Effect of entomopathogenic fungus <Emphasis Type="Italic">Metarhizium robertsii</Emphasis> on non-target organisms,water bugs (Heteroptera: Corixidae,Naucoridae, Notonectidae)

The influence of the fungus Metarhizium robertsii Bischoff, Rehner and Humber on the mortality of four water bug species, Cymatia coleoptrata (Fabricius), Sigara assimilis (Fieber), Ilyocoris cimicoides cimicoides (Linnaeus), and Notonecta reuteri Hungerford, and bloodsucking mosquito Anopheles messeae Falleroni, was investigated under various concentrations of conidia and different treatment types. We found that the mortality of adults of the water bug species was similar or higher than that of A. messeae, with C. coleoptrata and S. assimilis being more susceptible to M. robertsii than N. reuteri, I. c. cimicoides, and the mosquito A. messeae. Treatment with dry conidia at concentrations of 5 × 10⁴ and 5 × 10⁵ conidia/ml caused higher mortality of the water bug species than did treatment at the same concentrations with conidia in an aqueous suspension. In contrast, higher concentrations (5 × 10⁶ conidia/ml) led to higher mortality after treatment with the aqueous suspension, relative to treatment with dry conidia. Our studies showed that water bugs exhibited the classical development of a mycosis with hemocoel colonization, mummification, and conidia formation on cadavers directly on the surface of the water. Possible changes in invertebrate communities in aquatic ecosystems after treatment with Metarhizium are discussed.     

Effects of Metarhizium robertsii on the bloodsucking mosquito Aedes flavescens and non‐target predatory insects (Odonata)

The effects of different concentrations and methods of treatment with Metarhizium robertsii Bisch., Rehner & Humber conidia on the non‐target aquatic dragonfly larvae Lestes sponsa Hansemann, Lestes dryas Kirby and Aeshna affinis Vander Linden and on the target bloodsucking mosquito larvae Aedes (O.) flavescens (Muller) were analysed. We found that dragonflies are significantly less susceptible than mosquitoes to the fungus. Larvae of L. sponsa larvae were more susceptible to wet conidia than dry conidia. However, the mortality of the air‐breathing larvae of A. affinis was significantly higher after treatment with dry conidia relative to aqueous suspension. The results help to minimize the negative effects of entomopathogenic fungi on non‐target predator insects under the control of mosquito larvae.     

Comparative efficacy and pathogenicity of keratinophilic soil fungi against Culex quinquefasciatus larvae

Out of seven fungal species belonging to four genera isolated from pond and wallow soils using feathers of Pavo cristatus as bait, four species viz., Aspergillus flavus, Aspergillus niger, Chrysosporium pseudomerdarium and Trichophyton ajelloi were most frequent. Chrysosporium and Trichophyton spp. were more pathogenic on Culex quinquefasciatus larvae than Aspergillus and Penicillium. The bioefficacy tests conducted as per the protocol of World Health Organization and the LC₅₀ values calculated by the Probit analysis showed that 3^rd-instar C. quinquefasciatus were more susceptible to the conidia of above fungi. Highest mortality was observed in the larvae of C. quinquefasciatus when exposed to T. ajelloi. The density of fungal conidia was greatest on the ventral brush, palmate hair and anal region of the mosquito larvae after exposing for 72 hours. The potentiality of these fungi for use in the control of C. quinquefasciatus is discussed which can be exploited as a suitable biocontrol agent in the tropics.     

