Search

Entomopathogenic fungi can achieve important innovative outcomes for integrated mosquito control especially of Aedes aegypti, the key vector of arboviruses to humans in the tropics and subtropics. This study sought to design and to develop a simple dissemination device to attract and to infect gravid A. aegypti adults with a granular formulation of the ascomycete Metarhizium humberi IP 46, and to validate this device in the laboratory as well as in semi-field and field conditions. Hydrogel (polyacrylamide potassium polyacrylate) was confirmed to be a suitable substitute for water used in the device that attracted gravid females under field conditions. Females laid eggs on black polyethylene terephthalate carpet fixed in the device that also proved to be a suitable substrate for a granular formulation of fungal microsclerotia and/or conidia. The plastic device (29.5 cm high) was divided into a lower closed compartment with a water reservoir and an upper, laterally open but covered compartment with continuously hydrated gel and the fungal formulation attached to the carpet. The uppermost compartment permitted free circulation of mosquito adults. The device attracted both male and female A. aegypti. The fungal formulations of IP 46 propagules tested in the device were effective against adults in laboratory, semi-field, and field settings. Findings in the laboratory, semi-field, and especially in field conditions strengthen the value and utility of this innovative device for focal applications of a mycoinsecticide against this important mosquito vector. Key points • Low-cost and simple disseminating device for focal control of Aedes aegypti. • Granulized Metarhizium humberi IP 46 and hydrogel yield extended control. • Findings in field tests strengthen benefit of the device for focal application.

Rhipicephalus microplus (Canestrini 1888) causes severe economic losses to the cattle industry in Brazil. Products alternative to chemical acaricides have been investigated for tick control, and th...

This study was sought to devise pellets containing inorganic materials and microsclerotia of Metarhizium anisopliae strain IP 119 for biological control of Rhipicephalus microplus, the most economically important tick in Brazilian cattle industry. In addition, we evaluated the storage stability of the pellets, their tolerance to ultraviolet radiation (UV-B), and efficacy against ticks under laboratory conditions. Fungal microsclerotia were produced by liquid culture fermentation and mixed with pre-selected inorganic matrices: vermiculite powder, diatomaceous earth, and colloidal silicon dioxide (78:20:2, w/w/w). The microsclerotial pellets were then prepared by a two-stage process involving extrusion and spheronization. Pellet size averaged 525.53 ± 7.74 μm, with a sphericity index of 0.72 ± 0.01, while biomass constituents did not affect the wet mass properties. Conidial production from microsclerotial pellets upon rehydration ranged from 1.85 × 109 to 1.97 × 109 conidia g−1 with conidial viability ≥ 93%. Conidial production from pellets stored at 4 °C was invariable for up to 21 days. Unformulated microsclerotia and microsclerotial pellets were extremely tolerant to UV-B compared with aerial conidia. Engorged tick females exposed to conidia from sporulated pellets applied to soil samples and upon optimal rehydration exhibited shorter oviposition time length, shorter life span, and reduced number of hatched larvae. In summary, microsclerotial pellets of M. anisopliae IP 119 effectively suppressed R. microplus and showed outstanding UV-B tolerance in laboratory tests. Prospectively, this formulation prototype is promising for targeting the non-parasitic stage of this tick on outdoor pasture fields and may offer a novel mycoacaricide for its sustainable management. • Pellets with microsclerotia and inorganic materials are innovative for tick control. • Metarhizium microsclerotia show superior UV-B tolerance in relation to conidia. • Pellets of Metarhizium microsclerotia produce infective conidia against ticks.

The impact of ambient relative humidity (RH) on conidial production of Metarhizium humberi IP 46 microsclerotia (MS) formulated in pellets or granules was investigated, and a promising granular formulation was tested against Aedes aegypti adults to confirm its efficacy. Microcrystalline cellulose (MC) and diatomaceous earth (DE) or a combination of vermiculite (VE), DE and silicon dioxide (SD) were tested as carriers in granular formulations containing MS. A range of 93-96.5% RH was critical for fungal development, and at least 96.5-98.5% RH was required for high conidial production on pellets or granules. Conidial production was clearly higher on pellets and granules prepared with VE than MC as the main carrier. VE granules containing MS were highly active against A. aegypti adults. Most mosquitoes were killed within 6 days after treatment regardless of the exposure time of adults to the formulation (1 min-24 h) or ambient humidity (75 or >98%). Production of conidia on dead adults varied between 7.3 × 106 and 2.2 × 107 conidia/individual, when exposed to MS granules for 12 h and 1 min, respectively. Granular formulations containing VE as the main carrier and MS as the active ingredient of M. humberi have strong potential for use against A. aegypti. KEY POINTS: • High conidial production on granular microsclerotial formulations at >96.5% RH • Vermiculite is more appropriate as a carrier than microcrystalline cellulose • Granules with IP 46 microsclerotia are highly active against Aedes aegypti adults.

The aim of the present study was to analyze ten native Metarhizium spp. isolates as to their UV-B tolerances. Comparisons included: different fungal propagules (conidia, blastospores, or microsclerotia [MS]); conidia in aqueous suspensions or in 10% mineral oil-in-water emulsions; and conidia mixed with different types of soil. The UV-B effect was expressed as the germination of conidia or culturability of blastospores and MS relative to nongerminated propagules. Metarhizium anisopliae LCM S05 exhibited high tolerance as blastospores and/or MS, but not as conidia; LCM S10 and LCM S08 had positive results with MS or conidia but not blastospores. The formulations with 10% mineral oil did not always protect Metarhizium conidia against UV-B. Conidia of LCM S07, LCM S08, and LCM S10 exhibited the best results when in aqueous suspensions, 24 h after UV-B exposure. In general, conidia mixed with soil and exposed to UV-B yielded similar number of colony forming units as conidia from unexposed soil, regardless the soil type. It was not possible to predict which type of propagule would be the most UV-B tolerant for each fungal isolate; in conclusion, many formulations and propagule types should be investigated early in the development of new fungal biocontrol products.

