Search

Mosquitoes threaten over half of the world's population through vectored diseases such as malaria, zika, yellow fever, dengue, and chikungunya. Mosquitoes have a highly developed olfactory system attuned to chemotaxis relating to host-seeking, mating, and oviposition behavior. In this study, we aimed to determine the spatial efficacy of 2 plant-based repellent blends (Blend3 and Blend4 that had previously been found to successfully repel Aedes, Anopheles and Culex mosquitoes in wind tunnel assays) in excluding Aedes aegypti from the window entry. A new cage system was developed for parallel "no-choice" and "choice" olfactometric assays. In the no-choice trial, Blends 3 and 4, as well as commercial products (N, N-diethyl-3-methylbenzamide, p-menthane-3,8-diol [PMD], 3-(N-n-butyl-N-acetyl)-amino-propionic acid ethyl ester, and 2-(2-hydroxyethyl)-1-methylpropylstyrene 1-piperidine carboxylate), were adsorbed into filter papers of different sizes and placed in a window created between 2 attached bug dorms. Then, the number of mosquitoes entering the window was counted through a 6-min period. In choice olfactometric assays, Blends 3, 4, and PMD were adsorbed into filter paper and the number of mosquitoes moving away from Blend 3 and PMD were compared. No-choice assays showed that Blend3 (P < 0.001) and Blend4 (P = 0.0012) were more repellent than the best commercial product PMD. Additionally, while Blend 4 was significantly more repellent than Blend 3 (P = 0.012) in the choice assay, overall, these 2 blends show promise as new repellents for the spatial exclusion of Aedes aegypti from window entry alone or as part of a "push-pull'' strategy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Mosquitoes of the genera Aedes, Anopheles and Culex vector a wide range of pathogens seriously affecting humans and livestock on a global scale. Over-reliance on insecticides and repellents has driven research into alternative, naturally-derived compounds to fulfil the same objectives. Steam distilled extracts of four plants with strong, yet attractive, volatile profiles were initially assessed for repellency in a dual-port olfactometer using Aedes aegypti as the model species. Picea sitchensis was found to be the most repellent, proving comparable to leading products when applied at 100% (p = 1.000). Key components of conifer-derived volatile profiles were then screened via electroantennography before those components eliciting an electrophysiological response were assayed individually in the olfactometer; according to WHO protocol. The most promising 5 were selected for reductive analyses to produce an optimised semiochemical blend. This combination, and a further two variations of the blend, were then progressed to a multi-species analysis using the BG-test whereby bite-attempt frequency on hands was assessed under different repellent treatments; assays were compared between Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Efficacy was found against all three species, although it was found that Ae. aegypti was the most susceptible to the repellent, with An. gambiae being the least. Here, a novel, naturally-derived blend is presented with weak spatial repellency, as confirmed in laboratory assays. Further work will be required to assess the full extent of the potential of the products, both in terms of field application and species screening; however, the success of the products developed demonstrate that plant metabolites have great capacity for use in the repellent sector; both to improve upon known compounds and to reduce the usage of toxic products currently on the market., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Wood et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Olfactory systems are indispensable for insects as they, including Western Flower Thrips ( Frankliniella occidentalis ), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis . Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3 . Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3 . The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

Entomopathogenic fungi are promising as an environmentally benign alternative to chemical pesticides for mosquito control. The current study investigated the virulence of Metarhizium anisopliae blastospores against Aedes aegypti under both laboratory and field conditions. Virulence bioassays of conidia and blastospores were conducted in the laboratory, while field simulation bioassays were conducted under two conditions: totally shaded (TS) or partially shaded (PS). In the first bioassay (zero h), the larvae were added to the cups shortly after the preparation of the blastospores, and in the subsequent assays, larvae were added to the cups 3, 6, 9, and 12 days later. The survival of the larvae exposed to blastospores in the laboratory was zero on day two, as was the case for the larvae exposed to conidia on the sixth day. Under TS conditions, zero survival was seen on the third day of the bioassay. Under PS conditions, low survival rates were recorded on day 7. For the persistence bioassay under PS conditions, low survival rates were also observed. Metarhizium anisopliae blastospores were more virulent to Ae. aegypti larvae than conidia in the laboratory. Blastospores remained virulent under field simulation conditions. However, virulence rapidly declined from the third day of field bioassays. Formulating blastospores in vegetable oil could protect these propagules when applied under adverse conditions. This is the first time that blastospores have been tested against mosquito larvae under simulated field conditions, and the current study could be the basis for the development of a new biological control agent.

Aedes aegypti (Diptera: Culicidae) is the principal vector of dengue and other viruses that cause disease among 100 to 400 million people each year. The recent development of widespread insecticidal resistance has led to the rapid development of biological control solutions aimed at larval control. While the efficacy of Metarhizium brunneum has been shown against Aedes larvae, the impact of larval population dynamics will need to be determined to formulate effective control strategies. In this study, larvae were subjected to four concentrations of M. brunneum (105, 106, 107, 108 conidia ml-1). Larvae were found to be susceptible to M. brunneum with dose-dependent efficacy. When constant larval immigration was added as a parameter, peak mortality was consistently found to occur on the fourth day, before a significant reduction in control efficacy linked to a decline in conidial availability within the water column. This suggests that M. brunneum treatments should be applied at a concentration 1 × 107 conidia ml-1 every four days to effectively control mosquito larvae in the field, regardless of the fungal formulation, water volume, or larval density. Understanding fungal-mosquito dynamics is critical in developing appropriate control programs as it helps optimize the fungal control agent's dose and frequency of application., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

The efficient oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) catalyst materials are crucial in the energy research domain due to their tunability. Structural modification in perovskites such as lanthanum cerates (LaCeO 3 ) upon doping at A or B sites significantly affects the surface activity and enhances the catalysis efficacy. Herein, B-site nickel-doped lanthanum cerate (LaCe 1-x Ni x O 3±δ ) nanopowders were applied as ORR indicators in high-temperature electrochemical impedance spectroscopy for solid-oxide fuel cell (SOFC) tests and in cyclic voltammetric OER investigations in alkaline medium. The integration into SOFC applications, via solid-state EIS in a co-pressed three-layered cell with LCNiO as cathode, is investigated in an oxygen-methane environment and reveals augmented conductivity with temperatures of 700-850°C. The higher electrokinetic parameters-including diffusion coefficients, D o heterogeneous rate constant, k o , and peak current density for OER in KOH-methanol at a LCNiO-9-modified glassy carbon electrode-serve as robust gauges of catalytic performance. CV indicators and EIS conductivities of LaCe 1-x Ni x O 3±δ nanomaterials indicate promising potencies for electrocatalytic energy applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Butt, Erum, Mujtaba, Medvedev and Janjua.)

