NATURE BIOTECHNOLOGY VOLUME 30 NUMBER 1 JANUARY 2012 35 that participates in critical host physiological processes, thereby minimizing nontarget effects. Insect diuretic hormones participate in the regulation of water balance, and MSDH belongs to the corticotropin-releasing factor– related family of peptides. Synthetic MSDH has been shown to stimulate fluid excretion in vivo, resulting in pronounced loss of fluid through the gut and the epidermis, decreased feeding and ultimately insect death 8–10 . TMOFs are unblocked deca- and hexa-peptides that terminate trypsin biosynthesis in the insect gut and are found in the ovaries of insects that include both mosquitoes and flies. Aea-TMOF circulates in the hemolymph, binds to gut receptors on the hemolymph side of the gut and inhibits trypsin biosynthesis by exerting a translational control on trypsin mRNA 11 . Because TMOF resists proteolysis in the gut and easily traverses the gut epithelial cells into the hemolymph in adults and larvae, it was fed to different species of mosquito larvae in which it caused inhibition of food digestion and anorexia, ultimately leading to starvation and death 12 . TMOF is currently under development as an insecticide and appears to be very specific against mosquitoes with minimal nontarget effects 13 . Indeed, TMOFs from different insects have different peptide sequences (e.g., the Aea- TMOF sequence is YDPAPPPPPP, whereas the gray flesh fly, Sarcophaga bullata, Sb- TMOF sequence is NPTNLH 14 ). MSDH-Gly (42 amino acids) and the A. aegypti Aea-TMOF peptides were expressed in B. bassiana by transformation of expression vectors containing a constitutive B. bassiana-derived gpd promoter and the nucleotide sequence corresponding to the insecticides has resulted in substantial damage to ecosystems and the emergence of insecticide-resistant agricultural pests. Mosquitoes are vectors of many human and animal infectious diseases that cause death and economic hardship. World Health Organization (WHO; Geneva) recommendations suggest the use of different control strategies as part of integrated vector management control to prevent the emergence of insecticide-resistant mosquitoes. Effective strategies, however, for long-term reduction of mosquito populations remain elusive 2 . Entomopathogenic fungi are virulent to a wide range of Lepidoptera as well as mosquitoes and have been considered as possible candidates for reducing disease transmission of vector- borne infectious agents 3,4 . Methods have been developed for delivery of these agents in agricultural settings as well as to adult and larval mosquitoes, and the fungi appear to be equally (or more) effective against insecticide-resistant strains as compared with their insecticide-susceptible parental strains 5–7 . To test whether host molecules can be used to increase the virulence of entomopathogenic fungi, we engineered two insect peptides—Manduca sexta diuretic hormone (MSDH) and A. aegypti trypsin- modulating oostatic factor (Aea-TMOF)—in B. bassiana, which expresses and secretes these hormones as it infects its host. Our idea was that the exogenously produced host peptide hormone would disrupt the normal endocrine or neurological balance of the host, making it more susceptible to the invading fungus. As candidates, we chose a potential broad host range target (MSDH) as well as a more host-specific peptide (TMOF) target Exploiting host molecules to augment mycoinsecticide virulence To the Editor: A pressing need exists for additional tools in insect control, particularly as few new chemical pesticides are under development. Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, both US Environmental Protection Agency (EPA)-approved biological control agents, offer an environmentally friendly alternative to chemical insecticides. One limitation to the use of entomopathogenic fungi is the relatively long time (6–12 days) it takes for the fungus to kill target insects. Expression of a scorpion toxin in M. anisopliae increases fungal toxicity about ninefold toward the yellow fever mosquito, Aedes aegypti 1 ; however, expression of nonspecies-specific toxins to control mosquitoes may promote the development of toxin resistance. Ideally, a strain with enhanced virulence toward target insects with minimal nontarget effects, coupled to a decreased likelihood of the development of resistance to the agent is most desired. To meet this objective, we report a novel approach to insect control, in which expression of host molecules in an insect pathogen is exploited for augmentation of virulence. The major advantages of such a strategy are the following: first, the increase in virulence can be tailored to be host specific depending upon the host molecule (peptide) chosen; and second, the development of resistance can be minimized as the host peptide hormones regulate developmental processes that are species and tissue specific. Contrary to current approaches in insect control, mutations that arise and could compensate for the fungal-expressed product during infection, also would lead to developmental defects—a fitness cost far greater than developing resistance to a pesticide. Thus, unlike methods that use insecticidal compounds, the development of resistance using our approach faces an additional burden. In theory, multiple host molecules can be expressed in the same fungal strain to further augment the specificity and virulence of mycoinsecticides to produce the next generation of more effective and safer insect biological control agents. Lepidopteran species cause billions of dollars worth of crop losses worldwide and are the most destructive pests in agriculture. Overuse and reliance on chemical Table 1 Calculated LD 50 and LT 50 of wild-type, MSDH- and TMOF-expressing B. bassiana strains against G. mellonella and A. aegypti Strain Host LD 50 (conidia/ml) LT 50 (h) Wild-type B. bassiana G. mellonella 2.4 ± 0.3 × 10 7a 134.7 ± 5.5 b Bb::spMSDH G. mellonella 0.3 ± 0.10 × 10 7a 86.2 ± 3.8 b Wild-type B. bassiana A. aegypti (adult) 5.0 ± 0.6 × 10 8c 163.2 ± 12.0 d Bb::spAeaTMOF A. aegypti (adult) 6.25 ± 0.5 × 10 7c 123.2 ± 9.2 d Wild-type B. bassiana A. aegypti (larvae) 1 × 10 8e 156 ± 6.6 f Bb::spAeaTMOF A. aegypti (larvae) 1 × 10 8e 100.8 ± 5.0 f a LD 50 calculated from 96-h time point. b Bioassay performed using spore concentration of 1 × 10 7 conidia/ml. c LD 50 calculated from 120-h time point. d Bioassay performed using 0.25 μl of a 1 × 10 8 conidia/ml spore solution applied to mosquito abdomen. e Accurate LD 50 could not be determined in this case. f Bioassay performed using spore concentration of 1 × 10 8 conidia/ml. 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