Environmental stress conditions compromise the effectiveness of microorganisms for use in biological control. We investigated the thermotolerance of seven Metarhizium isolates from Central Brazil to 32 °C, and the thermotolerance of conidia of Metarhizium anisopliae s.str. IP 119 (Metchnikoff) (Hypocreales: Clavicipitaceae) and M. robertsii formulated in oil-in-water emulsion to 45 °C. We also evaluated the efficacy of oil-formulated conidia of M. anisopliaes.str. IP 119 against the tick Rhipicephalus microplus (Canestrini) (Acari: Ixodidae) under stress conditions (32 °C; 75% RH). The conidial germination on tick’s cuticle was assessed by Scanning Electron Microscopy. Conidia of M. anisopliaes.str. IP 119 formulated in oil-in-water emulsion caused significant control (59.4%) of R. microplus, even when treated ticks were incubated under stress (32 °C; 75% RH). Metarhizium spp. incubated at 32 °C had both decreased radial growth and conidial production. Conidia of IP 119 formulated in oil-in-water emulsion were more tolerant to 45 °C than conidia suspended in water. An oil-in-water emulsion protected M. anisopliaes.str. conidia against heat and dry conditions and may enhance its efficacy to control ticks by improving the tolerance of fungi against adverse environmental factors.

Musca domestica L. is a cosmopolitan nuisance of high sanitary importance. Entomopathogenic fungi are innovative and attractive tools for integrated control of the housefly to overcome insufficient levels of control caused by increasing resistance of this pest against chemical insecticides. High virulence of a fungal strain is a prerequisite to develop a mycoinsecticide, and the present study investigated the potential of hypocrealean fungi from the genera Beauveria, Clonostachys, Cordyceps, Akanthomyces, Metarhizium, and Tolypocladium, isolated from mosquitoes in Central Brazil against M. domestica. The highest mortalities (larvae, pupae, and adults) were caused by Metarhizium humberi IP 478 (98%) and IP 421 (90%), Metarhizium anisopliae IP 432 (85%), Beauveria bassiana IP 433 (82%), and Tolypocladium cylindrosporum IP 425 (68%) after a 23-day exposure of initially pre-pupating third instar larvae to conidia mixed with vermiculite. Lethal concentrations to kill 90% of adults of IP 433 and IP 478 were 5 × 10

Background Oil formulations of entomopathogenic fungi have interest for biological mosquito control. Objectives The activities of M. anisopliae s.l. IP 46 conidia were tested in Aedes aegypti adults either without any formulation or formulated with vegetable or mineral oil and in combination with diatomaceous earth. Findings IP 46 was highly active against adults, the vector of important arboviruses in the tropics and subtropics. At an exposure of adults to 3.3 × 107 conidia/cm2, values of lethal times TL50 and TL90 reached minimal 3.8 and 4.6 days, respectively, and lethal concentrations LC50 and LC90 were 2.7 × 105 and 2.4 × 106 conidia/cm2, respectively, after 10 days of exposure. Activity against adults was improved by diatomaceous earth (KeepDry® KD) combined with mineral oil (Naturol® N) or vegetable oil (Graxol® G). Additives KD or N separately (and G to a lesser extent) or in combination, KD + N and KD + G without conidia had also a clear adulticidal effect. Efficacy of conidia formulated or not with KD + N decreased somewhat at shorter exposure periods. Time of exposure (0.017, 12, 48, 72 or 120 h) of adults to KD and N or IP 46 or conidia and KD and N had no significant effect on mortality. M. anisopliae s.l. recycled on fungus-killed mosquitoes producing high quantities of new conidia regardless of the conidial concentrations or formulations tested. Additives tested had no clear effect on quantitative conidiogenesis on cadavers. Main conclusions Formulations of IP 46 conidia with mineral oil and diatomaceous earth represent a promising tool for the development of potent strategies of focal control of this important vector with entomopathogenic fungi.

Several filamentous fungi are known to produce macroscopic pigmented hyphal aggregates named sclerotia. In recent years, some entomopathogenic fungi were reported to produce small sclerotia termed 'microsclerotia', becoming new potential propagules for biocontrol strategies. In this study, we described the production of microsclerotia-like pellets by the entomopathogenic fungus Beauveria bassiana. The carbon: nitrogen ratio equal to or higher than 12.5:1 amended with Fe2+ induced the germination of conidia, producing hyphal aggregate that formed sclerotial structures in submerged liquid cultures. These aggregates were able to tolerate desiccation as they germinated and subsequently produced viable conidia. Conidia derived from microsclerotial aggregates formulated with diatomaceous earth effectively kill Tribolium castaneum larvae. Optical and transmission microscopical imaging, qPCR and spectrophotometric analysis revealed that an oxidative stress scenario is involved in conidial differentiation into microsclerotia-like pellets, inducing fungal antioxidant response with high peroxidase activity - mainly detected in peroxisomes and mitochondria - and progress with active peroxisome proliferation. The results provide clues about B. bassiana microsclerotial differentiation and indicate that these pigmented aggregates are promising propagules for production, formulation and potentially application in the control of soil-inhabiting arthropod pests.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

Metarhizium species fungi are able to produce resistant structures termed microsclerotia, formed by compacted and melanized threads of hyphae. These propagules are tolerant to desiccation and produce infective conidia, thus they are promising candidates to use in biological control programs. In this study, we investigated the tolerance to both UV-B radiation and heat of microsclerotia of Metarhizium robertsii strain ARSEF 2575. We also adapted the liquid medium and culture conditions to obtain mycelial pellets from the same isolate in order to compare these characteristics between both types of propagules. We followed the peroxisome biogenesis and studied the oxidative stress during differentiation from conidia to microsclerotia by transmission electron microscopy after staining with a peroxidase activity marker, and by the expression pattern of genes potentially involved in these processes. We found that despite their twice smaller size, microsclerotia exhibited higher dry biomass, yield, and conidial productivity than mycelial pellets, both with and without UV-B and heat stresses. From the sixteen genes measured, we found an induction after 96-h differentiation in the oxidative stress marker genes MrcatA, MrcatP, and Mrgpx, the peroxisome biogenesis factors Mrpex5 and Mrpex14/17, and the photoprotection genes Mrlac1, Mrlac2, and Mrlac3. We concluded that an oxidative stress scenario is induced during microsclerotia differentiation in M. robertsii, and confirmed that due to its tolerance to desiccation, heat, and UV-B, this fungal structure could be an excellent candidate for use in biological control of pests under tropical and subtropical climates where heat and UV radiation are detrimental to entomopathogenic fungi survival and persistence., Facultad de Ciencias Exactas, Instituto de Investigaciones Bioquímicas de La Plata

The tick Rhipicephalus microplus poses a serious threat to the cattle industry, resulting in economic losses aggravated by tick resistance to chemical acaricides. Strains of Metarhizium spp., a well-known group of entomopathogenic fungi, can contribute to managing this ectoparasite. We explored two novel granular, microsclerotia- or blastospores-based formulations of Metarhizium robertsii for R. microplus control under semi-field conditions. Fungal persistence in soil was also observed for 336 days. The experiment used pots of Urochloa decumbens cv. Basilisk grass, treated with 0.25 or 0.5 mg of granular formulation/cm2 (25 or 50 kg/ha) applied to the soil surface prior to transferring engorged tick females onto the treated soil. The fungal granules yielded more conidia with subsequent sporulation under controlled indoor conditions than in the outdoor environment, where the levels of fungus rapidly declined over time. Metarhizium-root colonization ranged from 25 to 66.7% depending on the propagule and rate. Fungal formulations significantly reduced the number of tick larvae during the humid season, reaching at least 64.8% relative efficacy. Microsclerotia or blastospores-granular formulations of M. robertsii can reduce the impact of R. microplus, and thus prove to be a promising tool in the control of ticks.

