Virulence Screening of Malaysia-Isolated Metarhizium anisopliae against Rhipicephalus microplus

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Traditionally, chemical acaricides have been the primary management strategy; however, their indiscriminate use has led to resistance, environmental contamination, and health risks. Therefore, there is growing interest in exploring alternative approaches, such as entomopathogenic fungi like Metarhizium anisopliae . This study aimed to evaluate the effectiveness of M. anisopliae isolates from Malaysia against R. microplus using the Adult Immersion Test protocol. Engorged female ticks were utilized in the bioassay. The experiment involved applying M. anisopliae isolates (1518, 1521, 1522, PR1, HSAH5, and GT3) at a concentration of 10 8 through tick immersion. Mortality rates were monitored for 14 days, with experiments conducted in triplicate. Result showed that PR1 exhibited the highest virulence, causing 83.33% mortality within 14 days. There was no significant difference between the isolates in their ability to cause tick mortality. However, probit analysis revealed that PR1 have the shortest LT 50 and LT 90 with 10.03 days and 14.69 days, respectively. Correlation analysis revealed a significant moderate negative correlation between tick size and mortality and not significant between germination rate and tick mortality. These findings emphasize the influence of tick size on tick mortality. Although no isolate achieved 100% mortality, PR1 was notably effective, killing the highest percentage of ticks quickly and significantly reducing egg production compared to the control and other isolates. Overall, this study underscores the potential of Malaysia-isolated M. anisopliae in the management of adult R. microplus , offering insights into alternative strategies for pest control in the livestock sector. Adult Immersion Test Biological control Cattle ticks Entomopathogenic fungus Livestock Metarhizium anisopliae Figures Figure 1 INTRODUCTION Livestock are pivotal in driving economic prosperity globally, serving as essential sources of meat, milk, eggs, leather, and wool, contributing significantly to agricultural income worldwide (Zayadi et al., 2021). In Malaysia, the bovine population has exhibited consistent growth, reflecting the sector's importance and potential for further development (DVS., 2023). However, the prevalence of tick infestations poses a significant threat to livestock production worldwide, leading to substantial economic and agricultural setbacks across regions such as China, Japan, Australia, Malaysia, Africa, Brazil, and Mexico (Aw et al., 2017). In Malaysia, several studies have identified Rhipicephalus sp. as one of the predominant tick species infesting cattle, contributing to a staggering 60% of the overall infestation rate (Tay et al., 2014 ; Kho et al., 2015 ; Khadijah et al., 2015 ; Ola-Fadunsin et al., 2021 ). Rhipicephalus microplus , commonly known as the southern cattle tick, is the primary vector transmitting various diseases such as Babesiosis, Theileriosis, Anaplasmosis and Cattle Tick Fever, resulting in significant economic losses (Hosseini-Chegeni et al., 2019 ; Barbieri et al., 2023 ). Tick-borne diseases have a significant impact on livestock health and productivity, with studies indicating widespread prevalence rates in Malaysia, such as anaplasmosis affecting a staggering 84.4% of cattle (Tay et al., 2014 ). These diseases also contribute to substantial economic losses globally, with countries like Brazil, Indonesia, and the Philippines facing significant financial burdens (Grisi et al., 2014 ; McLeod & Kristjanson, 1999 ). Besides, high infestations of R. microplus can also lead to adverse health effects in animals, including anaemia, reduced weight gain, diminished milk production, and increased occurrences of myiasis (Barbieri et al., 2023 ). Livestock farmers often resort to chemical acaricides to combat the proliferation of R. microplus . However, the prolonged use of these acaricides has led to the development of resistance in ticks, posing a significant challenge to effective pest control strategies (Muniz et al., 2020 ; Beys-da-Silva et al., 2020; Githaka et al., 2020; Barbieri et al., 2023 ). Consequently, there is a growing interest in exploring alternative methods, such as biological control, to manage tick populations sustainably. Biological control offers a promising approach to mitigate tick infestations, with entomopathogenic fungi emerging as effective biocontrol agents against arthropod pests. Among these fungi, Metarhizium anisopliae is a prominent candidate for tick management due to its ability to infect and kill ticks effectively (Dara, 2017). Despite extensive research on the pathogenicity of M. anisopliae against various tick species, there remains a notable gap in research regarding its efficacy specifically against cattle ticks in Malaysia. This is due to the effectiveness of this fungus depends on several factors including fungal isolates and host species tested (Hussien et al., 2021 ). Additionally, it is important to consider that the efficacy of M. anisopliae can vary depending on climate, humidity, and other environmental conditions, as different isolates may exhibit varying levels of efficacy towards ticks in different geographical locations (Camargo et al., 2012 ). Therefore, comprehensive evaluations of locally isolated M. anisopliae against native tick populations are necessary to determine the suitability and effectiveness of specific M. anisopliae isolates for tick management across various geographic regions. MATERIALS AND METHODS Ticks Collection Rhipicephalus microplus engorged female ticks were initially sourced from naturally infested cattle in Kota Bharu, Kelantan. A total of two hundred eighty engorged females were collected from ticks infesting cattle in Kelantan, and subsequently identified under a dissecting microscope based on Abdullah et al. ( 2016 ) and Walker et al. (2014). The collection of ticks was performed between 7 a.m. to 9 a.m. and tick sizes ranging more than 4mm were chosen. The tick size was estimated using a ruler to measure the length from the tip of its head to the end of its lower body. Each collected tick underwent immersion in 0.1% sodium hypochlorite followed by individual rinsing with distilled water for 10 seconds to eliminate potential environmental contaminants. Subsequently, the ticks were placed in Petri dishes containing moist filter paper and subjected to controlled environmental conditions with temperature (27 ± 2°C), relative humidity (80 ± 5%), and photoperiod (12 h light: 12 h dark). Fungus Preparation Locally isolated M. anisopliae from various locations (as shown in Table 1 ) were reactivated by inoculating them onto the engorged ticks. Conidia emerged from the tick cadavers and were subsequently isolated to acquire pure cultures of M. anisopliae isolates on Sabouraud Dextrose agar mixed with 1% yeast (SDAY). Metarhizium anisopliae was cultivated on rice grain for mass production for approximately 16 days. The rice packs underwent manual agitation every 2 days to prevent aggregation and enhance aeration. Subsequently, the rice grains were separated from the conidia using a 125-micron mesh sieve. The harvested conidia were then transferred into a plastic container and stored in the refrigerator for subsequent use. Table 1 List of local isolates of Metarhizium anisopliae and their localities. Code of isolates Locality 1518 Hutan Simpan Belum, Perak 1521 Batu Gangan, Cameron Highland 1522 Tringkap, Cameron Highland PR1 Pantai Remis, Selangor HSAH5 Hutan Simpan Air Hitam, Puchong GT3 Gunung Tahan, Perak Metarhizium anisopliae suspensions were prepared by weighing 0.1g of conidia and mixing them with 10 ml of sterile distilled water and 0.1% Tween 80. The suspension was vortexed for 3 minutes to ensure thorough mixing. Serial dilutions were then performed, and each dilution was vortexed for 30 seconds. The concentrations of conidia were counted using a haemocytometer and adjusted to final concentrations of 1x10 8 conidia/ml. Germination Test A volume of 20 µl of the fungal suspension with a concentration of 10 7 conidia/ml was inoculated by evenly spread onto a Sabouraud Dextrose Agar + 1% Yeast Extract (SDAY). The inoculated plates are then incubated under room conditions for 24 hours. After incubation, the percentage of germinated spores is determined by counting the number of germinated (A) and non-germinated (B) spores in three random fields of view under a microscope. The conidia were considered to be germinated if the germ tube was longer than the width of the conidia. The germination rate is calculated using the formula: % Germination = (A / (A + B)) x 100. Each isolate was repeated thrice to determine the germination rate of the conidia. Adults Immersion Test The size of ticks used in the bioassay was measured using body length and recorded. A total of 70 adult ticks (size > 4 mm) were randomly allocated into 7 groups for each replicate. The bioassay comprised six treatment groups (6 different isolates), and one control group. The adult ticks were individually immersed in a conidia suspension with a concentration of 1x10 8 for 10 seconds before being transferred to a Petri dish lined with moist filter paper. For the control groups, 10 adult ticks were dipped in distilled water with 1% Tween 80. All Petri dishes were sealed with parafilm and maintained under controlled conditions of temperature (27 ± 2°C) and relative humidity (80 ± 5%). Tick mortality was monitored for 14 days by observing tick movement under a microscope. Dead ticks were transferred to new Petri dishes and observed for fungal growth through the tick cuticle. Three replicates were performed to determine the virulence of M. anisopliae towards R. microplus adults. Statistical Analysis Statistical analyses were performed using SPSS software (ver. 29). Differences in tick sizes, germination rates and mortality rates between treatments were compared using one-way ANOVA. The median lethal time (LT 50 ) and LT 90 values representing the time required for 50% and 90% tick mortality, were determined using Probit regression analysis. Additionally, Pearson correlation analyses were conducted to evaluate the relationships between tick size, germination rates, and mortality rates, providing insights into potential associations between these variables. Mortality rates were adjusted using the Abbot formula: Corrected % = [1 - \(\frac{\left(\text{n}\right) \text{I}\text{n}\text{s}\text{e}\text{c}\text{t} \text{p}\text{o}\text{p}\text{u}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n} \text{i}\text{n} \text{T}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d} \text{G}\text{r}\text{o}\text{u}\text{p} \text{a}\text{f}\text{t}\text{e}\text{r} \text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}{\left(\text{n}\right)\text{I}\text{n}\text{s}\text{e}\text{c}\text{t} \text{p}\text{o}\text{p}\text{u}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n} \text{i}\text{n} \text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{G}\text{r}\text{o}\text{u}\text{p} \text{a}\text{f}\text{t}\text{e}\text{r} \text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}\) ) x 100 RESULTS Results in Table 2 showed that all fungal isolates used were able to achieve 80% germination rate, ranging between 84–89%. No significant difference was found between the isolates in terms of germination. Isolate 1521 was the isolate with the highest germination rate at 89.00 ± 4.93. Ticks used in this study were all with sizes ranging from 5.72–6.25 mm. The ticks being assigned to each treatment were not significantly different from each other in terms of size. All M. anisopliae isolates were pathogenic to R. microplus at the concentration of 10 8 conidia/ml in the laboratory. Mortality caused by M. anisopliae ranged between 36.67–83.33% of the 14-day experimental periods (Table 2 ). The highest mortality was observed in PR1, while the lowest mortality was in 1518. There was no significant difference between the isolates in their ability to cause tick mortality. However, only PR1 and HSAH5 caused significantly higher mortality in ticks compared to the control. Table 2 Fungus germination rate, tick size and tick mortality rate used in this study. Isolates Germination rate (%) Tick size (mm) Mortality (n) Corrected Mortality (%) 1518 84.33 ± 2.96 a 5.95 ± 0.14 a 4.67 ± 0.88 ab 36.67 1521 89.00 ± 4.93 a 5.82 ± 0.11 a 6.67 ± 0.88 ab 56.67 1522 87.66 ± 0.33 a 5.90 ± 0.13 a 5.00 ± 1.00 ab 40.00 PR1 88.00 ± 3.79 a 5.72 ± 0.12 a 9.33 ± 0.33 a 83.33 GT3 84.33 ± 2.33 a 5.85 ± 0.10 a 5.33 ± 2.60 ab 43.33 HSAH5 84.33 ± 2.96 a 5.80 ± 0.14 a 7.67 ± 1.33 a 66.67 Control - 6.25 ± 0.15 a 1.00 ± 0.00 b - Within the column, means followed by the same letter did not differ significantly at P < 0.05. All M. anisopliae isolates were capable of growing hyphae on the tick cadavers four days after the ticks died. Green conidia were then being observed fully covering the tick cadavers by day-8 (Fig. 1 ). Table 3 shows the lethal times (LT 50 and LT 90 ) and their associated confidence intervals for various M. anisopliae isolates tested against R. microplus . The results indicated that the LT 50 values ranged notably from as low as 10.03 days for isolate PR1 to as high as 15.59 days for the 1518. Similarly, the LT 90 values vary substantially, with the shortest being 14.69 days for PR1 and the longest being 29.28 days for the 1518. The associated confidence intervals highlight the consistency and reliability on the estimations of the LT 50 and LT 90 for each isolate. Isolate PR1, for instance, with a narrow confidence interval, indicating a high precision on the lethal time estimated. Conversely, the 1518 with a wider confidence interval, suggesting significant variability and reduced precision in estimating the LT 50 and LT 90 for this isolate. Table 