Differences in thermal tolerance and locomotion capacity support distinct dispersal strategies in marine larvae Halarachnidae mites

Differences in thermal tolerance and locomotion capacity support distinct dispersal strategies in marine larvae Halarachnidae mites | 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 Differences in thermal tolerance and locomotion capacity support distinct dispersal strategies in marine larvae Halarachnidae mites Marcela K Castelo, Lucía Pérez Zippilli, Juan Pablo Loureiro, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7592659/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Halarachnidae mites are obligate ectoparasites infesting the respiratory tract of marine mammals. Larvae disperse and it is generally accepted that infestation occurs during nose to nose host interaction. However, mite infested hosts can exhibit sneezing and catarrh that could act as a long-distance dispersal mechanism. It is currently unknown if larvae can survive the external to the hosts environmental conditions and locate new hosts. We evaluated the thermal tolerance of two species of Halarachnidae mites, Orthohalarachne attenuata and O. diminuata , and tested if they survive extreme temperature conditions exposing larvae to constant temperatures. Then, we tested if larval age influences thermal tolerance between these two species. Finally, we studied the locomotion capacities as it can be a complementary mechanism to survive external host conditions while searching for new hosts. Orthohalarachne attenuata has a higher thermal tolerance than O. diminuata when exposed to a combination of high and low temperatures and time of exposure, which is compatible with the hypothesis of a full larval development in the respiratory system organs and dispersion to the external environment. Age does not seem to influence thermal performance. Finally, O. diminuata has a much higher locomotion capacity than O. attenuata . Our experiments show that O. attenuata has an overall higher thermal tolerance than O. diminuata although a lower locomotion. Together, our results indicate that Halarachnidae mites larvae have different strategies to withstand the environmental conditions outside the host but compatible with the hypothesis that larvae can disperse far through host sneezing and survive. Acari marine mammals parasites behaviour thermotolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION As obligate endoparasites, mites of the respiratory tract of pinnipeds are a remarkable example of coevolution because they are highly adapted to their hosts and their particular way of living. Contrary to ectoparasite insects that are exposed to direct marine water conditions, endoparasitic mites must cope with a very different environment (Fain 1994 ). Mites are exposed to stable but high temperature coincident with the host internal medium. Also, mites are exposed to changing conditions of available oxygen in the nasal cavity whenever hosts dive. But colonization of a new host requires leaving the current host and exposure to external and variable conditions. Upon reaching a new host, larvae attach to nasal structures. After moulting, there can be two short non-feeding nymphal stages but these can be unobservable or suppressed (Furman and Smith 1973 ). Adult mites then attach to the respiratory tissues piercing the epithelium with the tarsal structures and chelicerae. Adults feed on loose cells, lymph, mucus, and other body fluids (Dowling 2006 ). Larvae are the dispersing stage responsible for the spread of the infestation on land among members of the colonies (Furman and Smith 1973 , Kim et al. 1980 , Porta et al. 2024 ). In fact, larvae have tarsi with protective structures as adaptation for the dispersal phase in the external environment (Porta et al. 2024 ). The current most accepted hypothesis regarding dispersion between hosts in nature states that the main mode of parasite transmission occurs when hosts interact nose-to-nose . According to this hypothesis, larvae infest new hosts via direct contact among individuals rendering them exposed to warm and humid internal host environmental conditions (Furman and Smith 1973 , Kim et al. 1980 ). Hence, larvae should have coevolved survival strategies like a finely tuned thermal tolerance with the best performance at a similar temperature as that experienced in the host (c.a. ~37 ºC). However, larvae could disperse through a different process. Mite infested hosts can manifest nasal inflammation and excessive mucus causing congestion of the upper respiratory mucosa evident by symptoms like frequent sneezing, catarrh, saccadic head movements, and nasal itching (Furman and Smith 1973 , Kim et al. 1980 ). So, the host's secretions can be an effective way of dispersion. Hence, larvae would be exposed to environmental conditions very different to those from the internal host like extreme temperatures, different levels of humidity and desiccation. Larvae should then be able to tolerate a wide range of temperatures in a dry environment for a variable amount of time while searching for a new host. Fur seals and sea lions are the most abundant marine mammals in South America. Mites from the family Halarachnidae Oudemans 1906 are the main endoparasites that attack Otariidae (Lindquist et al. 2009 , Newell 1947 ). Registries show that Orthohalarachne species infest Arctocephalus australis (Zimmermann, 1783) (South American fur seal) and Otaria flavescens (Shaw, 1800 ) (Southern sea lion) in South America (Duarte-Benvenuto et al. 2022 , Finnegan 1934 , Katz et al. 2012 , Gastal et. al., 2016 , Gómez-Puerta and Gonzales-Viera 2015, Porta et al. 2024 , Rivera-Luna et al. 2023 , Seguel et al. 2018 , Castelo et al., submitted). To date, two species of Orthohalarachne have been described that affect otariids: O. attenuata Banks, 1910 and O. diminuata Doetschman, 1944 , which can co-occur in the same host. Both species differ in size and the final anchorage location of the adult stage to the host. Orthohalarachne attenuata larvae measure around 1,440 um long and are bigger than O. diminuata larvae that measure between 620 and 775 um (Doetschman 1944 , Domrow 1974 ) (Fig. 1 C-D). Adults and larvae O. attenuata reside in the nasal cavity and nasopharyngeus, whereas O. diminuata adults are found in the lower respiratory organs, larvae in the upper ones, and nymphs are less observable and could migrate along the tract (Kim et al. 1980 ). Although both species present some morphological differences, there is almost no information on differences in behaviour or ecology between them. In this work, we explored if larvae of O. attenuata and O. diminuata have different adaptations to cope with the external to the host environmental conditions. In particular, we studied the thermal tolerance to high and low temperatures. We also studied the influence of larval age on thermal tolerance. Finally, we studied the locomotion capacity of both species in order to estimate potentiality for dispersion. We hypothesized that O. attenuata should tolerate more extreme temperatures than O. diminuata given its bigger size and more variable conditions of the experienced microhabitat in the upper respiratory organs. On the contrary, O. diminuata could show a higher level of locomotion because of its smaller size and migration behaviour occurring during development toward more stable microhabitat conditions in the lower part of the respiratory tract. MATERIALS AND METHODS Mites Arctocephalus australis hosts were found in Buenos Aires province coast, Argentine, taken for recovery to Fundación Mundo Marino (Fig. 1 A). Whenever a host died, they were kept in a fridge at 3°C until the performance of the necropsy to separate the respiratory system. After performing the necropsies, the turbinate and nasopharyngeal tissues were separated (Fig. 1 B). Then, O. attenuata and O. diminuata larvae were separated manually in glass Petri dishes with saline solution using a stereomicroscope. A piece of fabric was placed at the base of the Petri dish to allow mites to attach. Saline solution was replaced every two days. Mites were kept at a room temperature of 25 ± 1ºC and a 12:12 light/dark cycle until used in the experiments. In total, mites from seven hosts were used. Experimental design High thermal tolerance experiments To study the tolerance to high temperature in O. attenuata and O. diminuata we exposed 3 to 12-day old larvae for 1h to different temperatures ranging from 37 to 49 ºC (Table 1 ). The age of the larvae is expressed as days elapsed since the day of the host necropsy. These temperatures were chosen starting with the internal host temperature up to whenever all mites were dead. In order to control temperature a controllable thermal device was constructed. Nine 1cm diameter, 3cm depth wells were drilled in an aluminium block. Underneath the aluminium block, a peltier element (TEC 12706) was attached with thermal paste for thermal conduction. A water cooling