Toxoplasma gondii is an apicomplexan protozoan that is frequently found in both humans and animals worldwide. The aim of this review was to list important aspects of Toxoplasma gondii infection in cattle in Brazil. The frequency of occurrence of T. gondii antibodies in Brazilian cattle ranges from 1 to 89.1%, depending on the region evaluated, based on data from 1978 to 2018. However, some characteristics of T. gondii infection in cattle remain uncertain, such as the role of meat intake in transmitting the parasite to humans. Most information regarding T. gondii infection among Brazilian cattle is limited to evaluations of the frequency of occurrence of antibodies. About 70% of the diagnoses of infection in these ruminants in Brazil are made via the indirect fluorescence antibody test (IFAT). Nevertheless, little is known about the population structure of this protozoan in cattle. It is necessary to expand the studies on toxoplasmosis in cattle, in order to better understand T. gondii infection in these animals and its implications for Brazilian public health.

The current study compared the virulence of conidia and blastospores of Metarhizium robertsii (IP 146), M. anisopliae sensu lato (s.l.) (IP 363) and Beauveria bassiana s.l. (IP 361 and CG 307) against unfed larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae). In addition, the development of fungal propagules on tick cuticle was investigated. Tick larvae were treated with fungal suspensions at 106, 107 or 108 propagules mL-1, and percent mortality was assessed every two days. Engorged females were immersed in fungal suspensions (1.0 × 107 propagules mL-1) for 1 min, and their biological parameters monitored daily. The virulence of conidia and blastospores against larvae varied considerably among the isolates tested. Only females treated with blastospores of IP 146 or IP 361 presented lower nutrient and egg production indices than the control group; the higher percent control was reached when females were treated with blastospores of IP 146 (98%), IP 363 (79%), or IP 361 (93%) in comparison to the groups treated with conidia, 71%, 59%, or 63%, respectively. Engorged females treated with conidia or blastospores of IP 146 or IP 361 were also examined by scanning electron microscopy (SEM). Germination of blastospores of IP 146 and IP 361 was observed on tick cuticle after 4 h incubation at 27 ± 1 °C and RH > 90%, whereas germ tubes from conidia of both isolates were observed at 48 h. Appressoria in developing blastospores of B. bassiana IP 361 were observed after 4 h incubation, whereas no appressoria were seen in developing blastospores of M. robertsii IP 146. Blastospore penetrations by both fungal isolates through natural openings was also evidenced by SEM; fine sections of R. microplus engorged females treated with blastospores of IP 146 or IP 361 confirmed that these isolates penetrated through their cuticle and natural openings. Blastospores might be promising for use in biocontrol of ticks, since they are virulent against R. microplus and present rapid development on their cuticle.

Physically disturbed Triatoma infestans (Hemiptera: Reduviidae) adults, as well as adults of other Chagas' disease vectors, secrete a mix of volatile organic compounds (VOCs) with alarm and possible sexual and defence functions. The aim of the present research was to test whether infection with the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales: Clavicipitaceae) has an effect on VOC secretion in disturbed T. infestans and on the expression of two genes (Ti-brnq and Ti-bckdc) potentially involved in VOC biosynthesis. The volatiles released by insects at different time periods after fungal treatment were identified and their relative amounts measured. Isobutyric acid was the most abundant volatile found in both healthy and fungus-infected insects and underwent no significant relative changes through the infection process. The secretion of propionic acid, however, was significantly higher at 1-4 days post-infection (d.p. i.) compared with that in controls. A slight induction of both Ti-brnq and Ti-bckdc genes was found by real-time polymerase chain reaction at 4 d.p. i., with expression values reaching up to three-fold those in controls. The early stages of fungal infection seem to affect the composition of the alarm pheromone by changing the expression pattern of both genes analysed. These results help to elucidate the impact of fungal infections on the chemical ecology of triatomine bugs.

Benzaldehyde and 2-hexanone are allomones produced by beagle dogs that reduce infestation by Rhipicephalus sanguineus sensu lato on these animals and on susceptible dogs which artificially release these repellents. These observations were obtained in previous laboratory tests or artificial infestations of susceptible dogs under controlled conditions. Here we evaluated the efficacy of collars delivering these repellents for suppressing the loads of R. sanguineus s.l. on naturally infested mixed-breed dogs under field conditions. Thirty dogs naturally infested with R. sanguineus s.l. were separated into two groups with 15 dogs each. The dogs from the treatment group received the collars with slow-release formulations of the allomones, and the dogs from the control group received collars without such compounds. Collar effectiveness tests were carried out over 30 days. All ticks found were removed from each dog, identified, and counted every collection day. The density of the different life stages of R. sanguineus s.l. varied greatly between groups and across evaluation times. Adult ticks were the stage most abundant and prevalent on infested dogs. The counts for larvae, nymphs and adults were similar between the control and the treatment groups within each tick collection day. Conversely, when we considered the total number of ticks over 30 days and gathered all life stages collected in the same animal, the dogs from the control group had significantly higher loads (35.3 ± 4.27) of ticks than the dogs from the treatment group (21.8 ± 2.96) (P0.01). In addition, the efficacy of treatment with repellent collars was high for adult ticks (30.8%) but was extremely low for reducing larval or nymphal infestation (0 or 2.6%, respectively). The present study demonstrates, for the first time, the efficacy of these volatile compounds on naturally infested dogs under field conditions. The development of a technologically enhanced device for slow release of the allomones tested here can be an advantageous alternative for reducing R. sanguineus s.l. infestation on dogs in residential environments.

