Dose-response relationship of Beauveria bassiana (Balsamo) Vuillemin on Rhipicephalus microplus ticks

Dose-response relationship of Beauveria bassiana (Balsamo) Vuillemin on Rhipicephalus microplus ticks | 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 Dose-response relationship of Beauveria bassiana (Balsamo) Vuillemin on Rhipicephalus microplus ticks Ghirmay zeina, Mark Laing This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-832395/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 Background Isolation and optimization are the primary step in the development of fungal pathogens for biological control agents. In this study, three indigenous strains of Beauveria bassiana ( Bb -27, Bb -40 and Bb -41) and one commercial strain (Eco- Bb ) were tested for dose/response relationship of the fungus against Rhipicephalus microplus ticks. Results We used R. microplus larvae of 2 weeks of age, and exposed them to four conidial concentrations: 1x10 3 , 3x10 5 , 4x10 7 , 3.2x10 8 conidia.mL − 1 of each strain. Water was used as a control. Larval mortality was significantly affected by the type of formulation. There was a positive relationship between the concentration of the formulation and mortality of the treated ticks. All the ticks treated with the formulation containing 3.2x10 8 conidia.mL − 1 resulted in high levels of mortality. Conclusions From this study, it can be concluded that, because of the diversity in the performance of B. bassiana strains, optimization of the formulation for different Isolates of B. bassiana can be achieved through scrutinizing the virulence of the Isolates. Parasitology Biological control Beauveria bassiana Rhipicephalus microplus ticks dose virulence Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Background Ticks such as the Southern cattle tick, Rhipicephalus microplus (Ixodida: Amblyommidae) are blood-feeding arthropods that parasitize livestock worldwide (De León et al. 2020 ). They are major livestock parasites and can transmit pathogens that cause diseases of economic importance in cattle (Springer et al. 2020 ; Ghafar et al. 2020 ). Control of ticks is, therefore, a major concern for all livestock farmers. To date, the control of ticks has been largely dependent upon the use of acaricides (Rodriguez-Vivas et al. 2018 ; Nwanade et al. 2020 ). However, due to the emergence of tick populations resistant to the acaricides (Brito et al. 2011 ), and concerns on food and environmental pollutions (Harrison and Wilmshurst 1973; George and Davey 2004; Shanmuganath et al. 2021 ), new approaches are being sought for effective tick control. Recently entomopathogenic fungi, such as Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metchnikoff) Sorokin has been used as an alternative to acaricides. Entomopathogenic fungi are natural enemies of arthropods. These entomopathogenic fungi invade the host by penetrating the outer surface of the host, hence, they not only infect the blood-sucking stage of the tick but also parasitize the non-feeding stages of the arthropod. Besides, the fungi also have a wide host range (De Faria and Wraight 2007 ). The process of infection of the tick by the fungus starts with the formation of germ tubes that penetrate through the outer surface of the tick. However, factors such as the viability of the conidia and virulence factors that are specific to each isolate affect the pathogenicity of the isolate (Soetopo 2004 ). Therefore, isolating and selecting a largely efficacious strain is the first step in the development of a fungal biological control agent (BCA). Once the most virulent strain has been isolated, utilising the optimal mode of inoculation is necessary for successful treatment. Dipping is one of the most commonly used modes of application to control ticks. However, this method has its flaws because: (i) it is expensive because it requires a large number of conidia for the suspension to completely dip the cow in the formulation; and (ii) it is not practical when the pasture also has to be treated against the non-parasitic stage of the tick. One of the efficient and practical application methods to treat for all life cycle stages of the ticks is spraying using ultra-low volume application equipment. Therefore, during laboratory tests, it is important to adopt a suitable method to apply the formulation that can be successfully replicated in the field. To do so, the formulation of the BCA (fungus, water, oil and adjuvant) must be optimised to develop a cost-effective and virulent formulation against the targeted host. Dose-response relationship assessment is one of the effective method used to optimise the formulation. In previous studies, we isolated three strains of B. bassiana with a high degree of virulence against R. microplus . The aim of this study was to conduct several concentration-mortality tests to determine the optimal concentration of conidia of formulated B. bassiana needed to control R. microplus . 2 Methods 2.1 Ethics statement All experiments using live animals were performed in accordance with the University of KwaZul u- Natal guidelines on animal experimentation and were approved by the Committee of Ethics of Animal (REF: AREC/018/017D). Laboratory bioassays were carried out at Biocontrol Research Laboratory, College of Agriculture, Engineering and Science, School of Agricultural, Earth and Environmental Sciences, Pietermaritzburg campus, South Africa. 2.2 Ticks Engorged R. microplus ticks were collected from cattle in Ukulinga Research Farm in 2018. Engorged females were placed into Petri dishes and incubated under laboratory conditions, at 27 ± 1 ◦ C and RH of ≥ 80% to allow oviposition. Eggs were then transferred into plastic cups (30 mm radius, 20mm height) with perforated lids. Live larvae were used for the concentration-mortality virulence assessment. 2.3 Fungal isolates Four strains of B. bassiana were used in the present study. Three strains were locally isolated in our laboratory, whereas the fourth one - Eco- Bb ® - was a commercial strain obtained from Plant Health Products (Pty) Ltd. Fungal isolates were cultured in Potato Dextrose Agar (PDA) medium to provide sufficient conidia for further research. To prepare the PDA medium, 39 g of commercial Potato Dextrose Agar powder (Merck, Darmstadt, Germany) amended with 1 mL of Dodine (Merck, Darmstadt, Germany) and 0.05 g of Chloramphenicol (Merck, Darmstadt, Germany) was added into 1000 mL of distilled water. Then the mixture was boiled while mixing to dissolve completely. To sterilise the media the mixture was autoclaved at 121ºC for 15 minutes. Finally, the solution was aseptically dispensed into sterile Petri dishes. The petri dishes (90-mm) were incubated at 25 ± 2 ◦ C in complete darkness. Conidia were harvested from 3-week-old cultures with a sterile spatula. 