{"paper_id":"2d484fc7-fa01-4562-bd70-faecd39e841d","body_text":"Staying on board: synchronized behavioral switching allows phoretic nematodes to persist through host molting | 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 Staying on board: synchronized behavioral switching allows phoretic nematodes to persist through host molting Yusuke Katsumi, Mamoru Takata, Kenji Matsuura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9067626/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Habitat turnover can temporarily eliminate the environments that organisms depend on, posing a major challenge to their persistence. For epibionts living on animal body surfaces, periodic renewal of the host integument during molting causes a sudden and complete loss of habitat. How such epibionts persist on the same host individual through these drastic events has remained elusive. Here, we show that phoretic nematodes associated with the subterranean termite Reticulitermes speratus maintain attachment throughout host molting by adopting a synchronized behavioral strategy. We isolated two phylogenetically distant species of phoretic nematodes, Oigolaimella sp. and Diplogastrellus sp., from this termite. Both species were primarily attached to the mouthparts (labium and maxillae) of the worker caste, which undergoes periodic molting. Quantitative analyses of nematode abundance and attachment sites across defined worker molting stages revealed that nematodes temporarily migrated into the foregut during the period surrounding ecdysis to avoid being shed with the molted exoskeleton, which is subsequently consumed by nestmates. Our findings reveal a behavioral strategy that enables epibionts to persist despite complete host surface renewal. More broadly, this study highlights coordinated spatial reallocation as an adaptive mechanism facilitating persistence in environments characterized by cyclical disturbance. Habitat turnover Phoretic nematodes Termites Host molting Epibionts Behavioral synchronization Figures Figure 1 Figure 2 Figure 3 Significance Statement Recurring disturbance can repeatedly eliminate the very habitats on which organisms depend, yet some species maintain long-term associations under such conditions. Animal molting represents an extreme case: the host’s body surface is completely replaced, seemingly eliminating surface-dwelling epibionts. We show that phoretic nematodes of the termite Reticulitermes speratus circumvent this constraint through precise behavioral synchronization with the host’s molting process. Rather than being removed with the shed exoskeleton during molting, the nematodes transiently retreat into the host foregut and subsequently reattach to the newly exposed body surface of the same individual. This dynamic spatial reallocation allows uninterrupted host association despite complete and cyclical habitat renewal. By revealing how epibionts respond to host developmental transitions, our study identifies behavioral reallocation as an adaptive principle enabling persistence under repeated habitat loss, thereby extending general theory on disturbance, symbiosis, and ecological stability. Introduction Natural environments are inherently dynamic, fluctuating across temporal and spatial scales, from abrupt disturbances such as fires and droughts to periodic processes such as seasonal cycles (Sousa 1984; Bell 2010; Bernhardt et al. 2020). These processes generate habitat turnover, which involves the temporary loss and reorganization of living space and can constrain survival and reproduction (Turner 2010; Van Teeffelen et al. 2012; Haddad et al. 2015). Because persistence under habitat turnover represents a central problem in ecology, organisms have evolved diverse strategies to cope with both stochastic and predictable disturbance regimes (Southwood 1977; Wilcox et al. 2006; Bernhardt et al. 2020). Seasonal migration in birds, post-fire resprouting in plants, and dormancy or desiccation tolerance in microbes illustrate how species either endure local conditions or relocate when habitats become unsuitable (Salewski and Bruderer 2007; Rebecchi et al. 2007; Premoli and Steinke 2008). Explaining how organisms maintain persistence under recurrent habitat renewal remains fundamental to understanding ecological stability in fluctuating environments (Pascual and Guichard 2005; Fraterrigo and Rusak 2008). Animal body surfaces constitute biotic habitats that support a diverse assemblage of epibionts, yet they are intrinsically dynamic environments shaped by host behavior and physiology (Marshall 1981; Wahl 1989). Attachment to a host provides access to food resources, protection from natural enemies, and opportunities for dispersal, making persistence on the host critical for fitness (Wahl 1989; Bartlow and Agosta 2021). However, because the host itself undergoes behavioral and developmental transitions, epibionts experience repeated disturbance within this biotic habitat. Among the processes that disrupt attachment, renewal of the host integument imposes particularly strong constraints. Molting represents an extreme and cyclical form of habitat turnover, as the entire body surface is replaced and attached organisms are physically removed (Dyrynda 1986; Duneau and Ebert 2012). Once shed with the exuviae, epibionts are exposed to desiccation, predation, and other lethal stresses (Duneau and Ebert 2012; Bartlow and Agosta 2021). Thus, maintaining association with the same host individual across molting events constitutes a fundamental ecological challenge for organisms inhabiting animal body surfaces (Threlkeld et al. 1993). Termites provide an exceptionally stringent system for examining persistence under recurrent molting disturbance. As hemimetabolous insects, they undergo multiple molts while maintaining a broadly similar morphology across developmental stages (Howard and Thorne 2010). In particular, the worker caste, which constitutes the majority of the colony, remains developmentally juvenile and continues to molt periodically while performing essential colony tasks (Roisin and Korb 2010). For epibionts inhabiting the worker body surface, this creates a biotic habitat that undergoes repeated renewal within the same host individual. The social organization of termites further intensifies this constraint. Molting occurs under close assistance from nestmates, and shed exuviae are rapidly consumed (Xing et al. 2013; Tong et al. 2021). Consequently, detachment together with the exuviae is not merely a loss of attachment, but also entails an immediate risk of mortality through consumption by nestmates. Despite this combination of cyclical habitat replacement and elevated mortality risk, diverse epibionts have been documented on termite body surfaces (Wang et al. 2002; Wilson et al. 2021; Chen et al. 2022). Among these, phoretic nematodes have been repeatedly isolated from multiple termite genera, indicating a widespread and evolutionarily persistent association with their hosts (Kanzaki et al. 2012). In this study, we asked how phoretic nematodes associated with termites persist on the same host individual despite complete and recurrent habitat turnover caused by molting. Using the subterranean termite Reticulitermes speratus as a model system, we first isolated phoretic nematodes and characterized their species composition through molecular phylogenetic analyses, which revealed the coexistence of two distinct and phylogenetically distant species. Because termite colonies consist of multiple castes that differ in morphology, behavior, and developmental state, we next quantified nematode abundance across castes to determine whether association was caste specific. This analysis demonstrated a strong bias toward workers, the caste that undergoes periodic molting. To examine nematode responses to host integument renewal, we then defined discrete stages of the worker molting cycle based on detailed morphological criteria. Finally, using this staging framework, we quantified nematode abundance and attachment sites across molting stages to evaluate spatial and temporal dynamics in relation to host developmental transitions. Materials and methods Isolation and molecular identification of phoretic nematodes from Reticulitermes speratus Phoretic nematodes were isolated from workers of R. speratus (Fig. 1a) collected in Kyoto Prefecture, Japan, for molecular phylogenetic analyses. Each worker was dissected in 0.75% NaCl solution under a stereomicroscope (Olympus SZX16), and nematodes attached to the body surface were collected using fine needles. DNA was extracted from single nematodes following the protocol of Sata (2018) with minor modifications. Each nematode was incubated at 50 °C for 120 min and then at 95 °C for 20 min in a solution containing 1 μL of proteinase K (2 mg/mL) and 4 μL of lysis buffer (10 mM Tris–HCl, pH 8.0; 150 mM NaCl; 10 mM EDTA, pH 8.0; 0.1% SDS). After incubation, 4 μL of TE buffer (10 mM Tris–HCl, pH 8.0; 1 mM EDTA, pH 8.0) was added, and the samples were stored at 4 °C. The 18S rDNA fragment (~600 bp) was amplified using the primer pair 18S 965 (5′-GGCGATCAGATACCGCCCTAGTT-3′) and 18S 1573R (5′-TACAAAGGGCAGGGACGTAAT-3′) (Powers et al. 2009). The total PCR volume was 10 μL, including 5 μL of KOD One® PCR Master Mix (TOYOBO, Osaka, Japan), 0.3 μL of each primer (10 μM), 1 μL of DNA template, and 3.4 μL of nuclease-free water. A ProFlex™ PCR System (Applied Biosystems, MA, USA) was used for PCR amplification, and the amplification conditions were as follows: 98 °C for 2 min; 35 cycles of 98 °C for 10 s, 58 °C for 5 s, and 68 °C for 5 s; followed by a final extension at 68 °C for 1 min. PCR products were confirmed by 1% agarose gel electrophoresis and stored at 4 °C prior to sequencing. Sequencing reactions were performed in both directions using BigDye Terminator chemistry on a 3500 Genetic Analyzer (Applied Biosystems). Sequence chromatograms were inspected and aligned using the MUSCLE algorithm implemented in MEGA11 (Tamura et al. 2021). The resulting sequences were compared against GenBank sequences using BLAST, and the closest matches were selected as reference sequences. Phylogenetic trees were constructed using the Maximum Likelihood method under the General Time Reversible model with gamma-distributed rate variation and a proportion of invariant sites (GTR+G+I). Node support was assessed with 1000 bootstrap replicates in MEGA11. Mononchoides striatus (GenBank accession no. AY593924) was used as an outgroup. Quantification of nematode abundance across host castes and body regions To quantify the abundance of phoretic nematodes across host castes and body regions and to evaluate patterns of attachment, three colonies of R. speratus were examined. From each colony, one primary king and five individuals of each of the following castes, secondary queens, soldiers, and