Congo red induces trans-priming to UV-B radiation in Metarhizium robertsii.

Metarhizium spp. is used as a biocontrol agent but is limited because of low tolerance to abiotic stress. Metarhizium robertsii is an excellent study model of fungal pathogenesis in insects, and its tolerance to different stress conditions has been extensively investigated. Priming is the time-limited pre-exposure of an organism to specific stress conditions that increases adaptive response to subsequent exposures. Congo red is a water-soluble azo dye extensively used in stress assays in fungi. It induces morphological changes and weakens the cell wall at sublethal concentrations. Therefore, this chemical agent has been proposed as a stressor to induce priming against other stress conditions in entomopathogenic fungi. This study aimed to evaluate the capacity of Congo red to induce priming in M. robertsii. Conidia were grown on potato dextrose agar with or without Congo red.The tolerance of conidia produced from mycelia grown in these three conditions was evaluated against stress conditions, including osmotic, oxidative, heat, and UV-B radiation. Conidia produced on medium supplemented with Congo red were significantly more tolerant to UV-B radiation but not to the other stress conditions assayed. Our results suggest that Congo red confers trans-priming to UV-B radiation but not for heat, oxidative, or osmotic stress.

Entomopathogenic fungi and Schinus molle essential oil: The combination of two eco-friendly agents against Aedes aegypti larvae.

Aedes aegypti transmits arbovirus, which is a public health concern. Certain filamentous fungi have the potential to control the disease. Here, the effects of Metarhizium anisopliae s.l. CG 153, Beauveria bassiana s.l. CG 206 and Schinus molle L. were investigated against Aedes aegypti larvae. In addition, the effect of essential oil on fungal development was analyzed. Fungal germination was assessed after combination with essential oil at 0.0025 %, 0.0075 %, 0.005 %, or 0.01 %; all of the oil concentrations affected germination except 0.0025 % (v/v). Larvae were exposed to 0.0025 %, 0.0075 %, 0.005 %, or 0.01 % of the essential oil or Tween 80 at 0.01 %; however, only the essential oil at 0.0025 % achieved similar results as the control. Larvae were exposed to fungi at 107 conidia mL-1 alone or in combination with the essential oil at 0.0025 %. Regardless of the combination, M. anisopliae reduced the median survival time of mosquitoes more than B. bassiana. The cumulative survival of mosquitoes exposed to M. anisopliae alone or in combination with essential oil was 7.5 % and 2 %, respectively, and for B. bassiana, it was 75 % and 71 %, respectively. M. anisopliae + essential oil had a synergistic effect against larvae, whereas B. bassiana + essential oil was antagonistic. Scanning and transmission electron microscopy, and histopathology confirmed that the interaction of M. anisopliae was through the gut and hemocoel. In contrast, the mosquito's gut was the main route for invasion by B. bassiana. Results from gas chromatography studies demonstrated sabinene and bicyclogermacrene as the main compounds of S. molle, and the in-silico investigation found evidence that both compounds affect a wide range of biological activity. For the first time, we demonstrated the potential of S. molle and its interaction with both fungal strains against A. aegypti larvae. Moreover, for the first time, we reported that S. molle might be responsible for significant changes in larval physiology. This study provides new insights into host-pathogen interplay and contributes to a better understanding of pathogenesis in mosquitoes, which have significant consequences for biological control strategies.

Neem oil increases the persistence of the entomopathogenic fungus Metarhizium anisopliae for the control of Aedes aegypti (Diptera: Culicidae) larvae.