3 Lethal times (LT 50 and LT 90 ) and confidence interval (CI) of Malaysia-isolated M. anisopliae against R. microplus . Isolate LT 50 [CI] (day) LT 90 [CI] (day) 1518 15.59 [13.66–20.38] 29.28 [21.73–72.25] 1521 12.66 [11.93–13.80] 17.91 [15.71–24.45] 1522 13.79 [12.11–16.19] 26.07 [20.66–43.44] PR1 10.03 [8.00–10.89] 14.69 [13.41–19.10] GT3 13.02 [11.90–14.58] 21.63 [18.15–31.04] HSAH5 11.70 [ 10.77–12.45] 16.51 [14.96–20.25] Overall, there is a significant moderate negative correlation between tick size and mortality, indicating that larger tick sizes are associated with lower mortality rates. Meanwhile, the correlation between germination rate and tick mortality overall was not significant. Among the isolates, there is a significantly high correlation between tick size and mortality in isolate GT3. Conversely, in other isolates, the correlations between tick size and tick mortality, as well as germination rate and tick mortality, showed no significant differences. Table 4 Correlation between the variables Tick size vs tick mortality Germination rate vs tick mortality Isolates Pearson correlation (r) P-value Pearson correlation (r) P-value 1518 -0.19 0.88 0.32 0.79 1521 -0.95 0.21 -0.11 0.93 1522 0.00 1.00 0.57 0.61 PR1 0.92 0.26 -0.96 0.17 GT3 -1.00 0.04* 0.46 0.70 HSAH5 -0.33 0.79 0.62 0.58 Overall -0.52 0.03* 0.08 0.74 * Different significantly at P < 0.05 Very high positive correlation: 0.90 to 1.00; very high negative correlation: -0.90 to -1.00; high positive correlation: 0.70 to 0.90; high negative correlation: -0.70 to -0.90; moderate positive correlation: 0.50 to 0.70; moderate negative correlation: -0.50 to -0.70; low positive correlation: 0.30 to 0.50; low negative correlation: -0.30 to -0.50; very weak positive correlation: 0.00 to 0.30; very weak negative correlation: 0.00 to -0.30 (Jaadi., 2021). DISCUSSION This study aimed to assess the virulence of various M. anisopliae isolates from Malaysia against R. microplus . Among the six isolates tested, only two isolates, PR1 and HSAH5, exhibited the ability to cause significant mortality from the control group; while only PR1 achieved a mortality rate exceeding 80% within a 14-day observation period. This finding corroborates previous research demonstrating substantial variation in the virulence of different M. anisopliae isolates. For instance, Fernandez-Salas et al. (2017) evaluated 55 M. anisopliae strains, with only 34 strains inducing high mortality in engorged adult ticks. Similarly, Tavassoli et al. (2008) investigated three M. anisopliae strains, finding that only one strain exhibited significantly higher mortality rates compared to the others. Such disparities in virulence and pathogenicity among M. anisopliae strains may be attributed to factors such as the geographical origin of the fungi and inherent characteristics of the ticks involved (Fernandez-Salas et al., 2017). The successful development of entomopathogenic fungi as biological control agents significantly depend on the selection of highly efficient isolates, and the fungi must be adapted to the environmental conditions of the area where they are employed (Chen et al., 2014 ). Other than that, as highlighted by Fernandez-Salas et al. (2017) and Kirkland et al. ( 2004 ), differences in virulence can be influenced by the strain's ability to penetrate the tick cuticle and evade the host's immune system following penetration. Some strains may possess superior capabilities in these aspects, enabling them to kill ticks swiftly and effectively. This variability underscores the importance of selecting isolates with optimal traits for tick control applications. The primary limitation hindering the widespread adoption of entomopathogenic fungi as a replacement for chemical acaricides is their relatively slower killing speed compared to conventional treatments. To address this challenge, our research also focused on identifying the most efficient isolates capable of rapidly eliminating the ticks. Among the six isolates examined, PR1 stood out as the most promising candidate, demonstrating remarkable efficacy in achieving a 50% mortality rate within 10 days and a 90% mortality rate within 15 days. This rapid action distinguishes PR1 as the fastest-acting isolate compared to others, which required longer durations to achieve similar reductions in tick populations. The ability of PR1 to cause 83% tick mortality within 14 days shows the potential to be employed as the biocontrol since the average longevity of engorged female ticks typically ranges from 6 to 19 days with its oviposition started 5 to 8 days after fully engorged and laid maximum up to 1,535 eggs within 11 days (Bandaranayaka et al., 2021 ). Although 100% mortality was not achieved, PR1 was able to reduce the tick population by reducing eggs being successfully produced by the ticks during their lifetime. According to De Aquino et al. (2020), the ticks being used in this study were classified under partially engorged female ticks, which typically measure between 5–6mm; whereas fully engorged female ticks are those with a size range from 8–11mm. Our result demonstrated that tick size significantly influenced the tick mortality rate when being exposed to M. anisopliae . Although there were studies which reported engorged ticks were more susceptible to different fungal strains and formulations compared to unfed ticks (Tavassoli et al., 2011 ; Hedimbi et al., 2011 ). There were no previous studies examining the effect of size within the engorged ticks on tick mortality. Thus, the specific mechanisms of how larger-size ticks can resist the infection of M. anisopliae are not known. We hypothesize larger ticks might have better energy storage and innate immunity to fight off the entomopathogenic fungi that are trying to invade the ticks. This hypothesis is supported by a study from Shikano et al. (2014), which states that increased protein intake might indicate compensatory feeding to offset impaired conversion of dietary protein into bodily nitrogen or to meet the higher protein requirements necessary for sustaining an elevated immune response. Additionally, a study by Jensen et al. ( 2016 ) found that in cockroaches, highly resistant populations showed a greater dependence on rich nutritional conditions. Nutrition significantly affects insecticide susceptibility, as the availability and quality of nutritional resources impact cuticle composition and the upregulation of detoxifying enzymes. These enzymes are energetically costly to produce and require substantial investments in amino acids and other essential nutrients such as sulfur and iron. Further research is needed to clarify these mechanisms, which could lead to improved strategies for enhancing the efficacy of fungal biocontrol agents against ticks. Conclusion In conclusion, the present study revealed that under laboratory conditions all M. anisopliae isolates from Malaysia are capable of killing the ticks, but the time taken for tick mortality