workers, were collected. Because multiple secondary queens coexist within colonies of R. speratus due to asexual queen succession (Matsuura et al. 2009), secondary queens were treated as the representative queen caste in this study. Each termite was divided into the head, thorax, and abdomen and dissected in 0.75% NaCl solution under a stereomicroscope, and attached nematodes were counted for each caste and body region. Definition of molting stages based on morphological markers As a prerequisite for examining the adaptive responses of phoretic nematodes to the periodic molting of workers, the molting cycle of R. speratus was classified based on morphological characteristics. Molting stages were defined using external morphology, the presence or absence of gut contents, and the presence or absence of gut air bubbles as diagnostic indicators. Termite workers are known to alternate repeatedly between an intermolt period, during which they actively forage and feed while performing colony tasks, and a molting process (Raina et al. 2008; Xing et al. 2013) (Fig. 2a). The molting process is defined as the period during which feeding ceases in preparation for ecdysis, gut contents are expelled (gut purging), and feeding does not resume until after ecdysis, when cuticle hardening is completed and symbiotic microbiota are reacquired (Raina et al. 2008; Masuoka et al. 2013; Kakkar et al. 2016). Accordingly, intermolt individuals and individuals undergoing the molting process were distinguished based on the presence or absence of gut contents. Because the termite body is semi-transparent, individuals with gut contents exhibit a darker abdomen, whereas those that have undergone gut purging display a pale and translucent appearance. Therefore, non-molting and molting individuals could be visually distinguished based on abdominal coloration. The validity of this criterion was confirmed by microscopic examination and dissection, which verified that workers with a translucent abdomen had expelled their gut contents and lost their symbiotic flagellates. Furthermore, the cuticle is renewed during the molting process, and immediately after ecdysis, the exoskeleton lacks substantial melanin deposition (Xing et al. 2013; Kakkar et al. 2016). Therefore, exoskeletal coloration, particularly that of the mandibles, was used as a morphological marker to distinguish individuals before and after ecdysis. In R. speratus , workers that have expelled their gut contents are known to undergo ecdysis approximately 5–6 days later (Masuoka et al. 2013). Based on these morphological characteristics and temporal information, we defined four stages within the molting process of R. speratus : pre-molt, pre-ecdysis, post-ecdysis, and post-molt. Terminology and stage definitions followed previous studies (Raina et al. 2008; Xing et al. 2013). Identification of pre-molt and pre-ecdysis individuals To distinguish between pre-molt and pre-ecdysis stages among gut-purged individuals, we used the external appearance of the head capsule as a morphological indicator. Previous studies in other termite genera have reported the formation of wrinkles on the head capsule immediately prior to ecdysis (Xing et al. 2013). We therefore tested whether this characteristic could be applied to R. speratus . Individuals lacking gut contents and exhibiting darkly pigmented mandibles were selected. Among them, those showing visible wrinkles on the head capsule were designated as pre-ecdysis candidates. Selected individuals were marked on the abdomen with enamel paint and placed with 50 unmarked workers from the same colony in a 30-mm Petri dish lined with moist filter paper. The occurrence of ecdysis was assessed 12 h after marking, based on the operational definition that the pre-ecdysis stage lasts approximately 12 h before exoskeleton shedding (Xing et al. 2013). Ecdysis was confirmed by the disappearance of the paint mark. For each colony, 10 marked individuals were examined, and the experiment was replicated across three colonies. As a control, gut-purged individuals with dark mandibles but without head capsule wrinkles were marked and reared under identical conditions. Ecdysis was assessed after 12 h. Based on these results, the presence of head capsule wrinkles was validated as a reliable indicator of the pre-ecdysis stage, and gut-purged individuals lacking this characteristic were classified as pre-molt. Identification of post-ecdysis and post-molt individuals To distinguish between post-ecdysis and post-molt stages, we used the presence of gut air bubbles as a morphological indicator. Because gut air bubbles appear after the completion of ecdysis, we first verified their temporal dynamics following molting. Workers in which ecdysis was confirmed were marked on the abdomen with enamel paint and housed with 50 unmarked workers from the same colony in a 30-mm Petri dish lined with moist filter paper. Marked individuals were collected at 3-h intervals after ecdysis, and dissections were performed to assess the presence or absence of gut air bubbles. At each time point, five individuals per colony were examined, and the experiment was replicated across three colonies. Using this procedure, we recorded the time elapsed from ecdysis until the disappearance of gut air bubbles. Based on these results, individuals exhibiting gut air bubbles were classified as post-ecdysis, whereas those lacking air bubbles were classified as post-molt. Time to feeding resumption after ecdysis To estimate the duration of the molting process, we quantified the time required for workers to resume feeding after ecdysis. Workers immediately after ecdysis were marked on the body surface with enamel paint and housed with 50 unmarked workers from the same colony in a 30-mm Petri dish containing brown-rotted pinewood mixed cellulose (BPC) medium (Mitaka et al. 2023). Marked individuals were collected and dissected at 12-h intervals to assess the presence or absence of gut contents. At each time point, five individuals per colony were examined, with three colonies used as replicates. Individuals with detectable gut contents were considered to have resumed feeding. Comparison of nematode abundance before and after host ecdysis under individual isolation To examine the effects of host ecdysis on phoretic nematodes, we compared nematode abundance on hosts before and after ecdysis and on exuviae. Workers in the pre-molt and pre-ecdysis stages were individually placed in 30-mm Petri dishes lined with moistened filter paper and maintained in isolation for 24 h at 25 °C under dark, humid conditions. Molted individuals, non-molted individuals, and exuviae were then collected and individually dissected in 0.75% NaCl solution under a stereomicroscope. The total number of associated nematodes was recorded for each sample. For each colony and sample category, at least 10 samples were examined, and the experiment was replicated across three colonies. The final sample sizes were 30 individuals before ecdysis, 31 individuals after ecdysis, and 31 exuviae. Quantification of nematode abundance and attachment sites during the molting cycle To examine nematode responses to host molting, we quantified nematode abundance and attachment sites at each stage of the molting cycle. Workers were collected from three independent colonies and assigned to one of five stages: intermolt, pre-molt, pre-ecdysis, post-ecdysis, and post-molt, based on the criteria defined in this study. Each worker was dissected individually in 0.75% NaCl solution under a stereomicroscope, and both the total number of nematodes and their attachment sites were recorded. At least 15 individuals per stage were examined for each colony. The final sample sizes were 56 individuals for intermolt, 67 for pre-molt, 62 for pre-ecdysis, 67 for post-ecdysis, and 68 for post-molt. In addition, a subset of nematodes was subjected to molecular phylogenetic analysis based on partial 18S rDNA gene sequences. Statistical analysis All statistical analyses were performed in R (version 4.4.1) (R Core Team 2024). For the analysis of caste-dependent differences in nematode abundance, differences among host castes were tested using a Kruskal–Wallis test followed by Dunn’s post hoc test with Bonferroni correction. For the individual molting experiment examining nematode persistence across host ecdysis, nematode abundance among molting states (before- and after-ecdysis individuals) and exuviae was analyzed using a generalized linear model (GLM) with a Poisson error distribution, including molting state and colony as explanatory variables. The effects of molting state and colony were evaluated using likelihood ratio tests (LRTs). The interaction between molting state and colony was tested by comparing models with and without the interaction using an LRT. Tukey-adjusted pairwise comparisons were conducted for post hoc comparisons among molting states and exuviae. For the analysis of nematode abundance across stages of the host molting cycle, we fitted a GLM with a negative binomial error distribution including molting stage and colony as explanatory variables. The effect of molting stage was assessed using an LRT. Tukey-adjusted pairwise comparisons were conducted for post hoc comparisons among molting stages. The interaction between molting stage and colony was tested by comparing models with and without the interaction using an LRT. For the analysis of nematode localization within hosts across stages of the host molting cycle, we analyzed the proportion of nematodes located in the foregut using a generalized linear model (GLM) with a binomial error distribution and logit link function. The response variable was the number of foregut-localized nematodes relative to the total number of nematodes recovered from each worker, and molting stage and colony were included as explanatory variables. Because the distribution of nematodes among host compartments differed strongly among molting stages, the data could exhibit separation (i.e., some stages showing exclusively foregut or non-foregut occurrences). To address this issue, model parameters were estimated using bias-reduced maximum likelihood estimation implemented in the brglmFit method. The effects of molting stage, colony, and their interaction were evaluated using likelihood ratio tests (LRTs). Sidak-adjusted pairwise comparisons were conducted for post hoc comparisons among molting stages. Results Molecular identification of phoretic nematodes associated with Reticulitermes speratus Two phoretic nematode taxa were isolated from the body surface of worker termites of R. speratus . Molecular phylogenetic analysis based on partial 18S rDNA sequences identified these nematodes as belonging to the genera Oigolaimella and Diplogastrellus (Fig. 1b). The obtained sequences showed high similarity (>99%) to previously