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See detailInsect pathogenic Aspergillus species in mosquito (Diptera: Culicidae) control compared to Metarhizium anisopliae
Bawin, Thomas ULg

Doctoral thesis (2016)

Many mosquito (Diptera: Culicidae) species are vectors responsible for the transmission of infectious diseases of medical and veterinary importance. Risk for infection considerably increased during the ... [more ▼]

Many mosquito (Diptera: Culicidae) species are vectors responsible for the transmission of infectious diseases of medical and veterinary importance. Risk for infection considerably increased during the last decades due to climate changes and increasing global trade. Vector control is essential for public health management. Integrated pest management is now promoted due to harmful side effects of the chemical insecticides classically used for mosquito control and insect resistance development. This PhD thesis takes part in the selection process of insect pathogenic fungi. Three biological models (Aspergillus clavatus Desmazieres, Aspergillus flavus Link, and Metarhizium anisopliae (Metschnikoff) Sorokin) were selected and investigated regarding their spore insecticidal activity, mode of action, and selectivity. Throughout this scheme, the use of agricultural materials and derivatives in fermentation processes was emphasized. Production methods and insecticidal potential of the fungi were first investigated. On the one hand, the two Aspergillus species were compared to M. anisopliae regarding their spore production when cultured on agro-industrial substrates (white rice and wheat bran) and their insecticidal effect on larvae of the southern house mosquito, Culex quinquefasciatus Say. Our results showed that both substrates are suitable to culture the three insect pathogenic fungal species; but wheat bran-based media increased from 3 to 7-fold the spore yields in solid-state compared to white rice-based media. Also, insect pathogenic Aspergillus species were suggested to yield in similar spore levels in such conditions (in a range of 10^9 spores per g of substrate) and be as virulent against mosquito larvae compared to M. anisopliae. On the other hand, a bioreactor design intended to support large-scale production on agricultural materials by combining the technological advantages of submerged and solid-state fermentations was implemented using A. clavatus. This system allowed facility in recuperation and purification of spores (confined on a packed solid substrate) that retained virulence against mosquito larvae; but also metabolites (contained in a circulating liquid medium) that showed insecticidal effect. The mechanisms responsible for the insecticidal effects of the spores, and their selectivity were then investigated. On the one hand, the invasion routes of A. clavatus spores on mosquito larvae were investigated by light and electron microscopy. Histological observations suggest that toxins secreted by active germinating spores of A. clavatus in the digestive tract altered the larval tissues, leading to necrosis and causing larval death. Fungal proliferation and sporulation then occurred during a saprophytic phase. Such action mode also probably occurred in the case of the two other species. On the other hand, the insecticidal activity of the fungi was assessed against the pea aphid, Acyrthosiphon pisum Linnaeus. Adult A. pisum aphids were susceptible (increased mortality and decreased reproductive potential) to the three fungal species. As a consequence, these fungi would probably be able to infect a broad spectrum of insect hosts (whatever terrestrial or aquatic) once released in the environment. The implications of the results for mosquito control and application strategies are finally discussed, and perspectives for future works proposed. [less ▲]

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See detailHistopathological effects of Aspergillus clavatus (Ascomycota: Trichocomaceae) on larvae of the southern house mosquito, Culex quinquefasciatus (Diptera: Culicidae)
Bawin, Thomas ULg; Seye, Fawrou; Boukraa, Slimane ULg et al

in Fungal Biology (2016), 120(4), 489-499

Aspergillus clavatus (Ascomycota: Trichocomaceae) was previously found to be an opportunistic pathogen of mosquitoes (Diptera: Culicidae). In the present study, the mechanism leading to its insecticidal ... [more ▼]