Identification of Metarhizium strains highly efficacious against Aedes,Anopheles and Culex larvae

Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, have been shown to be efficacious in killing mosquito larvae of different mosquito species. The current study compared the pathogenicity and efficacy of two formulations of three fungal strains against different instars of three mosquito species with the aim of identifying the most virulent strain for use under field conditions. Three strains of Metarhizium, ARESF 4556, ARSEF 3297 and V275, were assayed against early (L_2?3) and late (L_3–4) instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Two formulations of the fungi were tested, dry conidia and aqueous suspensions (i.e. ‘wet’ conidia). Effects of all combinations of conidia, mosquito species, instar, fungal strain and concentration on mosquito mortality were analysed using Cox regression and Kaplan–Meier analyses. Strain ARSEF 4556 was more virulent than ARSEF 3297 and V275, with LT₅₀ values ranging from 0.3 to 1.1 days, with Anopheles and Culex being more susceptible than Aedes. Early and late instars were equally susceptible independent of species. Although the formulation did influence mortality rates, both ‘wet’ and ‘dry’ conidia applications were highly effective in killing mosquito larvae. Viable spores were more efficacious than heat killed spores. The latter did cause mortality but only at high concentrations. Metarhizium sp. has proved to be effective in reducing survivability of all larval stages of Aedes, Anopheles and Culex under laboratory conditions. Aedes larvae were generally more tolerant than Anopheles and Culex irrespective of fungal strain.     

Aquatain AMF efficacy on juvenile mosquito stages in control of Culex pipiens complex and Aedes albopictus

Aquatain^® mosquito formulation (AMF) is a silicone-based monomolecular film, which has recently been approved for use in the European Union. The physical mode of action based on lowering water surface tension prevents mosquito larvae/pupae respiration. Additionally, AMF disables gravid females from landing on the water surface and obstructs the natural oviposition process. Due to multistage effects on mosquitoes, AMF could be a product of choice for defined water body and container breeders such as Culex pipiens L. complex, principal vector of West Nile virus in Europe, and the invasive Aedes albopictus (Skuse) (both Diptera: Culicidae), vector of dengue and chikungunya viruses. The primary objectives of this study were to evaluate the efficacy of AMF, to determine the susceptibility of the immature forms of C. pipiens and A. albopictus, and the persistence/longevity of the product to suppress the eclosion of adults. AMF achieved high mortality rates of juvenile A. albopictus and C. pipiens under laboratory conditions. However, in the field C. pipiens larvae showed higher susceptibility to AMF than A. albopictus. Pupae of the two mosquito species were highly susceptible to the presence of AMF. When C. pipiens juveniles were exposed to AMF in the wild, effects lasted for 21 days in densely covered water bodies and 56 days in water recipients with less vegetation. In both breeding sites, natural habitat and artificial water recipient, the two mosquito species with high impact on public health in Europe could successfully be suppressed by application of AMF (1 ml m⁻²).     

Romanomermis culicivorax parasitism and the development, growth, and feeding rates of two mosquito species

The dry weight, development, and feeding rates of Culex pipiens and Toxorhynchites amboinensis larvae infected with the nematode, Romanomermis culicivorax, were measured and gross conversion efficiences were calculated. The weight of C. pipiens larvae infected at two different inoculum levels did not differ significantly from controls until day 6 postinfection (PI). Infected larvae of T. amboinensis were significantly lighter than controls at days 2, 4, and 6 PI. The rate of larval mosquito development was slowed after day 3 in parasitized individuals of both mosquito species. Infection significantly retarded the interval feeding rate of the filter-feeding C. pipiens throughout development. Infected T. amboinensis larvae consumed significantly fewer prey larvae of C. pipiens than controls. Calculation of gross conversion efficiency (GCE) showed that lightly infected C. pipiens larvae had an elevated GCE early in the infection but were less efficient relative to controls after 4 days PI. Lightly parasitized T. amboinensis had a lower total GCE than controls.     

An effective entomopathogenic fungus Metarhizium anisopliae for the simultaneous control of Aedes albopictus and Culex pipiens mosquito adults

