Predatory activity of Macrochelidae mites (Acari: Mesostigmata) against larvae of Haemonchus contortus (Strongylida: Trichostrongylidae) under field conditions

Predatory activity of Macrochelidae mites (Acari: Mesostigmata) against larvae of Haemonchus contortus (Strongylida: Trichostrongylidae) under field conditions | 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 Predatory activity of Macrochelidae mites (Acari: Mesostigmata) against larvae of Haemonchus contortus (Strongylida: Trichostrongylidae) under field conditions Karina Araújo dos Anjos, Elianai Ribeiro de Souza, Bruna Gonçalves Santos Costa, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8457739/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The gastrointestinal nematode Haemonchus contortus (Rudolphi, 1803) is the most prevalent endoparasite in sheep worldwide, causing serious economic losses due to resistance to commercial anthelmintics, which necessitates alternative and sustainable control strategies. Biological control using predatory mites, naturally present in the environment, emerges as a promising approach. This study evaluated the predatory activity of two mite species, Macrocheles merdarius (Berlese, 1889) and Holostaspella bifoliata (Trägårdh, 1952), on H. contortus larvae under simulated field conditions in Brachiaria sp. pastures. Two independent trials were conducted: in the first, M. merdarius was applied at different densities (12, 25, and 50 mites); in the second, both species were tested individually and in combination. The experimental plots were delimited in 50 cm² quadrants. Larval recovery data (e) were transformed by log₁₀(n + 1) for normalization and analysed using generalized linear models (PROC GLM, SAS), with t-tests for comparisons between treatments and controls (p < 0.05). In the first trial, M. merdarius significantly reduced larval recovery, reaching up to 84% effectiveness at the highest density. In the second trial, H. bifoliata showed the greatest reduction in infective larvae (87.7%), surpassing M. merdarius (56.5%) and the combined treatment (58.5%). It has been shown that both mite species can reduce the population of H. contortus in its free-living stage, and their use can enhance integrated pest management strategies and reduce reliance on chemical anthelmintics. Biological control Macrocheles Holostaspella Integrated parasite management Haemonchus Gastrointestinal nematode Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Gastrointestinal nematode (GIN) infections represent one of the major constraints to livestock production, particularly in small ruminants. In tropical and subtropical regions, these infections are among the primary causes of mortality in young animals (Almeida et al. 2005). The life cycle of these helminths is typically direct, with infective third-stage larvae (L 3 ) migrating from faeces to pasture in response to environmental stimuli, where they subsequently infect grazing hosts. During this free-living stage, nematodes are exposed to a range of biotic and abiotic factors that may either promote or inhibit their development (Costa 1982 ). Among the biotic factors that negatively influence nematode survival are natural antagonists that share the same microhabitat as parasitic larvae. Several of these organisms have been explored as potential biological control agents, including bacteria, viruses, beetles, predatory nematodes, fungi, and soil mites (Gaugler and Bilgrami 2004 ; Pinto 2024 ). Among these antagonists, edaphic mites have attracted increasing attention in recent years; however, research on their role in controlling gastrointestinal nematodes remains limited. Despite the growing number of studies, the available evidence is still emerging. Aguilar-Marcelino et al. ( 2014 ) first reported the predatory behaviour of Lasioseius penicilliger (Berlese, 1916) (Blattisociidae) on H. contortus L 3 larvae. Subsequent investigations have expanded this knowledge: Grisez et al. ( 2023 ) described the predatory potential of two Macrochelidae species on the same parasite, while Dos Anjos et al. ( 2024 ) and Bamière et al. ( 2025 ) demonstrated, through in vitro assays, that members of Macrochelidae family exhibit high efficacy as biological control agents against H. contortus larvae. Supporting these findings, Barros ( 2025 ) reported that Gamasellodes lavafesii Castro, Azevedo & Castilho, 2020 and Cosmolaelaps mediocuspis (Karg, 1981) consumed 40.8% and 29.8% of H. contortus L 3 , respectively, indicating substantial predatory efficiency. Cano (2025) reported that, of 500 H. contortus L 3 larvae, Asca sp. and Macrocheles sp. consumed 57.2% and 66% larvae per day, respectively, also indicating substantial predatory efficiency. In this context, sustainable and innovative alternatives for parasite control become essential in livestock systems to reduce productivity losses and to prevent chemical contamination of animal product and the environment. Therefore, this study assessed the potential of two Macrochelidae mite species to control H. contortus larvae, specifically during their free-living stages, under simulated field conditions. Material and Method Two trials were conducted at the Institute of Animal Science (Instituto de Zootecnia) in Nova Odessa, São Paulo, Brazil. The first at paddocks area of Laboratory of Forage and Animal Nutrition in December 2022 (area 1; 22°46′14″S, 47°18′08″W). The second trial was conducted at the paddocks area of Laboratory of Parasitology in March 2023 (area 2; 22°46′28″S, 47°17′47″W). The experiments were performed in two distinct areas located 1 km apart, designated as trial 1 and trial 2, which were subdivided into quadrants for treated and control plots. The pastures had remained free of animals for one year prior to the start of the trials. Climate data, including temperature and humidity, were obtained from the Agronomic Institute (IAC) database, which maintains continuous meteorological records for the municipality of Nova Odessa, São Paulo, Brazil. Mites In the first trial, M. merdarius was used as a macrobiological agent to control H. contortus larvae during their free-living stages in pasture. In the second trial, both M. merdarius and H. bifoliata (Fig. 1 ) were employed to evaluate the control of H. contortus . The mites were obtained from the stock colony maintained at the Animal Parasitology Laboratory of the Biological Institute, São Paulo (23°35′13″S, 46°38′54″W), and subsequently released into the treatment area. Haemonchus contortus Two Santa Inês lambs free of helminths were infected with a single dose of 5,000 L 3 larvae of a multidrug resistant strain of H. contortus , resistant to moxidectin, closantel, trichlorfon, levamisole phosphate, albendazole, and ivermectin (Almeida et al., 2010). The lambs were kept at the Laboratory of Parasitology, Institute of Animal Science (Instituto de Zootecnia), Nova Odessa, São Paulo. The faecal samples used in this study were macerated and homogenised, and the number of eggs per gram (EPG) was quantified as the mean of three counts using the Gordon and Whitlock (1948) technique, modified by Ueno and Gonçalves (1998). This procedure