Efeito da temperatura e da formulação oleosa na germinação e virulência de conídios de Metarhizium anisopliae a Rhipicephalus sanguineus
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2014-10-08
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Universidade Federal de Goiás
Resumo
Resistence of the tick Rhipicephalus sanguineus to chemicals is commonly reported. Entomopathogenic fungi emerge
as an alternative to the exclusive use of chemical acaricides for tick control; however, heat and other environmental
factors may limit the efficacy of fungi. Thus, the current study evaluated the effect of heat on conidial germination of
Metarhizium anisopliae IP 119 when suspended in pure mineral oil (Impex®) or in water (Tween 80, 0.01%) and
applied to the cuticle of R. sanguineus (in vivo) or in artificial medium PDAY (in vitro). Then, the results obtained
with tests in vivo were compared to the results obtained with tests conducted in vitro, and finally, experiments were
conducted to verify the virulence of M. anisopliae IP 119 for R. sanguineus engorged females. Conidial suspensions of
M. anisopliae in water or oil, adjusted to 108 conidia ml-1, were exposed to 0 (control) or 4 h to 45 ± 0.5 °C in a water
bath, and then, individually inoculated onto the dorsal surface of R. sanguineus engorged females. The samples were
incubated at 27 ± 1 °C and RH > 80% for 0, 12, 18, 24, 36, 48 or 72 h. In parallel, aliquots of the aqueous conidial
suspensions (control or 45 °C) were inoculated onto PDAY medium in Petri plates. The samples were incubated at the
same temperature and for the same time periods described above. After each incubation time, ticks were fixed and
dissected, i.e., a fragment of the dorsal cuticle was removed. Cuticles that received conidia suspended in water,
exposed or not to heat, were processed for fluorescence microscopy for evaluation of conidial germination. Cuticles
that received conidia suspended in water or oil, exposed or not to heat, were processed for scanning electron
microscopy for evaluation of conidial germination. Conidial germination on PDAY plates was assessed using a phasecontrast microscope. A minimum of 300 conidia per cuticle or plate was evaluated for calculation of percent
germination. When conidia were exposed to heat, a high percentage of germination was observed in PDAY medium
(61.5%) in comparison to the tick cuticle (13%), 72 h after inoculation. Appressoria were observed 36 h after
inoculation, only in conidia that were not exposed to heat. On tick cuticle, the percentage of germination of conidia
suspended in mineral oil and exposed to heat was high in comparison to the percentage of germination of conidia
suspended in water, at all incubation times investigated. At 36 h of incubation, the mean percentage of germination of
conidia suspended in oil reached 16.3%, whereas conidia suspended in water reached only 2.2%. Conversely, in the
control groups (ticks treated with conidia not exposed to heat), the percentage of germination of conidia suspended in
water or oil differed at 48 and 72 h of incubation, where germination was also high for conidia suspended in oil and
inoculated on the tick cuticle. M. anisopliae conidia suspended in oil exposed or not to heat showed control percentage
greater than the observed with aqueous suspensions. Thus, the results showed a significant delay in germination for
conidia suspended in water and exposed to 45 °C in comparison to conidia suspended in mineral oil; in this sense, the
oil formulation of conidia when exposed or not to heat proved to be more effective for controlling R. sanguineus
engorged females. Moreover, conidia suspended in water germinated faster when inoculated in PDAY than when they
were applied to the tick cuticle. Therefore, the results indicated that the negative effect of heat on conidial germination
was more expressive when conidia were suspended in water and applied to the arthropod cuticle than it would be
predicted by the in vitro thermotolerance tests. Also, conidia suffered less interference from heat exposures when
suspended in oil. In conclusion, mineral oil protects conidia against the heat effects and enhances the fungal
germination on the cuticle of R. sanguineus.
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Barreto, L. P. Efeito da temperatura e da formulação oleosa na germinação e virulência de conídios de Metarhizium anisopliae a Rhipicephalus sanguineus. 2014. 75 f. Dissertação (Mestrado em Medicina Tropical e Saúde Publica) - Universidade Federal de Goiás, Goiânia, 2014.