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Item Efficacy of entomopathogenic nematodes in insect cadaver formulation against engorged females of Rhipicephalus microplus (Acari: Ixodidae) in semi-field conditions(2020) Monteiro, Caio Marcio de Oliveira; Brito, Letícia Coelho Miranda; Paula, Luiza Gabriella Ferreira de; Fernandes, Éverton Kort Kamp; Dolinski, Claudia de Melo; Bittencourt, Vânia Rita Elias Pinheiro; Furlong, John; Prata, Márcia Cristina de AzevedoThe 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.Item Inorganic pellets containing microsclerotia of Metarhizium anisopliae: a new technological platform for the biological control of the cattle tick Rhipicephalus microplus(2021) Santos, Thainá Rodrigues; Paixão, Flávia Regina Santos da; Catão, Alaine Maria Lopes; Muniz, Elen Regozino; Silva, Cárita de Souza Ribeiro e; Taveira, Stephânia Fleury; Luz, Wolf Christian; Mascarin, Gabriel Moura; Fernandes, Éverton Kort Kamp; Marreto, Ricardo NevesThis 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.Item Relative humidity impacts development and activity against Aedes aegypti adults by granular formulations of Metarhizium humberi microsclerotia(2021) Rodrigues Filho, Juscelino; Catão, Alaine Maria Lopes; Santos, Amanda Soares dos; Paixão, Flávia Regina Santos da; Santos, Thainá Rodrigues; Mercado Martinez, Juan Gabriel; Marreto, Ricardo Neves; Mascarin, Gabriel Moura; Fernandes, Éverton Kort Kamp; Humber, Richard Alan; Luz, Wolf ChristianThe 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.Item UV-B tolerances of conidia, blastospores, and microsclerotia of Metarhizium spp. entomopathogenic fungi(2021) Corval, Amanda Rocha da Costa; Silva, Emily Mesquita da; Corrêa, Thaís Almeida; Silva, Cárita de Souza Ribeiro e; Bitencourt, Ricardo de Oliveira Barbosa; Fernandes, Éverton Kort Kamp; Bittencourt, Vânia Rita Elias Pinheiro; Roberts, Donald Wilson; Golo, Patrícia SilvaThe 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.Item Activity against Musca domestica of hypocrealean fungi isolated from culicids in central Brazil and formulated in vermiculite(2022) Páramo, Manuel Enrique Rueda; Santos, Karine Rodrigues dos; Gomes, Marcos Daniel Filgueiras; Fernandes, Éverton Kort Kamp; Montalva, Cristian; Humber, Richard Alan; Luz, Wolf ChristianMusca 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 × 107 and 108 conidia g−1 substrate, respectively. Fifty percent of adults were killed within 4 to 5 days of exposure initially as pupae close to emergence to substrate treated with conidia of IP 478 or IP 433 at 1.1 × 108 conidia g−1, respectively. The other fungal strains tested were less virulent. The results demonstrate high potentials for conidial preparations in vermiculite of IP 433 and IP 478 as candidates for the biological control of both pre-pupating larvae, pupae, and emerging adults of houseflies.Item First report of Leptolegnia chapmanii (Peronosporomycetes: Saprolegniales) affecting mosquitoes in central Brazil(2016) Montalva, Cristian; Santos, Karine Rodrigues dos; Collier, Karin Ferretto Santos; Rocha, Luiz Fernando Nunes; Fernandes, Éverton Kort Kamp; Castrillo, Louela A.; Luz, Wolf Christian; Humber, Richard AlanNumerous 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 bod ies 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 bet ter 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.Item Phenotype responses to abiotic stresses, asexual reproduction and virulence among isolates of the entomopathogenic fungus Cordyceps javanica (Hypocreales: Cordycipitaceae)(2018) Mascarin, Gabriel Moura; Pereira Junior, Ronaldo Alves; Fernandes, Éverton Kort Kamp; Quintela, Eliane Dias; Dunlap, Christopher A.; Arthurs, Steven PaulSelecting entomopathogenic fungal isolates with resilience to environmental stresses, optimal mass production characteristics, and with high virulence to target pests favors the development of mycopesticides. A case in point, Cordyceps (= Isaria) javanica has been extensively investigated for non-chemical control of whiteflies worldwide. We phylogenetically characterized 11 native C. javanica isolates from Northeastern and Central Brazil. These isolates were screened for tolerance to heat-shock, UV-B radiation, osmotic and oxidative stresses, as well as conidial production on cereal grain and insecticidal activity against the whitefly Bemisia tabaci (MEAM 1) in the laboratory. All isolates were pathogenic to whiteflies and significant (3-fold) differences in median lethal con centration were observed among isolates. Furthermore, pronounced differences among isolates were found for stress factors and