varied among the isolates. Among all, only PR1 can kill more than 80% of tick mortality within 14 days post-inoculation. It is also demonstrated that the tick size influenced the mortality rate caused by the fungus. The estimated LC 50 values for PR1 also showed that it had promising potential as a biocontrol agent of R. microplus . Therefore, further investigations are necessary to validate the effectiveness of this isolate under field conditions. Declarations Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This study is supported by funding from the Ministry of Higher Education (MOHE) through the Fundamental Research Grant Scheme (FRGS) – FRGS/1/2022/WAB04/UMK/02/9. References Abdullah HHaM, El-Molla A, Salib FA, Allam NaT, Ghazy AA, Abdel-Shafy S (2016) Morphological and molecular identification of the brown dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii (Acari: Ixodidae) vectors of Rickettsioses in Egypt. Veterinary World/Veterinary World 9(10):1087–1101. https://doi.org/10.14202/vetworld.2016.1087-1101 Aw KMS, Hue SM (2017) Mode of Infection of Metarhizium spp. Fungus and Their Potential as Biological Control Agents. 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J Sustainable Nat Resour 2(2). https://doi.org/10.30880/jsunr.2021.12.02.001 Cite Share Download PDF Status: Published Journal Publication published 16 Dec, 2024 Read the published version in International Journal of Tropical Insect Science → Version 1 posted Editorial decision: Major revisions 20 Sep, 2024 Reviewers agreed at journal 02 Sep, 2024 Reviewers invited by journal 02 Sep, 2024 Editor assigned by journal 06 Jun, 2024 First submitted to journal 05 Jun, 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. 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. 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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-4532343","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348308706,"identity":"450ae09f-9fe9-4405-b31b-f11e54cf957d","order_by":0,"name":"Nurul Fatin Amirah Mohd Azmi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYLCCBDBiYDjwAUiwsZOi5eAMkBZm4i1iYGDmAZMElPK3H3664eEeuzz+/tOJh21+bZPnY2Zg/PAxB7cWiTNpZjcSniUXSxw4u+Fwbt9twzZmBmbJmdvwWHMgAajlAHNiw8FeoJae24xALWzMvHi0yJ9//g2opT5x/mHeDYcte27bE9RicCMHZMvhxA3HgFoYftxOJKjF8MabMqCW44kbz/BuONjbcDu5jZmxGa9f5M6nb7v540B14rzzZzd/+PHntu389uaDHz7i8z4KYGwDkw3EqgeBP6QoHgWjYBSMgpECANduXo1szd4fAAAAAElFTkSuQmCC","orcid":"","institution":"University of Malaysia Kelantan Faculty of Veterinary Medicine: Universiti Malaysia Kelantan Fakulti Perubatan Veterinar","correspondingAuthor":true,"prefix":"","firstName":"Nurul","middleName":"Fatin Amirah Mohd","lastName":"Azmi","suffix":""},{"id":348308707,"identity":"0d02a4e9-2fe3-4912-98d5-7102476a8189","order_by":1,"name":"Mohammed Dauda Goni","email":"","orcid":"","institution":"University of Malaysia Kelantan Faculty of Veterinary Medicine: Universiti Malaysia Kelantan Fakulti Perubatan Veterinar","correspondingAuthor":false,"prefix":"","firstName":"Mohammed","middleName":"Dauda","lastName":"Goni","suffix":""},{"id":348308708,"identity":"28538d18-ba83-4564-b78a-64c61d15860c","order_by":2,"name":"Ahmad Syazwan Samsuddin","email":"","orcid":"","institution":"Forest Research Institute Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"Syazwan","lastName":"Samsuddin","suffix":""},{"id":348308709,"identity":"de56acec-05bb-48e5-b8f1-c971f11dcc8d","order_by":3,"name":"Tan Li Peng","email":"","orcid":"https://orcid.org/0000-0003-1668-3224","institution":"University of Malaysia Kelantan Faculty of Veterinary Medicine: Universiti Malaysia Kelantan Fakulti Perubatan Veterinar","correspondingAuthor":false,"prefix":"","firstName":"Tan","middleName":"Li","lastName":"Peng","suffix":""}],"badges":[],"createdAt":"2024-06-05 08:06:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4532343/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4532343/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s42690-024-01391-6","type":"published","date":"2024-12-16T15:57:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65836209,"identity":"8a55f2dc-7507-4ce3-aaff-762f428beb5b","added_by":"auto","created_at":"2024-10-03 10:45:32","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":775946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRhipicephalus microplus\u003c/em\u003e infected with \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (1x10\u003csup\u003e8\u003c/sup\u003e conidia ml\u003csup\u003e-1\u003c/sup\u003e) (A) 1518; (B) 1521; (C) 1522; (D) PR1; (E) GT3; (F) HSAH5.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4532343/v1/3e081d4321d64dfdda3aca1d.jpeg"},{"id":72201682,"identity":"679d53cf-9a53-49de-bda8-dc1016b609a4","added_by":"auto","created_at":"2024-12-23 16:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1211156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4532343/v1/829cf34d-e093-43b2-9d03-de25e9b0f102.pdf"}],"financialInterests":"","formattedTitle":"Virulence Screening of Malaysia-Isolated Metarhizium anisopliae against Rhipicephalus microplus","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLivestock are pivotal in driving economic prosperity globally, serving as essential sources of meat, milk, eggs, leather, and wool, contributing significantly to agricultural income worldwide (Zayadi et al., 2021). In Malaysia, the bovine population has exhibited consistent growth, reflecting the sector's importance and potential for further development (DVS., 2023). However, the prevalence of tick infestations poses a significant threat to livestock production worldwide, leading to substantial economic and agricultural setbacks across regions such as China, Japan, Australia, Malaysia, Africa, Brazil, and Mexico (Aw et al., 2017). In Malaysia, several studies have identified \u003cem\u003eRhipicephalus sp.\u003c/em\u003e as one of the predominant tick species infesting cattle, contributing to a staggering 60% of the overall infestation rate (Tay et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kho et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Khadijah et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ola-Fadunsin et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eRhipicephalus microplus\u003c/em\u003e, commonly known as the southern cattle tick, is the primary vector transmitting various diseases such as Babesiosis, Theileriosis, Anaplasmosis and Cattle Tick Fever, resulting in significant economic losses (Hosseini-Chegeni et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barbieri et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Tick-borne diseases have a significant impact on livestock health and productivity, with studies indicating widespread prevalence rates in Malaysia, such as anaplasmosis affecting a staggering 84.4% of cattle (Tay et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These diseases also contribute to substantial economic losses globally, with countries