CRISPR-based gene drive (GD) systems bias allele inheritance during meiosis, enabling transgenes to spread at rates exceeding Mendel's law of segregation. This capability underlies their potential as powerful tools for controlling mosquito-borne diseases. GDs can be engineered either to suppress mosquito populations or to modify them by introducing traits that block pathogen transmission. Recent advances have focused on improving evolutionary stability, with modeling studies providing insights into expected population dynamics. With a focus on the most current population modification GDs, we discuss advances in GD architectures - including integral and allelic drives, combined modification-suppression systems, and both homing and non-homing toxin-antidote designs - that expand the range of possible strategies and address limitations of early homing drives. Numerous antipathogen effectors with strong pathogen-blocking activity can now be coupled to these systems, with current efforts assessing their durability against genetically diverse pathogens. Key challenges remain, including resistance evolution, ecological impacts, and long-term stability. Nonetheless, GDs offer a promising approach for reducing disease transmission, especially in regions where conventional interventions are difficult to sustain.

Insect larval pigmentation is a key aspect of their ecology and development and often involves complex biochemical pathways. While carotenoids and flavonoids are well-studied contributors, the molecular mechanisms underlying biliverdin-associated pigmentation, responsible for striking blue-green hues in many insects, remain largely unknown. The Chinese oak silkworm, Antheraea pernyi, exhibits remarkable natural variation in larval epidermal coloration across strains, making it an excellent model for insect pigmentation. Here we report the discovery of six biliverdin-binding protein (BBP) genes (ApBBP1-6) in A. pernyi. Integrated transcriptomic and qRT-PCR analyses show that ApBBP1 gene activity is uniquely predominant in the larval epidermis, midgut, and fat body of the blue-skinned Lan strain but completely absent in the white-skinned Bai strain. Genomic comparisons revealed an ∼3.9 kb deletion in the Bai ApBBP1 locus that results in 214 bp truncation in the coding sequence. Cas9/sgRNA-mediated gene ablations of the Lan ApBBP1 locus resulted in significantly reduced ApBBP1 mRNA levels concomitant with a decreased biliverdin content in larval epidermis confirmed by metabolite profiling. The ablation converted the characteristic Lan strain blue larval skin to white. This work resolves a long-standing question regarding the molecular basis of biliverdin-derived coloration in insects and establishes A. pernyi as a powerful model for investigating the evolution and genetic regulation of complex pigmentary traits. Identification of ApBBP1 creates opportunities for exploring the conservation of this mechanism across Lepidoptera and the potential for engineering insect pigmentation.

Malaria remains a significant global health challenge with an estimated 282 million cases reported in 2024. CRISPR/Cas9-based gene-drive systems have emerged as promising tools to block Plasmodium transmission by mosquito vectors. The TP13 drive system targets the Anopheles gambiae cardinal (Agcd) gene and carries two engineered monoclonal antibodies to achieve rapid population modification to prevent parasite transmission. Previous cage trials demonstrated complete drive introduction in three to six generations and supported modeling predicting a potential >90% reduction in malaria incidence under optimal conditions. However, naturally-occurring genetic polymorphisms in wild mosquito populations, particularly single nucleotide polymorphisms (SNPs) within Cas9/guide RNA target sites, pose a potential barrier to drive efficiency. High genetic diversity in An. gambiae results in drive-system target-site variants, including an A→T transversion in the Agcd gene, which occurs at high frequencies in African populations and could affect TP13 drive dynamics. The impact of this and other SNPs on TP13 performance was assessed by establishing three An. gambiae Ndokayo lines, one with the wild-type Agcd and two with homozygous SNP haplotypes. We evaluated drive conversion rates in vivo, population dynamics in cage trials, fitness costs and parasite suppression efficacy. No negative effects on drive performance and parasite suppression were observed. The results provide insights into the influence of naturally-occurring polymorphisms on gene drive propagation, informing safety, efficacy and target product profile requirements for advancing gene-drive mosquitoes toward field trials.