Aspergillus clavatus (Ascomycota: Trichocomaceae) was previously found to be an opportunistic pathogen of mosquitoes (Diptera: Culicidae). In the present study, the mechanism leading to its insecticidal activity was investigated regarding histological damages on Culex quinquefasciatus larvae exposed to A. clavatus spores. Multiple concentration assays using spore suspensions (0.5 x 10^8 to 2.5 x 10^8 spores/ml) revealed 17.0% to 74.3% corrected mortalities after 48 h exposure. Heat-deactivated spores induced a lower mortality compared to non-heated spores suggesting that insecticidal effects are actively exerted. Spore-treated and untreated larvae were prepared for light microscopy as well as for scanning and transmission electron microscopy. Spores failed to adhere to the external body surface (except the mouth parts) of these aquatic immature stages but progressively filled the digestive tract where their metabolism seemed to activate. In parallel, the internal tissues of the larvae, i.e. the midgut wall, the skeletal muscles, and the cuticle-secreting epidermis, were progressively destroyed between 8 and 24 h of exposure. These observations suggest that toxins secreted by active germinating spores of A. clavatus in the digestive tract altered the larval tissues, leading to their necrosis and causing larval death. Fungal proliferation and sporulation then occurred during a saprophytic phase. A. clavatus enzymes or toxins responsible for these pathogenic effects need to be identified in further studies before any use of this fungus in mosquito control. [less ▲]

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See detailProduction of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae
Bawin, Thomas ULg; Seye, Fawrou; Boukraa, Slimane ULg et al

in Biocontrol Science & Technology (2016), 26(5), 617-629

Entomopathogenic micro-organisms including fungi have become increasingly studied for integrated pest management. The spore productivity and insecticidal activity of two opportunistic insect pathogenic ... [more ▼]

Entomopathogenic micro-organisms including fungi have become increasingly studied for integrated pest management. The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media were suitable for spore production and increased the spore yield in solid-state from 3 to 7 fold: A. clavatus produced 48.4 ± 5.2 and 15.7 ± 1.6 x 10^8 spores/g, A. flavus produced 22.3 ± 4.1 and 3.1 ± 2.5 x 10^8 spores/g, and M. anisopliae produced 39.6 ± 6.5 and 13.1 ± 2.6 x 10^8 spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC50) of 1.1, 1.8, and 1.3 x 10^8 spores/ml against Culex quinquefasciatus Say larvae in 72 h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognized entomopathogenic fungus. Wheat bran could advantageously be used in large-scale fermentation for a possible cost-effective pest control using these fungi. [less ▲]

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See detailDiversity and ecology survey of mosquitoes potential vectors in Belgian equestrian farms: A threat prevention of mosquito-borne equine arboviruses
Boukraa, Slimane ULg; de la Grandière, Maria Ana ULg; Bawin, Thomas ULg et al

in Preventive Veterinary Medicine (2016), 124

Emergence of West Nile Virus was recently recorded in several European countries, which can lead to severe health problems in horse populations. Europe is also at risk of introduction of mosquito-borne ... [more ▼]

Emergence of West Nile Virus was recently recorded in several European countries, which can lead to severe health problems in horse populations. Europe is also at risk of introduction of mosquito-borne equine alphavirus from Americas. Prevention of these arboviruses requires a clear understanding of transmission cycles, especially their vectors. To characterize mosquito fauna, their ecology and identify potential vectors of equine arboviruses in Belgium, entomological surveys of six equestrian farms located in the Wolloon Region were conducted during 2011–2012. The harvest of mosquitoes was based on larval sampling (272 samples from 111 breeding sites) and monthly adults trapping (CO2-baited traps, Mosquito Magnet Liberty Plus). Among 51,493 larvae and 319 adult mosquitoes collected, morphological identification showed the presence of 11 species: Anopheles claviger (Meigen), An. maculipennis s.l. (Meigen), An. plumbeus (Stephens), Culex hortensis (Ficalbi), Cx. territans (Walker), Cx. pipiens s.l. L., Cx. torrentium (Martini), Coquillettidia richiardii (Ficalbi), Culiseta annulata (Schrank), Aedes cantans (Meigen), Ae. geniculatus (Olivier). Molecular identification of Cx. pipiens species complex allowed the detection of three molecular forms, Pipiens (92.3%), Molestus (4.6%) and Hybrid (3.1%). Larvae of Cx. pipiens sl and Cx. torrentium were omnipresent and the most abundant species. Water troughs, ponds and slurry (liquid manure) were the most favorable breeding sites of mosquito larvae. Based upon behavior and ecology of the identified mosquito species, Studied Belgian equestrian farms seem to provide a suitable environment and breeding sites for the proliferation of potential vectors of arboviruses and those being a real nuisance problem for horses and neighboring inhabitants. [less ▲]

