Multi-Locus Assortment (MLA) for Transgene Dispersal and Elimination in Mosquito Populations

BACKGROUND:Replacement of wild-type mosquito populations with genetically modified versions is being explored as a potential strategy to control vector-borne diseases. Due to lower expected relative fitness of transgenic individuals, transgenes must be driven into populations for these scenarios to be successful. Several gene drive mechanisms exist in a theoretical sense but none are currently workable in mosquitoes. Even if strategies were workable, it would be very difficult to recall released transgenes in the event of unforeseen consequences. What is needed is a way to test transgenes in the field for feasibility, efficacy and safety prior to releasing an active drive mechanism. METHODOLOGY/PRINCIPAL FINDINGS:We outline a method, termed Multi-locus assortment (MLA), to spread transgenes into vector populations by the release of genetically-modified mosquitoes carrying multiple stable transgene inserts. Simulations indicate that [1] insects do not have to carry transgenes at more than 4 loci, [2] transgenes can be maintained at high levels by sequential small releases, the frequency of which depends on the construct fitness cost, and [3] in the case of unforeseen negative non-target effects, transgenes can be eliminated from the population by halting transgenic releases and/or mass releases of wild-type insects. We also discuss potential methods to create MLA mosquito strains in the laboratory. CONCLUSIONS/SIGNIFICANCE:While not as efficient as active drive mechanisms, MLA has other advantages: [1] MLA strains can be constructed for some mosquito species with currently-available technology, [2] MLA will allow the ecological components of transgenic mosquito releases to be tested before actual gene drive mechanisms are ready to be deployed, [3] since MLA is not self-propagating, the risk of an accidental premature release into nature is minimized, and [4] in the case that active gene drive mechanisms prove impossible to develop, the MLA approach can be used as a back-up transgene dispersal mechanism for disease control efforts in some systems

Manipulating insulin signaling to enhance mosquito reproduction

<p>Abstract</p> <p>Backgrond</p> <p>In the mosquito <it>Aedes aegypti </it>the insulin/insulin growth factor I signaling (IIS) cascade is a key regulator of many physiological processes, including reproduction. Two important reproductive events, steroidogenesis in the ovary and yolk synthesis in the fat body, are regulated by the IIS cascade in mosquitoes. The signaling molecule phosphatase and tensin homolog (PTEN) is a key inhibitor of the IIS cascade that helps modulate the activity of the IIS cascade. In <it>Ae. aegypti</it>, six unique splice variants of AaegPTEN were previously identified, but the role of these splice variants, particularly AaegPTEN3 and 6, were unknown.</p> <p>Results</p> <p>Knockdown of AaegPTEN or its specific splice variant AaegPTEN6 (the splice variant thought to regulate reproduction in the ovary and fat body) using RNAi led to a 15–63% increase in egg production with no adverse effects on egg viability during the first reproductive cycle. Knockdown of AaegPTEN3, expressed predominantly in the head, had no effect on reproduction. We also characterized the protein expression patterns of these two splice variants during development and in various tissues during a reproductive cycle.</p> <p>Conclusion</p> <p>Previous studies in a range of organisms, including <it>Drosophila melanogaster </it>and <it>Caenorhabditis elegans</it>, have demonstrated that disruption of the IIS cascade leads to decreased reproduction or sterility. In this study we demonstrate that knockdown of the IIS inhibitor PTEN can actually increase reproduction in the mosquito, at least during the first reproductive cycle.</p

The Anopheles gambiae Insecticidal Targets Made Bare by In-silica Analysis

seveml wot·ks had attempted to use genomics to explain the mode of mosquito t·esistance and pt·edict dmg tat·get. The use of insecticides in val'ious ways has been the majm· malal'ia vectot· conti'Ol stmtegy being deployed lately, mostly pyt·ethi'Oid, the majm· t·ecommended compound class fot· IRS, ITNs and LLITNs. Resistance to dmgs and insecticides has continually obstmcted vectm·/malal'ia contt·ol stntegies. The advet·t effect is so enonnous in the Sub-Sahamn Afl'ican; its socioeconomic impact is unquantifiable in evet·y measm·e. Thus, the quick necessity fm· the development and elucidation of potent, cheap and efficient new potential insecticidal tat·gets, especially those in the class pyt·ethi'Oid fm· the malal'ia vectot·, A. gambiae. In this wm·k, an updated Anopheles gambiae biochemical metabolic netwm·k (AnoCyc vel'l.O), othet·wise known as pathway genome database (PGDB) was extmcted, the database was t·econstt·ucted by developing a computational gmph model in an appi'Oach that modeled the metabolic netwot·k of the m·ganism as a bipat·tite gmph, deployed the concept of choke point, load point and t·eaction without deviation to detet·mine the essential enzymatic t·eactions in the netwm·ks. Each potential dmg tat·get to theit· coiTesponding gene/pi'Otein and such encoding pi'Otein sequences wet·e extmcted. (PDB) was blasted fot· genes that have stmctm·e m· homologue of >= 30 sequence identity. Finally, we deployed Ovet·ton and Bation Scm·e (OB-Scm·e) and Pat·Ct·ys pt·ediction to mnk pi'Oteins by theit' likely success in ct·ystallization. 61 potential insecticidal candidate tat·gets was made bat·e, one clinically validated insecticidal tat·get and othet·s with biological evidence in the litemtnt·e. Seven of these tat·gets ideally stand out and have no homology with othet· vetiebmtes. These in depth dissection of the biochemical metabolic netwm·ks of the Anopheles effectively identified the ideal gene pi'Oducts and specifically extmct essential enzymes as new potential insecticidal tat·get against A. gambiae