system was attached on the other side of the peltier element for heat dissipation when needed. As a controller, we used a Peltier Controller QC-PC-PID-01 (Quick-Ohm Küpper and Co. GmbH). The temperature at each well was measured with an external type-K thermocouple in order to account for any difference in temperature between the wells. In each assay, the wells were filled with water to keep the temperature inside stable. Then, two individuals of the same species were placed inside an eppendorf type tube of 0.5 ml filled with saline solution and then submerged in the well. After the experimentation time, each tube was removed and placed in a rack for status evaluation. Each individual was evaluated immediately after exposition and 20h post exposition. Individuals were counted as alive if they showed any movement of any part of the body or dead if no movement was observed after a gentle touch with a paintbrush. Effect of time of exposition on high thermal tolerance In order to evaluate the influence of time of exposition on thermal tolerance, we exposed 9 to 18-day old larvae to 40 or 41 ºC for 1,2,3 or 4h. Using the same apparatus as before, individuals were exposed to the different treatments and evaluated immediately after or 20h post exposition (Table 2 ). Temperatures were chosen because both mite species were tolerant to those temperatures at 1h in the previous experiment. Low thermal tolerance To study the tolerance to low temperature we exposed 6 to 17-day old larvae for 5h to different temperatures ranging from 0 to -7.5ºC (Table 1 ). In order to control temperature, we generated a mix of ice and water or salty water at different concentrations. Individuals were placed in vials with 2ml of saline solution and a piece of fabric. Vials were then submerged in the mixed iced water. After the experimentation time, vials were left 30m at room temperature to allow recovery of mites. Then, each individual was observed under a stereomicroscope to assess status. Each individual was evaluated immediately after exposition. Individuals were registered as alive if they showed any movement of any part of the body or dead if no movement was observed after a gentle touch with a paintbrush. Influence of age on thermal tolerance In order to study the effect of age on thermal tolerance, we repeated the high and low thermal tolerance experiments with larvae of 20 to 23-days old. Each larva was exposed to a constant temperature during 1h. The rationale behind this experiment was to evaluate the amount of time mite larvae can tolerate extreme temperatures to search for a host after dispersion in land. For these experiments with aged larvae we tested temperatures between 40 and 44 ºC as young larvae showed high tolerance to those temperatures in the previous experiments. Locomotion and dispersion capacity In order to study the locomotion capacity, mites were placed on a circle filter paper covered by a 6cm diameter petri dish. Half of the petri dish was covered with a black plastic to generate two distinct zones in the experimental arena. In each assay, a mite larva was placed either on the illuminated half or the dark part as starting point in order to discard any bias. For three minutes, the crosses between the zones were registered. Half of the experiments were done with a 2.5 watt led light (wavelength 430–680 nm, peak 460nm) on top of the petri dish and the other half were done only with the natural light (Table 3 ). Table 3 Experimental design for the locomotion capacity in larvae of Orthohalarachne attenuata and O. diminuata . N indicates the amount of replicates for each species and light condition. Species Light condition N O. attenuata O. diminuata On 24 Off Statistical analysis In order to study the tolerance to extreme temperatures different GLMM models were used. The probability of mite larvae to survive was analysed with a generalised linear mixed model (GLMM) with logit link function and binomial distribution. The logit link function ensures fitted values between 0 and 1, and the binomial distribution is typically used for probability data. If the mite larva showed any movement it was counted as alive with a 1, otherwise it was assumed dead and a 0 was registered. To study the response to high temperature ( High temp model ), predictors used were temperature (fixed categorical predictor with nine levels) and species (fixed categorical predictor with two levels). In the case of response to low temperature ( Low temp model ), the models were constructed with temperature as a predictor (fixed categorical predictor with four levels) and species (fixed categorical predictor with two levels). All the models included fixed factors and their interaction. Then, in order to study the influence of age on thermal tolerance ( age temp models ) the models included temperature as a predictor (fixed categorical predictor with five and three levels levels for high and low age temp models), age of larvae (fixed categorical predictor with two levels) and species (categorical fixed predictor with two levels). All the models included fixed factors and their interaction. To study the effect of time of exposition on high thermal tolerance another model was constructed ( Time high temp model ). In this case, the model included temperature as a predictor (fixed categorical predictor with two levels), time of exposition (fixed categorical predictor with four levels) and species (fixed categorical predictor with two levels). All the models included the fixed factors and their interaction. Since much data showed perfect discrimination, a Bayesian generalized linear model was implemented. The probability of mite larvae survival was analyzed with a Bernoulli distribution model using logit link function. Bayesian regularization was applied using normal (0,1) priors on all regression coefficients to improve estimation stability and handle potential complete separation issues. Post-hoc comparisons were conducted using estimated marginal means with pairwise contrasts between time points for each species-temperature combination, providing median posterior estimates with 95% highest density intervals for survival probability differences. Finally, in order to study the locomotion capacity of mite larvae we analyzed the number of crosses in three minutes through a GLMM with a log link function and negative binomial distribution as there was overdispersion detected with a poisson distribution. As predictors, light condition (fixed categorical predictor with two levels) and species (fixed categorical predictor with two levels) were included. The model included fixed factors and their interaction. All the analyses were done using the R v4.1.2 "Bird Hippie'' software (R Core Team 2021 ). The packages glmmTMB and brms were used to fit the models (Brooks et al. 2017 , Bürkner 2017 ). For testing model assumptions, we used the package DHARMa (Hartig and Lohse 2021 ). To test overdispersion we used the function “check_overdispersion” of the package “performance” (Lüdecke et al. 2021 ). Graphs were done using the package ggplot2 (Wickham et al. 2020 ). Tukey contrasts were performed with the emmeans function of the package emmeans (Lenth et al. 2020 ). RESULTS High thermal tolerance Both mite species, O. attenuata and O. diminuata , had similar thermal tolerance (Chisq sp x temp(1) = 2.6, P = 0.105, Fig. 2 A). As expected, the models showed that as temperature increases, the odds for the proportion of larvae alive diminishes (Chisq temp = 27.6, P < 0.001, Table 4 ). Both species showed a high tolerance up to 44ºC (Fig. 2 A). Temperatures above 44ºC generated high damage increasing the proportion of dead larvae (Fig. 2 A). Table 4 Estimated effect of high temperature on the log-odds for the probability of survival immediately after exposition in larvae of O. attenuata and O. diminuata . SE: Standard error for the temperature trend. LCI and UCI: lower and upper confidence intervals. P: p-value. Species Temperature trend SE LCI UCI P Trend difference [LCI : UCI] O. attenuata -1.867 0.522 -2.89 -0.84 < 0.001 [-2.04 : 0.19] O. diminuata -0.945 0.226 -1.39 -0.50 < 0.001 Time exposition at 40 and 41ºC revealed differences in tolerance between O. attenuata and O. diminuata . On one side, O. attenuata did not evince any significant effect of exposition time neither for 40 nor 41ºC and tolerated the conditions with a high probability of survival (Fig. 2 B, Table S1 ). However, survival in O. diminuata was dependent both on temperature and time of exposition. At every time of exposition, O. diminuata larvae showed a lower survival at 41ºC compared to 40ºC (Fig. 2 B, Table S1 ). Low thermal tolerance Orthohalarachne attenuata had a higher thermal tolerance to low temperature compared to O. diminuata but only at a very low temperature. For 0ºC, -2ºC and − 5ºC both species recovered almost completely after 30 minutes post exposure. However, at -7.5ºC exposure, a lower proportion of O. diminuata larvae had recovered