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See detailMolecular detection of six (endo-) symbiotic bacteria in Belgian mosquitoes: first step towards the selection of appropriate paratransgenesis candidates
Raharimalala, Fara Nantenaina; Boukraa, Slimane ULg; Bawin, Thomas ULg et al

in Parasitology Research (2016)

Actually, the use of symbiotic bacteria is one of alternative solution to avoid vector resistance to pesticides. In Belgium, among 31 identified mosquito species, 10 were considered as potential vectors ... [more ▼]

Actually, the use of symbiotic bacteria is one of alternative solution to avoid vector resistance to pesticides. In Belgium, among 31 identified mosquito species, 10 were considered as potential vectors. Given to introduction risks of arbovirosis, the purpose of this study was to investigate the presence of symbiosis bacteria in potential mosquito vectors. Eleven species caught from 12 sites in Belgium were used: Culex pipiens s.l., Culex torrentium, Culex hortensis, Anopheles claviger, Anopheles maculipennis s.l., Anopheles plumbeus, Culiseta annulata, Ochlerotatus geniculatus, Ochlerotatus dorsalis, Aedes albopictus, and Coquillettidia richiardii. Six genera of symbiotic bacteria were screened: Wolbachia sp., Comamonas sp, Delftia sp., Pseudomonas sp., Acinetobacter sp., and Asaia sp.Atotal of 173 mosquito individuals (144 larvae and 29 adults) were used for the polymerase chain reaction screening. Wolbachia was not found in any Anopheles species nor Cx. torrentium. A total absence of Comamonas and Delftia was observed in all species. Acinetobacter, Pseudomonas, and Asaia were found in most of species with a high prevalence for Pseudomonas. These results were discussed to develop potential strategy and exploit the variable occurrence of symbiotic bacteria to focus on them to propose biological ways of mosquito control. [less ▲]

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See detailAbility of Tuta absoluta (Lepidoptera: Gelechiidae) to develop on alternative host plant species
Bawin, Thomas ULg; Dujeu, David; De Backer, Lara ULg et al

in Canadian Entomologist (2015)

The tomato leafminer, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae), is a widespread devastating pest reported to develop on economically important solanaceous crops. The characterization of its host ... [more ▼]

The tomato leafminer, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae), is a widespread devastating pest reported to develop on economically important solanaceous crops. The characterization of its host range could help to understand and prevent the dispersion behavior of the insect in the environment. In this study, the ability of T. absoluta to develop on 12 cultivated or non-cultivated plants including Solanaceae, Chenopodiaceae, Convolvulaceae, Fabaceae and Malvaceae species under laboratory conditions was assessed. For each plant species, we monitored the development times of immature stages, survival, sex ratios and adult fecundity rates. All the 6 tested non-solanaceous plants, including Chenopodium, Convolvulus and Malva species, were not able to sustain (i.e. allow growth and development) T. absoluta larvae. Among Solanaceae, Solanum species were the most suitable host plants for the pest, but others could be opportunistically colonized with fewer incidences. T. absoluta appears to be strongly related to solanaceous plants which would predominantly support self-sustaining field populations. Preventing crop infestation by removing potential host plants in the immediate field vicinity and culture rotations with non-solanaceous crops is of primary importance. [less ▲]

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See detailTuta absoluta (Lepidoptera: Gelechiidae) development on wild and cultivated plant species
Bawin, Thomas ULg; Dujeu, David ULg; Fagan, Maud et al

Conference (2015, August 24)

Introduction: The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread invasive species damaging economically important solanaceous crop plants, including tomatoes and potatoes ... [more ▼]

