First record of the terrestrial isopod Porcellionides pruinosus as an ant guest along foraging trails and inside Messor ebeninus nests in the Negev Desert

First record of the terrestrial isopod Porcellionides pruinosus as an ant guest along foraging trails and inside Messor ebeninus nests in the Negev Desert | 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 Short Report First record of the terrestrial isopod Porcellionides pruinosus as an ant guest along foraging trails and inside Messor ebeninus nests in the Negev Desert Karmi Oxman, Kamila M.D. Kuabara, Sean O’Donnell This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4784632/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Dec, 2024 Read the published version in Insectes Sociaux → Version 1 posted 5 You are reading this latest preprint version Abstract We report field observations of the cosmopolitan terrestrial isopod species Porcellionides pruinosus both inside multiple nests and following foraging trails of the seed harvester ant Messor ebeninus in the spring of 2022 and 2023. To our knowledge, this is the first observation of P. pruinosus in association with any ant species. Isopods inside the nest either travelled to deeper tunnels or joined foraging trails. The density of isopods along foraging trails was as high as sixty individuals along one 22meter foraging trail. Results from twelve preliminary homing assays revealed that the distance isopods were displaced did not affect whether they returned to the trail, but the farther away they were displaced the longer it took them to return to the trail. Two of the four isopods introduced into a foraging trail of a new nest continued walking with the foreign trail. Isopods were met with minimal aggression from the ants along the trail, and there were no instances of nest guarding observed. The presence of P. pruinosus inside M. ebeninus . nests could be driven by a need for thermal refuge and access to subterranean granaries. The known ability of isopods to cue into ant pheromones is a plausible mechanism for allowing isopods to follow foraging trails. The benefit of trail following behavior needs further investigation. We hypothesize a benefit to walking on a path clear of obstacles, and efficient detection of patchy desert food sources. Ants Hymenoptera Isopoda Israel Oniscidea trail following Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction A great diversity of guests, known as myrmecophiles, have been found in the nests of ants (Hölldobler and Wilson 1990). There is evidence in the fossil record that these relationships began early in the radiation of social insects (Parmentier 2020). Among the many arthropod taxa that co-inhabit ant nests are terrestrial crustaceans in the order Isopoda. Isopods in the genus Platyarthrus Brandt, 1833, found throughout the Mediterranean region of the Middle East, are almost exclusively myrmecophilic (Donisthorpe 1943; Williams 1988; Schmalfuss 2003; Dekoninck 2007; Garcia et al. 2018). This includes Platyarthrus schoblii Budde-Lund, 1885 , found in ant nests in Israel (Warburg 2007). Platyarthrus hoffmannseggii Brandt, 1833 for example, are blind, but when ants leave their nests, these isopods show positive chemotaxis towards an increasing gradient of formic acid laid by ant workers and use this to emigrate to the new nest site (Parmentier 2020). Here we report the first observation of the abundant and cosmopolitan isopod species Porcellionides pruinosus (Brandt, 1833) in association with ant colonies. P. pruinosus is found in all habitats throughout Israel, and Messor spp. ants are one of the most prominent genera of ants in the Mediterranean Basin and highly diverse within Israel (Warburg 2007; Maya et al. 2023). We observed P. pruinosus not only inside the nests of multiple Messor ebeninus Santschi, 1927, colonies over two years, but the isopods were also present along the entire length of M. ebeninus foraging trails. Methods Field site and dates of observations The field site was located on a highly disturbed man-made terrace in Be’er Sheva, Israel (31.2724ºN, 34.8259ºE). This part of the Be’er Sheva Valley is characterized by its typical sloping loess plains of the arid northern Negev Desert. Five M. ebeninus nests were monitored (Fig. 1B). Observations, collections, and assays were made from April 12 th through 26 th in 2022 between 5am and 9am. Additional monitoring of M. ebeninus trails with P. pruinosus guests took place subsequently while performing other fieldwork in the same area from April 30 th through May 25 th , 2023 during the same morning hours. A 1080P EFUTONPRO borescope inspection camera with a 3.99mm diameter and LED lights was used to view the inside of M. ebeninus nests. M. ebeninus nests that were checked for presence inside the nest had P. pruinosus actively walking along the foraging trails. A DS1920 iButton temperature logger recorded soil surface temperature and humidity. Trail density The number of individual P. pruinosus isopods along a 22-meter branch of the foraging trail of the same M. ebeninus nest was quantified while the ants were active on April 23 rd and 25 th , 2022. Videos were taken from above with an Apple iPhone SE (2 nd generation) and later used to quantify the number of isopods. The density of ants along the trail were quantified on nine occasions, at different spots, but from the same trails and nests, on April 12 th , 14 th , 20 th , 24 th , and 25 th , 2022. The width of the trail was calculated using the homing videos. Part of each video has a US-unit ruler in-view, so the width of each trail could be calculated by taking a still frame image and creating a unique conversion factor for the number of pixels in 0.025mm for each video. The limits of the trail were considered to be within the cleared foliage that the ants walk within the bounds of. The number of ants within a randomly placed 0.05m 2 square was calculated using a similar method and averaged across the nine quantification occasions. Removal of isopods from trails Homing assays were performed by removing 12 isopods from foraging trails (ESM_3.mov). The isopods were individually released between 0 and 25.4 centimeters perpendicular distance from the trail they were removed from. For trials with a distance of 0 centimeters, the isopod was picked up and placed back on the trail in the same location. If the isopod returned to the trail after being a removed, and continued walking in the trails for at least 30 seconds or 0.25 meters, whichever occurred first, then it was considered to have returned to the foraging trail. Introduction of isopods from trails of one nest to trails of another Four isopods were taken from the foraging trail of one M. ebeninus colony nest to the foraging trail of another ant colony. The criteria from the homing assays were used to determine whether an isopod joined the new foraging trail. Sample collection, DNA extraction and amplification To confirm the identity of each species, we utilized DNA barcoding. This approach uses short and standardized segments of DNA for species identification (Magoga et al. 2022). As many other invertebrate groups, Oniscidea, the almost entirely terrestrial suborder of Isopoda, desperately needs taxonomic revision. We aim to take a small step in this direction by depositing the sequence results from our species identification using the mitochondrial marker COI into the NCBI database (Dimitriou et al. 2018). Isopod and ant specimens were collected with forceps or an aspirator directly into 100% ethanol in April 2022 (Fig. 2). They were kept in a -20°C freezer until April 3, 2024, when they were removed for DNA extraction. For DNA extraction from the isopod, its head and six of its legs were used. For the ant, its entire body except for the gaster was used. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germany). Polymerase chain reaction (PCR) was used to amplify the cytochrome c oxidase subunit I(COX1) gene, with the primer pair LCO1480/HCO2198 (Folmer 1994). The PCR mix included 7µl diH2O, 1µl 10 mol LCO1480, 1µl 10 µmol HCO2198, 10µL One Taq Quick Load. PCR reactions were run on a BioRad C1000 Touch Thermal Cycler (Bio-Rad Laboratories Inc.). Thermocycling program for the ant consisted of an initial denaturation at 94°C (10 min), followed by 35 cycles at 94°C (denaturation, 30s), 50°C (annealing, 30s), 65°C (extension, 30s), and an extension step at 65°C. For the isopod, the program used was an initial denaturation at 95°C (180s), followed by 35 cycles at 94°C (denaturation, 60s), 50°C (annealing, 60s), 72°C (extension, 60s), and an extension step at 72°C. A negative control for the extraction and negative control for PCR were included in the thermocycling. Five µl of the PCR product was verified by electrophoresis in an agarose gel stained with 1µl 5X red loading dye. The PCR products were sent to Functional Biosciences Inc. for DNA purification and sequencing. COX1 sequences were deposited in the GenBank Library (isopod: PQ007511-PQ007512; ant: PQ007629-PQ007630). Results Observations and behavioral assays Porcellionides pruinosus were first spotted foraging on M. ebeninus trails on the morning of April 11 th , 2022. Isopods were seen in foraging trails belonging to at least five nests between April 11 th and April 26 th , 2022, and between April 30 th and May 25 th , 2023 (Fig. 1B, C, E-G). P. pruinosus was observed inside at least one M. ebeninus nest on three occasions on April 13 th , 21 st , and 23 rd , 2022 at 6:05, 7:10, and 9:30 respectively (ESM_1.mov). Inside the nest isopods either traveled from one internal tunnel to another internal tunnel or exited the nest towards the foraging trails. Trail density The highest density of isopods on April 23 rd and 25 th was observed between 6:25 and 8:00 (Fig. 3). One observation of a trail with an unusually high density of isopods was quantified on April 12 th , 2022. There were at least 60 individual isopods along this 22m foraging trail, and a density of approximately 8 ants per 0.05m 2 (estimate was scaled to be roughly 3000 or more ants along the whole length) (ESM_2.mov). Over the instances of quantification during the homing assays, the average trail width was 0.10m, with an average of 2.89 ants per 0.05m 2 . Removal of isopods from trails Some of the isopods did not walk back to the trail once they were removed for the assay. The distance the isopods were displaced did not appear to affect whether they returned (Table 1, X 2 (1, N = 12) = 0.2, p = 0.634). Among those isopods that did return to the trail, the ones that were removed farther away took more time to return (Fig. 4). This was nearly statistically significant given our sample size of 7 isopods (F 3,6 = 6.35, p = 0.082). Considering only the dates and times that were during homing assays, soil surface temperature ranged from 15.1° C to 30.6° C , increasing over the course of the day, while humidity decreased (Fig. 5) Introduction of isopods from trails of one nest to trails of another Two out of the four isopods introduced into new trails continued walking with the new trail, and the other two immediately strayed from the new trail. Aversion of M. ebeninus towards isopods For the most part, ants and isopods either walked side by side along the foraging trail or brushed past each other without altering either of their trajectories of movement. However, ants were also observed responding aggressively towards isopods. Aggression behavior was not quantified but consisted of a brief mandible-nip from ants towards an isopod leg, propelling the isopod forwards. These interactions, while assumed to be ants defending their trails, was never observed to cause P. pruinosus to leave the foraging trail. Isopods remained on the trail and usually proceeded to walk in the same direction even after the confrontation. A different species of isopod Hemilepistus reaumuri (Milne-Edwards, 1840), which is at least two-fold larger in size than P. pruinosus, did immediately leave the boundaries of the foraging trail after being snapped at by M. ebeninus. This was observed on several occasions but not quantified. No observations were made of nest guarding by ants towards isopods, as they were observed going in and out of the nest without ant reaction. Species identification The isopod was identified as Porcellionides pruinosus , based on location, morphology, body size, and DNA barcode (Lefebvre 2005). DNA sequences were blasted with the BLASTN algorithm (Altschul et al. 1997), against the GenBank Library. There are two species of Messor spp. commonly referred to in this region: Messor arenarius (Fabricius, 1787) and Messor ebeninus Santschi, 1927. Based on locality, morphology, behavior and nest architecture, we refer to the species in this study as Messor ebeninus (Avgar et al. 2008; Vonshak and Ionescu-Hirsch 2009; Levine et al. 2019; O'Donnell et al. 2020; Maya et al. 2023). Our BLAST results for COI however, returned other species with high percent identities. These included Messor wassmanii Krausse, 1910 and Messor concolor Santschi, 1927 . Both species are thought to be color variants of Messor semirufus (André, 1883), but are found in other parts of Israel. The next best hit was for Messor minor (André, 1883), which is geographically rare in Israel (Vonshak and Ionescu-Hirsch 2009). We did return one hit for Messor ebeninus , but the sample is from a large insect survey in India. We believe that the lack of genetic hits for M. ebeninus may be due to the lack of samples deposited into the NCBI GenBank. Discussion In a review of the distribution and relative abundance of isopods within Israel, Armadillo albus Schmalfuss, 1996, was the only species reported to be associated with Messor seed harvesting ant nests specifically (Warburg and Cohen 1991 ). Isopods of the species Lucasius andalusicus Garcia, 2019 have been found inside of Messor barbarus (Linnaeus, 1767) nests in Spain, and are cited as being “coexistent” (Socias et al. 2019 ). Another Porcellionides species, P. myrmecophilus (Stein, 1859), was sighted inside Messor spp. nests in Malta (Cassar 2023). Our observations of P. pruinosus following M. ebeninus trails, and their presence inside nests, suggest that there may be more to the “coexistence” of ants and isopods than previously noted. Most remarkably, isopods often returned to the trail when removed and were met with minimal aggression from the ants along the trail. To our knowledge, this is the first published observation of P. pruinosus in association with any ant species. Platyarthrus hoffmannseggii follow emigration paths when ants leave (Hermann 1982 ). It is important to note that this behavior differs from the observation in the current study, in which P. pruinosus followed foraging trails in both directions, to and from the nest. The ability of another terrestrial isopod to follow chemical cues to emigrate to new ant nests does suggest, however, that isopods could use a similar mechanism to follow ants along foraging trails. Isopods are known to be gregarious cluster-seekers to avoid desiccation (Allee 1926 ; Friedlander 1965 ; Brockett and Hassall 2005 ). Porcellionides pruinosus is widespread across a range of habitats, but the Negev desert is an extreme among their arid habitat limits (Warburg 1968 ). Their affinity for grouping together to avoid dehydration might explain their presence inside the temperate conditions found inside the M. ebeninus nests, which are suitable homes for a range of accidental ant-guests as well as true myrmecophiles likely due to their nest size and presence of stored plant matter harvested by the ants (Cassar 2023). Why are isopods following ant trails? Other than the emigration trails followed by Platyarthrus isopods, to our knowledge this trail-following behavior was not observed in other myrmecophilous isopods. Our hypotheses include that isopods are foraging on stored plant material inside the ant nest and follow foraging trails from the field into the subterranean M. ebeninus nest granaries. It is plausible that the isopods follow ants to a highly rewarding food source in the field. The spatial distribution of arthropod soil-surface activity is influenced by the patchy distribution of perennial shrubs in the Negev desert (Liu and Steinberger 2017 ). Perhaps following collective trails directly to shrub microhabitats is more efficient than searching for the sparsely distributed food patches as a solitary isopod (Fig. 5 ). The grasses, low-lying foliage and spikey seed riddled soil surface is difficult for some invertebrates to maneuver around. From personal observation, Hemilepistus reaumuri , the largest isopod in this habitat, was spotted fumbling over plants, flipping onto their backs, and dropping food items that were held in their front pereopods. It could be that isopods walk the M. ebeninus paths because they have already been cleared of obstacles. This still leaves the question of why the M. ebeninus ants do not aggress towards the isopods as they do towards other invertebrates that cross their path. Further investigation is needed to discern whether trail following behavior by isopods is accidental or indicative of some other type of relationship that may provide benefits or drawbacks upon one or both species. Declarations Author contributions KO: conceptualization, fieldwork, methodology, data curation, DNA analysis for species identification, writing of the original draft, review, and editing. KMDK: methodology for DNA extraction, amplification, and sequencing, sequence data analysis, review, and editing. SO: project administration, analysis, review, and editing. Acknowledgements Fieldwork was funded by Drexel University Stein Family Fellowship to KO. Sequencing was funded by Drexel University William L. McLean III Fellowship to KO. We thank Dr. Itamar Giladi for his mentorship in the field, hosting, assistance with species identification, and knowledge of the local Negev ecology. Data availability Data will be published as a supplementary file with the paper; to be uploaded after final acceptance. References Allee WC (1926) Studies in animal aggregations: causes and effects of bunching in land isopods. J Exp Zool 45(1):255–277. https://doi.org/10.1002/jez.1400450108 Altschul SF et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402. https://doi.org/10.1093/nar/25.17.3389 Avgar T, Giladi I, Nathan R (2008) Linking traits of foraging animals to spatial patterns of plants: social and solitary ants generate opposing patterns of surviving seeds. Ecol Lett 11224–11234. https://doi.org/10.1111/j.1461-0248.2007.01140.x Brockett BFT, Hassall M (2005) The existence of an Allee effect in populations of Porcellio scaber (Isopoda: Oniscidea). Eur J Soil Biol 41(3–4):123–127. https://doi.org/10.1016/j.ejsobi.2005.09.004 Cassar T, Lapeva-Gjonova A, Mifsud D (2023) The Intranidal Myrmecophiles of the Maltese Islands with Notes on Messor Nests as Repositories of Biodiversity. Insects 14(1):45. https://doi.org/10.3390/insects14010045 Dekoninck W, Lock K, Janssens F (2007) Acceptance of two native myrmecophilous species, Platyarthrus hoffmannseggii (Isopoda: Oniscidea) and Cyphoderus albinus (Collembola: Cyphoderidae) by the introduced invasive garden ant Lasius neglectus (Hymenoptera: Formicidae) in Belgium. Eur J Entomol 104(1):159–161. https://doi.org/10.14411/eje.2007.023 Dimitriou AC, Taiti S, Schmalfuss H, Sfenthourakis S (2018) A molecular phylogeny of Porcellionidae (Isopoda, Oniscidea) reveals inconsistencies with present taxonomy. ZooKeys 801:163–176. https://doi.org/10.3897/zookeys.801.23566 Donisthorpe H (1943) A Myrmecophilous Woodlouse. Nature 151:675–676. https://doi.org/10.1038/151675a0 Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(5):294–299 Friedlander CP (1965) Aggregation in Oniscus asellus Linn. Anim Behav 13(2–3):342–346. https://doi.org/10.1016/0003-3472(65)90054-0 Garcia L, Baena M, Pérez-Gómez Á, Rojas D (2018) First record of the terrestrial isopod Platyarthrus parisii Arcangeli, 1930 (Crustacea: Oniscidea: Platyarthridae) for the European continent, with remarks on Platyarthrus schoblii Budde-Lund, 1885. Bolletí de la Societat D’història. Nat de Les Balears 61:113–119 Hermann HR (1982) Social Insects. Academic, London Hölldobler B, Wilson EO (1990) The Ants. Belknap Press of Harvard University Lefebvre F, Isabelle Marcadé (2005) New insights in the Porcellionides pruinosus complex (Isopoda, Oniscidea): biological, behavioural, and morphological approaches. Crustaceana 78(4):465–480 Levine N, Ben-Zvi G, Seifan M, Giladi I (2019) Investment in reward by ant-dispersed plants consistently selects for better partners along a geographic gradient. AoB PLANTS 11(3):plz027. https://doi.org/10.1093/aobpla/plz027 Liu R, Steinberger Y (2017) Seasonal distribution and diversity of ground-active arthropods between shrub microhabitats in the Negev Desert, Israel. Arid Land Research and Management 32(1): 1–20. https://doi.org/110.1080/15324982.2017.1389774 Magoga G et al (2022) Curation of a reference database of COI sequences for insect identification through DNA metabarcoding: COins Database. https://doi.org/10.1093/database/baac055 . Database baac055 Maya S et al (2023) Systematics of harvester ants (Messor) in Israel based on integrated morphological, genetic, and ecological data. https://doi.org/10.1101/2023.07.16.549226 . bioRxiv 2023.07.16.549226 O’Donnell S, Bulova S, Caponera V, Oxman K, Giladi I (2020) Species differ in worker body size effects on critical thermal limits in seed-harvesting desert ants (Messor ebeninus and M. arenarius). Insectes Sociaux 67(4):473–479. https://doi.org/10.1007/s00040-020-00782-5 Parmentier T (2020) Guests of Social Insects. In: Starr C (ed) Encyclopedia of Social Insects. Springer, Cham. https://doi.org/10.1007/978-3-319-90306-4_164-1 Schmalfuss H (2003) World catalog of terrestrial isopods. Isopoda, Crustacea Socias LG, Gómez AP, Luque FR (2019) A new species of Lucasius (Isopoda: Oniscidea: Porcellionidae) from Southern Spain, with remarks on Lucasius myrmecophilus Kinahan, 1859. Boletín de la Sociedad Entomológica Aragonesa 64:11–20 Vonshak M, Ionescu-Hirsch A (2009) A checklist of the ants of Israel (Hymenoptera: Formicidae). Isr J Entomol 39:33–55 Warburg MR, Cohen N (1991) Reproductive Pattern, Allocation, and Potential in a Semelparous Isopod from the Mediterranean Region of Israel. J Crustac Biol 11(3):368–374. https://doi.org/10.2307/1548463 Warburg MR (1968) Behavioral adaptations of terrestrial isopods. Am Zool 8(3):545–559 Warburg MR (2007) Distribution, reproduction, and relative abundance of Oniscids: a long-term study on isopods (Isopoda, Oniscidea) in Israel. Crustaceana 80(10):1223–1252. https://doi.org/10.1163/156854007782321218 Williams T, Franks NR (1988) Population size and growth rate, sex ratio and behaviour in the ant isopod, Platyarthrus hoffmannseggi. J Zool 215(4):703–717. https://doi.org/10.1111/j.1469-7998.1988.tb02405.x Tables Table 1 Individuals that either returned or did not return to the ant foraging trail Distance removed (cm) #Individuals that returned #Individuals that did not return 0.00 2 12.70 2 4 20.32 2 25.40 1 1 Supplementary Files ESM1.mov ESM2.mov ESM3.mov ESM4.xlsx Cite Share Download PDF Status: Published Journal Publication published 09 Dec, 2024 Read the published version in Insectes Sociaux → Version 1 posted Editorial decision: Major Revisions Needed 03 Sep, 2024 Reviewers agreed at journal 28 Jul, 2024 Reviewers invited by journal 27 Jul, 2024 Editor assigned by journal 26 Jul, 2024 First submitted to journal 22 Jul, 2024 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. 