2.4 Concentration-mortality virulence bioassays of selected isolates Fungal virulence towards ticks was determined using formulations of varying conidial concentrations. We used R. microplus larvae of 2 weeks of age, and exposed them to four conidial concentrations: 1x10 3 , 3x10 5 , 4x10 7 , 3.2x10 8 conidia.mL − 1 of each strain; distilled water containing 0.05% breackthrou® and 10% avocado oil was used as a control. All the formulations were prepared in sterile distilled water containing 0.05% breackthrou® and 10% avocado oil. Conidial counts were adjusted to the desired concentration after counting the conidia using a haemocytometer (Neubauer-improved: Hirschmann®). Fifteen larvae of R. microplus were placed in plastic cups with perforated lids. Each cup was sprayed using a hand-held, finger-tip sprayer from a 60-mL, travel-size bottle (Sprayco, Livonia, MI, USA) affixed to a 15-mL conical Fisherbrand centrifuge tube. Trials at each concentration were replicated three times. All treatments were then incubated at 27 ± 1 ◦ C and RH ≥ 80%. All larvae were individually examined under a dissecting microscope for the detection of fungal infection. Larvae were considered to be dead from the absence of movement after stimulation, and observation of mycelium emerging from the cuticle. Post-treatment mortality was recorded daily. Dead larvae from all treatment groups were incubated at 25 ± 1◦C and RH ≥ 80% for 20 days to allow fungal growth. 2.5 Statistical Analysis The mortality caused by each treatment was recorded daily for 12 days. Corrected mortality was calculated using the formula developed by Abbott recommended by the FAO (Abbott, 1925 ; FAO, 2004). Then the data were subjected to one way ANOVA. Values of p < 0.05 were considered significant. Where there is an overall statistically significant difference among groups, post hoc analysis (Tukey) was performed to indicate the difference between groups. The LC 50 and LC 90 were determined using probit analysis and the data were subjected to two-way ANOVA. Means were considered to be statistically different at P < 0.05. 3 Results 3.1 Virulence of four strains of Beauveria bassiana against Rhipicephalus microplus larvae The virulence of 4 strains of B. bassiana applied at varying concentration and control groups to larvae of R. microplus are presented in Table 1 . All isolates of B. bassiana were pathogenic to R. microplus at all concentrations but with varying degree of virulence. Generally, the pathogenicity of the fungus for all strains increased as the concentration of the conidia increased (Fig. 1 to 4 ). Those ticks treated with Eco- Bb at the concentration of 10 3 conidia.mL − 1 recorded the lowest mortality. Overall, the mortality of the treated ticks increased 12 days post-treatment. However, treatment with Bb -40 at the concentration of 10 8 conidia.mL − 1 was the best performing formulation 12 days after treatment. Mortality in the control group was less than 10%. Larval mortality was significantly was significantly affected by the formulation at 12 days post-treatment. Twenty days after incubation many of the infected larvae of R. microplus were covered with fungal hyphae. Table 1 Virulence of four strains of Beauveria bassiana against Rhipicephalus microplus larvae Treatment % Mortality (Mean) Control 3.5 a 1x10 3 Eco- Bb 12.33 a 1x10 3 Bb -27 17.67 ab 1x10 3 Bb -40 25.67 ab 3x10 5 Eco- Bb 26.67 abc 3x10 5 Bb -27 39.83 bcd 1x10 3 Bb -41 40.0 bcd 4x10 7 Eco- Bb 41.17 bcd 3x10 5 Bb -41 42.17 bcd 3x10 5 Bb -40 52.17 cde 4x10 7 Bb -27 52.17 cde 3.2x10 8 Bb -27 61.0 def 3.2x10 8 Eco- Bb 63.17 def 4x10 7 Bb -41 64.5 def 4x10 7 Bb -40 74.5 ef 3.2x10 8 Bb -41 78.67 f 3.2x10 8 Bb -40 92.17 g P Value 0.001 F Value 10.59 SE 2.87 DF 16 3.2 Dose-response assessment for four strains of Beauveria bassiana in virulence bioassays against unfed larvae of Rhipicephalus microplus The lethal concentrations, LC 50 and LC 90 are shown in Table 2 . The lethal concentration (LC 50 ) was not significantly affected by isolate at a 95% confidence level. Generally all isolates needed the lowee concentration to kill 50% of the larvae. The results of LC 90 shows that substantially higher concentration of the conidia is required to achieve 90% mortality of R. microplus larvae in the short term. However, it is not clear why there was no significant difference among groups. Table 2 The lethal concentrations of Beaveria bassiana conidia required to cause 50 and 90% mortality (LC 50 and LC 90 ) of unfed larvae of R. microplus 12 days after treatment LC Isolate Mean SD 95% Confidence Interval for Mean P-value Lower Bound Upper Bound LC 50 Bb -27 1.86 x10 7 1.58 x 10 7 1.99 x 10 6 3.52 x 10 7 0.059 Bb -40 4.82 x 10 6 1.17 x 10 7 -7.42 x 10 6 1.71 x 10 7 Bb -41 1.16 x 10 6 1.16 x 10 6 -5.91 x 10 4 2.37 x 10 6 Eco- Bb 1.13 x 10 8 1.51 x 10 8 -4.51 x 10 7 2.71 x 10 8 LC 90 Bb -27 1.48 x 10 17 3.58 x 10 17 -2.28 x 10 17 5.23 x 10 17 0.404 Bb -40 3.24 x 10 9 4.80 x 10 9 -1.81 x 10 9 8.28 x 10 9 Bb -41 2.67 x 10 13 3.81 x 10 13 -1.33 x 10 13 6.67 x 10 13 Eco- Bb 7.12 x 10 13 7.19 x 10 13 -4.24 x 10 12 1.47 x 10 14 4 Discussion Many laboratory studies have been conducted on the use of entomopathogenic fungi for the biological control for livestock ticks (Frazzon et al. 2000 ; Sun et al. 2011 ; Camargo et al. 2012 ; Ma et al. 2013 ). In the current study, we observed that the fungus is highly virulent on unfed larvae of R. microplus but with varying degrees of pathogenicity among the different isolates tested. All isolates caused more than 60% mortality of unfed larvae of R. microplus , 12 days after treatment with 10 8 conidia.mL − 1 . However, no isolate caused mortality greater than 50%, six days after application with the same concentration. This means that all the tested isolates need more time to infect the larvae. Furthermore, the mortality of the larvae increased as the concentration of the conidia in the formulation increased, demonstrating dose-dependent mortality in R. microplus larvae. The question then is whether a higher conidial concentration was required to overcome the tick defence system during germination and proliferation stage, or just a large number of conidia are required to increase the chance of conidia to stick on the cuticle of the tick. The same trend in the dose/response relationship was observed by Frazzon et al. ( 2000 ) who found that all B. bassiana isolates tested were pathogenic to ticks, but the isolates had varying degrees of virulence against R. microplus larvae. Samish et al. ( 2001 ) also observed variation in the pathogenicity of various entomopathogenic fungi to different developmental stages of R. microplus . Under laboratory conditions, the variation in the virulence of the fungus within the species could be explained by the ability of the