Gene-drive population modification strategies are being developed to control the transmission by anopheline mosquitoes of the parasites that cause human malaria. These approaches are designed to reduce disease prevalence and incidence by spreading dominant antiparasite effector genes throughout vector populations. The strains must sustain drive and parasite suppression properties over extended periods of time to have an epidemiological impact. Three gene-drive strains, AcTP13 and AcTP43 in Anopheles coluzzii and AgTP13 in Anopheles gambiae, carrying autonomous Cas9/guide RNA-based drive systems linked to multiple antiparasite effector genes were remarkably stable in all A. coluzzii replicates over a 2-y (35 generation) period in laboratory cage trials. Two of three A. gambiae replicates performed equally well. Stability was assessed as a function of population dynamics (size), molecular integrity of the gene-drive cassettes, maintenance of drive efficiency (gene conversion), generation and accumulation of mutant drive-resistant target alleles, drive system-generated off-target effects, and effector gene parasite suppression activity. All lines met stability requirements with the exception of one AgTP13 cage replicate that was affected by drive-resistant target-site mutations. Notably, all strains retained parasite suppression activity and high drive efficiencies throughout the duration of the trials. These results support the further development and deployment of these strains for malaria control.

Malaria remains a major global health burden and is caused by protozoan parasites in the genus Plasmodium. Parasites are transmitted to humans during blood feeding by anopheline mosquitoes, and members of the Anopheles gambiae species complex are important vectors in sub-Saharan Africa. Gene-drive technologies offer promising options for disease control by enabling the spread of genetic traits through mosquito populations that block parasite transmission. We report here the development and characterization of four population modification gene-drive strains in Anopheles gambiae s.s. and An. coluzzii carrying compound effector genes. We sought to enhance the effectiveness of existing gene-drive strains to block Plasmodium transmission, thereby reducing vector competence and minimizing the opportunities for selection of resistant parasites. Two compound effector gene modules, TP24 and TP43, were introduced using Cas9 endonuclease and dual guide RNAs into TP13-based gene-drive strains to produce the An. gambiae AgTP24 and AgTP43 strains. The gene-drive cassettes were then introgressed into An. coluzzii to produce AcTP24 and AcTP43. Gene-drive dynamics, gene conversion, and inheritance were high in all strains, with 95% to 100% inheritance of the gene-drive constructs. Life table analyses showed mixed impacts on fitness dependent on the species and copy number (hemi- or homozygosity) of the gene-drive systems. The compound effector molecule gene complexes significantly reduced both parasite prevalence and infection intensities in An. gambiae and An. coluzzii following challenge assays with the human malaria parasite, P. falciparum. These findings highlight the potential of compound effector strategies in gene-drive systems to achieve durable malaria transmission control.

Light serves as a primary zeitgeber for regulating circadian rhythms in mosquitoes. As important vector insects, the diurnal Aedes albopictus and the nocturnal Culex quinquefasciatus are not only nuisance biters, but also transmit viruses such as dengue, Zika, and West Nile. We conducted a comparative analysis of the oscillating molecular rhythms in these two mosquito species. Light-blocking paint application experiments reveal species-specific photic pathways. CRISPR/Cas9 knockouts of the Aalopsin1, Cxqopsin6, and Cry1 genes revealed that Ae. albopictus relies principally on the compound eye pathway, whereas Cx. quinquefasciatus depends more on the non-visual ocelli-like pathway for circadian rhythm regulation. In addition, different light conditions can affect the lifespan of adult mosquitoes. These findings reveal the light-regulated circadian rhythm in diurnal and nocturnal vector mosquitoes. Preferential differences in the compound eye versus ocelli-like light-sensing pathways underlie the distinct diurnal activity of Aedes albopictus and the nocturnal activity of Culex quinquefasciatus.

The development of mosquito transgenesis technologies was driven by the need to make stable and heritable modifications to the genomes of these important insects for a variety of basic and applied objectives. While a number of transient assay systems for gene expression analyses were developed, transposable elements (TEs) were the first tools that allowed the production of genetically manipulated strains for studies that involve the complex biology of these insects and their ability to transmit pathogens. TEs have been replaced more recently for most applications by the adaptation of Cas9/guide RNA techniques, but they are still useful in randomly sampling genomes as enhancer traps and identifying neutral regions in the genome free of insertion site effects. Coupled with the Cas9/guide RNA technologies, precise editing and engineering of the genetic mechanisms responsible for much of the biology of these interesting and important insects is now possible.