Introduction: The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread invasive species damaging economically important solanaceous crop plants, including tomatoes and potatoes. Little is known about the ability of the microlepidoptera to encounter and develop on alternative wild and agricultural plant species. These plants could provide refuges and have to be identified for more efficient integrated management strategies. Objectives: In the present study, we assessed under laboratory conditions the ability of T. absoluta to develop on such plant species referred as potential hosts in the literature, including Solanaceae, Chenopodiaceae, Convolvulaceae, Fabaceae, and Malvaceae. Materials and methods: For each plant species, fitness tests were performed in Petri dishes by isolating single individuals with excised leaf. Two choice behavioral assays were performed in flying tunnels. Volatile organic compounds released by solanaceous plants were trapped using a dynamic collection system, and analyzed by GC-MS. Results: We found that Solanum species allowed higher larval survivability and shorter development time (from egg to adult emergency) compared to the other plants. Non-solanaceous plants were not able to sustain T. absoluta larvae. Two choice behavioral assays revealed that adult distribution and female oviposition did not differ between Solanum species, which were preferred to other tested solanaceous plants. The hypothesis that female host plant choice is influenced by plant volatile organic compounds was tested. Solanum volatile profiles showed similarities, and were presenting quantitative and qualitative differences with the other tested solanaceous plants, providing some explanations in the observed behavioral discrimination. Further electrophysiological and behavioral assays are required to confirm the effect of specific chemicals on the choice of the oviposition site in T. absoluta. Conclusion: It can be concluded that Solanum species are the more suitable hosts for T. absoluta development. Other solanaceous plant species could be opportunistically colonized with little incidence but care should be taken in these results as genetic variability in insects and plants, as well as plant physiological state, might have an impact on the pest survivability. [less ▲]

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See detailLa lutte contre les moustiques (Diptera: Culicidae): diversité des approches et application du contrôle biologique
Bawin, Thomas ULg; Seye, Fawrou; Boukraa, Slimane ULg et al

in Canadian Entomologist (2015), 147(4), 476-500

Many mosquito (Diptera: Culicidae) species are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance. Currently, changes in the vectors’ geographical ... [more ▼]

Many mosquito (Diptera: Culicidae) species are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance. Currently, changes in the vectors’ geographical distribution induced chiefly by anthropogenic factors are accompanied by emerging and reemerging infectious diseases in Europe and North America. Since the advent of synthetic insecticides during the Second World War, mosquitoes are the object of considerably expanded and deepened research. In an integrated pest management context, means of control are now mainly classified as: (1) environmental management and physical control, (2) chemical control, (3) genetic control, and (4) biological control by means of entomophagous predators and entomopathogenic microorganisms. In this context, these last have significant potential because of their ability to infect and kill their host with more or less targeted selectivity. This article proposes to emphasize biological control among other techniques in mosquito control, and to assess the potential and the opportunities offered by entomopathogenic bacteria, viruses and fungi. Finally, their use as biopesticides is discussed. [less ▲]

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See detailCould alternative solanaceous hosts act as refuges for the tomato leafminer, Tuta absoluta?
Bawin, Thomas ULg; Dujeu, David ULg; De Backer, Lara ULg et al

in Arthropod-Plant Interactions (2015), 9(4), 425-435

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread devastating pest reported to develop on economically important solanaceous plants. The characterization of its effective ... [more ▼]

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread devastating pest reported to develop on economically important solanaceous plants. The characterization of its effective host range could help to understand and prevent the dispersion behavior of the insect in the environment. In this study, the ability of T. absoluta to locate and develop on wild (Solanum nigrum, Atropa belladonna, Datura stramonium) and cultivated (Solanum tuberosum) solanaceous plant species under laboratory conditions was assessed. Dual-choice behavioral assays performed in flying tunnels (S. tuberosum versus another plant) revealed that adult distribution and female oviposition did not differ between Solanum species, which were preferred to the other tested plants. The volatile molecules released by each tested plant species provide some explanations in the observed behavioral discrimination: S. nigrum and S. tuberosum volatile profiles were similar, and were presenting quantitative and qualitative differences with the other tested Solanaceous plants. To determine whether the host plant choice was adaptive or not, we have finally conducted fitness assays, by rearing T. absoluta larvae on each plant species and have shown that Solanum species allowed higher larval survivability and lower development time (from egg to adult emergency) compared to the other plants. We conclude that Solanum species are suitable host plants for T. absoluta, but other Solanaceous plant species could be opportunistically colonized with fewer incidences. [less ▲]

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See detailTuta absoluta (Lepidoptera: Gelechiidae) development on wild and cultivated plant species
Bawin, Thomas ULg; Dujeu, David ULg; Fagan, Maud ULg et al

Conference (2015, May 19)

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread invasive species damaging economically important cultivated solanaceous crop plants, including tomatoes and potatoes. Little ... [more ▼]

