk D, Moons MJ, Diest PJ van. 2011b. Formaldehyde substitute fixatives: effects on nucleic acid preservation. Journal of Clinical Pathology 64:960–967. Moreno-Martín P, Mourín M, Verdes A, Álvarez-Campos P. 2023. Morphological and molecular study of Syllinae (Annelida, Syllidae) from Bermuda, with the description of five new species. Royal Society Open Science 10:230638. Moreno-Martínez L, Martín GS, Riesgo A, Giribet G, Álvarez-Campos P. 2023. Molecular analysis of Indo-Pacific Syllinae annelids with the description of five new species from the Philippine Islands. Systematics and Biodiversity 21:2227630. Mutter GL, Zahrieh D, Liu C, Neuberg D, Finkelstein D, Baker HE, Warrington JA. 2004. Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays. BMC Genomics 5:88. Özpolat BD, Sloane ES, Zattara EE, Bely AE. 2016. Plasticity and regeneration of gonads in the annelid Pristina leidyi . EvoDevo 7:22. Padmanaban A, Salowsky R, Cher C. 2012. RNA quality control using the agilent 2200 TapeStation system–assessment of the RIN e quality metric. Agilent technologies application notes. Phan HV, van Gent M, Drayman N, Basu A, Gack MU, Savaş T. 2021. High-throughput RNA sequencing of paraformaldehyde-fixed single cells. Nat Commun 12, 5636. Preusser M, Plumer S, Dirnberger E, Hainfellner JA, Mannhalter C. 2010. Fixation of Brain Tumor Biopsy Specimens with RCL2 Results in Well-Preserved Histomorphology, Immunohistochemistry and Nucleic Acids. Brain Pathology 20:1010–1020. Salehi Z, Najafi M. 2014. RNA Preservation and Stabilization. Biochem Physiol 3:126. Salgado de Oliveira S, Ruiz Lopes KA, Leite PM, Villaça Morais F, Rodrigues de Campos Velho ND. 2018. Physiological evaluation of the behavior and epidermis of freshwater planarians ( Girardia tigrina and Girardia sp.) exposed to stressors. Biol Open 7 (6): bio029595. Sebé-Pedrós A, Saudemont B, Chomsky E, Plessier F, Mailhé M-P, Renno J, Loe-Mie Y, Lifshitz A, Mukamel Z, Schmutz S, Novault S, Steinmetz PRH, Spitz F, Tanay A, Marlow H. 2018. Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq. Cell 173:1520-1534.e20. Stefanits H, Bieńkowski M, Galanski M, Mitulović G, Ströbel T, Gelpi E, Ribalta T, Broholm H, Hartmann C, Kros JM, Preusser M, Hainfellner JA. 2016. KINFix – A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens. Clin Neuropathol 35:3–12. Strand M, Herrera-Bachiller A, Nygren A, Kånneby T. 2014. A new nemertean species: what are the useful characters for ribbon worm descriptions? Journal of the Marine Biological Association of the United Kingdom 94:317–330. Sundberg P, Vodoti ET, Zhou H, Strand M. 2009. Polymorphism hides cryptic species in Oerstedia dorsalis (Nemertea, Hoplonemertea). Biological Journal of the Linnean Society 98:556–567. Tanaka N, Kanatani S, Kaczynska D, Fukumoto K, Louhivuori L, Mizutani T, Kopper O, Kronqvist P, Robertson S, Lindh C, Kis L, Pronk R, Niwa N, Matsumoto K, Oya M, Miyakawa A, Falk A, Hartman J, Sahlgren C, Clevers H, Uhlén P. 2020. Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies. Nat Biomed Eng 4:875–888. Tilic E, Neunzig N, Bartolomaeus T. 2022. Hairy and iridescent chaetae of the sea mouse Aphrodita (Annelida, Errantia). Acta Zoologica 103:479–491. Undheim EAB, Hamilton BR, Kurniawan ND, Bowlay G, Cribb BW, Merritt DJ, Fry BG, King GF, Venter DJ. 2015. Production and packaging of a biological arsenal: Evolution of centipede venoms under morphological constraint. Proceedings of the National Academy of Sciences 112:4026–4031. Undheim EAB, Sunagar K, Hamilton BR, Jones A, Venter DJ, Fry BG, King GF. 2014. Multifunctional warheads: Diversification of the toxin arsenal of centipedes via novel multidomain transcripts. Journal of Proteomics 102:1–10. Verdes A, Álvarez-Campos P, Nygren A, Martín GS, Deheyn DD, Gruber DF, Holford M, Verdes A, Álvarez-Campos P, Nygren A, Martín GS, Deheyn DD, Gruber DF, Holford M. 2022. Molecular phylogeny and evolution of bioluminescence in Odontosyllis (Annelida, Syllidae). Invert Systematics 36:622–630. Winsor L, Sluys R. 2018. Basic Histological Techniques for Planarians. In: Rink JC, (eds). Planarian Regeneration. Methods in Molecular Biology, vol 1774. Humana Press, New York, NY. Yáñez E, Cuadrado S, Martínez-Ansemil E. 2006. External sense organs in freshwater oligochaetes (Annelida, Clitellata) revealed by scanning electron microscopy. J Morphol 198–207. Author Contributions I.dO. and P.M.-M.: data curation, formal analysis, investigation, visualization, writing—original draft, writing—review and editing. P.Á.