reported phoretic nematode sequences from R. speratus , including Oigolaimella sp. (Susoy et al. 2015) and Diplogastrellus sp. (GenBank accession no. AB597239). Both taxa exhibited morphological characteristics typical of the dauer stage while attached to their termite hosts. The two genera were detected in worker individuals from the same colonies. Under stereomicroscopic observation, the two taxa were highly similar in external morphology, making reliable discrimination based solely on morphology difficult. For subsequent quantitative analyses, Oigolaimella sp. and Diplogastrellus sp. were treated collectively as a single phoretic nematode assemblage. Caste-dependent differences in nematode abundance The abundance of phoretic nematodes differed significantly among host castes (Kruskal–Wallis test, χ ² = 38.74, df = 3, p < 0.001; Fig. 1c). Post hoc pairwise comparisons using Dunn’s test with Bonferroni correction showed that workers carried significantly more nematodes than primary kings ( p = 0.010), secondary queens ( p < 0.001), and soldiers ( p < 0.001). In contrast, no significant differences were detected among the non-worker castes (primary kings, secondary queens, and soldiers). Across all castes examined, nematodes were detected exclusively on the head and were absent from the thorax and abdomen. On the head, nematodes were observed attached to the mouthparts, particularly to the labium and maxillae. Establishment of temporal and morphological markers of the worker molting cycle Workers within the molting process that had completed gut purging and exhibited wrinkles on the head capsule all underwent ecdysis within 12 h after observation (Fig. 2b). In contrast, individuals without wrinkles on the head capsule did not undergo ecdysis within the same period. Based on these results, individuals with wrinkles on the head capsule were defined as pre-ecdysis, whereas gut-purged individuals showing pronounced mandibular pigmentation but without wrinkles on the head capsule were defined as pre-molt. Analysis of the time from ecdysis to gut air bubble disappearance showed that all individuals examined at the 15-h post-ecdysis time point lacked gut air bubbles (Fig. 2c). Based on this result, the presence of gut air bubbles was defined as a diagnostic marker of the immediate post-ecdysis stage. Individuals lacking gut air bubbles, lacking pronounced mandibular pigmentation, and not yet having resumed feeding were defined as post-molt. Examination of the time from ecdysis to feeding resumption showed that all individuals examined at the 48-h post-ecdysis time point had resumed feeding (Fig. 2d). In contrast, some individuals examined at earlier time points had not yet resumed feeding. These results indicate that workers enter the intermolt stage within 2 days after ecdysis. By integrating these temporal and morphological markers, we established a detailed framework of the worker molting cycle in R. speratus (Fig. 2e). The molting process lasted approximately 8 days and was operationally divided into four stages: pre-molt, pre-ecdysis, post-ecdysis, and post-molt. Persistence and redistribution of nematodes across the host molting cycle In the individual molting experiment, nematodes were detected on workers after ecdysis. Nematode abundance did not differ between individuals before and after ecdysis (Tukey-adjusted comparison: p = 0.691; Fig. S1). In contrast, nematode abundance was significantly higher on both before-ecdysis and after-ecdysis individuals than on exuviae ( p < 0.001 for both comparisons; Fig. S1). Overall, nematode abundance differed significantly among the three groups (before-ecdysis individuals, after-ecdysis individuals, and exuviae) (GLM, LRT: χ ² = 197.609, df = 2, p < 0.001), whereas colony had no significant effect (GLM, LRT: χ ² = 0.573, df = 2, p = 0.751), and the interaction between group and colony was not significant (GLM, LRT: χ ² = 2.194, df = 4, p = 0.700). These results indicate that most nematodes remain attached to the host and are rarely left on the exuviae during ecdysis. Observations of attachment sites at different stages of the molting cycle revealed that nematodes were detected in the foregut primarily during the pre-ecdysis and post-ecdysis stages (Fig. 3a, b). In contrast, at the other stages, nematodes were attached predominantly to the mouthparts, and foregut localization was rare. Total nematode abundance per host individual did not differ among molting stages (Fig. 3c). Molting stage had no significant effect on total nematode abundance (GLM, LRT: χ ² = 2.301, df = 4, p = 0.687), whereas colony had a significant effect (GLM, LRT: χ ² = 6.301, df = 2, p = 0.045). Post hoc pairwise comparisons detected no significant differences between any pair of molting stages (Tukey-adjusted comparisons). The interaction between molting stage and colony was not significant (GLM, LRT: χ ² = 3.886, df = 8, p = 0.870). However, nematode localization within termite individuals differed significantly among molting stages. The proportion of foregut-localized nematodes was significantly affected by molting stage (GLM, LRT: χ ² = 861.56, df = 4, p < 0.001), whereas neither colony (GLM, LRT: χ ² = 2.39, df = 2, p = 0.302) nor the interaction between molting stage and colony (GLM, LRT: χ ² = 3.494, df = 8, p = 0.900) had significant effects. Post hoc pairwise comparisons showed that the proportions of foregut-localized nematodes were significantly higher at the pre-ecdysis and post-ecdysis stages than at the intermolt, pre-molt, and post-molt stages (Sidak-adjusted comparisons, p < 0.001). The proportion was also significantly higher at the pre-ecdysis stage than at the post-ecdysis stage ( p < 0.001). In contrast, no significant differences were detected between any pair of the intermolt, pre-molt, and post-molt stages. These results indicate that nematodes temporarily relocate from the mouthparts to the foregut in synchrony with host ecdysis. Molecular analysis of nematodes detected in the foregut revealed that they belonged to Oigolaimella sp. and Diplogastrellus sp., the same taxa as those isolated from the body surface (Fig. S2). Discussion We demonstrate that phoretic nematodes associated with the subterranean termite Reticulitermes speratus persist on the same host individual despite complete and recurrent habitat turnover caused by host molting. Social insects form large, long-lived colonies (Wilson 1971) with relatively stable internal environments (Oster and Wilson 1978), making them persistent and resource-rich habitats for a wide range of parasites, mutualists, and phoronts (Hughes et al. 2008). However, in addition to social immunity mechanisms represented by frequent mutual grooming and nest sanitation (Meunier 2015), periodic molting in termites (Howard and Thorne 2010) creates strong removal pressures, making it a particularly difficult environment for attached organisms to maintain their association. Despite these constraints, our results showed no decline in nematode abundance across molting events, indicating continuous attachment to the same host individual. Moreover, during a restricted period immediately before and after ecdysis, the number of nematodes on the body surface temporarily decreased, while the number detected in the foregut increased. These patterns indicate that nematodes relocate from the body surface to the foregut in synchrony with host molting. Together, our findings show that termite-phoretic nematodes maintain attachment during molting through spatial reallocation between the body surface and the gut, thereby continuously exploiting a high-benefit but high-risk social host environment. The transient migration to the foregut, which lies at the core of this persistence strategy, appears to exploit the anatomical dynamics of insect molting. The foregut is an ectodermally derived structure lined with cuticle, and like the external exoskeleton it is renewed during ecdysis (Hoskins and Craig 1935). However, during molting, the foregut cuticle separates from the external cuticle and is subsequently transported posteriorly through the gut lumen, as suggested for other insects (Rowland and Goodman 2016). We confirmed a similar foregut renewal pattern in R. speratus (Fig. S3). By migrating to the foregut, which constitutes the region where the exoskeletal cuticle separates from the foregut lining and where sloughing of the old cuticle is initiated, nematodes can gain early access to the renewed host cuticle (Fig. S4). This relocation likely enables them to avoid being shed with the exuviae and thereby maintain attachment to the same host individual. Notably, nematodes began migrating into the foregut before ecdysis, suggesting that they respond in advance to physiological changes in the host associated with the molting process. Termite molting is accompanied by cessation of feeding, assistance from nestmates, and cuticular softening (Raina et al. 2008; Xing et al. 2013), any of which could serve as cues triggering behavioral switching. Compared with previously documented responses of epibionts to host molting, this strategy is distinctive. In decapod crustaceans, branchial isopods attach to newly exposed internal surfaces during ecdysis (Cash and Bauer 1993), whereas other symbionts temporarily detach and reattach after molting is completed (Shiozaki and Itani 2020). In aquatic insects, water mites transfer directly from the exuviae of pupae to newly emerged adults (Jalil and Mitchell 1972). All of these strategies rely on repositioning within the external space of the host individual. In contrast, the nematodes documented here achieve continuous association by switching between external and internal host compartments, exploiting the host’s own anatomical renewal process. The fact that this spatial reallocation strategy was observed in two phylogenetically distant lineages further suggests that recurrent molting constitutes a strong and predictable selective pressure capable of promoting convergent behavioral solutions. More broadly, our results provide a novel adaptive mechanism by which epibionts can overcome the structural constraint of complete host surface renewal. The observation that phoretic nematodes were localized on the body surface of periodically molting workers is central to understanding their life history. Within termite colonies, soldiers and reproductives have completed their final molt (Roisin and Korb 2010), and attachment to these castes would eliminate the risk of removal through molting. Nevertheless, nematodes were biased toward workers, suggesting that workers provide benefits that compensate for the risks associated with recurrent habitat turnover. Previous studies on other termite species and their associated nematodes have shown that dauer larvae can be isolated from termite hosts, whereas adult stages occur in nest