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread invasive species damaging economically important cultivated solanaceous crop plants, including tomatoes and potatoes. Little is known about the ability of this microlepidoptera to encounter and develop on alternative wild and agricultural plant species. These plants could provide refuges and have to be identified for more efficient integrated management strategies. In the present study, we assessed under laboratory conditions the ability of T. absoluta to develop on such plant species referred as potential hosts in the literature, including Solanaceae, Chenopodiaceae, Convolvulaceae, Fabaceae, and Malvaceae. For each plant species, fitness tests were performed in Petri dishes by isolating single individuals with excised leaf. We found that Solanum species allowed higher larval survivability and shorter development time (from egg to adult emergency) compared to the other plants. Non-solanaceous plants were not able to sustain T. absoluta larvae. Two choice behavioral assays performed in flying tunnels revealed that adult distribution and female oviposition did not differ between Solanum species, which were preferred to other tested solanaceous plants. These results appeared to be consistent with survival rates and development times. Because larval survivability depends on the female’s oviposition choice, the hypothesis that host plant choice is influenced by plant volatile organic compounds has to be tested. Volatile organic compounds released by solanaceous plants were trapped using a dynamic collection system, and analyzed by GC-MS. Solanum volatile profiles showed similarities, and were presenting quantitative and qualitative differences with the other tested solanaceous plants, providing some explanations in the observed behavioral discrimination. Further electrophysiological and behavioral assays are required to confirm the effect of specific chemicals on the choice of the oviposition site in T. absoluta. It can be concluded that Solanum species are the more suitable hosts for T. absoluta development. Other solanaceous plant species could be opportunistically colonized with little incidence but care should be taken in these results as genetic variability in insects and plants, as well as plant physiological state, might have an impact on the pest survivability. [less ▲]

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See detailA fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae
Zune, Quentin ULg; Delepierre, Anissa ULg; Gofflot, Sebastien et al

in Applied Microbiology and Biotechnology (2015)

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state related physiological mechanisms. This work is based on the comparative analysis of classical ... [more ▼]

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state related physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilm reactor for the production of a Gla::GFP fusion protein by Aspergillus oryzae. The biofilm reactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2D-gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e. with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniform colonization of the packing by the fungal biomass and leads to a better quality of the fusion protein. [less ▲]

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See detailTuta absoluta (Lepidoptera: Gelechiidae) ability to localize and develop on wild and cultivated solanaceous plant species
Bawin, Thomas ULg; Dujeu, David ULg; Fagan, Maud ULg et al

Conference (2014, December 13)

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread invasive species damaging economically important cultivated solanaceous crop plants, including tomatoes and potatoes. Little ... [more ▼]

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a widespread invasive species damaging economically important cultivated solanaceous crop plants, including tomatoes and potatoes. Little is known about the ability of this microlepidoptera to encounter and develop on alternative wild and agricultural plant species. These plants could provide refuges and have to be identified for more efficient integrated management strategies. In the present study, we assessed the ability of T. absoluta to develop on wild (Solanum nigrum, Atropa belladonna, Datura stramonium) and cultivated (Solanum tuberosum) solanaceous plant species under laboratory conditions. Fitness tests were performed in Petri dishes by isolating single individuals with excised leaf from one of the host plants. We found that Solanum species allowed higher larval survivability and shorter development time (from egg to adult emergency) compared to the other plants. Two choice behavioral assays performed in flying tunnels (S. tuberosum versus another plant) revealed that adult distribution and female oviposition did not differ between Solanum species, which were preferred to the other tested plants. These results appeared to be consistent with survival rates and development times. Because larval survivability depends on the female’s oviposition choice, the hypothesis that host plant choice is influenced by plant volatile organic compounds has to be tested. It can be concluded that Solanum species remain the more suitable hosts for T. absoluta development among the tested plants. Other plant species could be opportunistically colonized with little incidence but care should be taken in these results as genetic variability in insects and plants, as well as plant physiological state, might have an impact on the pest survivabilty. [less ▲]

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See detailPathogenicity of Aspergillus clavatus produced in a fungal biofilm bioreactor toward Culex quinquefasciatus (Diptera: Culicidae)
Seye, Fawrou; Bawin, Thomas ULg; Boukraa, Slimane ULg et al

in Journal of Pesticide Science (2014), 39(3), 127-132

Many entomopathogenic fungi have been demonstrated to be potential agents for efficiently controlling mosquito populations. In the present study, we investigated a bioreactor system to produce metabolites ... [more ▼]