conidial production. Using principal component analysis, our results highlighted three major clusters formed by isolates (i) resistant to osmotic and oxidative stress, (ii) resilient to UV-B, and (iii) with high virulence, conidial production and heat tolerance. Overall, isolate CG1228 performed best based on multi-stress resistance, mass production and virulence attributes in the laboratory. This study highlights the importance of exploring natural variation in entomopathogenic fungi for selection of appropriate isolates for effective bio control of insect pests coupled with mass production characteristics and abiotic stress tolerances.Item Riboflavin induces Metarhizium spp. to produce conidia with elevated tolerance to UV-B, and upregulates photolyases, laccases and polyketide synthases genes(2018) Pereira Júnior, Ronaldo Alves; Bonnet, Carla Huarte; Paixão, Flávia Regina Santos da; Roberts, Donald Wilson; Luz, Wolf Christian; Pedrini, Nicolás; Fernandes, Éverton Kort KampThe 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 programsItem Activity of additives and their effect in formulations of Metarhizium anisopliae s.l. IP 46 against Aedes aegypti adults and on post mortem conidiogenesis(2019) Rodrigues Filho, Juscelino; Borges, Priscilla Rodrigues; Fernandes, Éverton Kort Kamp; Luz, Wolf ChristianBackground: 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 sub tropics. 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.Item Carnauba wax enhances the insecticidal activity of entomopathogenic fungi against the blowfly Lucilia sericata (Diptera: Calliphoridae)(2020) Muniz, Elen Regozino; Bedini, Stefano; Sarrocco, Sabrina; Vannacci, Giovanni; Mascarin, Gabriel Moura; Fernandes, Éverton Kort Kamp; Conti, BarbaraBlowfly, 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 en tomopathogenic fungi, Beauveria bassiana, Beauveria pseudobassiana and Akanthomyces muscarius against L. ser icata. 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 sus pension (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 sus tainable tool to reduce the over-reliance on chemical insecticides and their risk of resistance.Item Toxoplasma gondii in cattle in Brazil: a review(2020) Gomes, Daniella Ferreira Cordeiro; Krawczak, Felipe da Silva; Oliveira, Cairo Henrique Sousa de; Ferreira Júnior, Álvaro; Fernandes, Éverton Kort Kamp; Lopes, Welber Daniel Zanetti; Sevá, Anaiá da Paixão; Gennari, Solange MariaToxoplasma 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.Item The Msn2 transcription factor regulates acaricidal virulence in the fungal pathogen Beauveria bassiana(2021) Muniz, Elen Regozino; Silva, Cárita de Souza Ribeiro e; Arruda, Walquíria; Keyhani, Nemat O.; Fernandes, Éverton Kort KampBeauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress–response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress–response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout (DBbmsn2) and complemented mutant (DBbmsn2/Bbmsn2) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and DBbmsn2 treatments, whereas treatment with the wild-type or DBbmsn2/ Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with DBbmsn2 was lower than in the groups treated with wild-type (56.1%) or DBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or DBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of DBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with DBbmsn2 than those treated with the wild-type or DBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.Item New insights into the infection of the American cockroach Periplaneta americana nymphs with Metarhizium anisopliae s.l. (Ascomycota: Hypocreales)(2016) Concepción Gutierrez, Alejandra; Machado, Jose Arimateia Rabelo; Hubner-Campos, Rayssa Fátima; Pennisi, M. A.; Lopez Lastra, Claudia Cristina; García, Juan José; Fernandes, Éverton Kort Kamp; Luz, Wolf Christian; Rodrigues Filho, JuscelinoAims 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.Item UV‐B radiation reduces in vitro germination of Metarhizium anisopliae s.l. but does not affect virulence in fungus‐treated Aedes aegypti adults and development on dead mosquitoes(2016) Falvo, Marianel Lucía; Pereira Junior, Ronaldo Alves; Rodrigues Filho, Juscelino; Lopez Lastra, Claudia Cristina; García, Juan José; Fernandes, Éverton Kort Kamp; Luz, Wolf ChristianAims 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 Metarhizium 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 fibreglass nettings and exposed from 0 to 16·6 kJ m−2 did not differ significantly 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 the adulticidal activity of IP 46 