established the number of eggs released in the first trial at 100,000 and in the second trial at 200,000. Predation test under simulated field conditions First trial Each treatment received faces containing 100,000 H. contortus eggs, except for the negative control (NC), which was used to assess any pre-existing nematode contamination in the pasture. The mites were released 24 hours after the faces were placed. Six replicates were conducted for five treatments, as follows: T1–12 mites; T2–25 mites; T3–50 mites; T0–0 mites (positive control); and 0 mites (negative control). The second release was performed seven days later with 6, 12, and 25 mites in treatments 1, 2, and 3, respectively. The PC did not receive mites, as its purpose was to compare larval recovery. Second trial In this trial, nine replicates were performed for four treatments: control (C), treatment with the two macrochelid mite species M. merdarius (Tmerd) and H. bifoliata (Tbifo), and a mixed treatment containing equal proportions of both species (Tmix). A total of 100 mites were released in each treatment (Tmerd, Tbifo, and Tmix), divided into two releases, as previously described in the first trial (Table 1 ). Table 1 Number of mites released per application in each treatment Control M. merdarius H. bifoliata Mixed * 1st application (day 2) 0 50 50 25/25 2nd application (day 6) 0 50 50 25/25 *Mixed = 25 M. merdarius + 25 H. bifoliata per application. Larvae Recovery The recovery of H. contortus L 3 larvae from pasture grasses was performed following a technique adapted from Taylor ( 1939 ) and Raynauld and Gruner (1982). Briefly, 50 cm² quadrants were delineated in pastures containing Brachiaria sp, with an average height of approximately 50 cm with the of a 50 cm × 50 cm hollow iron square frame was used to delimit the experimental area. Each treatment was spaced 1 m apart, with 50 cm between replicates (Fig. 2 ). The experiment continued for a further nine days after the final release of mites, for a total duration of 15 days. On day 15, the grasses from each quadrant were cut close to the ground using pruning shears and placed into labelled plastic bags. Subsequently, the samples were transferred to buckets containing 10 L of water and 1 mL of neutral detergent, together with a sieve to separate the plant material from the larvae that settled at the bottom. The material was left overnight to allow larval detachment from the grasses and sedimentation. After this period, the grasses were removed, weighed, and oven-dried at 65°C to determine the dry matter content (Fig. 3 a and 3 b). The excess water in the buckets was removed using a pump until approximately 2 L remained. This volume was transferred to a graduated cylinder and left to stand overnight to allow further larval sedimentation. Excess water was again removed using a pump until 50 mL remained. The remaining suspension was transferred to Falcon tubes and stored at 4 ± 1°C for subsequent counting of H. contortus L 3 larvae, according to Ueno and Gonçalves (1998) and Keith ( 1953 ). Statistical analysis The data obtained from the simulated field test were log10-transformed (n + 1) to approximate a normal distribution. Subsequently, the transformed data were analysed using a generalised linear model (PROC GLM, SAS Institute, Cary, NC, USA), in which the model included the treatment effect (treatment × control). Mean comparisons between treated and control groups were performed using the t -test at a significance level of P < 0.05 for each mite species evaluated. Results First trial In the first simulated field trial with the mite species M. merdarius , the mean numbers of H. contortus L 3 larvae recovered were 4,260, 1,120, 2,880 and 660 for plots T0, T1, T2 and T3, respectively (Fig. 3 ). Based on these values, treatment efficacy compared with the control was estimated at 73.7% for T1, 32.4% for T2 and 84.5% for T3. No larvae were recovered from the negative control (NC) plot, ruling out the possibility of prior contamination of the field with H. contortus larvae and thereby eliminating potential interference with the experimental outcomes. Although the differences were not statistically significant, the data indicate that the application of mites to the plots exerted a predatory effect on H. contortus larval populations. When comparing the positive control (PC, without mites) with the mite-treated plots, a marked reduction in the number of L 3 larvae recovered was observed. This finding suggests that M. merdarius interferes with nematode recovery, possibly through predation, competition, or disruption of larval development. Treatment T1 (18 mites) resulted in the recovery of 1,120 L 3 larvae, whereas T3 (75 mites) showed the lowest larval count (660 L 3 ). Interestingly, T2 (37 mites) did not present an intermediate recovery (2,880 L 3 ), suggesting that the relationship between mite density and L 3 reduction is not strictly linear. This pattern may reflect experimental variability, complex ecological interactions within the system, or a non–dose-dependent effect. Environmental conditions during the 15-day trial were relatively stable, with an average minimum temperature of 18.6°C and a maximum of 30.7°C. Relative humidity ranged from 50.0% to 96.3%, and cumulative precipitation was 4.38 mm (Instituto Agronômico [IAC] 2022). Second trial The logarithmic transformation of the data and the statistical significance of the results reinforce the robustness of the analysis, allowing direct comparisons among treatments and supporting the assessment of potential synergistic effects in the predatory activity of the mites. The application of M. merdarius and H. bifoliata significantly reduced the introduced population of H. contortus . Both individual and mixed applications were effective in decreasing the number of larvae present in the faeces of the test plots (P < 0.05) (Fig. 4 ). Although no significant difference was detected between treatments, Tbifo showed the greatest reduction in larvae of H. contortus (87.7%), compared with Tmerd (56.5%) and the Tmix treatment (58.5%). The reduction in the mean values of Tmerd, Tbifo and Tmix relative to the control group indicates that the application of mites restricted the recovery of L 3 larvae. The Tmerd treatment showed a considerable reduction compared with the control, confirming the predatory activity of M. merdarius . Tbifo produced the lowest mean, suggesting that H. bifoliata exerted an even stronger effect on larval reduction. The standard deviations, however, indicate substantial variability among replicates, which should be considered when interpreting these results. Overall, H. bifoliata exhibited the highest efficacy, achieving the lowest mean larval recovery, while M. merdarius also significantly reduced larval counts, though to a lesser extent. Interestingly, the combined treatment (Tmix) did not produce an additive or synergistic effect beyond that observed for the individual application of H. bifoliata , resulting instead in values closer to those of Tmerd. During the second experimental period, moderate temperature fluctuations were recorded. The mean minimum temperature was 19.8°C, while