The purpose of this study was to select the most effective Metarhizium anisopliae isolates for the simultaneous control of Aedes albopictus and Culex pipiens mosquito adults. The selection was performed using 65 isolates of M. anisopliae, an entomopathogenic fungus isolated from Korean soils. The selection process was carried out through evaluations of conidial thermotolerance, UV-B tolerance, pathogenicity and virulence against adults of two mosquito species and conidial production on grain medium. The thermotolerance of 65 M. anisopliae isolates ranged from 0% to 99%, and their UV-B tolerance ranged from 7.1% to 94.8%. Among them, 30 isolates with high thermotolerance were selected for evaluation of their pathogenicity and virulence against adults of the two mosquito species. Metarhizium anisopliae isolates were more susceptible to Cx. pipiens adults than Ae. albopictus adults because 11 isolates were pathogenic to Ae. albopictus and 21 isolates were pathogenic to Cx. pipiens. Virulence and conidial production were evaluated using 6 isolates with similar pathogenicity against the two species of mosquito. As a result, the accumulated mortality was 74–100% for Ae. albopictus and 78–100% for Cx. pipiens. LT₅₀ values ranged from 2.3 to 5.2 days for Ae. albopictus and from 3.2 − 4.7 days for Cx. pipiens. Comparison of conidial production using three kinds of grain media showed various results according to isolate and medium. Based on the above results, M. anisopliae CN6S1W1 was finally selected as the most effective fungal isolate for the simultaneous control of Ae. albopictus and Cx. pipiens mosquito adults.     

Pathogenesis of infection by the hyphomycetous fungus,Tolypocladium cylindrosporum inAedes sierrensis andCulex tarsalis [Dip.: Culicidae]

The mode of infection and cycle of development ofTolypocladium cylindrosporum Gams was examined inAedes sierrensis andCulex tarsalis. Larvae were found to be infected through the external cuticle, the pharynx and the midgut. Blastospores and conidia were both infective although for equal numerical concentrations blastospores proved more virulent causing high mortality within the first 48 h after inoculation (80 % for L2 larvae exposed to 5×10⁵ spores/ml), while conidia generally took 7–10 days to produce the same results. Sporulation did not occur on submerged cadavers. Conidia were produced only on floating cadavers in contact with air. Conidial production on floating 4th instar larvae was found to average 1.8×10⁷ conidia/larva. Invasion of the haemocoele and fairly extensive growth of the fungus almost invariably occurred before larvae were killed. This was particularly true forAedes sierrensis larvae. Details are presented of growth within the host and post-mortem penetration of the fungus out of the cadaver. AdultA. sierrensis sprayed with a conidial suspension proved susceptible to infection with 100 % mortality being recorded at 10 days. Infections originated in the thorax, suggesting, the integument or possibly the thoracic spiracles to be the most probable site of infection.     

Semi-field and Field Evaluations of Floating Briquette Formulation of Bacillus thuringiensis var. israelensis against Three Mosquito Species in Republic of Korea

A floating briquette formulation was developed to allow slow releasing which is useful for the spreading of Bacillus thuringiensis var. israelensis (B.t.i.) into water. A semi-field confined assessment was conducted to investigate the effectiveness mosquito control using the briquettes (37.5% AI) against Anopheles sinensis, Culex pipiens, and Ochlerotatus togoi in small plots. The treatment produced continuously 100% larval reductions against An. sinensis and Cx. pipiens at an over 2.0g briquette until 20 and 25 days, respectively. After 25 days from the treatment, the mortality rates of An. sinensis were 55% and 80% at 4.0g and 8.0g briquettes, respectively. After 30 days from the treatment, the mortality rates of Cx. pipiens were 71% and 86% at 4.0g and 8.0g briquettes, respectively. The mortality rates of Oc. togoi were 99% at an 2.0g briquettes after 20 days, with killing of 75% and 99% at 4.0 and 8.0g briquettes after 30 days, respectively. In field, mosquito control evaluations of the briquettes were performed in four marshes with sewage effluent waters, Busan during the period of June through September, 2002. Culex pipiens larvae were treated with the B.t.i. briquettes at the rate of a briquette per 4m² in marsh. Each treatment of the briquettes resulted in satisfactory level of mosquito control up to 21-33 days. The treatment made the mosquito reduction in total averages of 91.7%, 91.3%, 82.4% 82.0%, and 100.0% in 1, 2, 3, 4 and 5 weeks probably due to be long released period of the briquette.     