BACKGROUND: The entomopathogenic fungus Metarhizium anisopliae is a candidate for the integrated management of the disease vector mosquito Aedes aegypti. Metarhizium anisopliae is pathogenic and virulent against Ae. aegypti larvae; however, its half-life is short without employing adjuvants. Here, we investigated the use of neem oil to increase virulence and persistence of the fungus under laboratory and simulated field conditions. METHODS: Neem was mixed with M. anisopliae and added to recipients. Larvae were then placed in recipients at 5-day intervals for up to 50 days. Survival rates were evaluated 7 days after exposing larvae to each treatment. The effect of neem on conidial germination following exposure to ultraviolet radiation was evaluated under laboratory conditions. Statistical tests were carried out using ANOVA and regression analysis. RESULTS: Laboratory bioassays showed that the fungus alone reduced survival to 30% when larvae were exposed to the treatment as soon as the suspension had been prepared (time zero). A mixture of fungus + neem resulted in 11% survival at time zero. The combination of fungus + neem significantly reduced larval survival rates even when suspensions had been maintained for up to 45 days before adding larvae. For simulated-field experiments 1% neem was used, even though this concentration is insecticidal, resulting in 20% survival at time zero. However, this toxic effect was reduced over time. When used alone under simulated-field conditions the fungus rapidly lost virulence. The formulation fungus + neem effectively maintained fungal virulence, with larval survival rates significantly reduced for up to 45 days after preparation of the suspensions. The effective half-life of the fungus or neem when used separately was 6 and 13 days, respectively. The half-life of fungus formulated in 1% neem was 34 days. Conidia suspended in neem maintained high levels of germination even following a 2-h exposure to ultraviolet radiation. CONCLUSIONS: A combination of the entomopathogenic fungus M. anisopliae with neem oil effectively increases the half-life and virulence of the fungus when tested against Ae. aegypti larvae, even under simulated field conditions. Neem oil also protected the fungus from the damaging effects of ultraviolet radiation.

Increased virulence in the locust-specific fungal pathogen Metarhizium acridum expressing dsRNAs targeting the host F1 F0 -ATPase subunit genes.

BACKGROUND: Metarhizium acridum is a host-specific fungal pathogen with great potential for locust control. However, the slow killing action of M. acridum has impeded its widespread application. To enhance fungal virulence, we constructed transgenic M. acridum strains that express double-stranded (ds)RNAs targeting the genes of the F1 F0 -ATP synthase α and ß subunits in Locusta migratoria. RESULTS: The two host genes were transcriptionally suppressed in L. migratoria nymphs (instar V) infected by RNA interference (RNAi) strains targeting one or two subunit genes of the host ATP synthase, followed by reduced ATPase activity and ATP synthesis. Consequently, the RNAi strain targeting both subunit genes displayed high virulence that was 3.7-fold that in the wild-type strain. CONCLUSION: Our results demonstrate that dsRNA expression in M. acridum can cause host RNA silencing during infection and greatly enhances the fungal virulence through interference with critical host genes, highlighting a new strategy for augmentation of fungal virulence against insect pests. © 2018 Society of Chemical Industry.

Persistence of Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria bassiana (Hypocreales: Clavicipitaceae) in Tobacco Soils and Potential as Biocontrol Agents of Spodoptera litura (Lepidoptera: Noctuidae).

Entomopathogenic fungi (EPF), such as Metarhizium spp. and Beauveria bassiana, are widely used in the biocontrol of many species of insect pests. Tobacco is an economically important crop in Guangdong Province of China, but insect pests, such as Spodoptera litura Fabricius, are a major threat to production. Here, we tested the persistence of five Metarhizium species and B. bassiana in glasshouse pot and field experiments and assessed their long-term efficacy against S. litura. We found that the colony forming units of these EPF decreased by c. 93% by 180 d in the pot soils declines tended to be exponential. In contrast, declines of c. 99% in field soils were more gradual (linear), occurring throughout the 360 d experiment. Metarhizium anisopliae Ma09 had the longest estimated half-life of 41 d, while that of B. bassiana was the shortest (9 d). Fungal density in the upper soil layer (0-5 cm) decreased rapidly and was undetectable after 150 d, whereas density was consistently greatest in the mid-layer (10-15 cm). At 180 d after inoculation, strain Ma09 elicited highest rates of mortality in S. litura. We conclude that soils in Guangdong Province are all suitable for the use of Metarhizium as a biocontrol agent, where M. anisopliae Ma09 offers greatest residual activity.

