LA CIENCIA LOGRA CAMBIAR EL MOSQUITO HEMBRA A MACHO EN BÚSQUEDA DEL CONTROL DE INSECTOS

Los mosquitos machos no pican y no pueden transmitir patógenos a los humanos. Los mosquitos hembras, por otro lado, pueden picar. Efectivamente, los mosquitos hembras Aedes aegypti requieren sangre para producir huevos, lo que los convierte en los principales portadores de los patógenos que causan el zika y el dengue en los humanos

Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9.

BACKGROUND:Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS:Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. CONCLUSION AND SIGNIFICANCE:The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions

TALEN-based gene disruption in the dengue vector Aedes aegypti.

In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20-40% of fertile survivors produced kmo alleles that failed to complement an existing kh(w) mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1-7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector

Generation of new mutant <i>kmo</i> alleles from pooled G₀ populations.

*<p>wt, wild-type; we, white-eyed.</p

Aedes aegypti D7 long salivary proteins modulate blood feeding and parasite infection

Mosquito saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis and immune responses. Here, we generated two knockout (KO) mosquito lines by CRISPR/Cas9 to functionally characterize D7L1 and D7L2, two abundantly expressed salivary proteins from the yellow fever mosquito vector Aedes aegypti. The D7s bind and scavenge biogenic amines and eicosanoids involved in hemostasis at the bite site. The absence of D7 proteins in the salivary glands of KO mosquitoes was confirmed by mass spectrometry, enzyme-linked immunosorbent assay, and fluorescence microscopy of the salivary glands with specific antibodies. D7-KO mosquitoes had longer probing times than parental wildtypes. The differences in probing time were abolished when mutant mice resistant to inflammatory insults were used. These results confirmed the role of D7 proteins as leukotriene scavengers in vivo. We also investigated the role of D7 salivary proteins in Plasmodium gallinaceum infection and transmission. Both KO lines had significantly fewer oocysts per midgut. We hypothesize that the absence of D7 proteins in the midgut of KO mosquitoes might be responsible for creating a harsh environment for the parasite. The information generated by this work highlights the biological functionality of salivary gene products in blood feeding and pathogen infection. IMPORTANCE: During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti, the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.This research was supported by the Division of Intramural Research Program of the NIH/NIAID (AI001246, to E.C.) and by a subcontract from grant 1R01AI099483 (to Z.N.A.)S

TALEN-induced deletions in AAEL008879, the <i>Ae. aegypti kmo</i> gene.

<p>Sequenced amplicons obtained from white-eyed individuals were aligned and compared to the wt <i>kmo</i> sequence in the Lvp/<i>kh^w</i> hybrid genetic background (<b>A</b>) or the Lvp background alone (<b>B</b>). The DNA-binding regions of the right (RH) and left (LH) TALENs are indicated. The three in-frame deletions are indicated (*).</p

Identification of new <i>kmo</i> mutant alleles in the LVP genetic background.

<p>Identification of new <i>kmo</i> mutant alleles in the LVP genetic background.</p

Plasmid-based SSA assay for TALEN activity in <i>Ae. aegypti</i> embryos.

<p>(<b>A</b>) cDNA structure of the <i>Ae. aegypti kmo</i> gene (AAEL008879). Exons (roman numerals), initiation and termination (white vertical bars) codons, and TALEN recognition site (black vertical bar) are indicated. The exon skipped in <i>kh^w</i> strain is indicated (white, cross-hatched arrow). The KMO ORF, with predicted extracellular (grey), transmembrane (black) and intracellular (white) domains are indicated below. (<b>B</b>) Schematic representation of the SSA test plasmid. TALEN recognition sites for <i>Ae. aegypti kmo</i> were located between two direct repeats (cross-hatched boxes) of the initial 298 bp of the Firefly luciferase (FF-luc) coding region. Stop codons (denoted by *) in the +1 (7), +2 (10) and +3 (7) reading frames in the spacer are indicated. Transcription from the <i>polyubiquitin</i> (<i>PUb</i>) promoter is expected to lead to translation in the +1 ORF at the FF-luc AUG in the first repeat, resulting in a truncated protein. Fourteen additional AUG codons are present prior to the full-length +2 frame FF-luc ORF to minimize read-through translation. Double-stranded DNA break induction by the introduced TALEN pair (lightning shape) followed by SSA-mediated repair restores the FF-luc ORF. (<b>C</b>) Relative levels of FF-luc activity in the presence or absence of the KMO-targeted TALEN pair 24 hours following injection into <i>Ae. aegypti</i> embryos. Statistical significance following the Mann-Whitney test is indicated.</p

TALEN-generated <i>kmo</i> alleles phenocopy <i>kh^w</i> strain mosquitoes.

<p>LVP, <i>kh^w</i> and LVP<i>^kmo</i> mosquitoes imaged as larvae (L4), pupae (P) and adults (A).</p