Mean Lethal Time Research Articles

In vitro evaluation of the pathogenicity of fungi isolated from the Urabá region (Antioquia, Colombia) against Aedes aegypti larvae

Introduction. Aedes aegypti is an important vector of arboviral diseases like dengue among others. Traditional control strategies, such as the use of insecticides, have lost effectiveness due to the emergence of resistance in mosquito populations. Biological control and fungi applied for biocontrol are presented as viable and ecological alternatives. Objective. To evaluate in vitro pathogenicity of Trichoderma sp. isolates obtained from Urabá (Antioquia) on larvae of Ae. aegypti, and to determine the mean lethal concentration and mean lethal time of the most pathogenic isolate. Materials and methods. Using the sentinel larvae method of Ae. aegypti, fungi were isolated from water bodies in the Urabá region (Antioquia). The isolates were characterized morphologically and molecularly to determine their taxonomic identity. Pathogenicity tests were performed in vitro on Ae. aegypti larvae in the L2/L3 stages. Subsequently, a strain was selected to establish its mean lethal concentration and mean lethal time. Results. Trichoderma sp. strain AP-91 caused high mortality in larval populations of Ae. aegypti. We estimated a mean lethal concentration of 1.8 × 107 conidia/ml and a mean lethal time of 20.67 hours. Conclusion. The strain AP-91 showed potential for its use as biological control of Ae. aegypti, making it a suitable candidate for scale-up cultures applied to integrated vector management. This research suggests exploring compounds and enzymes produced by the AP-91 strain to understand better its pathogenicity.

Conserved fungal effector NLS1 suppresses Lepidoptera insect immunity by targeting the host defense protein Hdd11.

Entomopathogenic fungi have been widely used as the main mycoinsecticide for controlling agricultural and forest pests. The effector molecules of these mycopathogens have evolved to adapt to their hosts. The role of fungal effectors in evading the host immune system in insects remains mainly unclear. We characterized the widely distributed fungal effector necrosis-inducing-like secreted protein 1 (NLS1) in the entomopathogenic fungus Metarhizium robertsii. Our findings revealed the presence of M. robertsii NLS1 (MrNLS1) in host hemocytes during the early stage of hemocoel infection. MrNLS1 knock down (ΔMrNLS1) reduced fungal pathogenicity during infection and altered the expression of host immune genes. The molecular docking results and the yeast 2-hybrid assay confirmed that MrNLS1 interacts with the host defense protein Hdd11. The phylogenetic analysis indicated that Hdd11 is conserved across a broad range of Lepidoptera species. Knock down of hdd11 in Helicoverpa armigera, Bombyx mori, and Galleria mellonella markedly suppressed their immune responses against M. robertsii. However, no significant difference was observed in the mean lethal time between hdd11-knockdown Lepidoptera species infected with ΔMrNLS1 and those infected with wild-type M. robertsii. Therefore, in Lepidoptera insects, Hdd11 is essential for fungal defense. In conclusion, M. robertsii infects Lepidoptera insects by targeting host Hdd11 through its protein MrNLS1, thereby suppressing the host immune response. Our findings clarify the molecular mechanisms underlying fungal infection pathogenesis.

Thiamethoxam toxicity on the stingless bee Friesiomelitta varia: LC50, survival time, and enzymatic biomarkers assessment

Bees play a crucial role as pollinating insects in both natural and cultivated areas. However, the use of pesticides, such as thiamethoxam, has been identified as a contributing factor compromising bee health. The current risk assessment primarily relies on the model species Apis mellifera, raising concerns about the applicability of these assessments to other bee groups, including stingless bees. In this study, we investigated the acute toxicity of thiamethoxam on the stingless bee Frieseomelitta varia by determining the average lethal concentration (LC50) and mean lethal time (LT50). Additionally, we evaluated the enzymatic profile of Acetylcholinesterase (AChE), Carboxylesterase-3 (CaE-3), and Glutathione S-Transferase (GST), in the heads and abdomens of F. varia after exposure to thiamethoxam (LC50/10). The LC50 of thiamethoxam was determined to be 0.68 ng ai/μL, and the LT50 values were 37 days for the control group, 25 days at LC50/10, and 27 days at LC50/100. The thiamethoxam significantly decreased the survival time of F. varia. Furthermore, the enzymatic profile exhibited differences in CaE3 activity within one day in the heads and ten days in the abdomen. GST activity showed differences in the abdomen after one and five days of thiamethoxam exposure. These findings suggests that the abdomen is more affected than the head after oral exposure to thiamethoxam. Our study provides evidence of the toxicity of thiamethoxam at both the cellular and organismal levels, reinforcing the need to include non-Apis species in pollinator risk assessments.and provide solid arguments for bee protection.