Involvement of MaSom1, a downstream transcriptional factor of cAMP/PKA pathway, in conidial yield, stress tolerances, and virulence in Metarhizium acridum.

Flo8/Som1, which functions downstream from the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway, plays important roles in hyphal development, spore formation, and virulence in yeast and several filamentous fungi. However, the functions of Som1 in entomopathogenic fungi are still a mystery. In this study, MaSom1, a Flo8/Som1 homolog, was identified and functionally characterized in a model entomopathogenic fungus Metarhizium acridum. Similar to Flo8/Som1 in other fungi, MaSom1 mainly localized to the nucleus in M. acridum. Disruption of MaSom1 reduced conidial yield, delayed conidial germination, and impaired the fungal tolerances to heat and UV-B. The expression levels of some genes involved in defenses of heat shock and UV-B radiation were significantly reduced in ΔMaSom1. MaSom1 is also important for cell wall integrity and conidial surface structures in M. acridum. Some genes related to fungal cell wall synthesis were downregulated in ΔMaSom1. Bioassays showed that ΔMaSom1 had a dramatically decreased virulence after both topical inoculation and intrahemocoel injection of the fungus in locusts. Moreover, inactivation of MaSom1 reduced appressorium formation, diminished fungal growth in locust hemolymph in vitro, and enhanced insect immune responses. Taken together, these results indicate that disruption of MaSom1 leads to a decline of fungal virulence because of impairments in conidial germination and appressorium formation, reduction of fungal growth in host hemolymph, and enhancement of insect immune responses owing to the changes in conidial surface structures.

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of Metarhizium anisopliae on Rhipicephalus microplus tick larvae.

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 106, 107 and 108 conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT50 values obtained were 134.6, 27.2 and 24.8 days for concentrations of 106, 107 and 108 conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT50 values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 106, 107 and 108 conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.

Estrogen-mediated protection of the organotin-degrading strain Metarhizium robertsii against oxidative stress promoted by monobutyltin.

Dibutyltin (DBT) is a global pollutant characterized by pro-oxidative properties. The fungal strain Metarhizium robertsii can eliminate high levels of DBT efficiently. In this study, induction of oxidative stress as well as its alleviation through the application of natural estrogens during the elimination of DBT by M. robertsii were evaluated. During the first 24 h of incubation, the initial concentration of DBT (20 mg l-1) was reduced to 3.1 mg l-1, with simultaneous formation of a major byproduct - monobutyltin (MBT). In the presence of estrone (E1) or 17ß-estradiol (E2), the amounts of dibutyltin residues in the fungal cultures were found to be approximately 2-fold higher compared to cultures without estrogens, which was associated with the simultaneous utilization of the compounds by cytochrome P450 enzymes. On the other hand, MBT levels were approximately 2.5 times lower in the fungal cultures with the addition of one of the estrogens. MBT (not DBT) promotes the generation of O2-, H2O2, and NO at levels 65.89 ± 18.08, 4.04 ± 3.62, and 27.92 ± 1.95, respectively. Superoxide dismutase and catalase activities did not show any response of the M. robertsii strain against the overproduction of superoxide anion and hydrogen peroxide. Application of E1 as well as E2 ensured non-enzymatic defense against nitrosative and oxidative stress through scavenging of nitrogen and oxygen reactive species, and limited their levels from 1.5-fold to 21-fold, depending on the used estrogen.

Neem oil increases the efficiency of the entomopathogenic fungus Metarhizium anisopliae for the control of Aedes aegypti (Diptera: Culicidae) larvae.