the mean maximum reached 32.9°C. Relative humidity ranged from 40.0% to 98.1%, and total precipitation over the 15-day period was 2.1 mm (Instituto Agronômico [IAC], 2022). Discussion This study represents the first evaluation of the effect of a predatory mite on gastrointestinal nematode larvae under simulated field conditions. The findings provide valuable evidence of the potential of these mites to suppress helminths during their free-living stages in pasture environments. In the first simulated field trial, the release of 75 M. merdarius mites resulted in the greatest reduction of H. contortus larvae. Interestingly, even the lowest release rate produced a significant effect, reducing the larval population by 74%. Conversely, the intermediate dose yielded the lowest reduction (67%), which may reflect experimental variability, complex ecological interactions, or a non-linear dose–response relationship. Another possible explanation is that M. merdarius did not establish effectively in the environment because individuals were dispersed through phoresy. Rodrigues et al. ( 2001 ) reported that M. merdarius disperses via seven species of coprophagous beetles among the 11 species collected in a region of Brazil, supporting this interpretation. Regardless of the underlying mechanism, the application of M. merdarius demonstrated promising potential to reduce the ascent of infective L 3 larvae onto pasture herbage, indicating its potential usefulness for the biological control of gastrointestinal nematodes. These findings suggest that although higher numbers of mites (as in T3) may enhance nematode control, the relationship between mite density and control efficacy is likely influenced by multiple factors that warrant further investigation to be fully elucidated. In the second trial, although no significant differences were observed among treatments, H. bifoliata achieved greater control of first-instar H. contortus larvae, with an 87.7% reduction, compared to M. merdarius , which achieved a 56% reduction. This outcome contrasts with previous findings, as in the in vitro trial conducted by Dos Anjos et al. ( 2024 ) using the same species, M. merdarius exhibited the highest reduction rate of H. contortus larvae (93.6%), compared to 70.8% for H. bifoliata . Lan et al. ( 2025 ) obtained similar results in their studies with Stratiolaelaps scimitus (Laelapidae) and Macrocheles glaber (Macrochelidae) for the control of Lycoriella sp., with S. scimitus showing the best performance. Several factors may account for this discrepancy, with population growth being one of the main explanations. In their study, Bamière et al. ( 2025 ) reported that Macrocheles robustulus laid eggs in the units supplied with H. contortus . Likewise, Dos Anjos et al. ( 2024 ) observed that H. bifoliata exhibited a greater population increase than M. merdarius when feeding on H. contortus larvae. In a simulated field study, Azevedo et al. ( 2020 ) reported that the release of the predatory mite S. scimitus , together with a supplemental food source ( Rhabditella axei (Cobbold)), resulted in improved control of the plant-parasitic nematode Meloidogyne incognita (Kofoid and White, 1919) on tomato plants compared to releasing the predator alone. According to the authors, the increase in the predatory mite population in the presence of R. axei was a key factor in nematode suppression. These findings indicate that the availability of alternative prey can enhance the efficacy of natural enemies in the biological management of nematodes, suggesting that the provision of a supplemental food source could intensify the predatory activity of M. merdarius . Regarding the mixed treatment, the observed effect was similar to that of M. merdarius alone, with reductions of 58.5% and 56.5%, respectively. Lan et al. ( 2025 ) reported a comparable outcome for S. scimitus and M. glaber when released together. This reduced effectiveness may be attributed to interspecific antagonism, a phenomenon also observed during mite colony maintenance. When both species coexisted, H. bifoliata eventually outcompeted M. merdarius . Nevertheless, despite this apparent dominance, field surveys have shown that M. merdarius occurs more frequently and exhibits greater dominance than H. bifoliata (Marchiori et al. 2001 ; Azevedo et al. 2017 ; Borges et al. 2021 ). Conclusion These results suggest that the application of mite species from the family Macrochelidae may represent an effective strategy for reducing populations of the nematode H. contortus , as evidenced by the decrease in the number of recovered L 3 larvae. A comparison between single-species treatments and mixed treatments (Tmix) may help determine whether a positive (synergistic) interaction occurs in their predatory activity or, conversely, whether the two species interfere with each other’s performance. Although the present study demonstrates the predatory capacity of the tested mites against H. contortus , further studies under field conditions are required to confirm the efficacy and feasibility of applying these species. Declarations Acknowledgements We thank Dr. Helder Louvandini, from the Animal Nutrition Laboratory at the Center for Nuclear Energy in Agriculture, University of São Paulo (USP), for providing the sheep feces infected with H. contortus used in the second assay of this study. We also thank all the staff and students from the Parasitology Laboratory at the Institute of Animal Science, Nova Odessa, and the Animal Parasitology Laboratory at the Biological Institute, São Paulo, for their support and collaboration during the development of this research. This study was supported by a grant from the Coordination for the Improvement of Higher Education Personnel (CAPES) - Código de Financiamento 001. Author contributions K.A.A: Conceptualization, Data Curation, Investigation, Methodology, Visualization, Weiting - original draft, Writing - review & editing. E.R.S.: Investigation. B.G.S.C.: Investigation. L.M.K.: Conceptualization, Data Curation, Methodology, Resouces, Supervision, Writing - review & editing. Rodrigo Giglioti: Formal Analysis. W.T.M.: Methodology. F.C.D.: Conceptualization, Writing - review & editing. M.C.M.: Project administration, supervision, writing - review & editing. Competing interests The authors declare that they have no competing interests. Ethical approval All the procedures involving these animals had previously been approved by the institution’s ethics and animal welfare committee (CEUA-IZ 338-2021). References DE-Almeida LR, Castro AA, Da Silva FJM, Da Fonseca AH (2005) Desenvolvimento, sobrevivência e distribuição de larvas infectantes de nematóides gastrintestinais de ruminantes, na estação seca da Baixada Fluminense, RJ. Rev. Bras. Parasitol. 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Manual para diagnóstico das helmintoses de ruminantes. JIICA, Tokyo. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-8457739","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":570682098,"identity":"cf212590-e048-4e34-acea-9e5c0a130d3c","order_by":0,"name":"Karina Araújo dos 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07:21:10","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68884,"visible":true,"origin":"","legend":"","description":"","filename":"1fb1b7e274854cbea0a930d9c5fa2f9b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8457739/v1/70a746de63c143e939de1089.xml"},{"id":99884952,"identity":"5790100f-3071-47e2-b2b7-42f578c1247d","added_by":"auto","created_at":"2026-01-09 12:17:10","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78075,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8457739/v1/8cdb6e201db0f0a4903dd0ab.html"},{"id":99884936,"identity":"f1c7647d-5926-446d-a508-721403e21659","added_by":"auto","created_at":"2026-01-09 12:17:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":673815,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies of mites used in the tests: (a) \u003cem\u003eMacrocheles merdarius\u003c/em\u003e; (b) \u003cem\u003eHolostaspella bifoliata\u003c/em\u003e; (c) ventral shields of \u003cem\u003eM. merdarius\u003c/em\u003e; (d) ventral shields of \u003cem\u003eH. bifoliata\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8457739/v1/d17056e22a523ce89d21564d.png"},{"id":100358743,"identity":"8559419c-e9d8-414e-9d70-150b28d56efa","added_by":"auto","created_at":"2026-01-16 07:21:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":345402,"visible":true,"origin":"","legend":"\u003cp\u003eDelimitation of the area for the field simulation test, showing the layout of quadrants. Source: personal archive\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8457739/v1/c78ad8b15a13f4f117c016f9.png"},{"id":100357966,"identity":"9de4628e-2aba-4d4b-a38f-6e487c65f1c8","added_by":"auto","created_at":"2026-01-16 07:20:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26529,"visible":true,"origin":"","legend":"\u003cp\u003eRecovery of \u003cem\u003eHaemonchus contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e under different treatments with the predatory mite \u003cem\u003eM. merdarius\u003c/em\u003e: (a) number of L\u003csub\u003e3 \u003c/sub\u003erecovered (raw data); (b) percentage reduction of L\u003csub\u003e3\u003c/sub\u003e compared to the control group (C = 0% reduction). Bars represent mean values of larvae recovered per treatment\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8457739/v1/d99ffa796de0d5c3a8625e5e.png"},{"id":99884940,"identity":"0fd5b40e-5f38-4624-b739-ce4717d70bcf","added_by":"auto","created_at":"2026-01-09 12:17:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28221,"visible":true,"origin":"","legend":"\u003cp\u003eMean distribution of \u003cem\u003eHaemonchus contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e (log₁₀-transformed; t-test, p \u0026lt; 0.05) recovered for evaluating predatory activity under simulated field conditions (C = control; Tmerd = \u003cem\u003eM. merdarius\u003c/em\u003e; Tbifo = \u003cem\u003eH. bifoliata\u003c/em\u003e; Tmix = mixed \u003cem\u003eM. merdarius\u003c/em\u003e + \u003cem\u003eH. bifoliata\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8457739/v1/887c2d28226904f70d2d534a.png"},{"id":105564362,"identity":"de3d8130-0ad2-4677-8ff0-e27e5060bbad","added_by":"auto","created_at":"2026-03-27 12:49:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1884233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8457739/v1/46f0e6bf-4a66-4b77-88fb-31a999dcd7b8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Predatory activity of Macrochelidae mites (Acari: Mesostigmata) against larvae of Haemonchus contortus (Strongylida: Trichostrongylidae) under field conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastrointestinal nematode (GIN) infections represent one of the major constraints to livestock production, particularly in small ruminants. In tropical and subtropical regions, these infections are among the primary causes of mortality in young animals (Almeida et al. 2005). The life cycle of these helminths is typically direct, with infective third-stage larvae (L\u003csub\u003e3\u003c/sub\u003e) migrating from faeces to pasture in response to environmental stimuli, where they subsequently infect grazing hosts. During this free-living stage, nematodes are exposed to a range of biotic and abiotic factors that may either promote or inhibit their development (Costa \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Among the biotic factors that negatively influence nematode survival are natural antagonists that share the same microhabitat as parasitic larvae. Several of these organisms have been explored as potential biological control agents, including bacteria, viruses, beetles, predatory nematodes, fungi, and soil mites (Gaugler and Bilgrami \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pinto \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong these antagonists, edaphic mites have attracted increasing attention in recent years; however, research on their role in controlling gastrointestinal nematodes remains limited. Despite the growing number of studies, the available evidence is still emerging. Aguilar-Marcelino et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) first reported the predatory behaviour of \u003cem\u003eLasioseius penicilliger\u003c/em\u003e (Berlese, 1916) (Blattisociidae) on \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e larvae. Subsequent investigations have expanded this knowledge: Grisez et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) described the predatory potential of two Macrochelidae species on the same parasite, while Dos Anjos et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and Bami\u0026egrave;re et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) demonstrated, through \u003cem\u003ein vitro\u003c/em\u003e assays, that members of Macrochelidae family exhibit high efficacy as biological control agents against \u003cem\u003eH. contortus\u003c/em\u003e larvae. Supporting these findings, Barros (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported that \u003cem\u003eGamasellodes lavafesii\u003c/em\u003e Castro, Azevedo \u0026amp; Castilho, 2020 and \u003cem\u003eCosmolaelaps mediocuspis\u003c/em\u003e (Karg, 1981) consumed 40.8% and 29.8% of \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e, respectively, indicating substantial predatory efficiency. Cano (2025) reported that, of 500 \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e larvae, \u003cem\u003eAsca\u003c/em\u003e sp. and \u003cem\u003eMacrocheles\u003c/em\u003e sp. consumed 57.2% and 66% larvae per day, respectively, also indicating substantial predatory efficiency.\u003c/p\u003e \u003cp\u003eIn this context, sustainable and innovative alternatives for parasite control become essential in livestock systems to reduce productivity losses and to prevent chemical contamination of animal product and the environment. Therefore, this study assessed the potential of two Macrochelidae mite species to control \u003cem\u003eH. contortus\u003c/em\u003e larvae, specifically during their free-living stages, under simulated field conditions.