Metarhizium brunneum is a highly effective entomopathogenic fungus that also functions as a plant biostimulant. It can act as both an endophyte and rhizosphere colonizer; however, the mechanisms driving biostimulation are multifactorial. In this work, oilseed rape ( Brassica napus ) seeds were grown in composts treated with different concentrations of M. brunneum strains ARSEF 4556 or V275, or the M. brunneum -derived volatile organic compounds 1-octen-3-ol and 3-octanone. Biostimulation efficacy was found to be strongly dose dependent. Concentrations of 1 × 10 6 conidia g -1 compost were found to be most effective for the M. brunneum , whereas dosages of 1 µL 100 g -1 compost were found to be efficacious for the volatiles. These optimized doses were assessed individually and in combined formulations with a hydrogel against oilseed rape ( Brassica napus ), sitka spruce ( Picea sitchensis ), maize ( Zea mays ) and strawberry ( Fragaria annanassa ). Both volatile compounds were highly effective biostimulants and were found to increase in biostimulatory efficiency when combined with M. brunneum conidia. Hydrogels were not found to interact with the growth process and may offer avenues for novel formulation technologies. This study demonstrates that Metarhizium -derived volatile organic compounds are actively involved in plant growth promotion and have potential for use in novel formulations to increase the growth of a wide range of commercially relevant crops.

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds' population at risk from such diseases. Development of combined predator-parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite-predator treatments may be related to additive stress factors induced within the target host larvae., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

Hexanary high-entropy oxides (HEOs) were synthesized through the mechanochemical sol-gel method for electrocatalytic water oxidation reaction (WOR). As-synthesized catalysts were subjected to characterization, including X-ray diffraction (XRD), Fourier transforms infrared (FTIR) analysis, and scanning electron microscopy (SEM). All the oxide systems exhibited sharp diffraction peaks in XRD patterns indicating the defined crystal structure. Strong absorption between 400-700 cm -1 in FTIR indicated the formation of metal-oxide bonds in all HEO systems. WOR was investigated via cyclic voltammetry using HEOs as electrode platforms, 1M KOH as the basic medium, and 1M methanol (CH 3 OH) as the facilitator. Voltammetric profiles for both equiatomic (EHEOs) and non-equiatomic (NEHEOs) were investigated, and NEHEOs exhibited the maximum current output for WOR. Moreover, methanol addition improved the current profiles, thus leading to the electrode utility in direct methanol fuel cells as a sequential increase in methanol concentration from 1M to 2M enhanced the OER current density from 61.4 to 94.3 mA cm -2 using NEHEO. The NEHEOs comprising a greater percentage of Al, ([Al 0.35 (Mg, Fe, Cu, Ni, Co) 0.65 ] 3 O 4 ) displayed high WOR catalytic performance with the maximum diffusion coefficient, D° (10.90 cm 2 s -1 ) and heterogeneous rate constant, k° (7.98 cm s -1 ) values. These primary findings from the EC processes for WOR provide the foundation for their applications in high-energy devices. Conclusively, HEOs are proven as novel and efficient catalytic platforms for electrochemical water oxidation.

Fungal volatile organic compounds (VOCs) represent promising candidates for biopesticide fumigants to control crop pests and pathogens. Herein, VOCs produced using three strains of the entomopathogenic fungus Metarhizium brunneum were identified via GC-MS and screened for antimicrobial activity. The VOC profiles varied with fungal strain, development state (mycelium, spores) and culture conditions. Selected VOCs were screened against a range of rhizosphere and non-rhizosphere microbes, including three Gram-negative bacteria ( Escherichia coli , Pantoea agglomerans , Pseudomonas aeruginosa ), five Gram-positive bacteria ( Micrococcus luteus , Staphylococcus aureus , Bacillus subtilis , B. megaterium , B. thuringiensis ), two yeasts ( Candida albicans , Candida glabrata ) and three plant pathogenic fungi ( Pythium ultimum , Botrytis cinerea , Fusarium graminearum ). Microbes differed in their sensitivity to the test compounds, with 1-octen-3-ol and isovaleric acid showing broad-spectrum antimicrobial activity. Yeasts and bacteria were inhibited by the same VOCs. Cryo-SEM showed that both yeasts and bacteria underwent some form of "autolysis", where all components of the cell, including the cell wall, disintegrated with little evidence of their presence in the clear, inhibition zone. The oomycete ( P. ultimum ) and ascomycete fungi ( F. graminearum , B. cinerea ) were sensitive to a wider range of VOCs than the bacteria, suggesting that eukaryotic microbes are the main competitors to M. brunneum in the rhizosphere. The ability to alter the VOC profile in response to nutritional cues may assist M. brunneum to survive among the roots of a wide range of plant species. Our VOC studies provided new insights as to how M. brunneum may protect plants from pathogenic microbes and correspondingly promote healthy growth.

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi., Competing Interests: The authors have declared that no competing interests exist.

Abstract: This study aimed to evaluate the decontamination effects of steam-ultrasound application, through specially designed nozzles installed inside a constructed machine, with a capacity of 10,500 birds per h on naturally contaminated broilers. Using three different skin-sampling areas-back, breast, and neck skin-microbial analysis of Campylobacter, Enterobacteriaceae, and total viable count (TVC) was performed before and after steam-ultrasound treatment. In total, 648 skin samples were analyzed for Campylobacter, and 216 samples were analyzed for Enterobacteriaceae and TVC. Results showed Campylobacter reductions (P < 0.001) of 0.8, 1.1, and 0.7 log, analyzed from back, breast, and the neck skin samples, respectively. Furthermore, reductions of Enterobacteriaceae (P < 0.001) by 1.6, 1.9, and 1.1 log and reductions of TVC (P < 0.001) by 2.0, 2.4, and 1.3 log were found on back, breasts, and neck, respectively. Campylobacter levels were evaluated after 8 days of refrigeration at 4°C in control and steam-ultrasound-treated broilers to determine contamination stability in a small 12-sample trial. The results showed no changes in reductions during refrigeration, indicating that reduced Campylobacter numbers remained stable in treated broilers. This study showed significant bacterial reduction was achieved in three different broiler surface areas at a slaughter speed of 10,500 birds per h at temperatures more than 80°C. The rapid treatment of less than 1.5-s exposure time inside the chamber makes this technology potentially suitable for modern and fast poultry processing lines., (Copyright ©, International Association for Food Protection.)

