Susceptibility of laboratory colonies of members of the Anopheles gambiae complex to entomopathogenic fungi Beauveria bassiana

Control of the major African malaria vectors of the Anopheles gambiae complex continues to rely heavily on the application of insecticides either by indoor residual house spraying (IRS) or bednet impregnation (ITNs). However, growing concerns about their negative impact on human health, their potential impact on the environment, and the ever-increasing spread of insecticide resistance across sub- Saharan Africa has diverted attention to alternative strategies that are not based on chemicals. Therefore, alternatives such as vector control with biological agents may be effective. Levels of susceptibility to the entomopathogenic fungus Beauveria bassiana were assessed against insecticide-resistant and insecticide-susceptible laboratory colonies as well as a wild-caught sample of the An. gambiae complex of which most members are major or localised malaria vectors. Insecticide susceptibility tests against all four classes of insecticides recommended by WHO for vector control was performed according to the standard WHO bioassay protocol. Fungal susceptibility tests using suspensors dusted with dry conidia of B. bassiana isolate IMI 391510 were performed on laboratory-reared colonies of An. gambiae complex members as well as on a sample of wild caught, insecticide susceptible An. arabiensis from Malawi. Our data showed that there is no interaction between insecticide susceptibility and fungal susceptibility. Survival of both insecticide susceptible and insecticide resistant colonies as well as wild mosquitoes was significantly affected (P< 0.001) by fungal infection. All infected mosquitoes succumbed to fungal infection within 7-20 days post-exposure compared to control groups for which 70-85% of mosquitoes were still alive at that time. The evidence of fungal infection was confirmed with more than 90% of the mosquito cadavers sporulating 3-5 days after incubation. These results provide hope for alternative control strategies for adult anopheline mosquitoes other than those that rely entirely on insecticides. There was evidence that insecticide susceptible and resistant mosquitoes can be killed by the fungus B. bassiana within the 14 day period before they become infective with the malaria Plasmodium parasite. However, conidia formulations and concentrations as well as a suitable delivery method for efficient coverage remain the areas of focus for field implementation

The infectivity of the entomopathogenic fungus Beauveria bassiana to insecticide-resistant and susceptible Anopheles arabiensis mosquitoes at two different temperatures

BACKGROUND: Control of the major African malaria vector species continues to rely extensively on the application of residual insecticides through indoor house spraying or bed net impregnation. Insecticide resistance is undermining the sustainability of these control strategies. Alternatives to the currently available conventional chemical insecticides are, therefore, urgently needed. Use of fungal pathogens as biopesticides is one such possibility. However, one of the challenges to the approach is the potential influence of varied environmental conditions and target species that could affect the efficacy of a biological 'active ingredient'. An initial investigation into this was carried out to assess the susceptibility of insecticide-susceptible and resistant laboratory strains and wild-collected Anopheles arabiensis mosquitoes to infection with the fungus Beauveria bassiana under two different laboratory temperature regimes. METHODS: Insecticide susceptibility to all four classes of insecticides recommended by WHO for vector control was tested on laboratory and wild-caught An. arabiensis, using standard WHO bioassay protocols. Mosquito susceptibility to fungus infection was tested using dry spores of B. bassiana under two temperature regimes (21 +/- 1 degrees C or 25 +/- 2 degrees C) representative of indoor conditions observed in western Kenya. Cox regression analysis was used to assess the effect of fungal infection on mosquito survival and the effect of insecticide resistance status and temperature on mortality rates following fungus infection. RESULTS: Survival data showed no relationship between insecticide susceptibility and susceptibility to B. bassiana. All tested colonies showed complete susceptibility to fungal infection despite some showing high resistance levels to chemical insecticides. There was, however, a difference in fungus-induced mortality rates between temperature treatments with virulence significantly higher at 25 degrees C than 21 degrees C. Even so, because malaria parasite development is also known to slow as temperatures fall, expected reductions in malaria transmission potential due to fungal infection under the cooler conditions would still be high. CONCLUSIONS: These results provide evidence that the entomopathogenic fungus B. bassiana has potential for use as an alternative vector control tool against insecticide-resistant mosquitoes under conditions typical of indoor resting environments. Nonetheless, the observed variation in effective virulence reveals the need for further study to optimize selection of isolates, dose and use strategy in different eco-epidemiological setting