The biocidal potential of Brevibacillus laterosporus against mosquitoes of major medical importance has been widely documented, but its effects on non-target invertebrates are still poorly known. In this study, we determined the lethal and sub-lethal effects of B. laterosporus strain UNISS 18, an entomopathogenic bacterium known for its effectiveness against synanthropic Diptera, on the larvae of the Asian tiger mosquito Aedes albopictus, a vector of several pathogens to humans. Moreover, we compared the larvicidal activity with the lethal action on the invasive snail Physella acuta and on two non-target water-dwelling species: the mayfly Cloeon dipterum, and the harlequin fly Chironomus riparius. B. laterosporus exhibited significant lethal effects on all the tested species with a concentration-dependent activity. However, the susceptibility varied among species, with a higher susceptibility of Ae. albopictus (LC50 = 0.16 × 107 spores mL−1) than the other species (LC50 = 0.31, 0.33, and 0.30 × 107 spores mL−1 for C. dipterum, C. riparius, and P. acuta, respectively). While 1st instar mosquito larvae were very susceptible to the bacterial infection, no effects on preimaginal development stages and adult emergence were observed at sub-lethal spores’ concentrations. Even if the efficacy of B. laterosporus against Ae. albopictus and the invasive freshwater snail P. acuta is promising for their control, the susceptibility of non-target beneficial aquatic insects, highlights the need of accurate evaluations before applying B. laterosporus for pest management in water environments.

The thermotolerance of oil-based conidial formulations of Metarhizium anisopliae s.l. (IP 46) and Metarhizium robertsii (ARSEF 2575) were investigated. Conidia of IP 46 or ARSEF 2575 were suspended in different adjuvants and exposed to 45 ± 0.2°C for 4, 6, 8 or 24 h; their viability was then assessed after 48 h incubation at 27 ± 1°C. Conidia heated in pure mineral or vegetable oil exhibited mean relative viability exceeding 70% after 8 h of heat exposure, whereas low germination (≤20%) was observed when conidia were heated in water (Tween 80® 0.01%), carboxymethyl cellulose gel or emulsifiable oils (Graxol® or Assist®) and exposed to heat for 6 or 8 h. In addition, conidia of IP 46 suspended in either pure mineral or canola oil and exposed to heat for 48 h had moderate viability, 57% or 41%, respectively. Unstable oil-in-water emulsions showed a higher percentage of conidia incorporated into oil micellae, while the stable emulsions had higher percentage of conidia outside the oil micellae. The thermotolerance of conidia formulated in stable emulsions, however, did not differ from that of conidia formulated in unstable emulsions. The present study highlights possibilities to alleviate the deleterious effects of heat stress towards Metarhizium spp. conidia applied for controlling arthropod pests and vectors through oil-based formulations. Fil: Santos Da Paixao, Flavia Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Universidade Federal de Goiás; Brasil Fil: Muniz, E.R.. Universidade Federal de Goiás; Brasil Fil: Barreto, L. P.. Universidade Federal de Goiás; Brasil Fil: Bernardo, C. C.. Universidade Federal de Goiás; Brasil Fil: Mascarin, G. M.. Universidade Federal de Goiás; Brasil Fil: Luz, C.. Universidade Federal de Goiás; Brasil Fil: Fernandes, Éverton. Universidade Federal de Goiás; Brasil

Nematophagous fungi are usually investigated and used in biocontrol of phytonematodes; recently, these fungi were found in association to the rhizosphere of crop plants colonizing their roots. Associated with plants, nematophagous fungi may also synthesize phytohormones or benefit the absorption of phosphorus by the plants. On the other hand, entomopathogenic fungi, especially Metarhizium spp. and Beauveria spp., frequently investigated for biological control of pests, have been reported as naturally occurring endophytes as well. These fungi positively affect plant growth, plant-disease antagonism, and rhizosphere colonization. In an increasing demand for more sustainable solutions to raise yields, plant productivity may be enhanced by exploring the potential of beneficial plant-entomopathogen interaction, as growth promoters. Accordingly, several entomopathogenic fungal species have the capacity to transfer nitrogen from dead insects to plants. The mode of action of entomopathogenic fungi as plant growth promoters shall be fully elucidated before these fungi being used in the field.

The aim of this study was to evaluate the effect of riboflavin supplementation of culture medium on conidial UV-A tolerance of M. acridum (Driver & Milner) (Hypocreales: Clavicipitaceae) and M. robertsii (Bischoff, Rehner & Humber) (Hypocreales: Clavicipitaceae). These fungi were produced on culture medium supplemented, or not supplemented, with riboflavin. Relative germination and expression patterns of some photoprotection-related genes were evaluated after irradiating with artificial UV-A, or with filtered solar radiation (> 320 nm; UV-A and visible radiation). M. acridum conidia harvested from riboflavin-supplemented culture medium demonstrated enhanced UV-A tolerance when irradiated with artificial UV-A. Nevertheless, relative germination of conidia of both species produced on riboflavin-supplemented medium and exposed to filtered solar radiation was significantly higher than those produced on medium not supplemented with riboflavin. Riboflavin increased the transcription of photolyases, laccases and polyketide synthase genes. However, each fungal species induced different genes patterns involved in DNA repair and photoprotection. The addition of riboflavin to the substrate used for mass production of Metarhizium spp. and the resulting enhancement of conidial tolerance to solar radiation may improve the effectiveness of these fungi in biological control programs., Instituto de Investigaciones Bioquímicas de La Plata

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.

The present study evaluated, in the laboratory, the virulence of Heterorhabditis bacteriophora LPP30 against engorged females of Rhipicephalus microplus; in addition, we evaluated, in semi-field tests, the effects of four isolates of Heterorhabditis spp. (i.e., H. bacteriophora HP88, Heterorhabditis baujardi LPP7, Heterorhabditis indica LPP1 and H. bacteriophora LPP30) in insect cadaver formulation against the non-parasitic phase of R. microplus. In the first experiment (in vitro), engorged females were exposed, in Petri dish, to H. bacteriophora LPP30 at 75, 150, 300, 600, 1200 and 2400 nematodes/tick (10 ticks per concentration tested). In the second experiment (semi-field), five engorged females and four cadavers of Galleria mellonella infected with nematodes were placed in pots with soil and grown Brachiaria decumbens; the pots were hold outdoor, exposed to natural environment conditions during the tests. In the third experiment (semi-field), 65 days after the cadavers had been placed in the pots for the second experiment, new engorged females (five per pot) were placed in the pots of the groups treated with H. bacteriophora HP88 or H. baujardi LPP7, to assess their persistence in the soil. In the first test, the percent control was higher than 95% in all treatment groups. In the second experiment, at day 22, the mean mortality rate was 78% in the groups treated with H. bacteriophora LPP30 or H. indica LPP1, and reached 100% and 98% when treated with H. bacteriophora HP88 and H. baujardi LPP7, respectively. In this experiment, the egg-laying inhibition index was higher than 90% in the groups treated with H. bacteriophora HP88 (97.2%) or H. baujardi LPP7 (91.9%). In the third experiment with H. bacteriophora HP88 and H. baujardi LPP7, the egg-laying inhibition index was 59.1% and 43.1%, respectively. We concluded that the isolate LPP30 was highly virulent under laboratory conditions, whereas in semi-field tests, HP88 and LPP7 were the most effective isolates. Moreover, HP88 and LPP7 remained active against engorged females of R. microplus in the soil for 65 days after application of EPN-infected cadavers of G. mellonella.