Efficacy of Nile tilapia (Oreochromis niloticus) juveniles and spinosyns bioinsecticides against aquatic stages of Culex pipiens: An experimental study

The mosquito Culex pipiens is the most widely distributed dipteran species in all regions of Egypt and the principal vector of Wuchereria bancrofti and certain arboviruses in human beings. For controlling C. pipiens vector, biological tools (e.g., larvivorous fish and bioinsecticides) are more potent and safer options to the environment, human beings, and beneficial organisms than chemical pesticides. The efficiency of O. niloticus juveniles as predatory fish species and two bioinsecticides, spinosad 24% and spinetoram 12%, was investigated against the C. pipiens developmental stages in the laboratory. The first trial evaluated the predatory efficacy of small-sized O. niloticus (2.1–2.6 cm; 250–315 mg) and large-sized O. niloticus (2.5–3.2 cm; 250–315 mg) against the 3rd larvae and pupae of C. pipiens. This is the first report in Egypt confirming the predation potential of O. niloticus as efficient predatory fish against the immature C. pipiens. Large-sized O. niloticus predated a greater number of 3rd of C. pipiens larvae and pupae than the small-sized ones. Furthermore, the daily consumption of C. pipiens larvae by small- and large-sized O. niloticus was significantly higher than the pupae. The second trial assessed the toxicity efficacy of spinosad 24% and spinetoram 12% against C. pipiens larvae and pupae. The results confirmed that the tested bioinsecticides showed higher potency toward C. pipiens larvae than pupae after exposure for 24 h and 48 h. Spinosad was more toxic toward 3rd C. pipiens larvae (LC₅₀ = 0.013 and 0.003 mg/L) and pupae (LC₅₀ = 320.69 and 44.28 mg/L) than spinetoram after 24 and 48 h. Herein, O. niloticus juveniles (as promising native predatory fish) and spinosyns bioinsecticides were more effective against C. pipiens in the larval stage than in the pupal stage. In conclusion, Nile tilapia juveniles and biorational compounds, spinosad 24% and spinetoram 12%, might be considered as promising and favorable environmental biological agents for controlling C. pipiens in Egypt. However, further trials are needed to investigate the potential of these agents in the control of this mosquito vector under field conditions.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Bioassays for comparative infectivity of mermithid nematodes (Romanomermis iyengari,Romanomermis culicivorax and Strelkovimermis spiculatus) for culicine mosquito larvae

Two improved bioassays were developed to establish infectivity baselines for selection experiments using mermithid nematode variants. Comparative infectivity of Romanomermis iyengari, Romanomermis culicivorax and Strelkovimermis spiculatus using larvae of three mosquito spp. Aedes sierrensis, Aedes aegypti and Culex pipiens were evaluated with “plate” and “tray” bioassays at selected intensity of infections. Using the “plate” bioassay, single mosquito larvae were immersed in 2 ml of water within individual depressions of 12-well, polystyrene tissue culture plates. One, three, or five preparasitic juveniles (J2) were added to each well. In the “tray” bioassay, polyethylene trays containing 500 ml water and 100 mosquito larvae were exposed to 500 (5:1, nematode:insect host) or 1000 (10:1) J2s. Percentage infection (PINF, infectivity) and intensity of infection (IINF, #nematodes per infected larvae) number were determined only after emergence of post-parasitic J3 juveniles. Under the bioassay conditions, all three species of nematodes resulted in infections in all mosquito hosts, but R. iyengari exhibited better effectiveness in the parasitism of mosquito larvae. The three species of mosquitoes presented high levels of susceptibility to each of the three species of nematodes, but in general Cx. pipiens and Ae. sierrensis were slightly more susceptible than Ae. aegypti. The “plate” bioassay was more efficient in measurement of infectivity of the mermithid species and in establishing baseline characteristics for these mosquito-parasitic nematodes. The “tray” bioassay was an effective bioassay for large cohorts of both infective juveniles and host larvae and, potential for field interactions.     

