Transgenic Mosquitoes Research Articles

Studies of Transgenic Mosquitoes in Disease-Endemic Countries: Preparation of Containment Facilities.

Novel approaches to area-wide control of vector species offer promise as additional tools in the fight against vectored diseases. Evaluation of transgenic insect strains aimed at field population control in disease-endemic countries may involve international partnerships and should be done in a stepwise approach, starting with studies in containment facilities. The preparations of both new-build and renovated facilities are described, including working with local and national regulations regarding land use, construction, and biosafety requirements, as well as international guidance to fill any gaps in regulation. The examples given are for containment categorization at Arthropod Containment Level 2 for initial facility design, classification of wastes, and precautions during shipping. Specific lessons were derived from preparations to evaluate transgenic (non-gene drive) mosquitoes in West and East African countries. Documented procedures and the use of a non-transgenic training strain for trial shipments and culturing were used to develop competence and confidence among the African facility staff, and along the chain of custody for transport. This practical description is offered to support other research consortia or institutions preparing containment facilities and operating procedures in conditions where research on transgenic insects is at an early stage.

Maintaining Quality of Candidate Strains of Transgenic Mosquitoes for Studies in Containment Facilities in Disease Endemic Countries.

Transgenic mosquitoes are being developed as novel components of area-wide approaches to vector-borne disease control. Best practice is to develop these in phases, beginning with laboratory studies, before moving to field testing and inclusion in control programs, to ensure safety and prevent costly field testing of unsuitable strains. The process of identifying and developing good candidate strains requires maintenance of transgenic colonies over many generations in containment facilities. By working in disease endemic countries with target vector populations, laboratory strains may be developed and selected for properties that will enhance intended control efficacy in the next phase, while avoiding traits that introduce unnecessary risks. Candidate strains aiming toward field use must consistently achieve established performance criteria, throughout the process of scaling up from small study colonies to production of sufficient numbers for field testing and possible open release. Maintenance of a consistent quality can be demonstrated by a set of insect quality and insectary operating indicators, measured over time at predetermined intervals. These indicators: inform comparability of studies using various candidate strains at different times and locations; provide evidence of conformity relevant to compliance with terms of approval for regulated use; and can be used to validate some assumptions related to risk assessments covering the contained phase and for release into the environment.

Population modification of Anopheline species to control malaria transmission

Vector control strategies based on population modification of Anopheline mosquitoes may have a significant role in the malaria eradication agenda. They could consolidate elimination gains by providing barriers to the reintroduction of parasites and competent vectors, and allow resources to be allocated to new control sites while maintaining treated areas free of malaria. Synthetic biological approaches are being used to generate transgenic mosquitoes for population modification. Proofs-of-principle exist for mosquito transgenesis, the construction of anti-parasite effector genes and gene-drive systems for rapidly introgressing beneficial genes into wild populations. Key challenges now are to develop field-ready strains of mosquitoes that incorporate features that maximize safety and efficacy, and specify pathways from discovery to development. We propose three pathways and a framework for target product profiles that maximize safety and efficacy while meeting the demands of the complexity of malaria transmission, and the regulatory and social diversity of potential end-users and stakeholders.

The bacterium Wolbachia exploits host innate immunity to establish a symbiotic relationship with the dengue vector mosquito Aedes aegypti

A host’s immune system plays a central role in shaping the composition of the microbiota and, in return, resident microbes influence immune responses. Symbiotic associations of the maternally transmitted bacterium Wolbachia occur with a wide range of arthropods. It is, however, absent from the dengue and Zika vector mosquito Aedes aegypti in nature. When Wolbachia is artificially forced to form symbiosis with this new mosquito host, it boosts the basal immune response and enhances the mosquito’s resistance to pathogens, including dengue, Zika virus and malaria parasites. The mechanisms involved in establishing a symbiotic relationship between Wolbachia and A. aegypti, and the long-term outcomes of this interaction, are not well understood. Here, we have demonstrated that both the immune deficiency (IMD) and Toll pathways are activated by the Wolbachia strain wAlbB upon its introduction into A. aegypti. Silencing the Toll and IMD pathways via RNA interference reduces the wAlbB load. Notably, wAlbB induces peptidoglycan recognition protein (PGRP)-LE expression in the carcass of A. aegypti, and its silencing results in a reduction of symbiont load. Using transgenic mosquitoes with stage-specific induction of the IMD and Toll pathways, we have shown that elevated wAlbB infection in these mosquitoes is maintained via maternal transmission. These results indicate that host innate immunity is utilized to establish and promote host-microbial symbiosis. Our results will facilitate a long-term projection of the stability of the Wolbachia–A. aegypti mosquito system that is being developed to control dengue and Zika virus transmission to humans.