BACKGROUND: Entomopathogenic fungi are potential candidates for use in integrated vector management and many isolates are compatible with synthetic and natural insecticides. Neem oil was tested separately and in combination with the entomopathogenic fungus Metarhizium anisopliae against larvae of the dengue vector Aedes aegypti. Our aim was to increase the effectiveness of the fungus for the control of larval mosquito populations. METHODS: Commercially available neem oil was used at concentrations ranging from 0.0001 to 1%. Larval survival rates were monitored over a 7 day period following exposure to neem. The virulence of the fungus M. anisopliae was confirmed using five conidial concentrations (1 × 10(5) to 1 × 10(9) conidia mL(-1)) and survival monitored over 7 days. Two concentrations of fungal conidia were then tested together with neem (0.001%). Survival curve comparisons were carried out using the Log-rank test and end-point survival rates were compared using one-way ANOVA. RESULTS: 1% neem was toxic to A. aegypti larvae reducing survival to 18% with S50 of 2 days. Neem had no effect on conidial germination or fungal vegetative growth in vitro. Larval survival rates were reduced to 24% (S50 = 3 days) when using 1 × 10(9) conidia mL(-1). Using 1 × 10(8) conidia mL(-1), 30% survival (S50 = 3 days) was observed. We tested a "sub-lethal" neem concentration (0.001%) together with these concentrations of conidia. For combinations of neem + fungus, the survival rates were significantly lower than the survival rates seen for fungus alone or for neem alone. Using a combination of 1 × 10(7) conidia mL(-1) + neem (0.001%), the survival rates were 36%, whereas exposure to the fungus alone resulted in 74% survival and exposure to neem alone resulted in 78% survival. When using 1 × 10(8) conidia mL(-1), the survival curves were modified, with a combination of the fungus + neem resulting in 12% survival, whilst the fungus alone at this concentration also significantly reduced survival rates (28%). CONCLUSIONS: The use of adjuvants is an important strategy for maintaining/increasing fungal virulence and/or shelf-life. The addition of neem to conidial suspensions improved virulence, significantly reducing larval survival times and percentages.

Cuticle Fatty Acid Composition and Differential Susceptibility of Three Species of Cockroaches to the Entomopathogenic Fungi Metarhizium anisopliae (Ascomycota, Hypocreales).

Differences in free fatty acids (FFAs) chemical composition of insects may be responsible for susceptibility or resistance to fungal infection. Determination of FFAs found in cuticular lipids can effectively contribute to the knowledge concerning insect defense mechanisms. In this study, we have evaluated the susceptibility of three species of cockroaches to the entomopathogenic fungi Metarhizium anisopliae (Metschnikoff) Sorokin by topical application. Mortality due to M. anisopliae was highly significant on adults and nymphs of Blattella germanica L. (Blattodea: Blattellidae). However, mortality was faster in adults than in nymphs. Adults of Blatta orientalis L. (Blattodea: Blattidae) were not susceptible to the fungus, and nymphs of Blaptica dubia Serville (Blattodea: Blaberidae) were more susceptible to the fungus than adults. The composition of cuticular FFAs in the three species of cockroaches was also studied. The analysis indicated that all of the fatty acids were mostly straight-chain, long-chain, saturated or unsaturated. Cuticular lipids of three species of cockroaches contained 19 FFAs, ranging from C14:0 to C24:0. The predominant fatty acids found in the three studied species of cockroaches were oleic, linoleic, palmitic, and stearic acid. Only in adults of Bl. orientalis, myristoleic acid, γ-linolenic acid, arachidic acid, dihomolinoleic acid, and behenic acid were identified. Lignoceric acid was detected only in nymphs of Bl. orientalis. Heneicosylic acid and docosahexaenoic acid were identified in adults of Ba. dubia.

An ENA ATPase, MaENA1, of Metarhizium acridum influences the Na(+)-, thermo- and UV-tolerances of conidia and is involved in multiple mechanisms of stress tolerance.