after 30 minutes of exposure (Fig. 2 C, Table S2). Influence of age on thermal tolerance Larvae of different ages of both species did not show any difference in thermal tolerance when exposed to high temperatures (Chisq sp x temp x age = 0.582, P = 0.446, Fig. 3 A). Also, when exposed to low temperatures, larvae of both species showed similar recovery for every temperature (Chisq sp x temp x age = 3.377, P = 0.066, Fig. 3 B). Interestingly, at -7.5ºC old O. diminuata larvae showed similar recovery as when exposed to the other temperatures. Locomotion and dispersion capacity Locomotion was different between species. Orthohalarachne diminuata had between 54% and 328% increase in the mean number of crossings per minute compared to O. attenuata at both conditions (lights on or off) since no influence of light was detected (Fig. 4 , Table 5 ). Table 5 Results for the locomotion experiments in larvae of O. attenuata and O. diminuata . SE: Standard error for the mean number of crossings. LCI and UCI: lower and upper confidence intervals. P: p-value. Ratio shows the difference in the number of crosses between both species. Species Mean number of crossings SE LCI UCI P Ratio [LCI : UCI] O. attenuata 0.816 0.167 0.547 1.220 0.321 2.57 [1.54 : 4.28] O. diminuata 2.099 0.337 1.532 2.870 < 0.001 DISCUSSION The present study aimed to investigate differences in thermal tolerance in O. attenuata and O. diminuata , the two known species of Orthohalarachne that infest southamerican Otariidae. We studied the capacity of larvae to withstand thermal exposures to high and low temperatures. Additionally, we explored the effect of age post host necropsy on thermal tolerance performance. Also, we explored the effect of long exposures to high and low temperatures. Finally, we explored differences in locomotion between both species as a means of estimating potential dispersion of larvae. Arthropod parasites of semi-aquatic mammals, such as pinnipeds, face the challenge of completing their life cycles in habitats that alternate between terrestrial and aquatic. The most critical phase is the dispersal of free-living, mobile individuals, which occur during the terrestrial stage of these marine mammals, typically corresponding to the reproductive or molting season (Berta 2018 ). At this stage, hosts are gathered together and they typically interact with each other through nose to nose contacts. Although these contacts might be an important way of infection, larvae could be expelled and released to the environment when hosts sneeze. So, larvae should then survive to temperature conditions of the beach, track down and move until reaching a new host. One of the main results of our work is that larvae of both O. attenuata and O. diminuata show similar thermal tolerance between 37 and 46 ºC. These temperatures are in line with the temperature of the inner host but also higher temperatures that could be frequent when larvae are expelled in the austral summer. If mites can withstand temperatures that they are regularly exposed to, then adults should not be able to survive temperatures above 37 ºC because they are only exposed to the conditions deep in the respiratory tract of the hosts. We ran a small test performed with adults that confirmed this hypothesis (adults exposed to 37ºC recovered while adults exposed to 44ºC not, data not shown). Thermal tolerance is an important mechanism that can assure that larvae can survive a long time after being expelled from the host and searching for a new one. According to our results, thermal tolerance seems not to be affected by age of larvae post-necropsy which can be taken as an indicator of their ability to wait for a new host. Hence, larvae might be able to maintain a stable physiological state, and under starvation, increase their chances of survival and host colonization. These adaptive strategies would explain why these mites are highly successful in a highly variable environment. If larval infection mode were only via nose-to-nose strategy, larvae would not need to withstand high temperatures. However, we propose expelling larvae as an important infection mechanism so larvae could need to survive long exposures to high temperature. In order to further test this idea, we exposed larvae to high but not lethal temperatures for longer periods of time. We then found that O. attenuata can still withstand 40 and 41ºC for long periods of time. On the contrary, O. diminuata survival was reduced after long exposures to 41ºC. Together, these results indicate that O. diminuata larvae are less tolerant to high temperatures than O. attenuata . High temperatures could be experienced by these larvae if they were exposed to environmental conditions in the beach and if dispersion by sneezing. However, larvae could be exposed also to low temperature environments since the resting and mating places of the hosts experience large thermal daily variations. Hence, our low temperature tolerance experiments showed that O. attenuata has a higher tolerance to low temperatures compared to O. diminuata . Together, both results seem to indicate that O. attenuata has a higher thermal tolerance compared to O. diminuata , rendering it a more capable species to survive the harsh environmental conditions. Thermal tolerance allows different species to withstand harsh environmental conditions. However, mite larvae may not only rely on physiological capacities when searching for new hosts. Although a worse thermal performance could render O. diminuata as a species with less infection capabilities, higher locomotion could allow them to reach their hosts earlier. Our results, indeed, show that O. diminuata has a much higher locomotion activity than O. attenuata . So, our results support the hypothesis that O. attenuata is a species with a lower capacity of locomotion but that can survive the external environmental conditions for a long time until finding a new host. On the contrary, O. diminuata larvae would rely more on a higher locomotion capacity that would allow them to find a host faster. Once mite larvae are expelled, they must then crawl along surfaces until they reach the nostrils of a new host (Furman and Smith 1973 , Porta et al. 2024 ). Different cues might be involved in the process. For instance, it has been proposed that larvae orient to different host cues mainly from breathing (Furman and Smith 1973 ). If this were the case, controlled experiments testing orientation of these larvae to different host cues are needed. Host location and the different cues involved are a complete intrigue in mites from marine mammals. But also, the physiological capabilities that mites have to cope with the host inner environment while diving. Host diving is a huge challenge for their obligate parasites. External parasites such as lice have a repertoire of mechanisms to survive huge pressures but also hypoxic sea water (Leonardi and Lazzari 2014). As external parasites, lice have been much more explored than internal parasites. Hence, it should be interesting to test if mite larvae can survive low oxygen conditions that would be experienced during apnea while hosts dive. In conclusion, our study shows for the first time the ecophysiological capacities of endoparasites of pinnipeds that were until now unknown. Both O. attenuata and O. diminuata seem to have different mechanisms that support the hypothesis of external infection. It remains open to test whether larvae are capable of orienting to host cues. Together, our results shed light for the first time on the ecophysiology of these largely unknown marine parasites. Declarations Acknowledgements We thank the veterinarians and technicians of the Fundación Mundo Marino for their help and collaboration in collecting the samples. This work was supported by the University of Buenos Aires, Argentina (grant UBACyT 2020 N° 20020190100059BA, to Marcela K. Castelo), the Fundación Mundo Marino, and the Consejo Nacional de Investigaciones Científicas y Técnicas. Funding This work was supported by the University of Buenos Aires, Argentina (grant UBACyT 2020 N° 20020190100059BA, to Marcela K. Castelo), the Fundación Mundo Marino, and the Consejo Nacional de Investigaciones Científicas y Técnicas. Competing interests The authors have no relevant financial or non-financial interests to disclose. Author contributions Conceptualization [MKC, JEC]; methodology [MKC, JEC]; visualization [MKC, JEC]; funding acquisition [MKC, JPL]; resources [MKC, JPL, DE]; project administration [MKC]; supervision [MKC]; Writing – original draft preparation [MKC, JEC]; Lucía Pérez Zippilli: investigation [LPZ, JCAdN, GAM]; data curation [LPZ, JCAdN, GAM]; formal analysis [JEC]; methodology and software [JEC]; supervision [JEC]. Data availability All data can be found in the Harvard Dataverse repository at https://doi.org/doi:10.7910/DVN/ZYQVLR. Ethics approval No licences or permits were required for this research. Hosts used during the experiments were only those that died naturally in Fundación Mundo Marino. References Berta A (2018) Pinnipeds. In Encyclopedia of marine mammals (pp. 733-740). Academic Press. 