Many entomopathogenic fungi have been demonstrated to be potential agents for efficiently controlling mosquito populations. In the present study, we investigated a bioreactor system to produce metabolites and conidia by combining technological advantages of submerged and solid-state fermentations. The efficiency of fungal products was tested toward mosquitoes. Aspergillus clavatus (Eurotiales: Trichocomaceae) was grown by semi-solid-state fermentation in a bioreactor for up to 7 days. Depending on conidial doses (2.5×10^7, 5×10^7, 7.5×10^7, 10×10^7 and 12.5×10^7 conidia/mL), mortality ranged from 37.2±15.0 to 86.3±5.0% toward larvae and from 35.8±2.0 to 85.2±1.5% toward adults. The metabolites (10, 20, 40, 60, 80 and 100% v/v) yielded mortality from 23.7±15.0 to 100.0±0.1% toward larvae, and two sprayed volumes (5 and 10 mL) reached 45.5±1.4 and 75.6±2.6% mortality, respectively, toward adults. [less ▲]

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See detailDevelopment of entomopathogenic fungi in mosquito control: which kind of production for which efficiency?
Bawin, Thomas ULg; Delvigne, Frank ULg; Francis, Frédéric ULg

Poster (2014, August 06)

Mosquitoes (Diptera: Culicidae) are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance such as filariasis, malaria and encephalitis. As part of an ... [more ▼]

Mosquitoes (Diptera: Culicidae) are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance such as filariasis, malaria and encephalitis. As part of an integrated vector control, entomopathogenic fungi could be developed as biopesticides in two ways: spores and metabolites recognized as effective virulence factors. Solid-state fermentation enhances spore production and induces the secretion of metabolites quantitatively and qualitatively different from submerged fermentation, which impairs fungal metabolic efficiency. In this context, we showed high spore productivity of solid-state media based on agro-industrial substrates as wheat bran. Spores remained pathogenic, as revealed by classical toxicity tests and electron microscopy. However, the absence of free water makes culture parameter variations difficult to control in large-scale. Recently, we performed a bioreactor design intended for simultaneous spore and metabolite production, combining the technological advantages of submerged and solid-state fermentations. Biofilm fermentation (i.e. growth of fungal biomass on an inert support immerged in a nutrient medium) is a tremendous production system favouring the secretion of insecticidal metabolites in the liquid medium as we showed recently. This is also an interesting tool to provide an overview of the complexity of the metabolic pathways involved in the regulation of extracellular metabolites secretion because corresponding genes are reported to be differentially expressed from classical fermentation systems. Researches in vector control are currently intensified. In this context, the identification of genes and metabolites specifically expressed during biofilm fermentation will help to develop new technologies related both to the design of bioreactor and the production of insecticidal proteins. [less ▲]

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See detailMolecular detection of six high importance endosymbiotic bacteria in Belgian wild-caught mosquitoes
Raharimalala, Fara Nantenaina ULg; Boukraa, Slimane ULg; Bawin, Thomas ULg et al

Poster (2014, July)

Introduction Several disease vectors presented a resistance to various pesticides currently used. One of an alternative solution was to use endosymbiotic bacteria because their probably interactive ... [more ▼]

Introduction Several disease vectors presented a resistance to various pesticides currently used. One of an alternative solution was to use endosymbiotic bacteria because their probably interactive effects with their host. According to the introduction risks of mosquito born disease and their dispersion, we propose to investigate the prevalence of six endosymbiontic bacteria in wild-caught Culicidae in Belgium. Methods Eleven species of Belgian fields mosquitoes (Culex pipiens s.l., Cx. torrentium, Cx. hortensis, Anopheles claviger, An. maculipennis s.l., An. plumbeus, Culiseta annulata, Ochlerotatus geniculatus, Oc. dorsalis, Aedes albopictus and Coquillettidia richiardii) were used for the screening of six genera endosymbiotic bacteria (Wolbachia sp., Commamonas sp., Delftia sp., Pseudomonas sp., Acinetobacter sp. and Asaia sp.) according to their possible impact in mosquito biology. PCR was done for the screening and positives bands were sequenced and deposited in GenBank. Results Total of 144 larvae and 32 adults were used. Wolbachia, Pseudomonas, Acinetobacter and Asaia were found in mosquitoes with different proportions, according to stages (adults, larvae) with a predominance of Pseudomonas in all species, as far as Acinetobacter and Asaia also have a high prevalence. Commamonas and Delftia were absent from all species tested, either in larvae and in adults. Discussion Choice of endosymbiotic bacteria studied here was allowing of their importance in literature. For Pseudomonas, it showed that this bacteria could produced ovipositon attractants for mosquito. Acinetobacter was suggested efficient in transmission and maintenance within host populations. Asaia was capable of efficiently crossing body barriers and colonizing different organs. Wolbachia was currently the most studied bacteria which plays an important role in the genetic manipulation of the host. Present advances in understanding the mosquito–microbiota relationships may have a great impact in a better understanding of some traits of mosquito biology and in the development of innovative mosquito-borne disease-control strategies. [less ▲]

