Synthesis and secretion of volatile short-chain fatty acids in Triatoma infestans infected with Beauveria bassiana.

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.

Riboflavin induces Metarhizium spp. to produce conidia with elevated tolerance to UV-B, and upregulates photolyases, laccases and polyketide synthases genes.

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.

First report of Coelomomyces santabrancae sp. nov. (Blastocladiomycetes: Blastocladiales) infecting mosquito larvae (Diptera: Culicidae) in central Brazil.

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±2.3µm, 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.

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.

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 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.

New insights into the infection of the American cockroach Periplaneta americana nymphs with Metarhizium anisopliae s.l. (Ascomycota: Hypocreales).

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.

Efficacy of Tolypocladium cylindrosporum against Aedes aegypti eggs, larvae and adults.

AIMS: The aim of this study was to test the activity of seven Tolypocladium cylindrosporum isolates against different developmental stages of Aedes aegypti. METHODS AND RESULTS: Ovicidal activity (≤30% of eclosion; at a 84% eclosion of control eggs) and lowest cumulative emergence of adults (≤30%; at a 66·3% control emergence) originating from eclosing larvae was found 15 and 30 days, respectively, after applying conidia of ARSEF 962 or 1580 onto the eggs. All isolates induced larval mortality when third-instar larvae were treated with conidia (≤5% control mortality); the lowest lethal concentration (LC50) at 5 days after treatment was found with ARSEF 1580 (9·2 × 10(5) conidia ml(-1)), and the shortest lethal time (LT50 ) with ARSEF 2912 (2·8 days at 3·3 × 10(6) conidia ml(-1)). The pathogenicity of T. cylindrosporum to A. aegypti adults is corroborated. ARSEF 1027, 1580 and 2912 induced the highest cumulative mortality in adults (≥90%) 15 days after application (≤5% control mortality). CONCLUSIONS: Findings emphasized an isolate- and developmental stage-related virulence of T. cylindrosporum. SIGNIFICANCE AND IMPACT OF THE STUDY: ARSEF 1580, in the group of tested isolates, is the most promising isolate for use against terrestrial eggs, aquatic larvae and adults of this important vector.

Nomuraea rileyi as biological control agents of Rhipicephalus microplus tick.

Nomuraea rileyi, a fungus pathogenic to insects, has been widely used for biological control of agricultural pests in Brazil. This study investigates the effects of N. rileyi, isolates Nr 138, Nr 151, and Nr 177, to eggs, larvae, and engorged females of Rhipicephalus microplus tick. Specimens were immersed in 1 ml of conidial suspension for 3 min, whereas the control group was immersed in 0.01% Tween 80 water solution. The isolate Nr 138 controlled 67.37% of ticks when the highest conidial concentration was used, 10(8) conidia ml(-1). The isolate Nr 177 significantly reduced the percentage of hatch of larvae from eggs treated with 10(8) conidia ml(-1). Conversely, the isolate Nr 151 was not virulent to eggs, larvae, or adults. Variability in virulence was observed among the N. rileyi isolates investigated in the current study-Nr 138 was more virulent to engorged females, while Nr 177 was more virulent to unfed larvae. Although N. rileyi proved to be virulent to several stages of R. microplus, the results obtained in this study indicate that N. rileyi does not appear to be a remarkable biological control agent for R. microplus.

Characterization of Metarhizium species and varieties based on molecular analysis, heat tolerance and cold activity.

AIMS: The genetic relationships and conidial tolerances to high and low temperatures were determined for isolates of several Metarhizium species and varieties. METHODS AND RESULTS: Molecular-based techniques [AFLP and rDNA (ITS1, ITS2 and 5.8S) gene sequencing] were used to characterize morphologically identified Metarhizium spp. isolates from a wide range of sources. Conidial suspensions of isolates were exposed to wet heat (45 + or - 0.2 degrees C) and plated on potato dextrose agar plus yeast extract (PDAY) medium. After 8-h exposure, the isolates divided clearly into two groups: (i) all isolates of Metarhizium anisopliae var. anisopliae (Ma-an) and Metarhizium from the flavoviride complex (Mf) had virtually zero conidial relative germination (RG), (ii) Metarhizium anisopliae var. acridum (Ma-ac) isolates demonstrated high heat tolerance (c. 70-100% RG). Conidial suspensions also were plated on PDAY and incubated at 5 degrees C for 15 days, during which time RGs for Ma-an and Ma-ac isolates were virtually zero, whereas the two Mf were highly cold active (100% RG). CONCLUSIONS: Heat and cold exposures can be used as rapid tools to tentatively identify some important Metarhizium species and varieties. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of Metarhizium spp. currently relies primarily on DNA-based methods; we suggest a simple temperature-based screen to quickly obtain tentative identification of isolates as to species or species complexes.

Genetic diversity among Brazilian isolates of Beauveria bassiana: comparisons with non-Brazilian isolates and other Beauveria species.

AIMS: The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp. METHODS AND RESULTS: The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp. CONCLUSION: Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu. The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil.