Entomopathogenic Fungus Metarhizium Anisopliae Research Articles

Molecular evolution and transcriptional profile of GH3 and GH20 β-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae

Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of β-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study explored the transcriptional profile and evolution of putative GH20 NAGases (MaNAG1 and MaNAG2) and GH3 NAGases (MaNAG3 and MaNAG4) identified in M. anisopliae. While MaNAG2 orthologs are conserved in several ascomycetes, MaNAG1 clusters only with Aspergilllus sp. and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 were phylogenetically related with bacterial GH3 NAGases. The transcriptional profiles of M. anisopliae NAGase genes were evaluated in seven culture conditions showing no common regulatory patterns, suggesting that these enzymes may have specific roles during the Metarhizium life cycle. Moreover, the expression of MaNAG3 and MaNAG4 regulated by chitinous substrates is the first evidence of the involvement of putative GH3 NAGases in physiological cell processes in entomopathogens, indicating their potential influence on cell differentiation during the M. anisopliae life cycle.

TUNGRO DISEASE CONTROL THROUGH THE ELIMINATION VECTOR ROLE OF GREEN LEAF HOPPER WITH ENVIRONMENT FRIENDLY CONTROL

Green leafhopper (GLH)plays an important role in tungro disease epidemics. Reduce the activity of GLHsuckasvectorsof tungro virus was effective to limit transmission of the virus. Integrated control of tungro disease may involve multiple components at once including using sambilata with entomopathogenicfungus Metarhiziumanisopliae. This research was conducted to determine the effect of sambilata and M.anisopliaein controlling the GLH as tungro virus vectors. The experiment was conducted in tungro endemic areas in Tanjungsiang,Subang District at dry season 2013 and wet season 2013/2014. Experiments using split plot design with four replications. The main plot was consists of GLH resistant varieties(IR66), tungro resistant varieties (Inpari 9), and check varieties(Ciherang). The subplots were M.anisopliae applications, sambilata, and control. Application was done on rice plant age 14, 28 and 42 days after planting (DAP).The results showed that the intensity of tungro on Ciherang showed the highest intensity compared toIR66 and Inpari9. Effect of entomopathogenic fungus M.anisopliae application to tungro disease showed a lower intensity compared with sambilata extracts and control. The intensity of tungro disease in farmers’ fields as a comparison of experiment was high enough on average between 1 until 69%. In general, the density of GLH population began to increase on the observation of 14 to 28 DAP. GLH population density was highest at 28 DAP. However, the population density of GLH decreased at 42 until 56 DAP.

Integrated Control of Nymphal Ixodes scapularis: Effectiveness of White-Tailed Deer Reduction, the Entomopathogenic Fungus Metarhizium anisopliae, and Fipronil-Based Rodent Bait Boxes.

Pathogens transmitted by ticks are the leading cause of arthropod-associated human diseases in the United States and managing the risk of exposure to potentially infected ticks is of vital public health importance. A 3-year integrated tick management program to control blacklegged ticks, Ixodes scapularis, the primary vector for the pathogenic agents of Lyme disease, human anaplasmosis, and babesiosis, was implemented in the town of Redding in southwestern Connecticut beginning in 2013. Combinations of white-tailed deer, Odocoileus virginianus, reduction, area application of the entomopathogenic fungus Metarhizium anisopliae, and fipronil-based rodent bait boxes were evaluated for their ability to reduce nymphal I. scapularis over 3 years. Interference from local hunters prevented sufficient, sustained deer removal previously reported to negatively impact I. scapularis abundances (i.e., <5 deer/km2). The combination of fipronil-based bait boxes and broadcast application of M. anisopliae had the most impact of any treatment combination; questing nymphs were reduced 78-95% within each year and Borrelia burgdorferi-infected questing nymphal I. scapularis encounter potential was reduced by 66% as compared with no treatment in the third year of the study. A combination of the broadcast application of M. anisopliae and small rodent-targeted fipronil-based bait boxes is an effective low-toxicity integrated approach that significantly reduced encounters with B. burgdorferi-infected questing nymphal I. scapularis on individual properties.