Pyrethroid resistance in Anopheles gambiae leads to increased susceptibility to the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana

<p>Abstract</p> <p>Background</p> <p>Entomopathogenic fungi are being investigated as a new mosquito control tool because insecticide resistance is preventing successful mosquito control in many countries, and new methods are required that can target insecticide-resistant malaria vectors. Although laboratory studies have previously examined the effects of entomopathogenic fungi against adult mosquitoes, most application methods used cannot be readily deployed in the field. Because the fungi are biological organisms it is important to test potential field application methods that will not adversely affect them. The two objectives of this study were to investigate any differences in fungal susceptibility between an insecticide-resistant and insecticide-susceptible strain of <it>Anopheles gambiae sensu stricto</it>, and to test a potential field application method with respect to the viability and virulence of two fungal species</p> <p>Methods</p> <p>Pieces of white polyester netting were dipped in <it>Metarhizium anisopliae </it>ICIPE-30 or <it>Beauveria bassiana </it>IMI391510 mineral oil suspensions. These were kept at 27 ± 1°C, 80 ± 10% RH and the viability of the fungal conidia was recorded at different time points. Tube bioassays were used to infect insecticide-resistant (VKPER) and insecticide-susceptible (SKK) strains of <it>An. gambiae s.s</it>., and survival analysis was used to determine effects of mosquito strain, fungus species or time since fungal treatment of the net.</p> <p>Results</p> <p>The resistant VKPER strain was significantly more susceptible to fungal infection than the insecticide-susceptible SKK strain. Furthermore, <it>B. bassiana </it>was significantly more virulent than <it>M. anisopliae </it>for both mosquito strains, although this may be linked to the different viabilities of these fungal species. The viability of both fungal species decreased significantly one day after application onto polyester netting when compared to the viability of conidia remaining in suspension.</p> <p>Conclusions</p> <p>The insecticide-resistant mosquito strain was susceptible to both species of fungus indicating that entomopathogenic fungi can be used in resistance management and integrated vector management programmes to target insecticide-resistant mosquitoes. Although fungal viability significantly decreased when applied to the netting, the effectiveness of the fungal treatment at killing mosquitoes did not significantly deteriorate. Field trials over a longer trial period need to be carried out to verify whether polyester netting is a good candidate for operational use, and to see if wild insecticide-resistant mosquitoes are as susceptible to fungal infection as the VKPER strain.</p

Synergy in Efficacy of Fungal Entomopathogens and Permethrin against West African Insecticide-Resistant Anopheles gambiae Mosquitoes

Background Increasing incidences of insecticide resistance in malaria vectors are threatening the sustainable use of contemporary chemical vector control measures. Fungal entomopathogens provide a possible additional tool for the control of insecticide-resistant malaria mosquitoes. This study investigated the compatibility of the pyrethroid insecticide permethrin and two mosquito-pathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, against a laboratory colony and field population of West African insecticide-resistant Anopheles gambiae s.s. mosquitoes. Methodology/Findings A range of fungus-insecticide combinations was used to test effects of timing and sequence of exposure. Both the laboratory-reared and field-collected mosquitoes were highly resistant to permethrin but susceptible to B. bassiana and M. anisopliae infection, inducing 100% mortality within nine days. Combinations of insecticide and fungus showed synergistic effects on mosquito survival. Fungal infection increased permethrin-induced mortality rates in wild An. gambiae s.s. mosquitoes and reciprocally, exposure to permethrin increased subsequent fungal-induced mortality rates in both colonies. Simultaneous co-exposure induced the highest mortality; up to 70.3±2% for a combined Beauveria and permethrin exposure within a time range of one gonotrophic cycle (4 days). Conclusions/Significance Combining fungi and permethrin induced a higher impact on mosquito survival than the use of these control agents alone. The observed synergism in efficacy shows the potential for integrated fungus-insecticide control measures to dramatically reduce malaria transmission and enable control at more moderate levels of coverage even in areas where insecticide resistance has rendered pyrethroids essentially ineffective