Effect of interruption of over‐flooding releases of transgenic mosquitoes over wild population of Aedes aegypti: two case studies in Brazil

AbstractThe number of mosquito populations resistant to insecticides is increasing along with the reemerging of vector‐borne diseases. New technologies are under evaluation to complement the strategies used against these mosquitoes. Transgenic mosquitoes are one approach that some countries are considering and they are being evaluated to control the wild population. Although they have achieved success in population suppression of Aedes aegypti (L.) (Diptera: Culicidae), these studies have not demonstrated what the outcomes are when releases are interrupted (ceased). In this study, after demonstrating suppression of Ae. aegypti using transgenic technology, changes in the spatial distribution of the infestation and the abundance of the vector Ae. aegypti were assessed in the post‐release period, along with fluctuation of transgenic mosquitoes in two areas of Brazil. In both pilot trials, there was an average suppression of ca. 70% of the wild population due to the release of transgenic males compared to the pre‐release period. In Juazeiro (Mandacaru), in the post‐release phase, the number of eggs per trap ranged between 0.06 and 14.41 (mean ± SE = 4.44 ± 0.44), and the ovitrap index (OI = number of ovitraps with eggs/total number of ovitraps recovered) ranged from 0.01 to 0.43 (0.13 ± 0.01). In Jacobina (Pedra Branca), during the post‐release phase, the number of eggs per trap ranged between 1 and 7.2 (1.72 ± 0.72), and the OI ranged from 1 to 0.83 (0.095 ± 0.032). The mosquito population in Juazeiro (Mandacaru) remained suppressed for 17 weeks after the release interruption, whereas in Jacobina (Pedra Branca) suppression lasted 32 weeks. In Juazeiro, transgenic larvae were detected up to 5 months after the interruption of the over‐flooding releases of transgenic males. In Jacobina, they were found up to 2 months after the release interruption. The number of eggs collected increased 4–5 months after the release interruption, which indicated that the Ae. aegypti population had been re‐established after the interruption of releases. The results demonstrate that the technique requires a continuous release in the treated areas, and after suppression, the release rate can be decreased and used as a barrier against external migration.

MicroRNA-275 targets sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) to control key functions in the mosquito gut.

The yellow fever mosquito Aedes aegypti is the major vector of arboviruses, causing numerous devastating human diseases, such as dengue and yellow fevers, Chikungunya and Zika. Female mosquitoes need vertebrate blood for egg development, and repeated cycles of blood feeding are tightly linked to pathogen transmission. The mosquito’s posterior midgut (gut) is involved in blood digestion and also serves as an entry point for pathogens. Thus, the mosquito gut is an important tissue to investigate. The miRNA aae-miR-275 (miR-275) has been shown to be required for normal blood digestion in the female mosquito; however, the mechanism of its action has remained unknown. Here, we demonstrate that miR-275 directly targets and positively regulates sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase, which is implicated in active transport of Ca2+ from the cytosol to the sarco/endoplasmic reticulum. We utilized a combination of the gut-specific yeast transcription activator protein Gal4/upstream activating sequence (Gal4/UAS) system and miRNA Tough Decoy technology to deplete the endogenous level of miR-275 in guts of transgenic mosquitoes. This gut-specific reduction of miR-275 post blood meal decreased SERCA mRNA and protein levels of the digestive enzyme late trypsin. It also resulted in a significant reduction of gut microbiota. Moreover, the decrease of miR-275 and SERCA correlated with defects in the Notch signaling pathway and assembly of the gut actin cytoskeleton. The adverse phenotypes caused by miR-275 silencing were rescued by injections of miR-275 mimic. Thus, we have discovered that miR-275 directly targets SERCA, and the maintenance of its level is critical for multiple gut functions in mosquitoes.

Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan

Presently three major groups of proteins from Aedes aegypti, cadherin, alkaline phosphatases (ALP) and aminopeptidases N (APN), have been identified as Cry11Aa toxin receptors. To further characterize their role on toxicity, transgenic mosquitoes with silenced Aedes cadherin expression were previously generated and the role of cadherin in mediating the toxicity of four different mosquitocidal toxins (Cry11Aa, Cry11Ba, Cry4Aa and Cry4Ba) was demonstrated. Here, we investigated the role of another reported Cry11Aa receptor, ALP1. As with Aedes cadherin, this protein is localized in the apical cell membrane of distal and proximal gastric caecae and the posterior midgut. We also successfully generated transgenic mosquitoes that knockdowned ALP1 transcript levels using an inducible Aedes heat shock promoter, Hsp70A driving dsALP1RNA. Four different mosquitocidal toxins were used for larval bioassays against this transgenic mosquito. Bioassay results show thatCry11Aa toxicity to these transgenic larvae following a heat shock decreased (4.4 fold) and Cry11Ba toxicity is slightly attenuated. But Cry4Aa and Cry4Ba toxicity to ALP1 silenced larvae is unchanged. Without heat shock, toxicity of all four toxins does not change, suggesting this heat shock promoter is heat-inducible. Notably, transgenic mosquitoes with ALP1 knockdown are about 3.7 times less resistant to Cry11Aa toxin than those with Aedes cadherin knockdown. These results demonstrate that the ALP1 is an important secondary receptor for Cry11Aa and Cry11Ba, but it might not be involved in Cry4Aa and Cry4Ba toxicity.

Why is Aedes aegypti Linnaeus so Successful as a Species?

Diseases transmitted by mosquitoes impose enormous burden towards human morbidity and mortality. Over the last three decades, Brazil has suffered from severe Dengue epidemics. In September 2014, this situation is further complicated by the introduction of two other viruses, Zika and Chikungunya, placing Brazil in a triple epidemic. In this article, we discuss the biology of Aedes aegypti Linnaeus, and the principal initiatives currently used to control mosquito populations and the diseases they transmit. Aedes aegypti has broad global distribution and is involved in the transmission of various arboviral diseases such as Dengue, Zika, and Chikungunya. Several factors contribute to the success of the species, particularly behavioral plasticity, rapid development, desiccation-resistant eggs, resistance to the principle insecticide classes currently available on the market, preference for the urban environment, and proximity to humans. Vector control programs are the best way to reduce the burden of mosquito-borne diseases. Chemical control is most commonly used in recent times, and unfortunately, the results have not been satisfactory but instead, there is increased vector dispersal and, subsequently, the spread of disease epidemics. Investigations of alternative control methods such as release of Wolbachia-infected mosquitoes for blocking vector-borne pathogens, release of transgenic mosquitoes carrying a lethal gene for offspring, and the use of insecticide-dispersing mosquitoes are under way in Brazil, and some have shown promising results. Special emphasis should be placed on integrated management of all available tactics, so as to maximize efforts towards mosquito control. Finally, we emphasize that continuous actions and community participation control initiatives are critically important for success.

Zika Virus and Transgenic Mosquitoes: Potentials of a Public Health Tool

<p>With transgenic mosquitoes now being deployed as a major solution to the pandemic Zika virus disease, more curious questions have arisen about this public health technology. This essay reviews the philosophical considerations that presaged the development, by WHO, of the genetically modified mosquitoes. It focuses more specifically on the inter-disciplinary approach in formulating the clinical trials process. While it is still early to determine the full merits of this health intervention tool, it is nonetheless timely to examine the rich vein of form that constitutes the clinical trial protocol. I argue that the steps it suggests should be a gold standard for all clinical trial procedures including those that use human subjects and those with both direct and indirect environmental implications. Also, the early signs portend that its application could indeed be widespread. For instance, malaria and dengue fever were the original targets, but now the development of transgenic mosquito technology is deemed a potent option in the fight against Zika virus disease. In addition, this clinical trial protocol has remarkably gone beyond the four pillars of principlism to recognize the possibility that a new technology (such as GMM), can have the potential to simultaneously impact human individuals, the community and the ecosystem in tandem. In “Guidance framework for testing of genetically modified mosquito,” the WHO has set an example that calls for emulation. With the right awareness, biomedical researchers, scholars, policy makers, and students stand to benefit immensely from this reformulated approach.</p>

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites.

