Isabele da Costa Angelo

An intensive search for promising fungal biological control agents of ticks, particularly Rhipicephalus microplus.

Entomopathogenic fungi have been investigated worldwide as promising biological control agents of the cattle tick Rhipicephalus microplus. The current study evaluates the virulence of several fungal isolates to R. microplus larva in the laboratory as part of an effort to identify isolates with promise for effective biocontrol of R. microplus in the field. Sixty fungal isolates, encompassing 5 Beauveria spp. and 1 Engyodontium albus (=Beauveria alba), were included in this study. In addition to bioassays, the isolates were characterized morphologically and investigated as to their potential for conidial mass production. These findings were correlated with previous reports on the same fungal isolates of their natural UV-B tolerance (Fernandes et al., 2007), thermotolerance and cold activity (Fernandes et al., 2008), and genotypes (Fernandes et al., 2009). R. microplus larvae obtained from artificially infested calves were less susceptible to Beauveria bassiana infection than ticks acquired from naturally infested cattle from a different location. Isolates CG 464, CG 500 and CG 206 were among the most virulent Beauveria isolates tested in this study. All fungal isolates presented morphological features consistent with their species descriptions. Of the 53 B. bassiana isolates, five (CG 481, CG 484, CG 206, CG 235 and CG 487) had characteristics that qualified them as promising candidates for biological control agents of R. microplus, viz., mean LC(50) between 10(7) and 10(8)conidiaml(-1); produced 5000 conidia or more on 60mm(2) surface area of PDAY medium; and, in comparison to untreated (control) conidia, had the best conidial tolerances to UV-B (7.04 kJ m(-2)) and heat (45°C, 2h) of 50% or higher, and conidial cold (5°C, 15d) activity (mycelial growth) higher than 60%. The current study of 60 Beauveria spp. isolates, therefore, singles out a few (five) with high potential for controlling ticks under field conditions.

Susceptibility of different populations of ticks to entomopathogenic fungi

This study aimed to evaluate the in vitro effect of the entomopathogenic fungi Metarhizium anisopliae sensu lato (s.l.) and Beauveria bassiana sensu lato (s.l.) on two distinct populations of Rhipicephalus microplus, from two different experimental farms. Bioassays were performed with engorged females, eggs and larvae. Fungal infection was evaluated based on biological parameters of treated engorged females, percentage of hatch from treated eggs, and percentage of mortality and mean lethal time (LT90) of treated larvae. When the treatments were compared between the two populations, there were significant differences in the following parameters: pre-oviposition period, hatching period and egg production index. Moreover, the results showed that B. bassiana s.l., isolate Bb 986, was more virulent than M. anisopliae s.l., isolate 959, for engorged females, showing a control percentage of 49%. In the bioassay with eggs, the hatching percentage ranged from 3.1% to 49.5% in one population and from 3.4% to 42.7% in the other, with no significant difference between the two populations. In the bioassay with unfed larvae, the mortality percentage ranged from 91.8% to 98.7% in one population and from 71.0% to 94.0% in the other. The LT(90) varied from 19.52 to 27.51 days in one of the populations and 22.89 to 37.31 days in the other. These results suggest that populations of R. microplus show distinct variation in their susceptibility to B. bassiana s.l. and M. anisopliae s.l.

Effect of oil-based formulations of acaripathogenic fungi to control Rhipicephalus microplus ticks under laboratory conditions

The formulations of acaripathogenic fungi to control ticks have been widely studied. The present study evaluated the efficacy of oil-based formulations of Metarhizium anisopliae sensu lato (s.l.), isolate Ma 959, and Beauveria bassiana, isolate Bb 986, on different Rhipicephalus microplus stages, comparing the efficacy between aqueous suspensions and 10, 15 and 20% mineral oil formulations. Twelve groups were formed: one aqueous control group; three mineral oil control groups, at 10, 15 or 20%; two aqueous fungal suspensions of M. anisopliae s.l. or B. bassiana; and three formulations of M. anisopliae (s.l.) or B. bassiana containing 10, 15, and 20% mineral oil. To prepare aqueous suspensions and oily formulations, fungal isolates were cultivated on rice grains in polypropylene bags. The conidial suspensions and formulations had a concentration of 10(8)conidia/mL. Bioassays were repeated twice. After treatment, the following biological parameters of engorged females were evaluated: hatching percentage, egg production index, nutritional index, and percentage of tick control. The following parameters were evaluated in the bioassays with eggs: period of incubation, period of hatch, and hatching percentage. Mortality was evaluated in bioassays with larvae. M. anisopliae s.l. and B. bassiana oil-based formulations were more effective than aqueous suspensions against R. microplus eggs, larvae and engorged females, however, there was no significant difference between the three oil concentrations used. M. anisopliae s.l. and B. bassiana formulated in mineral oil reached 93.69% and 21.67% efficacy, respectively, while M. anisopliae s.l. and B. bassiana aqueous suspensions attained 18.70% and 1.72% efficacy, respectively. M. anisopliae s.l. oil-based formulations caused significant effects in all biological parameters of engorged females while B. bassiana oil-based formulations modified significantly the nutritional index only. Eggs treated with M. anisopliae s.l. and B. bassiana oil-based formulations showed hatching rates that decreased 102.5 and 3.65 times, respectively. In the bioassay with larvae, M. anisopliae s.l. oil-based formulations caused nearly 100% mortality five days after treatment, while larva treated with B. bassiana oil-based formulations reached 100% mortality at day 20 after treatment. Larva from oil-based control groups showed mortality at day 15 after treatment, which indicated a possible toxic effect of the oil for this R. microplus stage. The results showed that the fungal mineral oil formulations tested were more effective than the aqueous suspension. Oil-based formulations at 10, 15 and 20% enhanced the activity of M. anisopliae s.l. Ma 959, and B. bassiana Bb 986, isolates against R. microplus eggs, larvae, and engorged females tick. Mineral oil was effective as an adjuvant in formulations of M. anisopliae s.l., Ma 959, and B. bassiana, Bb 986, for the control of R. microplus under laboratory conditions.