Wolbachia Induces Male-Specific Mortality in the Mosquito Culex pipiens (LIN Strain)

Background: Wolbachia are maternally inherited endosymbionts that infect a diverse range of invertebrates, including insects, arachnids, crustaceans and filarial nematodes. Wolbachia are responsible for causing diverse reproductive alterations in their invertebrate hosts that maximize their transmission to the next generation. Evolutionary theory suggests that due to maternal inheritance, Wolbachia should evolve toward mutualism in infected females, but strict maternal inheritance means there is no corresponding force to select for Wolbachia strains that are mutualistic in males. Methodology/Principal findings: Using cohort life-table analysis, we demonstrate that in the mosquito Culex pipiens (LIN strain), Wolbachia-infected females show no fitness costs due to infection. However, Wolbachia induces up to a 30% reduction in male lifespan. Conclusions/significance: These results indicate that the Wolbachia infection of the Culex pipiens LIN strain is virulent in a sex-specific manner. Under laboratory situations where mosquitoes generally mate at young ages, Wolbachia strains that reduce male survival could evolve by drift because increased mortality in older males is not a significant selective force

Duplication, concerted evolution and purifying selection drive the evolution of mosquito vitellogenin genes

<p>Abstract</p> <p>Background</p> <p>Mosquito vitellogenin (<it>Vtg</it>) genes belong to a small multiple gene family that encodes the major yolk protein precursors required for egg production. Multiple <it>Vtg </it>genes have been cloned and characterized from several mosquito species, but their origin and molecular evolution are poorly understood.</p> <p>Results</p> <p>Here we used <it>in silico </it>and molecular cloning techniques to identify and characterize the evolution of the <it>Vtg </it>gene family from the genera <it>Culex</it>, <it>Aedes/Ochlerotatus</it>, and <it>Anopheles</it>. We identified the probable ancestral <it>Vtg </it>gene among different mosquito species by its conserved association with a novel gene approximately one kilobase upstream of the start codon. Phylogenetic analysis indicated that the <it>Vtg </it>gene family arose by duplication events, but that the pattern of duplication was different in each mosquito genera. Signatures of purifying selection were detected in <it>Culex</it>, <it>Aedes </it>and <it>Anopheles</it>. Gene conversion is a major driver of concerted evolution in <it>Culex</it>, while unequal crossover is likely the major driver of concerted evolution in <it>Anopheles</it>. In <it>Aedes</it>, smaller fragments have undergone gene conversion events.</p> <p>Conclusions</p> <p>The study shows concerted evolution and purifying selection shaped the evolution of mosquito <it>Vtg </it>genes following gene duplication. Additionally, similar evolutionary patterns were observed in the <it>Vtg </it>genes from other invertebrate and vertebrate organisms, suggesting that duplication, concerted evolution and purifying selection may be the major evolutionary forces driving <it>Vtg </it>gene evolution across highly divergent taxa.</p

Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species

Towards a method for cryopreservation of mosquito vectors of human pathogens

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes

Viral Paratransgenesis in the Malaria Vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae

Wolbachia Infections Are Virulent and Inhibit the Human Malaria Parasite Plasmodium Falciparum in Anopheles Gambiae

Endosymbiotic Wolbachia bacteria are potent modulators of pathogen infection and transmission in multiple naturally and artificially infected insect species, including important vectors of human pathogens. Anopheles mosquitoes are naturally uninfected with Wolbachia, and stable artificial infections have not yet succeeded in this genus. Recent techniques have enabled establishment of somatic Wolbachia infections in Anopheles. Here, we characterize somatic infections of two diverse Wolbachia strains (wMelPop and wAlbB) in Anopheles gambiae, the major vector of human malaria. After infection, wMelPop disseminates widely in the mosquito, infecting the fat body, head, sensory organs and other tissues but is notably absent from the midgut and ovaries. Wolbachia initially induces the mosquito immune system, coincident with initial clearing of the infection, but then suppresses expression of immune genes, coincident with Wolbachia replication in the mosquito. Both wMelPop and wAlbB significantly inhibit Plasmodium falciparum oocyst levels in the mosquito midgut. Although not virulent in non-bloodfed mosquitoes, wMelPop exhibits a novel phenotype and is extremely virulent for approximately 12–24 hours post-bloodmeal, after which surviving mosquitoes exhibit similar mortality trajectories to control mosquitoes. The data suggest that if stable transinfections act in a similar manner to somatic infections, Wolbachia could potentially be used as part of a strategy to control the Anopheles mosquitoes that transmit malaria