No abstract

To overcome burden of mosquito-borne diseases, multiple control strategies are needed. Population replacement with genetically modified mosquitoes carrying antipathogen effector genes is one of the possible approaches for controlling disease transmission. However, transgenic mosquitoes with antipathogen phenotypes based on overexpression of a single type effector molecule are not efficient in interrupting pathogen transmission. Here, we show that co-overexpression of two antimicrobial peptides (AMP), Cecropin A, and Defensin A, in transgenic Aedes aegypti mosquitoes results in the cooperative antibacterial and antiPlasmodium action of these AMPs. The transgenic hybrid mosquitoes that overexpressed both Cecropin A and Defensin A under the control of the vitellogenin promoter exhibited an elevated resistance to Pseudomonas aeruginosa infection, indicating that these AMPs acted cooperatively against this pathogenic bacterium. In these mosquitoes infected with P. gallinaceum, the number of oocysts was dramatically reduced in midguts, and no sporozoites were found in their salivary glands when the mosquitoes were fed twice to reactivate transgenic AMP production. Infection experiments using the transgenic hybrid mosquitoes, followed by sequential feeding on naive chicken, and then naive wild-type mosquitoes showed that the Plasmodium transmission was completely blocked. This study suggests an approach in generating transgenic mosquitoes with antiPlasmodium refractory phenotype, which is coexpression of two or more effector molecules with cooperative action on the parasite.vector-borne disease | malaria | immunity | antimicrobial peptide | vitellogenin

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5 flanking sequence (؊855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5 flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor͞Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.insect immunity ͉ gene regulation ͉ development ͉ melanization T he melanizing effects of phenoloxidases were identified nearly 60 years ago in grasshopper eggs (1). Since then, tyrosinase-type phenoloxidases (monophenol, L-dopa: oxidoreductase; EC 1.14.18.1), which are widely distributed in both prokaryotes and eukaryotes, have been characterized as coppercontaining enzymes catalyzing two key reactions in the synthesis of melanin (2). Tyrosinase-type phenoloxidase (PO)-mediated melanin synthesis plays a major role in wound healing and the formation of melanotic capsules that sequestrate parasites, parasitoids, and pathogens as they breach the cuticular exoskeleton or midgut epithelium or enter into the hemocoel of the insect (3). The role of PO in melanotic encapsulation has bestowed upon this enzyme the status of ''immune protein.'' Because melanization͞encapsulation can lead to refractoriness of certain Plasmodium species, the genes controlling melanization͞encapsu-lation in Anopheles gambiae are considered to be prime candidates for genetic manipulation of mosquito vectors as a future means of malaria control (4, 5). At present, six prophenoloxidase cDNAs have been cloned and sequenced from A. gambiae (6)(7)(8).Ashida (9) demonstrated that PO from Bombyx mori exists in a zymogen form and is activated via a serine proteinase cascade, which is set into motion by injury or microbial challenge (3, 10). To date, all insect prophenoloxidases (PPOs) seem to follow this activation pattern. However, apart from studies on the hormonal regulation of granular cuticular phenoloxidase synthesis (11) and a very recent study by Müller et al. (8), in which the authors show that 20-hydroxyecdysone (20E) is able to modulate PPO gene expression in A. gambiae cells, little is known about arthropod PPO gene regulation or direct hormonal regulation of insect immune ...