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Wickham H, Chang W, Henry L, Pedersen TL, Takahashi K, Wilke C, Woo K, Yutani H, Dunnington D (2020) ggplot2: create elegant data visualisations using the grammar of graphics (3.3.2) [Computer software]. https://CRAN.R-project.org/package=ggplot2 Zimmermann, E.A.W. Von. 1783. Geographische Geschichte des Menschen, und der allgemein verbreiteten vierfussigen Thiere - Phoca australis. Leipzig, 3: 276. Table 1 and 2 Table 1 and 2 are available in the Supplementary Files section. Supplementary Files Table12.docx Supplementarytables.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revise and Resubmit 22 Dec, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 10 Oct, 2025 Editor assigned by journal 15 Sep, 2025 First submitted to journal 11 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7592659","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":527477780,"identity":"4e0c0035-c1d4-450a-a040-c6ba22e371c7","order_by":0,"name":"Marcela K 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12:43:20","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100390,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/3867429e965fa2853b992fd4.html"},{"id":94194464,"identity":"3bc19d8e-732a-4890-b496-c819d122b08c","added_by":"auto","created_at":"2025-10-23 12:43:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":704629,"visible":true,"origin":"","legend":"\u003cp\u003eObtention of the Halarachnidae larvae used in this work. A) \u003cem\u003eArctocephalus australis\u003c/em\u003e, host. B) Extracted turbinates with attached larvae. C) Dorsal view of \u003cem\u003eOrthohalarachne attenuata\u003c/em\u003e. D) Dorsal view of \u003cem\u003eO. diminuata\u003c/em\u003e. Bar 1 mm.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/0a97bccee126bc8c118deab3.png"},{"id":94194466,"identity":"345e62fa-0b57-4c86-a48b-b04997642008","added_by":"auto","created_at":"2025-10-23 12:43:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":136987,"visible":true,"origin":"","legend":"\u003cp\u003eThermal tolerance of \u003cem\u003eOrthohalarachne \u003c/em\u003emites. A) Proportion of \u003cem\u003eO. attenuata\u003c/em\u003e (green bars) and \u003cem\u003eO. diminuata\u003c/em\u003e (blue bars) larvae alive after exposure to different high temperatures. B) Survival of \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e larvae after exposure to 40ºC or 41ºC for 1h (black), 2h (dark grey), 3h (grey), or 4h (light grey). C) Proportion of \u003cem\u003eO. attenuata\u003c/em\u003e (green bars) and \u003cem\u003eO. diminuata\u003c/em\u003e (blue bars) larvae alive after 5h of exposure to different low temperatures.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/436d77ebbcd56dd57c26be2d.png"},{"id":94194468,"identity":"9d2c87f3-b223-4664-92db-3ff70744cef1","added_by":"auto","created_at":"2025-10-23 12:43:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":143643,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of age in thermal tolerance of \u003cem\u003eOrthohalarachne \u003c/em\u003emites. Proportion of young (black bars) and old (grey bars) \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003elarvae alive. A) Results when larvae were exposed to high temperature. B) Results when larvae were exposed to low temperature.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/f1cee67b8490a387e5afa11b.png"},{"id":94194717,"identity":"c4e1031f-fbcc-4077-af16-4b8901f40779","added_by":"auto","created_at":"2025-10-23 12:51:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":149682,"visible":true,"origin":"","legend":"\u003cp\u003eLocomotion and dispersal capacity of \u003cem\u003eOrthohalarachne \u003c/em\u003emites. Mean number ± SE of crossings per minute of \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e larvae with the light off (green points) or the lights on (blue points).\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/8eb830de2fea5c98d1153570.png"},{"id":94196274,"identity":"e6b88e89-269a-485d-ac1d-4f459c0199a2","added_by":"auto","created_at":"2025-10-23 13:07:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1884281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/326e4b2f-4436-43f7-b0c4-79798275c653.pdf"},{"id":94194716,"identity":"4934feac-d3fb-45ab-b419-0018b1e03aee","added_by":"auto","created_at":"2025-10-23 12:51:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16680,"visible":true,"origin":"","legend":"","description":"","filename":"Table12.docx","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/b43e63a4e1f691445e59cce2.docx"},{"id":94194721,"identity":"6889faa0-0e45-4d60-b479-940175df91e4","added_by":"auto","created_at":"2025-10-23 12:51:20","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":9600,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7592659/v1/66633a68405c41f92bb348cd.docx"}],"financialInterests":"","formattedTitle":"Differences in thermal tolerance and locomotion capacity support distinct dispersal strategies in marine larvae Halarachnidae mites","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAs obligate endoparasites, mites of the respiratory tract of pinnipeds are a remarkable example of coevolution because they are highly adapted to their hosts and their particular way of living. Contrary to ectoparasite insects that are exposed to direct marine water conditions, endoparasitic mites must cope with a very different environment (Fain \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Mites are exposed to stable but high temperature coincident with the host internal medium. Also, mites are exposed to changing conditions of available oxygen in the nasal cavity whenever hosts dive. But colonization of a new host requires leaving the current host and exposure to external and variable conditions.\u003c/p\u003e\u003cp\u003eUpon reaching a new host, larvae attach to nasal structures. After moulting, there can be two short non-feeding nymphal stages but these can be unobservable or suppressed (Furman and Smith \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). Adult mites then attach to the respiratory tissues piercing the epithelium with the tarsal structures and chelicerae. Adults feed on loose cells, lymph, mucus, and other body fluids (Dowling \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Larvae are the dispersing stage responsible for the spread of the infestation on land among members of the colonies (Furman and Smith \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e, Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1980\u003c/span\u003e, Porta et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In fact, larvae have tarsi with protective structures as adaptation for the dispersal phase in the external environment (Porta et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The current most accepted hypothesis regarding dispersion between hosts in nature states that the main mode of parasite transmission occurs when hosts interact \u003cem\u003enose-to-nose\u003c/em\u003e. According to this hypothesis, larvae infest new hosts via direct contact among individuals rendering them exposed to warm and humid internal host environmental conditions (Furman and Smith \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e, Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Hence, larvae should have coevolved survival strategies like a finely tuned thermal tolerance with the best performance at a similar temperature as that experienced in the host (c.a. ~37 \u0026ordm;C).\u003c/p\u003e\u003cp\u003eHowever, larvae could disperse through a different process. Mite infested hosts can manifest nasal inflammation and excessive mucus causing congestion of the upper respiratory mucosa evident by symptoms like frequent sneezing, catarrh, saccadic head movements, and nasal itching (Furman and Smith \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e, Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). So, the host's secretions can be an effective way of dispersion. Hence, larvae would be exposed to environmental conditions very different to those from the internal host like extreme temperatures, different levels of humidity and desiccation. Larvae should then be able to tolerate a wide range of temperatures in a dry environment for a variable amount of time while searching for a new host.