Chilodonella uncinata is Not Pathogenic to Culex pipiens Mosquitoes

Chilodonella uncinata was previously reported to be pathogenic to Culex and other mosquitoes based on the observation of high densities of ciliates inside of dead mosquito larvae. Here, we tested whether co‐culturing Cx. pipiens with Ch. uncinata resulted in decreased survival of the mosquito larvae. We find no evidence that Ch. uncinata is pathogenic to Cx. pipiens. We suggest that Ch. uncinata may instead feed on already deceased larvae.     

Larvicidal,ovicidal activities and histopathological alterations induced by Carum copticum (Apiaceae) extract against Culex pipiens (Diptera: Culicidae)

An experiment was carried out, firstly, to determine the possible toxicity of Carum copticum (Apiaceae) extract against Culex pipiens (Diptera: Culicidae), and, secondly, to study the histopathological alterations in the midgut of Cx. pipiens as a result of treatment with C. copticum extract. Larvicidal and ovicidal activities of C. copticum extract against the larvae of Cx. pipiens was determined according to World health organization (WHO). The inhibition effect of C. copticum was assessed by determining the mortality of the treated larvae and eggs. The histopathological effect of the C. copticum extracts on midgut epithelium of the larvae was examined under both light and transmission electron microscopy. The crude extract of C. copticum exerted 100% mortality for Cx. pipiens after 24 h at 200 μm/ml, and zero hatchability (100% mortality) at 150 μm/ml for Cx. pipiens. The histopathological study showed that larvae treated with C. copticum extract had cytopathological alterations of the midgut epithelium. The study provided information on various effects of C. copticum extract against Cx. pipiens.     

Susceptibility of Megalurothrips sjostedti developmental stages to Metarhizium anisopliae and the effects of infection on feeding, adult fecundity, egg fertility and longevity

The susceptibility of immature stages of the legume flower thrips, Megalurothrips sjostedti, to the entomopathogenic fungus Metarhizium anisopliae, was investigated under laboratory conditions. The adult stage was found to be more susceptible to infection than the larval and pupal stages. Mortality at all stages was dose-dependent, with the highest concentration of 1×10⁸ conidia ml^–1 producing the highest mortality (26, 46 and 100% for larvae, pupae and adults, respectively) at 8 days post-inoculation. At the same concentration, daily pollen consumption was significantly reduced at 2 days after treatment in infected adults but more slowly in infected larvae. Fecundity, egg fertility and longevity in adults surviving infection as larvae were significantly reduced compared to the control.     

Insecticidal effects of a novel polyherbal formulation (HF7) against Culex pipiens L. (Diptera: Culicidae)

Plant secondary metabolites represent the most efficient and convenient method to control and overcome environmental pollution and insecticidal resistance. This study explored the mosquitocidal activity of the combined extract of seven plants, (HF7) extracted using a Soxhlet extractor against Culex pipiens under laboratory conditions. Exposure of the 3rd instars of Cx. pipiens to HF7 hexane extract resulted in LC₅₀:114.5 μg/mL and LC₉₀:117.0 μg/mL values after 24 h. The ovicidal activities of hexane extract against Cx. pipiens eggs were 21.6%, 48.3%, and 71.6% at 187.5, 93.7, and 46.88 μg/mL, respectively. HF7-treated larvae showed the formation of irregular blebbing of epithelial cells toward the lumen and sloughing into the gut lumen. HF7 extract resulted in 100% adulticidal mortality at the concentration of 3.7 mg/test tube after 30 min of exposure. The IC₅₀ of HF7 extract was 97.03 µg/ml against larvae, at which nuclear and morphological changes were observed. The spectroscopy spectrum of HF7 hexane extract disclosed the presence of 57 different secondary metabolites, among which the dominant compound was eugenol (32.3%). HF7 hexane extract could serve as a botanical insecticide for controlling Cx. pipiens and potentially other mosquito species.     