materials, indicating that phoresy functions as a dispersal stage to termite nests (Fürst von Lieven and Sudhaus 2008). Therefore, in the nematodes examined in this study, phoresy on workers is likewise suggested to serve as a means of dispersal into nest substrates. Members of the genera Oigolaimella and Diplogastrellus have been shown to be bacteria feeders (Steel et al. 2010; Kanzaki et al. 2012), and termite nest materials rich in bacteria (Manjula et al. 2014) are considered to constitute a major feeding resource base. Workers perform labor within the colony and frequently engage in foraging activities at the colony periphery, making them more mobile than other castes (Traniello and Leuthold 2000). Therefore, the bias toward workers together with the molting-associated persistence strategy can be interpreted as a coordinated adaptation that maximizes dispersal opportunities. These findings suggest that phoresy is not merely a means of transport, but rather a highly integrated life-history strategy closely linked to host ecological roles and developmental dynamics. Taken together, our findings identify synchronized spatial reallocation between host compartments as a distinct adaptive solution that allows epibionts to persist despite recurrent host surface renewal. These nematodes possess a remarkable capacity to detect subtle physiological changes associated with host ecdysis from outside the host body and to adjust their behavior accordingly, underscoring the finely tuned nature of host–epibiont interactions. Elucidating the mechanisms underlying this sensory and behavioral coordination will provide deeper insight into the coevolutionary dynamics between hosts and their surface-associated organisms. More broadly, our study illustrates an adaptive mode in which persistence is achieved not by avoiding disturbance, but by evolving strategies synchronized with cyclical environmental transformation. This perspective offers a conceptual framework for understanding how organisms persist in dynamic environments characterized by predictable structural change. Declarations Funding This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP23H00332. Competing Interests The authors declare no competing interests. Ethics approval Not applicable. Consent to participate Not applicable. Additional Information No additional information. Data availability The datasets supporting the conclusions of this article are included in the supplementary file. Code availability The code used for graphing and statistical analyses is provided in the supplementary file. Acknowledgements We thank Soshi Araki and Yusei Takaishi for providing photographs, and Kiyotaka Yabe and Daichi Gomi for assistance with termite collection and for helpful discussions. Author contributions Conceptualization: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Methodology: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Validation: Yusuke Katsumi; Formal Analysis: Yusuke Katsumi; Investigation: Yusuke Katsumi; Resources: Yusuke Katsumi, Mamoru Takata; Data Curation: Yusuke Katsumi; Writing – Original Draft: Yusuke Katsumi, Kenji Matsuura; Writing – Review & Editing: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Visualization: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Supervision: Mamoru Takata, Kenji Matsuura; Project Administration: Yusuke Katsumi, Mamoru Takata; Funding Acquisition: Kenji Matsuura. 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Ann Entomol Soc Am 106:619–625. https://doi.org/10.1603/AN13007 Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.zip Supplementaryfigures.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers invited by journal 23 Mar, 2026 Editor assigned by journal 18 Mar, 2026 Submission checks completed at journal 18 Mar, 2026 First submitted to journal 08 Mar, 2026 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. 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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-9067626\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":624499776,\"identity\":\"2aa37078-1aa2-4019-890f-699e3eb4a720\",\"order_by\":0,\"name\":\"Yusuke Katsumi\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"\",\"institution\":\"Kyoto University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Yusuke\",\"middleName\":\"\",\"lastName\":\"Katsumi\",\"suffix\":\"\"},{\"id\":624499777,\"identity\":\"008cec39-a22c-4182-829b-24464852a234\",\"order_by\":1,\"name\":\"Mamoru Takata\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Kyoto University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mamoru\",\"middleName\":\"\",\"lastName\":\"Takata\",\"suffix\":\"\"},{\"id\":624499778,\"identity\":\"72c4104a-727a-457e-a605-41f7f422024f\",\"order_by\":2,\"name\":\"Kenji Matsuura\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Kyoto University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kenji\",\"middleName\":\"\",\"lastName\":\"Matsuura\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-03-09 02:53:18\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9067626/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9067626/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":107482895,\"identity\":\"a13830c8-b5e2-46f2-8346-0378ac5ec1e1\",\"added_by\":\"auto\",\"created_at\":\"2026-04-22 02:25:23\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":11055218,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhoretic nematodes associated with the subterranean termite \\u003cem\\u003eReticulitermes speratus\\u003c/em\\u003e. (a) A colony of \\u003cem\\u003eR. speratus\\u003c/em\\u003e showing the different castes. PK, primary king; SQ, secondary queen; S, soldier; W, worker. (b) Maximum likelihood phylogenetic tree showing the relationships of phoretic nematodes associated with \\u003cem\\u003eR. speratus\\u003c/em\\u003e based on partial 18S rDNA sequences. Numbers on branches indicate bootstrap support values (%) from 1000 replicates. Micrographs show phoretic nematodes (\\u003cem\\u003eDiplogastrellus\\u003c/em\\u003e sp. and \\u003cem\\u003eOigolaimella\\u003c/em\\u003e sp.). Scale bars = 100 µm. (c) Number of phoretic nematodes per termite in each caste of \\u003cem\\u003eR. speratus\\u003c/em\\u003e. Bars represent mean ± SE, and points represent individual termites. Different letters indicate significant differences among castes (Kruskal–Wallis test followed by Dunn’s test with Bonferroni correction, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9067626/v1/f387a93d11add8a138d98c75.png\"},{\"id\":107163522,\"identity\":\"1ee83fd9-a562-4dda-873d-f7f7b55692f6\",\"added_by\":\"auto\",\"created_at\":\"2026-04-17 13:30:15\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":18178383,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMorphological indicators of molting stages in \\u003cem\\u003eReticulitermes speratus\\u003c/em\\u003e. (a) Schematic representation of the termite molting cycle showing the repeated alternation of the intermolt period and the molting process. (b) Morphological features distinguishing the pre-molt and pre-ecdysis stages. Workers exhibiting wrinkles on the head capsule molted within 12 h after observation. These individuals were defined as pre-ecdysis, whereas gut-purged individuals with pronounced mandibular pigmentation but lacking head-capsule wrinkles were defined as pre-molt. (c) Morphological features distinguishing the post-ecdysis and post-molt stages. Gut air bubbles disappeared within 15 h after ecdysis and were therefore used as a diagnostic marker of the immediate post-ecdysis stage. Individuals lacking gut air bubbles, showing no pronounced mandibular pigmentation, and not yet having resumed feeding were defined as post-molt. The graph shows the proportion of individuals possessing gut air bubbles at each time point after ecdysis. (d) Time required for workers to resume feeding after ecdysis. Gut contents were observed in all individuals examined at 48 h after ecdysis. The graph shows the proportion of individuals possessing gut contents at each time point after ecdysis. (e) Timeline of the worker molting process showing the stages defined in this study.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9067626/v1/446e36726c81f871ee08c804.png\"},{\"id\":107163523,\"identity\":\"c9895d57-1736-487d-b617-f6ef2a0c7be3\",\"added_by\":\"auto\",\"created_at\":\"2026-04-17 13:30:16\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":35377631,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDynamics of phoretic nematode abundance and attachment site across the host molting cycle. (a) Schematic diagram showing the position of the foregut in a termite worker. (b) Micrograph of phoretic nematodes in the foregut of a worker termite at the pre-ecdysis stage. H, hypopharynx; Fg, foregut. Scale bar = 200 µm. (c) Total nematode abundance and attachment sites at different molting stages in termite workers. The upper panel shows the total number of nematodes per host individual (mean ± SE). Total nematode abundance did not differ among molting stages (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 2.27, df = 4, \\u003cem\\u003ep\\u003c/em\\u003e = 0.687). The lower panel shows the numbers of nematodes on the mouthparts and in the foregut at each molting stage. The proportion of foregut-localized nematodes differed significantly among molting stages (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 861.56, df = 4, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001). Foregut localization was significantly higher at the pre-ecdysis and post-ecdysis stages than at the intermolt, pre-molt, and post-molt stages (Sidak-adjusted comparisons, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001), and was also significantly higher at the pre-ecdysis stage than at the post-ecdysis stage (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001). No significant differences were detected between any pair of the intermolt, pre-molt, and post-molt stages.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9067626/v1/c176f29f0645914efe983531.png\"},{\"id\":107485626,\"identity\":\"b4290341-e138-4d71-86bf-60c9710f73e6\",\"added_by\":\"auto\",\"created_at\":\"2026-04-22 02:35:40\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":77146604,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9067626/v1/c7bdee38-f4af-4f2c-91f9-5b1587333134.pdf\"},{\"id\":107163520,\"identity\":\"c1ba15da-e623-474d-9a16-a652772d8340\",\"added_by\":\"auto\",\"created_at\":\"2026-04-17 13:30:15\",\"extension\":\"zip\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":27431,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarydata.zip\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9067626/v1/b242362f457f423077bbe7c9.zip\"},{\"id\":107481494,\"identity\":\"cc3ad40b-b013-434c-bed3-5f95fb63681d\",\"added_by\":\"auto\",\"created_at\":\"2026-04-22 02:18:30\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":493971,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementaryfigures.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9067626/v1/eccefd1cd3b1ec144e65f333.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Staying on board: synchronized behavioral switching allows phoretic nematodes to persist through host molting\",\"fulltext\":[{\"header\":\"Significance Statement\",\"content\":\"\\u003cp\\u003eRecurring disturbance can repeatedly eliminate the very habitats on which organisms depend, yet some species maintain long-term associations under such conditions. Animal molting represents an extreme case: the host\\u0026rsquo;s body surface is completely replaced, seemingly eliminating surface-dwelling epibionts. We show that phoretic nematodes of the termite \\u003cem\\u003eReticulitermes speratus\\u003c/em\\u003e circumvent this constraint through precise behavioral synchronization with the host\\u0026rsquo;s molting process. Rather than being removed with the shed exoskeleton during molting, the nematodes transiently retreat into the host foregut and subsequently reattach to the newly exposed body surface of the same individual. This dynamic spatial reallocation allows uninterrupted host association despite complete and cyclical habitat renewal. By revealing how epibionts respond to host developmental transitions, our study identifies behavioral reallocation as an adaptive principle enabling persistence under repeated habitat loss, thereby extending general theory on disturbance, symbiosis, and ecological stability.\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eNatural environments are inherently dynamic, fluctuating across temporal and spatial scales, from abrupt disturbances such as fires and droughts to periodic processes such as seasonal cycles (Sousa 1984; Bell 2010; Bernhardt et al. 2020). These processes generate habitat turnover, which involves the temporary loss and reorganization of living space and can constrain survival and reproduction (Turner 2010; Van Teeffelen et al. 2012; Haddad et al. 2015). Because persistence under habitat turnover represents a central problem in ecology, organisms have evolved diverse strategies to cope with both stochastic and predictable disturbance regimes (Southwood 1977; Wilcox et al. 2006; Bernhardt et al. 2020). Seasonal migration in birds, post-fire resprouting in plants, and dormancy or desiccation tolerance in microbes illustrate how species either endure local conditions or relocate when habitats become unsuitable (Salewski and Bruderer 2007; Rebecchi et al. 2007; Premoli and Steinke 2008). Explaining how organisms maintain persistence under recurrent habitat renewal remains fundamental to understanding ecological stability in fluctuating environments (Pascual and Guichard 2005; Fraterrigo and Rusak 2008).\\u003c/p\\u003e\\n\\u003cp\\u003eAnimal body surfaces constitute biotic habitats that support a diverse assemblage of epibionts, yet they are intrinsically dynamic environments shaped by host behavior and physiology (Marshall 1981; Wahl 1989). Attachment to a host provides access to food resources, protection from natural enemies, and opportunities for dispersal, making persistence on the host critical for fitness (Wahl 1989; Bartlow and Agosta 2021). However, because the host itself undergoes behavioral and developmental transitions, epibionts experience repeated disturbance within this biotic habitat. Among the processes that disrupt attachment, renewal of the host integument imposes particularly strong constraints. Molting represents an extreme and cyclical form of habitat turnover, as the entire body surface is replaced and attached organisms are physically removed (Dyrynda 1986; Duneau and Ebert 2012). Once shed with the exuviae, epibionts are exposed to desiccation, predation, and other lethal stresses (Duneau and Ebert 2012; Bartlow and Agosta 2021). Thus, maintaining association with the same host individual across molting events constitutes a fundamental ecological challenge for organisms inhabiting animal body surfaces (Threlkeld et al. 1993).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTermites provide an exceptionally stringent system for examining persistence under recurrent molting disturbance. As hemimetabolous insects, they undergo multiple molts while maintaining a broadly similar morphology across developmental stages (Howard and Thorne 2010). In particular, the worker caste, which constitutes the majority of the colony, remains developmentally juvenile and continues to molt periodically while performing essential colony tasks (Roisin and Korb 2010). For epibionts inhabiting the worker body surface, this creates a biotic habitat that undergoes repeated renewal within the same host individual. The social organization of termites further intensifies this constraint. Molting occurs under close assistance from nestmates, and shed exuviae are rapidly consumed (Xing et al. 2013; Tong et al. 2021). Consequently, detachment together with the exuviae is not merely a loss of attachment, but also entails an immediate risk of mortality through consumption by nestmates. Despite this combination of cyclical habitat replacement and elevated mortality risk, diverse epibionts have been documented on termite body surfaces (Wang et al. 2002; Wilson et al. 2021; Chen et al. 2022). Among these, phoretic nematodes have been repeatedly isolated from multiple termite genera, indicating a widespread and evolutionarily persistent association with their hosts (Kanzaki et al. 2012).\\u003c/p\\u003e\\n\\u003cp\\u003eIn this study, we asked how phoretic nematodes associated with termites persist on the same host individual despite complete and recurrent habitat turnover caused by molting. Using the subterranean termite \\u003cem\\u003eReticulitermes speratus\\u003c/em\\u003e as a model system, we first isolated phoretic nematodes and characterized their species composition through molecular phylogenetic analyses, which revealed the coexistence of two distinct and phylogenetically distant species. Because termite colonies consist of multiple castes that differ in morphology, behavior, and developmental state, we next quantified nematode abundance across castes to determine whether association was caste specific. This analysis demonstrated a strong bias toward workers, the caste that undergoes periodic molting. To examine nematode responses to host integument renewal, we then defined discrete stages of the worker molting cycle based on detailed morphological criteria. Finally, using this staging framework, we quantified nematode abundance and attachment sites across molting stages to evaluate spatial and temporal dynamics in relation to host developmental transitions.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eIsolation and molecular identification of phoretic nematodes from\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eReticulitermes speratus\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePhoretic nematodes were isolated from workers of \\u003cem\\u003eR. speratus\\u003c/em\\u003e (Fig. 1a) collected in Kyoto Prefecture, Japan, for molecular phylogenetic analyses. Each worker was dissected in 0.75% NaCl solution under a stereomicroscope (Olympus SZX16), and nematodes attached to the body surface were collected using fine needles. DNA was extracted from single nematodes following the protocol of Sata (2018) with minor modifications. Each nematode was incubated at 50 °C for 120 min and then at 95 °C for 20 min in a solution containing 1 μL of proteinase K (2 mg/mL) and 4 μL of lysis buffer (10 mM Tris–HCl, pH 8.0; 150 mM NaCl; 10 mM EDTA, pH 8.0; 0.1% SDS). After incubation, 4 μL of TE buffer (10 mM Tris–HCl, pH 8.0; 1 mM EDTA, pH 8.0) was added, and the samples were stored at 4 °C. The 18S rDNA fragment (~600 bp) was amplified using the primer pair 18S 965 (5′-GGCGATCAGATACCGCCCTAGTT-3′) and 18S 1573R (5′-TACAAAGGGCAGGGACGTAAT-3′) (Powers et al. 2009). The total PCR volume was 10 μL, including 5 μL of KOD One® PCR Master Mix (TOYOBO, Osaka, Japan), 0.3 μL of each primer (10 μM), 1 μL of DNA template, and 3.4 μL of nuclease-free water. A ProFlex™ PCR System (Applied Biosystems, MA, USA) was used for PCR amplification, and the amplification conditions were as follows: 98 °C for 2 min; 35 cycles of 98 °C for 10 s, 58 °C for 5 s, and 68 °C for 5 s; followed by a final extension at 68 °C for 1 min. PCR products were confirmed by 1% agarose gel electrophoresis and stored at 4 °C prior to sequencing. Sequencing reactions were performed in both directions using BigDye Terminator chemistry on a 3500 Genetic Analyzer (Applied Biosystems). Sequence chromatograms were inspected and aligned using the MUSCLE algorithm implemented in MEGA11 (Tamura et al. 2021). The resulting sequences were compared against GenBank sequences using BLAST, and the closest matches were selected as reference sequences. Phylogenetic trees were constructed using the Maximum Likelihood method under the General Time Reversible model with gamma-distributed rate variation and a proportion of invariant sites (GTR+G+I). Node support was assessed with 1000 bootstrap replicates in MEGA11. \\u003cem\\u003eMononchoides striatus\\u003c/em\\u003e (GenBank accession no. AY593924) was used as an outgroup.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eQuantification of nematode abundance across host castes and body regions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo quantify the abundance of phoretic nematodes across host castes and body regions and to evaluate patterns of attachment, three colonies of \\u003cem\\u003eR. speratus\\u003c/em\\u003e were examined. From each colony, one primary king and five individuals of each of the following castes, secondary queens, soldiers, and workers, were collected. Because multiple secondary queens coexist within colonies of \\u003cem\\u003eR. speratus\\u003c/em\\u003e due to asexual queen succession (Matsuura et al. 2009), secondary queens were treated as the representative queen caste in this study. Each termite was divided into the head, thorax, and abdomen and dissected in 0.75% NaCl solution under a stereomicroscope, and attached nematodes were counted for each caste and body region.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDefinition of molting stages based on morphological markers\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAs a prerequisite for examining the adaptive responses of phoretic nematodes to the periodic molting of workers, the molting cycle of \\u003cem\\u003eR. speratus\\u003c/em\\u003e was classified based on morphological characteristics. Molting stages were defined using external morphology, the presence or absence of gut contents, and the presence or absence of gut air bubbles as diagnostic indicators.