Gene-drive systems are under development for the population modification of anopheline vectors of human malaria parasites. The key to their success is the fixation of genes in target mosquito populations that encode molecules preventing parasite transmission. High-efficiency Cas9/guide RNA (gRNA)-based gene-drive systems can facilitate this objective. A potential challenge to these systems is the presence of naturally-occurring or drive system-induced sequence polymorphisms in the genomic target site that could impede Cas9/gRNA-mediated cleavage and negatively impact gene-drive dynamics and fixation. Careful choice of the target site can mitigate the impact of natural variation, and here we analyze drive system-mediated, target-site mutagenesis in the outcross and testcross progeny of an Anopheles gambiae strain homo- and hemizygous for the TP13-based gene-drive system. The resulting data allow for estimation of the rates at which drive-system activity generates mutant target-site alleles in the germline and the impact of inherited paternal- and maternal-effect mutations. Functional and nonfunctional mutant alleles are recovered from the germlines at average rates per target gene/generation of 0.08% for paternal and 0.33% for maternal testcross lineages, with an overall average rate of 0.21%. Clustering effects amplify the potential inheritance frequencies of the mutant alleles. Mutations originating in the germlines represent 47% of the total inherited in testcross progeny, with the balance coming from mutant alleles generated by paternal and maternal effects inherited through the respective parental lineages. This approach allows the estimation of potential cleavage-resistant allele formation and inheritance for this drive system in this species and provides empirically derived values to inform more realistic data-driven gene-drive modeling.

Background Oviposition site selection is an important component of vector mosquito reproductive biology. The Asian Tiger mosquito, Aedes albopictus, is a major and important vector of arboviruses including Dengue. Previous studies documented the preference of gravid females for small, dark-colored water containers as oviposition sites, which they sought during the twilight period (dusk) of their locomotor activity. Vision plays an important role in this behavior, and factors such as the shape, size, and color of the container, light intensity, polarization, spectrum, and other visual cues guide the search for suitable oviposition sites, but the mechanistic factors driving this behavior are unclear. Methodology/Principal findings We blindfolded adult female compound eyes and observed the effects of a lack of vision on the ability to discriminate and utilize preferred oviposition sites. Furthermore, the transcriptomes of blindfolded mosquitoes were screened to identify genes with vision-sensitive expression profiles and gene-editing was used to create non-functional mutations in two of them, rhodopsin-like (mutation designated ‘rho-l△807’) and kynurenine hydroxylase (mutation designated ‘khw’). Behavioral tests of both mutant and control strains revealed that the rho-l△807 mutant mosquitoes had a significant decrease in their ability to search for preferred oviposition sites that correlated with a reduced ability to recognize long-wavelength red light. The khw mutant mosquitoes also had a reduced ability to identify preferred oviposition sites that correlated with reductions in their ability to respond to variations in daily brightness and their ability to discriminate among different color options of the containers and background monochromatic light. Conclusions/Significance This study underscores the importance of visual cues in the oviposition site selection behavior of adult female Ae. albopictus. We demonstrate that wild-type rho-l and kh gene products play a crucial role in this behavior, as mutants exhibit altered sensitivity or recognition of light intensity and substrate colors.

BackgroundNovel technologies are needed to combat anopheline vectors of malaria parasites as the reductions in worldwide disease incidence has stalled in recent years. Gene drive-based approaches utilizing Cas9/guide RNA (gRNA) systems are being developed to suppress anopheline populations or modify them by increasing their refractoriness to the parasites. These systems rely on the successful cleavage of a chromosomal DNA target site followed by homology-directed repair (HDR) in germline cells to bias inheritance of the drive system. An optimal drive system should be highly efficient for HDR-mediated gene conversion with minimal error rates. A gene-drive system, AgNosCd-1, with these attributes has been developed in the Anopheles gambiae G3 strain and serves as a framework for further development of population modification strains. To validate AgNosCd-1 as a versatile platform, it must perform well in a variety of genetic backgrounds.ResultsWe introduced or introgressed AgNosCd-1 into different genetic backgrounds, three in geographically-diverse Anopheles gambiae strains, and one each in an An. coluzzii and An. arabiensis strain. The overall drive inheritance, determined by presence of a dominant marker gene in the F2 hybrids, far exceeded Mendelian inheritance ratios in all genetic backgrounds that produced viable progeny. Haldane's rule was confirmed for AgNosCd-1 introgression into the An. arabiensis Dongola strain and sterility of the F1 hybrid males prevented production of F2 hybrid offspring. Back-crosses of F1 hybrid females were not performed to keep the experimental design consistent across all the genetic backgrounds and to avoid maternally-generated mutant alleles that might confound the drive dynamics. DNA sequencing of the target site in F1 and F2 mosquitoes with exceptional phenotypes revealed drive system-generated mutations resulting from non-homologous end joining events (NHEJ), which formed at rates similar to AgNosCd-1 in the G3 genetic background and were generated via the same maternal-effect mechanism.ConclusionsThese findings support the conclusion that the AgNosCd-1 drive system is robust and has high drive inheritance and gene conversion efficiency accompanied by low NHEJ mutation rates in diverse An. gambiae s.l. laboratory strains.