Re-establishing fish migration channel of large reservoirs in Jinsha River Basin of China by using an eco-friendly reservoir operation method

Study regionA deep reservoir between Xiluodu (XLD) dam and Xiangjiaba (XJB) dam, located in the lower reaches of Jinsha River. Study focusDam construction, especially high dam construction, normally caused the supersaturated total dissolved gas (TDG) downstream of the dam and reduced the flow velocity of the reservoir, which hindered the safe migration of fish. In this research, an eco-friendly reservoir operation model with a multi-objective optimization method was established to reduce supersaturated TDG level and increase reservoir flow velocity in this key ecological zone to restore fish migration pathways. The model consisted of three modules: supersaturated TDG prediction module, reservoir velocity prediction module and multi-objective optimization module. In order to re-establish suitable migratory habitat for fish migration, ecological constraints were added to the reservoir multi-objective operational method including minimum supersaturated TDG level and maximum reservoir flow velocity. And, a 2-D TDG transport diffusion model was adopted to verify the effectiveness of eco-friendly reservoir operation results. The study aims to re-establish fish migration channel of large reservoirs by using eco-friendly reservoir operation methods. New hydrological insightsThe proposed reservoir operation model was efficient in reducing the maximum residence time of supersaturated TDG and increasing the reservoir flow velocity. The area between Xiluodu (XLD) dam and Xiangjiaba (XJB) dam was used as a studying case to analyze the usability of the developed method. The best simulation result was obtained under the scenario with comprehensive consideration of power generation and ecological objectives, and the maximum residence time of 140% supersaturated TDG level was 4 h, which was consistent with the designed worst Mean Lethal Time (LT50) of 4 h for fish. The worst simulation result was obtained in the pattern targeting power generation only, with a maximum residence time of 8 h for supersaturated TDG level at 140%, which exceeded the corresponding LT50 of fish. Under all three scenarios, the XLD hydropower station was generating at full capacity and the minimum flow velocity in the XJB reservoir would be above 0.2 m/s. Overall, this study provides an opportunity to reconstruct the fish migration habitat in the reservoir area.

Beauveria bassiana exhibits strong virulence against Dendroctonus ponderosae in greenhouse and field experiments

The mountain pine beetle (MPB) has infested over 16 million hectares of pine forests in western Canada, killing over 50% of mature lodgepole pine, Pinus contorta, in British Columbia alone. There are few tools available to manage irruptive bark beetle populations and to mitigate tree mortality. Beauveria bassiana is an entomopathogenic fungus that causes mortality to several bark beetle species. However, the potential for B. bassiana as a biocontrol agent against pine beetle populations is unknown. We selected three strains of B. bassiana from several culture collections and evaluated their conidial stability under cold storage, in planta (greenhouse, and pine bolts) and in natura (forest stand, pine bolts, and live pines) conditions. The stability assays showed that all fungal strains maintained a minimum effective conidial yield through the assay durations (3-12weeks). In addition, we adapted a biphasic liquid-solid fermentation approach for the large-scale production of conidial biomass, yielding up to a 100-fold increase in production. In greenhouse virulence assays, the mean lethal time of MPBs was reduced to 3-4days upon treatment with B. bassiana, where high B. bassiana-associated mycosis was also observed. Furthermore, the application of B. bassiana formulation substantially affected the gallery network of MPBs in bolts in the field, resulting in shorter larval galleries and significantly reduced offspring production. Indeed, high titer treatments reduced the mean larvae per gallery to virtually zero. Together these results demonstrate that B. bassiana may be a viable biocontrol tool to reduce mountain pine beetle populations in pine forests in western Canada. KEY POINTS: • Three B. bassiana strains identified to be stable at various test conditions. • Large-scale conidial biomass production using liquid-solid biphasic fermentation. • Reproductive success of D. ponderosae significantly reduced by B. bassiana formulation.