The Mormon cricket (MC) is an important pest in the western United States. This study evaluated the virulence in the laboratory of 32 isolates of Metarhizium spp. towards field-collected MCs. Additionally, four isolates were tested in outdoor field-cage studies. All 32 Metarhizium isolates were pathogenic towards the MC (could induce some mortality in the laboratory), including four isolates of the grasshopper-specialist species M. acridum. Virulence varied considerably among the isolates. Field studies, conducted in 2008–2009, showed a statistically significant effect of fungal treatments in both years. Pairwise comparison of the survival curves, however, revealed that, in 2008, three of the isolates did not differ statistically from the non-fungus control treatment. In 2009, all three of the isolates tested had significantly lower survival rates than the control treatment: MCs exposed to M. robertsii isolate DWR 346 had the lowest survival with a LT50 of 16 days. We hypothesize that the poor field performance resulted from a combination of negative environmental effects and isolate selection, and propose that further field studies with additional isolates are needed to identify an efficacious fungal agent for MC biocontrol.

Aims Control of diurnal Aedes aegypti with mycoinsecticides should consider the exposure of fungus-treated adults to sunlight, and especially to UV-B radiation that might affect activity of conidia applied on the mosquito′s surface. Methods and results Germination of M. anisopliae s.l. IP 46 conidia on SDAY medium was not affected at the lowest level of radiation with UV-B, 0.69 kJ m−2, but was retarded and reduced at higher 2.075 and 4.15 kJ m−2, and completely inhibited at ≥ 8.3 kJ m−2. In contrast, germination of conidia applied onto fiberglass nettings and exposed from 0–16.6 kJ m−2 did not differ significantly among among levels of irradiance exposure and the controls. There was also no significant impact of UV-B up to 16.6 kJ m−2 on adulticidal activity of IP and on the subsequent conidiogenesis on cadavers. The Quaite weighted UV-B irradiance in the laboratory (1152 mW m−2) was higher than the natural sunlight irradiance observed in the city of Goiânia in Central Brazil on midday (706 mW m−2 in August to 911 mW m−2 in October 2015). Conclusions UV-B does not impair the activity of IP 46 conidia applied previously to radiation on A. aegypti adults. Significance and Impact of Study Findings contribute to a better understanding of the effectiveness of M. anisopliae against day-active A. aegypti and its potential for biological mosquito control. This article is protected by copyright. All rights reserved.

The ability of aquatic stages of Aedes aegypti (Diptera: Culicidae), key vector of dengue, chikungunya and Zika viruses, to survive in minimal residues of water has remained unstudied. We found that larvae eclosed from up to 50% of eggs set in permanent water films on water agar (at 1–2% agar), and some (≤9.5%) of these first-instar larvae developed to second instar (L2). Mean larval survival was about 2 weeks; the longest periods of survival were 31.6 and 38.7 days in a water film and in free water controls, respectively. In another test, L3 moved in the films, with the greatest displacements of the larvae being up to 45 mm per day in the first 3 days; subsequent displacements in the water film were distinctly reduced during the next 20 days by which time all L3 had died. L3 did not detect or move in any directed manner in the film towards nearby small quantities of free water. Most pupae (≥93.7%) placed into water films or in water survived and developed to adults within 3 days. The unexpected survival of aquatic immature stages in desiccating breeding sites might be a reason for both the tenacious persistence of mosquito populations in seasons with infrequent rainfall and failures of classical control strategies against this important vector.

A new fungal pathogen of Culicinae (Diptera: Culicidae) adults, Conidiobolus macrosporus (Entomophthorales: Ancylistaceae), was detected and isolated during a survey of mosquito pathogens close to the city of Aruana, Goias State, in December 2014. The morphological characteristics of C. macrosporus are presented, and reasons for some uncertainty about this identification are discussed. The pathogenicity and high virulence of this fungus for Aedes aegypti were confirmed in laboratory conditions. Mortality of adults exposed to conidia was observed within 24 h of exposure to the pathogen, and increased to 100% as quickly as 3 days after inoculation (with the highest conidial concentration tested, 8.3 × 104 conidia/cm2). Repeated attempts to obtain genomic sequence data failed despite confirmations that the DNA extraction methods were themselves successful.

The effect of heat stress (45°C) versus non-heat stress (27°C) on germination of Metarhizium anisopliae sensu stricto (s.s.) isolate IP 119 was examined with conidia formulated (suspended) in pure mineral oil or in water (Tween 80, 0.01%), and then applied onto the cuticle of Rhipicephalus sanguineus sensu lato (s.l.) engorged females or onto culture medium (PDAY). In addition, bioassays were performed to investigate the effect of conidia heated while formulated in oil, then applied to blood-engorged adult R. sanguineus females. Conidia suspended in water then exposed to 45°C, in comparison to conidia formulated in mineral oil and exposed to the same temperature, germinated less and more slowly when incubated on either PDAY medium or tick cuticle. Also, conidial germination on tick cuticle was delayed in comparison to germination on artificial culture medium; for example, germination was 13% on tick cuticle 72h after inoculation, in contrast to 61.5% on PDAY medium. Unheated (27°C) conidia suspended in either water or oil and applied to tick cuticle developed appressoria 36h after treatment; whereas only heat-stressed conidia formulated in oil developed appressoria on tick cuticle. In comparison to conidia heated in mineral oil, there was a strong negative effect of heat on germination of conidia heated in water before being applied to arthropod cuticle. Nevertheless, bioassays [based primarily on egg production (quantity) and egg hatchability] exhibited high percentages of tick control regardless of the type of conidial suspension; i.e., water- or oil-formulated conidia, and whether or not conidia were previously exposed to heat. In comparison to aqueous conidial preparations, however, conidia formulated in oil reduced egg hatchability irrespective of heat or no-heat exposure. In conclusion, mineral-oil formulation protected conidia against heat-induced delay of both germination and appressorium production when applied to the cuticle of R. sanguineus.

