Blocking of Plasmodium transmission by cooperative action of Cecropin A and Defensin A in transgenic Aedes aegypti mosquitoes

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

The sex locus is tightly linked to factors conferring sex-specific lethal effects in the mosquito Aedes aegypti

“…The pBac[3xP3-EGFP, afm] vector containing an EGFP transformation marker under the control of the 3xP3 eye-specific promoter (Horn and Wimmer, 2000) was used to subclone a DNA fragment containing the vitellogenin (Vg) gene promoter linked to the cecropin A (CecA) gene complementary DNA and SV40 polyadenylation element (Kokoza et al, 2010). The resulting pBac [3xP3-EGFP afm, Vg-CecA] donor plasmid was mixed with the phsp-pBac helper plasmid at final concentrations of 0.35 and 0.25 μg ml − 1 , respectively, in a 5 mM KCl and 0.1 mM NaH 2 PO 4 (pH 6.8) buffer.…”

Pathogen-insect interaction candidate molecules for transmission-blocking control strategies of vector borne diseases

“…35 These studies indicate the need for improving the efficacy of the effector molecule expression; for instance, the simultaneous expression of cecropin and defensin A completely blocked infection. 36 Dengue. From an infected blood meal, DENV invade and multiply within the mosquito midgut epithelial cells, to later disseminate to other organs, reaching the salivary glands, from where they are inoculated to new human hosts in subsequent blood meals 37 (figure1).…”

Transgenic Mosquitoes to Control Vector Borne Diseases

“…Red fluorescent protein (dsRED) [7,8] are used to select transgenic mosquitoes from non-transgenic mosquitoes. Using fluorescent markers as reporter genes saved the time and effort of rearing nontransgenic mosquitoes to adults because the fluorescent protein expressing mosquitoes could be observed and separated even in early larval stages, however, if insecticide resistance genes were used as reporter genes both transgenic and non-transgenic larvae had to be reared until adult stage.4 Some of the pathogen effector molecules used when producing transgenic mosquitoes were antiparasitic peptides3, antimicrobial peptides [9], antisense RNA, and shRNA [2]. As promoters for the DNA constructs, salivary gland specific promoters from Maltase-like I gene (MalI) and Apyrase gene (Apy) of Ae.…”

“…The strong promoters such as Ae. aegypti carboxypeptidase A (AeCPA) [2] and Vitellogenin (Vg) [9] genes promoters that drives expression in stage, tissue and sex specific manner, Anopheles gambiae carboxy peptidase (AgCP) [3] and β2 [10] genes promoters, Ae. aegypti Act5C [7] and Actin-4 (AeAct-4)8 genes promoters have been used to drive the transgene expression in strong manner in TMs.…”

“…gambiae having infection of either P. falciparum or P. berghei as a result of non activation of defensins or cecropins in the latter mosquitoes. However, based on this result it was concluded that combinatorial over expression of several antiparasitic effector molecules in TMs would completely block the transmission of Plasmodium in Anopheles genus mosquitoes to prevent malaria [9].…”