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 the 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.Item Conidiobolus macrosporus (Entomophthorales), a mosquito pathogen in central Brazil(2016) Montalva, Cristian ; Rocha, Luiz Fernando Nunes; Fernandes, Éverton Kort Kamp; Humber, Richard Alan; Luz, Wolf ChristianA 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 Aruanã, Goiás 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.Item Tolerance to abiotic factors of microsclerotia and mycelial pellets from Metarhizium robertsii, and molecular and ultrastructural changes during microsclerotial differentiation(2021) Paixão, Flávia Regina Santos da; Huarte Bonnet, Carla; Silva, Cárita de Souza Ribeiro e; Mascarin, Gabriel Moura; Fernandes, Éverton Kort Kamp; Pedrini, NicolásMetarhizium species fungi are able to produce resistant structures termed microsclerotia, formed by compact 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 ultraviolet B (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 16 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 and Mrlac2; and Mrlac3. We concluded that an oxidative stress scenario is induced during microsclerotia differentiation in M. robertsii and confirmed that because of 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.Item Innovative granular formulation of Metarhizium robertsii microsclerotia and blastospores for cattle tick control(2021) Marciano, Allan Felipe; Mascarin, Gabriel Moura; Franco, Renato Felipe Ferreira; Golo, Patrícia Silva; Jaronski, Stefan T.; Fernandes, Éverton Kort Kamp; Bittencourt, Vânia Rita Elias PinheiroThe 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-feld 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 signifcantly reduced the number of tick larvae during the humid season, reaching at least 64.8% relative efcacy. 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.Item Cuticular lipids as a first barrier defending ixodid ticks against fungal infection(2022) Silva, Cárita de Souza Ribeiro e; Muniz, Elen Regozino; Lima, Valesca Henrique; Bernardo, Cíntia das Chagas; Arruda, Walquíria; Castro, Rosane Nora; Golo, Patrícia Silva; Angelo, Isabele da Costa; Fernandes, Éverton Kort KampThe chemical composition of tick cuticles acts as a barrier to pathogens and may limit infection by entomopathogenic fungi. This study characterized the cuticular neutral lipids (NL) and hydrocarbons (HCs) of four ixodid ticks that are widely distributed in Brazil. HC extracts were analyzed by gas chromatography-mass spectrometry and used to challenge Beauveria bassiana IP361 and Metarhizium robertsii IP146; the effect of cuticular extracts in fungal growth were evaluated by disk diffusion and conidial viability assays. In addition, conidial germination on the tick cuticle was evaluated by scanning electron microscopy, and NL from ticks treated with fungi were assessed by thin layer chromatography. Six HCs were exclusively identified in Amblyomma sculptum. Additionally, cuticle extracts from Dermacentor nitens and A. sculptum inhibited the growth of M. robertsii IP146 and reduced conidial germination of B. bassiana IP361 to 70% and 49%, respectively; the same extracts also produced cytotoxic effects, with conidial death above 30% and 60%. Electron micrographs showed a delayed germination of conidia incubated for 48 h or 72 h on D. nitens and A. sculptum. The lipid profile of A. sculptum treated with fungi was not significantly altered; triacylglycerol was not detected in the cuticle extracts of any other tick species. Finally, A. sculptum and D. nitens cuticles have lipid components that may limit the development of M. robertsii.Item Increased heat tolerance afforded by oil-based conidial formulations of Metarhizium anisopliae and Metarhizium robertsii(2017) Paixão, Flávia Regina Santos da; Muniz, Elen Regozino; Barreto, Lucas Prado; Bernardo, Cíntia das Chagas; Mascarin, Gabriel Moura; Luz, Wolf Christian; Fernandes, Éverton Kort KampThe 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.Item Differential susceptibility of blastospores and aerial conidia of entomopathogenic fungi to heat and UV-B stresses(2020) Bernardo, Cíntia das Chagas; Pereira Junior, Ronaldo Alves; Luz, Wolf Christian; Mascarin, Gabriel Moura; Fernandes, Éverton Kort KampWe investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores were produced in solid or liquid Adámek-modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m−2 under artificial UV-B radiation. Our results showed that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in regions where heat and UV-B are limiting environmental factors.