\\u003c/p\\u003e\\n\\u003cp\\u003eTermite workers are known to alternate repeatedly between an intermolt period, during which they actively forage and feed while performing colony tasks, and a molting process (Raina et al. 2008; Xing et al. 2013) (Fig. 2a). The molting process is defined as the period during which feeding ceases in preparation for ecdysis, gut contents are expelled (gut purging), and feeding does not resume until after ecdysis, when cuticle hardening is completed and symbiotic microbiota are reacquired (Raina et al. 2008; Masuoka et al. 2013; Kakkar et al. 2016). Accordingly, intermolt individuals and individuals undergoing the molting process were distinguished based on the presence or absence of gut contents.\\u003c/p\\u003e\\n\\u003cp\\u003eBecause the termite body is semi-transparent, individuals with gut contents exhibit a darker abdomen, whereas those that have undergone gut purging display a pale and translucent appearance. Therefore, non-molting and molting individuals could be visually distinguished based on abdominal coloration. The validity of this criterion was confirmed by microscopic examination and dissection, which verified that workers with a translucent abdomen had expelled their gut contents and lost their symbiotic flagellates.\\u003c/p\\u003e\\n\\u003cp\\u003eFurthermore, the cuticle is renewed during the molting process, and immediately after ecdysis, the exoskeleton lacks substantial melanin deposition (Xing et al. 2013; Kakkar et al. 2016). Therefore, exoskeletal coloration, particularly that of the mandibles, was used as a morphological marker to distinguish individuals before and after ecdysis. In \\u003cem\\u003eR. speratus\\u003c/em\\u003e, workers that have expelled their gut contents are known to undergo ecdysis approximately 5–6 days later (Masuoka et al. 2013). Based on these morphological characteristics and temporal information, we defined four stages within the molting process of \\u003cem\\u003eR. speratus\\u003c/em\\u003e: pre-molt, pre-ecdysis, post-ecdysis, and post-molt. Terminology and stage definitions followed previous studies (Raina et al. 2008; Xing et al. 2013).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eIdentification of pre-molt and pre-ecdysis individuals\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo distinguish between pre-molt and pre-ecdysis stages among gut-purged individuals, we used the external appearance of the head capsule as a morphological indicator. Previous studies in other termite genera have reported the formation of wrinkles on the head capsule immediately prior to ecdysis (Xing et al. 2013). We therefore tested whether this characteristic could be applied to \\u003cem\\u003eR. speratus\\u003c/em\\u003e. Individuals lacking gut contents and exhibiting darkly pigmented mandibles were selected. Among them, those showing visible wrinkles on the head capsule were designated as pre-ecdysis candidates. Selected individuals were marked on the abdomen with enamel paint and placed with 50 unmarked workers from the same colony in a 30-mm Petri dish lined with moist filter paper. The occurrence of ecdysis was assessed 12 h after marking, based on the operational definition that the pre-ecdysis stage lasts approximately 12 h before exoskeleton shedding (Xing et al. 2013). Ecdysis was confirmed by the disappearance of the paint mark. For each colony, 10 marked individuals were examined, and the experiment was replicated across three colonies. As a control, gut-purged individuals with dark mandibles but without head capsule wrinkles were marked and reared under identical conditions. Ecdysis was assessed after 12 h. Based on these results, the presence of head capsule wrinkles was validated as a reliable indicator of the pre-ecdysis stage, and gut-purged individuals lacking this characteristic were classified as pre-molt.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eIdentification of post-ecdysis and post-molt individuals\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo distinguish between post-ecdysis and post-molt stages, we used the presence of gut air bubbles as a morphological indicator. Because gut air bubbles appear after the completion of ecdysis, we first verified their temporal dynamics following molting. Workers in which ecdysis was confirmed were marked on the abdomen with enamel paint and housed with 50 unmarked workers from the same colony in a 30-mm Petri dish lined with moist filter paper. Marked individuals were collected at 3-h intervals after ecdysis, and dissections were performed to assess the presence or absence of gut air bubbles. At each time point, five individuals per colony were examined, and the experiment was replicated across three colonies. Using this procedure, we recorded the time elapsed from ecdysis until the disappearance of gut air bubbles. Based on these results, individuals exhibiting gut air bubbles were classified as post-ecdysis, whereas those lacking air bubbles were classified as post-molt.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eTime to feeding resumption after ecdysis\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo estimate the duration of the molting process, we quantified the time required for workers to resume feeding after ecdysis. Workers immediately after ecdysis were marked on the body surface with enamel paint and housed with 50 unmarked workers from the same colony in a 30-mm Petri dish containing brown-rotted pinewood mixed cellulose (BPC) medium (Mitaka et al. 2023). Marked individuals were collected and dissected at 12-h intervals to assess the presence or absence of gut contents. At each time point, five individuals per colony were examined, with three colonies used as replicates. Individuals with detectable gut contents were considered to have resumed feeding.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eComparison of nematode abundance before and after host ecdysis under individual isolation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo examine the effects of host ecdysis on phoretic nematodes, we compared nematode abundance on hosts before and after ecdysis and on exuviae. Workers in the pre-molt and pre-ecdysis stages were individually placed in 30-mm Petri dishes lined with moistened filter paper and maintained in isolation for 24 h at 25 °C under dark, humid conditions. Molted individuals, non-molted individuals, and exuviae were then collected and individually dissected in 0.75% NaCl solution under a stereomicroscope. The total number of associated nematodes was recorded for each sample. For each colony and sample category, at least 10 samples were examined, and the experiment was replicated across three colonies. The final sample sizes were 30 individuals before ecdysis, 31 individuals after ecdysis, and 31 exuviae.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eQuantification of nematode abundance and attachment sites during the molting cycle\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo examine nematode responses to host molting, we quantified nematode abundance and attachment sites at each stage of the molting cycle. Workers were collected from three independent colonies and assigned to one of five stages: intermolt, pre-molt, pre-ecdysis, post-ecdysis, and post-molt, based on the criteria defined in this study. Each worker was dissected individually in 0.75% NaCl solution under a stereomicroscope, and both the total number of nematodes and their attachment sites were recorded. At least 15 individuals per stage were examined for each colony. The final sample sizes were 56 individuals for intermolt, 67 for pre-molt, 62 for pre-ecdysis, 67 for post-ecdysis, and 68 for post-molt. In addition, a subset of nematodes was subjected to molecular phylogenetic analysis based on partial 18S rDNA gene sequences.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStatistical analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll statistical analyses were performed in R (version 4.4.1) (R Core Team 2024). For the analysis of caste-dependent differences in nematode abundance, differences among host castes were tested using a Kruskal–Wallis test followed by Dunn’s post hoc test with Bonferroni correction.\\u003c/p\\u003e\\n\\u003cp\\u003eFor the individual molting experiment examining nematode persistence across host ecdysis, nematode abundance among molting states (before- and after-ecdysis individuals) and exuviae was analyzed using a generalized linear model (GLM) with a Poisson error distribution, including molting state and colony as explanatory variables. The effects of molting state and colony were evaluated using likelihood ratio tests (LRTs). The interaction between molting state and colony was tested by comparing models with and without the interaction using an LRT. Tukey-adjusted pairwise comparisons were conducted for post hoc comparisons among molting states and exuviae.\\u003c/p\\u003e\\n\\u003cp\\u003eFor the analysis of nematode abundance across stages of the host molting cycle, we fitted a GLM with a negative binomial error distribution including molting stage and colony as explanatory variables. The effect of molting stage was assessed using an LRT. Tukey-adjusted pairwise comparisons were conducted for post hoc comparisons among molting stages. The interaction between molting stage and colony was tested by comparing models with and without the interaction using an LRT.\\u003c/p\\u003e\\n\\u003cp\\u003eFor the analysis of nematode localization within hosts across stages of the host molting cycle, we analyzed the proportion of nematodes located in the foregut using a generalized linear model (GLM) with a binomial error distribution and logit link function. The response variable was the number of foregut-localized nematodes relative to the total number of nematodes recovered from each worker, and molting stage and colony were included as explanatory variables. Because the distribution of nematodes among host compartments differed strongly among molting stages, the data could exhibit separation (i.e., some stages showing exclusively foregut or non-foregut occurrences). To address this issue, model parameters were estimated using bias-reduced maximum likelihood estimation implemented in the brglmFit method. The effects of molting stage, colony, and their interaction were evaluated using likelihood ratio tests (LRTs). Sidak-adjusted pairwise comparisons were conducted for post hoc comparisons among molting stages.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eMolecular identification of phoretic nematodes associated with \\u003cem\\u003eReticulitermes speratus\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwo phoretic nematode taxa were isolated from the body surface of worker termites of \\u003cem\\u003eR. speratus\\u003c/em\\u003e. Molecular phylogenetic analysis based on partial 18S rDNA sequences identified these nematodes as belonging to the genera\\u0026nbsp;\\u003cem\\u003eOigolaimella\\u003c/em\\u003e and \\u003cem\\u003eDiplogastrellus\\u003c/em\\u003e (Fig. 1b).