Christian Luz

Selection of Beauveria bassiana and Metarhizium anisopliae isolates to control Triatoma infestans

Twenty three isolates of Beauveria bassiana and 13 isolates of Metarhizium anisopliae were tested on third instar nymphs of Triatoma infestans, a serious vector of Chagas disease. Pathogenicity tests at saturated humidity showed that this insect is very susceptible to fungal infection. At lower relative humidity (50%), conditions expected in the vector microhabitat, virulence was significantly different among isolates. Cumulative mortality 15 days after treatment varied from 17.5 to 97.5%, and estimates of 50% survival time varied from 6 to 11 days. Maintaining lower relative humidity, four B. bassiana and two M. anisopliae isolates were selected for analysis of virulence at different conidial concentrations and temperatures. Lethal concentrations sufficient to kill 50% of insects (LC50) varied from 7.1 x 10(5) to 4.3 x 10(6) conidia/ml, for a B. bassiana isolate (CG 14) and a M. anisopliae isolate (CG 491) respectively. Most isolates, particularly B. bassiana isolates CG 24 and CG 306, proved to be more virulent at 25 and 30 degrees C, compared to 15 and 20 degrees C. The differential virulence at 50% humidity observed among some B. bassiana isolates was not correlated to phenetic groups in cluster analysis of RAPD markers. In fact, the B. bassiana isolates analyzed presented a high homogeneity (> 73% similarity).

Ovicidal Activity of Entomopathogenic Hyphomycetes on Aedes aegypti (Diptera: Culicidae) Under Laboratory Conditions

Abstract The ovicidal activity of 21 hyphomycete fungi species against Aedes aegypti (L.) (Diptera: Culicidae) was tested. Fungi with ovicidal activity developed on high numbers of eggs (≥70%) during 25 d of exposure. A clear ovicidal activity with low values of hatch (1.3–40%) was observed after 25 d of incubation with Isaria farinosa (Holm: Fries) Fries, Paecilomyces carneus (Duché & Heim) Brown & Smith, Paecilomyces marquandii (Massee) Hughes, Isaria fumosorosea (Wize), Metarhizium anisopliae (Metschnikoff) Sorokin, Penicillium sp., Paecilomyces lilacinus (Thom) Samson, Beauveria bassiana (Balsamo) Vuillemin, and Evlachovaea kintrischica Borisov & Tarasov. More than 63% of eggs hatched after 25-d exposures to 11 other fungi species deemed as ineffective. These are the first results to show the effects of entomopathogenic fungi against eggs of Ae. aegypti, and they suggest their potential as control agents of this vector.

Impact of moisture on survival of Aedes aegypti eggs and ovicidal activity of Metarhizium anisopliae under laboratory conditions

The effect of relative humidity (43%, 75%, 86% and > 98%) on Aedes aegypti eggs treated with Metarhizium anisopliae or water only was tested for up to a six months exposure at 25 degrees C. Survival of larvae inside eggs was clearly affected by the lowest humidity (43%) tested, and eclosion diminished at all humidities after increasing periods of exposure. M. anisopliae showed to have a strong ovicidal activity only at humidity close to saturation. No difference of activity was found between conidia and hyphal bodies tested. This fungus affected larvae inside eggs and has potential as a control agent of this important vector in breeding sites with high moisture.

Occurrence of Metarhizium spp. in Central Brazilian soils.

The biodiversity of entomopathogenic fungi in tropical ecosystems is still little investigated, and the objective of this study was to isolate and identify fungi of the entomopathogenic genus Metarhizium (Hypocreales: Clavicipitaceae) present in undisturbed soils of the Central Brazilian Cerrado. A total of 107 Metarhizium isolates was obtained from soils collected from Cerrado sites in the state of Goiás; gene sequences from 63 of these were obtained and compared. Among these, one was confirmed to be M. anisopliae sensu stricto; 53 were very closely allied to M. anisopliae but require more extensive genetic characterization to determine if they might represent a new taxon in the M. anisopliae species complex. Eight of these Cerrado isolates were referable to M. robertsii, and the remaining isolate is the first South American (and Southern Hemisphere) collection of M. flavoviride var. pemphigi. These findings underline the need for better characterization of the diversity of these widely distributed fungi in Brazil.

Potential of oil-based formulations of Beauveria bassiana to control Triatoma infestans

The in vitro development of Beauveria bassiana conidia was monitored when immersed in six concentrations of seven non-ionic (MP 6400, MP 600, Renex 60, Renex 95, Span 80, Tween 20 and Tween 80) and three anionic (DOS 75, Hostapaval BVQ 9 and Surfax 220) surfactants and 11 vegetable oils (linseed, soybean, groundnut, rapeseed, thistle, sunflower, olive, sesame, corn, castor, and babassu). The influence of the oils on the settling behavior of Triatoma infestans nymphs and the activity of an oil–water formulation of the fungus against this vector under laboratory and simulated field conditions were also determined. With exception of DOS 75 and Surfax 220 germination of conidia on complete medium was >98% at 24 h after exposure to surfactants up to 10%. Elevated rates of germination (>25%) were observed in 10% corn, thistle and linseed oil 8 days after incubation. Pure oils had a significant repellent effect to T. infestans. Repellency decreased generally at 10% of the oil and some oils showed some attractiveness for nymphs when tested at 1%. Nymphs were highly susceptible to oil–water formulated conidia, even at unfavorable moisture for extra-tegumental development of the fungus on the insect cuticle.

Activity of oil-formulated Beauveria bassiana against Triatoma sordida in peridomestic areas in Central Brazil.

