quantifying-fitness-costs-in-transgenic-aedes-aegypti-mosquitoes

Quantifying Fitness Costs in Transgenic <em>Aedes aegypti</em> Mosquitoes

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life ...

transgenic-manipulation-arthropod-vectors-tools-to-study-vector-borne

Transgenic Manipulation of Arthropod Vectors: Tools to Study Vector-Borne Diseases

Colorado State University

National Institutes of Health

We recently characterized Pfs47, a protein expressed on the surface of sexual stages and ookinetes of Plasmodium falciparum, as a malaria transmission-blocking vaccine (TBV) target. Mice immunization induced antibodies that conferred strong transmission-reducing activity (TRA) at a concentration of 200 μg/mL. Here, we sought to optimize the Pfs47 vaccine to elicit higher titers of high-affinity antibodies, capable of inducing strong TRA at a lower concentration. We report the development and evaluation of a Pfs47-based virus-like particle (VLP) vaccine generated by conjugating our 58 amino acid Pfs47 antigen to Acinetobacter phage AP205-VLP using the SpyCatcher:SpyTag adaptor system. AP205-Pfs47 complexes (VLP-P47) formed particles of ~22 nm diameter that reacted with polyclonal anti-Pfs47 antibodies, indicating that the antigen was accessible on the surface of the particle. Mice immunized with VLP-P47 followed by a boost with Pfs47 monomer induced significantly higher antibody titers, with higher binding affinity to Pfs47, than mice that received two immunizations with either VLP-P47 (VLP-P47/VLP-P47) or the Pfs47 monomer (P47/P47). Purified IgG from VLP-P47/P47 mice had strong TRA (83-98%) at concentrations as low as 5 μg/mL. These results indicate that conjugating the Pfs47 antigen to AP205-VLP significantly enhanced antigenicity and confirm the potential of Pfs47 as a TBV candidate.

Engineering a Virus-Like Particle as an Antigenic Platform for a Pfs47-Targeted Malaria Transmission-Blocking Vaccine.

The mosquito vector  transmits arthropod-borne viruses (arboviruses) of medical importance, including Zika, dengue, and yellow fever viruses. Controlling mosquito populations remains the method of choice to prevent disease transmission. Novel mosquito control strategies based on genetically manipulating mosquitoes are being developed as additional tools to combat arbovirus transmission. Genetic control of mosquitoes includes two basic strategies: population suppression and population replacement. The former aims to eliminate mosquito populations while the latter aims to replace wild populations with engineered, pathogen-resistant mosquitoes. In this review, we outline suppression strategies being applied in the field, as well as current antiviral effector genes that have been characterized and expressed in transgenic  for population replacement. We discuss cutting-edge gene drive technologies that can be used to enhance the inheritance of effector genes, while highlighting the challenges and opportunities associated with gene drives. Finally, we present currently available models that can estimate mosquito release numbers and time to transgene fixation for several gene drive systems. Based on the recent advances in genetic engineering, we anticipate that antiviral transgenic  exhibiting gene drive will soon emerge; however, close monitoring in simulated field conditions will be required to demonstrate the efficacy and utility of such transgenic mosquitoes.

Antiviral Effectors and Gene Drive Strategies for Mosquito Population Suppression or Replacement to Mitigate Arbovirus Transmission by .

The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015-2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic  engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito's siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands ( = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that  innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.

The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic .

Tsetse flies are vectors of parasitic African trypanosomes, the etiological agents of human and animal African trypanosomoses. Current disease control methods include fly-repelling pesticides, fly trapping, and chemotherapeutic treatment of infected people and animals. Inhibiting tsetse's ability to transmit trypanosomes by strengthening the fly's natural barriers can serve as an alternative approach to reduce disease. The peritrophic matrix (PM) is a chitinous and proteinaceous barrier that lines the insect midgut and serves as a protective barrier that inhibits infection with pathogens. African trypanosomes must cross tsetse's PM in order to establish an infection in the fly, and PM structural integrity negatively correlates with trypanosome infection outcomes. Bloodstream form trypanosomes shed variant surface glycoproteins (VSG) into tsetse's gut lumen early during the infection establishment, and free VSG molecules are internalized by the fly's PM-producing cardia. This process results in a reduction in the expression of a tsetse microRNA (miR275) and a sequential molecular cascade that compromises PM integrity. miRNAs are small non-coding RNAs that are critical in regulating many physiological processes. In the present study, we investigated the role(s) of tsetse miR275 by developing a paratransgenic expression system that employs tsetse's facultative bacterial endosymbiont, Sodalis glossinidius, to express tandem antagomir-275 repeats (or miR275 sponges). This system induces a constitutive, 40% reduction in miR275 transcript abundance in the fly's midgut and results in obstructed blood digestion (gut weights increased by 52%), a significant increase (p-value < 0.0001) in fly survival following infection with an entomopathogenic bacteria, and a 78% increase in trypanosome infection prevalence. RNA sequencing of cardia and midgut tissues from paratransgenic tsetse confirmed that miR275 regulates processes related to the expression of PM-associated proteins and digestive enzymes as well as genes that encode abundant secretory proteins. Our study demonstrates that paratransgenesis can be employed to study microRNA regulated pathways in arthropods that house symbiotic bacteria.