\\u0026nbsp;The obtained sequences showed high similarity (\\u0026gt;99%) to previously reported phoretic nematode sequences from \\u003cem\\u003eR. speratus\\u003c/em\\u003e, including Oigolaimella sp.\\u0026nbsp;(Susoy et al. 2015)\\u0026nbsp;and Diplogastrellus sp. (GenBank accession no. AB597239).\\u003c/p\\u003e\\n\\u003cp\\u003eBoth taxa exhibited morphological characteristics typical of the dauer stage while attached to their termite hosts. The two genera were detected in worker individuals from the same colonies. Under stereomicroscopic observation, the two taxa were highly similar in external morphology, making reliable discrimination based solely on morphology difficult. For subsequent quantitative analyses, \\u003cem\\u003eOigolaimella\\u003c/em\\u003e sp. and \\u003cem\\u003eDiplogastrellus\\u003c/em\\u003e sp. were treated collectively as a single phoretic nematode assemblage.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCaste-dependent differences in nematode abundance\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe abundance of phoretic nematodes differed significantly among host castes (Kruskal–Wallis test, \\u003cem\\u003eχ\\u003c/em\\u003e² = 38.74, df = 3, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001; Fig. 1c). Post hoc pairwise comparisons using Dunn’s test with Bonferroni correction showed that workers carried significantly more nematodes than primary kings (\\u003cem\\u003ep\\u003c/em\\u003e = 0.010), secondary queens (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001), and soldiers (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001). In contrast, no significant differences were detected among the non-worker castes (primary kings, secondary queens, and soldiers). Across all castes examined, nematodes were detected exclusively on the head and were absent from the thorax and abdomen. On the head, nematodes were observed attached to the mouthparts, particularly to the labium and maxillae.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEstablishment of temporal and morphological markers of the worker molting cycle\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWorkers within the molting process that had completed gut purging and exhibited wrinkles on the head capsule all underwent ecdysis within 12 h after observation (Fig. 2b). In contrast, individuals without wrinkles on the head capsule did not undergo ecdysis within the same period. Based on these results, individuals with wrinkles on the head capsule were defined as pre-ecdysis, whereas gut-purged individuals showing pronounced mandibular pigmentation but without wrinkles on the head capsule were defined as pre-molt.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAnalysis of the time from ecdysis to gut air bubble disappearance showed that all individuals examined at the 15-h post-ecdysis time point lacked gut air bubbles (Fig. 2c). Based on this result, the presence of gut air bubbles was defined as a diagnostic marker of the immediate post-ecdysis stage. Individuals lacking gut air bubbles, lacking pronounced mandibular pigmentation, and not yet having resumed feeding were defined as post-molt.\\u003c/p\\u003e\\n\\u003cp\\u003eExamination of the time from ecdysis to feeding resumption showed that all individuals examined at the 48-h post-ecdysis time point had resumed feeding (Fig. 2d). In contrast, some individuals examined at earlier time points had not yet resumed feeding. These results indicate that workers enter the intermolt stage within 2 days after ecdysis.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eBy integrating these temporal and morphological markers, we established a detailed framework of the worker molting cycle in \\u003cem\\u003eR. speratus\\u003c/em\\u003e (Fig. 2e). The molting process lasted approximately 8 days and was operationally divided into four stages: pre-molt, pre-ecdysis, post-ecdysis, and post-molt.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePersistence and redistribution of nematodes across the host molting cycle\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn the individual molting experiment, nematodes were detected on workers after ecdysis. Nematode abundance did not differ between individuals before and after ecdysis (Tukey-adjusted comparison: \\u003cem\\u003ep\\u003c/em\\u003e = 0.691; Fig. S1). In contrast, nematode abundance was significantly higher on both before-ecdysis and after-ecdysis individuals than on exuviae (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001 for both comparisons; Fig. S1). Overall, nematode abundance differed significantly among the three groups (before-ecdysis individuals, after-ecdysis individuals, and exuviae) (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 197.609, df = 2, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001), whereas colony had no significant effect (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 0.573, df = 2, \\u003cem\\u003ep\\u003c/em\\u003e = 0.751), and the interaction between group and colony was not significant (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 2.194, df = 4, \\u003cem\\u003ep\\u003c/em\\u003e = 0.700). These results indicate that most nematodes remain attached to the host and are rarely left on the exuviae during ecdysis.\\u003c/p\\u003e\\n\\u003cp\\u003eObservations of attachment sites at different stages of the molting cycle revealed that nematodes were detected in the foregut primarily during the pre-ecdysis and post-ecdysis stages (Fig. 3a, b). In contrast, at the other stages, nematodes were attached predominantly to the mouthparts, and foregut localization was rare.\\u003c/p\\u003e\\n\\u003cp\\u003eTotal nematode abundance per host individual did not differ among molting stages (Fig. 3c). Molting stage had no significant effect on total nematode abundance (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 2.301, df = 4, \\u003cem\\u003ep\\u003c/em\\u003e = 0.687), whereas colony had a significant effect (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 6.301, df = 2, \\u003cem\\u003ep\\u003c/em\\u003e = 0.045). Post hoc pairwise comparisons detected no significant differences between any pair of molting stages (Tukey-adjusted comparisons). The interaction between molting stage and colony was not significant (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 3.886, df = 8, \\u003cem\\u003ep\\u003c/em\\u003e = 0.870).\\u003c/p\\u003e\\n\\u003cp\\u003eHowever, nematode localization within termite individuals differed significantly among molting stages. The proportion of foregut-localized nematodes was significantly affected by molting stage (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 861.56, df = 4, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001), whereas neither colony (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 2.39, df = 2, \\u003cem\\u003ep\\u003c/em\\u003e = 0.302) nor the interaction between molting stage and colony (GLM, LRT: \\u003cem\\u003eχ\\u003c/em\\u003e² = 3.494, df = 8, \\u003cem\\u003ep\\u003c/em\\u003e = 0.900) had significant effects. Post hoc pairwise comparisons showed that the proportions of foregut-localized nematodes were significantly higher at the pre-ecdysis and post-ecdysis stages than at the intermolt, pre-molt, and post-molt stages (Sidak-adjusted comparisons, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001). The proportion was also significantly higher at the pre-ecdysis stage than at the post-ecdysis stage (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001). In contrast, no significant differences were detected between any pair of the intermolt, pre-molt, and post-molt stages. These results indicate that nematodes temporarily relocate from the mouthparts to the foregut in synchrony with host ecdysis.\\u003c/p\\u003e\\n\\u003cp\\u003eMolecular analysis of nematodes detected in the foregut revealed that they belonged to \\u003cem\\u003eOigolaimella\\u003c/em\\u003e sp. and\\u0026nbsp;\\u003cem\\u003eDiplogastrellus\\u003c/em\\u003e sp., the same taxa as those isolated from the body surface (Fig. S2).\\u003cbr clear=\\\"all\\\"\\u003e\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eWe demonstrate that phoretic nematodes associated with the subterranean termite \\u003cem\\u003eReticulitermes speratus\\u003c/em\\u003e persist on the same host individual despite complete and recurrent habitat turnover caused by host molting. Social insects form large, long-lived colonies (Wilson 1971) with relatively stable internal environments (Oster and Wilson 1978), making them persistent and resource-rich habitats for a wide range of parasites, mutualists, and phoronts (Hughes et al. 2008). However, in addition to social immunity mechanisms represented by frequent mutual grooming and nest sanitation (Meunier 2015), periodic molting in termites (Howard and Thorne 2010) creates strong removal pressures, making it a particularly difficult environment for attached organisms to maintain their association. Despite these constraints, our results showed no decline in nematode abundance across molting events, indicating continuous attachment to the same host individual. Moreover, during a restricted period immediately before and after ecdysis, the number of nematodes on the body surface temporarily decreased, while the number detected in the foregut increased. These patterns indicate that nematodes relocate from the body surface to the foregut in synchrony with host molting. Together, our findings show that termite-phoretic nematodes maintain attachment during molting through spatial reallocation between the body surface and the gut, thereby continuously exploiting a high-benefit but high-risk social host environment.