Field tests were carried out during the rainy season of 2001/2002 in São Luís de Montes Belos, Goiás, Brazil, to evaluate the potential of the entomopathogenic fungus, Beauveria bassiana, against peridomestic Triatoma sordida. An oil-water formulation of the isolate CG 14 (Embrapa) was applied in triatomine infested hen houses of four farms at a final concentration of 10(6) conidia/cm2. Numbers of T. sordida decreased over the next 25 days, after application of the fungus, and B. bassiana developed on dead insects in one hen house. A high number of B. bassiana colonies was detected in substrates collected in treated hen houses 24 h after application of CG 14. In the following three months the presence of B. bassiana declined to values found before treatment.

First report of Metarhizium anisopliae IP 46 pathogenicity in adult Anopheles gambiae s.s. and An. arabiensis (Diptera; Culicidae)

The entomopathogenic fungus Metarhizium anisopliae isolate IP 46, originating from a soil sample collected in 2001 in the Cerrado of Central Brazil, was tested for its ability to reduce the survival of adult male and female Anopheles gambiae s.s. and An. arabiensis mosquitoes. A 6-h exposure to the fungus coated on test paper at a concentration of 3.3 × 106 conidia cm-2 reduced the daily survival of both mosquito species (HR = 3.14, p < 0.001), with higher risk of dying in An. gambiae s.s relative to An. arabiensis (HR = 1.38, p < 0.001). Fungal sporulation was observed in >95% of mosquito cadavers in the treatment groups. The results indicate that M. anisopliae IP 46 has the potential to be a bio-control agent for African malaria vector species, and is a suitable candidate for further research and development.

Development of Rhodnius prolixus (Hemiptera: Reduviidae) under Constant and Cyclic Conditions of Temperature and Humidity

Development of Rhodnius prolixus after eclosion until the adult stage was studied at constant temperatures (T), 15, 20, 25, 28, 35 C, and relative humidities (RH), 75, 86 and 97%, and fluctuating (16/8 hr) temperatures, T I/II, 15/28 C, 20/25 C, 25/28 C and 25/35 C, and relative humidities, RH I/II, 86/75% and 97/75%. Eclosion or molting were not observed at 15 C and 86 or 97% RH, respectively. At 35 C and 75% RH only few insects molted. By alternating T I/II, 15/28 C and 25/35 C, insects developed at high frequency. Cumulating the average lengths of the interphases within independent groups for each instar, R. prolixus reached the adult stage most rapidly (86.7 days) and at highest frequency per instar (mean: 91.8%) at 28 C and 75% RH. Under fluctuating T I/II, development was completed within 100 days or less at 25/28 C and 25/35 C with high rates of hatch and molting. Development was slowest at fluctuating TI/II, 15/28 C and 20/25 C (>185 days), and at constant 20 C (>300 days). Mortality was higher at constant 97% RH or fluctuating RH I, 97%, than at constant or fluctuating 86% RH. Refeeding was minimal at optimal conditions of T and RH for development. The most refeeding was observed at a constant 35 C.

Enhanced ovicidal activity of an oil formulation of the fungus Metarhizium anisopliae on the mosquito Aedes aegypti

Abstract The effect of humidity on the activity of Metarhizium anisopliae IP 46 (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) formulated in sunflower oil against Aedes aegypti (L.) (Diptera: Culicidae) eggs was examined. After exposure of eggs at 75% relative humidity (RH) for ≤ 25 days, ovicidal activity was not increased by oil‐in‐water formulated conidia, hyphal bodies or pure‐oil formulated conidia, compared with conidia or hyphal bodies prepared in water only. At optimal > 98% RH, eclosion was ≤ 13.7% after treatment with oil‐in‐water formulated propagules in ≤ 10% oil, and it was completely inhibited when conidia were applied in pure oil. At 86–100% RH, new conidia were found on eggs treated with oil‐formulated conidia and incubated down to 91% RH. Ovicidal activity was still detected at 93% RH and was augmented with increasing humidity and time of exposure of eggs. Eclosion of larvae was distinctly reduced by IP 46 pure‐oil formulated conidia after a minimal initial exposure of 3 days at > 98% RH, followed by: (a) a 12‐day exposure at 75% RH before submersion in water; (b) a minimal 5‐day exposure at > 98% RH and direct subsequent transfer of treated eggs to water, or (c) a minimal daily 20‐h exposure at > 98% RH alternating with 4 h at 75% RH for 10 days. We demonstrate that oil‐based formulations of conidia of M. anisopliae enhance ovicidal activity at high humidities and conclude that these formulations have potential in the integrated control of Ae. aegypti.

Detection of entomopathogenic fungi in peridomestic triatomine-infested areas in central Brazil and fungal activity against Triatoma infestans (Klug) (Hemiptera: Reduviidae)

From 148 substrate samples collected in peridomestic triatomine-infested habitats of 24 farms in Central Brazil, 31 isolates of Metarhizium anisopliae (Metsch.) Sorok. and 15 isolates of Beauveria bassiana (Bals.) Vuill. were obtained. Most of the isolates were found in substrates that consisted of soil mixed with animal feces or soil only in areas of trees where poultry roost and pig houses. Fungi were detected with an insect bait method using Triatoma infestans (Klug), and combined in vitro and in vivo techniques with modified Chase medium and T. infestans. All isolates were highly virulent to T. infestans third instar nymphs at a relative humidity > 98% and temperature of 25 ± 0.5°C. However, activity against nymphs was reduced at 75% relative humidity. Results underline the potential of B. bassiana and M. anisopliae as agents for biological control of vectors of Chagas disease. Both species occur naturally in peridomestic habitats of triatomines in Central Brazil and may contribute to control these vectors and to reduce the risk of reinfestation of houses after eliminating domestic vector species.