EFFECT OF TEMPERATURE AND ULTRAVIOLET RADIATION ON GROWTH AND PATHOGENICITY OF Metarhizium anisopliae

The efficacy of entomopathogenic fungi in the field depends on the influence of various environmental factors, including temperature and level of exposure to UV light. It is hypothesized that temperature and level of UV light exposure of Metarhizium anisopliae (Hypocreales, Clavicipiceae) may affect its efficiency in controlling the sweetpotato weevil (Cylas formicarius, Fabricius; Coleoptera, Brentidae). The objective of this study was to evaluate the influence of temperature and ultraviolet light radiation on the growth and pathogenicity of different native strains of Cuban origin of the M. anisopliae Sorokin complex with biological activity against C. formicarius. Colony diameter was measured at 28, 30, 32 and 34 °C temperature and exposed to ultraviolet light (254 nm) for 10, 15, 20, 30, 30, 60 and 120 minutes. Under laboratory conditions, the percentage germination of conidia of strains LBMa-11, LBM-30, LBM-41 and LBM-146 was determined, as well as their pathogenic capacity by means of bioassays with adults of C. formicarius in terms of the percentage of cumulative mortality and the mean lethal time (TL50). Results proved that temperature and UV light affect the ability of M. anisopliae to control C. formicarius. A delay in cumulative mortality was detected for all strains exposed to UV light, with an increase in TL50. The optimum temperature range was between 28 and 30 °C. The strain LBM-127 showed the highest sensitivity to temperature, LBMa-11 the highest tolerance to UV light, and LBM-146 was the most virulent according to Probit regression analysis.

Low Concentration of Azadirachtin Has the Same Toxic Effect as Imidacloprid + Lambda-Cyhalothrin in Workers of Two Species of Leaf-Cutter Ants1

Leaf-cutter ants of the genus Atta are pests controlled by broad-spectrum insecticides that adversely affect the environment and human health. Toxicity of 20 concentrations of chemical imidacloprid + λ-cyhalothrin and biological insecticides azadirachtin and spinetoram were evaluated on workers of A. mexicana and A. cephalotes ants. Initial evaluation determined optimal concentrations to kill 10-90%. Mean lethal concentrations of imidacloprid and azadirachtin were determined, and mean lethal times were through 3 × 10-1 µl/liter concentration of each insecticide. Azadirachtin and imidacloprid + λ-cyhalothrin at 3 × 10-1 µl/liter concentration killed 100% of both species. LC50 values of imidacloprid + λ-cyhalothrin and azadirachtin were similar against both species. But, the TL50 of imidacloprid + λ-cyhalothrin was less (28-29 hours) in both species compared to the TL50 by azadirachtin that was 36 to 41 hours. Insecticide based on extracts of neem (azadirachtin) was as toxic as imidacloprid + λ-cyhalothrin against A. mexicana and A. cephalotes worker ants. Azadirachtin is an environmentally friendly alternative to manage the two ant species, and probably other species of Atta and other ant pests.

Efficacy and horizontal transmission of Beauveria bassiana and its synergistic activity with diflubenzuron against the house fly, Musca domestica L.

ABSTRACT Resistance of house flies to chemical insecticides is increasing in the world, thus calling for sustainable control methods of flies. In this study, larvicidal and adulticidal activities of Beauveria bassiana strain DEBI 008 against the house fly were evaluated. Also, we examined the horizontal transmission of the B. bassiana between adult house flies of both sexes. Then, the susceptibility level of immature house flies to diflubenzuron was determined. In addition to synergistic effects, we investigated the combining low doses of B. bassiana and diflubenzuron on the house fly larvae. In the pathogenicity assay using direct application, the mean lethal concentration values (LC50) were estimated for larvae (4.71×106, conidia/ml) and adults (5.65×107, conidia/ml) after 10 days. Based on mean lethal time (LT50) of adult house flies, it was observed that LT50 at the high concentration of 1 × 108 conidia was in the range of 4.89 days with 100% mortality; LT50 at the lowest concentration of 105 conidia ranged from 10 to 15 days with 48% mortality. The results of B. bassiana in horizontal transmission assays on adults also showed that LT50 for the females infected by horizontal transmission was 5.33 days with 100% mortality; meanwhile, for males horizontally infected with 100% mortality, this was 6.11 days. The susceptibility of the house fly population to diflubenzuron (LC50) was 1.21 (mg/ kg), and high mortality was recorded at the highest concentration treated (6 mg/ kg) with 89.33% mortality. Synergistic interactions between B. bassiana and diflubenzuron were observed with the increased mortality rates of the combined treatment. The most significant synergy based on Finney’s method was the combination of LC25 (B. bassiana)+ LC20 (diflubenzuron) (χ 2 = 60.75). Therefore, combined use in a synergistic manner could result in the enhanced biological control by accelerating the B. bassiana strain and diflubenzuron. The results of this study could be, therefore, helpful for integrated pest management to control the house fly.