Lethal and Pre-Lethal Effects of a Fungal Biopesticide Contribute to Substantial and Rapid Control of Malaria Vectors

Rapidly emerging insecticide resistance is creating an urgent need for new active ingredients to control the adult mosquitoes that vector malaria. Biopesticides based on the spores of entomopathogenic fungi have shown considerable promise by causing very substantial mortality within 7–14 days of exposure. This mortality will generate excellent malaria control if there is a high likelihood that mosquitoes contact fungi early in their adult lives. However, where contact rates are lower, as might result from poor pesticide coverage, some mosquitoes will contact fungi one or more feeding cycles after they acquire malaria, and so risk transmitting malaria before the fungus kills them. Critics have argued that ‘slow acting’ fungal biopesticides are, therefore, incapable of delivering malaria control in real-world contexts. Here, utilizing standard WHO laboratory protocols, we demonstrate effective action of a biopesticide much faster than previously reported. Specifically, we show that transient exposure to clay tiles sprayed with a candidate biopesticide comprising spores of a natural isolate of Beauveria bassiana, could reduce malaria transmission potential to zero within a feeding cycle. The effect resulted from a combination of high mortality and rapid fungal-induced reduction in feeding and flight capacity. Additionally, multiple insecticide-resistant lines from three key African malaria vector species were completely susceptible to fungus. Thus, fungal biopesticides can block transmission on a par with chemical insecticides, and can achieve this where chemical insecticides have little impact. These results support broadening the current vector control paradigm beyond fast-acting chemical toxins

Insect pathogens as biological control agents: back to the future

The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 15 years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of MCAs, particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for medically important pests including dipteran vectors,. These pathogens combine the advantages of chemical pesticides and microbial control agents (MCAs): they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systemics (i.e. in transgenic plants). Unlike broad spectrum chemical pesticides, B. thuringiensis toxins are selective and negative environmental impact is very limited. Of the several commercially produced MCAs, B. thuringiensis (Bt) has more than 50% of market share. Extensive research, particularly on the molecular mode of action of Bt toxins, has been conducted over the past two decades. The Bt genes used in insect-resistant transgenic crops belong to the Cry and vegetative insecticidal protein families of toxins. Bt has been highly efficacious in pest management of corn and cotton, drastically reducing the amount of broad spectrum chemical insecticides used while being safe for consumers and non-target organisms. Despite successes, the adoption of Bt crops has not been without controversy. Although there is a lack of scientific evidence regarding their detrimental effects, this controversy has created the widespread perception in some quarters that Bt crops are dangerous for the environment. In addition to discovery of more efficacious isolates and toxins, an increase in the use of Bt products and transgenes will rely on innovations in formulation, better delivery systems and ultimately, wider public acceptance of transgenic plants expressing insect-specific Bt toxins. Fungi are ubiquitous natural entomopathogens that often cause epizootics in host insects and possess many desirable traits that favor their development as MCAs. Presently, commercialized microbial pesticides based on entomopathogenic fungi largely occupy niche markets. A variety of molecular tools and technologies have recently allowed reclassification of numerous species based on phylogeny, as well as matching anamorphs (asexual forms) and teleomorphs (sexual forms) of several entomopathogenic taxa in the Phylum Ascomycota. Although these fungi have been traditionally regarded exclusively as pathogens of arthropods, recent studies have demonstrated that they occupy a great diversity of ecological niches. Entomopathogenic fungi are now known to be plant endophytes, plant disease antagonists, rhizosphere colonizers, and plant growth promoters. These newly understood attributes provide possibilities to use fungi in multiple roles. In addition to arthropod pest control, some fungal species could simultaneously suppress plant pathogens and plant parasitic nematodes as well as promote plant growth. A greater understanding of fungal ecology is needed to define their roles in nature and evaluate their limitations in biological control. More efficient mass production, formulation and delivery systems must be devised to supply an ever increasing market. More testing under field conditions is required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly, greater attention must be paid to their use within integrated pest management programs; in particular, strategies that incorporate fungi in combination with arthropod predators and parasitoids need to be defined to ensure compatibility and maximize efficacy. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are potent MCAs. Substantial progress in research and application of EPNs has been made in the past decade. The number of target pests shown to be susceptible to EPNs has continued to increase. Advancements in this regard primarily have been made in soil habitats where EPNs are shielded from environmental extremes, but progress has also been made in use of nematodes in above-ground habitats owing to the development of improved protective formulations. Progress has also resulted from advancements in nematode production technology using both in vivo and in vitro systems; novel application methods such as distribution of infected host cadavers; and nematode strain improvement via enhancement and stabilization of beneficial traits. Innovative research has also yielded insights into the fundamentals of EPN biology including major advances in genomics, nematode-bacterial symbiont interactions, ecological relationships, and foraging behavior. Additional research is needed to leverage these basic findings toward direct improvements in microbial control