like Brazil, Indonesia, and the Philippines facing significant financial burdens (Grisi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; McLeod \u0026amp; Kristjanson, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Besides, high infestations of \u003cem\u003eR. microplus\u003c/em\u003e can also lead to adverse health effects in animals, including anaemia, reduced weight gain, diminished milk production, and increased occurrences of myiasis (Barbieri et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLivestock farmers often resort to chemical acaricides to combat the proliferation of \u003cem\u003eR. microplus\u003c/em\u003e. However, the prolonged use of these acaricides has led to the development of resistance in ticks, posing a significant challenge to effective pest control strategies (Muniz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Beys-da-Silva et al., 2020; Githaka et al., 2020; Barbieri et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consequently, there is a growing interest in exploring alternative methods, such as biological control, to manage tick populations sustainably. Biological control offers a promising approach to mitigate tick infestations, with entomopathogenic fungi emerging as effective biocontrol agents against arthropod pests. Among these fungi, \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e is a prominent candidate for tick management due to its ability to infect and kill ticks effectively (Dara, 2017).\u003c/p\u003e \u003cp\u003eDespite extensive research on the pathogenicity of \u003cem\u003eM. anisopliae\u003c/em\u003e against various tick species, there remains a notable gap in research regarding its efficacy specifically against cattle ticks in Malaysia. This is due to the effectiveness of this fungus depends on several factors including fungal isolates and host species tested (Hussien et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, it is important to consider that the efficacy of \u003cem\u003eM. anisopliae\u003c/em\u003e can vary depending on climate, humidity, and other environmental conditions, as different isolates may exhibit varying levels of efficacy towards ticks in different geographical locations (Camargo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, comprehensive evaluations of locally isolated \u003cem\u003eM. anisopliae\u003c/em\u003e against native tick populations are necessary to determine the suitability and effectiveness of specific \u003cem\u003eM. anisopliae\u003c/em\u003e isolates for tick management across various geographic regions.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eTicks Collection\u003c/p\u003e \u003cp\u003e \u003cem\u003eRhipicephalus microplus\u003c/em\u003e engorged female ticks were initially sourced from naturally infested cattle in Kota Bharu, Kelantan. A total of two hundred eighty engorged females were collected from ticks infesting cattle in Kelantan, and subsequently identified under a dissecting microscope based on Abdullah et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Walker et al. (2014). The collection of ticks was performed between 7 a.m. to 9 a.m. and tick sizes ranging more than 4mm were chosen. The tick size was estimated using a ruler to measure the length from the tip of its head to the end of its lower body. Each collected tick underwent immersion in 0.1% sodium hypochlorite followed by individual rinsing with distilled water for 10 seconds to eliminate potential environmental contaminants. Subsequently, the ticks were placed in Petri dishes containing moist filter paper and subjected to controlled environmental conditions with temperature (27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), relative humidity (80\u0026thinsp;\u0026plusmn;\u0026thinsp;5%), and photoperiod (12 h light: 12 h dark).\u003c/p\u003e \u003cp\u003eFungus Preparation\u003c/p\u003e \u003cp\u003eLocally isolated \u003cem\u003eM. anisopliae\u003c/em\u003e from various locations (as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were reactivated by inoculating them onto the engorged ticks. Conidia emerged from the tick cadavers and were subsequently isolated to acquire pure cultures of \u003cem\u003eM. anisopliae\u003c/em\u003e isolates on Sabouraud Dextrose agar mixed with 1% yeast (SDAY). \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e was cultivated on rice grain for mass production for approximately 16 days. The rice packs underwent manual agitation every 2 days to prevent aggregation and enhance aeration. Subsequently, the rice grains were separated from the conidia using a 125-micron mesh sieve. The harvested conidia were then transferred into a plastic container and stored in the refrigerator for subsequent use.\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\u003eList of local isolates of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e and their localities.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCode of isolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocality\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHutan Simpan Belum, Perak\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBatu Gangan, Cameron Highland\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTringkap, Cameron Highland\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePantai Remis, Selangor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSAH5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHutan Simpan Air Hitam, Puchong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGunung Tahan, Perak\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 \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e suspensions were prepared by weighing 0.1g of conidia and mixing them with 10 ml of sterile distilled water and 0.1% Tween 80. The suspension was vortexed for 3 minutes to ensure thorough mixing. Serial dilutions were then performed, and each dilution was vortexed for 30 seconds. The concentrations of conidia were counted using a haemocytometer and adjusted to final concentrations of 1x10\u003csup\u003e8\u003c/sup\u003e conidia/ml.\u003c/p\u003e \u003cp\u003eGermination Test\u003c/p\u003e \u003cp\u003eA volume of 20 \u0026micro;l of the fungal suspension with a concentration of 10\u003csup\u003e7\u003c/sup\u003e conidia/ml was inoculated by evenly spread onto a Sabouraud Dextrose Agar\u0026thinsp;+\u0026thinsp;1% Yeast Extract (SDAY). The inoculated plates are then incubated under room conditions for 24 hours. After incubation, the percentage of germinated spores is determined by counting the number of germinated (A) and non-germinated (B) spores in three random fields of view under a microscope. The conidia were considered to be germinated if the germ tube was longer than the width of the conidia. The germination rate is calculated using the formula: % Germination = (A / (A\u0026thinsp;+\u0026thinsp;B)) x 100. Each isolate was repeated thrice to determine the germination rate of the conidia.