Pathogenicity of Metarhizium anisopliae isolates on Nezara viridula and Dichelops melacanthus in wheat crop

ABSTRACT: Bugs such as Nezara viridula (Linnaeus) and Dichelops melacanthus (Dallas) are considered the main insect pests of wheat crop in Brazil. The use of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) may be an alternative for the management of these insects in the crop. The objective of this work was to verify the pathogenicity of different isolates of M. anisopliae on adults of N. viridula and D. melacanthus under laboratory and greenhouse conditions. In the laboratory, isolates 05RA, 11RA, 08RA and 02RA were obtained from N. viridula and D. melacanthus infested with M. anisopliae. Also, a high pathogenicity (100% of mortality) of both species was recorded in a bioassay of the topical application 8 Days After Application (DAA). However, compared to the other isolates, the 08RA isolate showed the highest pathogenicity in a shorter time interval for N. viridula (Mean Time “MT” = 2.8 days) and D. melacanthus (MT = 4.0 days). Under greenhouse conditions, the 08RA isolate provided a mortality of 44.9% (N. viridula) and 35.7% (D. melacanthus) in the same evaluation period. However, at 14 DAA, the mortality was 100% for both species, with the MT values of N. viridula and D. melacanthus being obtained at 8 days and 10 days, respectively. The fungus M. anisopliae is a promising alternative for the control of adult N. viridula and D. melacanthus in wheat crop.

&lt;b&gt;Compatibility of polymers to fungi &lt;i&gt;Beauveria bassiana&lt;/i&gt; and &lt;/i&gt;Metarhizium anisopliae&lt;/i&gt; and their formulated products stability

Stability during storage is a limiting factor in the development and use of bioinsecticides. This study aims to evaluate the compatibility of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana with components used in encapsulated formulations and to evaluate shelf-life in formulations with B. bassiana. Sodium alginate and maltodextrin, hemicellulose, and dimethyl sulfoxide solvents were evaluated. Compatibility was evaluated by mixing the components with PDA, and the parameters evaluated were vegetative growth, sporulation and viability. The formulations of fungi were prepared in various concentrations of sodium alginate and then added drop-wise to calcium chloride. To evaluate the stability, formulations were stored at 26.5, 4.0, and -20.0°C. Monthly, samples were taken, and the capsules were placed on Petri dishes with PDA to verify the growth after seven days of incubation. These evaluations were performed by the 12th month. Biopolymers evaluated were compatible with both fungi, exception maltodextrin at 1.5%, which was classified as moderately toxic to B. bassiana. Dimethyl sulfoxide was classified as moderately toxic to B. bassiana at 3.0% and to M. anisopliae at 2.0% and 3.0%. The formulations were stable throughout the 12 months in the conditions evaluated, while for the pure conidia of the fungus, 46% viability in the 6th month at 26.5°C was observed.

Species diversity of the entomopathogenic fungi Metarhizium anisopliae and M. flavoviride species complexes isolated from insects in Japan

Phylogenetic analyses of insect-derived isolates of the Metarhizium anisopliae and M. flavoviride species complexes in Japan were conducted to reveal their species diversity. Fifty-seven isolates were identified as nine species, including one species first reported for Japan. Metarhizium pingshaense was the most frequently isolated species from this genus, and the 29 isolates of M. pingshaense came from six orders and 14 families of insects. New host–pathogen associations were found for two species with relatively narrow host ranges: Hymenoptera-M. pemphigi, Orthoptera- and Phasmatodea-M. majus.

Direct and Indirect Effects of Pesticides on the Insidious Flower Bug (Hemiptera: Anthocoridae) Under Laboratory Conditions.

Greenhouse producers are interested in integrating natural enemies along with pesticides to suppress western flower thrips, Frankliniella occidentalis (Pergande), populations. The insidious flower bug, Orius insidiosus (Say), is a commercially available natural enemy of western flower thrips. We conducted a series of laboratory experiments to determine the direct and indirect effects of 28 pesticides (insecticides, miticides, and fungicides), 4 pesticide mixtures, and 4 surfactants (36 total treatments plus a water control) on the adult O. insidiosus survival and predation on western flower thrips adults under laboratory conditions. The number of live and dead O. insidiosus adults was recorded after 24, 48, 72, and 96 h. The results of the study indicate that the fungicides (aluminum tris, azoxystrobin, fenhexamid, and kresoxim-methyl), insect growth regulators (azadirachtin, buprofezin, kinoprene, and pyriproxyfen), botanicals (Capsicum oleoresin extract, garlic oil, soybean oil; and rosemary, rosemary oil, peppermint oil, and cottonseed oil), and entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae) were minimally directly harmful to adult O. insidiosus, with 80% to 100% adult survival. However, abamectin, spinosad, pyridalyl, chlorfenapyr, tau-fluvalinate, imidacloprid, dinotefuran, acetamiprid, and thiamethoxam directly affected O. insidiosus survival after 96 h (0-60% adult survival). The pesticide mixtures of abamectin + spinosad and chlorfenapyr + dinotefuran reduced adult survival (20% and 0%, respectively, after 48 h). Furthermore, the surfactants were not directly harmful to O. insidiosus adults. All western flower thrips adults were killed by the surviving adult O. insidiosus after 48 h, indicating no indirect effects of the pesticides on predation.