\u003c/p\u003e\u003cp\u003eFur seals and sea lions are the most abundant marine mammals in South America. Mites from the family Halarachnidae Oudemans 1906 are the main endoparasites that attack Otariidae (Lindquist et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Newell \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1947\u003c/span\u003e). Registries show that \u003cem\u003eOrthohalarachne\u003c/em\u003e species infest \u003cem\u003eArctocephalus australis\u003c/em\u003e (Zimmermann, 1783) (South American fur seal) and \u003cem\u003eOtaria flavescens\u003c/em\u003e (Shaw, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1800\u003c/span\u003e) (Southern sea lion) in South America (Duarte-Benvenuto et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Finnegan \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1934\u003c/span\u003e, Katz et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Gastal et. al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, G\u0026oacute;mez-Puerta and Gonzales-Viera 2015, Porta et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Rivera-Luna et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Seguel et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Castelo et al., submitted). To date, two species of \u003cem\u003eOrthohalarachne\u003c/em\u003e have been described that affect otariids: \u003cem\u003eO. attenuata\u003c/em\u003e Banks, 1910 and \u003cem\u003eO. diminuata\u003c/em\u003e Doetschman, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1944\u003c/span\u003e, which can co-occur in the same host. Both species differ in size and the final anchorage location of the adult stage to the host. \u003cem\u003eOrthohalarachne attenuata\u003c/em\u003e larvae measure around 1,440 um long and are bigger than \u003cem\u003eO. diminuata\u003c/em\u003e larvae that measure between 620 and 775 um (Doetschman \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1944\u003c/span\u003e, Domrow \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Adults and larvae \u003cem\u003eO. attenuata\u003c/em\u003e reside in the nasal cavity and nasopharyngeus, whereas \u003cem\u003eO. diminuata\u003c/em\u003e adults are found in the lower respiratory organs, larvae in the upper ones, and nymphs are less observable and could migrate along the tract (Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Although both species present some morphological differences, there is almost no information on differences in behaviour or ecology between them.\u003c/p\u003e\u003cp\u003eIn this work, we explored if larvae of \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e have different adaptations to cope with the external to the host environmental conditions. In particular, we studied the thermal tolerance to high and low temperatures. We also studied the influence of larval age on thermal tolerance. Finally, we studied the locomotion capacity of both species in order to estimate potentiality for dispersion. We hypothesized that \u003cem\u003eO. attenuata\u003c/em\u003e should tolerate more extreme temperatures than \u003cem\u003eO. diminuata\u003c/em\u003e given its bigger size and more variable conditions of the experienced microhabitat in the upper respiratory organs. On the contrary, \u003cem\u003eO. diminuata\u003c/em\u003e could show a higher level of locomotion because of its smaller size and migration behaviour occurring during development toward more stable microhabitat conditions in the lower part of the respiratory tract.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eMites\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eArctocephalus australis\u003c/em\u003e hosts were found in Buenos Aires province coast, Argentine, taken for recovery to Fundaci\u0026oacute;n Mundo Marino (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Whenever a host died, they were kept in a fridge at 3\u0026deg;C until the performance of the necropsy to separate the respiratory system. After performing the necropsies, the turbinate and nasopharyngeal tissues were separated (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Then, \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e larvae were separated manually in glass Petri dishes with saline solution using a stereomicroscope. A piece of fabric was placed at the base of the Petri dish to allow mites to attach. Saline solution was replaced every two days. Mites were kept at a room temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026ordm;C and a 12:12 light/dark cycle until used in the experiments. In total, mites from seven hosts were used.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eHigh thermal tolerance experiments\u003c/h2\u003e\n \u003cp\u003eTo study the tolerance to high temperature in \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e we exposed 3 to 12-day old larvae for 1h to different temperatures ranging from 37 to 49 \u0026ordm;C (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The age of the larvae is expressed as days elapsed since the day of the host necropsy. These temperatures were chosen starting with the internal host temperature up to whenever all mites were dead. In order to control temperature a controllable thermal device was constructed. Nine 1cm diameter, 3cm depth wells were drilled in an aluminium block. Underneath the aluminium block, a peltier element (TEC 12706) was attached with thermal paste for thermal conduction. A water cooling system was attached on the other side of the peltier element for heat dissipation when needed. As a controller, we used a Peltier Controller QC-PC-PID-01 (Quick-Ohm K\u0026uuml;pper and Co. GmbH). The temperature at each well was measured with an external type-K thermocouple in order to account for any difference in temperature between the wells.\u003c/p\u003e\n \u003cp\u003eIn each assay, the wells were filled with water to keep the temperature inside stable. Then, two individuals of the same species were placed inside an eppendorf type tube of 0.5 ml filled with saline solution and then submerged in the well. After the experimentation time, each tube was removed and placed in a rack for status evaluation. Each individual was evaluated immediately after exposition and 20h post exposition. Individuals were counted as alive if they showed any movement of any part of the body or dead if no movement was observed after a gentle touch with a paintbrush.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eEffect of time of exposition on high thermal tolerance\u003c/h3\u003e\n\u003cp\u003eIn order to evaluate the influence of time of exposition on thermal tolerance, we exposed 9 to 18-day old larvae to 40 or 41 \u0026ordm;C for 1,2,3 or 4h. Using the same apparatus as before, individuals were exposed to the different treatments and evaluated immediately after or 20h post exposition (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Temperatures were chosen because both mite species were tolerant to those temperatures at 1h in the previous experiment.\u003c/p\u003e\n\u003ch3\u003eLow thermal tolerance\u003c/h3\u003e\n\u003cp\u003eTo study the tolerance to low temperature we exposed 6 to 17-day old larvae for 5h to different temperatures ranging from 0 to -7.5\u0026ordm;C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In order to control temperature, we generated a mix of ice and water or salty water at different concentrations. Individuals were placed in vials with 2ml of saline solution and a piece of fabric. Vials were then submerged in the mixed iced water. After the experimentation time, vials were left 30m at room temperature to allow recovery of mites. Then, each individual was observed under a stereomicroscope to assess status. Each individual was evaluated immediately after exposition. Individuals were registered as alive if they showed any movement of any part of the body or dead if no movement was observed after a gentle touch with a paintbrush.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eInfluence of age on thermal tolerance\u003c/h2\u003e\u003cp\u003eIn order to study the effect of age on thermal tolerance, we repeated the high and low thermal tolerance experiments with larvae of 20 to 23-days old. Each larva was exposed to a constant temperature during 1h. The rationale behind this experiment was to evaluate the amount of time mite larvae can tolerate extreme temperatures to search for a host after dispersion in land. For these experiments with aged larvae we tested temperatures between 40 and 44 \u0026ordm;C as young larvae showed high tolerance to those temperatures in the previous experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLocomotion and dispersion capacity\u003c/h3\u003e\n\u003cp\u003eIn order to study the locomotion capacity, mites were placed on a circle filter paper covered by a 6cm diameter petri dish. Half of the petri dish was covered with a black plastic to generate two distinct zones in the experimental arena. In each assay, a mite larva was placed either on the illuminated half or the dark part as starting point in order to discard any bias. For three minutes, the crosses between the zones were registered. Half of the experiments were done with a 2.5 watt led light (wavelength 430\u0026ndash;680 nm, peak 460nm) on top of the petri dish and the other half were done only with the natural light (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental design for the locomotion capacity in larvae of \u003cem\u003eOrthohalarachne attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e. N indicates the amount of replicates for each species and light condition.