\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cp\u003eTwo trials were conducted at the Institute of Animal Science (Instituto de Zootecnia) in Nova Odessa, S\u0026atilde;o Paulo, Brazil. The first at paddocks area of Laboratory of Forage and Animal Nutrition in December 2022 (area 1; 22\u0026deg;46\u0026prime;14\u0026Prime;S, 47\u0026deg;18\u0026prime;08\u0026Prime;W). The second trial was conducted at the paddocks area of Laboratory of Parasitology in March 2023 (area 2; 22\u0026deg;46\u0026prime;28\u0026Prime;S, 47\u0026deg;17\u0026prime;47\u0026Prime;W). The experiments were performed in two distinct areas located 1 km apart, designated as trial 1 and trial 2, which were subdivided into quadrants for treated and control plots. The pastures had remained free of animals for one year prior to the start of the trials.\u003c/p\u003e \u003cp\u003eClimate data, including temperature and humidity, were obtained from the Agronomic Institute (IAC) database, which maintains continuous meteorological records for the municipality of Nova Odessa, S\u0026atilde;o Paulo, Brazil.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMites\u003c/h2\u003e \u003cp\u003eIn the first trial, \u003cem\u003eM. merdarius\u003c/em\u003e was used as a macrobiological agent to control \u003cem\u003eH. contortus\u003c/em\u003e larvae during their free-living stages in pasture. In the second trial, both \u003cem\u003eM. merdarius\u003c/em\u003e and \u003cem\u003eH. bifoliata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were employed to evaluate the control of \u003cem\u003eH. contortus\u003c/em\u003e. The mites were obtained from the stock colony maintained at the Animal Parasitology Laboratory of the Biological Institute, S\u0026atilde;o Paulo (23\u0026deg;35\u0026prime;13\u0026Prime;S, 46\u0026deg;38\u0026prime;54\u0026Prime;W), and subsequently released into the treatment area.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHaemonchus contortus\u003c/h3\u003e\n\u003cp\u003eTwo Santa In\u0026ecirc;s lambs free of helminths were infected with a single dose of 5,000 L\u003csub\u003e3\u003c/sub\u003e larvae of a multidrug resistant strain of \u003cem\u003eH. contortus\u003c/em\u003e, resistant to moxidectin, closantel, trichlorfon, levamisole phosphate, albendazole, and ivermectin (Almeida et al., 2010). The lambs were kept at the Laboratory of Parasitology, Institute of Animal Science (Instituto de Zootecnia), Nova Odessa, S\u0026atilde;o Paulo.\u003c/p\u003e \u003cp\u003eThe faecal samples used in this study were macerated and homogenised, and the number of eggs per gram (EPG) was quantified as the mean of three counts using the Gordon and Whitlock (1948) technique, modified by Ueno and Gon\u0026ccedil;alves (1998). This procedure established the number of eggs released in the first trial at 100,000 and in the second trial at 200,000.\u003c/p\u003e\n\u003ch3\u003ePredation test under simulated field conditions\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFirst trial\u003c/h2\u003e \u003cp\u003eEach treatment received faces containing 100,000 \u003cem\u003eH. contortus\u003c/em\u003e eggs, except for the negative control (NC), which was used to assess any pre-existing nematode contamination in the pasture. The mites were released 24 hours after the faces were placed. Six replicates were conducted for five treatments, as follows: T1\u0026ndash;12 mites; T2\u0026ndash;25 mites; T3\u0026ndash;50 mites; T0\u0026ndash;0 mites (positive control); and 0 mites (negative control). The second release was performed seven days later with 6, 12, and 25 mites in treatments 1, 2, and 3, respectively. The PC did not receive mites, as its purpose was to compare larval recovery.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSecond trial\u003c/h3\u003e\n\u003cp\u003eIn this trial, nine replicates were performed for four treatments: control (C), treatment with the two macrochelid mite species \u003cem\u003eM. merdarius\u003c/em\u003e (Tmerd) and \u003cem\u003eH. bifoliata\u003c/em\u003e (Tbifo), and a mixed treatment containing equal proportions of both species (Tmix). A total of 100 mites were released in each treatment (Tmerd, Tbifo, and Tmix), divided into two releases, as previously described in the first trial (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \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 \u003cp\u003eNumber of mites released per application in each treatment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eM. merdarius\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eH. bifoliata\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMixed\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1st application (day 2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25/25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2nd application (day 6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25/25\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\u003e*Mixed\u0026thinsp;=\u0026thinsp;25 \u003cem\u003eM. merdarius\u003c/em\u003e\u0026thinsp;+\u0026thinsp;25 \u003cem\u003eH. bifoliata\u003c/em\u003e per application.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLarvae Recovery\u003c/h2\u003e \u003cp\u003eThe recovery of \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e larvae from pasture grasses was performed following a technique adapted from Taylor (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1939\u003c/span\u003e) and Raynauld and Gruner (1982). Briefly, 50 cm\u0026sup2; quadrants were delineated in pastures containing \u003cem\u003eBrachiaria\u003c/em\u003e sp, with an average height of approximately 50 cm with the of a 50 cm \u0026times; 50 cm hollow iron square frame was used to delimit the experimental area. Each treatment was spaced 1 m apart, with 50 cm between replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The experiment continued for a further nine days after the final release of mites, for a total duration of 15 days.\u003c/p\u003e \u003cp\u003eOn day 15, the grasses from each quadrant were cut close to the ground using pruning shears and placed into labelled plastic bags. Subsequently, the samples were transferred to buckets containing 10 L of water and 1 mL of neutral detergent, together with a sieve to separate the plant material from the larvae that settled at the bottom. The material was left overnight to allow larval detachment from the grasses and sedimentation. After this period, the grasses were removed, weighed, and oven-dried at 65\u0026deg;C to determine the dry matter content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe excess water in the buckets was removed using a pump until approximately 2 L remained. This volume was transferred to a graduated cylinder and left to stand overnight to allow further larval sedimentation. Excess water was again removed using a pump until 50 mL remained. The remaining suspension was transferred to Falcon tubes and stored at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for subsequent counting of \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e larvae, according to Ueno and Gon\u0026ccedil;alves (1998) and Keith (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1953\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data obtained from the simulated field test were log10-transformed (n\u0026thinsp;+\u0026thinsp;1) to approximate a normal distribution. Subsequently, the transformed data were analysed using a generalised linear model (PROC GLM, SAS Institute, Cary, NC, USA), in which the model included the treatment effect (treatment \u0026times; control). Mean comparisons between treated and control groups were performed using the \u003cem\u003et\u003c/em\u003e-test at a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for each mite species evaluated.