Response of peanut Arachis hypogaea roots to the presence of beneficial and pathogenic fungi by transcriptome analysis

Entomopathogenic fungus Metarhizium anisopliae obtain survival benefit meanwhile promote the nutrient absorption of root as an endophyte. However, little is known concerning molecular mechanisms in the process. We performed the transcriptome sequencing of A. hypogaea roots inoculated M. anisopliae and pathogenic Fusarium axysporum, respectively. There were 81323 unigenes from 132023 transcripts. Total 203 differentially expressed genes (DEGs) respond to the two fungi, including specific 76 and 34 DEGs distributed respectively in M. anisopliae and F. axysporum treatment. KEGG pathway enrichment for DEGs showed the two top2 were signal transductions of plant-pathogen interaction and plant hormone. By qRT-PCR, the mRNA level of 26 genes involved in plant-fungus interaction confirmed the reliability of the RNA-Seq data. The expression pattern of the key DEGs on jasmonic acid (JA) or salicylic acid (SA) signaling pathway presented regulating consistency with JA or SA concentration detected by HPLC-MS. Those significantly stronger down-regulated DEGs by M. anisopliae thanby F. axysporum linking to hypersensitive response and negative regulation of defense, and those specific up-regulated genes in M. anisopliae treatment may predict that the less immunity is conducive to symbiosis F. axysporum may trigger JA-mediated defense regulated by ERF branch of JA signaling pathway, whereas M. anisopliae does not.

Efecto de Hongos Entomopatogenos Sobre Larvas de Mosca Sierra (Monoctenus sanchezi Smith)

Abstract. Monoctenus sanchezi is a sawfly of economic importance in forest management because of damage to the foliage of white cedar. Mortality by entomopathogenic fungi Metarhizium anisopliae, Beauveria bassiana, and Trichothecium sp. on M. sanchezi larvae was 98, 60, and 36% on the sixth day. Metarhizium anisopliae may be an alternative for biological control of the sawfly in Mexico.

Identification of double-stranded RNA viruses in Brazilian strains of Metarhizium anisopliae and their effects on fungal biology and virulence

Brazil is the leading country in the use of the entomopathogenic fungus Metarhizium anisopliae against agricultural pests. Very little information has been documented about the diversity and the impact of mycoviruses on M. anisopliae compared to M. robertsii and M. brunneum. This study reports the discovery of mycoviruses associated with native Brazilian Metarhizium spp. strains from the entomopathogen collection of the University of São Paulo, including strains used for mass-production of aerial conidia in sugar-alcohol factories to meet their own demand for controlling sugarcane spittlebugs and used in commercial mycoinsecticides. We also investigated the potential effects of viral infection on the biological traits of some of these strains. Viral-like double-stranded RNA (dsRNA) was found in 61% of the 36 Metarhizium spp. strains analyzed and showed a large variation of different electrophoretic banding patterns consisting of 2 to 8 dsRNA bands in polyacrylamide gels (PAGE). No dsRNA was detected in any of the strains from commercial products used in this study. Efforts to “cure” strains with dsRNA revealed that the inhibitor of protein synthesis cycloheximide was not efficient in eradicating dsRNA viruses from Metarhizium spp. strains. Some dsRNA segments of M. anisopliae ESALQ 26 were lost following hyphal tip subculturing. The dsRNA-variants of M. anisopliae strains ESALQ 866, ESALQ 1256 and ESALQ 1595 were healed after either monoconidial or hyphal tip subculturing. Single spore cultures of M. anisopliae ESALQ 1256 exhibited significant differences in vegetative growth, conidia production, and virulence against Tenebrio molitor larvae, although these differences were not linked with dsRNA. Moreover, fungal variants free of or positive for dsRNA had similar heat and UV tolerance responses. Our results suggest that viral-like dsRNA are very common among Brazilian M. anisopliae strains, but there is sparse evidence that these mycoviruses are harmful to fungal biological traits.