-C. and A.V.: conceptualization, data curation, funding acquisition, investigation, project administration, resources, supervision, writing—review and editing. All authors gave final approval for publication and agreed to be held accountable for the work performed therein. Tables and Figures Figure 1 . Invertebrate models used in this study. (a) Overview of the phylogenetic relationships of the selected species; (b–d) Representative specimens of (b) planarian Dugesia subtentaculata (Platyhelminthes), (c) annelid Pristina leidyi (Annelida), (d) nemertean Lineus lacteus (Nemertea); (e) Number of samples analyzed per species and type of fixative used. Figure 2 . Scanning electron microphotography of Pristina leidyi fixed with different solutions. (a) Individual fixed with KINFix, lateral view of the whole worm; (b) Individual fixed with KINFix, detail of the midbody segments; (c) Individual fixed with an ascending ethanol concentration, detail of the anterior part; (d) Individual fixed with 10% formalin, detail of the anterior part; (e) Individual fixed with 3% glutaraldehyde, detail of the midbody segments. All scale bars are 100 µm. Figure 3. Nanodrop quantification of the total RNA extracted from the samples preserved in KFNew, KFOld, and RNAlater for the studied species: Lineus lacteus (a, d, g), Dugesia subtentaculata (b, e, h), and Pristina leidyi. (c, f, i); (a–c) RNA concentration; (d–f) 280/260 ratio values; (g–i) 260/230 ratio values. Mean values for each condition are indicated above the bars. Significative differences between samples are indicated by an asterisk (* P ≤ 0.05, ** P ≤ 0.01, * P ≤ 0.001, ** P ≤ 0.0001). Figure 4 . TapeStation profiles of the total RNA extracted from the three studied species, the nemertean L. lacteus , the planarian D. subtentaculata and the annelid P. leidyi . Three replicates for each condition (KFNew, KFOld, and RNAlater ) are showed. (a) Statistical analysis of the total RNA concentration for each condition; (b) Gel images of the different samples analyzed; (c) Statistical analysis of the RIN e values for each condition. Mean values for each condition are indicated above the bars; (d) Representative electropherogram of total RNA from one replicate per condition. Significative differences between samples are indicated by an asterisk (* P ≤ 0.05, ** P ≤ 0.01). Figure 5 . Comparison of ACME-dissociated cells in live and KINFix-fixed Pristina leidyi individuals for single-cell transcriptomic experiments. Flow cytometry profiles of ungated and gated profiles of ACME-fixed cells from (a) living organisms, used as control samples, and (b) KINFix-fixed samples, stained with DRAQ5 (nucleus) and Concanavalin-A (cytoplasm). For each comparison (a, b), ungated cytoplasm positive singlets (blue) and nucleus positive gated population (orange) in the upper-left corner, gated nucleus-positive cells resulting in G1 (magenta) and G2 (green) populations in the upper-right corner, histogram of the DNA content (linear scale) showing the relative proportions of G1 and G2 cells in the bottom-left corner, table showing the number and percentage of events of the gated populations in the bottom-right corner. Table 1. Statistical comparison of the concentration, absorbance and RIN e values measured with the Nanodrop and TapeStation for all samples fixed with KFNew, KFOld and RNAlater. Comparisons were performed separately for each species. Only p-values for statistically significant differences (p < 0.05) are shown in parentheses. Unless otherwise specified, statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Comparisons marked with an asterisk were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparisons, applied when data did not follow a normal distribution. Supplementary Table S1 . Nanodrop and TapeStation measures of all samples used in the study. Average values are in bold at the end of the corresponding column. Supplementary Material File (fig1_3especies_av.pdf) Download 33.36 MB File (fig4_allspecies_pa-c_av_ido.pdf) Download 24.10 MB Information & Authors Information Version history V1 Version 1 18 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Ecology and Evolution Keywords comparative freshwater invertebrate laboratory marine none of the above statistical terrestrial Authors Affiliations Olmo Irene del Universidad Autonoma de Madrid Departamento de Biologia View all articles by this author Moreno Paula Universidad Autonoma de Madrid Departamento de Biologia View all articles by this author Patricia Alvarez-campos Universidad Autonoma de Madrid Departamento de Biologia View all articles by this author Aida Verdes 0000-0002-9193-9253 [email protected] Museo Nacional de Ciencias Naturales View all articles by this author Metrics & Citations Metrics Article Usage 235 views 56 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Olmo Irene del, Moreno Paula, Patricia Alvarez-campos, et al. A field-friendly, non-toxic fixative for integrated morphological and molecular research in non-model invertebrates. Authorea . 18 September 2025. 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