Numerous isolates of an oomycete 'fungus', Leptolegnia chapmanii, are reported from Brazil for the first time. This aquatic pathogen was baited with Aedes aegypti sentinel larvae from stagnant, temporary bodies of water in selected locations under secondary tropical forest in and near the central Brazilian city of Goiânia and from more distant sites in the western and northern regions of the state of Goiás. Isolates were identified based on their morphological and developmental characters, comparative sequence data for the ITS and TEF loci, as well as their rapid activity against A. aegypti larvae. Taxonomic issues affecting the application of the name L. chapmanii and its typification are rectified. This study contributes to a better understanding of the presence and distribution of this oomycete in Brazil, its sequence-based identification, and of its potential as a biological agent against mosquito vectors.

Conidia of entomopathogenic fungi (EF) are the propagules most frequently used in arthropod biocontrol programs. This anamorphic form is essential for the infection process, including spore germination, penetration, vegetative growth, conidiogenesis and dissemination. Most EF are mesophilic and can develop between 10 and 40 °C, but optimal growth is between 25 and 35 °C. Abiotic factors, especially temperature (high or low) can determine their viability, virulence and success or failure of infection process. Temperature has the highest impact on conidial stress inhibiting metabolic processes, such as decreased morphogenesis during germination, protein denaturation and membrane disorganization. Several studies show that some strains of Beauveria spp., Metarhizium spp., and Isaria spp. exhibit conidial survival even when grown at high temperatures, indicating a relationship between conidial thermotolerance and their geographical isolation origin. Moreover, the high variability in fungal thermotolerance is also dependent of the culture media composition and growth condition. EF that grow at high temperatures do not grow at low temperatures and vice versa. Moreover, when growth conditions are not set at optimal temperatures, EF development is affected and their effectiveness in biological control programs of arthropods is reduced. Thermal stress directly impacts on fungal strains ability to target arthropods and their environmental activity performance. The screening for fungal strains with a higher thermotolerance and the improvement on conidial formulations may aid in optimizing the conditions for biocontrol agent application.

Aims: The effect of nutritional supplementation of two Metarhizium species with riboflavin (Rb) during production of conidia was evaluated on (i) conidial tolerance (based on germination) to UV-B radiation and on (ii) conidial expression following UV-B irradiation, of enzymes known to be active in photoreactivation, viz., photolyase (Phr), laccase (Lac) and polyketide synthase (Pks). Methods and Results: Metarhizium acridum (ARSEF 324) and Metarhizium robertsii (ARSEF 2575) were grown either on (i) potato dextrose agar medium (PDA), (ii) PDA supplemented with 1% yeast extract (PDAY), (iii) PDA supplemented with Rb (PDA+Rb), or (iv) PDAY supplemented with Rb (PDAY+Rb). Resulting conidia were exposed to 866·7 mW m−2 of UV-B Quaite-weighted irradiance to total doses of 3·9 or 6·24 kJ m−2. Some conidia also were exposed to 16 klux of white light (WL) after being irradiated, or not, with UV-B to investigate the role of possible photoreactivation. Relative germination of conidia produced on PDA+Rb (regardless Rb concentration) or on PDAY and exposed to UV-B was higher compared to conidia cultivated on PDA without Rb supplement, or to conidia suspended in Rb solution immediately prior to UV-B exposure. The expression of MaLac3 and MaPks2 for M. acridum, as well as MrPhr2, MrLac1, MrLac2 and MrLac3 for M. robertsii was higher when the isolates were cultivated on PDA+Rb and exposed to UV-B followed by exposure to WL, or exposed to WL only. Conclusions: Rb in culture medium increases the UV-B tolerance of M. robertsii and M. acridum conidia, and which may be related to increased expression of Phr, Lac and Pks genes in these conidia. Significance and Impact of the Study: The enhanced UV-B tolerance of Metarhizium spp. conidia produced on Rb-enriched media may improve the effectiveness of these fungi in biological control programs., Instituto de Investigaciones Bioquímicas de La Plata

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. Fil: Muniz, Elen R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina. Universidade Federal de Goiás; Brasil Fil: Catão, Alaine M. L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina. Universidade Federal de Goiás; Brasil Fil: Rueda Páramo, Manuel Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina. Universidade Federal de Goiás; Brasil Fil: Rodrigues, Juscelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina. Universidade Federal de Goiás; Brasil Fil: Rodrigues, Juscelino. Universidade Federal de Goiás; Brasil Fil: Lopez Lastra, Claudia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Garcia, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Fernandes, Éverton. Universidade Federal de Goiás; Brasil Fil: Luz, Wolf Christian. Universidade Federal de Goiás; Brasil

Ultraviolet (UV) radiation is a key limiting factor for biological pest control with entomopathogenic fungi. While littleis known about the impact of UV on Metarhizium anisopliae Metchnikoff (Sorokin) (Hypocreales: Clavicipitaceae)conidia in aquatic mosquito-breeding sites, this study determined the effect of UV-B on the viability and virulenceof M. anisopliae sensu lato (s.l.) strain IP 46 in the laboratory against Aedes aegypti (L.) (Diptera: Culicidae) larvae.Conidia were treated in cups under defined water depths (0, 1, 2, and 3 cm) to six different UV-B doses (0, 0.657,1.971, 3.942, 7.884, 11.826, or 15.768 kJ m−2) at 27 ± 2°C. The ability of treated conidia to germinate up to 24 hpostexposure on PDAY + benomyl + chloramphenicol medium at 25 ± 1°C was adversely affected by higher dosesof UV-B radiation regardless of the water depth. Germination, however, did not fall below 70% regardless of thetest conditions. In fact, conidial virulence against second-instar larvae was not affected by either the water depth(F3,84 = 0.3, P = 0.85) or any tested levels of UV-B radiation (F6,21 ≤ 1.2, P ≥ 0.39) including those distinctly higher thanmight be expected for tropical sites. These findings strengthen previous observations that IP 46 has significantpotential for use against A. aegypti larvae, even when exposed to elevated UV-B irradiance levels in the smallbreeding sites that are common for this important vector. Fil: Falvo, Marianel Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Albornoz Medina, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Rodrigues Juscelino. Universidade Federal de Goiás; Brasil Fil: Lopez Lastra, Claudia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Juan José Garcia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Fernandes, Éverton. Universidade Federal de Goiás; Brasil Fil: Luz, Christian. Universidade Federal de Goiás; Brasil

Conidia of the entomopathogenic fungus Metarhizium anisopliae (Ascomycota: Clavicipitaceae) were assessed against Rhipicephalus sanguineus (Arachnida: Ixodidae) eggs under laboratory conditions. Clusters of 25 eggs were applied either directly with the fungal conidial formulations or set on previously fungus-treated filter paper. Treatments consisted of conidia formulated in water or an oil-in-water emulsion at final concentrations of 3.3 × 10(3), 10(4), 3.3 × 10(4), 10(5), or 3.3 × 10(5) conidia/cm(2). The development of mycelium and new conidia on egg clusters incubated at 25 °C and humidity close to saturation depended on conidial concentration, formulation, and application technique. No larvae eclosed from eggs after direct applications of conidia regardless of the formulation. The eclosion and survival of larvae from indirectly treated egg clusters depended on the type of formulation and conidial concentration applied. Oil-in-water formulations of conidia demonstrated the highest activity against eggs of R. sanguineus.