Paratransgenic manipulation of a tsetse microRNA alters the physiological homeostasis of the fly's midgut environment.

 mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, including Zika (ZIKV), dengue (DENV), and yellow fever (YFV). Vector competence studies with  often involve challenging mosquitoes with an artificial bloodmeal containing virus and later quantifying viral titer or infectious plaque-forming units (PFU) in various mosquito tissues at relevant time points post-infection. However,  mosquitoes are known to exhibit midgut infection and escape barriers (MIB and MEB, respectively), which influence the prevalence and titer of a disseminated infection and can introduce unwanted variability into studies analyzing tissues such as the salivary glands. To surmount this challenge, we describe herein a protocol for the intrathoracic inoculation of ZIKV in  This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut, or are analyzing salivary gland infection or escape barriers. Graphic abstract:  Figure made with Biorender.com.

Intrathoracic Inoculation of Zika Virus in .

The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from  studies to maintain the integrity of the germline genome by silencing transposable elements (TE).  mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of  Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3' ends of piRNAs, bound to mature (3' 2' O-methylated) and unmethylated RNAs with similar micromolar affinities (K = 1.7 ± 0.8 μM and K of 5.0 ± 2.2 μM, respectively;  = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.

 Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization.

Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.

Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology.

Studying protein localization in mosquito salivary glands provides novel insights on the function and physiological relevance of salivary proteins and also provides an avenue to study interactions between mosquitoes and pathogens. Salivary proteins display compartmentalization. For example, proteins involved in blood feeding are stored in the medial and distal lateral lobes, whereas proteins related to sugar metabolism localize to the proximal portion of the lateral lobes. Immunohistochemistry assays use antibodies raised against recombinant salivary proteins to reveal the protein localization and interactions within the tissue. In this assay, permeabilization of the salivary glands allows the antibodies to enter the cells and bind their target proteins. The primary antibody-antigen complexes are later marked with fluorescently labeled secondary antibodies. Antibodies that recognize pathogen-specific proteins can also be incorporated in these assays, providing information about pathogen localization within the salivary glands or pathogen interactions with mosquito salivary proteins. Here, we introduce immunohistochemistry assays for use in mosquito salivary glands.

Performing Immunohistochemistry in Mosquito Salivary Glands.

Immunohistochemistry is a valuable technique that provides information on protein localization and interactions in tissues. Mosquito salivary gland immunohistochemistry requires the meticulous dissection of a delicate tissue. The integrity of the salivary glands must be closely monitored throughout the entire process to prevent structural damage and loss of saliva. This protocol describes a series of simple steps to perform salivary gland immunohistochemistry including tissue dissection, permeabilization, immunostaining, mounting, and imaging by confocal microscopy.

A Simple Method for Immunohistochemistry and Imaging of Mosquito Salivary Glands.

The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika viruses. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus is amenable to both gene drive and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target 2 intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of 3 promoters (nanos, β2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single-generation crossings, measured maternal effects, and assessed fitness costs associated with various transgenic lines to model the rate of gene drive fixation. Overall, MGDrivE modeling suggested that when an autonomous gene drive is placed into an intergenic locus, the gene drive system will eventually be blocked by the accrual of gene drive blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous gene drive, it was the fitness of the gene drive line that most strongly influenced the persistence of the gene drive in the simulated population. As such, we propose that when autonomous CRISPR/Cas9-based gene drive systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as gene drive-blocking indels accrue, the gene drive becomes exhausted due to the fixation of CRISPR resistance alleles.

Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling.