In fungi, ENA ATPases play key roles in osmotic and alkaline pH tolerance, although their functions in thermo- and UV-tolerances have not been explored. Entomopathogenic fungi are naturally widespread and have considerable potential in pest control. An ENA ATPase gene, MaENA1, from the entomopathogenic fungus Metarhizium acridum was functionally analyzed by deletion. MaENA1-disruption strain (ΔMaENA1) was less tolerant to NaCl, heat, and UV radiation than a wild-type strain (WT). Digital Gene Expression profiling of conidial RNAs resulted in 281 differentially expressed genes (DEGs) between the WT and ΔMaENA1 strains. Eighty-five DEGs, 56 of which were down-regulated in the ΔMaENA1 strain, were shown to be associated with heat/UV tolerance, including six cytochrome P450 superfamily genes, 35 oxidoreductase genes, 24 ion-binding genes, seven DNA repair genes, and five other genes. In addition, eight genes were components of stress responsive pathways, including the Ras-cAMP PKA pathway, the RIM101 pathway, the Ca(2+)/calmodulin pathway, the TOR pathway, and the HOG/Spc1/Sty1/JNK pathway. These results demonstrated that MaENA1 influences fungal tolerances to Na(+), heat, and UV radiation in M. acridum, and is involved in multiple mechanisms of stress tolerance. Therefore, MaENA1 is required for the adaptation and survival of entomopathogenic fungi in stressful conditions in the environment and in their hosts.

Nest sanitation through defecation: antifungal properties of wood cockroach feces.

The wood cockroach Cryptocercus punctulatus nests as family units inside decayed wood, a substrate known for its high microbial load. We tested the hypothesis that defecation within their nests, a common occurrence in this species, reduces the probability of fungal development. Conidia of the entomopathogenic fungus, Metarhizium anisopliae, were incubated with crushed feces and subsequently plated on potato dextrose agar. Relative to controls, the viability of fungal conidia was significantly reduced following incubation with feces and was negatively correlated with incubation time. Although the cockroach's hindgut contained abundant ß-1,3-glucanase activity, its feces had no detectable enzymatic function. Hence, these enzymes are unlikely the source of the fungistasis. Instead, the antifungal compound(s) of the feces involved heat-sensitive factor(s) of potential microbial origin. When feces were boiled or when they were subjected to ultraviolet radiation and subsequently incubated with conidia, viability was "rescued" and germination rates were similar to those of controls. Filtration experiments indicate that the fungistatic activity of feces results from chemical interference. Because Cryptocercidae cockroaches have been considered appropriate models to make inferences about the factors fostering the evolution of termite sociality, we suggest that nesting in microbe-rich environments likely selected for the coupling of intranest defecation and feces fungistasis in the common ancestor of wood cockroaches and termites. This might in turn have served as a preadaptation that prevented mycosis as these phylogenetically related taxa diverged and evolved respectively into subsocial and eusocial organizations.

Altered immunity in crowded locust reduced fungal (Metarhizium anisopliae) pathogenesis.

The stress of living conditions, similar to infections, alters animal immunity. High population density is empirically considered to induce prophylactic immunity to reduce the infection risk, which was challenged by a model of low connectivity between infectious and susceptible individuals in crowded animals. The migratory locust, which exhibits polyphenism through gregarious and solitary phases in response to population density and displays different resistance to fungal biopesticide (Metarhizium anisopliae), was used to observe the prophylactic immunity of crowded animals. We applied an RNA-sequencing assay to investigate differential expression in fat body samples of gregarious and solitary locusts before and after infection. Solitary locusts devoted at least twice the number of genes for combating M. anisopliae infection than gregarious locusts. The transcription of immune molecules such as pattern recognition proteins, protease inhibitors, and anti-oxidation proteins, was increased in prophylactic immunity of gregarious locusts. The differentially expressed transcripts reducing gregarious locust susceptibility to M. anisopliae were confirmed at the transcriptional and translational level. Further investigation revealed that locust GNBP3 was susceptible to proteolysis while GNBP1, induced by M. anisopliae infection, resisted proteolysis. Silencing of gnbp3 by RNAi significantly shortened the life span of gregarious locusts but not solitary locusts. By contrast, gnbp1 silencing did not affect the life span of both gregarious and solitary locusts after M. anisopliae infection. Thus, the GNBP3-dependent immune responses were involved in the phenotypic resistance of gregarious locusts to fungal infection, but were redundant in solitary locusts. Our results indicated that gregarious locusts prophylactically activated upstream modulators of immune cascades rather than downstream effectors, preferring to quarantine rather than eliminate pathogens to conserve energy meanwhile increasing the "distance" of infectious and target individuals. Our study has obvious implications for bio-pesticides management of crowded pests, and for understanding disease epidemics and adaptiveness of pathogens.