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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-4784632","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":332582716,"identity":"74f7f3b7-ad84-4864-a676-7c140db91bbc","order_by":0,"name":"Karmi Oxman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACCRCRAGEzPgASPHykaGE2AGlhI0oLFLCBOQS1SLaffbrhAYNN4tr2s8cqv+bYybAxMD98dAOPFmmedLMbCQxpxmZn8tJuy25LBjqMzdg4B48WOYY0NqCWw3JmB3LMbktuYwZq4WGTxquF/xlYC4/Z+TdmxZLb6glrkZaA2XIjx4zx47bDhLVIzgDZYgD0y403xtKM247zsDET8IvE+TS2mz8qbBK3nc8x/PhzW7U9P3vzw8f4tECAAYRi5gGTBJUjAcYfpKgeBaNgFIyCEQMAlFJBv6lTQUYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0001-0651-9239","institution":"Drexel University","correspondingAuthor":true,"prefix":"","firstName":"Karmi","middleName":"","lastName":"Oxman","suffix":""},{"id":332582717,"identity":"176c7e35-a41c-471b-a49a-6774d6066ee0","order_by":1,"name":"Kamila M.D. Kuabara","email":"","orcid":"","institution":"Drexel University","correspondingAuthor":false,"prefix":"","firstName":"Kamila","middleName":"M.D.","lastName":"Ku","suffix":"M.D."},{"id":332582718,"identity":"3e17355d-c6eb-4d05-bb12-32e1973871de","order_by":2,"name":"Sean O’Donnell","email":"","orcid":"","institution":"Drexel University","correspondingAuthor":false,"prefix":"","firstName":"Sean","middleName":"","lastName":"O’Donnell","suffix":""}],"badges":[],"createdAt":"2024-07-22 23:38:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4784632/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4784632/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00040-024-01013-x","type":"published","date":"2024-12-09T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63071793,"identity":"c96e5fa0-d647-4e25-b1f4-47578ee7d70d","added_by":"auto","created_at":"2024-08-22 20:09:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":753752,"visible":true,"origin":"","legend":"\u003cp\u003eAdult isopods in the photos are approximately 10mm in length from head to uropod, and ants are approximately 6mm from head to gaster. (A) Still image from borescope video (snake camera) \u003cem\u003einside\u003c/em\u003e \u003cem\u003eM. ebeninus\u003c/em\u003e nest, with \u003cem\u003eP. pruinosus \u003c/em\u003ein view. (B) Approximate locations of five \u003cem\u003eM. ebeninus \u003c/em\u003enests that had \u003cem\u003eP. pruinosus \u003c/em\u003ein their foraging trails in the spring of 2022 and 2023. (C) \u003cem\u003eP. pruinosus \u003c/em\u003ewalking in the same direction as \u003cem\u003eM. ebeninus\u003c/em\u003e as they carry food items. (D) Removal assay, isopod (circled) was removed from the trail, and placed a known distance from the trail boundary (within the dotted lines). (E-G) Additional examples of \u003cem\u003eP. pruinosus \u003c/em\u003ewalking in foraging trails of \u003cem\u003eM. ebeninus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/9908e7d7e591ff7168e6bb3d.jpg"},{"id":63071781,"identity":"ceb77c1d-35ed-4b35-b395-4beb936342c5","added_by":"auto","created_at":"2024-08-22 20:09:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":513130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePorcellionides pruinosus\u003c/em\u003e specimen before DNA extraction:\u003cem\u003e \u003c/em\u003e(A) Lateral view, automatically and manually stitched microscope images to scale in Adobe Photoshop; (B) Ventral view of caudal genitalia and pleopods, male; (C) Ventral view of caudal uropod. \u003cem\u003eMessor ebeninus\u003c/em\u003e specimen before DNA extraction: (D) Lateral view, automatically stitched microscope images to scale; (E) Anterior view of mandibles; (F) Antennae; (G) Tarsal claw.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/427ab6f4f90e81d711d6ba6c.jpg"},{"id":63071782,"identity":"c016d0ee-e79e-428d-8929-b412ceba77a2","added_by":"auto","created_at":"2024-08-22 20:09:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":88327,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of individual \u003cem\u003ePorcellionides pruinosus \u003c/em\u003eforaging on the same 22 meter branch of the foraging trail of an \u003cem\u003eM. ebeninus \u003c/em\u003enest on April 23\u003csup\u003erd\u003c/sup\u003e and 25\u003csup\u003eth\u003c/sup\u003e, 2022.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/c0b058b588d5495d41faff7c.jpg"},{"id":63071815,"identity":"abc95794-d0f3-4749-97df-5b5918f90943","added_by":"auto","created_at":"2024-08-22 20:09:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73550,"visible":true,"origin":"","legend":"\u003cp\u003eIsopod removal assay: Scatter plot showing the amount of time in seconds it took for the isopods that did return to the trail to rejoin it. Each point represents one individual, except on 25-April-22 there were two individuals used for the 0 cm assay.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/8fbbe6645c97bb1a40ac35f8.jpg"},{"id":63071783,"identity":"e0312665-e38e-4d77-911f-307d73d6db4c","added_by":"auto","created_at":"2024-08-22 20:09:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87389,"visible":true,"origin":"","legend":"\u003cp\u003eSurface temperature and humidity during the twelve homing assays. Each point represents the time and temperature or humidity value during one homing assay. Assays occurred between April 12\u003csup\u003eth\u003c/sup\u003e to 25\u003csup\u003eth\u003c/sup\u003e, 2022.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/e206e07a5b9694d0f6665187.jpg"},{"id":71552514,"identity":"31107ca8-017d-4d2f-a718-0d47b1505112","added_by":"auto","created_at":"2024-12-16 16:07:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1963604,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/9e7e7e1e-b1a2-4b88-b930-f5d63c37bcf7.pdf"},{"id":63071955,"identity":"208dd9b4-6097-4831-88f1-092efe911d98","added_by":"auto","created_at":"2024-08-22 20:10:18","extension":"mov","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":22562127,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.mov","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/57d43e3716866dd913ce698a.mov"},{"id":63071821,"identity":"6eb58fda-7950-4467-901a-a5d222a91551","added_by":"auto","created_at":"2024-08-22 20:10:06","extension":"mov","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":312201999,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.mov","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/689d00aa7f7bd8cb9aec8e3d.mov"},{"id":63071785,"identity":"fbedb678-94f3-43a9-bb0e-4c24e4a4b7ad","added_by":"auto","created_at":"2024-08-22 20:09:46","extension":"mov","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":258633792,"visible":true,"origin":"","legend":"","description":"","filename":"ESM3.mov","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/37c9538d87d80ef60d82d66c.mov"},{"id":63071956,"identity":"5a6b88aa-42c7-4b3f-9ee9-172b8e29dddb","added_by":"auto","created_at":"2024-08-22 20:10:18","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":20625,"visible":true,"origin":"","legend":"","description":"","filename":"ESM4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4784632/v1/0ec52041e5af82aeeb57140b.xlsx"}],"financialInterests":"","formattedTitle":"First record of the terrestrial isopod Porcellionides pruinosus as an ant guest along foraging trails and inside Messor ebeninus nests in the Negev Desert","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA great diversity of guests, known as myrmecophiles, have been found in the nests of ants\u0026nbsp;(H\u0026ouml;lldobler and Wilson 1990). There is evidence in the fossil record that these relationships began early in the radiation of social insects\u0026nbsp;(Parmentier 2020). Among the many arthropod taxa that co-inhabit ant nests are terrestrial crustaceans in the order Isopoda. Isopods in the genus \u003cem\u003ePlatyarthrus\u003c/em\u003e Brandt, 1833, found throughout the Mediterranean region of the Middle\u003cem\u003e\u0026nbsp;\u003c/em\u003eEast, are almost exclusively myrmecophilic\u0026nbsp;(Donisthorpe 1943; Williams 1988; Schmalfuss 2003; Dekoninck 2007; Garcia et al. 2018). This\u0026nbsp;includes \u003cem\u003ePlatyarthrus schoblii\u0026nbsp;\u003c/em\u003eBudde-Lund, 1885\u003cem\u003e,\u0026nbsp;\u003c/em\u003efound in ant nests in Israel\u0026nbsp;(Warburg 2007). \u003cem\u003ePlatyarthrus hoffmannseggii\u003c/em\u003e Brandt, 1833 for example, are blind, but when ants leave their nests, these isopods show positive chemotaxis towards an increasing gradient of formic acid laid by ant workers and use this to emigrate to the new nest site\u0026nbsp;(Parmentier 2020).