Responsible Innovation and political accountability: genetically modified mosquitoes in Brazil

ABSTRACTIn this paper, we analyse the introduction of genetically modified (GM) mosquitoes in Brazil and use this case to probe the notion of Responsible Innovation and its applicability to the development of new public health biotechnologies in the global South. OX513A, a strain of GM Aedes aegypti mosquitoes developed by the British firm Oxitec, has been used experimentally in Brazil since 2009, when it was imported into the country as a promising new tool in the fight against dengue. We discuss the regulatory history of OX513A in Brazil, as well as the forms of ‘community engagement’ that have accompanied the release of transgenic mosquitoes. We argue that the conduct of a scientific research project is only part of a broader effort to localise insect biotechnology in Brazil, an effort that has enjoyed very visible support from political authorities across the country. We conclude by arguing that if the framework of Responsible Innovation is to have purchase on this sort of transnational and multifaceted innovation trajectory, it has to include at its centre a strong notion of political accountability.

The Anopheles FBN9 immune factor mediates Plasmodium species-specific defense through transgenic fat body expression

Mosquitoes have a multifaceted innate immune system that is actively engaged in warding off various pathogens, including the protozoan malaria parasite Plasmodium. Various immune signaling pathways and effectors have been shown to mediate a certain degree of defense specificity against different Plasmodium species. A key pattern recognition receptor of the Anopheles gambiae immune system is the fibrinogen domain-containing immunolectin FBN9, which has been shown to be transcriptonally induced by Plasmodium infection, and to mediate defense against both rodent and human malaria parasites and bacteria. Here we have further studied the defense specificity of FBN9 using a transgenic approach, in which FBN9 is overexpressed in the fat body tissue after a blood meal through a vitellogenin promoter. Interestingly, the Vg-FBN9 transgenic mosquitoes showed increased resistance only to the rodent parasite P. berghei, and not to the human parasite P. falciparum, pointing to differences in the mosquito's defense mechanisms against the two parasite species. The Vg-FBN9 transgenic mosquitoes were also more resistant to infection with both Gram-positive and Gram-negative bacteria and showed increased longevity when infected with P. berghei. Our study points to the importance of both experimentally depleting and enriching candidate anti-Plasmodium effectors in functional studies in order to ascertain their suitability for the development of transgenic mosquito–based malaria control strategies.

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells.

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.

Use of transgenic Aedes aegypti in Brazil: risk perception and assessment.

The OX513A strain of Aedes aegypti, which was developed by the British company Oxitec, expresses a self-limiting transgene that prevents larvae from developing to adulthood. In April 2014, the Brazilian National Technical Commission on Biosafety completed a risk assessment of OX513A and concluded that the strain did not present new biological risks to humans or the environment and could be released in Brazil. At that point, Brazil became the first country to approve the unconstrained release of a genetically modified mosquito. During the assessment, the commission produced a comprehensive list of – and systematically analysed – the perceived hazards. Such hazards included the potential survival to adulthood of immature stages carrying the transgene – should the transgene fail to be expressed or be turned off by exposure to sufficient environmental tetracycline. Other perceived hazards included the potential allergenicity and/or toxicity of the proteins expressed by the gene, the potential for gene flow or increased transmission of human pathogens and the occupation of vacant breeding sites by other vector species. The Zika epidemic both elevated the perceived importance of Ae. aegypti as a vector – among policy-makers and regulators as well as the general public – and increased concerns over the release of males of the OX513A strain. We have therefore reassessed the potential hazards. We found that release of the transgenic mosquitoes would still be both safe and of great potential value in the control of diseases spread by Ae. aegypti, such as chikungunya, dengue and Zika.