Hematophagous arthropods are animals that feed on vertebrate blood for egg production. Mosquitoes must pierce the host skin, locate blood vessels, and extract blood without being noticed. Mosquito stylets lacerate host tissues, triggering the activation of the three branches of hemostasis, or stopping of blood flow: vasoconstriction, platelet aggregation, and coagulation. Mosquitoes inject saliva into the host skin during their intradermal search for blood (also called probing), and salivary proteins counteract hemostasis. Blood feeding dynamics have been traditionally described by observational studies, in which researchers using magnifying glasses watched mosquitoes in the act of blood feeding. These studies provided the foundation for protocols to evaluate mosquito blood feeding in a more quantitative manner. Here, we introduce mosquito blood feeding biology with a focus on the feeding steps, which include penetration, probing, and feeding. Understanding blood feeding dynamics is crucial for evaluating probing time and other relevant parameters derived from blood feeding, such as blood meal size, fecundity, and fertility. Other considerations, including the relationship between probing and pathogen transmission and novel technologies to address blood feeding, are also discussed.

Determination of Mosquito Probing and Feeding Time to Evaluate Mosquito Blood Feeding.

Female mosquitoes need vertebrate blood for egg development. Evaluating mosquito behavior is essential for determining the ability of a mosquito to blood feed. Blood feeding experiments are often performed using artificial membrane feeders; however, such experiments do not represent realistic scenarios in which a mosquito injects saliva into the host to prevent host hemostatic responses. Vertebrate animal models are therefore more representative of a natural blood feeding event. Here, we describe a methodology to evaluate mosquito blood feeding success that can be used to compare blood feeding between mosquito groups-for instance, wild-type versus transgenic mosquitoes lacking salivary proteins or field-collected versus laboratory-reared mosquitoes. We also include a simple procedure to measure blood meal size, allowing for a more quantitative assessment of feeding status. The volume of ingested blood directly affects mosquito fecundity and fertility, important markers of fitness. The methods described herein can be used to evaluate transmission-blocking vaccines, insecticides, or fitness costs associated with transgenic mosquitoes.

Evaluation of  Feeding Status.

In mosquitoes, the intradermal search for vertebrate blood (probing time) corresponds to the time taken from initial insertion of the mouthparts in the skin until visualization of the initial engorgement of blood in the midgut. Probing time evaluation provides useful information on the ability of a mosquito to initiate successful blood feeding. In this protocol, we describe how to determine feeding parameters in , a widely distributed mosquito that transmits several deadly pathogens, including yellow fever, dengue, Zika, and Chikungunya viruses. We focus on the different steps of a blood feeding event, including penetration, probing, interprobing, and feeding time. Penetration time corresponds to the insertion of the stylets into the host skin and usually lasts <10 sec. Probing time or intradermal search for blood involves saliva secretion into the skin. Some researchers group penetration and probing time as the exploratory phase for blood. Feeding time is an active phase of blood ingestion and engorgement. Feeding parameters depend on mosquito behaviors and these measurements are visually taken by the investigator. We include a video that provides a close look at a mosquito feeding event in which penetration, probing, and feeding times can be observed. To record these experimental times, one must closely watch the mosquito feeding behavior including stylet penetration in the host skin, visualization of the first traces of blood in the midgut, engorgement of the midgut, and removal of stylets from the skin.

ArticleTotal : 2	Year
Publication title Cited by 3	2023
Publication title	2023

Article	Year
Engineering a Virus-Like Particle as an Antigenic Platform for a Pfs47-Targeted Malaria Transmission-Blocking Vaccine. Scientific reports\| PubMed ID: 31727945
Antiviral Effectors and Gene Drive Strategies for Mosquito Population Suppression or Replacement to Mitigate Arbovirus Transmission by . Insects\| PubMed ID: 31940960
The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic . Viruses\| PubMed ID: 33142991	2020
Paratransgenic manipulation of a tsetse microRNA alters the physiological homeostasis of the fly's midgut environment. PLoS pathogens\| PubMed ID: 34107000	2021
Intrathoracic Inoculation of Zika Virus in . Bio-protocol\| PubMed ID: 34692914	2021
Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization. International journal of molecular sciences\| PubMed ID: 34884537	2021
Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology. Cell reports\| PubMed ID: 35417706	2022
Performing Immunohistochemistry in Mosquito Salivary Glands. Cold Spring Harbor protocols\| PubMed ID: 35960615	2022
A Simple Method for Immunohistochemistry and Imaging of Mosquito Salivary Glands. Cold Spring Harbor protocols\| PubMed ID: 35960629	2022
Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling. G3 (Bethesda, Md.)\| PubMed ID: 36250791	2022
Determination of Mosquito Probing and Feeding Time to Evaluate Mosquito Blood Feeding. Cold Spring Harbor protocols\| PubMed ID: 36669861	2023
Evaluation of Feeding Status. Cold Spring Harbor protocols\| PubMed ID: 36690389	2023
Evaluation of Penetration, Probing, and Feeding Times on Mice. Cold Spring Harbor protocols\| PubMed ID: 36690390	2023

Adeline E. Williams

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