strain to penetrate the cuticle of the tick. Enzymes such as proteases (Joshi et al. 1995 ) and chitinase (Fang et al., 2005 ) are important virulence factors for B. bassiana . As compared to the mortality observed in our previous study (unpublished), the mortality induced by isolate Bb -27 was low (61 vs 91) for same concentration (10 8 conidia.mL − 1 ) 12 days post-treatment. This discrepancy could have resulted from either culturing conditions or attenuation of virulence due to successive subculturing on artificial media (Butt and Goettel 2000 ) The process of infection started after about four days. But four days post-treatment the mobility of the infected larvae was significantly affected while the non-infected larvae were moving vigorously. Unlike our observation during the isolation of entomopathogenic fungi using Galleria mellonella , the cadavers did not show any change in colour in contrast to the non-infected larvae. But five days after further incubation, some of the cadavers start to be covered by white hyphae. However, even after incubation for 20 days, not all larvae were covered by the hyphae. In contrast, Samish et al. ( 2001 ) reported that two days post-treatment the colour of the larvae darkened, followed by immobilisation of the treated ticks indicating an infection in larvae. After incubation for 72 hours, sporulation started to appear on the surface of the larvae. In another study, Abdigoudarzi et al. ( 2009 ) reported that sporulation from the tick surface was not observed. In our studies case, since some of the cadavers showed sporulation in their surface, Secondary epizootics or horizontal infection can increase the overall infection level of a tick population by the entomopathogenic fungi. Klinger et al. ( 2006 ), determined the likelihood of secondary infection of adult Colorado potato beetles ( Leptinotarsa decemlineata ) (Coleoptera: Chrysomelidae) in the presence of Beauveria bassiana sporulating on cadavers. Their results indicated that significant likelihood of horizontal infection of adults (30–70% of horizontal infection) from the cadavers with the presence of sporulation. 5 Conclusions Given the diversity in the performance of B. bassiana strains, a selection of virulent isolates is crucial for the development of efficacious bio-acaricides. The present study also demonstrated a positive dose/response relationship. Further studies are required to validate whether there is indeed a horizontal infection between cadavers and new uninfected tick populations. Additionally, field trials are required to confirm these results under field conditions. 6 List Of Abbreviations B. bassiana Beauveria bassiana L Liter mL Milliliter RH Relative Humidity g Gram mg Milligram R. microplus Rhipicephalus microplus Declarations We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal Ethical approval and consent to participate: All experiments using live animals were performed following the University of KwaZulu-Natal guidelines on animal experimentation and were approved by the Committee of Ethics of Animal (REF: AREC/018/017D). Consent for Publication: All authors have approved the manuscript and agree with its submission to Egyptian Journal of Biological Pest Control. Availability of data and material : Available upon request Competing interests: There was no conflict of interest. Funding: Not Applicable Authors Contribution: Prof Laing Conceived the research and reviewed the manuscript. Dr. Zeina designed the analysis; Collected the data; Performed the analysis and wrote the paper. Acknowledgements : The authors acknowledge the assistance of Luwam Weldegabr Ziena during the lab work References Abbott WS (1925) A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18(2), 265-267 Abdigoudarzi M, Esmaeilnia K, Shariat N (2009) Laboratory study on biological control of ticks (Acari: Ixodidae) by entomopathogenic indigenous fungi ( Beauveria bassiana ). 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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-832395","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":47379824,"identity":"47944c02-847b-4599-94f0-e38bfa8a66bc","order_by":0,"name":"Ghirmay zeina","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYDACCcYGEGXHz8x8gAHCJlJLsmQ7WwKxWiAU44bzPAbEaZGPbm77dKPmMDPDYZ5vEj932MgxsB8+ugGfFsM7B5tn5xw7zMfYzLtNsvdMmjEDT1raDbxaZiQ2M+ewHWZmZubdJsHbdjixQYLHjAgt/w4ztjHzPJP8S4wWeQmglty2w4w9zDxs0kTZYgDW0peeLMHMZmwt25ZmzEbIL/Iz0h8z53yztrM/f/jhzbdtNnL87IeP4bflAJhqBhEs4Dhiw6ccbEsDmKoDEcwfCKkeBaNgFIyCkQkAY11JbPONVaMAAAAASUVORK5CYII=","orcid":"","institution":"University of KwaZulu-Natal - Pietermaritzburg Campus","correspondingAuthor":true,"prefix":"","firstName":"Ghirmay","middleName":"","lastName":"zeina","suffix":""},{"id":47379825,"identity":"58dce4bd-c01f-4110-a0f8-bd351cbac79b","order_by":1,"name":"Mark Laing","email":"","orcid":"","institution":"University of KwaZulu-Natal - Pietermaritzburg Campus","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Laing","suffix":""}],"badges":[],"createdAt":"2021-08-21 10:27:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-832395/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-832395/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12722523,"identity":"242554ba-76ad-4b33-b29e-5b16a93d1d14","added_by":"auto","created_at":"2021-08-24 16:31:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92898,"visible":true,"origin":"","legend":"Mortality of R. microplus larvae treated with formulations of four strains of Beauveria bassiana: Bb-27 (From Day 1 to day 12)","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-832395/v1/ddd32cf28d6eac17d5f3fe5d.png"},{"id":12722522,"identity":"116092ef-be3b-4aa4-8897-6d44ebc7ca5d","added_by":"auto","created_at":"2021-08-24 16:31:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39978,"visible":true,"origin":"","legend":"Mortality of R. microplus larvae treated with formulations of four strains of Beauveria bassiana: Bb-40 (From Day 1 to day 12)","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-832395/v1/89fa7e277fcdba0722493b30.png"},{"id":12722525,"identity":"2b669f83-153f-4341-af7c-a9eaf6246f77","added_by":"auto","created_at":"2021-08-24 16:31:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98657,"visible":true,"origin":"","legend":"Mortality of R. microplus larvae treated with formulations of four strains of Beauveria bassiana: Bb-41 (From Day 1 to day 12)","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-832395/v1/eea02a7cd978d27a59093a1a.png"},{"id":12722524,"identity":"44410098-3969-4895-9742-8ce3907cfadf","added_by":"auto","created_at":"2021-08-24 16:31:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77623,"visible":true,"origin":"","legend":"Mortality of R. microplus larvae treated with formulations of four strains of Beauveria bassiana: Eco-Bb (From Day 1 to day 12)","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-832395/v1/a68a88ab7e690a6889ddb9f6.png"},{"id":15674774,"identity":"d409a870-e28e-4923-9fcb-539ad95dbf19","added_by":"auto","created_at":"2021-11-18 14:24:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":597287,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-832395/v1/5c00db43-1b89-4ce6-9350-f107e50dab7c.pdf"},{"id":12722526,"identity":"b26c1fdd-114e-47c0-85ea-b3e74110a6c4","added_by":"auto","created_at":"2021-08-24 16:31:42","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":845986,"visible":true,"origin":"","legend":"","description":"","filename":"AdjuvantAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-832395/v1/b95a9b3c496ef26d686b48ae.