Background: The use of entomopathogenic fungi (EPF) for the control of adult mosquitoes is a promising alternative to synthetic insecticides. Previous studies have only evaluated conidiospores against adult mosquitoes. However, blastospores, which are highly virulent against mosquito larvae and pupae, could also be effective against adults., Methods: Metarhizium anisopliae (ESALQ 818 and LEF 2000) blastospores and conidia were first tested against adult Aedes aegypti by spraying insects with spore suspensions. Blastospores were then tested using an indirect contact bioassay, exposing mosquitoes to fungus-impregnated cloths. Virulence when using blastospores suspended in 20% sunflower oil was also investigated., Results: Female mosquitoes sprayed with blastospores or conidia at a concentration of 10 8  propagules ml -1 were highly susceptible to both types of spores, resulting in 100% mortality within 7 days. However, significant differences in virulence of the isolates and propagules became apparent at 10 7  spores ml -1 , with ESALQ 818 blastospores being more virulent than LEF 2000 blastospores. ESALQ 818 blastospores were highly virulent when mosquitoes were exposed to black cotton cloths impregnated with blastospores shortly after preparing the suspensions, but virulence declined rapidly 12 h post-application. The addition of vegetable oil to blastospores helped maintain virulence for up to 48 h., Conclusion: The results showed that blastospores were more virulent to adult female Ae. aegypti than conidia when sprayed onto the insects or applied to black cloths. Vegetable oil helped maintain blastospore virulence. The results show that blastospores have potential for use in integrated vector management, although new formulations and drying techniques need to be investigated., (© 2021. The Author(s).)

DNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several predicted signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics., (© 2021. The Author(s).)

The co-evolutionary arms race between disease-causing agents and their insect victims is ancient and complex - leading to the development of specialised attack and defence strategies. Among such strategies is the capacity of fungal and oomycete pathogens to deploy degradative enzymes, notably proteases, to facilitate infection directly across the integument. To counter these proteases, insects such as the greater wax moth Galleria mellonella release metalloprotease inhibitors and other immune factors to thwart the invading fungus. To date, molecular-based confirmation of insect metalloprotease inhibitor's incontrovertible role in antifungal defence has been lacking. We targeted the IMPI gene for suppression using RNAi and exposed those insects to the entomopathogenic fungus Metarhizium brunneum ARSEF4556. Levels of IMPI were reduced significantly in the integument (10-fold) and fat body (5-fold) of RNAi-treated insects when compared to control larvae, and displayed a significantly higher mortality rate. We also surveyed candidate immune/detoxification gene expression levels (e.g., DOPA decarboxylase, galiomycin) in three tissues (integument, midgut, fat body) in order to gauge any potential non-target effects of RNAi. The loss of IMPI via RNAi compromises antifungal defences and leaves G. mellonella vulnerable to infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Preparations of the fungus Cordyceps sinensis and bovine colostrum are considered nutraceuticals due to their anti-inflammatory, repair and gut alimentation properties in mammalian models. To reduce the reliance on rodents in routine experimentation, we gauged the capacity of nutraceuticals to alleviate gastric damage in an insect surrogate, Galleria mellonella. Larvae were reared on standard or supplemented diets - 10% (w/w) colostrum, 10% (w/w) C. sinensis, or 5% + 5% each - prior to receiving an oral dose of the NSAID indomethacin (30 mg/kg) or challenged with the bacterial pathogen Campylobacter jejuni (1-3 x10 6 ) via two inoculation routes. Insects reared on a cordyceps-supplemented diet proved most resistant to indomethacin-induced gut leakiness, and displayed stable health indices after C. jejuni challenge (~77% survival). Insects reared on a colostrum-supplemented diet also showed recalcitrance in the gut, but were more sensitive to C. jejuni when injected directly into the body cavity (50% survival). The nutraceutical blend yielded improved health outcomes when compared to the standard diet, but was not as effective as either nutraceutical alone. Our findings represent clear evidence that insects were more resistant to known chemical and microbial agitators when reared on nutraceutical-supplemented diets - toxicological endpoints that are shared with vertebrate studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum., Results: The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis., Conclusions: The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

Objective: To assess knowledge and practices regarding hand-washing among mothers of children aged ≤5 years., Methods: The cross-sectional study was conducted from March to July 2015 in rural areas of Union Council Charwa, Tehsil Pasrur in District Sialkot, Pakistan, and comprised mothers of children aged ≤5 years. Data was collected using a pre-tested and validated structured questionnaire developed in line with World health Organisation guidelines on hand-washing technique. Data was analysed using SPSS 17., Results: Of the 422 subjects, 319 (75.4%) had sufficient knowledge and 104 (24.6%) showed good practices regarding hand-washing. However, only 59 (14%) mothers were found adherent to the appropriate technique of hand-washing., Conclusions: Health education and behaviour-changing communication approaches must be implemented to educate the masses about proper hand-washing technique.

Three strains of fungi belonging to the genus Metarhizium Sorokīn (ARSEF 4556, ARSEF 3297, native strain) were assayed against adults and nymphs of the Neotropical brown stinkbug Euschistus heros (F.) and the green-belly stinkbug Dichelops furcatus (F.). The most virulent strain, ARSEF 4556, caused over 90% mortality. The average survival time of the second and fifth instar nymphs and adults following immersion in 1 × 108 conidia ml-1 was 4.8, 5.7, and 5.2 d, respectively. The second instar nymphs were more susceptible than the adults. The LC50 values and median survival times for second instar and adult E. heros were 1.6 × 107 and 3.1 × 107 conidia ml-1 and 6 and 8 d, respectively. Eggs of E. heros and the closely related stinkbug, D. furcatus, were highly susceptible to ARSEF 4556 with the mean mortality of eggs immersed in 1 × 108 conidia ml-1 being 77.4% and 89.7%, respectively. The strain 3297 showed also good aptitudes for stinkbugs control with mortalities higher than 80% against nymphs and adults and eggs mortalities of 75.5% for E. heros and 79.6% for D. furcatus. This study has shown that it is possible to have a two-pronged control strategy, targeting adults and to reduce oviposition and targeting egg clusters to prevent emergence and dispersal of nymphs. Besides early instars of nymphs have been shown to be more susceptible to the fungal strains than late instars and adults., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The genus Metarhizium is composed of entomopathogenic fungal biological control agents (BCAs) used for invertebrate pest control. The phylogenetic relationships of species within this genus are still under scrutiny as several cryptic species can be found. In this work, the mitochondrial (mt) genome of Metarhizium brunneum ARSEF 4556 was fully sequenced and a comparative genome analysis was conducted with 7 other available mt genomes, belonging to 5 Metarhizium species: M. anisopliae, M. brunneum, M. robertsii, M. guizhouense and M. majus. Results showed that Metarhizium demonstrates greater conserved stability than other fungal mt genomes. Furthermore, this analysis located 7 diverse regions in both intergenic domains and gene fragments which were ideal for species/strain discrimination. The sequencing of these regions revealed several SNPs among 38 strains tested, 11 of which were uncharacterized. Single gene phylogenies presented variable results which may be used further for intra-species discrimination. Phylogenetic trees based on the concatenation of mt domains and the nuclear ITS1-5.8S-ITS2 region showed discrimination of the species studied and allowed the identification of uncharacterized strains. These were mostly placed within species M. anisopliae and M. brunneum. Five strains clustered together in a clade related to M. brunneum, suggesting that they comprise a cryptic species., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