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a ‘causal pathway’ would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Aedes albopictus is an important vector of arboviruses and prefers small containers of stagnant water as oviposition sites. One of the mechanisms mosquitoes use to search for suitable oviposition sites is relying on odor cues from prospective sites and their surroundings. The genetic and molecular bases of this behavior are not known for Ae. albopictus. Oviposition site-searching behavior can be separated into 2 stages: container location and water detection. We applied a glue compound to the antennae and the maxillary palps of adult females to mask their ability to detect molecules that may guide them to preferred oviposition sites. Treatment of the antennae significantly reduces the location index (P < 0.001), indicating a decreased ability to find oviposition sites, whereas no significant difference was observed in mosquitoes with maxillary palps treated with the same glue compound (P > 0.05). The detection time, measured as the duration from contact with the water surface to the deposition of the first egg, was extended in mosquitoes with treated antennae or maxillary palps, supporting the conclusion that olfaction is involved in the detection of oviposition site. Transcriptomic analysis identified differentially expressed olfactory-related genes, including obp67, obp56d-like, obp19d-like and obp67-like. RNA interference (RNAi)-mediated knockdown of obp67 and obp56d-like significantly affected the location index and detection time, respectively. Cas9/guide RNA-mediated knockout of obp56d-like resulted in a prolonged detection time, compared with the wild type (P < 0.05). These findings help to elucidate aspects of the olfactory mechanisms involved in Ae. albopictus oviposition site selection, and provide a basis for the development of mosquito surveillance and control strategies.

The vast majority of all global species have circadian rhythm cycles that allow them to adapt to natural environments. These regular rhythms are regulated by core clock genes and recent studies have also implicated roles for microRNAs in this regulation. Oviposition is an important circadian behavior in the reproductive cycle of insect vectors of diseases, and little is known about the rhythm or its regulation in mosquitoes. Aedes albopictus is a diurnal mosquito that transmits arboviruses and is the major cause of outbreaks of dengue fever in China. We analyzed the oviposition rhythm patterns of A. albopictus under different light/dark conditions and show that the mosquitoes have an oviposition peak between zeitgeber time 9 (ZT 9) and ZT 12. Furthermore, the antagomir-mediated knockdown of expression of the microRNA miR-2940-1 affected the oviposition rhythm of A. albopictus. These data support the conclusion that miR-2940-1 is involved in the regulation of oviposition rhythm in A. albopictus and provide a foundation for using oviposition rhythms as a new target for vector mosquito control.

Transposon-mediated transgenesis of mosquito vectors of disease pathogens followed the early success of transgenesis in the vinegar fly, Drosophila melanogaster . The P transposable element used in Drosophila does not function canonically in mosquitoes, and repeatable, routine transgenesis in mosquitoes was not accomplished until new transposable elements were discovered and validated. A number of distinct transposons were subsequently identified that mediate the introduction of exogenous DNA in a stable and heritable manner in mosquito species, including members of the genera Aedes , Anopheles , and Culex . The most versatile element, piggyBac , is functional in all of these mosquito genera, as well as in many other insects in diverse orders, and has been used extensively outside the class. Transposon-mediated transgenesis of recessive and dominant marker genes and reporter systems has been used to define functional fragments of gene control sequences, introduce exogenous DNA encoding products beneficial to medical interests, and act as “enhancer traps” to identify endogenous genes with specific expression characteristics.