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDose-response relationship of \u003cem\u003eBeauveria bassiana \u003c/em\u003e(Balsamo) Vuillemin on \u003cem\u003eRhipicephalus microplus \u003c/em\u003eticks\u003c/p\u003e","fulltext":[{"header":"1 Background","content":"\u003cp\u003eTicks such as the Southern cattle tick, \u003cem\u003eRhipicephalus microplus\u003c/em\u003e (Ixodida: Amblyommidae) are blood-feeding arthropods that parasitize livestock worldwide (De Le\u0026oacute;n et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They are major livestock parasites and can transmit pathogens that cause diseases of economic importance in cattle (Springer et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ghafar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Control of ticks is, therefore, a major concern for all livestock farmers. To date, the control of ticks has been largely dependent upon the use of acaricides (Rodriguez-Vivas et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nwanade et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, due to the emergence of tick populations resistant to the acaricides (Brito et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and concerns on food and environmental pollutions (Harrison and Wilmshurst 1973; George and Davey 2004; Shanmuganath et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), new approaches are being sought for effective tick control.\u003c/p\u003e \u003cp\u003eRecently entomopathogenic fungi, such as \u003cem\u003eBeauveria bassiana\u003c/em\u003e (Balsamo) Vuillemin and \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (Metchnikoff) Sorokin has been used as an alternative to acaricides. Entomopathogenic fungi are natural enemies of arthropods. These entomopathogenic fungi invade the host by penetrating the outer surface of the host, hence, they not only infect the blood-sucking stage of the tick but also parasitize the non-feeding stages of the arthropod. Besides, the fungi also have a wide host range (De Faria and Wraight \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The process of infection of the tick by the fungus starts with the formation of germ tubes that penetrate through the outer surface of the tick. However, factors such as the viability of the conidia and virulence factors that are specific to each isolate affect the pathogenicity of the isolate (Soetopo \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Therefore, isolating and selecting a largely efficacious strain is the first step in the development of a fungal biological control agent (BCA).\u003c/p\u003e \u003cp\u003eOnce the most virulent strain has been isolated, utilising the optimal mode of inoculation is necessary for successful treatment. Dipping is one of the most commonly used modes of application to control ticks. However, this method has its flaws because: (i) it is expensive because it requires a large number of conidia for the suspension to completely dip the cow in the formulation; and (ii) it is not practical when the pasture also has to be treated against the non-parasitic stage of the tick. One of the efficient and practical application methods to treat for all life cycle stages of the ticks is spraying using ultra-low volume application equipment. Therefore, during laboratory tests, it is important to adopt a suitable method to apply the formulation that can be successfully replicated in the field. To do so, the formulation of the BCA (fungus, water, oil and adjuvant) must be optimised to develop a cost-effective and virulent formulation against the targeted host. Dose-response relationship assessment is one of the effective method used to optimise the formulation. In previous studies, we isolated three strains of \u003cem\u003eB. bassiana\u003c/em\u003e with a high degree of virulence against \u003cem\u003eR. microplus\u003c/em\u003e. The aim of this study was to conduct several concentration-mortality tests to determine the optimal concentration of conidia of formulated \u003cem\u003eB. bassiana\u003c/em\u003e needed to control \u003cem\u003eR. microplus\u003c/em\u003e.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethics statement\u003c/h2\u003e \u003cp\u003eAll experiments using live animals were performed in accordance with the University of KwaZul\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eu-\u003c/span\u003eNatal guidelines on animal experimentation and were approved by the Committee of Ethics of Animal (REF: AREC/018/017D). Laboratory bioassays were carried out at Biocontrol Research Laboratory, College of Agriculture, Engineering and Science, School of Agricultural, Earth and Environmental Sciences, Pietermaritzburg campus, South Africa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Ticks\u003c/h2\u003e \u003cp\u003eEngorged \u003cem\u003eR. microplus\u003c/em\u003e ticks were collected from cattle in Ukulinga Research Farm in 2018. Engorged females were placed into Petri dishes and incubated under laboratory conditions, at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u003csup\u003e◦\u003c/sup\u003eC and RH of \u0026ge;\u0026thinsp;80% to allow oviposition. Eggs were then transferred into plastic cups (30 mm radius, 20mm height) with perforated lids. Live larvae were used for the concentration-mortality virulence assessment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fungal isolates\u003c/h2\u003e \u003cp\u003eFour strains of \u003cem\u003eB. bassiana\u003c/em\u003e were used in the present study. Three strains were locally isolated in our laboratory, whereas the fourth one - Eco-\u003cem\u003eBb\u003c/em\u003e\u0026reg; - was a commercial strain obtained from Plant Health Products (Pty) Ltd. Fungal isolates were cultured in Potato Dextrose Agar (PDA) medium to provide sufficient conidia for further research. To prepare the PDA medium, 39 g of commercial Potato Dextrose Agar powder (Merck, Darmstadt, Germany) amended with 1 mL of Dodine (Merck, Darmstadt, Germany) and 0.05 g of Chloramphenicol (Merck, Darmstadt, Germany) was added into 1000 mL of distilled water. Then the mixture was boiled while mixing to dissolve completely. To sterilise the media the mixture was autoclaved at 121\u0026ordm;C for 15 minutes. Finally, the solution was aseptically dispensed into sterile Petri dishes. The petri dishes (90-mm) were incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003e◦\u003c/sup\u003eC in complete darkness. Conidia were harvested from 3-week-old cultures with a sterile spatula.