Infection of silkworm larvae by the entomopathogenic fungus Metarhizium anisopliae

ABSTRACT: The isolate E9 of Metarhizium anisopliae was used in commercial hybrids of Bombyx mori larvae to evaluate its biological effect. Symptomatological analyses showed typical signs of fungal infection. Histopathology revealed the presence of large numbers of hemocytes in the hemocoel, and on the sixth dpi the bodies of the insects appeared to be colonised by the fungus. The isolate E9 is pathogenic to larvae B. mori and; therefore, death of the insects was caused by the colonization of fungus in the epidermal and mesodermal tissues.

Cuticular colour reflects underlying architecture and is affected by a limiting resource

Central to the basis of ecological immunology are the ideas of costs and trade-offs between immunity and life history traits. As a physical barrier, the insect cuticle provides a key resistance trait, and Tenebrio molitor shows phenotypic variation in cuticular colour that correlates with resistance to the entomopathogenic fungus Metarhizium anisopliae. Here we first examined whether there is a relationship between cuticular colour variation and two aspects of cuticular architecture that we hypothesised may influence resistance to fungal invasion through the cuticle: its thickness and its porosity. Second, we tested the hypothesis that tyrosine, a semi-essential amino acid required for immune defence and cuticular melanisation and sclerotisation, can act as a limiting resource by supplementing the larval diet and subsequently examining adult cuticular colouration and thickness. We found that stock beetles and beetles artificially selected for extremes of cuticular colour had thicker less porous cuticles when they were darker, and thinner more porous cuticles when they were lighter, showing that colour co-varies with two architectural cuticular features. Experimental supplementation of the larval diet with tyrosine led to the development of darker adult cuticle and affected thickness in a sex-specific manner. However, it did not affect two immune traits. The results of this study provide a mechanism for maintenance of cuticular colour variation in this species of beetle; darker cuticles are thicker, but their production is potentially limited by resource constraints and differential investments in resistance mechanisms between the sexes.

Persistence of Brazilian isolates of the entomopathogenic fungi Metarhizium anisopliae and M. robertsii in strawberry crop soil after soil drench application

Establishment, persistence and local dispersal of the entomopathogenic fungi Metarhizium anisopliae (ESALQ1037) and M. robertsii (ESALQ1426) (Ascomycota: Hypocreales) were investigated in the soil and rhizosphere following soil drench application in strawberries between 2012 and 2013 at a single location in Inconfidentes, Minas Gerais, Brazil. Metarhizium spp. isolates (n=108) were collected using selective agar media and insect bait methods, and characterized by sequence analyses of the 5’–end of the translation elongation factor 1-α and the MzFG543igs intergenic region and by multilocus simple sequence repeat analysis. Both applied fungal isolates were frequently recovered from bulk soil and rhizosphere samples of the treated plots, suggesting that they were able to establish and disperse within the soil. Persistence within the soil and strawberry rhizosphere for both fungal isolates was observed up to 12 months after application with frequencies of 25% of haplotypes similar to isolate ESALQ1037 and 87.5% of haplotypes similar to isolate ESALQ1426, respectively. Overall, M. robertsii was the most abundant species in the agroecosystem studied representing 77.8% of the isolates recovered across all sample dates.

The effect of combined application of the entomopathogenic fungus Metarhizium anisopliae and the release of predatory mite Phytoseiulus longipes for the control of the spider mite Tetranychus evansi on tomato

The entomopathogenic fungus Metarhizium anisopliae (Ma) and the predatory mite Phytoseiulus longipes (Pl) are both potential biocontrol agents of the tomato spider mite Tetranychus evansi. The combination of the two agents may enhance biological control of T. evansi if there is no antagonistic effect. Here, we evaluated the effect of combining the application of Ma and Pl in the control of T. evansi under screenhouse and field conditions on tomato crops. The acaricide abamectin was included as a control. Spray applications of Ma and abamectin, and release of Pl in the screenhouse significantly reduced mite population density among motile (P < 0.0001) and egg stages (P < 0.0001) of T. evansi as compared to the control. There was also significant reduction in mite population density in the field but only with spray applications of Ma and abamectin, and Ma + Pl treatments among motile (P < 0.0001) and egg stages (P < 0.0001). In the screenhouse experiment, leaf damage was significantly lower in all the treatments than in the control. In the field, leaf damage was significantly higher in the control and Pl alone treatments than in the other treatments. The combination of both Ma and Pl resulted in a reduction of T. evansi numbers in the screenhouse and field, but did not consistently differ significantly from Ma alone. Based on these results, we conclude that there is no benefit of combining M. anisopliae and P. longipes for the control of T. evansi in tomato crops.

