Cuticular Lipids as a First Barrier Defending Ixodid Ticks against Fungal Infection.

The chemical composition of tick cuticles acts as a barrier to pathogens and may limit infection by entomopathogenic fungi. This study characterized the cuticular neutral lipids (NL) and hydrocarbons (HCs) of four ixodid ticks that are widely distributed in Brazil. HC extracts were analyzed by gas chromatography-mass spectrometry and used to challenge Beauveria bassiana IP361 and Metarhizium robertsii IP146; the effect of cuticular extracts in fungal growth were evaluated by disk diffusion and conidial viability assays. In addition, conidial germination on the tick cuticle was evaluated by scanning electron microscopy, and NL from ticks treated with fungi were assessed by thin layer chromatography. Six HCs were exclusively identified in Amblyomma sculptum. Additionally, cuticle extracts from Dermacentor nitens and A. sculptum inhibited the growth of M. robertsii IP146 and reduced conidial germination of B. bassiana IP361 to 70% and 49%, respectively; the same extracts also produced cytotoxic effects, with conidial death above 30% and 60%. Electron micrographs showed a delayed germination of conidia incubated for 48 h or 72 h on D. nitens and A. sculptum. The lipid profile of A. sculptum treated with fungi was not significantly altered; triacylglycerol was not detected in the cuticle extracts of any other tick species. Finally, A. sculptum and D. nitens cuticles have lipid components that may limit the development of M. robertsii.

The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria bassiana.

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress-response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress-response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout (ΔBbmsn2) and complemented mutant (ΔBbmsn2/Bbmsn2) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and ΔBbmsn2 treatments, whereas treatment with the wild-type or ΔBbmsn2/Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with ΔBbmsn2 was lower than in the groups treated with wild-type (56.1%) or ΔBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or ΔBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of ΔBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with ΔBbmsn2 than those treated with the wild-type or ΔBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.

Carnauba wax enhances the insecticidal activity of entomopathogenic fungi against the blowfly Lucilia sericata (Diptera: Calliphoridae).

Blowfly, Lucilia sericata (Diptera: Calliphoridae), is a problematic synanthropic insect pest, a vector of microbial pathogens, and the causal agent of secondary myiasis. Fungal biopesticides are considered eco-friendly tools, alternative to synthetic pesticides, for the control of arthropod pests; however, to date, little is known about their bioactivity against blowflies. In this study, we assessed the insecticidal activity of three well-known entomopathogenic fungi, Beauveria bassiana, Beauveria pseudobassiana and Akanthomyces muscarius against L. sericata. In addition, we tested powdered carnauba wax as an electrically charged dust carrier in an attempt to enhance the virulence of fungal spores. Pathogenicity tests on adult flies, by adult immersion in conidial suspension (108 conidia mL-1), showed that the median lethal time (LT50) was 5.3, 5.9, and 6.2 days for B. bassiana, A. muscarius and B. pseudobassiana, respectively. In topical tests, when 108 dry conidia were mixed with or without carnauba wax, the LT50 was 7.7, 10.2, and 14 days without this carrier and 6.9, 8.6, and 13.8 days with it for B. bassiana, B. pseudobassiana and A. muscarius, respectively. Overall, our findings showed that, among the tested fungi, B. bassiana was the most virulent when formulated as a dry powder with carnauba wax, which greatly improved fungal efficacy against the blowfly. We discuss the utility of carnauba wax for electrostatic formulation powder of fungal spores in the integrated management of blowflies as an environmentally sustainable tool to reduce the over-reliance on chemical insecticides and their risk of resistance.

Impact of short-term temperature challenges on the larvicidal activities of the entomopathogenic watermold Leptolegnia chapmanii against Aedes aegypti, and development on infected dead larvae.

The oomycete Leptolegnia chapmanii is among the most promising entomopathogens for biological control of Aedes aegypti. This mosquito vector breeds in small water collections, where this aquatic watermold pathogen can face short-term scenarios of challenging high or low temperatures during changing ambient conditions, but it is yet not well understood how extreme temperatures might affect the virulence and recycling capacities of this pathogen. We tested the effect of short-term exposure of encysted L. chapmanii zoospores (cysts) on A. aegypti larvae killed after infection by this pathogen to stressful low or high temperatures on virulence and production of cysts and oogonia, respectively. Cysts were exposed to temperature regimes between -12 °C and 40 °C for 4, 6 or 8 h, and then their infectivity was tested against third instar larvae (L3) at 25 °C; in addition, production of cysts and oogonia on L3 killed by infection exposed to the same temperature regimes as well as their larvicidal activity were monitored. Virulence of cysts to larvae and the degree of zoosporogenesis on dead larvae under laboratory conditions were highest at 25 °C but were hampered or even blocked after 4 up to 8 h exposure of cysts or dead larvae at both the highest (35 °C and 40 °C) and the lowest (-12 °C) temperatures followed by subsequent incubation at 25 °C. The virulence of cysts was less affected by accelerated than by slow thawing from the frozen state. The production of oogonia on dead larvae was stimulated by short-term exposure to freezing temperatures (-12 °C and 0 °C) or cool temperatures (5 °C and 10 °C) but was not detected at higher temperatures (25 °C-40 °C). These findings emphasize the susceptibility of L. chapmanii to short-term temperature stresses and underscore its interest as an agent for biocontrol of mosquitoes in the tropics and subtropics, especially A. aegypti, that breed preferentially in small volumes of water that are generally protected from direct sunlight.