Mutation of a prenyltransferase results in accumulation of subglutinols and destruxins and enhanced virulence in the insect pathogen, Metarhizium anisopliae.

The insect pathogenic fungus, Metarhizium anisopliae is a commercialized microbial agent used in biological control efforts targeting a diverse range of agricultural and other insect pests. The second step in the synthesis of a group of M. anisopliae α-pyrone diterpenoids (termed subglutinols) involves the activity of a prenyltransferase family geranylgeranyl diphosphate synthase (product of the subD/MaGGPPS5 gene). Here, we show that targeted gene disruption of MaGGPPS5 results in earlier conidial germination and faster greater vegetative growth compared to the wild type (WT) parent and complemented strains. In addition, insect bioassays revealed that the ΔMaGGPPS5 mutant strain displayed significantly increased virulence, with a ~50% decrease in the mean lethal time (LT50 , from 6 to 3 days) to kill (50% of) target insects, and an ~15-40-fold decrease in the mean lethal dose (LC50 ). Metabolite profiling indicated increased accumulation in the ΔMaGGPPS5 mutant of select subglutinols (A, B and C) and destruxins (A, A2, B and B2), the latter a set of fungal secondary metabolites that act as insect toxins, with a concomitant loss of production of subglutinol 'analogue 45'. These data suggest that the increased virulence phenotype seen for the ΔMaGGPPS5 strain can, at least in part, be attributed to a combination of faster growth and increased insect toxin production, linking the production of two different secondary metabolite pathways, and represent a novel approach for the screening of isolates with enhanced virulence via modulation of terpenoid secondary metabolite biosynthesis.

Evaluation of Native Entomopathogenic Fungi Isolates for Microbial Control of the Mediterranean Fruit Fly (Ceratitis Capitata (Diptera: Tephritidae)) Pupae and Adults

Two isolates of the entomopathogenic fungi (EPF) Beauveria bassiana (Hypocreales: Cordycipitaceae), an isolate of Aspergillus flavus (Eurotiales: Trichocomaceae), of Verticillium sp. (Glomerellales: Plectosphaerellaceae) and of Aschersonia sp. (Hypocreales: Clavicipitaceae) were evaluated for their pathogenicity against the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae) pupae and adults under laboratory conditions, using four different concentrations. Pathogenicity towards pupae and adults was tested via spraying and body contact bioassays respectively. Average mortality of treated individuals ranged from 60 to 100% for pupae and from 39 to 100% for adults depending on the fungal isolates and concentrations. The highest mortality levels were observed for individuals treated by Verticillium sp., A. flavus and Aschersonia sp., while the B. bassiana isolates were the less effective against both adult and pupae. Estimated lethal concentrations 90 ranged from 3x105 to 3.8 x 107 conidia/ml for adults and from 6.58 x 103 to 2.5 x 105 conidia/ml for pupae with lower values calculated for A. flavus, Verticillium sp. and Aschersonia sp. Mean lethal times 90 were less than 79h for C. capitata adults and ranged from 94 to 273 hours for pupae according to the fungal isolates. Microscopic observation of treated pupae showed a mycelium growth on the bodies of death individuals, while visible signs of mycosis are less observed for adults. Results of these experiments demonstrated that native EPF isolates could be a promising biocontrol agent to manage safely C. capitata and might be used against pupae by soil application or against adults by cover or bait sprays.