Efficiency of Lecanicillium lecanii to control the tick Rhipicephalus microplus.

Rhipicephalus microplus, known as the cattle tick, causes serious economic losses in the Brazilian cattle industry each year. Traditional parasite control is primarily based on the use of chemical acaricides, which unfortunately have many negative side effects. Biological control is seen as a promising alternative to chemical acaricide use. This study evaluates the entomopathogenic fungus Lecanicillium lecanii for effectiveness in controlling engorged females, eggs, and larvae of R. microplus. Conidial formulations of L. lecanii, isolate CG 420, were prepared in both oil (15% mineral oil) and aqueous suspensions. Ticks were immersed in a 1ml oil-based conidial suspension at 1x10(8)conidiaml(-1) or one of several aqueous conidial suspensions at 1x10(5), 10(6), 10(7) or 10(8)conidiaml(-1). The control groups were immersed in water or oil solutions with no conidia. Treatments with aqueous conidial suspensions were conducted with 10 ticks per group (1x10(5), 10(6), 10(7), 10(8)conidiaml(-1) or control) whereas the oil treatments used 30 ticks per group (1x10(8)conidiaml(-1) or control). Bioassays were repeated twice on different days with different batches of conidia. After treatment, the biological parameters of engorged females were evaluated, while eggs and larvae were evaluated taking into consideration hatchability and mortality, respectively. The results indicate that L. lecanii has the potential to control engorged females, eggs and larvae of R. microplus. Better results were observed when conidial oil-based suspension was used. In general, engorged females treated with 1x10(8)conidiaml(-1) oil suspensions died before laying eggs, resulting in 97.6% of tick control. As far as we know, this is the first report of the effects of L. lecanii on R. microplus tick.

Virulence potential of Metarhizium anisopliae s.l. isolates on Rhipicephalus (Boophilus) microplus larvae.

The use of entomopathogenic fungi to control arthropods has been reported worldwide for decades. The aim of the present study was to evaluate the virulence of 30 Metarhizium anisopliae s.l. Brazilian isolates from different geographical regions, hosts or substrates on the larvae of Rhipicephalus (Boophilus) microplus ticks under in vitro conditions to the selection of virulent isolates in order to be further used in biological control programs. The current study confirmed the lethal action of M. anisopliae s.l. isolates on R. (B.) microplus larvae with different mortality levels, usually directly proportional to the conidia concentration. No relationship was found between the origin of the isolate and its virulence potential or between the virulence potential and conidia production. Three isolates (CG 37, CG 384 and IBCB 481) caused a high percentage of larval mortality, reaching LC(50) at 10(6) conidia ml(-1), thus requiring a lower conidia concentration to cause an approximately 100% larval mortality. The results of this study suggest that these three isolates are the most promising for use in programs aimed at microbial control in the field.

Haemolymph protein and lipid profile of Rhipicephalus (Boophilus) microplus infected by fungi.

The current study evaluates the protein and lipid profile of haemolymph of Rhipicephalus (Boophilus) microplus engorged females infected by Metarhizium anisopliae, Beauveria bassiana or Fusarium oxysporum. Ticks were immersed or inoculated with conidial suspension. Haemolymph was collected from the dorsal surface of engorged females. The results showed altered total protein amounts; however, no significant difference was observed on electrophoretic profile among haemolymph samples. In addition, altered lipid profile was detected in haemocyte samples from ticks treated with Beauveria and Metarhizium.