\u003c/p\u003e \u003cp\u003eAdults Immersion Test\u003c/p\u003e \u003cp\u003eThe size of ticks used in the bioassay was measured using body length and recorded. A total of 70 adult ticks (size\u0026thinsp;\u0026gt;\u0026thinsp;4 mm) were randomly allocated into 7 groups for each replicate. The bioassay comprised six treatment groups (6 different isolates), and one control group. The adult ticks were individually immersed in a conidia suspension with a concentration of 1x10\u003csup\u003e8\u003c/sup\u003e for 10 seconds before being transferred to a Petri dish lined with moist filter paper. For the control groups, 10 adult ticks were dipped in distilled water with 1% Tween 80. All Petri dishes were sealed with parafilm and maintained under controlled conditions of temperature (27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and relative humidity (80\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). Tick mortality was monitored for 14 days by observing tick movement under a microscope. Dead ticks were transferred to new Petri dishes and observed for fungal growth through the tick cuticle.\u003c/p\u003e \u003cp\u003eThree replicates were performed to determine the virulence of \u003cem\u003eM. anisopliae\u003c/em\u003e towards \u003cem\u003eR. microplus\u003c/em\u003e adults.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS software (ver. 29). Differences in tick sizes, germination rates and mortality rates between treatments were compared using one-way ANOVA. The median lethal time (LT\u003csub\u003e50\u003c/sub\u003e) and LT\u003csub\u003e90\u003c/sub\u003e values representing the time required for 50% and 90% tick mortality, were determined using Probit regression analysis. Additionally, Pearson correlation analyses were conducted to evaluate the relationships between tick size, germination rates, and mortality rates, providing insights into potential associations between these variables. Mortality rates were adjusted using the Abbot formula:\u003c/p\u003e \u003cp\u003eCorrected % = [1 - \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{\\left(\\text{n}\\right) \\text{I}\\text{n}\\text{s}\\text{e}\\text{c}\\text{t} \\text{p}\\text{o}\\text{p}\\text{u}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\text{i}\\text{n} \\text{T}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d} \\text{G}\\text{r}\\text{o}\\text{u}\\text{p} \\text{a}\\text{f}\\text{t}\\text{e}\\text{r} \\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}}{\\left(\\text{n}\\right)\\text{I}\\text{n}\\text{s}\\text{e}\\text{c}\\text{t} \\text{p}\\text{o}\\text{p}\\text{u}\\text{l}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\text{i}\\text{n} \\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l} \\text{G}\\text{r}\\text{o}\\text{u}\\text{p} \\text{a}\\text{f}\\text{t}\\text{e}\\text{r} \\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e) x 100\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eResults in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed that all fungal isolates used were able to achieve 80% germination rate, ranging between 84\u0026ndash;89%. No significant difference was found between the isolates in terms of germination. Isolate 1521 was the isolate with the highest germination rate at 89.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93. Ticks used in this study were all with sizes ranging from 5.72\u0026ndash;6.25 mm. The ticks being assigned to each treatment were not significantly different from each other in terms of size.\u003c/p\u003e \u003cp\u003eAll \u003cem\u003eM. anisopliae\u003c/em\u003e isolates were pathogenic to \u003cem\u003eR. microplus\u003c/em\u003e at the concentration of 10\u003csup\u003e8\u003c/sup\u003e conidia/ml in the laboratory. Mortality caused by \u003cem\u003eM. anisopliae\u003c/em\u003e ranged between 36.67\u0026ndash;83.33% of the 14-day experimental periods (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest mortality was observed in PR1, while the lowest mortality was in 1518. There was no significant difference between the isolates in their ability to cause tick mortality. However, only PR1 and HSAH5 caused significantly higher mortality in ticks compared to the control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFungus germination rate, tick size and tick mortality rate used in this study.\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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 \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGermination rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTick size\u003c/p\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMortality\u003c/p\u003e \u003cp\u003e(n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCorrected Mortality (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e83.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSAH5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\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\u003eWithin the column, means followed by the same letter did not differ significantly at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eAll \u003cem\u003eM. anisopliae\u003c/em\u003e isolates were capable of growing hyphae on the tick cadavers four days after the ticks died. Green conidia were then being observed fully covering the tick cadavers by day-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the lethal times (LT\u003csub\u003e50\u003c/sub\u003e and LT\u003csub\u003e90\u003c/sub\u003e) and their associated confidence intervals for various \u003cem\u003eM. anisopliae\u003c/em\u003e isolates tested against \u003cem\u003eR. microplus\u003c/em\u003e. The results indicated that the LT\u003csub\u003e50\u003c/sub\u003e values ranged notably from as low as 10.03 days for isolate PR1 to as high as 15.59 days for the 1518. Similarly, the LT\u003csub\u003e90\u003c/sub\u003e values vary substantially, with the shortest being 14.69 days for PR1 and the longest being 29.28 days for the 1518. The associated confidence intervals highlight the consistency and reliability on the estimations of the LT\u003csub\u003e50\u003c/sub\u003e and LT\u003csub\u003e90\u003c/sub\u003e for each isolate. Isolate PR1, for instance, with a narrow confidence interval, indicating a high precision on the lethal time estimated. Conversely, the 1518 with a wider confidence interval, suggesting significant variability and reduced precision in estimating the LT\u003csub\u003e50\u003c/sub\u003e and LT\u003csub\u003e90\u003c/sub\u003e for this isolate.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLethal times (LT\u003csub\u003e50\u003c/sub\u003e and LT\u003csub\u003e90\u003c/sub\u003e) and confidence interval (CI) of Malaysia-isolated \u003cem\u003eM. anisopliae\u003c/em\u003e against \u003cem\u003eR. microplus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eLT\u003csub\u003e50\u003c/sub\u003e [CI]\u003c/p\u003e \u003cp\u003e(day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eLT\u003csub\u003e90\u003c/sub\u003e [CI]\u003c/p\u003e \u003cp\u003e(day)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[13.66\u0026ndash;20.38]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e29.