Xavier Nirmala

Engineered Resistance to Plasmodium falciparum Development in Transgenic Anopheles stephensi

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.

Engineering Plasmodium-refractory phenotypes in mosquitoes

A remarkable number of effector mechanisms have been developed for interfering with malaria parasite development in mosquitoes. These effector mechanisms affect different aspects of parasite biology and therefore could be targeted synergistically to reduce the probability of emergence of parasite resistance to any one mechanism. The use of these mechanisms will depend on how efficiently and safely they can be introduced into existing mosquito populations.

The accumulation of specific mRNAs following multiple blood meals in Anopheles gambiae

One approach to genetic control of transmission of the parasites that cause human malaria is based on expressing effector genes in mosquitoes that disable the pathogens. Endogenous mosquito promoter and other cis‐acting DNA sequences are needed to direct the optimal tissue‐, stage‐ and sex‐specific expression of the effector molecules. The mRNA accumulation profiles of eight different genes expressed specifically in the midgut, salivary glands or fat body tissues of the malaria vector, Anopheles gambiae, were characterized as a measure of their suitability to direct the expression of effector molecules designed to disable specific stages of the parasites. RT‐PCR techniques were used to determine the abundance of the gene products and their duration following multiple blood meals. Transcription from the midgut‐expressed carboxypeptidase‐encoding gene, AgCP, follows a cyclical, blood‐inducible expression pattern with maximum accumulation every 3 h post blood meal. Other midgut‐expressed genes encoding a trypsin and chymotrypsin, Antryp2 and Anchym1, respectively, and the fat body‐expressed genes, Vg1 and Cathepsin, also show a blood‐inducible pattern of expression with maximum accumulation 24 h after every blood meal. Expression of the Lipophorin gene in the fat body and apyrase and D7‐related genes (AgApy and D7r2) in the salivary glands is constitutive and not significantly affected by blood meals. Promoters of the midgut‐ and fat body‐expressed genes may lead to maximum accumulation of antiparasite effector molecule transcripts after multiple blood meals. The multiple feeding behaviour of An. gambiae thus can be an advantage to express high levels of antiparasite effector molecules to counteract the parasites throughout most of adult development.

Pro-apoptotic cell death genes, hid and reaper, from the tephritid pest species, Anastrepha suspensa

Pro-apoptotic proteins from the reaper, hid, grim (RHG) family are primary regulators of programmed cell death in Drosophila due to their antagonistic effect on inhibitor of apoptosis (IAP) proteins, thereby releasing IAP-inhibition of caspases that effect apoptosis. Using a degenerate PCR approach to conserved domains from the 12 Drosophila species, we have identified the first reaper and hid orthologs from a tephritid, the Caribfly Anastrephasuspensa. As-hid is the first identified non-drosophilid homolog of hid, and As-rpr is the second non-drosophilid rpr homolog. Both genes share more than 50% amino acid sequence identity with their Drosophila homologs, suggesting that insect pro-apoptotic peptides may be more conserved than previously anticipated. Importantly, both genes encode the conserved IBM and GH3 motifs that are key for IAP-inhibition and mitochondrial localization. Functional verification of both genes as cell death effectors was demonstrated by cell death assays in A. suspensa embryonic cell culture, as well as in heterologous Drosophila melanogaster S2 cells. Notably, heterologous cell death activity was found to be higher for Anastrepha genes than their Drosophila counterparts. In common with the Drosophila cognates, As-hid and As-rpr negatively regulated the Drosophila inhibitor of apoptosis (DIAP1) gene to promote apoptosis, and both genes when used together effected increased cell death activity, indicating a co-operative function for As-hid and As-rpr. We show that these tephritid cell death genes are functional and potent as cell death effectors, and could be used to design improved transgenic lethality systems for insect population control.

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac-based transposon vector system using independent vector and transposase helper plasmids. Minimum estimated germ-line transformation frequencies were approximately 13–21% per fertile G0 individual, similar to previously reported frequencies using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control. The first allows post-integration stabilization of a transposon-vector by deletion of a terminal sequence necessary for mobilization. The complete pB[L1-EGFP-L2-DsRed-R1] vector was integrated into the Chiapas wild type strain with subsequent deletion of the L2-DsRed-R1 sub-vector carrying the piggyBac 3′ terminal sequence. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5–10% in most tests, however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release. The second vector, pB[XL-EGFP, Asß2-tub-DsRed.T3], has the DsRed.T3 fluorescent protein reporter gene regulated by the A. suspensaAsß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm bundles were unambiguously observed in the spermathecae of non-transgenic females mated to transgenic males. One transgenic line apparently had a male-specific Y-chromosome insertion, having potential use for sexing by fluorescent-embryo sorting. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.

An EST database of the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae)

Invasive tephritid fruit flies are a great threat to agriculture worldwide and warrant serious pest control measures. Molecular strategies that promote embryonic lethality in these agricultural pests are limited by the small amount of nucleotide sequence data available for tephritids. To increase the dataset for sequence mining, we generated an EST database by 454 sequencing of the caribfly, Anastrepha suspensa, a model tephritid pest. This database yielded 95,803 assembled sequences with 24% identified as independent transcripts. The percentage of caribfly sequences with hits to the closely related tephritid, Rhagoletis pomonella, transcriptome was higher (28%) than to Drosophila proteins/genes (18%) in NCBI. The database contained genes specifically expressed in embryos, genes involved in the cell death, sex-determination, and RNAi pathways, and transposable elements and microsatellites. This study significantly expands the nucleotide data available for caribflies and will be a valuable resource for gene isolation and genomic studies in tephritid insects.

A DsRed fluorescent protein marker under polyubiquitin promoter regulation allows visual and amplified gene detection of transgenic Caribbean fruit flies in field traps

Field population surveillance of a targeted insect pest species is critical in evaluating management programs such as the sterile insect technique. Fluorescent powder dyes currently used to distinguish released tephritids from the field population are not optimal in terms of reliability and human health issues. Genetically transformed tephritid species present the possibility of using fluorescent transgenes for marking. Here we studied the stability of DsRed fluorescence in transgenic flies maintained in aqueous torula yeast borax and propylene glycol. DsRed was stable in both solutions for three weeks by visual microscopic observations and could be used to unambiguously distinguish them from non-fluorescent wild type flies. To compensate for any potential ambiguity in visual identification a diagnostic PCR was developed that could specifically amplify the exotic heterologous marker gene. Therefore, the use of sterile transgenic insect strains carrying stably integrated fluorescent protein marker genes in biologically-based control programs could greatly improve released fly identification in pest management programs.