Commercial formulation of Metarhizium anisopliae for the control of Rhipicephalus microplus in a pen study

The present study evaluated, for the first time, the effect of the commercial formulation Metarril(®) SP Organic of Metarhizium anisopliae plus 10% mineral oil to control Rhipicephalus microplus in a pen study. Three groups were formed with six animals each: the first group was exposed to Metarril(®) plus 10% mineral oil and 1% Tween 80; the second group was exposed to sterile distilled water, mineral oil and Tween 80 (oil control group); and the third group received no treatment (control group). The fungal formulation contained 1 × 10(8)conidiaml(-1). Each animal was sprayed with 3L of formulation. Fallen ticks were counted daily and a sample of 20 engorged females per day was incubated for assessment of biological parameters. Throughout the study period, Metarril(®) oil-based formulation showed an efficacy ranging from 19.20% to 67.39% in comparison with the control group; and from 8.18% to 61.38% in comparison with the oil control group. The average efficacy of Metarril(®) oil-based formulation was 47.74% and 40.89% in comparison with control and oil control groups, respectively. Changes in the biological parameters of engorged R. microplus females were observed in the first three days after treatment, with a significant reduction in hatching percentage and egg production index. We concluded that Metarril(®) SP Organic plus 10% mineral oil was efficient against R. microplus in pen studies. However, further in vivo studies are required to increase the efficacy and to establish a protocol for the use of this product in the field against the cattle tick.

Virulence of Metarhizium anisopliae to eggs and immature stages of Stomoxys calcitrans.

Stomoxys calcitrans control is primarily based on the use of chemical products; however, new methods for control have been investigated. Biological control has been proposed as a promising alternative to traditional chemicals. The current study evaluated three methods in vitro to treat S. calcitrans eggs with the fungus Metarhizium anisopliae and assessed the virulence of this fungus to immature stages of this fly. In the first method of egg exposure, eggs were immersed in conidial suspensions and transferred to Petri plates with high humidity. A high mortality was observed in both treated and control groups. In the second method, the eggs were transferred to rearing medium right after immersion in conidial suspensions; the suspensions with highest conidial concentrations, 107 and 108 conidia mL−1, caused 96.25% and 100% mortality, respectively. In the third method, the eggs were sprayed with conidial suspensions on the rearing medium. A high mortality was observed at 107 and 108 conidia mL−1, 78.3% and 100%, respectively. The second method had the lowest mortality in the control group and was considered the most useful method for evaluating the effect of entomopathogenic fungi on S. calcitrans eggs. While eggs were very susceptible to M. anisopliae infection, larvae and pupae were not.

Performance of Metarhizium anisopliae and Its Combination with Deltamethrin against a Pyrethroid‐Resistant Strain of Boophilus microplus in a Stall Test

The use of the entomopathogenic fungus, Metarhizium anisopliae, to control ticks is a promising alternative to chemical acaricides, but both chemical and biological products could also work together in an integrated management. The current study evaluated the performance of M. anisopliae and its combination with deltamethrin to control a Boophilus microplus strain resistant to pyrethroid. Calves were held in individual pens and artificially infested with B. microplus. They were treated with an aqueous conidial suspension of M. anisopliae, a deltamethrin solution, or both fungus and deltamethrin combined. After treatment, engorged females that naturally dropped off from calves were counted daily and evaluated in the laboratory. The mean mortality rate was 32.57% in the groups treated with M. anisopliae, 38.58% in those groups treated with deltamethrin, and 30.92% in those treated with fungus and chemical combined. Additionally, ticks that dropped off from calves after 2 days had a lower nutrient index (NI) and egg production index (EPI) than those that dropped off at a later day after treatment. This indicates that M. anisopliae may reduce B. microplus population only for a few days after treatment when a simple aqueous conidial suspension was used.

Metarhizium anisopliae for controlling Rhipicephalus microplus ticks under field conditions

Metarril SP Organic is a product based on the fungus Metarhizium anisopliae, which was developed for controlling agricultural pests. The present study evaluated the effect of Metarril SP Organic plus 10% mineral oil, for controlling Rhipicephalus microplus under field conditions. Three groups were formed: Control group, which received no treatment; Oil control group, which was bathed only with water, Tween 80 and mineral oil; and Metarril group, bathed in the oil-based formulation Metarril SP Organic. Two treatments per group were performed and to verify the effect of the treatments, all R. microplus ticks between 4.5 and 8.0mm in length on the left side of the cattle were counted on days +7, +14 and +21 after each treatment, and a sample of engorged females was collected for evaluation of biological parameters. The Metarril SP Organic oil formulation showed efficacy ranging from 8.53 to 90.53%. The average efficacy of the oil-based formulation of Metarril SP Organic was 75.09 and 46.59% compared with the groups Control and Oil control, respectively. There were no significant changes in biological parameters of engorged R. microplus females collected from animals. Although there was no significant difference in the amount of ticks between the Oil control and Metarril groups, it is believed that the association of mineral oil with Metarril SP Organic product is effective in R. microplus tick control in field. Thus, this association has potential to be used in strategic control programs of cattle tick.