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLight condition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eO. attenuata\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eO. diminuata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOff\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eIn order to study the tolerance to extreme temperatures different GLMM models were used. The probability of mite larvae to survive was analysed with a generalised linear mixed model (GLMM) with logit link function and binomial distribution. The logit link function ensures fitted values between 0 and 1, and the binomial distribution is typically used for probability data. If the mite larva showed any movement it was counted as alive with a 1, otherwise it was assumed dead and a 0 was registered.\u003c/p\u003e\u003cp\u003eTo study the response to high temperature (\u003cem\u003eHigh temp model\u003c/em\u003e), predictors used were temperature (fixed categorical predictor with nine levels) and species (fixed categorical predictor with two levels). In the case of response to low temperature (\u003cem\u003eLow temp model\u003c/em\u003e), the models were constructed with temperature as a predictor (fixed categorical predictor with four levels) and species (fixed categorical predictor with two levels). All the models included fixed factors and their interaction.\u003c/p\u003e\u003cp\u003eThen, in order to study the influence of age on thermal tolerance (\u003cem\u003eage temp models\u003c/em\u003e) the models included temperature as a predictor (fixed categorical predictor with five and three levels levels for high and low age temp models), age of larvae (fixed categorical predictor with two levels) and species (categorical fixed predictor with two levels). All the models included fixed factors and their interaction.\u003c/p\u003e\u003cp\u003eTo study the effect of time of exposition on high thermal tolerance another model was constructed (\u003cem\u003eTime high temp model\u003c/em\u003e). In this case, the model included temperature as a predictor (fixed categorical predictor with two levels), time of exposition (fixed categorical predictor with four levels) and species (fixed categorical predictor with two levels). All the models included the fixed factors and their interaction. Since much data showed perfect discrimination, a Bayesian generalized linear model was implemented. The probability of mite larvae survival was analyzed with a Bernoulli distribution model using logit link function. Bayesian regularization was applied using normal (0,1) priors on all regression coefficients to improve estimation stability and handle potential complete separation issues. \u003cem\u003ePost-hoc\u003c/em\u003e comparisons were conducted using estimated marginal means with pairwise contrasts between time points for each species-temperature combination, providing median posterior estimates with 95% highest density intervals for survival probability differences.\u003c/p\u003e\u003cp\u003eFinally, in order to study the locomotion capacity of mite larvae we analyzed the number of crosses in three minutes through a GLMM with a log link function and negative binomial distribution as there was overdispersion detected with a poisson distribution. As predictors, light condition (fixed categorical predictor with two levels) and species (fixed categorical predictor with two levels) were included. The model included fixed factors and their interaction.\u003c/p\u003e\u003cp\u003eAll the analyses were done using the R v4.1.2 \"Bird Hippie'' software (R Core Team \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The packages glmmTMB and brms were used to fit the models (Brooks et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, B\u0026uuml;rkner \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For testing model assumptions, we used the package DHARMa (Hartig and Lohse \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To test overdispersion we used the function \u0026ldquo;check_overdispersion\u0026rdquo; of the package \u0026ldquo;performance\u0026rdquo; (L\u0026uuml;decke et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Graphs were done using the package ggplot2 (Wickham et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Tukey contrasts were performed with the emmeans function of the package emmeans (Lenth et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHigh thermal tolerance\u003c/h2\u003e\u003cp\u003eBoth mite species, \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e, had similar thermal tolerance (Chisq \u003csub\u003esp x temp(1)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.6, P\u0026thinsp;=\u0026thinsp;0.105, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As expected, the models showed that as temperature increases, the odds for the proportion of larvae alive diminishes (Chisq \u003csub\u003etemp\u003c/sub\u003e = 27.6, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both species showed a high tolerance up to 44\u0026ordm;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Temperatures above 44\u0026ordm;C generated high damage increasing the proportion of dead larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEstimated effect of high temperature on the log-odds for the probability of survival immediately after exposition in larvae of \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e. SE: Standard error for the temperature trend. LCI and UCI: lower and upper confidence intervals. P: p-value.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemperature trend\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLCI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUCI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTrend difference [LCI : UCI]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eO. attenuata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-1.867\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.522\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-2.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e[-2.04 : 0.19]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eO. diminuata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-0.945\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.226\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-1.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTime exposition at 40 and 41\u0026ordm;C revealed differences in tolerance between \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e. On one side, \u003cem\u003eO. attenuata\u003c/em\u003e did not evince any significant effect of exposition time neither for 40 nor 41\u0026ordm;C and tolerated the conditions with a high probability of survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, survival in \u003cem\u003eO. diminuata\u003c/em\u003e was dependent both on temperature and time of exposition. At every time of exposition, \u003cem\u003eO. diminuata\u003c/em\u003e larvae showed a lower survival at 41\u0026ordm;C compared to 40\u0026ordm;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eLow thermal tolerance\u003c/h2\u003e\u003cp\u003e\u003cem\u003eOrthohalarachne attenuata\u003c/em\u003e had a higher thermal tolerance to low temperature compared to \u003cem\u003eO. diminuata\u003c/em\u003e but only at a very low temperature. For 0\u0026ordm;C, -2\u0026ordm;C and \u0026minus;\u0026thinsp;5\u0026ordm;C both species recovered almost completely after 30 minutes post exposure. However, at -7.5\u0026ordm;C exposure, a lower proportion of \u003cem\u003eO. diminuata\u003c/em\u003e larvae had recovered after 30 minutes of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table S2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eInfluence of age on thermal tolerance\u003c/h2\u003e\u003cp\u003eLarvae of different ages of both species did not show any difference in thermal tolerance when exposed to high temperatures (Chisq \u003csub\u003esp x temp x age\u003c/sub\u003e = 0.582, P\u0026thinsp;=\u0026thinsp;0.446, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Also, when exposed to low temperatures, larvae of both species showed similar recovery for every temperature (Chisq \u003csub\u003esp x temp x age\u003c/sub\u003e = 3.377, P\u0026thinsp;=\u0026thinsp;0.066, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Interestingly, at -7.5\u0026ordm;C old \u003cem\u003eO. diminuata\u003c/em\u003e larvae showed similar recovery as when exposed to the other temperatures.