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See detailEffect of entomopathogenic Aspergillus strains against the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae)
Seye, Fawrou; Bawin, Thomas ULg; Boukraa, Slimane ULg et al

in Applied Entomology and Zoology (2014)

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a ... [more ▼]

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a selection process, laboratory bioassays were carried with five different concentrations of Aspergillus clavatus (Desmazières), Aspergillus flavus (Link) and Metarhizium anisopliae ((Metschnikoff) Sorokin) spores against the pea aphid, Acyrthosiphon pisum (Harris). Aspergillus isolates induced higher mortalities than M. anisopliae which is a well-known entomopathogen in the literature. Lethal concentrations (LC50 and LC90) were 1.23 x 10^3 and 1.34 x 10^7 spores/ml for A. flavus, 4.95 x 10^2 and 5.65 x 10^7 spores/ml for A. clavatus, and 3.67 x 10^3 and 9.71 x 10^7 spores/ml for M. anisopliae five days after treatment. Mycelia development and sporulation on adult cadavers was observed 48 hours after incubation. The intrinsic growth rate of A. pisum decreased with increased spore concentration for all fungal strains suggesting an increase in pathogen fitness related to a consumption of host resources. In conclusion, Aspergillus species could be useful in aphid control as pest control agents despite their saprophytic lifestyle. This is also in our knowledge the first report of A. clavatus and A. flavus strains pathogenic to aphids. [less ▲]

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See detailPhylogenetic study of Aedes albopictus and Aedes koreicus (Diptera, Culicidae) origin, two invasive mosquito species in Belgium
Raharimalala, Fara Nantenaina ULg; Boukraa, Slimane ULg; Bawin, Thomas ULg et al

Poster (2014, April 10)

The main ways of the introduction of invasive mosquito species in Europe were greatly facilitated by the importation of used tires and plants from the South of China (Dracaena sanderiana, called "Lucky ... [more ▼]

The main ways of the introduction of invasive mosquito species in Europe were greatly facilitated by the importation of used tires and plants from the South of China (Dracaena sanderiana, called "Lucky bamboo"), which are a very good breeding sites of these mosquitoes (www.eidatlantique.eu). For Belgium, several species of exotic mosquitoes have already been listed such as Ochlerotatus japonicus japonicus, Aedes albopictus and A. koreicus. We have captured one male of A. albopictus in Belgium in July 2013 (Boukraa et al, 2013), and teams from other entomologist have been able to find several individuals in the months that have followed our discovery (personal communication). However, his presence was reported first in 2000 (Schaffner et al. 2004) but until 2012, no individuals have been found (Versteirt et al., 2013). We also could find larvae of A. koreicus in abundance. These discovery suggest that these two invasive species are being established in Belgium. Methods: Adult mosquitoes were collected by CO2-baited traps Mosquito Magnet Liberty Plus (MMLP) and immature stages by the dipping method from 8 sites of Belgium. Each individual was then determined morphologically. For A. albopictus and A. koreicus, reconfirmation by molecular method was performed with COI and ND5 mitochondrial primers and sequences were then aligned with those of mosquitoes available in databases by using BioEdit and Multialn softwares. The two gene sequences were concatenated to improve the reliability of the phylogenetic analysis and were carried out by using the Seaview software based on maximum likelihood (ML) methods. Trees were then constructed with the general time reversible (GTR) model, and branch supports were estimated by bootstrapping with 1000 replicates. Result: The aim of this work will focus on the study of the probable origin of these two species of Aedes, which are for the moment safe yet, but that might become dangerous in the event of massive outbreak. The knowledge of the probably origin of the two invasive mosquitoes allow take more protection against their way to enter in the country. [less ▲]