In this study, the binding tendency of bisnitrophenoxy compounds (BN) having different methylene (-CH 2 -) n spacer groups (n = 8-11) with fish sperm double stranded deoxyribonucleic acid (dsDNA) was explored. Cyclic voltammetry (CV) was used to evaluate various kinetic and binding parameters (K s,h , D o , K b and binding site sizes). Performed electrochemical studies designated strong contact of these symmetric molecules with dsDNA in threading intercalation mode of binding. The number (n) of methylene spacer group in the molecular structure of bisnitrophenoxy compounds, e .g., BN-8 (1-nitro-4-(8-(4-nitrophenoxy)octyloxy)benzene, was observed to have a strong influence on their binding affinity. Decreased peak current values and positively shifted peak potentials recorded via cyclic voltammetry clearly depicted that bisnitrophenoxy compounds can intercalate with dsDNA. Results demonstrated the following order of binding constants; K b (M -1 ): BN-8 (2.32 × 10 4 ) < BN-9 (5.73 × 10 4 ) < BN-10 (8.97 × 10 4 ) < BN-11 (17.34 × 10 4 ). The order of increasing binding sites from BN-8 (0.13) to BN-11 (1.38), revealed the maximum threading intercalation strength by bisnitrophenoxy compound having the longest methylene spacer (n = 11). Thermodynamic studies augmented the strong binding of BN-11 with dsDNA as compared to BN-8 because of the long-chain, -CH 2 - spacer in its structure. The spontaneity of dsDNA-binding was revealed by the negative ΔG values for interaction of all the compounds. Moreover, binding parameters from thermodynamic and kinetic studies also corresponded to the threading intercalation mode of interaction, which itself points to the potency of the envisioned drug-like molecules., (© 2020 Published by Elsevier Ltd.)

Camelpox virus is the causative agent of Camelpox, a highly contagious disease of camels. A high passage Camelpox virus strain has previously been reported to contain several genes which more closely resemble Vaccinia, a virus species with no known natural host, encompassing various strains that show high inter-strain genomic variation. In this study, we demonstrate that yet another high passage, live attenuated vaccine, comprising a different strain of Camelpox virus, contains genomic sequences that match a differing strain of Vaccinia virus. These results are discussed in the context of hypotheses put forward to explain the unknown origins of Vaccinia virus, suggesting further studies to elucidate evolutionary trajectories of Orthopoxviruses through passaging., Competing Interests: Declaration of competing interest None., (Copyright © 2020 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Under different pathological conditions, high levels of reactive oxygen species (ROS) cause substantial damage to multiple organs. To counter these ROS levels in multiple organs, we have engineered highly potent novel terpolymers. We found that combination of FDA-approved polyethylene glycol, fumaric acid moieties and electroactive tetra(aniline) by varying the content of tetra(aniline) results into a novel drug composition with biologically active and tunable intrinsic antioxidant properties. To test the intrinsic antioxidative properties of these novel terpolymers, we used alloxan to induce diabetes in rats where ROS generation is known to be higher. The systemic administration of terpolymers to the diabetic rats showed strong electroactive antioxidant behavior which not only normalized ROS levels, but also improved the levels of enzymatic antioxidants including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). As a proof-of-principle, we here show TANI based novel drug composition of terpolymers with tunable intrinsic antioxidant properties in multiple organs., (Copyright © 2019. Published by Elsevier B.V.)

The corrosion inhibitive capabilities of some ferrocene-based Schiff bases on aluminium alloy AA2219-T6 in acidic medium were investigated using Tafel polarization, electrochemical impedance spectroscopy (EIS), weight loss measurement, FT-IR spectroscopy and scanning electron microscopic (SEM) techniques. The influence of molecular configuration on the corrosion inhibition behavior has been explored by quantum chemical calculation. Ferrocenyl Schiff bases 4,4'-((((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methaneylylidene))bis(azaneylylidene))bisferrocene (Fcua), 4,4'-((((ethane-1,2-diylbis(oxy))bis(2-methoxy-1,4-phenylene))bis(methaneylylidene))bis(azaneylylidene))bisferrocene (Fcub) and 4,4'-((((ethane-1,2-diylbis(oxy))bis(2-ethoxy-1,4-phenylene))bis(methaneylylidene))bis(azaneylylidene))bisferrocene (Fcuc) have been synthesized and characterized by FT-IR, 1 H and 13 C NMR spectroscopic studies. These compounds showed a substantial corrosion inhibition against aluminium alloy in 0.1 M of HCl at 298 K. Fcub and Fcuc showed better anticorrosion efficiency as compared with Fcua due to the electron donating methoxy and ethoxy group substitutions, respectively. Polarization curves also indicated that the studied biferrocenyl Schiff bases were mixed type anticorrosive materials. The inhibition of the aluminium alloy surface by biferrocenyl Schiff bases was evidenced through scanning electron microscopy (SEM) studies. Semi-empirical quantum mechanical studies revealed a correlation between corrosion inhibition efficiency and structural functionalities., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In order for entomopathogenic fungi to colonize an insect host, they must first attach to, and penetrate, the cuticle layers of the integument. Herein, we explored the interactions between the fungal pathogen Metarhizium brunneum ARSEF 4556 and two immunologically distinct morphs, melanic (M) and non-melanic (NM), of the greater wax moth Galleria mellonella . We first interrogated the cuticular compositions of both insect morphs to reveal substantial differences in their physiochemical properties. Enhanced melanin accumulation, fewer hydrocarbons, and higher L -dihydroxyphenylalanine (DOPA) decarboxylase activity were evident in the cuticle of the M larvae. This "hostile" terrain proved challenging for M. brunneum - reflected in poor conidial attachment and germination, and elevated expression of stress-associated genes ( e.g., Hsp30, Hsp70 ). Lack of adherence to the cuticle impacted negatively on the speed of kill and overall host mortality; a dose of 10 7 conidia killed ~30% of M larvae over a 12-day period, whereas a 100-fold lower dose (10 5 conidia) achieved a similar result for NM larvae. Candidate gene expression patterns between the insect morphs indicated that M larvae are primed to "switch-on" immunity-associated genes (e.g., phenoloxidase) within 6-12 h of conidia exposure and can sustain a "defense" response. Critically, M. brunneum responds to the distinct physiochemical cues of both hosts and adjusts the expression of pathogenicity-related genes accordingly (e.g., Pr2, Mad1, Mad2 ). We reveal previously uncharacterized mechanisms of attack and defence in fungal-insect antibiosis.