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Concentration-mortality virulence bioassays of selected isolates\u003c/h2\u003e \u003cp\u003eFungal virulence towards ticks was determined using formulations of varying conidial concentrations. We used \u003cem\u003eR. microplus\u003c/em\u003e larvae of 2 weeks of age, and exposed them to four conidial concentrations: 1x10\u003csup\u003e3\u003c/sup\u003e, 3x10\u003csup\u003e5\u003c/sup\u003e, 4x10\u003csup\u003e7\u003c/sup\u003e, 3.2x10\u003csup\u003e8\u003c/sup\u003e conidia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of each strain; distilled water containing 0.05% breackthrou\u0026reg; and 10% avocado oil was used as a control. All the formulations were prepared in sterile distilled water containing 0.05% breackthrou\u0026reg; and 10% avocado oil. Conidial counts were adjusted to the desired concentration after counting the conidia using a haemocytometer (Neubauer-improved: Hirschmann\u0026reg;).\u003c/p\u003e \u003cp\u003eFifteen larvae of \u003cem\u003eR. microplus\u003c/em\u003e were placed in plastic cups with perforated lids. Each cup was sprayed using a hand-held, finger-tip sprayer from a 60-mL, travel-size bottle (Sprayco, Livonia, MI, USA) affixed to a 15-mL conical Fisherbrand centrifuge tube. Trials at each concentration were replicated three times. All treatments were then incubated at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003e◦\u003c/sup\u003eC and RH\u0026thinsp;\u0026ge;\u0026thinsp;80%. All larvae were individually examined under a dissecting microscope for the detection of fungal infection. Larvae were considered to be dead from the absence of movement after stimulation, and observation of mycelium emerging from the cuticle. Post-treatment mortality was recorded daily. Dead larvae from all treatment groups were incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1◦C and RH\u0026thinsp;\u0026ge;\u0026thinsp;80% for 20 days to allow fungal growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe mortality caused by each treatment was recorded daily for 12 days. Corrected mortality was calculated using the formula developed by Abbott recommended by the FAO (Abbott, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1925\u003c/span\u003e; FAO, 2004). Then the data were subjected to one way ANOVA. Values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. Where there is an overall statistically significant difference among groups, post hoc analysis (Tukey) was performed to indicate the difference between groups. The LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e were determined using probit analysis and the data were subjected to two-way ANOVA. Means were considered to be statistically different at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Virulence of four strains of \u003cem\u003eBeauveria bassiana\u003c/em\u003e against \u003cem\u003eRhipicephalus microplus\u003c/em\u003e larvae\u003c/h2\u003e \u003cp\u003eThe virulence of 4 strains of \u003cem\u003eB. bassiana\u003c/em\u003e applied at varying concentration and control groups to larvae of \u003cem\u003eR. microplus\u003c/em\u003e are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All isolates of \u003cem\u003eB. bassiana\u003c/em\u003e were pathogenic to \u003cem\u003eR. microplus\u003c/em\u003e at all concentrations but with varying degree of virulence. Generally, the pathogenicity of the fungus for all strains increased as the concentration of the conidia increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e to \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Those ticks treated with Eco-\u003cem\u003eBb\u003c/em\u003e at the concentration of 10\u003csup\u003e3\u003c/sup\u003e conidia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e recorded the lowest mortality. Overall, the mortality of the treated ticks increased 12 days post-treatment. However, treatment with \u003cem\u003eBb\u003c/em\u003e-40 at the concentration of 10\u003csup\u003e8\u003c/sup\u003e conidia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was the best performing formulation 12 days after treatment. Mortality in the control group was less than 10%. Larval mortality was significantly was significantly affected by the formulation at 12 days post-treatment. Twenty days after incubation many of the infected larvae of \u003cem\u003eR. microplus\u003c/em\u003e were covered with fungal hyphae.\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\u003eVirulence of four strains of \u003cem\u003eBeauveria bassiana\u003c/em\u003e against \u003cem\u003eRhipicephalus microplus\u003c/em\u003e larvae\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% Mortality (Mean)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1x10\u003csup\u003e3\u003c/sup\u003e Eco-\u003cem\u003eBb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1x10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1x10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3x10\u003csup\u003e5\u003c/sup\u003e Eco-\u003cem\u003eBb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.67\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3x10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.83\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1x10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.0\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4x10\u003csup\u003e7\u003c/sup\u003e Eco-\u003cem\u003eBb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.17\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3x10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.17\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3x10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.17\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4x10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.17\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.2x10\u003csup\u003e8\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.0\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.2x10\u003csup\u003e8\u003c/sup\u003e Eco-\u003cem\u003eBb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.17\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4x10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.5\u003csup\u003edef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4x10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74.5\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.2x10\u003csup\u003e8\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.67\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.2x10\u003csup\u003e8\u003c/sup\u003e \u003cem\u003eBb\u003c/em\u003e-40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.17\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\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\u003e3.2 Dose-response assessment for four strains of \u003cem\u003eBeauveria bassiana\u003c/em\u003e in virulence bioassays against unfed larvae of \u003cem\u003eRhipicephalus microplus\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe lethal concentrations, LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) was not significantly affected by isolate at a 95% confidence level. Generally all isolates needed the lowee concentration to kill 50% of the larvae. The results of LC\u003csub\u003e90\u003c/sub\u003e shows that substantially higher concentration of the conidia is required to achieve 90% mortality of \u003cem\u003eR. microplus\u003c/em\u003e larvae in the short term. However, it is not clear why there was no significant difference among groups.\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\u003eThe lethal concentrations of \u003cem\u003eBeaveria bassiana\u003c/em\u003e conidia required to cause 50 and 90% mortality (LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e) of unfed larvae of \u003cem\u003eR. microplus\u003c/em\u003e 12 days after treatment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e95% Confidence Interval for Mean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLower Bound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUpper Bound\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBb\u003c/em\u003e-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.86 x10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.58 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.99 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.52 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.059\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBb\u003c/em\u003e-40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.82 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.17 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.42 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.71 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBb\u003c/em\u003e-41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.16 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.16 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5.91 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.37 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEco-\u003cem\u003eBb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.13 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.51 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.51 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.71 x 10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBb\u003c/em\u003e-27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.48 x 10\u003csup\u003e17\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.58 x 10\u003csup\u003e17\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.28 x 10\u003csup\u003e17\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.23 x 10\u003csup\u003e17\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.404\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBb\u003c/em\u003e-40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.24 x 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.80 x 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.81 x 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.28 x 10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBb\u003c/em\u003e-41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.67 x 10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.81 x 10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.33 x 10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.67 x 10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEco-\u003cem\u003eBb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.12 x 10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.19 x 10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.24 x 10\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.47 x 10\u003csup\u003e14\u003c/sup\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 \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eMany laboratory studies have been conducted on the use of entomopathogenic fungi for the biological control for livestock ticks (Frazzon et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Camargo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the current study, we observed that the fungus is highly virulent on unfed larvae of \u003cem\u003eR. microplus\u003c/em\u003e but with varying degrees of pathogenicity among the different isolates tested. All isolates caused more than 60% mortality of unfed larvae of \u003cem\u003eR. microplus\u003c/em\u003e, 12 days after treatment with 10\u003csup\u003e8\u003c/sup\u003e conidia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, no isolate caused mortality greater than 50%, six days after application with the same concentration. This means that all the tested isolates need more time to infect the larvae. Furthermore, the mortality of the larvae increased as the concentration of the conidia in the formulation increased, demonstrating dose-dependent mortality in \u003cem\u003eR. microplus\u003c/em\u003e larvae. The question then is whether a higher conidial concentration was required to overcome the tick defence system during germination and proliferation stage, or just a large number of conidia are required to increase the chance of conidia to stick on the cuticle of the tick. The same trend in the dose/response relationship was observed by Frazzon et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) who found that all \u003cem\u003eB. bassiana\u003c/em\u003e isolates tested were pathogenic to ticks, but the isolates had varying degrees of virulence against \u003cem\u003eR. microplus\u003c/em\u003e larvae. Samish et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) also observed variation in the pathogenicity of various entomopathogenic fungi to different developmental stages of \u003cem\u003eR. microplus\u003c/em\u003e. Under laboratory conditions, the variation in the virulence of the fungus within the species could be explained by the ability of the strain to penetrate the cuticle of the tick. Enzymes such as proteases (Joshi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and chitinase (Fang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) are important virulence factors for \u003cem\u003eB. bassiana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAs compared to the mortality observed in our previous study (unpublished), the mortality induced by isolate \u003cem\u003eBb\u003c/em\u003e-27 was low (61 vs 91) for same concentration (10\u003csup\u003e8\u003c/sup\u003e conidia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 12 days post-treatment. This discrepancy could have resulted from either culturing conditions or attenuation of virulence due to successive subculturing on artificial media (Butt and Goettel \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe process of infection started after about four days. But four days post-treatment the mobility of the infected larvae was significantly affected while the non-infected larvae were moving vigorously. Unlike our observation during the isolation of entomopathogenic fungi using \u003cem\u003eGalleria mellonella\u003c/em\u003e, the cadavers did not show any change in colour in contrast to the non-infected larvae. But five days after further incubation, some of the cadavers start to be covered by white hyphae. However, even after incubation for 20 days, not all larvae were covered by the hyphae. In contrast, Samish et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) reported that two days post-treatment the colour of the larvae darkened, followed by immobilisation of the treated ticks indicating an infection in larvae. After incubation for 72 hours, sporulation started to appear on the surface of the larvae. In another study, Abdigoudarzi et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported that sporulation from the tick surface was not observed. In our studies case, since some of the cadavers showed sporulation in their surface, Secondary epizootics or horizontal infection can increase the overall infection level of a tick population by the entomopathogenic fungi. Klinger et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), determined the likelihood of secondary infection of adult Colorado potato beetles (\u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e) (Coleoptera: Chrysomelidae) in the presence of \u003cem\u003eBeauveria bassiana\u003c/em\u003e sporulating on cadavers. Their results indicated that significant likelihood of horizontal infection of adults (30\u0026ndash;70% of horizontal infection) from the cadavers with the presence of sporulation.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eGiven the diversity in the performance of \u003cem\u003eB. bassiana\u003c/em\u003e strains, a selection of virulent isolates is crucial for the development of efficacious bio-acaricides. The present study also demonstrated a positive dose/response relationship. Further studies are required to validate whether there is indeed a horizontal infection between cadavers and new uninfected tick populations. Additionally, field trials are required to confirm these results under field conditions.\u003c/p\u003e "},{"header":"6 List Of Abbreviations ","content":"\u003cp\u003e\u003cem\u003eB. bassiana \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Beauveria bassiana\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Liter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Milliliter\u003c/p\u003e\n\u003cp\u003eRH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Relative Humidity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eg \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gram\u003c/p\u003e\n\u003cp\u003emg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Milligram\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eR. microplus \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Rhipicephalus microplus\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eWe confirm that this manuscript has not been published elsewhere and is not under consideration by another journal\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate:\u003c/strong\u003e All experiments using live animals were performed following the University of KwaZulu-Natal guidelines on animal experimentation and were approved by the Committee of Ethics of Animal (REF: AREC/018/017D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e All authors have approved the manuscript and agree with its submission to Egyptian Journal of Biological Pest Control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: \u0026nbsp; Available upon request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e There was no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Not Applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution:\u003c/strong\u003e Prof Laing Conceived the research and reviewed the manuscript. Dr. Zeina designed the analysis; Collected the data; Performed the analysis and wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors acknowledge the assistance of Luwam Weldegabr Ziena during the lab work\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAbbott WS (1925) A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18(2), 265-267\u003c/p\u003e\n\u003cp\u003eAbdigoudarzi M, Esmaeilnia K, Shariat N (2009) Laboratory study on biological control of ticks (Acari: Ixodidae) by entomopathogenic indigenous fungi (\u003cem\u003eBeauveria bassiana\u003c/em\u003e). 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Veterinary Parasitology 188(1\u0026ndash;2), 140-147\u003c/p\u003e\n\u003cp\u003eDe Faria MR, Wraight SP (2007) Mycoinsecticides and mycoacaricides: a comprehensive list with worldwide coverage and international classification of formulation types. Biological Control 43(3), 237-256\u003c/p\u003e\n\u003cp\u003eDe Le\u0026oacute;n AAP, Mitchell III RD, Watson DW (2020). Ectoparasites of cattle. Veterinary Clinics of North America: Food Animal Practice, 36(1), 173-185.\u003c/p\u003e\n\u003cp\u003eFang W, Leng B, Xiao Y, Jin K, Ma J, Fan Y, Pei Y (2005) Cloning of \u003cem\u003eBeauveria bassiana\u003c/em\u003e chitinase gene Bbchit1 and its application to improve fungal strain virulence. Applied and Environmental Microbiology 71(1), 363-370\u003c/p\u003e\n\u003cp\u003eFood and Agriculture Organization of the United Nations (FAO) (2004) Resistance management and integrated parasites control in ruminants/ guidelines. In: Module 1-Ticks: Acaricide Resistance, Diagnosis, Management and Prevention. Animal Production and Health, Division Food and Agriculture Organization, Rome, pp. 25\u0026ndash;77\u003c/p\u003e\n\u003cp\u003eFrazzon APG, Junior IDSV, Masuda A, Schrank A, Vainstein MH (2000) \u003cem\u003eIn vitro\u003c/em\u003e assessment of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e isolates to control the cattle tick \u003cem\u003eBoophilus microplus\u003c/em\u003e. Veterinary Parasitology 94(1-2), 117-125\u003c/p\u003e\n\u003cp\u003eGeorge JE, Pound JM, Davey RB (2004) Chemical control of ticks on cattle and the resistance of these parasites to acaricides. Parasitology 129(S1) S353-S366\u003c/p\u003e\n\u003cp\u003eGhafar A, Cabezas-Cruz A, Galon C, Obregon D, Gasser RB, Moutailler S, Jabbar A (2020). Bovine ticks harbour a diverse array of microorganisms in Pakistan. Parasites \u0026amp; vectors, 13(1), 1-15.