Incorporating Fish Tolerance to Supersaturated Total Dissolved Gas for Generating Flood Pulse Discharge Patterns Based on a Simulation‐Optimization Approach

AbstractSupersaturated total dissolved gas (STDG) caused by the flood discharge can result in bubble disease and even fish death, posing potential risks to aquatic ecosystems. In order to reduce the impacts of STDG on such ecosystem, a dynamic multi‐objective STDG management model (DMO‐STDGM) with the flood pulse discharge pattern is developed based on the prototype observation and laboratory test analysis, which consists of STDG prediction, fish safety assessment, multi‐objective optimization and 2‐D STDG transport diffusion simulation modules. The proposed model can support generation of flood pulse discharge patterns to reduce the level of STDG and minimize the maximum residence time of STDG water. The developed model is applied to optimize the discharge pattern of Xiluodu project to reduce the effects of STDG on fish. Simulation results of the worst scenario with the flood pulse discharge pattern show that the maximum residence time of STDG for T120%, T125%, T130%, and T140% are 12, 9, 4, 3 and 1 h, which are less than the corresponding LT50s (Mean Lethal Time). It means that the physiological function of fish can recover from the affect of STDG, thus fish can survive in the worst scenario. The established model can also be employed for increasing power generation. Simulation results indicate that the flood pulse discharge pattern with equal weight of ecology and power generation can ensure full load operation of hydropower station with 12,600 MW. The established model is widely applicable and can provide an important theoretical and technical support for water quality and ecosystem management under multiple complexities.

Susceptibility of adult and larval stages of housefly, Musca domestica to entomopathogenic fungal biopesticides

ABSTRACT The current study assessed the efficacy of entomopathogenic fungal (EPF) biopesticides against adult and larval stages of houseflies, Musca domestica L. under laboratory conditions. Six commercial EPF biopesticides were selected for virulence tests; three strains of Beauveria bassiana (HF23 in the product balEnce™, GHA in the product Botanigard ES and ATCC 74040 in the product Naturalis-L), one strain of Metarhizium brunneum (F52 in the product Met52®), one strain of Lecanicillium muscarium (KV01 in the product Mycotal®), and one strain of Isaria fumosorosea (Apopka 97, in the product Preferal WG). Adult and larval stages of houseflies were sprayed with 2 ml of a conidial concentration (108 conidia ml−1) of each EPF biopesticide individually using a Potter tower. The results showed that balEnce™ and Botanigard ES were significantly the highest efficacy against adults and larvae of M. domestica, compared to other EPF biopesticides. The mean conidial concentration (LC50) for adults and larvae of M. domestica exposed to balEnce™ was 4.7 × 105 conidia ml−1 and 4.8 × 105 conidia ml−1 respectively, which was not significantly different from LC50 value of Botanigard ES (6.0 × 105 and 4.6 × 105 conidia ml−1). The mean lethal time (LT50) values of balEnce™ and Botanigard ES were significantly shorter at 109 and 108 than other conidial concentrations. balEnce™ or Botanigard ES caused 44–94% reduction in daily fecundity of the adult females from day 3 to day 10 post-treatment. Results demonstrated that EPF biopesticides balEnce™ and Botanigard ES have potential as biocontrol agents of M. domestica but further research under operational conditions is required.

Behavioural Responses and Mortality of Clarias gariepinus Juveniles Exposed to Acute Concentrations of Paraquat

Paraquat is the most common contact and non- selective herbicide for exterminating vegetative pest. Fish are ideal sentinels for detecting aquatic pollutants and are largely used as bio indicators of environmental pollution. This study is aimed to determine the behavioural changes, lethal concentrations (LCs) and mean lethal time (MLT) of paraquat exposed to Clarias gariepinus. A 96 hours renewable bioassay was conducted with various paraquat concentrations 0.0, 0.25, 0.50, 0.75, 1.0 and 1.25 mg/l. Behavioural changes and cumulative mortality were observed and recorded at 12, 24, 48, 72 and 96 hour. Behavioural changes such as air gulping, erratic swimming, loss of balance, excessive mucus secretion, discolouration and death were observed with severity increasing as concentration and duration of exposure increases. The LC50 values were decreased from 0.191mg/l (0.171 – 0.222) in 12 hour to 0.107mg/l (0.065 – 0.150) in 96 hour, while relative toxicity factor (TF) was increased from 1 to 1.79 times respectively. The 96hr MLT values were decreased from 91.18 hours (54.09-105.64) at the lowest concentration to 16.22 hours (9.06 – 25.15) at the highest concentration with relative toxicity time (RTT) increasing from 1 to 5.62 times. Herbicide should be apply with caution and studies on the sub lethal effects of paraquat on the haematological, biochemical and histological parameters of C. gariepinus juveniles will be necessary.

