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 1years. 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 microbial control agents (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 control of medically important pests including dipteran vectors. These pathogens combine the advantages of chemical pesticides and 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.

Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics.

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

Behavioral thermoregulation in the migratory locust: a therapy to overcome fungal infection.

We examined under laboratory conditions the thermopreference of the migratory locust, Locusta migratoria migratorioides, following infection by the entomopathogenic fungus Metarhizium anisopliae var. acridum and its influence on mycosis. Infected locusts raised their body temperature more frequently than healthy conspecifics through selection of high temperatures in a heat gradient. Thermoregulation did not, however, alter the frequency of feeding events nor the amount of food eaten by infected L. migratoria. A thermoregulation regime of a minimum of 4 h/day substantially increased survival of inoculated insects (by 85%). However, the therapeutic effect decreased when thermoregulation was delayed following inoculation of the pathogen. Thermoregulation reduced locust mortality but did not completely eliminate the fungus from infected hosts; the fungus grew and killed the insects when thermoregulation was interrupted. We suggest that periodic, short bouts of thermoregulation, when performed from the onset of infection and for an extended period of time, are sufficient to provide a therapeutic effect to infected hosts. Such thermoregulatory capacity of locusts may limit the potential of fungal pathogens as biological control agents under certain ecological conditions.

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride.

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32 degrees C; several isolates grew at 8 and 37 degrees C. None of the isolates grew at 40 degrees C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 degrees C with several isolates exhibiting optimal growth at temperatures as high as 32 degrees C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 degrees C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 degrees C. However, the two most cold tolerant isolates at 8 degrees C were M. flavoviride and the three most heat tolerant at 35 degrees C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.

Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes.

The influence of simulated sunlight on survival of conidia of 4 species of entomopathogenic Hyphomycetes was investigated. Conidia from 65 isolates ofBeauveria bassiana, 23 ofMetarhizium anisopliae, 14 ofMetarhizium flavoviride and 33 isolates ofPaecilomyces fumosoroseus were irradiated by artificial sunlight (295 to 1,100 nm at an ultraviolet-B irradiance of 0.3 W m(-2)) for 0, 1, 2, 4 and 8 h. Survival was estimated by comparing the number of colony forming units (CFU) produced by conidia exposed to irradiation to the number of CFUs produced by an unexposed control. Survival decreased with increased exposure to simulated sunlight; exposure for 2 h or more was detrimental to all isolates tested. Overall, isolates ofM. flavoviride were the most resistant to irradiation followed byB. bassiana andM. anisopliae. Conidia ofP. fumosoroseus were most susceptible. In addition to the large interspecies differences in susceptibility to irradiation, there was also an intraspecies variation indicating that strain selection to irradiation tolerance may be important in the development of microbial control agents where increased persistence in an insolated environment is desirable.

Studies on bioassay of the entomopathogenic hyphomycete fungus Tolypocladium cylindrosporum in mosquitoes.

Larvae of Aedes aegypti, Ae. vexans and Culiseta inornata were used to study the pathogenicity of the hyphomycete fungus Tolypocladium cylindrosporum. Experiments consisted of continuous exposure of second-instar larvae to 5 concentrations of conidia at 10, 15, 20 and 25 degrees C. The 3 test species were susceptible at all temperatures tested. There was generally a direct relationship between dosage and pathogenicity in the host population, however linear relationships between probit mortality at 10 days and log dosage occurred in only 11 of 37 assays performed. In these 11 assays, median lethal concentrations were in the order of 10(4)-10(5) conidia/ml. Median lethal times were between 3 and 14 days. It was concluded that the present bioassay method for T. cylindrosporum is inadequate and needs major improvement.

Field incidence of mosquito pathogens and parasites in central Alberta.

Ten pools and ponds were monitored on a weekly basis for presence of mosquito pathogens and parasites over a three year period near Edmonton, Alberta. Acari, fungi, Microsporidia and Peritrichida were found associated with mosquitoes. Percentage of collections with pathogens and parasites (prevalence) followed by the mean percentage infection within the samples, in parentheses (estimated % incidence) for the three year period were as follows: Coelomomyces psorophorae var. psorophorae 0.9(0.01-0.02), Culicinomyces clavisporus 1.2(0.09-0.2), Saprolegniales 50.1(15), Smittium sp. 23.0(4), Microsporidia 10.9(0.6-1.4), Peritrichida 43.4(30) and Acari 3.2(0.04). The high incidence of Saprolegniales may be a result of attack on stressed individuals under laboratory conditions. Several host and country records are reported. It is concluded that pathogens and parasites generally had little effect on the mosquito populations.

Preliminary field trials with the entomopathogenic hyphomycete Tolypocladium cylindrosporum in central Alberta.

Conidia and blastoconidia of the pathogenic hyphomycete Tolypocladium cylindrosporum were applied to temporary and semi-permanent pools in central Alberta on 6 occasions. Infections were detected in the field only following application of blastoconidia; indications were that infections did not occur in the field following application of conidia. Most infections were detected only after holding field-collected immatures under laboratory conditions for up to 24 days. Five species of mosquitoes were found infected. Infections occurred in collections up to 29 days post-application with most mosquitoes dying as 4th instars. No infections were detected during subsequent years. Infection rates of up to 33% for blastoconidia and 55% for conidia were estimated. It is speculated that field-collected mosquitoes succumbed to the fungus as a result of the stress of transfer to laboratory conditions.

The urban mosquitoes of Suva, Fiji: seasonal incidence and evaluation of environmental sanitation and ULV spraying for their control.

Larval surveys and oviposition traps were used to monitor urban mosquito populations in two adjacent transects in Suva, Fiji between May 1978 and August 1979. Populations of Aedes aegypti and Ae. pseudoscutellaris fluctuated seasonally with changes in rainfall, the latter species being most prevalent throughout the year. Populations of these two species were highest between December and July and lowest between August and October. Larval populations of Culex quinquefasciatus did not show a seasonal variation and larval populations of Cx. annulirostris were too low for any conclusions to be made. All species were found breeding most often in miscellaneous containers, with tyres, plant containers and flower vases also being important sources for Ae. aegypti breeding. Through environmental sanitation the Breteau Index for all species was reduced by 88%; Premise Index by 72% and the Container Index by 83%, when compared to a control area. ULV applied malathion was effective in temporarily reducing Ae. pseudoscutellaris populations from 50--100%. Effects on Ae. aegypti were inconclusive. It is concluded that through enforcement of the existing laws and strict monthly surveillance during the periods of highest seasonal density, urban Aedes and Culex populations can be maintained at an acceptable level.