The infectivity of the entomopathogenic fungus <it>Beauveria bassiana </it>to insecticide-resistant and susceptible <it>Anopheles arabiensis </it>mosquitoes at two different temperatures

Abstract Background Control of the major African malaria vector species continues to rely extensively on the application of residual insecticides through indoor house spraying or bed net impregnation. Insecticide resistance is undermining the sustainability of these control strategies. Alternatives to the currently available conventional chemical insecticides are, therefore, urgently needed. Use of fungal pathogens as biopesticides is one such possibility. However, one of the challenges to the approach is the potential influence of varied environmental conditions and target species that could affect the efficacy of a biological 'active ingredient'. An initial investigation into this was carried out to assess the susceptibility of insecticide-susceptible and resistant laboratory strains and wild-collected Anopheles arabiensis mosquitoes to infection with the fungus Beauveria bassiana under two different laboratory temperature regimes. Methods Insecticide susceptibility to all four classes of insecticides recommended by WHO for vector control was tested on laboratory and wild-caught An. arabiensis, using standard WHO bioassay protocols. Mosquito susceptibility to fungus infection was tested using dry spores of B. bassiana under two temperature regimes (21 ± 1°C or 25 ± 2°C) representative of indoor conditions observed in western Kenya. Cox regression analysis was used to assess the effect of fungal infection on mosquito survival and the effect of insecticide resistance status and temperature on mortality rates following fungus infection. Results Survival data showed no relationship between insecticide susceptibility and susceptibility to B. bassiana. All tested colonies showed complete susceptibility to fungal infection despite some showing high resistance levels to chemical insecticides. There was, however, a difference in fungus-induced mortality rates between temperature treatments with virulence significantly higher at 25°C than 21°C. Even so, because malaria parasite development is also known to slow as temperatures fall, expected reductions in malaria transmission potential due to fungal infection under the cooler conditions would still be high. Conclusions These results provide evidence that the entomopathogenic fungus B. bassiana has potential for use as an alternative vector control tool against insecticide-resistant mosquitoes under conditions typical of indoor resting environments. Nonetheless, the observed variation in effective virulence reveals the need for further study to optimize selection of isolates, dose and use strategy in different eco-epidemiological settings.</p

Fungal infection counters insecticide resistance in African malaria mosquitoes

The evolution of insecticide resistance in mosquitoes is threatening the effectiveness and sustainability of malaria control programs in various parts of the world. Through their unique mode of action, entomopathogenic fungi provide promising alternatives to chemical control. However, potential interactions between fungal infection and insecticide resistance, such as cross-resistance, have not been investigated. We show that insecticide-resistant Anopheles mosquitoes remain susceptible to infection with the fungus Beauveria bassiana. Four different mosquito strains with high resistance levels against pyrethroids, organochlorines, or carbamates were equally susceptible to B. bassiana infection as their baseline counterparts, showing significantly reduced mosquito survival. Moreover, fungal infection reduced the expression of resistance to the key public health insecticides permethrin and dichlorodiphenyltrichloroethane. Mosquitoes preinfected with B. bassiana or Metarhizium anisopliae showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Our results show a high potential utility of fungal biopesticides for complementing existing vector control measures and provide products for use in resistance management strategies