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Here we report the first observation of the abundant and cosmopolitan isopod species \u003cem\u003ePorcellionides pruinosus\u003c/em\u003e (Brandt, 1833) in association with ant colonies.\u0026nbsp;\u003cem\u003eP. pruinosus\u0026nbsp;\u003c/em\u003eis found in all habitats throughout Israel, and \u003cem\u003eMessor\u0026nbsp;\u003c/em\u003espp. ants are one of the most prominent genera of ants in the Mediterranean Basin and highly diverse within Israel\u0026nbsp;(Warburg 2007; Maya et al. 2023). We observed\u0026nbsp;\u003cem\u003eP. pruinosus\u003c/em\u003e not only inside the nests of multiple\u0026nbsp;\u003cem\u003eMessor\u0026nbsp;\u003c/em\u003e\u003cem\u003eebeninus\u003c/em\u003e Santschi, 1927, colonies over two years, but the isopods were also present along the entire length of\u0026nbsp;\u003cem\u003eM. ebeninus\u003c/em\u003e foraging trails.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eField site and dates of observations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The field site was located on a highly disturbed man-made terrace in Be\u0026rsquo;er Sheva, Israel (31.2724\u0026ordm;N, 34.8259\u0026ordm;E). This part of the Be\u0026rsquo;er Sheva Valley is characterized by its typical sloping loess plains of the arid northern Negev Desert. Five \u003cem\u003eM. ebeninus\u003c/em\u003e nests were monitored (Fig. 1B). Observations, collections, and assays were made from April 12\u003csup\u003eth\u003c/sup\u003e through 26\u003csup\u003eth\u003c/sup\u003e in 2022 between 5am and 9am. Additional monitoring of \u003cem\u003eM. ebeninus\u003c/em\u003e trails with \u003cem\u003eP. pruinosus\u0026nbsp;\u003c/em\u003eguests took place subsequently while performing other fieldwork in the same area from April 30\u003csup\u003eth\u003c/sup\u003e through May 25\u003csup\u003eth\u003c/sup\u003e, 2023 during the same morning hours. A 1080P EFUTONPRO borescope inspection camera with a 3.99mm diameter and LED lights was used to view the inside of \u003cem\u003eM. ebeninus\u003c/em\u003e nests. \u003cem\u003eM. ebeninus\u003c/em\u003e nests that were checked for presence inside the nest had \u003cem\u003eP. pruinosus\u0026nbsp;\u003c/em\u003eactively walking along the foraging trails.\u0026nbsp;A DS1920 iButton temperature logger recorded\u0026nbsp;soil surface temperature and humidity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrail density\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe number of individual \u003cem\u003eP. pruinosus\u0026nbsp;\u003c/em\u003eisopods along a 22-meter branch of the foraging trail of the same \u003cem\u003eM. ebeninus\u0026nbsp;\u003c/em\u003enest was quantified while the ants were active on April 23\u003csup\u003erd\u003c/sup\u003e and 25\u003csup\u003eth\u003c/sup\u003e, 2022. Videos were taken from above with an Apple iPhone SE (2\u003csup\u003end\u003c/sup\u003e generation) and later used to quantify the number of isopods. The density of ants along the trail were quantified on nine occasions, at different spots, but from the same trails and nests, on April 12\u003csup\u003eth\u003c/sup\u003e, 14\u003csup\u003eth\u003c/sup\u003e, 20\u003csup\u003eth\u003c/sup\u003e, 24\u003csup\u003eth\u003c/sup\u003e, and 25\u003csup\u003eth\u003c/sup\u003e, 2022. The width of the trail was calculated using the homing videos. Part of each video has a US-unit ruler in-view, so the width of each trail could be calculated by taking a still frame image and creating a unique conversion factor for the number of pixels in 0.025mm for each video. The limits of the trail were considered to be within the cleared foliage that the ants walk within the bounds of. The number of ants within a randomly placed 0.05m\u003csup\u003e2\u003c/sup\u003e square was calculated using a similar method and averaged across the nine quantification occasions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemoval of isopods from trails\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHoming assays were performed by removing 12 isopods from foraging trails (ESM_3.mov). The isopods were individually released between 0 and 25.4 centimeters perpendicular distance from the trail they were removed from. For trials with a distance of 0 centimeters, the isopod was picked up and placed back on the trail in the same location.\u0026nbsp;If the isopod returned to the trail after being a removed, and continued walking in the trails for at least 30 seconds or 0.25 meters, whichever occurred first, then it was considered to have returned to the foraging trail.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntroduction of isopods from trails of one nest to trails of another\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour isopods were taken from the foraging trail of one \u003cem\u003eM. ebeninus\u003c/em\u003e colony nest to the foraging trail of another ant colony. The criteria from the homing assays were used to determine whether an isopod joined the new foraging trail.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection, DNA extraction and amplification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the identity of each species, we utilized DNA barcoding. This approach uses short and standardized segments of DNA for species identification\u0026nbsp;(Magoga et al. 2022). As many other invertebrate groups, Oniscidea, the almost entirely terrestrial suborder of Isopoda, desperately needs taxonomic revision. We aim to take a small step in this direction by depositing the sequence results from our species identification using the mitochondrial marker COI into the NCBI database\u0026nbsp;(Dimitriou et al. 2018).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Isopod and ant specimens were collected with forceps or an aspirator directly into 100% ethanol in April 2022 (Fig. 2). They were kept in a -20\u0026deg;C freezer until April 3, 2024, when they were removed for DNA extraction. For DNA extraction from the isopod, its head and six of its legs were used. For the ant, its entire body except for the gaster was used. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germany).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Polymerase chain reaction (PCR) was used to amplify the cytochrome c oxidase subunit I(COX1) gene, with the primer pair LCO1480/HCO2198\u0026nbsp;(Folmer 1994). The PCR mix included 7\u0026micro;l diH2O, 1\u0026micro;l 10 mol LCO1480, 1\u0026micro;l 10 \u0026micro;mol HCO2198, 10\u0026micro;L One Taq Quick Load. PCR reactions were run on a BioRad C1000 Touch Thermal Cycler (Bio-Rad Laboratories Inc.). Thermocycling program for the ant consisted of an initial denaturation at 94\u0026deg;C (10 min), followed by 35 cycles at 94\u0026deg;C (denaturation, 30s), 50\u0026deg;C (annealing, 30s), 65\u0026deg;C (extension, 30s), and an extension step at 65\u0026deg;C. For the isopod, the program used was an initial denaturation at 95\u0026deg;C (180s), followed by 35 cycles at 94\u0026deg;C (denaturation, 60s), 50\u0026deg;C (annealing, 60s), 72\u0026deg;C (extension, 60s), and an extension step at 72\u0026deg;C. A negative control for the extraction and negative control for PCR were included in the thermocycling. Five \u0026micro;l of the PCR product was verified by electrophoresis in an agarose gel stained with 1\u0026micro;l 5X red loading dye. The PCR products were sent to Functional Biosciences Inc. for DNA purification and sequencing. COX1 sequences were deposited in the GenBank Library (isopod: PQ007511-PQ007512; ant: PQ007629-PQ007630).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eObservations and behavioral assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cem\u003ePorcellionides\u003c/em\u003e\u003cem\u003e\u0026nbsp;pruinosus\u0026nbsp;\u003c/em\u003ewere first spotted foraging on \u003cem\u003eM. ebeninus\u003c/em\u003e trails on the morning of April 11\u003csup\u003eth\u003c/sup\u003e, 2022. Isopods were seen in foraging trails belonging to at least five nests between April 11\u003csup\u003eth\u003c/sup\u003e and April 26\u003csup\u003eth\u003c/sup\u003e, 2022, and between April 30\u003csup\u003eth\u003c/sup\u003e and May 25\u003csup\u003eth\u003c/sup\u003e, 2023 (Fig. 1B, C, E-G). \u003cem\u003eP. pruinosus\u0026nbsp;\u003c/em\u003ewas observed inside at least one \u003cem\u003eM. ebeninus\u003c/em\u003e nest on three occasions on April 13\u003csup\u003eth\u003c/sup\u003e, 21\u003csup\u003est\u003c/sup\u003e, and 23\u003csup\u003erd\u003c/sup\u003e, 2022 at 6:05, 7:10, and 9:30 respectively (ESM_1.mov). Inside the nest isopods either traveled from one internal tunnel to another internal tunnel or exited the nest towards the foraging trails.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrail density\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe highest density of isopods on April 23\u003csup\u003erd\u003c/sup\u003e and 25\u003csup\u003eth\u003c/sup\u003e was observed between 6:25 and 8:00 (Fig. 3). One observation of a trail with an unusually high density of isopods was quantified on April 12\u003csup\u003eth\u003c/sup\u003e, 2022. There were at least 60 individual isopods along this 22m foraging trail, and a density of approximately 8 ants per 0.05m\u003csup\u003e2\u003c/sup\u003e (estimate was scaled to be roughly 3000 or more ants along the whole length) (ESM_2.mov). Over the instances of quantification during the homing assays, the average trail width was 0.10m, with an average of 2.89 ants per 0.05m\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemoval of isopods from trails\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSome of the isopods did not walk back to the trail once they were removed for the assay. The distance the isopods were displaced did not appear to affect whether they returned (Table 1, X\u003csup\u003e2\u003c/sup\u003e (1, N = 12) = 0.2, p = 0.634). Among those isopods that \u003cem\u003edid\u0026nbsp;\u003c/em\u003ereturn to the trail, the ones that were removed farther away took more time to return (Fig. 4). This was nearly statistically significant given our sample size of 7 isopods (F\u003csub\u003e3,6\u003c/sub\u003e = 6.35, p = 0.082). Considering only the dates and times that were during homing assays, soil surface temperature ranged from 15.1\u0026deg;\u003csup\u003eC\u003c/sup\u003e to 30.6\u0026deg;\u003csup\u003eC\u003c/sup\u003e, increasing over the course of the day, while humidity decreased (Fig. 5)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntroduction of isopods from trails of one nest to trails of another\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo out of the four isopods introduced into new trails continued walking with the new trail, and the other two immediately strayed from the new trail.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAversion of \u003cem\u003eM. ebeninus\u003c/em\u003e towards isopods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the most part, ants and isopods either walked side by side along the foraging trail or brushed past each other without altering either of their trajectories of movement. However, ants were also observed responding aggressively towards isopods. Aggression behavior was not quantified but consisted of a brief mandible-nip from ants towards an isopod leg, propelling the isopod forwards. These interactions, while assumed to be ants defending their trails, was never observed to cause \u003cem\u003eP. pruinosus\u0026nbsp;\u003c/em\u003eto leave the foraging trail. Isopods remained on the trail and usually proceeded to walk in the same direction even after the confrontation. A different species of isopod \u003cem\u003eHemilepistus reaumuri\u0026nbsp;\u003c/em\u003e(Milne-Edwards, 1840), which is at least two-fold larger in size than \u003cem\u003eP. pruinosus,\u003c/em\u003e did immediately leave the boundaries of the foraging trail after being snapped at by \u003cem\u003eM. ebeninus.\u003c/em\u003e This was observed on several occasions but not quantified. No observations were made of nest guarding by ants towards isopods, as they were observed going in and out of the nest without ant reaction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecies identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isopod was identified as \u003cem\u003ePorcellionides pruinosus\u003c/em\u003e, based on location, morphology, body size, and DNA barcode\u0026nbsp;(Lefebvre 2005). DNA sequences were blasted with the BLASTN algorithm\u0026nbsp;(Altschul et al. 1997), against the GenBank Library.\u003c/p\u003e\n\u003cp\u003eThere are two species of \u003cem\u003eMessor\u003c/em\u003e spp. commonly referred to in this region: \u003cem\u003eMessor arenarius\u003c/em\u003e (Fabricius, 1787) and \u003cem\u003eMessor ebeninus\u003c/em\u003e Santschi, 1927. Based on locality, morphology, behavior and nest architecture, we refer to the species in this study as \u003cem\u003eMessor ebeninus\u003c/em\u003e (Avgar et al. 2008; Vonshak and Ionescu-Hirsch 2009; Levine et al. 2019; O\u0026apos;Donnell et al. 2020; Maya et al. 2023). Our BLAST results for COI however, returned other species with high percent identities. These included \u003cem\u003eMessor wassmanii\u003c/em\u003e Krausse, 1910 and \u003cem\u003eMessor concolor\u003c/em\u003e Santschi, 1927\u003cem\u003e.\u003c/em\u003e Both species are thought to be color variants of \u003cem\u003eMessor semirufus\u003c/em\u003e (Andr\u0026eacute;, 1883), but are found in other parts of Israel. The next best hit was for \u003cem\u003eMessor minor\u003c/em\u003e (Andr\u0026eacute;, 1883), which is geographically rare in Israel\u0026nbsp;(Vonshak and Ionescu-Hirsch 2009). We did return one hit for \u003cem\u003eMessor ebeninus\u003c/em\u003e, but the sample is from a large insect survey in India. We believe that the lack of genetic hits for \u003cem\u003eM. ebeninus\u0026nbsp;\u003c/em\u003emay be due to the lack of samples deposited into the NCBI GenBank.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn a review of the distribution and relative abundance of isopods within Israel, \u003cem\u003eArmadillo albus\u003c/em\u003e Schmalfuss, 1996, was the only species reported to be associated with \u003cem\u003eMessor\u003c/em\u003e seed harvesting ant nests specifically (Warburg and Cohen \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Isopods of the species \u003cem\u003eLucasius andalusicus\u003c/em\u003e Garcia, 2019 have been found inside of \u003cem\u003eMessor barbarus\u003c/em\u003e (Linnaeus, 1767) nests in Spain, and are cited as being \u0026ldquo;coexistent\u0026rdquo; (Socias et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Another \u003cem\u003ePorcellionides\u003c/em\u003e species, \u003cem\u003eP. myrmecophilus\u003c/em\u003e (Stein, 1859), was sighted inside \u003cem\u003eMessor\u003c/em\u003e spp. nests in Malta (Cassar 2023). Our observations of \u003cem\u003eP. pruinosus\u003c/em\u003e following \u003cem\u003eM. ebeninus\u003c/em\u003e trails, and their presence inside nests, suggest that there may be more to the \u0026ldquo;coexistence\u0026rdquo; of ants and isopods than previously noted. Most remarkably, isopods often returned to the trail when removed and were met with minimal aggression from the ants along the trail. To our knowledge, this is the first published observation of \u003cem\u003eP. pruinosus\u003c/em\u003e in association with any ant species. \u003cem\u003ePlatyarthrus hoffmannseggii\u003c/em\u003e follow emigration paths when ants leave (Hermann \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). It is important to note that this behavior differs from the observation in the current study, in which \u003cem\u003eP. pruinosus\u003c/em\u003e followed foraging trails in both directions, to and from the nest. The ability of another terrestrial isopod to follow chemical cues to emigrate to new ant nests does suggest, however, that isopods could use a similar mechanism to follow ants along foraging trails.