This study examines how the dynamics of fungus-insect interactions can be modulated by temperature. The wax moth, Galleria mellonella, is a well-studied and important model insect whose larvae in the wild develop optimally at around 34 °C in beehives. However, surprisingly little research on wax moths has been conducted at relevant temperatures. In this study, the entomopathogenic fungus Metarhizium robertsii inflicted rapid and substantial mortality on wax moth larvae maintained at a constant temperature of 24 °C, but at 34 °C a 10 fold higher dose was required to achieve an equivalent mortality. The cooler temperature favored fungal pathogenicity, with condial adhesion to the cuticle, germination and hemocoel invasion all significantly enhanced at 24 °C, compared with 34 °C. The wax moth larvae immune responses altered with the temperature, and with the infective dose of the fungus. Enzyme-based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. A dramatic upregulation in the basal expression of galiomicin and gallerimycin was triggered by cooling, and this was augmented in the presence of the fungus. Profiling of the predominant insect epicuticular fatty acids revealed a 4-7 fold increase in palmetic, oleic and linoleic acids in larvae maintained at 24 °C compared with those at 34 °C, but these failed to exert fungistatic effects on topically applied fungus. This study demonstrates the importance of choosing environmental conditions relevant to the habitat of the insect host when determining the dynamics and outcome of insect/fungus interactions, and has particular significance for the application of entomopathogens as biocontrol agents., (© 2016 Institute of Zoology, Chinese Academy of Sciences.)

Background: Entomopathogenic fungi are highly promising agents for controlling Aedes aegypti mosquitoes. Deploying fungus-impregnated black cloths in PET traps efficiently reduced Ae. aegypti female survival rates under intra-domicile conditions. With the aim of further increasing the effectiveness of the traps, the addition of attractive lures to fungus-impregnated traps was evaluated., Methods: Black cloths were suspended inside 2 l plastic bottles called "PET traps". These traps were placed in rooms simulating human residences. The first experiments evaluated the attraction of mosquitoes to PET traps with black cloths covered in adhesive film with and without synthetic lures (AtrAedes™). Traps were left in the test rooms for either 24 or 48 h. The attractiveness of the lures over time was also evaluated. The efficiency of PET traps with fungus-impregnated black cloths associated with lures was compared to that of traps without lures., Results: The highest percentage of captured mosquitoes (31 and 66%) were observed in PET traps with black cloths covered in adhesive film + attractive lure maintained in test rooms for 24 h and 48 h, respectively. Black cloths covered in adhesive film captured 17 or 36% of the mosquitoes at 24 h and 48 h, respectively. The attractiveness of the lures fell gradually over time, capturing 37% after 5 days on the bench and 22% of the mosquitoes after 30 days exposure to ambient conditions. Associating attractive synthetic lures with black cloths impregnated with M. anisopliae placed in test rooms for 120 h reduced mean survival to 32%, whilst black cloths impregnated with M. anisopliae without lures resulted in a 48% survival rate. Using Beauveria bassiana in the traps resulted in a 52% reduction in mosquito survival, whilst combining Beauveria and AtrAedes resulted in a 36% survival rate. PET traps impregnated with fungus + AtrAedes resulted in similar reductions in survival when left in the rooms for 24, 48, 72 or 120 h., Conclusions: AtrAedes increased attractiveness of PET traps with black cloths under intra-domicile conditions and when associated with M. anisopliae or B. bassiana, significantly reduced Aedes survival. This strategy will reduce the number of PET traps necessary per household.

Mosquitoes transmit several diseases, which are of global significance (malaria, dengue, yellow fever, Zika). The geographic range of mosquitoes is increasing due to climate change, tourism and trade. Both conidial and blastospore formulations of the entomopathogenic fungus, Metarhizium brunneum ARSEF 4556, are being investigated as mosquito larvicides. However, concerns have been raised over possible non-target impacts to arthropod mosquito predators such as larvae of Toxorhynchites brevipalpis which feed on larvae of mosquito vector species. Laboratory-based, small container bioassays showed, that T. bevipalpis larvae are susceptible to relatively high concentrations (i.e. ≥10 7  spores ml -1 ) of inoculum with blastospores being significantly more virulent than conidia. At lower concentrations (e.g. <10 7  spores ml -1 ), it appears that M. brunneum complements T. brevipalpis resulting in higher control than if either agent was used alone. At a concentration of 10 5  spores ml -1 , the LT 50 of for conidia and blastospores alone was 5.64 days (95% CI: 4.79-6.49 days) and 3.89 days (95% CI: 3.53-4.25 days), respectively. In combination with T. brevipalpis, this was reduced to 3.15 days (95% CI: 2.82-3.48 days) and 2.82 days (95% CI: 2.55-3.08 days). Here, combined treatment with the fungus and predator was beneficial but weaker than additive. At 10 7 and 10 8  blastospores ml -1 , mosquito larval mortality was mostly due to the fungal pathogen when the predator was combined with blastospores. However, with conidia, the effects of combined treatment were additive/synergistic at these high concentrations. Optimisation of fungal concentration and formulation will reduce: (1) risk to the predator and (2) application rates and costs of M. brunneum for control of mosquito larvae., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.