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFirst trial\u003c/h2\u003e \u003cp\u003eIn the first simulated field trial with the mite species \u003cem\u003eM. merdarius\u003c/em\u003e, the mean numbers of \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e larvae recovered were 4,260, 1,120, 2,880 and 660 for plots T0, T1, T2 and T3, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Based on these values, treatment efficacy compared with the control was estimated at 73.7% for T1, 32.4% for T2 and 84.5% for T3.\u003c/p\u003e \u003cp\u003eNo larvae were recovered from the negative control (NC) plot, ruling out the possibility of prior contamination of the field with \u003cem\u003eH. contortus\u003c/em\u003e larvae and thereby eliminating potential interference with the experimental outcomes. Although the differences were not statistically significant, the data indicate that the application of mites to the plots exerted a predatory effect on \u003cem\u003eH. contortus\u003c/em\u003e larval populations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen comparing the positive control (PC, without mites) with the mite-treated plots, a marked reduction in the number of L\u003csub\u003e3\u003c/sub\u003e larvae recovered was observed. This finding suggests that \u003cem\u003eM. merdarius\u003c/em\u003e interferes with nematode recovery, possibly through predation, competition, or disruption of larval development.\u003c/p\u003e \u003cp\u003eTreatment T1 (18 mites) resulted in the recovery of 1,120 L\u003csub\u003e3\u003c/sub\u003e larvae, whereas T3 (75 mites) showed the lowest larval count (660 L\u003csub\u003e3\u003c/sub\u003e). Interestingly, T2 (37 mites) did not present an intermediate recovery (2,880 L\u003csub\u003e3\u003c/sub\u003e), suggesting that the relationship between mite density and L\u003csub\u003e3\u003c/sub\u003e reduction is not strictly linear. This pattern may reflect experimental variability, complex ecological interactions within the system, or a non\u0026ndash;dose-dependent effect.\u003c/p\u003e \u003cp\u003eEnvironmental conditions during the 15-day trial were relatively stable, with an average minimum temperature of 18.6\u0026deg;C and a maximum of 30.7\u0026deg;C. Relative humidity ranged from 50.0% to 96.3%, and cumulative precipitation was 4.38 mm (Instituto Agron\u0026ocirc;mico [IAC] 2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSecond trial\u003c/h2\u003e \u003cp\u003eThe logarithmic transformation of the data and the statistical significance of the results reinforce the robustness of the analysis, allowing direct comparisons among treatments and supporting the assessment of potential synergistic effects in the predatory activity of the mites.\u003c/p\u003e \u003cp\u003eThe application of \u003cem\u003eM. merdarius\u003c/em\u003e and \u003cem\u003eH. bifoliata\u003c/em\u003e significantly reduced the introduced population of \u003cem\u003eH. contortus\u003c/em\u003e. Both individual and mixed applications were effective in decreasing the number of larvae present in the faeces of the test plots (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Although no significant difference was detected between treatments, Tbifo showed the greatest reduction in larvae of \u003cem\u003eH. contortus\u003c/em\u003e (87.7%), compared with Tmerd (56.5%) and the Tmix treatment (58.5%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe reduction in the mean values of Tmerd, Tbifo and Tmix relative to the control group indicates that the application of mites restricted the recovery of L\u003csub\u003e3\u003c/sub\u003e larvae. The Tmerd treatment showed a considerable reduction compared with the control, confirming the predatory activity of \u003cem\u003eM. merdarius\u003c/em\u003e. Tbifo produced the lowest mean, suggesting that \u003cem\u003eH. bifoliata\u003c/em\u003e exerted an even stronger effect on larval reduction. The standard deviations, however, indicate substantial variability among replicates, which should be considered when interpreting these results.\u003c/p\u003e \u003cp\u003eOverall, \u003cem\u003eH. bifoliata\u003c/em\u003e exhibited the highest efficacy, achieving the lowest mean larval recovery, while \u003cem\u003eM. merdarius\u003c/em\u003e also significantly reduced larval counts, though to a lesser extent. Interestingly, the combined treatment (Tmix) did not produce an additive or synergistic effect beyond that observed for the individual application of \u003cem\u003eH. bifoliata\u003c/em\u003e, resulting instead in values closer to those of Tmerd.\u003c/p\u003e \u003cp\u003eDuring the second experimental period, moderate temperature fluctuations were recorded. The mean minimum temperature was 19.8\u0026deg;C, while the mean maximum reached 32.9\u0026deg;C. Relative humidity ranged from 40.0% to 98.1%, and total precipitation over the 15-day period was 2.1 mm (Instituto Agron\u0026ocirc;mico [IAC], 2022).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study represents the first evaluation of the effect of a predatory mite on gastrointestinal nematode larvae under simulated field conditions. The findings provide valuable evidence of the potential of these mites to suppress helminths during their free-living stages in pasture environments.\u003c/p\u003e \u003cp\u003eIn the first simulated field trial, the release of 75 \u003cem\u003eM. merdarius\u003c/em\u003e mites resulted in the greatest reduction of \u003cem\u003eH. contortus\u003c/em\u003e larvae. Interestingly, even the lowest release rate produced a significant effect, reducing the larval population by 74%. Conversely, the intermediate dose yielded the lowest reduction (67%), which may reflect experimental variability, complex ecological interactions, or a non-linear dose\u0026ndash;response relationship. Another possible explanation is that \u003cem\u003eM. merdarius\u003c/em\u003e did not establish effectively in the environment because individuals were dispersed through phoresy. Rodrigues et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) reported that \u003cem\u003eM. merdarius\u003c/em\u003e disperses via seven species of coprophagous beetles among the 11 species collected in a region of Brazil, supporting this interpretation. Regardless of the underlying mechanism, the application of \u003cem\u003eM. merdarius\u003c/em\u003e demonstrated promising potential to reduce the ascent of infective L\u003csub\u003e3\u003c/sub\u003e larvae onto pasture herbage, indicating its potential usefulness for the biological control of gastrointestinal nematodes.