Diversity and genetic population structure of fungal pathogens infecting white grub larvae in agricultural soils.

White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and ß-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.

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

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

Subterranean termite prophylactic secretions and external antifungal defenses.

Termites exploit environments that make them susceptible to infection and rapid disease transmission. Gram-negative bacteria binding proteins (GNBPs) signal the presence of microbes and in some insects directly damage fungal pathogens with ß-1,3-glucanase activity. The subterranean termites Reticulitermes flavipes and Reticulitermes virginicus encounter soil entomopathogenic fungi such as Metarhizium anisopliae, which can evade host immune responses after penetrating the cuticle. An external defense that prevents invasion of fungal pathogens could be crucial in termites, allowing them to thrive under high pathogenic pressures. We investigated the role of secreted ß-1,3-glucanases in Reticulitermes defenses against M. anisopliae. Our results show that these termites secrete antifungal ß-1,3-glucanases on the cuticle, and the specific inhibition of GNBP associated ß-1,3-glucanase activity with d-δ-gluconolactone (GDL) reduces this activity and can cause significant increases in mortality after exposure to M. anisopliae. Secreted ß-1,3-glucanases appear to be essential in preventing infection by breaking down fungi externally.

Pathogen resistance in the moth Orgyia antiqua: direct influence of host plant dominates over the effects of individual condition.

The role of pathogens in insect ecology is widely appreciated but remains insufficiently explored. Specifically, there is little understanding about the sources of the variation in the outcome of insect-pathogen interactions. This study addresses the extent to which immune traits of larvae and pupae of the moth Orgyia antiqua L. (Lepidoptera: Lymantriidae) depend on the host plant species and individual condition of the insects. The two host plants, Salix myrsinifolia Salisb. and S. viminalis L., were chosen because they differ in the concentration of phenolic glycosides, harmful to most polyphagous insects. Individual condition was assumed to be reflected in body weight and development time, and was manipulated by rearing larvae either singly or in groups of four. The resistance traits recorded were survival and time to death after fungal infection in the larval stage and the efficiency of encapsulating a nylon implant by the pupae. The survival of the infected larvae was mainly determined by the species of the host plant. Encapsulation response was not associated with the resistance to the pathogen, suggesting that the host plant affected the pathogen rather than the immune system of the insect. Interestingly, the host plant supporting better larval growth led to inferior resistance to the pathogen, indicating a trade-off between different aspects of host plant quality.

Seletividade de Beauveria bassiana e Metarhizium anisopliae a Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) / Selectivity of Beauveria bassiana and Metarhizium anisopliae to Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae)

Trichogramma pretiosum Riley and the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae are efficient biological control agents and are thought to be used jointly. In here, we investigated if these entomopathogens could have any side-effects on T. pretiosum. Therefore, 1 x 8 cards containing sterilized eggs of Anagasta kuehniella (Zeller) that were sprayed with 0.2 ml of B. bassiana or M. anisopliae (1.0 × 10(9) conidia/ml) were offered to a T. pretiosum female for 24h (30 cards/fungus = 30 replicates). Afterwards, females were isolated in glass tubes. The control group was sprayed with sterile distillated water + Tween 80 (0.01 percent). In addition, 60 cards with sterilized eggs of A. kuehniella were submitted to parasitism by females of T. pretiosum for 24h. Of these cards, 30 were sprayed with B. bassiana or M. anisopliae and 30 with distillated water + Tween 80 (0.01 percent), and observed daily until parasitoid emergence. Metarhizium anisopliae decreased parasitoid emergence and caused confirmed mortality. Therefore, field and semi-field experiments should be conducted for a final assessment of the side-effects of these entomopathogens on Trichogramma as a ways to develop a control strategy in which both can be used.