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eLocomotion and dispersion capacity\u003c/h2\u003e\u003cp\u003eLocomotion was different between species. \u003cem\u003eOrthohalarachne diminuata\u003c/em\u003e had between 54% and 328% increase in the mean number of crossings per minute compared to \u003cem\u003eO. attenuata\u003c/em\u003e at both conditions (lights on or off) since no influence of light was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults for the locomotion experiments in larvae of \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e. SE: Standard error for the mean number of crossings. LCI and UCI: lower and upper confidence intervals. P: p-value. Ratio shows the difference in the number of crosses between both species.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean number of crossings\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLCI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUCI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRatio [LCI : UCI]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eO. attenuata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.816\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.547\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.321\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2.57 [1.54 : 4.28]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eO. diminuata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.099\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.337\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.532\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.870\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study aimed to investigate differences in thermal tolerance in \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e, the two known species of \u003cem\u003eOrthohalarachne\u003c/em\u003e that infest southamerican Otariidae. We studied the capacity of larvae to withstand thermal exposures to high and low temperatures. Additionally, we explored the effect of age post host necropsy on thermal tolerance performance. Also, we explored the effect of long exposures to high and low temperatures. Finally, we explored differences in locomotion between both species as a means of estimating potential dispersion of larvae.\u003c/p\u003e\u003cp\u003eArthropod parasites of semi-aquatic mammals, such as pinnipeds, face the challenge of completing their life cycles in habitats that alternate between terrestrial and aquatic. The most critical phase is the dispersal of free-living, mobile individuals, which occur during the terrestrial stage of these marine mammals, typically corresponding to the reproductive or molting season (Berta \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). At this stage, hosts are gathered together and they typically interact with each other through nose to nose contacts. Although these contacts might be an important way of infection, larvae could be expelled and released to the environment when hosts sneeze. So, larvae should then survive to temperature conditions of the beach, track down and move until reaching a new host.\u003c/p\u003e\u003cp\u003eOne of the main results of our work is that larvae of both \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e show similar thermal tolerance between 37 and 46 \u0026ordm;C. These temperatures are in line with the temperature of the inner host but also higher temperatures that could be frequent when larvae are expelled in the austral summer. If mites can withstand temperatures that they are regularly exposed to, then adults should not be able to survive temperatures above 37 \u0026ordm;C because they are only exposed to the conditions deep in the respiratory tract of the hosts. We ran a small test performed with adults that confirmed this hypothesis (adults exposed to 37\u0026ordm;C recovered while adults exposed to 44\u0026ordm;C not, data not shown).\u003c/p\u003e\u003cp\u003eThermal tolerance is an important mechanism that can assure that larvae can survive a long time after being expelled from the host and searching for a new one. According to our results, thermal tolerance seems not to be affected by age of larvae post-necropsy which can be taken as an indicator of their ability to wait for a new host. Hence, larvae might be able to maintain a stable physiological state, and under starvation, increase their chances of survival and host colonization. These adaptive strategies would explain why these mites are highly successful in a highly variable environment.\u003c/p\u003e\u003cp\u003eIf larval infection mode were only via \u003cem\u003enose-to-nose\u003c/em\u003e strategy, larvae would not need to withstand high temperatures. However, we propose expelling larvae as an important infection mechanism so larvae could need to survive long exposures to high temperature. In order to further test this idea, we exposed larvae to high but not lethal temperatures for longer periods of time. We then found that \u003cem\u003eO. attenuata\u003c/em\u003e can still withstand 40 and 41\u0026ordm;C for long periods of time. On the contrary, \u003cem\u003eO. diminuata\u003c/em\u003e survival was reduced after long exposures to 41\u0026ordm;C. Together, these results indicate that \u003cem\u003eO. diminuata\u003c/em\u003e larvae are less tolerant to high temperatures than \u003cem\u003eO. attenuata\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eHigh temperatures could be experienced by these larvae if they were exposed to environmental conditions in the beach and if dispersion by sneezing. However, larvae could be exposed also to low temperature environments since the resting and mating places of the hosts experience large thermal daily variations. Hence, our low temperature tolerance experiments showed that \u003cem\u003eO. attenuata\u003c/em\u003e has a higher tolerance to low temperatures compared to \u003cem\u003eO. diminuata\u003c/em\u003e. Together, both results seem to indicate that \u003cem\u003eO. attenuata\u003c/em\u003e has a higher thermal tolerance compared to \u003cem\u003eO. diminuata\u003c/em\u003e, rendering it a more capable species to survive the harsh environmental conditions.\u003c/p\u003e\u003cp\u003eThermal tolerance allows different species to withstand harsh environmental conditions. However, mite larvae may not only rely on physiological capacities when searching for new hosts. Although a worse thermal performance could render \u003cem\u003eO. diminuata\u003c/em\u003e as a species with less infection capabilities, higher locomotion could allow them to reach their hosts earlier. Our results, indeed, show that \u003cem\u003eO. diminuata\u003c/em\u003e has a much higher locomotion activity than \u003cem\u003eO. attenuata\u003c/em\u003e. So, our results support the hypothesis that \u003cem\u003eO. attenuata\u003c/em\u003e is a species with a lower capacity of locomotion but that can survive the external environmental conditions for a long time until finding a new host. On the contrary, \u003cem\u003eO. diminuata\u003c/em\u003e larvae would rely more on a higher locomotion capacity that would allow them to find a host faster.\u003c/p\u003e\u003cp\u003eOnce mite larvae are expelled, they must then crawl along surfaces until they reach the nostrils of a new host (Furman and Smith \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e, Porta et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Different cues might be involved in the process. For instance, it has been proposed that larvae orient to different host cues mainly from breathing (Furman and Smith \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). If this were the case, controlled experiments testing orientation of these larvae to different host cues are needed.\u003c/p\u003e\u003cp\u003eHost location and the different cues involved are a complete intrigue in mites from marine mammals. But also, the physiological capabilities that mites have to cope with the host inner environment while diving. Host diving is a huge challenge for their obligate parasites. External parasites such as lice have a repertoire of mechanisms to survive huge pressures but also hypoxic sea water (Leonardi and Lazzari 2014). As external parasites, lice have been much more explored than internal parasites. Hence, it should be interesting to test if mite larvae can survive low oxygen conditions that would be experienced during apnea while hosts dive.\u003c/p\u003e\u003cp\u003eIn conclusion, our study shows for the first time the ecophysiological capacities of endoparasites of pinnipeds that were until now unknown. Both \u003cem\u003eO. attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e seem to have different mechanisms that support the hypothesis of external infection. It remains open to test whether larvae are capable of orienting to host cues. Together, our results shed light for the first time on the ecophysiology of these largely unknown marine parasites.