Transposon-mediated transgenesis has revolutionized both basic and applied studies of mosquito vectors of disease. Currently, techniques such as enhancer traps and transposon tagging, which rely on remobilizable insertional mutagenesis, are only possible with transposon-based vector systems. Here, we provide general descriptions of methods and applications of transposon-based mosquito transgenesis. The exact procedures must be adapted to each mosquito species and comparisons of some differences among different mosquito species are outlined. A number of excellent publications showing detailed and specific protocols and methods are featured and referenced.

Plants produce various pigments that not only appear as attractive colors but also provide valuable resources in applications in daily life and scientific research. Biosynthesis pathways for these natural plant pigments are well studied, and most have multiple enzymes that vary among plant species. However, adapting these pathways to animals remains a challenge. Here, we describe successful biosynthesis of betalains, water-soluble pigments found only in a single plant order, Caryophyllales, in transgenic silkworms by coexpressing three betalain synthesis genes, cytochrome P450 enzyme CYP76AD1, DOPA 4,5-dioxygenase, and betanidin 5-O-glucosyltransferase. Betalains can be synthesized in various tissues under the control of the ubiquitous IE1 promoter but accumulate mainly in the hemolymph with yields as high as 274 μg/ml. Additionally, transformed larvae and pupae show a strong red color easily distinguishable from wild-type animals. In experiments in which expression is controlled by the promoter of silk gland-specific gene, fibroin heavy-chain, betalains are found predominantly in the silk glands and can be secreted into cocoons through spinning. Betalains in transformed cocoons are easily recovered from cocoon shells in water with average yields reaching 14.4 μg/mg. These data provide evidence that insects can synthesize natural plant pigments through a complex, multiple enzyme-mediated synthesis pathway. Such pigments also can serve as dominant visible markers in insect transgenesis applications. This study provides an approach to producing valuable plant-derived compounds by using genetically engineered silkworms as a bioreactor.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae . aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive (‘dead’) Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae . aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh) , resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1 , which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae . aegypti , such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

BACKGROUND:Worldwide invasion and expansion of Aedes albopictus, an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness.RESULTS:A number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534F/S) in the voltage gated sodium channel (vgsc) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534S/S) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity.CONCLUSIONS:This study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus. These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.

The initial signals governing sex determination vary widely among insects. Here we show that Armigeres subalbatus M factor (AsuMf), a male-specific duplication of an autosomal gene of the Drosophila behaviour/human splicing (DBHS) gene family, is the potential primary signal for sex determination in the human filariasis vector mosquito, Ar. subalbatus. Our results show that AsuMf satisfies two fundamental requirements of an M factor: male-specific expression and early embryonic expression. Ablations of AsuMf result in a shift from male- to female-specific splicing of doublesex and fruitless, leading to feminization of males both in morphology and general transcription profile. These data support the conclusion that AsuMf is essential for male development in Ar. subalbatus and reveal a male-determining factor that is derived from duplication and subsequent neofunctionalization of a member of the conserved DBHS family.

Plasmodium ookinetes use an invasive apparatus to invade mosquito midguts, and tubulins are the major structural proteins of this apical complex. We examined the role of tubulins in malaria transmission to mosquitoes. Our results demonstrate that the rabbit polyclonal antibodies (pAb) against human α-tubulin significantly reduced the number of P. falciparum oocysts in Anopheles gambiae midguts, while rabbit pAb against human β-tubulin did not. Further studies showed that pAb, specifically against P. falciparum α-tubulin-1, also significantly limited P. falciparum transmission to mosquitoes. We also generated mouse monoclonal antibodies (mAb) using recombinant P. falciparum α-tubulin-1. Out of 16 mAb, two mAb, A3 and A16, blocked P. falciparum transmission with EC50 of 12 μg/ml and 2.8 μg/ml. The epitopes of A3 and A16 were determined to be a conformational and linear sequence of EAREDLAALEKDYEE, respectively. To understand the mechanism of the antibody-blocking activity, we studied the accessibility of live ookinete α-tubulin-1 to antibodies and its interaction with mosquito midgut proteins. Immunofluorescent assays showed that pAb could bind to the apical complex of live ookinetes. Moreover, both ELISA and pull-down assays demonstrated that insect cell-expressed mosquito midgut protein, fibrinogen-related protein 1 (FREP1), interacts with P. falciparum α-tubulin-1. Since ookinete invasion is directional, we conclude that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 anchors and orients the ookinete invasive apparatus towards the midgut PM and promotes the efficient parasite infection in the mosquito.