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[21.73\u0026ndash;72.25]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[11.93\u0026ndash;13.80]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e17.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[15.71\u0026ndash;24.45]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[12.11\u0026ndash;16.19]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e26.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[20.66\u0026ndash;43.44]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[8.00\u0026ndash;10.89]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e14.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[13.41\u0026ndash;19.10]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[11.90\u0026ndash;14.58]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e21.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[18.15\u0026ndash;31.04]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSAH5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e[ 10.77\u0026ndash;12.45]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e16.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[14.96\u0026ndash;20.25]\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\u003eOverall, there is a significant moderate negative correlation between tick size and mortality, indicating that larger tick sizes are associated with lower mortality rates. Meanwhile, the correlation between germination rate and tick mortality overall was not significant. Among the isolates, there is a significantly high correlation between tick size and mortality in isolate GT3. Conversely, in other isolates, the correlations between tick size and tick mortality, as well as germination rate and tick mortality, showed no significant differences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between the variables\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTick size vs tick mortality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eGermination rate vs tick mortality\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePearson correlation (r)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePearson correlation (r)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSAH5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e* Different significantly at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eVery high positive correlation: 0.90 to 1.00; very high negative correlation: -0.90 to -1.00; high positive correlation: 0.70 to 0.90; high negative correlation: -0.70 to -0.90; moderate positive correlation: 0.50 to 0.70; moderate negative correlation: -0.50 to -0.70; low positive correlation: 0.30 to 0.50; low negative correlation: -0.30 to -0.50; very weak positive correlation: 0.00 to 0.30; very weak negative correlation: 0.00 to -0.30 (Jaadi., 2021).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to assess the virulence of various \u003cem\u003eM. anisopliae\u003c/em\u003e isolates from Malaysia against \u003cem\u003eR. microplus\u003c/em\u003e. Among the six isolates tested, only two isolates, PR1 and HSAH5, exhibited the ability to cause significant mortality from the control group; while only PR1 achieved a mortality rate exceeding 80% within a 14-day observation period. This finding corroborates previous research demonstrating substantial variation in the virulence of different \u003cem\u003eM. anisopliae\u003c/em\u003e isolates. For instance, Fernandez-Salas et al. (2017) evaluated 55 \u003cem\u003eM. anisopliae\u003c/em\u003e strains, with only 34 strains inducing high mortality in engorged adult ticks. Similarly, Tavassoli et al. (2008) investigated three \u003cem\u003eM. anisopliae\u003c/em\u003e strains, finding that only one strain exhibited significantly higher mortality rates compared to the others. Such disparities in virulence and pathogenicity among \u003cem\u003eM. anisopliae\u003c/em\u003e strains may be attributed to factors such as the geographical origin of the fungi and inherent characteristics of the ticks involved (Fernandez-Salas et al., 2017). The successful development of entomopathogenic fungi as biological control agents significantly depend on the selection of highly efficient isolates, and the fungi must be adapted to the environmental conditions of the area where they are employed (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Other than that, as highlighted by Fernandez-Salas et al. (2017) and Kirkland et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), differences in virulence can be influenced by the strain's ability to penetrate the tick cuticle and evade the host's immune system following penetration. Some strains may possess superior capabilities in these aspects, enabling them to kill ticks swiftly and effectively. This variability underscores the importance of selecting isolates with optimal traits for tick control applications.\u003c/p\u003e \u003cp\u003eThe primary limitation hindering the widespread adoption of entomopathogenic fungi as a replacement for chemical acaricides is their relatively slower killing speed compared to conventional treatments. To address this challenge, our research also focused on identifying the most efficient isolates capable of rapidly eliminating the ticks. Among the six isolates examined, PR1 stood out as the most promising candidate, demonstrating remarkable efficacy in achieving a 50% mortality rate within 10 days and a 90% mortality rate within 15 days. This rapid action distinguishes PR1 as the fastest-acting isolate compared to others, which required longer durations to achieve similar reductions in tick populations. The ability of PR1 to cause 83% tick mortality within 14 days shows the potential to be employed as the biocontrol since the average longevity of engorged female ticks typically ranges from 6 to 19 days with its oviposition started 5 to 8 days after fully engorged and laid maximum up to 1,535 eggs within 11 days (Bandaranayaka et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although 100% mortality was not achieved, PR1 was able to reduce the tick population by reducing eggs being successfully produced by the ticks during their lifetime.\u003c/p\u003e \u003cp\u003eAccording to De Aquino et al. (2020), the ticks being used in this study were classified under partially engorged female ticks, which typically measure between 5\u0026ndash;6mm; whereas fully engorged female ticks are those with a size range from 8\u0026ndash;11mm. Our result demonstrated that tick size significantly influenced the tick mortality rate when being exposed to \u003cem\u003eM. anisopliae\u003c/em\u003e. Although there were studies which reported engorged ticks were more susceptible to different fungal strains and formulations compared to unfed ticks (Tavassoli et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hedimbi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). There were no previous studies examining the effect of size within the engorged ticks on tick mortality. Thus, the specific mechanisms of how larger-size ticks can resist the infection of \u003cem\u003eM. anisopliae\u003c/em\u003e are not known. We hypothesize larger ticks might have better energy storage and innate immunity to fight off the entomopathogenic fungi that are trying to invade the ticks. This hypothesis is supported by a study from Shikano et al. (2014), which states that increased protein intake might indicate compensatory feeding to offset impaired conversion of dietary protein into bodily nitrogen or to meet the higher protein requirements necessary for sustaining an elevated immune response. Additionally, a study by Jensen et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that in cockroaches, highly resistant populations showed a greater dependence on rich nutritional conditions. Nutrition significantly affects insecticide susceptibility, as the availability and quality of nutritional resources impact cuticle composition and the upregulation of detoxifying enzymes. These enzymes are energetically costly to produce and require substantial investments in amino acids and other essential nutrients such as sulfur and iron. Further research is needed to clarify these mechanisms, which could lead to improved strategies for enhancing the efficacy of fungal biocontrol agents against ticks.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the present study revealed that under laboratory conditions all \u003cem\u003eM. anisopliae\u003c/em\u003e isolates from Malaysia are capable of killing the ticks, but the time taken for tick mortality varied among the isolates. Among all, only PR1 can kill more than 80% of tick mortality within 14 days post-inoculation. It is also demonstrated that the tick size influenced the mortality rate caused by the fungus. The estimated LC\u003csub\u003e50\u003c/sub\u003e values for PR1 also showed that it had promising potential as a biocontrol agent of \u003cem\u003eR. microplus\u003c/em\u003e. Therefore, further investigations are necessary to validate the effectiveness of this isolate under field conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis study is supported by funding from the Ministry of Higher Education (MOHE) through the Fundamental Research Grant Scheme (FRGS) \u0026ndash; FRGS/1/2022/WAB04/UMK/02/9.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdullah HHaM, El-Molla A, Salib FA, Allam NaT, Ghazy AA, Abdel-Shafy S (2016) Morphological and molecular identification of the brown dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii (Acari: Ixodidae) vectors of Rickettsioses in Egypt. 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J Sustainable Nat Resour 2(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.30880/jsunr.2021.12.02.001\u003c/span\u003e\u003cspan address=\"10.30880/jsunr.2021.12.02.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adult Immersion Test, Biological control, Cattle ticks, Entomopathogenic fungus, Livestock, Metarhizium anisopliae","lastPublishedDoi":"10.21203/rs.3.rs-4532343/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4532343/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eRhipicephalus microplus\u003c/em\u003e poses a significant challenge to the livestock industry, leading to substantial economic burdens. Traditionally, chemical acaricides have been the primary management strategy; however, their indiscriminate use has led to resistance, environmental contamination, and health risks. Therefore, there is growing interest in exploring alternative approaches, such as entomopathogenic fungi like \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e. This study aimed to evaluate the effectiveness of \u003cem\u003eM. anisopliae\u003c/em\u003e isolates from Malaysia against \u003cem\u003eR. microplus\u003c/em\u003e using the Adult Immersion Test protocol. Engorged female ticks were utilized in the bioassay. The experiment involved applying \u003cem\u003eM. anisopliae\u003c/em\u003e isolates (1518, 1521, 1522, PR1, HSAH5, and GT3) at a concentration of 10\u003csup\u003e8\u003c/sup\u003e through tick immersion. Mortality rates were monitored for 14 days, with experiments conducted in triplicate. Result showed that PR1 exhibited the highest virulence, causing 83.33% mortality within 14 days. There was no significant difference between the isolates in their ability to cause tick mortality. However, probit analysis revealed that PR1 have the shortest LT\u003csub\u003e50\u003c/sub\u003e and LT\u003csub\u003e90\u003c/sub\u003e with 10.03 days and 14.69 days, respectively. Correlation analysis revealed a significant moderate negative correlation between tick size and mortality and not significant between germination rate and tick mortality. These findings emphasize the influence of tick size on tick mortality. Although no isolate achieved 100% mortality, PR1 was notably effective, killing the highest percentage of ticks quickly and significantly reducing egg production compared to the control and other isolates. Overall, this study underscores the potential of Malaysia-isolated \u003cem\u003eM. anisopliae\u003c/em\u003e in the management of adult \u003cem\u003eR. microplus\u003c/em\u003e, offering insights into alternative strategies for pest control in the livestock sector.\u003c/p\u003e","manuscriptTitle":"Virulence Screening of Malaysia-Isolated Metarhizium anisopliae against Rhipicephalus microplus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-03 10:45:28","doi":"10.21203/rs.3.rs-4532343/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-09-20T14:10:31+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-09-02T09:49:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-02T09:47:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-07T02:15:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Tropical Insect Science","date":"2024-06-05T04:05:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dcdfbfc5-5de7-419b-b176-89a5656177ad","owner":[],"postedDate":"October 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:00:58+00:00","versionOfRecord":{"articleIdentity":"rs-4532343","link":"https://doi.org/10.1007/s42690-024-01391-6","journal":{"identity":"international-journal-of-tropical-insect-science","isVorOnly":false,"title":"International Journal of Tropical Insect Science"},"publishedOn":"2024-12-16 15:57:20","publishedOnDateReadable":"December 16th, 2024"},"versionCreatedAt":"2024-10-03 10:45:28","video":"","vorDoi":"10.1007/s42690-024-01391-6","vorDoiUrl":"https://doi.org/10.1007/s42690-024-01391-6","workflowStages":[]},"version":"v1","identity":"rs-4532343","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4532343","identity":"rs-4532343","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}