Biorational insecticides are being increasingly used in integrated pest management programs. In laboratory bioassays, the pathogenicity of blastospores and conidia of the entomopathogenic fungus Metarhizium brunneum ARSEF 4556 was evaluated against larvae of three mosquito species. Three propagule concentrations (1 × 106, 1 × 107, and 1 × 108 spores ml - 1) were used in the bioassays. Results showed that Aedes aegypti had lower survival rates when exposed to blastospores than when exposed to conidia, whereas the converse was true for Culex quinquefasciatus larvae. Anopheles stephensi larvae survival rates were similar when exposed to blastospores and conidia, except at the higher doses, where blastospores were more virulent. Several assays showed little difference in mortalities when using either 1 × 107 or 1 × 108 spores ml - 1, suggesting a threshold above which no higher control levels or economic benefit would be achieved. When tested at the lowest dose, the LT50 of Cx. quinquefasciatus using blastospores, wet conidia, and dry conidia was 3.2, 1.9, and 4.4 d, respectively. The LT50 of Ae. aegypti using blastospores, wet conidia, and dry conidia was 1.3, 3.3, and 6.2 d, respectively. The LT50 of An. stephensi using blastospores, wet conidia, and dry conidia was 2.0, 1.9, and 2.1 d, respectively. These observations suggest that for optimized control, two different formulations of the fungus may be needed when treating areas where there are mixed populations of Aedes, Anopheles, and Culex., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Microevolutionary mechanisms of resistance to a bacterial pathogen were explored in a population of the Greater wax moth, Galleria mellonella, selected for an 8.8-fold increased resistance against the entomopathogenic bacterium Bacillus thuringiensis (Bt) compared with a non-selected (suspectible) line. Defense strategies of the resistant and susceptible insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management and immune defense in the fat body. Following oral infection with Bt, the expression of these genes was further elevated in the fat body and midgut of both lines and to a greater extent some of them in resistant line than the susceptible line. This gene expression analysis reveals a pattern of resistance mechanisms targeted to sites damaged by Bt with the insect placing greater emphasis on tissue repair as revealed by elevated expression of these genes in both the fat body and midgut epithelium. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affects the viability of other gut bacteria. Remarkably the resistant line employs multifactorial adaptations for resistance to Bt without any detected negative trade off since the insects exhibited higher fecundity.

Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world's population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti.

Although many insects successfully live in dangerous environments exposed to diverse communities of microbes, they are often exploited and killed by specialist pathogens. Studies of host-pathogen interactions (HPI) provide valuable insights into the dynamics of the highly aggressive coevolutionary arms race between entomopathogenic fungi (EPF) and their arthropod hosts. The host defenses are designed to exclude the pathogen or mitigate the damage inflicted while the pathogen responds with immune evasion and utilization of host resources. EPF neutralize their immediate surroundings on the insect integument and benefit from the physiochemical properties of the cuticle and its compounds that exclude competing microbes. EPF also exhibit adaptations aimed at minimizing trauma that can be deleterious to both host and pathogen (eg, melanization of hemolymph), form narrow penetration pegs that alleviate host dehydration and produce blastospores that lack immunogenic sugars/enzymes but facilitate rapid assimilation of hemolymph nutrients. In response, insects deploy an extensive armory of hemocytes and macromolecules, such as lectins and phenoloxidase, that repel, immobilize, and kill EPF. New evidence suggests that immune bioactives work synergistically (eg, lysozyme with antimicrobial peptides) to combat infections. Some proteins, including transferrin and apolipophorin III, also demonstrate multifunctional properties, participating in metabolism, homeostasis, and pathogen recognition. This review discusses the molecular intricacies of these HPI, highlighting the interplay between immunity, stress management, and metabolism. Increased knowledge in this area could enhance the efficacy of EPF, ensuring their future in integrated pest management programs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Monitoring of pest populations is an essential component of integrated pest management. An early warning system helps growers decide when best to take control measures, or when to alter them, should a control method prove inadequate. Studies have shown that adding chemical attractants to sticky cards can increase trap catch of western flower thrips, Frankliniella occidentalis, a global pest of agriculture and horticulture, giving more accurate accounts of population size and dynamics, thus leading to more efficient monitoring. We identify a novel semiochemical to the species, (S)-(-)-verbenone, showing that addition of this compound to sticky traps significantly increased F. occidentalis catch in two geographically distinct populations, infesting two unrelated crops of global economic importance. We validate through field trials that (S)-(-)-verbenone is highly attractive to F.occidentalis and can be used with blue sticky traps to enhance trap catch, leading to better estimations of pest population densities. The compound may be used in other control methods against F.occidentalis such as lure and kill, mass trapping and push-pull.

Recent studies of healthy human airways have revealed colonization by a distinct commensal bacterial microbiota containing Gram-negative Prevotella spp. However, the immunological properties of these bacteria in the respiratory system remain unknown. Here we compare the innate respiratory immune response to three Gram-negative commensal Prevotella strains (Prevotella melaninogenica, Prevotella nanceiensis and Prevotella salivae) and three Gram-negative pathogenic Proteobacteria known to colonize lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma (Haemophilus influenzae B, non-typeable Haemophilus influenzae and Moraxella catarrhalis). The commensal Prevotella spp. and pathogenic Proteobacteria were found to exhibit intrinsic differences in innate inflammatory capacities on murine lung cells in vitro. In vivo in mice, non-typeable H. influenzae induced severe Toll-like receptor 2 (TLR2)-independent COPD-like inflammation characterized by predominant airway neutrophilia, expression of a neutrophilic cytokine/chemokine profile in lung tissue, and lung immunopathology. In comparison, P. nanceiensis induced a diminished neutrophilic airway inflammation and no detectable lung pathology. Interestingly, the inflammatory airway response to the Gram-negative bacteria P. nanceiensis was completely TLR2-dependent. These findings demonstrate weak inflammatory properties of Gram-negative airway commensal Prevotella spp. that may make colonization by these bacteria tolerable by the respiratory immune system., (© 2014 John Wiley & Sons Ltd.)