Nanoparticles for mosquito control: Challenges and constraints

Mosquito control programs are facing important and timely challenges, including the recent outbreaks of novel arbovirus, the development of resistance in several Culicidae species, and the rapid spreading of highly invasive mosquitoes worldwide. Current control tools mainly rely on the employment of (i) synthetic or microbial pesticides, (ii) insecticide-treated bed nets, (iii) adult repellents, (iv) biological control agents against mosquito young instars (mainly fishes, amphibians and copepods) (v) Sterile Insect Technique (SIT), (vi) “boosted SIT”, (vii) symbiont-based methods and (viii) transgenic mosquitoes. Currently, none of these single strategies is fully successful. Novel eco-friendly strategies to manage mosquito vectors are urgently needed. The plant-mediated fabrication of nanoparticles is advantageous over chemical and physical methods, since it is cheap, single-step, and does not require high pressure, energy, temperature, or the use of highly toxic chemicals. In the latest years, a growing number of plant-borne compounds have been proposed for efficient and rapid extracellular synthesis of metal nanoparticles effective against mosquitoes at very low doses (i.e. 1–30ppm). In this review, we focused on the promising potential of green-fabricated nanoparticles as toxic agents against mosquito young instars, and as adult oviposition deterrents. Furthermore, we analyzed current evidences about non-target effects of these nanocomposites used for mosquito control, pointing out their moderate acute toxicity for non-target aquatic organisms, absence of genotoxicity at the doses tested against mosquitoes, and the possibility to boost the predation rates of biological control agents against mosquitoes treating the aquatic environment with ultra-low doses (e.g. 1–3ppm) of green-synthesized nanoparticles, which reduce the motility of mosquito larvae. Challenges for future research should shed light on (i) the precise mechanism(s) of action of green-fabricated metal nanoparticles, (ii) their fate in the aquatic environment, and (iii) the possible toxicity of residual silver ions in the aquatic ecosystems, (iv) the standardization of chemical composition of botanical products used as sources of reducing and capping metabolites, (v) the optimization of the green nanosynthetic routes, in order to develop large-scale production of eco-friendly nanomosquitocides.

Determination of juvenile hormone titers by means of LC-MS/MS/MS and a juvenile hormone-responsive Gal4/UAS system in Aedes aegypti mosquitoes

In anautogenous mosquitoes, juvenile hormone III (JH) plays an essential role in female post-eclosion (PE) development, preparing them for subsequent blood feeding and egg growth. We re-examined the JH titer during the reproductive cycle of female Aedes aegypti mosquitoes. Using liquid chromatography coupled with triple tandem mass spectrometry (LC-MS/MS/MS), we have shown that it reaches its peak at 48–54 h PE in the female hemolymph and at 72 h PE in whole body extracts. This method represents an effective assay for determination of JH titers. The 2.1-kb 5’ promoter region of the Early Trypsin (ET) gene, which is specifically expressed in the female midgut under the control of JH during the PE phase, was utilized to genetically engineer the Ae. aegypti mosquito line with the ET-Gal4 activator. We then established the ET-GAL4>UAS-enhanced green fluorescent protein (EGFP) system in Ae. aegypti. In ET-Gal4>UAS-EGFP female mosquitoes, the intensity of the midgut-specific EGFP signal was observed to correspond to the ET gene transcript level and follow the JH titer during the PE phase. The EGFP signal and the EGFP transcript level were significantly diminished in midguts of transgenic female mosquitoes after RNA interference depletion of the JH receptor Methoprene-tolerant (Met), providing evidence of the control of ET gene expression by Met. Topical JH application caused premature enhancement of the EGFP signal and the EGFP transcript level in midguts of newly eclosed ET-Gal4>UAS-EGFP female mosquitoes, in which endogenous JH titer is still low. Hence, this novel ET-Gal4>UAS system permits JH-dependent gene overexpression in the midgut of Ae. aegypti female mosquitoes prior to a blood meal.