Entomopathogenic fungi secrete toxic secondary metabolites during the invasion of the insect hemocoel as part of the infection process. Although these compounds have been frequently mentioned as virulence factors, the roles of many of them remain poorly understood, including the question of whether they are expressed during the infection process. A major hurdle to this issue remains the low sensitivity of biochemical detection techniques (e.g., HPLC) within the complex samples that may contain trace quantities of fungal molecules inside the insect. In this study, quantitative reverse transcription real-time PCR (qRT-PCR) was used to measure the transcript levels within the insect fungal pathogen Beauveria bassiana, that encode for the synthetase enzymes of the secondary metabolites tenellin (BbtenS), beauvericin (BbbeaS) and bassianolide (BbbslS) during the infection of Triatoma infestans, a Chagas disease insect vector. Absolute quantification was performed at different time periods after insect treatment with various concentrations of propagules, either by immersing the insects in conidial suspensions or by injecting them with blastospores. Both BbtenS and BbbeaS were highly expressed in conidia-treated insects at days 3 and 12 post-treatment. In blastospore-injected insects, BbtenS and BbbeaS expression peaked at 24h post-injection and were also highly expressed in insect cadavers. The levels of BbbslS transcripts were much lower in all conditions tested. The expression patterns of insect genes encoding proteins that belong to the T. infestans humoral immune system were also evaluated with the same technique. This qPCR-based methodology can contribute to decifering the dynamics of entomopathogenic fungal infection at the molecular level.

Pr1 is a subtilisin-like protease produced by Metarhizium spp. entomopathogenic fungi, and it is recog- nized as heavily involved in the initial steps of the fungal invasion of arthropod-host cuticles. In the current study, cor- relation was sought between mortality of tick larvae and co- nidial Pr1 levels of one Metarhizium anisopliae senso latu (s.l.) isolate (CG 148). Conidia with different levels of pr1 gene expression and enzymatic activity were obtained by pro- ducing them on either artificial medium (to yield low Pr1 activity) or on Rhipicephalus microplus cadavers (to yield high Pr1 activity). Conidial proteolytic activity was assessed using N-suc-ala-ala-pro-phe-ρNA as the chromogenic sub- strate, and pr1 expression was profiled by qPCR using three genes (gpd, try, and tef) as reference genes. Pr1 enzymatic (proteolytic) activity on conidia obtained from tick cadavers was 36 U mg −1 in comparison to 4 U mg −1 on conidia from PDA medium. Also, pr1 gene expression level was ten times higher in conidia from tick cadavers compared to PDA medi- um. Bioassays of M. anisopliae s.l. CG 148 spores with ele- vated Pr1 proteolytic activity and gene expression levels did not demonstrate increased virulence (= significant changeper- cent mortality of tick larvae). The minimal levels of Pr1 on conidia produced on artificial medium was adequate to afford high levels of virulence, and the elevated amounts of the en- zyme on tick-cadaver-produced conidia did not induce elevat- ed larval mortality. As long as some Pr1 activity was present, fungal virulence of isolate CG 148 against tick larvae was not elevated by increased levels of conidial Pr1.

The air-breathing snail Biomphalaria glabrata proliferates in stagnant freshwater, and nothing is known about the survival of eggs in intermittently (rather than perpetually) wet habitats. In the present study their egg masses matured, and juveniles subsequently eclosed and were mobile in a stable water film of transitory habitats simulated by two different simple test devices described here. The viability of eggs maintained in an unstable film however, was diminished. The maturation of egg masses in a water film or in water was significantly prevented by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. The efficiency depended on the fungal propagule and test environment. Hyphal bodies were more effective against egg masses than conidia. This appears to be a first report of activity of either entomopathogen against a mollusc. Both devices offer accurate and reproducible conditions to test both biological questions and the effects of substances or pathogens against B. glabrata egg masses in water films.

A project from 2013 to 2017 sought to discover pathogenic fungi and oomycetes from dipteran species that are vectors of major diseases of humans and animals in central Brazil and to begin evaluating the potential of these pathogens as potential biological control agents concentrated on mosquito larvae. Some collecting sites proved to be especially productive for pathogens of naturally occurring mosquito species and for placements of healthy sentinel larvae of Aedes aegypti in various sorts of containers in a gallery forest in the Santa Branca Ecoturismo Private Reserve of Natural Patrimony (RPPN) near Terezópolis de Goiás (GO). Collections during May-April of 2016 and February 2017 yielded a few dead mosquito larvae of an undetermined Onirion sp. (Culicidae: Sabethini) whose hemocoels contained many ovoid, thick-walled, yellow-golden to golden-brown, ovoid thick-walled resistant sporangia, 38.3 ± 4 × 22.8 ± 2.3 µm, decorated by numerous, closely and randomly spaced punctations of variable size and shape. These were the first indisputable collections from Brazil of any Coelomomyces species. Comparisons of the morphology of these sporangia with those of other species of Coelomomyces, confirmed that this Brazilian fungus represented a new species that is described here as Coelomomyces santabrancae. Fil: Rueda Páramo, Manuel Enrique. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Montalva, C.. Universidade Federal de Goiás; Brasil. Universidad Austral de Chile; Chile Fil: Arruda, W.. Universidade Federal de Goias. Instituto de Ciencias Biologicas; Brasil Fil: Fernandes, Éverton. Universidade Federal de Goiás; Brasil Fil: Luz, Wolf Christian. Universidade Federal de Goiás; Brasil Fil: Humber, Richard A.. United States Department of Agriculture. Agricultural Research Service; Argentina