Food contact surfaces require rigorous sanitation procedures for decontamination, although these methods very often fail to efficiently clean and disinfect surfaces that are visibly contaminated with food residues and possible biofilms. In this study, the results of a short treatment (1 to 2 s) of combined steam (95°C) and ultrasound (SonoSteam) of industrial fish and meat transportation boxes and live-chicken transportation crates naturally contaminated with food and fecal residues were investigated. Aerobic counts of 5.0 to 6.0 log CFU/24 cm(2) and an Enterobacteriaceae spp. level of 2.0 CFU/24 cm(2) were found on the surfaces prior to the treatment. After 1 s of treatment, the aerobic counts were significantly (P < 0.0001) reduced, and within 2 s, reductions below the detection limit (<10 CFU) were reached. Enterobacteriaceae spp. were reduced to a level below the detection limit with only 1 s of treatment. Two seconds of steam-ultrasound treatment was also applied on two different types of plastic modular conveyor belts with hinge pins and one type of flat flexible rubber belt, all visibly contaminated with food residues. The aerobic counts of 3.0 to 5.0 CFU/50 cm(2) were significantly (P < 0.05) reduced, while Enterobacteriaceae spp. were reduced to a level below the detection limit. Industrial meat knives were contaminated with aerobic counts of 6.0 log CFU/5 cm(2) on the handle and 5.2 log CFU/14 cm(2) on the steel. The level of Enterobacteriaceae spp. contamination was approximately 2.5 log CFU on the handle and steel. Two seconds of steam-ultrasound treatment reduced the aerobic counts and Enterobacteriaceae spp. to levels below the detection limit on both handle and steel. This study shows that the steam-ultrasound treatment may be an effective replacement for disinfection processes and that it can be used for continuous disinfection at fast process lines. However, the treatment may not be able to replace efficient cleaning processes used to remove high loads of debris.

Stereochemistry plays a significant role in structure-activity relationships of messenger chemicals. The ability to distinguish between enantiomers and geometric isomers, however, may be limited to certain stereoisomeric substances, depending on the receiver. In this study, we assessed the preference of the peripheral olfactometry system of Western Flower Thrips, F. occidentalis towards ubiquitously expressed host compounds, with a goal of establishing whether particular stereoisomers enhance host odour recognition. We demonstrate that the peripheral olfactory system of a highly polyphagous thysanopteran insect has evolved to become highly sensitive to a type of green leaf volatile, which is highly ubiquitous in the plant kingdom. We show that there is a significantly greater antennal response to the cis isomer, more so than the isomerisation by-product trans -3-hexen-1-ol. We demonstrate that the antennae of a highly polyphagous insect are capable of detecting common plant secondary metabolites in both enantiomeric forms.

Adhesion of conidia of the insect pathogenic fungus, Metarhizium anisopliae, to the arthropod host cuticle initially involves hydrophobic forces followed by consolidation facilitated by the action of extracellular enzymes and secretion of mucilage. Gene expression analysis and atomic force microscopy were used to directly quantify recognition and adhesion between single conidia of M. anisopliae and the cuticle of the aquatic larval stage of Aedes aegypti and a representative terrestrial host, Tenebrio molitor. Gene expression data indicated recognition by the pathogen of both hosts; however, the forces for adhesion to the mosquito were approximately five times lower than those observed for Tenebrio. Although weak forces were recorded in response to Aedes, Metarhizium was unable to consolidate firm attachment. An analysis of the cuticular composition revealed an absence of long-chain hydrocarbons in Aedes larvae which are thought to be required for fungal development on host cuticle. This study provides, to our knowledge, the first evidence that Metarhizium does not form firm attachment to Ae. aegypti larvae in situ, therefore preventing the normal route of invasion and pathogenesis from occuring.

Pollinophagy is widely documented in the order Thysanoptera, with representative individuals from six of the nine divergent families known to feed on pollen. Various pollens of the genus Pinus increase the development time, fecundity, longevity, and settling preference of Western Flower Thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Certain species of flower thrips discriminate among pollen types, but no studies have elucidated the olfactory cues that play a role in their pollen preferences. In this study, the volatile organic compounds emitted by pollens of the genus Pinus were elucidated. Various chemicals from pollen headspace elicited electrophysiological responses from WFT antennae. The compound (S)-(-)-verbenone, identified in pollen headspace, attracted WFT in a 4-arm olfactometer. This compound has potential for use in integrated pest management programs against the pest. We present the hypothesis that this polyphagous insect may have retained ancestral 'relict' olfactory receptors through the course of evolution, to explain this attraction to pine pollen. This attraction has allowed the insect to find and exploit an unusual nutrient source that significantly increases its fitness. The study demonstrates how fossil record analysis and subsequent evolutionary knowledge can aid in explaining possibilities as to why some insects sense and respond to chemicals that would otherwise seem peculiar to their ecology, allowing insight into the evolutionary forces that may shape insect olfactory systems over time.

Background: Entomopathogenic fungi are potential candidates for use in integrated vector management, with recent emphasis aimed at developing adult mosquito control methods. Here we investigated the persistence of the fungus Metarhizium anisopliae when tested against female A. aegypti under field conditions., Methods: Black cotton cloths impregnated with M. anisopliae conidia, formulated in vegetable oil + isoparaffin, were maintained on a covered veranda for up to 30 days. At specific times, pieces of the cloths were removed, placed in Tween 80 and the resuspended conidia were sprayed directly onto mosquitoes. The persistence of conidia impregnated on black cloths using three different carriers was evaluated in test rooms. Fifty mosquitoes were released into each room and after a 5 day period, the surviving insects were captured. Another 50 insects were then released into each room. The capacity of the fungus at reducing mosquito survival was evaluated over a total of 35 days., Results: Conidia extracted from cloths maintained on the veranda for 2 to 18 days remained virulent, with 28 to 60% mosquito survival observed. Mosquito survival following exposure to fungus impregnated cloths showed that fungus + Tween caused similar reductions to that of fungus + vegetable oil. Mosquitoes exposed to the formulation fungus + vegetable oil had survival rates of 36% over the first 5 days of the experiment. Following the release of the second cohort of mosquitoes (6-11days), survival increased to 50%. The survival of the 12-17 day cohort (78%) was statistically equal to that of the controls (84%). Formulation of the fungus in vegetable oil + isoparaffin increased the persistence of the fungus, with the 18-23 day cohort (64% survival) still showing statistical differences to that of the controls (87% survival)., Conclusions: The potential of entomopathogenic fungi for the control of adult A. aegypti was confirmed under field conditions. Vegetable oil + isoparaffin formulations of M. anisopliae significantly increased the effectiveness of the fungus, thus reducing the need for frequent changes of black cloths in residences. Our future aim is to obtain effective control of mosquito populations, with cloths only needing to being replaced once a month.

Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host.