A review on the incidence, interaction, and future perspective on Zika virus

Zika virus (ZIKV) belongs to the family Flaviviridae and genus Flavivirus. It is a single‑stranded positive‑sense ribonucleic acid (RNA) virus, has its origin traced to Zika forest in Uganda. Its infection leads to ZIKV fever, characterized by arthralgia, myalgia, rash, conjunctivitis, and asthenia. Clinical presentation of the infection is nonspecific and may often be confused with symptoms of other flaviviral diseases (dengue, West Nile [WN], and chikungunya). Recently, ZIKV has been associated with congenital malformations and neurological complications such as microcephaly and Guillain–Barre’ syndrome. The viral tropism revealed an infection of the skin fibroblasts, keratinocytes, and immature dendritic cells through enhanced expression of dendritic cell‑specific intracellular adhesion molecule 3‑grabbing nonintegrin or anexelecto (Greekword: 'uncontrolled’) and tyrosine protein kinase receptor 3 systems. Silencing of T‑cell immunoglobulin (Ig) and mucin domain 1 (TIM ‑1 ) and AXL RNAs has shown blockage of viral entry through their anti‑TIM ‑1 and anti‑AXL antibodies, hence serving as a potential target for ZIKV drug development.Biotechnological approaches targeted toward ZIKV vector control include the development of transgenic mosquitoes to disrupt the genome pool of wild strains and use of an endosymbiotic bacterium to prevent replication of arboviruses within its vector. Other approaches include the use of gene drive and exploration of the genetic redundancy to disrupt the receptors used by the virus to gain entry into its host. It is also imperative to explore the modality through which neutralizing antibodies block this viral infection as this may prove as a potential target to arrest the viral life cycle. KEY WORDS: Flavivirus, microcephaly and Guillain–Barre’ syndrome, mosquito, receptors, Zika virus

Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi

Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

Periodic patterns and Pareto efficiency of state dependent impulsive controls regulating interactions between wild and transgenic mosquito populations

It is conceivable that genetically modified mosquitoes could stop the spread of malaria, by outcompeting the wild mosquitoes and interfering with their reproductive processes, and genetically inheriting and further transmitting a diminished potential to carry Plasmodium. To get insight into the possible outcomes, we formulate an ODE model for the interactions between wild and transgenic mosquito populations, which is subject to state-dependent impulsive perturbations. By first investigating the dynamics of the unperturbed system, we determine certain sufficient conditions for the existence and orbital stability of positive order-1 solution of the model system with state-dependent impulsive perturbations. Their feasibility is then illustrated by means of numerical simulations. In addition, to adequately control the wild mosquito population, we use a multi-target approach which, in addition to accounting for the total costs, keeps track of the total size of the wild mosquito population. To trade off these objectives, we consider the concept of Pareto efficiency to determine suitable control strategies which are near-optimal. Finally, we carry out numerical simulations to illustrate the Pareto frontier and then characterize the detailed Pareto efficient control regime.

Paratransgenesis: a promising new strategy for mosquito vector control.

The three main mosquito genera, Anopheles, Aedes and Culex, transmit respectively malaria, dengue and lymphatic filariasis. Current mosquito control strategies have proved unsuccessful, and there still is a substantial number of morbidity and mortality from these diseases. Genetic control methods have now arisen as promising alternative strategies, based on two approaches: the replacement of a vector population by disease-refractory mosquitoes and the release of mosquitoes carrying a lethal gene to suppress target populations. However, substantial hurdles and limitations need to be overcome if these methods are to be used successfully, the most significant being that a transgenic mosquito strain is required for every target species, making genetically modified mosquito strategies inviable when there are multiple vector mosquitoes in the same area. Genetically modified bacteria capable of colonizing a wide range of mosquito species may be a solution to this problem and another option for the control of these diseases. In the paratransgenic approach, symbiotic bacteria are genetically modified and reintroduced in mosquitoes, where they express effector molecules. For this approach to be used in practice, however, requires a better understanding of mosquito microbiota and that symbiotic bacteria and effector molecules be identified. Paratransgenesis could prove very useful in mosquito species that are inherently difficult to transform or in sibling species complexes. In this approach, a genetic modified bacteria can act by: (a) causing pathogenic effects in the host; (b) interfering with the host’s reproduction; (c) reducing the vector’s competence; and (d) interfering with oogenesis and embryogenesis. It is a much more flexible and adaptable approach than the use of genetically modified mosquitoes because effector molecules and symbiotic bacteria can be replaced if they do not achieve the desired result. Paratransgenesis may therefore become an important integrated pest management tool for mosquito control.