Entomopathogenic fungi play a central role in Brazil’s biopesticide market. Approximately 50% of registered microbial biopesticides comprise mycoinsecticides and/or mycoacaricides consisting of hypocrealean fungi, with most based on Metarhizium anisopliae sensu stricto (s. str.) and Beauveria bassiana s. str. These fungi are mainly used to control spittlebugs in sugarcane fields and whiteflies in row crops, respectively, with annual applications surpassing three million hectares. Research also emphasizes the potential of fungal entomopathogens to manage arthropod vectors of human diseases. Most registered fungal formulations comprise wettable powders or technical (non-formulated) products, with relatively few new developments in formulation technology. Despite the large area treated with mycoinsecticides (i.e., approx. 2 million ha of sugarcane treated with M. anisopliae and 1.5 million ha of soybean treated with B. bassiana), their market share remains small compared with the chemical insecticide market. Nevertheless, several major agricultural companies are investing in fungus-based products with the aim at achieving more sustainable IPM programs for major pests in both organic and conventional crops. Government and private research groups are pursuing innovative technologies for mass production, formulation, product stability and quality control, which will support cost-effective commercial mycoinsecticides. Here, we summarize the status of mycoinsecticides currently available in Brazil and discuss future prospects.

Little is known about the ovicidal effects of fungi that attack nymphs and adults of triatomine vectors. A combined formulation of Metarhizium anisopliae IP 46 conidia prepared with diatomaceous earth (DE) and vegetable oil was tested against eggs of Triatoma infestans. Eggs were highly susceptible to fungal infection at relative humidity close to saturation [>98% relative humidity (RH)] but not at 75% RH regardless of the formulation applied. Susceptibility of eggs decreased with longer post-ovipositional embryonation periods before treatments. The eventual eclosion of nymphs was best suppressed by application of conidia prepared with DE + oil and at a >98% RH incubation. Moreover, nymphs were less affected by the fungus when exposed for only a 24-h period after eclosion to a treated surface than individuals that were in constant contact with the conidia. These findings contribute to a better understanding of the potential of M. anisopliae as an agent against all developmental stages of T. infestans.

INTRODUCTION:The mosquito Aedes aegypti has evolved resistance to pyrethroid insecticides. The present study evaluated Ae. aegypti from Goiânia for the resistant phenotype and for mutations associated with resistance.METHODS:Insecticide dose-response bioassays were conducted on mosquitoes descended from field-collected eggs, and polymerase chain reaction (PCR) was used to genotype 90 individuals at sites implicated in pyrethroid resistance.RESULTS:All mosquito populations displayed high levels of resistance to deltamethrin, as well as high frequencies of the 1016Ile kdr and 1534Cys kdrmutations.CONCLUSIONS:Aedes aegypti populations in the Goiânia area are highly resistant to deltamethrin, presumably due to high frequencies of kdr(knockdown-resistance) mutations.

Impact of UV-radiation in entomopathogens in aquatic environments remains little investigated. The present study reports on the effect of UV-A on the larvicidal activity of Leptolegnia chapmanii zoospores in Aedes aegypti; on the production of zoospores in larvae killed by the pathogen and then exposed to UV-A; and on the activity of these zoospores against healthy larvae. Whereas the virulence of free zoospores in A. aegypti larvae was affected by a UV-A exposure time longer than 10min, production of zoospores in larvae and their virulence were not hampered at a maximal 8h exposure of dead larvae to UV-A. Findings suggest that dead larvae and zoosporangia provide a certain protection to zoospores against UV-A and emphasize the susceptibility of free encysted zoospores to such radiation.

Aims: To study the marked resistance of Periplaneta americana to entomopathogenic Metarhizium anisopliae. Methods and Results: The low susceptibility of 4th instar nymphs applied topically with conidia seemed to be related to an active removal of conidia by the cockroach and to a disabled or retarded germination and subsequent development of conidia on the cuticle (up to 80% germination in the next 7 days after application). Inhibitions or delays of germination were related to the composition of the epicuticular fatty acids (30·1% w/w oleic, 28·3% w/w linoleic, 24·5% w/w palmitic and 11·7% w/w stearic acid) reported here. Propagules invading the nymphs through the cuticle took at least 3 days to reach the haemocoel, and no propagules were found after day 8 post-treatment. Strain IP 46 infected >50% of nymphs treated with doses ≥2 × 104 hyphal bodies (HB) nymph−1 and reduced the survival of nymphs ≤50%. Most nymphs (>70%) survived after injection of 6 × 103 and 2 × 103 HB nymph−1. Conclusions: Findings emphasize a distinct resistance of nymphs of the American cockroach to infections by M. anisopliae. Significance and Impact of the Study: Our findings provide support for the development of biological control of this synanthropic cockroach pest. Fil: Gutierrez, Alejandra Concepción. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Machado, J. A. R.. Universidade Federal de Goiás; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Hubner Campos, R.. Universidade Federal de Goiás; Brasil Fil: Pennisi, M. A.. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina Fil: Rodrigues, J.. Universidade Federal de Goiás; Brasil Fil: Lopez Lastra, Claudia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Garcia, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Fernandes, É. K. K.. Universidade Federal de Goiás; Brasil Fil: Luz, C.. Universidade Federal de Goiás; Brasil

Rhipicephalus microplus (Canestrini) is an ectoparasite accountable for great economic losses. The use of entomopathogenic fungi to control arthropods has shown promising responses. The present study evaluated the virulence of Isaria farinosa (Holmsk.) Fr., Isaria fumosorosea (Wize) Brown and Smith, and Purpureocillium lilacinum (=Paecilomyces lilacinus) (Thom.) Samson to engorged females, eggs, and larvae of R. microplus. There were four treatment groups (10(5), 10(6), 10(7), and 10(8) conidia ml(-1)) and the control group (water and Tween 80, 0.1% v/v). The treatment was based on immersion of the specimen in 1 ml of the suspension or control solution. The study observed changes in egg viability and larval mortality after treatment. The results showed that I. farinosa, P. lilacinum, and I. fumosorosea caused alterations in the biological parameters of R. microplus ticks. I. fumosorosea presented the greatest potential to control R. microplus engorged females in vitro, causing a 49% decrease in nutritional index. All fungal isolates presented significant reduction in the egg production index. I. farinosa reduced the hatching percentage if the eggs were treated with the two highest conidial concentrations. All conidial concentrations of I. fumosorosea were able to reduce the hatching percentage significantly. All tested isolates showed pathogenicity toward unfed R. microplus larvae. As far as we know, this is the first study reporting the effect in vitro of I. farinosa, I. fumosorosea, and P. lilacinum to different developmental stages of R. microplus ticks.