Lethal and sublethal effects of mixtures of entomopathogenic fungi and synthetic insecticides on biological aspects of Musca domestica L.

The current study was performed to assess the effectiveness of the entomopathogenic fungi Metarhizium anisopliae var. anisopliae (Metschnikof) Sorokin and Isaria fumosorosea (Wize) Brown&Smith applied in combination with some synthetic insecticides against the house fly, Musca domestica L. (Diptera: Muscidae) at Department of Entomology, Bahauddin Zakariya University, Multan, Pakistan during 2015. An extensive, typically dose-dependent range of responses was shown by flies towards fungus-synthetic insecticide combinations using a bait method. The insecticides acetamiprid, emamectin benzoate, imidacloprid and lufenuron in combination with insect pathogenic fungi showed higher mortality than expected with significant synergistic interactions. In addition, the mixtures were also assessed for sublethal effects on biological parameters of M. domestica , namely adult longevity, fecundity, egg hatching, larval duration, percent pupation, pupal weight, pupal duration, adult emergence and sex ratio. The results showed a considerable impact of fungi and synthetic insecticide mixtures on all biological parameters of M. domestica (P<0.05) except adult emergence. A noteworthy reduction in adult longevity, fecundity, egg hatching, percent pupation and pupal weight was observed, while larval and pupal durations were prolonged. The results highlight the potential of combined use of entomopathogenic fungi and synthetic insecticides for the control of M. domestica . Moreover, the combination of entomopathogenic fungi and synthetic insecticides can reduce the concentrations of the active ingredient required to achieve the same result as when these are applied separately. However, field trials are required to validate the effects of entomopathogenic fungi and synthetic insecticides mixtures before they can be recommended as elements of an integrated pest management program for M. domestica .

Metarhizium anisopliae infection alters feeding and trophallactic behavior in the ant Solenopsis invicta

In social insects, social behavior may be changed in a way that preventing the spread of pathogens. We infected workers of the ant Solenopsis invicta with an entomopathogenic fungus Metarhizium anisopliae and then videotaped and/or measured worker feeding and trophallactic behavior. Results showed that fungal infected S. invicta enhanced their preference for bitter alkaloid chemical quinine on 3days after inoculation, which might be self-medication of S. invicta by ingesting more alkaloid substances in response to pathogenic infection. Furthermore, infected ants devoted more time to trophallactic behavior with their nestmates on 3days post inoculation, in return receiving more food. Increased interactions between exposed ants and their naive nestmates suggest the existence of social immunity in S. invicta. Overall, our study indicates that S. invicta may use behavioral defenses such as self-medication and social immunity in response to a M. anisopliae infection.

Belowground Ecology of Scarabs Feeding on Grass Roots: Current Knowledge and Future Directions for Management in Australasia.

Many scarab beetles spend the majority of their lives belowground as larvae, feeding on grass roots. Many of these larvae are significant pests, causing damage to crops and grasslands. Damage by larvae of the greyback cane beetle (Dermolepida albohirtum), for example, can cause financial losses of up to AU$40 million annually to the Australian sugarcane industry. We review the ecology of some scarab larvae in Australasia, focusing on three subfamilies; Dynastinae, Rutelinae, and Melolonthinae, containing key pest species. Although considerable research on the control of some scarab pests has been carried out in Australasia, for some species, the basic biology and ecology remains largely unexplored. We synthesize what is known about these scarab larvae and outline key knowledge gaps to highlight future research directions with a view to improve pest management. We do this by presenting an overview of the scarab larval host plants and feeding behavior; the impacts of abiotic (temperature, moisture, and fertilization) and biotic (pathogens, natural enemies, and microbial symbionts) factors on scarab larvae and conclude with how abiotic and biotic factors can be applied in agriculture for improved pest management, suggesting future research directions. Several host plant microbial symbionts, such as arbuscular mycorrhizal fungi and endophytes, can improve plant tolerance to scarabs and reduce larval performance, which have shown promise for use in pest management. In addition to this, several microbial scarab pathogens have been isolated for commercial use in pest management with particularly promising results. The entomopathogenic fungus Metarhizium anisopliae caused a 50% reduction in cane beetle larvae while natural enemies such as entomopathogenic nematodes have also shown potential as a biocontrol. Key abiotic factors, such as soil water, play an important role in affecting both scarab larvae and these control agents and should therefore feature in future multi-factorial experiments. Continued research should focus on filling knowledge gaps including host plant preferences, attractive trap crops, and naturally occurring pathogens that are locally adapted, to achieve high efficacy in the field.