\\u003c/p\\u003e\\n\\u003cp\\u003eThe transient migration to the foregut, which lies at the core of this persistence strategy, appears to exploit the anatomical dynamics of insect molting. The foregut is an ectodermally derived structure lined with cuticle, and like the external exoskeleton it is renewed during ecdysis (Hoskins and Craig 1935). However, during molting, the foregut cuticle separates from the external cuticle and is subsequently transported posteriorly through the gut lumen, as suggested for other insects (Rowland and Goodman 2016). We confirmed a similar foregut renewal pattern in \\u003cem\\u003eR. speratus\\u003c/em\\u003e (Fig. S3). By migrating to the foregut, which constitutes the region where the exoskeletal cuticle separates from the foregut lining and where sloughing of the old cuticle is initiated, nematodes can gain early access to the renewed host cuticle (Fig. S4). This relocation likely enables them to avoid being shed with the exuviae and thereby maintain attachment to the same host individual. Notably, nematodes began migrating into the foregut before ecdysis, suggesting that they respond in advance to physiological changes in the host associated with the molting process. Termite molting is accompanied by cessation of feeding, assistance from nestmates, and cuticular softening (Raina et al. 2008; Xing et al. 2013), any of which could serve as cues triggering behavioral switching. Compared with previously documented responses of epibionts to host molting, this strategy is distinctive. In decapod crustaceans, branchial isopods attach to newly exposed internal surfaces during ecdysis (Cash and Bauer 1993), whereas other symbionts temporarily detach and reattach after molting is completed (Shiozaki and Itani 2020). In aquatic insects, water mites transfer directly from the exuviae of pupae to newly emerged adults (Jalil and Mitchell 1972). All of these strategies rely on repositioning within the external space of the host individual. In contrast, the nematodes documented here achieve continuous association by switching between external and internal host compartments, exploiting the host’s own anatomical renewal process. The fact that this spatial reallocation strategy was observed in two phylogenetically distant lineages further suggests that recurrent molting constitutes a strong and predictable selective pressure capable of promoting convergent behavioral solutions. More broadly, our results provide a novel adaptive mechanism by which epibionts can overcome the structural constraint of complete host surface renewal.\\u003c/p\\u003e\\n\\u003cp\\u003eThe observation that phoretic nematodes were localized on the body surface of periodically molting workers is central to understanding their life history. Within termite colonies, soldiers and reproductives have completed their final molt (Roisin and Korb 2010), and attachment to these castes would eliminate the risk of removal through molting. Nevertheless, nematodes were biased toward workers, suggesting that workers provide benefits that compensate for the risks associated with recurrent habitat turnover. Previous studies on other termite species and their associated nematodes have shown that dauer larvae can be isolated from termite hosts, whereas adult stages occur in nest materials, indicating that phoresy functions as a dispersal stage to termite nests (Fürst von Lieven and Sudhaus 2008). Therefore, in the nematodes examined in this study, phoresy on workers is likewise suggested to serve as a means of dispersal into nest substrates. Members of the genera \\u003cem\\u003eOigolaimella\\u003c/em\\u003e and \\u003cem\\u003eDiplogastrellus\\u003c/em\\u003e have been shown to be bacteria feeders (Steel et al. 2010; Kanzaki et al. 2012), and termite nest materials rich in bacteria (Manjula et al. 2014) are considered to constitute a major feeding resource base. Workers perform labor within the colony and frequently engage in foraging activities at the colony periphery, making them more mobile than other castes (Traniello and Leuthold 2000). Therefore, the bias toward workers together with the molting-associated persistence strategy can be interpreted as a coordinated adaptation that maximizes dispersal opportunities. These findings suggest that phoresy is not merely a means of transport, but rather a highly integrated life-history strategy closely linked to host ecological roles and developmental dynamics.\\u003c/p\\u003e\\n\\u003cp\\u003eTaken together, our findings identify synchronized spatial reallocation between host compartments as a distinct adaptive solution that allows epibionts to persist despite recurrent host surface renewal. These nematodes possess a remarkable capacity to detect subtle physiological changes associated with host ecdysis from outside the host body and to adjust their behavior accordingly, underscoring the finely tuned nature of host–epibiont interactions. Elucidating the mechanisms underlying this sensory and behavioral coordination will provide deeper insight into the coevolutionary dynamics between hosts and their surface-associated organisms. More broadly, our study illustrates an adaptive mode in which persistence is achieved not by avoiding disturbance, but by evolving strategies synchronized with cyclical environmental transformation. This perspective offers a conceptual framework for understanding how organisms persist in dynamic environments characterized by predictable structural change.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP23H00332.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting Interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional Information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNo additional information.\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets supporting the conclusions of this article are included in the supplementary file.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCode availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe code used for graphing and statistical analyses is provided in the supplementary file.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Soshi Araki and Yusei Takaishi for providing photographs, and Kiyotaka Yabe and Daichi Gomi for assistance with termite collection and for helpful discussions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Methodology: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Validation: Yusuke Katsumi; Formal Analysis: Yusuke Katsumi; Investigation: Yusuke Katsumi; Resources: Yusuke Katsumi, Mamoru Takata; Data Curation: Yusuke Katsumi; Writing \\u0026ndash; Original Draft: Yusuke Katsumi, Kenji Matsuura; Writing \\u0026ndash; Review \\u0026amp; Editing: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Visualization: Yusuke Katsumi, Mamoru Takata, Kenji Matsuura; Supervision: Mamoru Takata, Kenji Matsuura; Project Administration: Yusuke Katsumi, Mamoru Takata; Funding Acquisition: Kenji Matsuura.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eBartlow AW, Agosta SJ (2021) Phoresy in animals: review and synthesis of a common but understudied mode of dispersal. 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Ann Entomol Soc Am 106:619–625. https://doi.org/10.1603/AN13007\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":true,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"behavioral-ecology-and-sociobiology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"beas\",\"sideBox\":\"Learn more about [Behavioral Ecology and Sociobiology](http://link.springer.com/journal/265)\",\"snPcode\":\"265\",\"submissionUrl\":\"https://www.editorialmanager.com/beas/default.aspx\",\"title\":\"Behavioral Ecology and Sociobiology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Habitat turnover, Phoretic nematodes, Termites, Host molting, Epibionts, Behavioral synchronization \",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9067626/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9067626/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Habitat turnover can temporarily eliminate the environments that organisms depend on, posing a major challenge to their persistence. For epibionts living on animal body surfaces, periodic renewal of the host integument during molting causes a sudden and complete loss of habitat. How such epibionts persist on the same host individual through these drastic events has remained elusive. Here, we show that phoretic nematodes associated with the subterranean termite Reticulitermes speratus maintain attachment throughout host molting by adopting a synchronized behavioral strategy. We isolated two phylogenetically distant species of phoretic nematodes, Oigolaimella sp. and Diplogastrellus sp., from this termite. Both species were primarily attached to the mouthparts (labium and maxillae) of the worker caste, which undergoes periodic molting. Quantitative analyses of nematode abundance and attachment sites across defined worker molting stages revealed that nematodes temporarily migrated into the foregut during the period surrounding ecdysis to avoid being shed with the molted exoskeleton, which is subsequently consumed by nestmates. Our findings reveal a behavioral strategy that enables epibionts to persist despite complete host surface renewal. More broadly, this study highlights coordinated spatial reallocation as an adaptive mechanism facilitating persistence in environments characterized by cyclical disturbance.\",\"manuscriptTitle\":\"Staying on board: synchronized behavioral switching allows phoretic nematodes to persist through host molting\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-04-17 13:30:09\",\"doi\":\"10.21203/rs.3.rs-9067626/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-13T16:31:54+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"46123624696155049645647463873409306779\",\"date\":\"2026-05-06T21:14:03+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"99001165069536003733216522937593250902\",\"date\":\"2026-05-06T09:49:25+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"158164994157237911560765929765795911189\",\"date\":\"2026-03-30T15:08:10+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-03-23T21:27:08+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-03-18T17:04:31+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-03-18T11:13:32+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Behavioral Ecology and Sociobiology\",\"date\":\"2026-03-09T02:36:51+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"behavioral-ecology-and-sociobiology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"beas\",\"sideBox\":\"Learn more about [Behavioral Ecology and Sociobiology](http://link.springer.com/journal/265)\",\"snPcode\":\"265\",\"submissionUrl\":\"https://www.editorialmanager.com/beas/default.aspx\",\"title\":\"Behavioral Ecology and Sociobiology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"97703e1f-ba65-49db-83b3-8a87a6ed5309\",\"owner\":[],\"postedDate\":\"April 17th, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-13T16:31:54+00:00\",\"index\":24,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"46123624696155049645647463873409306779\",\"date\":\"2026-05-06T21:14:03+00:00\",\"index\":23,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"99001165069536003733216522937593250902\",\"date\":\"2026-05-06T09:49:25+00:00\",\"index\":22,\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-17T13:30:09+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-04-17 13:30:09\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9067626\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9067626\",\"identity\":\"rs-9067626\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}