Candida albicans is a major human pathogen whose treatment is challenging due to antifungal drug toxicity, drug resistance and paucity of antifungal agents available. Myrocin (MYR) inhibits sphingosine synthesis, a precursor of sphingolipids, an important cell membrane and signaling molecule component. MYR also has dual immune suppressive and antifungal properties, potentially modulating mammalian immunity and simultaneously reducing fungal infection risk. Wax moth (Galleria mellonella) larvae, alternatives to mice, were used to establish if MYR suppressed insect immunity and increased survival of C. albicans-infected insects. MYR effects were studied in vivo and in vitro, and compared alone and combined with those of approved antifungal drugs, fluconazole (FLC) and amphotericin B (AMPH). Insect immune defenses failed to inhibit C. albicans with high mortalities. In insects pretreated with the drug followed by C. albicans inoculation, MYR+C. albicans significantly increased mortality to 93% from 67% with C. albicans alone 48 h post-infection whilst AMPH+C. albicans and FLC+C. albicans only showed 26% and 0% mortalities, respectively. MYR combinations with other antifungal drugs in vivo also enhanced larval mortalities, contrasting the synergistic antifungal effect of the MYR+AMPH combination in vitro. MYR treatment influenced immunity and stress management gene expression during C. albicans pathogenesis, modulating transcripts putatively associated with signal transduction/regulation of cytokines, I-kappaB kinase/NF-kappaB cascade, G-protein coupled receptor and inflammation. In contrast, all stress management gene expression was down-regulated in FLC and AMPH pretreated C. albicans-infected insects. Results are discussed with their implications for clinical use of MYR to treat sphingolipid-associated disorders.

A 'dark morph' melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25(th) generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host's own immune responses.

Background: Larvae of the crane fly Tipula paludosa (Diptera: Nematocera) are economically important pests of grasslands, nurseries and organically grown crops. This study was conducted to evaluate several entomopathogenic fungal strains, the entomopathogenic nematode Heterorhabditis bacteriophora and the organophosphate insecticide Dursban WG (75% chlorpyrifos) against larval stages of T. paludosa under laboratory and greenhouse conditions., Results: Metarhizium robertsii strain V1005 was the most virulent, causing 100% larval mortality 4 weeks post-inoculation. Eight other M. robertsii strains and M. brunneum (ARSEF 3297) caused mortality ranging between 0 and 60%, whereas strains of Beauveria bassiana, Isaria fumosorosea and Lecanicillium longisporum were non-pathogenic to T. paludosa. In greenhouse tests, medium (2 × 10(14) conidia ha(-1) ) and high (2 × 10(16) conidia ha(-1) ) concentrations of V1005 gave higher larval mortalities (90 and 100%) than lower (2 × 10(13) conidia ha(-1) ) concentrations (52 and 78%) at 4 and 8 weeks after treatment respectively. Dursban WG gave 100% larval mortality 4 weeks after treatment; H. bacteriophora UWS1 caused 28 and 65% mortality 4 and 8 weeks after treatment respectively., Conclusions: This study shows that M. robertsii V1005 has considerable potential for the control of T. paludosa, thereby reducing the use of chemical insecticides., (Copyright © 2012 Society of Chemical Industry.)

The large pine weevil, Hylobius abietis, is a major pest in European conifer forests causing millions of Euros of damage annually. Larvae develop in the stumps of recently felled trees; the emerging adults feed on the bark of seedlings and may kill them. This study investigated the susceptibility of different developmental stages of H. abietis to commercial and commercially viable isolates of entomopathogenic fungi, Metarhizium and Beauveria. All the developmental stages of H. abietis can be killed by Metarhizium robertsii, Metarhizium brunneum, and Beauveria bassiana. The most virulent isolate of M. robertsii ARSEF4556 caused 100% mortality of pupae, larvae and adults on day 4, 6 and 12, respectively. This strain was further tested against adult weevils in different concentrations (10(5)-10(8) conidia cm(-2) or ml(-1)) using two types of fungal formulation: 'dry' conidia and 'wet' conidia (suspended in 0.03% aq. Tween 80) applied on different substrates (tissue paper, peat and Sitka spruce seedlings). 'Dry' conidia were more effective than 'wet' conidia on tissue paper and on spruce or 'dry' conidia premixed in peat. The LC(50) value for 'dry' conidia of isolate ARSEF4556 was three folds lower than 'wet' conidia on tissue paper. This study showed that 'dry' conidia are more effective than 'wet' conidia, causing 100% adult mortality within 12 days. Possible strategies for fungal applications are discussed in light of the high susceptibility of larvae and pupae to fungal pathogen., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recent studies using culture-independent methods have characterized the human airway microbiota and report microbial communities distinct from other body sites. Changes in these airway bacterial communities appear to be associated with inflammatory lung disease, yet the pro-inflammatory properties of individual bacterial species are unknown. In this study, we compared the immune stimulatory capacity on human monocyte-derived dendritic cells (DCs) of selected airway commensal and pathogenic bacteria predominantly associated with lungs of asthma or COPD patients (pathogenic Haemophillus spp. and Moraxella spp.), healthy lungs (commensal Prevotella spp.) or both (commensal Veillonella spp. and Actinomyces spp.). All bacteria were found to induce activation of DCs as demonstrated by similar induction of CD83, CD40 and CD86 surface expression. However, asthma and COPD-associated pathogenic bacteria provoked a 3-5 fold higher production of IL-23, IL-12p70 and IL-10 cytokines compared to the commensal bacteria. Based on the differential cytokine production profiles, the studied airway bacteria could be segregated into three groups (Haemophilus spp. and Moraxella spp. vs. Prevotella spp. and Veillonella spp. vs. Actinomyces spp.) reflecting their pro-inflammatory effects on DCs. Co-culture experiments found that Prevotella spp. were able to reduce Haemophillus influenzae-induced IL-12p70 in DCs, whereas no effect was observed on IL-23 and IL-10 production. This study demonstrates intrinsic differences in DC stimulating properties of bacteria associated with the airway microbiota.

In this overview, some of the more significant recent developments in bioengineering natural products from insects with use or potential use in modern medicine are described, as well as in utilisation of insects as models for studying essential mammalian processes such as immune responses to pathogens. To date, insects have been relatively neglected as sources of modern drugs although they have provided valuable natural products, including honey and silk, for at least 4-7000 years, and have featured in folklore medicine for thousands of years. Particular examples of Insect Folk Medicines will briefly be described which have subsequently led through the application of molecular and bioengineering techniques to the development of bioactive compounds with great potential as pharmaceuticals in modern medicine. Insect products reviewed have been derived from honey, venom, silk, cantharidin, whole insect extracts, maggots, and blood-sucking arthropods. Drug activities detected include powerful antimicrobials against antibiotic-resistant bacteria and HIV, as well as anti-cancer, anti-angiogenesis and anti-coagulant factors and wound healing agents. Finally, the many problems in developing these insect products as human therapeutic drugs are considered and the possible solutions emerging to these problems are described., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests., Methods and Results: The conidial yields and the shelf-life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia-bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf-life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C., Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes., Significance and Impact of the Study:   This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)