Optimizing modes of inoculation of Rhipicephalus ticks (Acari: Ixodidae) with a mitosporic entomopathogenic fungus in the laboratory

Abstract

The process of strain selection is an important step in the development of insect pathogens for biological control. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using Rhipicephalus pulchellus Gersta¨cker (Acari: Ixodidae) as a model. Initially, an oil-based formulation of Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales) titred at 109 conidia ml-1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality), although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 9 104 ± 1.1 9 103 conidia ml-1) after spraying by Burgerjon’s spray tower compared to 1 9 106 conidia ml-1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC50 values following contamination of nymphs (LC50 = 1.4 9 107 conidia ml-1) and adults (LC50 = 6.7 9 107 conidia ml-1) in SS were significantly lower compared to SP; nymphs (LC50 = 5.7 9 108 conidia ml-1) and adults (LC50 = 5.3 9 109 conidia ml-1) and SW; nymphs (LC50 = 5 9 108 conidia ml-1). Although the LC50 value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at 2 weeks post-treatment) and (23.3% at 3 weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8%, respectively) and SW (0.0 and 0.0). Results show that among the modes of inoculation tested, SP was the most appropriate for inoculating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil.