Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis.

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Is Saglin a mosquito salivary gland receptor for Plasmodium falciparum?

BACKGROUND: Saglin, a 100 kDa protein composed of two 50 kDa homodimers, is present in the salivary glands of Anopheles gambiae and has been considered an essential receptor for sporozoites (SPZ) of Plasmodium berghei and Plasmodium falciparum (Pf), allowing SPZ to recognize, bind to, and infect mosquito salivary glands. Spatial and temporal patterns of Saglin expression reported here, however, suggest that this model does not fully describe the Saglin-SPZ interaction. RESULTS: Saglin protein was detected by indirect immunofluorescence microscopy only in the medial and proximal-lateral lobes, but not in the distal-lateral lobes, of the salivary glands of An. gambiae; the pattern of expression was independent of mosquito age or physiological state. These results were confirmed by steady-state Saglin transcript and protein expression using qRT-PCR and Western-blot analysis, respectively. Saglin was localized to the basal surface of the cells of the medial lobes and was undetectable elsewhere (intracellularly, on the lateral or apical membranes, the cells' secretory vacuoles, or in the salivary duct). In the cells of the proximal lateral lobes of the salivary glands, Saglin was distinctly intracellular and was not localized to any of the cell surfaces. Transgenic Anopheles stephensi were produced that expressed An. gambiae Saglin in the distal lateral lobes of the salivary gland. Additional Saglin expression did not enhance infection by PfSPZ compared to non-transgenic siblings fed on the same gametocyte-containing blood meal. CONCLUSIONS: The absence of Saglin in the distal lateral lobes of the salivary glands, a primary destination for SPZ, suggests Saglin is not an essential receptor for Plasmodium SPZ. The lack of any correlation between increased Saglin expression in the distal lateral lobes of the salivary glands of transgenic An. stephensi and PfSPZ infection is also consistent with Saglin not being an essential salivary gland receptor for Plasmodium SPZ.

An Anopheles stephensi Promoter-Trap: Augmenting Genome Annotation and Functional Genomics.

The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, Anopheles stephensi and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the tetameric AmCyan reporter as the fusion peptide displayed a preference for insertion into the 5'-end of transcripts. Notably 183 - 44882 bp upstream of the An. stephensi v1.0 ab initio gene models, which demonstrated that the promoter regions for the genes of An. stephensi are further upstream of the 5'-proximal regions of the genes in the ab inito models than may be otherwise predicted. RNA-Seq transcript coverage supported the insertion of the splice acceptor gene trap element into 5'-UTR introns for nearly half of all insertions identified. The use of a gene trap element that prefers insertion into the 5'-end of genes supports the use of this technology for the random generation of knock-out mutants, as well as the experimental confirmation of 5'-UTR introns in An. stephensi.

Gal4-based enhancer-trapping in the malaria mosquito Anopheles stephensi.

Transposon-based forward and reverse genetic technologies will contribute greatly to ongoing efforts to study mosquito functional genomics. A piggyBac transposon-based enhancer-trap system was developed that functions efficiently in the human malaria vector, Anopheles stephensi. The system consists of six transgenic lines of Anopheles stephensi, each with a single piggyBac-Gal4 element in a unique genomic location; six lines with a single piggyBac-UAStdTomato element; and two lines, each with a single Minos element containing the piggyBac-transposase gene under the regulatory control of the hsp70 promoter from Drosophila melanogaster. Enhancer detection depended upon the efficient remobilization of piggyBac-Gal4 transposons, which contain the yeast transcription factor gene Gal4 under the regulatory control of a basal promoter. Gal4 expression was detected through the expression of the fluorescent protein gene tdTomato under the regulatory control of a promoter with Gal4-binding UAS elements. From five genetic screens for larval- and adult-specific enhancers, 314 progeny were recovered from 24,250 total progeny (1.3%) with unique patterns of tdTomato expression arising from the influence of an enhancer. The frequency of piggyBac remobilization and enhancer detection was 2.5- to 3-fold higher in female germ lines compared with male germ lines. A small collection of enhancer-trap lines are described in which Gal4 expression occurred in adult female salivary glands, midgut, and fat body, either singly or in combination. These three tissues play critical roles during the infection of Anopheles stephensi by malaria-causing Plasmodium parasites. This system and the lines generated using it will be valuable resources to ongoing mosquito functional genomics efforts.

piggyBac transposon remobilization and enhancer detection in Anopheles mosquitoes.

Technical advances in mosquito biology are enabling the development of new approaches to vector control. Absent are powerful forward-genetics technologies, such as enhancer and gene traps, that permit determination of gene functions from the phenotypes arising from transposon insertion mutations. We show that the piggyBac transposon is highly active in the germline of the human malaria vector Anopheles stephensi. Up to 6% of the progeny from transgenic A. stephensi containing a single 6-kb piggyBac element with a marker gene expressing EGFP had the vector in new genomic locations when piggyBac transposase was provided in trans from a second integrated transgene. The active transposition of piggyBac resulted in the efficient detection of enhancers, with ~10% of the progeny with piggyBac in new locations with novel patterns of EGFP expression in third and fourth instar larvae and in adults. The availability of advanced transgenic capabilities such as efficient transposon-based forward-genetics technologies for Anopheles mosquitoes not only will accelerate our understanding of mosquito functional genomics and the development of novel vector and disease transmission control strategies, but also will enable studies by evolutionary developmental biologists, virologists, and parasitologists.

An active transposable element, Herves, from the African malaria mosquito Anopheles gambiae.

Transposable elements have proven to be invaluable tools for genetically manipulating a wide variety of plants, animals, and microbes. Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae, thereby disabling the mosquito's ability to transmit malaria. To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome. Endogenous, active class II transposable elements from An. gambiae have not been exploited as gene vectors/drivers because none have been isolated. We report the discovery of an active class II transposable element, Herves, from the mosquito An. gambiae. Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B. cucurbitae. Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D. melanogaster. Herves displays an altered target-site preference from the distantly related hAT elements, Hermes and hobo. Herves is also present in An. arabiensis and An. merus with copy numbers similar to that found in An. gambiae. Preliminary data from an East African population are consistent with the element being transpositionally active in mosquitoes.

Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti.

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.