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the veterinarians and technicians of the Fundaci\u0026oacute;n Mundo Marino for their help and collaboration in collecting the samples. This work was supported by the University of Buenos Aires, Argentina (grant UBACyT 2020 N\u0026deg; 20020190100059BA, to Marcela K. Castelo), the Fundaci\u0026oacute;n Mundo Marino, and the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the University of Buenos Aires, Argentina (grant UBACyT 2020 N\u0026deg; 20020190100059BA, to Marcela K. Castelo), the Fundaci\u0026oacute;n Mundo Marino, and the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization [MKC, JEC]; methodology [MKC, JEC]; visualization [MKC, JEC]; funding acquisition [MKC, JPL]; resources [MKC, JPL, DE]; project administration [MKC]; supervision [MKC]; Writing \u0026ndash; original draft preparation [MKC, JEC]; Luc\u0026iacute;a P\u0026eacute;rez Zippilli: investigation [LPZ, JCAdN, GAM]; data curation [LPZ, JCAdN, GAM]; formal analysis [JEC]; methodology and software [JEC]; supervision [JEC].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data can be found in the Harvard Dataverse repository at https://doi.org/doi:10.7910/DVN/ZYQVLR.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; No licences or permits were required for this research. Hosts used during the experiments were only those that died naturally in Fundaci\u0026oacute;n Mundo Marino.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerta A (2018) Pinnipeds. In Encyclopedia of marine mammals (pp. 733-740). Academic Press.\u003c/li\u003e\n\u003cli\u003eBrooks ME, Kristensen K, van Benthem KJ, Magnusson A, Berg CW, Nielsen A, Skaug HJ, M\u0026auml;chler M, Bolker BM (2017) glmmTMB balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. \u003cem\u003eR Journal\u003c/em\u003e 9, 378-400. https://doi.org/10.32614/rj-2017-066\u003c/li\u003e\n\u003cli\u003eB\u0026uuml;rkner PC (2017) brms: An R Package for Bayesian Multilevel Models Using Stan. \u003cem\u003eJournal of Statistical Software\u003c/em\u003e, 80(1), 1-28. doi:10.18637/jss.v080.i01\u003c/li\u003e\n\u003cli\u003eDoetschman WH (1944) A new s xpecies of endoparasitic mite of the family Halarachnidae (Acarina). 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Revista Peruana de Biolog\u0026iacute;a 22(2): 259\u0026ndash;262. https://doi.org/10.15381/rpb.v22i2.11360\u003c/li\u003e\n\u003cli\u003eHartig F, Lohse L (2021) DHARMa: residual diagnostics for hierarchical (multi-level / mixed) regression models (0.4.0) [Computer software]. https://CRAN.R-project.org/package=DHARMa\u003c/li\u003e\n\u003cli\u003eKatz H, Morgades D, Castro-Ramos M (2012) Pathological and Parasitological Findings in South American Fur Seal Pups (\u003cem\u003eArctocephalus australis\u003c/em\u003e) in Uruguay. ISRN Zoology 586079: 1\u0026ndash;7. https://doi.org/10.5402/2012/586079\u003c/li\u003e\n\u003cli\u003eKim KC, Haas VL, Keyes MC (1980) Populations, microh\u0026aacute;bitat preference and effects of infestations of two species of \u003cem\u003eOrthohalarachne \u003c/em\u003e(Halarachnidae: Acarina) in the northern fur seal. Journal of Wildlife Diseases 16: 45\u0026ndash;51. https://doi.org/10.7589/0090-3558-16.1.45\u003c/li\u003e\n\u003cli\u003eLenth R, Singmann H, Love J, Buerkner P, Herve M (2020) emmeans: estimated marginal means, aka least-squares means (1.4.8) [Computer software]. https://CRAN.R-project.org/package=emmeans\u003c/li\u003e\n\u003cli\u003eLindquist EE, Krantz GW, Walter DE (2009) Order Mesostigmata. In: Krantz GW, Walter DE (Eds) A Manual of Acarology. Texas Tech University Press, Lubbock, 124\u0026ndash;232.\u003c/li\u003e\n\u003cli\u003eL\u0026uuml;decke D, Ben-Shachar MS, Patil I, Waggoner P, Makowski D (2021) performance: An R Package for Assessment, Comparison and Testing of Statistical Models. Journal of Open Source Software, 6(60), 3139. https://doi.org/10.21105/joss.03139\u003c/li\u003e\n\u003cli\u003eNewell IM (1947) Studies on the morphology and systematics of the family Halarachnidae Oudemans 1906 (Acari, Parasitoidea). Bulletin of the Bingham Oceanographic Collection 10: 235\u0026ndash;266.\u003c/li\u003e\n\u003cli\u003ePorta AO, Loureiro JP, Castelo, MK (2024)  First record of \u003cem\u003eOrthohalarachne attenuata \u003c/em\u003ein \u003cem\u003eArctocephalus australis \u003c/em\u003ein mainland Argentina (Parasitiformes, Mesostigmata, Dermanyssoidea, Halarachnidae) with observations on its ambulacral morphology. ZooKeys, 1207, 355.\u003c/li\u003e\n\u003cli\u003eR Core Team (2021) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/\u003c/li\u003e\n\u003cli\u003eRivera-Luna H, Kniha E, Munoz P, Painean J, Balfanz F, Hering-Hagenbeck S, Prosl H, Walochnik J, Taubert A, Hermosilla C, Ebmer D (2023) Non-invasive detection of \u003cem\u003eOrthohalarachne attenuata \u003c/em\u003e(Banks, 1910) and \u003cem\u003eOrthohalarachne diminuata \u003c/em\u003e(Doetschman, 1944) (Acari: Halarachnidae) in free-ranging synanthropic South American sea lions \u003cem\u003eOtaria flavescens \u003c/em\u003e(Shaw, 1800). International Journal for Parasitology: Parasites and Wildlife, 21, 192-200.\u003c/li\u003e\n\u003cli\u003eSeguel M, Calder\u0026oacute;n K, Colegrove K, Adkesson M, C\u0026aacute;rdenas-Alayza S, Paredes E (2018) Helminth and respiratory mite lesions in Pinnipeds from Punta San Juan, Per\u0026uacute;. Acta Parasitologica 63(4): 839\u0026ndash;844. https://doi.org/10.1515/ap-2018-0103.\u003c/li\u003e\n\u003cli\u003eShaw G (1800) Seals. In: Kerasly G (ed) General Zoology or Systematic Natural History I(2), pp 266\u0026ndash;291.\u003c/li\u003e\n\u003cli\u003eWickham H, Chang W, Henry L, Pedersen TL, Takahashi K, Wilke C, Woo K, Yutani H, Dunnington D (2020) ggplot2: create elegant data visualisations using the grammar of graphics (3.3.2) [Computer software]. https://CRAN.R-project.org/package=ggplot2\u003c/li\u003e\n\u003cli\u003eZimmermann, E.A.W. Von. 1783. Geographische Geschichte des Menschen, und der allgemein verbreiteten vierfussigen Thiere - Phoca australis. Leipzig, 3: 276.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1 and 2","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acari, marine mammals, parasites, behaviour, thermotolerance","lastPublishedDoi":"10.21203/rs.3.rs-7592659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7592659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHalarachnidae mites are obligate ectoparasites infesting the respiratory tract of marine mammals. Larvae disperse and it is generally accepted that infestation occurs during nose to nose host interaction. However, mite infested hosts can exhibit sneezing and catarrh that could act as a long-distance dispersal mechanism. It is currently unknown if larvae can survive the external to the hosts environmental conditions and locate new hosts. We evaluated the thermal tolerance of two species of Halarachnidae mites, \u003cem\u003eOrthohalarachne attenuata\u003c/em\u003e and \u003cem\u003eO. diminuata\u003c/em\u003e, and tested if they survive extreme temperature conditions exposing larvae to constant temperatures. Then, we tested if larval age influences thermal tolerance between these two species. Finally, we studied the locomotion capacities as it can be a complementary mechanism to survive external host conditions while searching for new hosts. \u003cem\u003eOrthohalarachne attenuata\u003c/em\u003e has a higher thermal tolerance than \u003cem\u003eO. diminuata\u003c/em\u003e when exposed to a combination of high and low temperatures and time of exposure, which is compatible with the hypothesis of a full larval development in the respiratory system organs and dispersion to the external environment. Age does not seem to influence thermal performance. Finally, \u003cem\u003eO. diminuata\u003c/em\u003e has a much higher locomotion capacity than \u003cem\u003eO. attenuata\u003c/em\u003e. Our experiments show that \u003cem\u003eO. attenuata\u003c/em\u003e has an overall higher thermal tolerance than \u003cem\u003eO. diminuata\u003c/em\u003e although a lower locomotion. Together, our results indicate that Halarachnidae mites larvae have different strategies to withstand the environmental conditions outside the host but compatible with the hypothesis that larvae can disperse far through host sneezing and survive.\u003c/p\u003e","manuscriptTitle":"Differences in thermal tolerance and locomotion capacity support distinct dispersal strategies in marine larvae Halarachnidae mites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 12:43:15","doi":"10.21203/rs.3.rs-7592659/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revise and Resubmit","date":"2025-12-22T11:49:17+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-10-15T07:41:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T07:02:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-15T14:13:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biology","date":"2025-09-11T09:53:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1ebd67e5-a218-40b7-b176-11b52dffebf4","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T19:30:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 12:43:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7592659","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7592659","identity":"rs-7592659","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}