\u003c/p\u003e \u003cp\u003eThese findings suggest that although higher numbers of mites (as in T3) may enhance nematode control, the relationship between mite density and control efficacy is likely influenced by multiple factors that warrant further investigation to be fully elucidated.\u003c/p\u003e \u003cp\u003eIn the second trial, although no significant differences were observed among treatments, \u003cem\u003eH. bifoliata\u003c/em\u003e achieved greater control of first-instar \u003cem\u003eH. contortus\u003c/em\u003e larvae, with an 87.7% reduction, compared to \u003cem\u003eM. merdarius\u003c/em\u003e, which achieved a 56% reduction. This outcome contrasts with previous findings, as in the in vitro trial conducted by Dos Anjos et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) using the same species, \u003cem\u003eM. merdarius\u003c/em\u003e exhibited the highest reduction rate of \u003cem\u003eH. contortus\u003c/em\u003e larvae (93.6%), compared to 70.8% for \u003cem\u003eH. bifoliata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eLan et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) obtained similar results in their studies with \u003cem\u003eStratiolaelaps scimitus\u003c/em\u003e (Laelapidae) and \u003cem\u003eMacrocheles glaber\u003c/em\u003e (Macrochelidae) for the control of \u003cem\u003eLycoriella\u003c/em\u003e sp., with \u003cem\u003eS. scimitus\u003c/em\u003e showing the best performance. Several factors may account for this discrepancy, with population growth being one of the main explanations. In their study, Bami\u0026egrave;re et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported that \u003cem\u003eMacrocheles robustulus\u003c/em\u003e laid eggs in the units supplied with \u003cem\u003eH. contortus\u003c/em\u003e. Likewise, Dos Anjos et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) observed that \u003cem\u003eH. bifoliata\u003c/em\u003e exhibited a greater population increase than \u003cem\u003eM. merdarius\u003c/em\u003e when feeding on \u003cem\u003eH. contortus\u003c/em\u003e larvae.\u003c/p\u003e \u003cp\u003eIn a simulated field study, Azevedo et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that the release of the predatory mite \u003cem\u003eS. scimitus\u003c/em\u003e, together with a supplemental food source (\u003cem\u003eRhabditella axei\u003c/em\u003e (Cobbold)), resulted in improved control of the plant-parasitic nematode \u003cem\u003eMeloidogyne incognita\u003c/em\u003e (Kofoid and White, 1919) on tomato plants compared to releasing the predator alone. According to the authors, the increase in the predatory mite population in the presence of \u003cem\u003eR. axei\u003c/em\u003e was a key factor in nematode suppression. These findings indicate that the availability of alternative prey can enhance the efficacy of natural enemies in the biological management of nematodes, suggesting that the provision of a supplemental food source could intensify the predatory activity of \u003cem\u003eM. merdarius\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRegarding the mixed treatment, the observed effect was similar to that of \u003cem\u003eM. merdarius\u003c/em\u003e alone, with reductions of 58.5% and 56.5%, respectively. Lan et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported a comparable outcome for \u003cem\u003eS. scimitus\u003c/em\u003e and \u003cem\u003eM. glaber\u003c/em\u003e when released together. This reduced effectiveness may be attributed to interspecific antagonism, a phenomenon also observed during mite colony maintenance. When both species coexisted, \u003cem\u003eH. bifoliata\u003c/em\u003e eventually outcompeted \u003cem\u003eM. merdarius\u003c/em\u003e. Nevertheless, despite this apparent dominance, field surveys have shown that \u003cem\u003eM. merdarius\u003c/em\u003e occurs more frequently and exhibits greater dominance than \u003cem\u003eH. bifoliata\u003c/em\u003e (Marchiori et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Azevedo et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Borges et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThese results suggest that the application of mite species from the family Macrochelidae may represent an effective strategy for reducing populations of the nematode \u003cem\u003eH. contortus\u003c/em\u003e, as evidenced by the decrease in the number of recovered L\u003csub\u003e3\u003c/sub\u003e larvae. A comparison between single-species treatments and mixed treatments (Tmix) may help determine whether a positive (synergistic) interaction occurs in their predatory activity or, conversely, whether the two species interfere with each other\u0026rsquo;s performance. Although the present study demonstrates the predatory capacity of the tested mites against \u003cem\u003eH. contortus\u003c/em\u003e, further studies under field conditions are required to confirm the efficacy and feasibility of applying these species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Helder Louvandini, from the Animal Nutrition Laboratory at the Center for Nuclear Energy in Agriculture, University of S\u0026atilde;o Paulo (USP), for providing the sheep feces infected with \u003cem\u003eH. contortus\u003c/em\u003e used in the second assay of this study. We also thank all the staff and students from the Parasitology Laboratory at the Institute of Animal Science, Nova Odessa, and the Animal Parasitology Laboratory at the Biological Institute, S\u0026atilde;o Paulo, for their support and collaboration during the development of this research. This study was supported by a grant from the Coordination for the Improvement of Higher Education Personnel (CAPES) - C\u0026oacute;digo de Financiamento 001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eK.A.A:\u0026nbsp;\u003c/strong\u003eConceptualization, Data Curation, Investigation, Methodology, Visualization, Weiting - original draft, Writing - review \u0026amp; editing. \u003cstrong\u003eE.R.S.:\u003c/strong\u003e Investigation.\u003cstrong\u003e\u0026nbsp;B.G.S.C.:\u003c/strong\u003e Investigation. \u003cstrong\u003eL.M.K.:\u003c/strong\u003e Conceptualization, Data Curation, Methodology, Resouces, Supervision, Writing - review \u0026amp; editing. \u003cstrong\u003eRodrigo Giglioti:\u003c/strong\u003e Formal Analysis. \u003cstrong\u003eW.T.M.:\u003c/strong\u003e Methodology.\u003cstrong\u003e\u0026nbsp;F.C.D.:\u003c/strong\u003e Conceptualization, Writing - review \u0026amp; editing. \u003cstrong\u003eM.C.M.:\u003c/strong\u003e Project administration, supervision, writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eAll the procedures involving these animals had previously been approved by the institution\u0026rsquo;s ethics and animal welfare committee (CEUA-IZ 338-2021).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDE-Almeida LR, Castro AA, Da Silva FJM, Da Fonseca AH (2005) Desenvolvimento, sobreviv\u0026ecirc;ncia e distribui\u0026ccedil;\u0026atilde;o de larvas infectantes de nemat\u0026oacute;ides gastrintestinais de ruminantes, na esta\u0026ccedil;\u0026atilde;o seca da Baixada Fluminense, RJ. Rev. Bras. Parasitol. Vet. 14(3), 89\u0026ndash;94. Available online: https://www.redalyc.org/articulo.oa?id=397841455001 (accessed on 15 Sept. 2025).