\u003c/p\u003e\n\u003cp\u003eHarrison IR, Palmer BH, Wilmshurst, EC (1973) Chemical control of cattle ticks\u0026ndash;resistance problems. Pest Management Science 4(4), 531-542\u003c/p\u003e\n\u003cp\u003eJoshi L, St. Leger RJ, Bidochka MJ (1995) Cloning of a cuticle-degrading protease from the entomopathogenic fungus, \u003cem\u003eBeauveria bassiana\u003c/em\u003e. FEMS Microbiology Letters 125(2-3), 211-217\u003c/p\u003e\n\u003cp\u003eKirkland BH, Cho, EM, Keyhani NO (2004) Differential susceptibility of \u003cem\u003eAmblyomma maculatum\u003c/em\u003e and \u003cem\u003eAmblyomma americanum\u003c/em\u003e (Acari: Ixodidea) to the entomopathogenic fungi \u003cem\u003eBeauveria bassiana\u003c/em\u003e and \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e. Biological Control 31(3), 414-421\u003c/p\u003e\n\u003cp\u003eKlinger E, Groden E, Drummond F (2006) \u003cem\u003eBeauveria bassiana\u003c/em\u003e horizontal infection between cadavers and adults of the Colorado potato beetle, \u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e (Say). Environmental Entomology 35(4), 992-1000\u003c/p\u003e\n\u003cp\u003eLee SJ, Lee MR, Kim S, Kim JC, Park SE, Li D, Kim JS (2018) Genomic Analysis of the insect-killing fungus \u003cem\u003eBeauveria bassiana\u003c/em\u003e JEF-007 as a biopesticide. Scientific Reports 8(1), 12388\u003c/p\u003e\n\u003cp\u003eMa M, Guan G, Liu Q, Dang Z, Liu A, Ren Q, Yang J (2013) The \u003cem\u003ein vitro\u003c/em\u003e efficacy of deltamethrin and alpha-cypermethrin against engorged female \u003cem\u003eHaemaphysalis qinghaiensis\u003c/em\u003e ticks (Acari: Ixodidae). Experimental Parasitology 134(4), 405-408\u003c/p\u003e\n\u003cp\u003eNwanade CF, Wang M, Wang T, Yu Z, Liu J (2020). Botanical acaricides and repellents in tick control: current status and future directions. Experimental and Applied Acarology, 81(1), 1-35.\u003c/p\u003e\n\u003cp\u003eRodriguez-Vivas RI, Jonsson NN, Bhushan C (2018). Strategies for the control of Rhipicephalus microplus ticks in a world of conventional acaricide and macrocyclic lactone resistance. Parasitology research, 117(1), 3-29.\u003c/p\u003e\n\u003cp\u003eSamish M, Gindin G, Alekseev E, Glazer I (2001) Pathogenicity of entomopathogenic fungi to different developmental stages of \u003cem\u003eRhipicephalus sanguineus\u003c/em\u003e (Acari: Ixodidae). Journal of Parasitology 87(6), 1355-1359\u003c/p\u003e\n\u003cp\u003eShanmuganath C, Kumar S, Singh R, Sharma AK, Saminathan M, Saini M, Ghosh S (2021). Development of an efficient antitick natural formulation for the control of acaricide-resistant ticks on livestock. Ticks and Tick-borne Diseases, 12(3), 101655.\u003c/p\u003e\n\u003cp\u003eSoetopo D (2004) Efficacy of selected \u003cem\u003eBeauveria bassiana\u003c/em\u003e (Bals.) Vuill. isolates in combination with a resistant cotton variety (PSB-Ct 9) against the cotton bollworm, \u003cem\u003eHelicoverpa armigera\u003c/em\u003e (H\u0026uuml;bner) (Lepidoptera: Noctuidae). (Dissertation). Philippines: University of The Philippines Los Banos\u003c/p\u003e\n\u003cp\u003eSpringer A, Shuaib YA, Isaa MH, Ezz-Eldin MIE, Osman AY, Yagoub IA, Chitimia-Dobler L (2020). Tick fauna and associated Rickettsia, Theileria, and Babesia spp. in domestic animals in Sudan (north Kordofan and Kassala states). Microorganisms, 8(12), 1969.\u003c/p\u003e\n\u003cp\u003eSun M, Ren Q, Guan G, Liu Z, Ma M, Gou H, Yang J (2011) Virulence of \u003cem\u003eBeauveria bassiana\u003c/em\u003e, \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e and \u003cem\u003ePaecilomyces lilacinus\u003c/em\u003e to the engorged female \u003cem\u003eHyalomma anatolicum\u003c/em\u003e \u003cem\u003eanatolicum\u003c/em\u003e tick (Acari: Ixodidae). Veterinary Parasitology 180(3-4), 389-393\u003c/p\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, Beauveria bassiana, Rhipicephalus microplus, ticks, dose, virulence ","lastPublishedDoi":"10.21203/rs.3.rs-832395/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-832395/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIsolation and optimization are the primary step in the development of fungal pathogens for biological control agents. In this study, three indigenous strains of \u003cem\u003eBeauveria bassiana\u003c/em\u003e (\u003cem\u003eBb\u003c/em\u003e-27, \u003cem\u003eBb\u003c/em\u003e-40 and \u003cem\u003eBb\u003c/em\u003e-41) and one commercial strain (Eco-\u003cem\u003eBb\u003c/em\u003e) were tested for dose/response relationship of the fungus against \u003cem\u003eRhipicephalus microplus\u003c/em\u003e ticks.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe used \u003cem\u003eR. microplus\u003c/em\u003e larvae of 2 weeks of age, and exposed them to four conidial concentrations: 1x10\u003csup\u003e3\u003c/sup\u003e, 3x10\u003csup\u003e5\u003c/sup\u003e, 4x10\u003csup\u003e7\u003c/sup\u003e, 3.2x10\u003csup\u003e8\u003c/sup\u003e conidia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of each strain. Water was used as a control. Larval mortality was significantly affected by the type of formulation. There was a positive relationship between the concentration of the formulation and mortality of the treated ticks. All the ticks treated with the formulation containing 3.2x10\u003csup\u003e8\u003c/sup\u003e conidia.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e resulted in high levels of mortality.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eFrom this study, it can be concluded that, because of the diversity in the performance of \u003cem\u003eB. bassiana\u003c/em\u003e strains, optimization of the formulation for different Isolates of \u003cem\u003eB. bassiana\u003c/em\u003e can be achieved through scrutinizing the virulence of the Isolates.\u003c/p\u003e","manuscriptTitle":"Dose-response relationship of Beauveria bassiana (Balsamo) Vuillemin on Rhipicephalus microplus ticks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-24 16:31:40","doi":"10.21203/rs.3.rs-832395/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":"95469c58-7af3-4855-a6ff-dcc450df1199","owner":[],"postedDate":"August 24th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":6659118,"name":"Parasitology"}],"tags":[],"updatedAt":"2021-08-25T00:56:47+00:00","versionOfRecord":[],"versionCreatedAt":"2021-08-24 16:31:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-832395","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-832395","identity":"rs-832395","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}