\u003c/p\u003e \u003cp\u003eIsopods are known to be gregarious cluster-seekers to avoid desiccation (Allee \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1926\u003c/span\u003e; Friedlander \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1965\u003c/span\u003e; Brockett and Hassall \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003ePorcellionides pruinosus\u003c/em\u003e is widespread across a range of habitats, but the Negev desert is an extreme among their arid habitat limits (Warburg \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1968\u003c/span\u003e). Their affinity for grouping together to avoid dehydration might explain their presence inside the temperate conditions found inside the \u003cem\u003eM. ebeninus\u003c/em\u003e nests, which are suitable homes for a range of accidental ant-guests as well as true myrmecophiles likely due to their nest size and presence of stored plant matter harvested by the ants (Cassar 2023).\u003c/p\u003e \u003cp\u003eWhy are isopods following ant trails? Other than the emigration trails followed by \u003cem\u003ePlatyarthrus\u003c/em\u003e isopods, to our knowledge this trail-following behavior was not observed in other myrmecophilous isopods. Our hypotheses include that isopods are foraging on stored plant material inside the ant nest and follow foraging trails from the field into the subterranean \u003cem\u003eM. ebeninus\u003c/em\u003e nest granaries. It is plausible that the isopods follow ants to a highly rewarding food source in the field. The spatial distribution of arthropod soil-surface activity is influenced by the patchy distribution of perennial shrubs in the Negev desert (Liu and Steinberger \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Perhaps following collective trails directly to shrub microhabitats is more efficient than searching for the sparsely distributed food patches as a solitary isopod (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe grasses, low-lying foliage and spikey seed riddled soil surface is difficult for some invertebrates to maneuver around. From personal observation, \u003cem\u003eHemilepistus reaumuri\u003c/em\u003e, the largest isopod in this habitat, was spotted fumbling over plants, flipping onto their backs, and dropping food items that were held in their front pereopods. It could be that isopods walk the \u003cem\u003eM. ebeninus\u003c/em\u003e paths because they have already been cleared of obstacles.\u003c/p\u003e \u003cp\u003eThis still leaves the question of why the \u003cem\u003eM. ebeninus\u003c/em\u003e ants do not aggress towards the isopods as they do towards other invertebrates that cross their path. Further investigation is needed to discern whether trail following behavior by isopods is accidental or indicative of some other type of relationship that may provide benefits or drawbacks upon one or both species.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eKO: conceptualization, fieldwork, methodology, data curation, DNA analysis for species identification, writing of the original draft, review, and editing. KMDK: methodology for DNA extraction, amplification, and sequencing, sequence data analysis, review, and editing. SO: project administration, analysis, review, and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eFieldwork was funded by Drexel University Stein Family Fellowship to KO. Sequencing was funded by Drexel University William L. McLean III Fellowship to KO. We thank Dr. Itamar Giladi for his mentorship in the field, hosting, assistance with species identification, and knowledge of the local Negev ecology.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be published as a supplementary file with the paper; to be uploaded after final acceptance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllee WC (1926) Studies in animal aggregations: causes and effects of bunching in land isopods. J Exp Zool 45(1):255\u0026ndash;277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jez.1400450108\u003c/span\u003e\u003cspan address=\"10.1002/jez.1400450108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltschul SF et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389\u0026ndash;3402. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/25.17.3389\u003c/span\u003e\u003cspan address=\"10.1093/nar/25.17.3389\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvgar T, Giladi I, Nathan R (2008) Linking traits of foraging animals to spatial patterns of plants: social and solitary ants generate opposing patterns of surviving seeds. Ecol Lett 11224\u0026ndash;11234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1461-0248.2007.01140.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1461-0248.2007.01140.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrockett BFT, Hassall M (2005) The existence of an Allee effect in populations of Porcellio scaber (Isopoda: Oniscidea). Eur J Soil Biol 41(3\u0026ndash;4):123\u0026ndash;127. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejsobi.2005.09.004\u003c/span\u003e\u003cspan address=\"10.1016/j.ejsobi.2005.09.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCassar T, Lapeva-Gjonova A, Mifsud D (2023) The Intranidal Myrmecophiles of the Maltese Islands with Notes on Messor Nests as Repositories of Biodiversity. Insects 14(1):45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects14010045\u003c/span\u003e\u003cspan address=\"10.3390/insects14010045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDekoninck W, Lock K, Janssens F (2007) Acceptance of two native myrmecophilous species, Platyarthrus hoffmannseggii (Isopoda: Oniscidea) and Cyphoderus albinus (Collembola: Cyphoderidae) by the introduced invasive garden ant Lasius neglectus (Hymenoptera: Formicidae) in Belgium. Eur J Entomol 104(1):159\u0026ndash;161. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14411/eje.2007.023\u003c/span\u003e\u003cspan address=\"10.14411/eje.2007.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDimitriou AC, Taiti S, Schmalfuss H, Sfenthourakis S (2018) A molecular phylogeny of Porcellionidae (Isopoda, Oniscidea) reveals inconsistencies with present taxonomy. ZooKeys 801:163\u0026ndash;176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3897/zookeys.801.23566\u003c/span\u003e\u003cspan address=\"10.3897/zookeys.801.23566\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonisthorpe H (1943) A Myrmecophilous Woodlouse. Nature 151:675\u0026ndash;676. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/151675a0\u003c/span\u003e\u003cspan address=\"10.1038/151675a0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(5):294\u0026ndash;299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedlander CP (1965) Aggregation in Oniscus asellus Linn. Anim Behav 13(2\u0026ndash;3):342\u0026ndash;346. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0003-3472(65)90054-0\u003c/span\u003e\u003cspan address=\"10.1016/0003-3472(65)90054-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia L, Baena M, P\u0026eacute;rez-G\u0026oacute;mez \u0026Aacute;, Rojas D (2018) First record of the terrestrial isopod Platyarthrus parisii Arcangeli, 1930 (Crustacea: Oniscidea: Platyarthridae) for the European continent, with remarks on Platyarthrus schoblii Budde-Lund, 1885. Bollet\u0026iacute; de la Societat D\u0026rsquo;hist\u0026ograve;ria. Nat de Les Balears 61:113\u0026ndash;119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHermann HR (1982) Social Insects. Academic, London\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026ouml;lldobler B, Wilson EO (1990) The Ants. Belknap Press of Harvard University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLefebvre F, Isabelle Marcad\u0026eacute; (2005) New insights in the Porcellionides pruinosus complex (Isopoda, Oniscidea): biological, behavioural, and morphological approaches. Crustaceana 78(4):465\u0026ndash;480\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevine N, Ben-Zvi G, Seifan M, Giladi I (2019) Investment in reward by ant-dispersed plants consistently selects for better partners along a geographic gradient. AoB PLANTS 11(3):plz027. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aobpla/plz027\u003c/span\u003e\u003cspan address=\"10.1093/aobpla/plz027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu R, Steinberger Y (2017) Seasonal distribution and diversity of ground-active arthropods between shrub microhabitats in the Negev Desert, Israel. Arid Land Research and Management 32(1): 1\u0026ndash;20. https://doi.org/110.1080/15324982.2017.1389774\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagoga G et al (2022) Curation of a reference database of COI sequences for insect identification through DNA metabarcoding: COins Database. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/database/baac055\u003c/span\u003e\u003cspan address=\"10.1093/database/baac055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Database baac055\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaya S et al (2023) Systematics of harvester ants (Messor) in Israel based on integrated morphological, genetic, and ecological data. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2023.07.16.549226\u003c/span\u003e\u003cspan address=\"10.1101/2023.07.16.549226\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. bioRxiv 2023.07.16.549226\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Donnell S, Bulova S, Caponera V, Oxman K, Giladi I (2020) Species differ in worker body size effects on critical thermal limits in seed-harvesting desert ants (Messor ebeninus and M. arenarius). Insectes Sociaux 67(4):473\u0026ndash;479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00040-020-00782-5\u003c/span\u003e\u003cspan address=\"10.1007/s00040-020-00782-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParmentier T (2020) Guests of Social Insects. In: Starr C (ed) Encyclopedia of Social Insects. Springer, Cham. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-319-90306-4_164-1\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-90306-4_164-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmalfuss H (2003) World catalog of terrestrial isopods. Isopoda, Crustacea\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSocias LG, G\u0026oacute;mez AP, Luque FR (2019) A new species of \u003cem\u003eLucasius\u003c/em\u003e (Isopoda: Oniscidea: Porcellionidae) from Southern Spain, with remarks on \u003cem\u003eLucasius myrmecophilus\u003c/em\u003e Kinahan, 1859. Bolet\u0026iacute;n de la Sociedad Entomol\u0026oacute;gica Aragonesa 64:11\u0026ndash;20\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVonshak M, Ionescu-Hirsch A (2009) A checklist of the ants of Israel (Hymenoptera: Formicidae). Isr J Entomol 39:33\u0026ndash;55\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarburg MR, Cohen N (1991) Reproductive Pattern, Allocation, and Potential in a Semelparous Isopod from the Mediterranean Region of Israel. J Crustac Biol 11(3):368\u0026ndash;374. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/1548463\u003c/span\u003e\u003cspan address=\"10.2307/1548463\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarburg MR (1968) Behavioral adaptations of terrestrial isopods. Am Zool 8(3):545\u0026ndash;559\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarburg MR (2007) Distribution, reproduction, and relative abundance of Oniscids: a long-term study on isopods (Isopoda, Oniscidea) in Israel. Crustaceana 80(10):1223\u0026ndash;1252. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1163/156854007782321218\u003c/span\u003e\u003cspan address=\"10.1163/156854007782321218\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams T, Franks NR (1988) Population size and growth rate, sex ratio and behaviour in the ant isopod, Platyarthrus hoffmannseggi. J Zool 215(4):703\u0026ndash;717. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1469-7998.1988.tb02405.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1469-7998.1988.tb02405.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\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\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIndividuals that either returned or did not return to the ant foraging trail\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance removed (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#Individuals that \u003cem\u003ereturned\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#Individuals that \u003cem\u003edid not\u003c/em\u003e return\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\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"}],"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":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ants, Hymenoptera, Isopoda, Israel, Oniscidea, trail following","lastPublishedDoi":"10.21203/rs.3.rs-4784632/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4784632/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe report field observations of the cosmopolitan terrestrial isopod species \u003cem\u003ePorcellionides pruinosus\u003c/em\u003e both inside multiple nests and following foraging trails of the seed harvester ant \u003cem\u003eMessor ebeninus\u003c/em\u003e in the spring of 2022 and 2023. To our knowledge, this is the first observation of \u003cem\u003eP. pruinosus\u003c/em\u003e in association with any ant species. Isopods inside the nest either travelled to deeper tunnels or joined foraging trails. The density of isopods along foraging trails was as high as sixty individuals along one 22meter foraging trail. Results from twelve preliminary homing assays revealed that the distance isopods were displaced did not affect whether they returned to the trail, but the farther away they were displaced the longer it took them to return to the trail. Two of the four isopods introduced into a foraging trail of a new nest continued walking with the foreign trail. Isopods were met with minimal aggression from the ants along the trail, and there were no instances of nest guarding observed. The presence of \u003cem\u003eP. pruinosus\u003c/em\u003e inside \u003cem\u003eM. ebeninus\u003c/em\u003e. nests could be driven by a need for thermal refuge and access to subterranean granaries. The known ability of isopods to cue into ant pheromones is a plausible mechanism for allowing isopods to follow foraging trails. The benefit of trail following behavior needs further investigation. We hypothesize a benefit to walking on a path clear of obstacles, and efficient detection of patchy desert food sources.\u003c/p\u003e","manuscriptTitle":"First record of the terrestrial isopod Porcellionides pruinosus as an ant guest along foraging trails and inside Messor ebeninus nests in the Negev Desert","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 19:23:23","doi":"10.21203/rs.3.rs-4784632/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-09-03T08:54:59+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-07-28T21:15:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-27T16:36:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T05:55:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2024-07-22T19:38:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c0c0feda-88f9-436b-9238-409d1806eae5","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T16:03:22+00:00","versionOfRecord":{"articleIdentity":"rs-4784632","link":"https://doi.org/10.1007/s00040-024-01013-x","journal":{"identity":"insectes-sociaux","isVorOnly":false,"title":"Insectes Sociaux"},"publishedOn":"2024-12-09 15:57:56","publishedOnDateReadable":"December 9th, 2024"},"versionCreatedAt":"2024-08-22 19:23:23","video":"","vorDoi":"10.1007/s00040-024-01013-x","vorDoiUrl":"https://doi.org/10.1007/s00040-024-01013-x","workflowStages":[]},"version":"v1","identity":"rs-4784632","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4784632","identity":"rs-4784632","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}