Apis mellifera and Melipona scutellaris exhibit differential sensitivity to thiamethoxam

Apis mellifera is a pollinator insect model in pesticide risk assessment tests for bees. However, given the economic and ecological importance of stingless bees such as Melipona scutellaris in the Neotropical region, as well as the lack of studies on the effect of insecticides on these bees, toxicity tests for stingless bees should be carried out to understand whether insecticides affect both species of bees in the same manner. Thus, the present study quantified the differential sensitivity of the bees M.scutellaris and A.mellifera to the oral ingestion of the insecticide thiamethoxam by determining the mean lethal concentration (LC50), mean lethal time (LT50), and their effect on the insecticide target organ, the brain. The results showed that the stingless bee is more sensitive to the insecticide than A.mellifera, with a lower LC50 of 0.0543ng active ingredient (a.i.)/μL for the stingless bee compared to 0.227ng a.i./μL for A.mellifera. When exposed to a sublethal concentration, morphological and ultrastructural analyses were performed and evidenced a significant increase in spaces between nerve cells of both species. Thus, A.mellifera is not the most appropriate or unique model to determine the toxicity of insecticides to stingless bees.

Susceptibility of three species of the genus Ostrinia (Lepidoptera: Crambidae) to Nosema pyrausta (Microsporidia: Nosematida)

Microsporidia are obligate intracellular parasites that affect the population density of many insect pests. In particular, infection with Nosema pyrausta is one of the major mortality factors for the European corn borer Ostrinia nubilalis, the Asian corn borer Ostrinia furnacalis and the adzuki bean borer Ostrinia scapulalis. The purpose of the work is to compare the susceptibility to N. pyrausta and pathogenesis of three species of moths of the genus Ostrinia. Studies conducted over 2 years have shown that in all three species of host insects under laboratory conditions, both during oral infection and transovarian transmission of infection (in the daughter generations of experimentally infected insects), only diplokaryotic spores formed corresponding to the main morphotype of the genus Nosema. Mean lethal time increased with instar of larvae used for infection but didn’t differ between the three species. The rates of transovarial transmission of N. pyrausta were also similar. Thus, all the insect species examined may equally participate in the parasite persistence in nature and serve as model laboratory hosts for parasitological research and mass propagation of the microsporidium.

Insecticidal Action of Several Isolates of Entomopathogenic Fungi against The Granary Weevil Sitophilus granarius

The insecticidal virulence of various entomopathogenic fungal isolates retrieved from soil samples was tested on adults of the granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae). Bioassays were carried out in the laboratory where experimental adults were sprayed with 1 mL of conidial suspension (108 conidia/mL) from each isolate. Mortality was recorded at 7, 14, and 21 days after exposure. Mean mortality, mean lethal time, survival, and hazard effect were estimated for each isolate. Two isolates of Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae), one isolate of Aspergillus insuetus (Bainier) Thom & Church (Eurotiales: Trichocomaceae) and Metarhizium anisopliae (Metschinkoff) Sorokin (Hypocreales: Clavicipitaceae) resulted in the highest mortality (97–100%). The isolates with both the highest hazard effect and the lowest survival rate were Aspergillus sp. and M. anisopliae. Our results indicate that entomopathogenic fungi have the potential to become a very useful tool in reducing chemical applications in storage facilities.

Genetic Identification and Biological Characterization of Baculovirus Isolated from Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil

Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) larvae are polyphagous, aggressive, and have been found in many of Brazil's agricultural areas. Biological control with baculoviruses is part of an integrated pest management (IPM) strategy to manage this insect. Three isolates of nucleopolyhedrovirus (NPV) were collected from Brazilian populations of H. armigera, and compared genetically and biologically to Gemstar® (an imported nucleopolyhedrovirus), and to Nucleopolyedrovirus spodoptera (SPNPV) that was passed serially through H. armigera. Genetic sequencing of lef-8 and lef-9 genes revealed that the Brazilian isolates were closely related to nucleopolyhedrovirus species from Australia, South Africa, China, and India. The isolates caused high mortality rates in third instar H. armigera larvae. The mean lethal dose (LD50) and lethal time (LT50) differed between isolates, but was highest for HearNPV-BR2. This is the first report of HearNPV in Brazil, and the insecticidal properties of the BR2 isolate support its potential use in the production of biopesticides to manage H. armigera in Brazil.