Culex pipiens Development Is Greatly Influenced by Native Bacteria and Exogenous Yeast

Culex pipiens is the most cosmopolitan mosquito of the Pipiens Assemblage. By studying the nature of interactions between this species and microorganisms common to its breeding environment we can unravel important pitfalls encountered during development. We tested the survival rate of larval stages, pupae and adults of a Cx. pipiens colony exposed to a variety of microorganisms in laboratory conditions and assessed the transmission to offspring (F1) by those organisms that secured development up to adulthood. Three complementary experiments were designed to: 1) explore the nutritional value of yeasts and other microorganisms during Cx. pipiens development; 2) elucidate the transstadial transmission of yeast to the host offspring; and 3) to examine the relevance of all these microorganisms in female choice for oviposition-substratum. The yeast Saccharomyces cerevisiae proved to be the most nutritional diet, but despite showing the highest survival rates, vertical transmission to F1 was never confirmed. In addition, during the oviposition trials, none of the gravid females was attracted to the yeast substratum. Notably, the two native bacterial strains, Klebsiella sp. and Aeromonas sp., were the preferred oviposition media, the same two bacteria that managed to feed neonates until molting into 2^nd instar larvae. Our results not only suggest that Klebsiella sp. or Aeromonas sp. serve as attractants for oviposition habitat selection, but also nurture the most fragile instar, L1, to assure molting into a more resilient stage, L2, while yeast proves to be the most supportive diet for completing development. These experiments unearthed survival traits that might be considered in the future development of strategies of Cx. pipiens control. These studies can be extended to other members of the Pipiens Assemblage.     

Pine processionary moth (Thaumetopoea pityocampa,Lepidoptera: Thaumetopoeidae) larvae are highly susceptible to the entomopathogenic fungi Metarhizium brunneum and Beauveria bassiana

The larvae of the pine processionary moth (PPM), Thaumetopoea pityocampa, feed on the needles of pine and cedar. The urticating hairs of older instars pose a threat to human and animal health. Strains of the entomopathogenic fungi, Metarhizium brunneum (V275, ARSEF 4556) and Beauveria bassiana (KTU-24), were assayed against first to fourth instar T. pityocampa using doses ranging from 1?×?10⁵ to 1?×?10⁸ conidia mL^?1. The three strains differed slightly in their virulence but caused 100% mortality of all instars at the highest dose. The newly emerged or first instar larvae were extremely susceptible with 100% mortality being achieved 2–4 days post inoculation with V275 at all but the lowest dose. The fourth instar larvae appeared to be less susceptible than earlier instars. There was good horizontal transmission of conidia from treated to un-inoculated larvae. However, mortality was higher in third and fourth instars and where the ratio of inoculated versus untreated larvae was high. This we presume is due to spores being more readily trapped by the urticating hairs found on third and older instar larvae. Injection of the nests offers a simple and environmentally friendly way of controlling the pest with reduced risk to operators.     

Distribution and dynamics of esterase alleles in Culex pipiens complex in China

To investigate insecticide resistance and dynamic changes of carboxylesterase polymorphism in mosquitoes with time in the Culex pipiens complex (Diptera: Culicidae), nine field mosquito populations were collected in China. The resistance levels of fourth-instar larvae to organophosphate (dichlorvos, parathion, and chlorpyrifos), carbamate (fenobucarb and propoxur), and pyrethroid (permethrin, deltamethrin and tetramethrin) insecticides were determined by bioassay. Larvae had more resistance to organophosphate insecticides than to carbamate insecticides. A low but significant resistance was observed for carbamate insecticides. The resistance to pyrethroid insecticides varied from sensitive to high. Starch gel electrophoresis revealed the presence of the overproduced esterases B1, A2B2, A8B8, A9B9, B10 and A11B11. The frequency of each overproduced esterases varied depending on its regional localities. Compared with published surveys, the C. pipiens complex, which exhibited a high polymorphism of applied esterase alleles in China, showed dynamic evolution over time under local specific insecticide selection. The results are discussed in the context of recent alterations to insecticide campaigns, and in the evolution of resistance genes in Chinese C. pipiens populations.