O parasitóide Trichogramma pretiosum Riley e os fungos entomopatogênicos Beauveria bassiana e Metarhizium anisopliae são considerados eficientes agentes de controle biológico e podem ser empregados concomitantemente em algumas situações. O objetivo deste trabalho foi verificar quais os efeitos desses entomopatógenos sobre T. pretiosum. Foram usadas cartelas de 1 × 8 cm contendo ovos esterilizados de Anagasta kuehniella (Zeller) pulverizadas com 0,2 ml de B. bassiana ou M. anisopliae (1,0 × 10(9) conídios/ml) e mantidas individualmente com uma fêmea de T. pretiosum por 24h, totalizando 30 cartelas para cada fungo. Posteriormente, as fêmeas foram individualizadas em tubos de vidro. A testemunha consistiu na pulverização de água destilada estéril + Tween 80 (0,01 por cento). Paralelamente, 60 cartelas contendo ovos esterilizados de A. kuehniella foram submetidas ao parasitismo por fêmeas de T. pretiosum por 24h. Destas, 30 cartelas foram pulverizadas com B. bassiana ou M. anisopliae e 30 com água destilada estéril + 0,01 por cento, Tween 80, sendo os ovos observados diariamente até a emergência do parasitóide. Metarhizium anisopliae provocou diminuição na emergência de T. pretiosum e causou mortalidade confirmada. Assim, experimentos de campo e de semi-campo devem ser conduzidos para a avaliação final dos efeitos nocivos desses entomopatógenos a Trichogramma, para que estratégias de controle conjuntas possam ser desenvolvidas para esses agentes de controle.

Caracterização dos hemócitos de operários de Nasutitermes coxipoensis(Holmgren) (Isoptera: Termitidae) e avaliação hemocitária após parasitismo por Metarhizium anisopliae / Hemocyte characterization of Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae) workers and hemocyte evaluation after parasitism by Metarhizium anisopliae

O objetivo desta pesquisa foi caracterizar morfológica e ultraestruturalmente os hemócitos de operários de Nasutitermes coxipoensis (Holmgren) e quantificar os tipos celulares 24h, 48h e 72h após inoculação com Metarhizium anisopliae. Seis tipos de hemócitos foram identifi cados: plasmatócito, granulócito, esferulócito, prohemócito, adipohemócitos e oenocitóide. Nenhuma alteração na morfologia dessas células foi evidenciada durante os intervalos. Entretanto, houve variações na proporção dos hemócitos em relação à testemunha para esferulócitos, adipohemócitos e oenocitóides, nos três intervalos de avaliação, e para plasmatócitos e granulócitos no intervalo de 48h. As causas potenciais dessa variação e suas implicações são apresentadas e discutidas neste artigo.

We aimed to characterize the morphology and ultrastructure of hemocytes of Nasutitermes coxipoensis (Holmgren) workers and to quantify the cell types 24h, 48h and 72h after inoculation with Metarhizium anisopliae. Six hemocytes types were identified, plasmatocyte, granulocyte, spherulocyte, prohemocyte, adipohemocyte and eonocytoid Hemocytes did not present any morphological alteration at the several observation periods, but they did have a change in their abundance, as observed for spherulocytes, adipohemocytes and eonocytoids at all intervals, and for plasmatocytes and granulocytes at 48h after host inoculation. We argue on the possible reasons and implications of the observed changes.

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

The effect of humidity on the activity of Metarhizium anisopliae IP 46 (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) formulated in sunflower oil against Aedes aegypti (L.) (Diptera: Culicidae) eggs was examined. After exposure of eggs at 75% relative humidity (RH) for 98% RH, eclosion was 98% RH, followed by: (a) a 12-day exposure at 75% RH before submersion in water; (b) a minimal 5-day exposure at > 98% RH and direct subsequent transfer of treated eggs to water, or (c) a minimal daily 20-h exposure at > 98% RH alternating with 4 h at 75% RH for 10 days. We demonstrate that oil-based formulations of conidia of M. anisopliae enhance ovicidal activity at high humidities and conclude that these formulations have potential in the integrated control of Ae. aegypti.