MrSVP, a secreted virulence-associated protein, contributes to thermotolerance and virulence of the entomopathogenic fungus Metarhizium robertsii

BackgroundMetarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Unfortunately, its worldwide commercial use has been restricted by a short shelf life and inconsistencies in virulence. In our previous study, a gene (GenBank accession number EFZ01626) was found to be significantly upregulated in heat-treated conidia. In the present study, this gene was characterized via gene disruption and complementation strategies.ResultsThe gene (amplified by rapid amplification of cDNA ends PCR) was 1219 bp long and contained an open reading frame (ORF) of 777 bp. It encoded a protein of 234 amino acid residues with a 26-residue signal peptide. Bioinformatics analyses did not identify conserved functional domains; therefore, it was assumed to be a secreted virulence-associated protein according to its signal peptide and bioassay results. We found that the conidial germination rate of the ΔMrSVP mutant fungi dramatically decreased after heat shock treatment in a thermotolerance test. In addition, transcription levels of all tested heat shock–related genes were significantly lower in the mutant than in the wild type. We also demonstrated that the mean lethal time to death (LT50) of ΔMrSVP significantly increased relative to the wild type in insect bioassays (both topical inoculation and injection) involving Galleria mellonella. Moreover, similar rates of appressorium formation between ΔMrSVP and the wild type—and the significantly different expression of virulence-related genes such as acid trehalase and sucrose nonfermenting protein kinase in the haemocoel after injection—revealed that MrSVP is required for virulence in the insect haemocoel.ConclusionsOverall, our data suggest that the Mrsvp gene contributes to thermotolerance and virulence of M. robertsii. Furthermore, this gene is deeply involved in the mycosis of insect cadavers and in immune escape rather than insect cuticle penetration during infection.

Developing risk management treatments for taro from the Pacific Islands

Most taro imported from the Pacific are currently fumigated with toxic methyl bromide to kill pests, predominantly mites and nematodes that are generally found on the surface of taro. Combined high-pressure washing (HPW) and hot water treatment (HWT) were examined as alternative methods for disinfesting taro. Taro mites (Rhizoglyphus sp.) and root knot nematodes (Meloidogyne sp.) were exposed to a range of HWT and HPW conditions separately or together. At 47˚C, mean lethal times of 2.6—2.9 mins and 3.9—4.1 mins were estimated to control 99% and 99.99% of nymph and adult mites, respectively. Mite nymphs and adults were more tolerant to HWT than mite eggs. The mean lethal time estimate to control 99% of juvenile nematodes was 4.5 mins. Nematode eggs were the most tolerant life stage with only 10% mortality after a 4-min 47˚C HWT. HPW+HWT reduced heavy infestations of mobile pests on taro (n=30—117/taro) by 100%. HPW followed by HWT 50˚C for 12.5 mins reduced viable egg infestation by 95.8%. HPW followed by HWT can control surface pests on taro while maintaining taro quality.

Horizontal transmission of Metarhizium anisopliae (Hypocreales: Clavicipitacea) and the effects of infection on oviposition rate in laboratory populations of Musca domestica (Diptera: Muscidae).

Effective control of house fly, Musca domestica (L.), populations currently relies on the use of chemical insecticides in most situations. Entomopathogenic fungi such as Metarhizium anisopliae (Metschn.) Sorokin may provide an alternative to chemicals and their efficacy may be enhanced by autodissemination amongst flies. This study assessed the capacity of M. anisopliae for transmission between adult M. domestica flies and the effects of infection on the fecundity of females. Metarhizium anisopliae was transmitted between adult M. domestica with 91.67-100% mortality resulting across the three ratios of infected: non-infected flies tested (1:2, 1:5 and 1:10). The mean lethal time (LT50 ) for female recipients mixed with infected male donor flies at the three ratios was 3.95, 4.79 and 5.65 days, respectively, whereas for male recipients mixed with infected female donors at the same ratios the LT50 was 4.98, 5.98 and 7.44 days, respectively. Infection with M. anisopliae significantly reduced the reproductive capacity of female flies during the first 4 days of infection, with 25% fewer eggs oviposited by infected flies than by those that were uninfected. Autodissemination among house flies and reduction in oviposition in the early stages of infection could contribute significantly to the effectiveness of M. anisopliae used in biocontrol programmes. © 2017 Society of Chemical Industry.