\u003c/li\u003e\n\u003cli\u003eAzevedo LH, Castilho RC, Berto MM, Moraes GJ (2017) Macrochelid mites (Mesostigmata: Macrochelidae) from S\u0026atilde;o Paulo state, Brazil, with description of a new species of Macrocheles. Zootaxa. 4269(3):413-426. https://doi.org/10.11646/zootaxa.4269.3.5 \u003c/li\u003e\n\u003cli\u003eAzevedo LH, Moreira MFP, Pereira GG, Borges V, DE Moraes GJ, Inomoto MM, Vicente MH, De Siqueira Pinto M, Peres LEP, Rueda-Ram\u0026iacute;rez D, Carta L, Meyer SLF, Mowery J, Bauchan G, Ochoa R, Palevsky E (2020) Combined releases of soil predatory mites and provisioning of free-living nematodes for the biological control of root-knot nematodes on \u0026lsquo;Micro Tom\u0026rsquo; tomato. 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Tese de Doutorado, Escola Superior de Agricultura Luiz de Queiroz, Universidade de S\u0026atilde;o Paulo, Piracicaba. https://doi.org/10.11606/T.11.2025.tde-17092025-151046 \u003c/li\u003e\n\u003cli\u003eGordon HM, Whitlock HV (1939) A new technique for counting nematode eggs in sheep faeces. J. Counc. Sci. Ind. Res. Aust. 12(1), 50\u0026ndash;52.\u003c/li\u003e\n\u003cli\u003eGrisez C, Perrin W, Begou M, Jay-Robert P, Jacquiet P (2023) An initial investigation of the predatory activity of the phoretic mites of dung beetles, \u003cem\u003eMacrocheles\u003c/em\u003e sp. (Mesostigmata: Macrochelidae), on the gastrointestinal nematode of sheep \u003cem\u003eHaemonchus contortus\u003c/em\u003e (Strongylida: Trichostrongylidae). Biological control, 185, 105301 https://doi.org/10.1016/j.biocontrol.2023.105301.\u003c/li\u003e\n\u003cli\u003eInstituto Agron\u0026ocirc;mico (IAC) (2022) Boletim agrometeorol\u0026oacute;gico da cidade de Nova Odessa. Available from: https://clima.iac.sp.gov.br. Accessed 20 Jan 2023.\u003c/li\u003e\n\u003cli\u003eInstituto Agron\u0026ocirc;mico (IAC) (2022) Boletim agrometeorol\u0026oacute;gico da cidade de Nova Odessa. Available from: https://clima.iac.sp.gov.br. Accessed 15 May 2023.\u003c/li\u003e\n\u003cli\u003eKeith RK (1953). The differentiation of the infective larvae of some common nematode parasites of cattle. Australian Journal of Zoology, 1(2), 223-235. https://doi.org/10.1071/ZO9530223 \u003c/li\u003e\n\u003cli\u003eLan QX, Wen MF, Lu ZH, Ke BR, Fan QH, You MS (2025) Control of fungus gnats \u003cem\u003eLycoriella\u003c/em\u003e sp. in mushroom (\u003cem\u003eAgrocybe aegerita\u003c/em\u003e) cultivation with predatory mites \u003cem\u003eMacrocheles glaber\u003c/em\u003e (Acari: Macrochelidae) and \u003cem\u003eStratiolaelaps scimitus\u003c/em\u003e (Acari: Laelapidae). Insect Science. https://doi.org/10.1111/1744-7917.70141\u003c/li\u003e\n\u003cli\u003eMarchiori CH, Oliveira \u0026Acirc;TD, Linhares AX (2001). Artr\u0026oacute;podes associados a massas fecais bovinas no Sul do Estado de Goi\u0026aacute;s. Neotropical Entomology, 30, 19-24. https://doi.org/10.1590/S1519-566X2001000100004 \u003c/li\u003e\n\u003cli\u003ePinto SC (2024) Controle biol\u0026oacute;gico de nematoides gastrintestinais com a associa\u0026ccedil;\u0026atilde;o de fungos nemat\u0026oacute;fagos \u003cem\u003eDuddingtonia flagrans\u003c/em\u003e e \u003cem\u003ePochonia chlamydosporia\u003c/em\u003e em equinos mantidos a pasto [Universidade Estadual Paulista (Unesp)]. In PINTO. https://hdl.handle.net/11449/256231\u003c/li\u003e\n\u003cli\u003eRaynaud JP., Gruner L (1982). Feasibility of herbage sampling in large extensive pastures and availability of cattle nematode infective larvae in mountain pastures. Veterinary Parasitology, 10(1), 57-64. https://doi.org/10.1016/0304-4017(82)90007-3 \u003c/li\u003e\n\u003cli\u003eRodrigues SR, Marchini LC, Mendes MC (2001) \u0026Aacute;caros da fam\u0026iacute;lia Macrochelidae (Gamasida) associados com besouros copr\u0026oacute;fagos (Scarabaeidae). Revista Brasileira de Entomologia, 45(3), 207-214.\u003c/li\u003e\n\u003cli\u003eTaylor EL (1939) Technique for the estimation of pasture infestation by strongyloid larvae. Parasitology, 31(4), 473-478 https://doi.org/10.1017/S0031182000013007 \u003c/li\u003e\n\u003cli\u003eUeno H, Gutierres VC (1988). Manual para diagn\u0026oacute;stico das helmintoses de ruminantes. JIICA, Tokyo.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biological control, Macrocheles, Holostaspella, Integrated parasite management, Haemonchus, Gastrointestinal nematode","lastPublishedDoi":"10.21203/rs.3.rs-8457739/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8457739/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe gastrointestinal nematode \u003cem\u003eHaemonchus contortus\u003c/em\u003e (Rudolphi, 1803) is the most prevalent endoparasite in sheep worldwide, causing serious economic losses due to resistance to commercial anthelmintics, which necessitates alternative and sustainable control strategies. Biological control using predatory mites, naturally present in the environment, emerges as a promising approach. This study evaluated the predatory activity of two mite species, \u003cem\u003eMacrocheles merdarius\u003c/em\u003e (Berlese, 1889) and \u003cem\u003eHolostaspella bifoliata\u003c/em\u003e (Tr\u0026auml;g\u0026aring;rdh, 1952), on \u003cem\u003eH. contortus\u003c/em\u003e larvae under simulated field conditions in \u003cem\u003eBrachiaria\u003c/em\u003e sp. pastures. Two independent trials were conducted: in the first, \u003cem\u003eM. merdarius\u003c/em\u003e was applied at different densities (12, 25, and 50 mites); in the second, both species were tested individually and in combination. The experimental plots were delimited in 50 cm\u0026sup2; quadrants. Larval recovery data (e) were transformed by log₁₀(n\u0026thinsp;+\u0026thinsp;1) for normalization and analysed using generalized linear models (PROC GLM, SAS), with t-tests for comparisons between treatments and controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the first trial, \u003cem\u003eM. merdarius\u003c/em\u003e significantly reduced larval recovery, reaching up to 84% effectiveness at the highest density. In the second trial, \u003cem\u003eH. bifoliata\u003c/em\u003e showed the greatest reduction in infective larvae (87.7%), surpassing \u003cem\u003eM. merdarius\u003c/em\u003e (56.5%) and the combined treatment (58.5%). It has been shown that both mite species can reduce the population of \u003cem\u003eH. contortus\u003c/em\u003e in its free-living stage, and their use can enhance integrated pest management strategies and reduce reliance on chemical anthelmintics.\u003c/p\u003e","manuscriptTitle":"Predatory activity of Macrochelidae mites (Acari: Mesostigmata) against larvae of Haemonchus contortus (Strongylida: Trichostrongylidae) under field conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-09 12:16:59","doi":"10.21203/rs.3.rs-8457739/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6913271e-a9d6-4011-9a4f-027c7886679b","owner":[],"postedDate":"January 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T19:09:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-09 12:16:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8457739","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8457739","identity":"rs-8457739","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}