Biological control of Ixodes ricinus larvae and nymphs with Metarhizium anisopliae blastospores

The entomopathogenic fungus Metarhizium anisopliae is used as a biological pest control agent against various arthropod species, including ticks. However, the efficacy depends on tick species, tick stage and fungus strain. We studied the effect of M. anisopliae on engorged larvae and nymphs of Ixodes ricinus, the most abundant tick species in Europe, under laboratory and semi-field conditions. A significant reduction of engorged larvae and nymphs could be shown under laboratory as well as under semi-field conditions. Only 3.5% of the larvae treated in the lab and only 18.5% kept under semi-field conditions were able to develop into nymphs compared to the recovered nymphs of the control groups, which were regarded as 100%. Only 7.1% of nymphs were recovered as adult ticks after fungal treatment under semi-field conditions compared to the control (100%). The efficacy of blastospores of M. anisopliae against engorged larvae and nymphs of I. ricinus under semi-field conditions was demonstrated in this study, showing their high potential as a biological control agent of ticks. Further studies will have to investigate the effect of this agent against other stages of I. ricinus as well as other tick species before its value as a biological control agent against ticks can be fully assessed.

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

BackgroundEntomopathogenic 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.MethodsCommercially 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 × 105 to 1 × 109 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.Results1 % 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 × 109 conidia mL−1. Using 1 × 108 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 × 107 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 × 108 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 %).ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1280-9) contains supplementary material, which is available to authorized users.

Effects of a new outdoor mosquito control device, the mosquito landing box, on densities and survival of the malaria vector, Anopheles arabiensis, inside controlled semi-field settings.

BackgroundThe significance of malaria transmission occurring outdoors has risen even in areas where indoor interventions such as long-lasting insecticidal nets and indoor residual spraying are common. The actual contamination rates and effectiveness of recently developed outdoor mosquito control device, the mosquito landing box (MLB), on densities and daily survival of host-seeking laboratory Anopheles arabiensis, which readily bites humans outdoors was demonstrated.MethodsExperiments were conducted in large semi-field systems (SFS) with human volunteers inside, to mimic natural ecosystems, and using MLBs baited with natural or synthetic human odours and carbon dioxide. The MLBs were dusted with 10 % pyriproxyfen (PPF) or entomopathogenic fungi (Metarhizium anisopliae) spores to mark mosquitoes physically contacting the devices. Each night, 400 laboratory-reared An. arabiensis females were released in one SFS chamber with two MLBs, and another chamber without MLBs (control). Mosquitoes were individually recaptured while attempting to bite volunteers inside SFS or by aspiration from SFS walls. Mosquitoes from chambers with PPF-treated MLBs and respective controls were individually dipped in water-filled cups containing ten conspecific third-instar larvae, whose subsequent development was monitored. Mosquitoes recaptured from chambers with fungi-treated MLBs were observed for fungal hyphal growth on their cadavers. Separately, effects on daily survival were determined by exposing An. arabiensis in chambers having MLBs treated with 5 % pirimiphos methyl compared to chambers without MLBs (control), after which the mosquitoes were recaptured and monitored individually until they died.ResultsUp to 63 % (152/240) and 43 % (92/210) of mosquitoes recaptured inside treatment chambers were contaminated with pyriproxyfen and M. anisopliae, respectively, compared to 8 % (19/240) and 0 % (0/164) in controls. The mean number of larvae emerging from cups in which adults from chambers with PPF-treated MLBs were dipped was significantly lower [0.75 (0.50–1.01)], than in controls [28.79 (28.32–29.26)], P < 0.001). Daily survival of mosquitoes exposed to 5 % pirimiphos methyl was nearly two-fold lower than controls [hazard ratio (HR) = 1.748 (1.551–1.920), P < 0.001].ConclusionHigh contamination rates in exposed mosquitoes even in presence of humans, demonstrates potential of MLBs for controlling outdoor-biting malaria vectors, either by reducing their survival or directly killing host-seeking mosquitoes. The MLBs also have potential for dispensing filial infanticides, such as PPF, which mosquitoes can transmit to their aquatic habitats for mosquito population control.