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See detailBiofilms from entomopathogenic fungi in mosquito control
Bawin, Thomas ULg; Boukraa, Slimane; Seye, Fawrou et al

Poster (2014, February 07)

Mosquitoes (Diptera: Culicidae) are zoonotic vectors of medical and veterinary importance. As part of an integrated vector control, metabolites secreted by entomopathogenic fungi could be developed as ... [more ▼]

Mosquitoes (Diptera: Culicidae) are zoonotic vectors of medical and veterinary importance. As part of an integrated vector control, metabolites secreted by entomopathogenic fungi could be developed as biopesticides. In this context, filamentous microorganisms growing on a support as biofilm in a liquid medium would offer several advantages in bioreactor regarding performances and metabolites recovery. The production of toxic metabolites by an entomopathogenic fungus Aspergillus flavus in such conditions was assessed. Three initial inoculum levels, i.e. 10^1, 10^3 and 10^6 spores/ml of PYG medium, have been tested in shake flask with or without support. Toxicity tests were performed on Culex quinquefasciatus larvae using dilutions of 1, 2, 4, 6, 8 and 10% of liquid cultures. The results indicated that A. flavus tends to form pellets in submerged culture; the size and the amount of pellets was affected by the initial inoculum level of spores. Under similar conditions, the filaments fixed on a support and didn’t appear in free form in the liquid. Toxicity tests revealed differences between both free and fixed forms. All combined conditions, LC50s ranging up to dilutions of 2.2 and 4.8% were observed within 48 hours. Secretomes could be compared between these culture conditions by proteomic and metabolomic approaches. [less ▲]

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See detailUsing micro-injection technique to assess fungal toxicity in mosquito control
Bawin, Thomas ULg; Boukraa, Slimane; Seye, Fawrou et al

in Communications in Agricultural and Applied Biological Sciences (2014, February 07), 79(1), 181-185

Topical application of insecticidal compounds allows directly exposing these substances on insect tissues and measuring their toxicity while ignoring many factors. However, this technique remains ... [more ▼]

Topical application of insecticidal compounds allows directly exposing these substances on insect tissues and measuring their toxicity while ignoring many factors. However, this technique remains difficult to apply on mosquito larvae considering their aquatic lifestyle. Micro-injection could be used for the direct deposition of toxic compounds in the larvae. Capillaries exhibiting an injection tip with an external diameter of 0.5 mm have been designed from silica tubes. For each treatment, a capillary is mounted on a pump connected to a flow rate regulator. Culex quinquefasciatus larvae were injected with 10^7 spores/ml of entomopathogenic fungi (Aspergillus clavatus, Metarhizium anisopliae, Metarhizium sp.). Mortalities were recorded daily during 72h. The distribution of spores stained with methylene blue and injected into the body of larvae was also observed according to the system described. Results showed that spores were distributed over the whole body. The injection of Aspergillus clavatus, Metarhizium anisopliae and Metarhizium sp spores induced corrected mortalities of 62%, 53% and 57% after 72h, and differed statistically from control groups. Finally, post-mortem emergences of filaments from dead larvae were observed in the case of the three fungal strains confirming spore viability. Injection of inactivated spores (or inert bodies of similar size) could help to reject the hypothesis of a response due to the presence of foreign bodies. [less ▲]

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See detailInfestation Level Influences Oviposition Site Selection in the Tomato Leafminer Tuta absoluta (Lepidoptera: Gelechiidae)
Bawin, Thomas ULg; De Backer, Lara ULg; Dujeu, David ULg et al

in Insects (2014), 5

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a devastating pest that develops principally on solanaceous plants throughout South and Central America and Europe. In this study, we ... [more ▼]

The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae), is a devastating pest that develops principally on solanaceous plants throughout South and Central America and Europe. In this study, we tested the influence of three levels of T. absoluta infestations on the attraction and oviposition preference of adult T. absoluta. Three infestation levels (i.e., non-infested plants, plants infested with 10 T. absoluta larvae, and plants infested with 20 T. absoluta larvae) were presented by pairs in a flying tunnel to groups of T. absoluta adults. We found no differences in terms of adult attraction for either level of infestations. However, female oviposition choice is influenced by larvae density on tomato plants. We discuss the underlying mechanisms and propose recommendations for further research [less ▲]

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