Arthropod promoters for genetic control of disease vectors

Abstract

Vector-borne diseases (VBD) impose devastating effects on human health and a heavy financial burden. Malaria, Lyme disease, and dengue fever are just a few examples of VBDs that cause severe illnesses. The current strategies to control VBDs consist mainly of environmental modification and chemical use, and to a small extent, genetic approaches. The genetic approaches, including transgenesis/genome modification and gene-drive technologies, provide the basis for developing new tools for VBD prevention by suppressing vector populations or reducing their capacity to transmit pathogens. The regulatory elements such as promoters are required for a robust sex-, tissue- and stage-specific transgene expression. As discussed in this review, information on the regulatory elements is available for mosquito vectors but is scant for other vectors.

Need for innovation in vector control

Arthropod vectors are responsible for the transmission of many new and reemerging pathogens. These include causative agents of malaria, dengue fever, yellow fever, Zika fever, encephalitis, filariasis (mosquitoes); Leishmania parasites (sand flies); Lyme disease, anaplasmosis, and ehrlichiosis (ticks), Bartonella (fleas and lice); and rickettsia (fleas, lice, and ticks), among others. To put the risk posed by vectors into perspective, more than 80% of the world population is at risk for at least one vector-borne disease (VBD, see Glossary), contributing to 17% of all infectious diseases globally [1]. Environmental, climate and social factors can all influence the spread of VBDs, highlighting the importance of identifying strategies to control them.

In the absence of prophylactic drugs and/or vaccines to prevent or control many VBDs, chemical and environmental vector control methods remain a cornerstone in disease control and prevention [2] (Figure 1). Insecticides are widely used in public health to reduce vector populations. However, resistance has developed to every chemical class of insecticide. Insecticide resistance is expected to directly and profoundly affect the reemergence of VBDs [3]. Accelerated research and development of new tools that can be deployed alongside existing vector control strategies is key to VBD control. Methods such as gene drives that aim to genetically modify vector populations in the wild to either render them refractory to pathogens or impair their reproduction may prove invaluable tools [4]. These next-generation genetic vector control tools have been employed for disease prevention and mosquito control; however, these methods are still in the infancy for other vectors as described in the section below.

Figure 1. Vector control strategies.

Main strategies include environmental, chemical, and genetic approaches. The figure was made in Biorender.

Status of genetic vector control

Availability of ancillary methods such as the ability to rear the arthropods in the laboratory, transgene delivery protocols (embryo injection or related methods), and regulatory elements such as promoters for robust gene expression, are required for the development of genetic control methods. Most information about the biology of arthropods and regulatory elements is based on studies in the vinegar fly, Drosophila melanogaster. While model organisms provide a basic understanding, the knowledge cannot always be translated to the disease vectors because of the differences in biology. In recent years, progress has been made towards the availability of genomes and transcriptomes for the identification of suitable genes and their promoters, phenotypic genes for screening of modified organisms, embryo injection protocols or other transgene delivery methods, cell lines for testing promoters and transposases, and promoter-reporter systems to use genome editing methods for vector control. These efforts have been summarized in (Table 1).

Table 1.

Available genetic tools in different arthropod vectors

Arthropod vector	Available genetic tools	Commonly available promoters
Mosquitoes	Transposons, CRISPR/Cas, ReMOT, Binary systems	Actin5C, APY, CP, Vasa, IE1 and more
Black flies	none	none
Sand flies	CRISPR/Cas9 K-O	none
Tsetse flies	Paratransgenic	Identified computationally, none tested
Triatomine bugs	ReMOT	none
Black flies	none	none
Lice	none	none
Ticks	CRISPR/Cas9 K-O, ReMOT	CAG

Insect vectors and available genetic tools

Mosquitoes

Mosquitoes are the most common arthropod disease vectors. Mosquitoes in Anopheles, Aedes, and Culex genera can transmit pathogens that cause diseases such as malaria, dengue, lymphatic filariasis, yellow fever, Zika, Chikungunya, West Nile, and Japanese encephalitis. Chemical repellents, proper clothing, mosquito screens, and habitat modification are the mainstay of mosquito and mosquito-borne disease management. While genetic control is still limited, mosquito research is far ahead of other vectors [5]. For instance, ancillary methods for mosquito transgenesis, such as the ability to maintain in the laboratory, embryo injection, and genomics data, have been available for decades (Table 1), which allowed the transgenic development using transposable elements as well as genome modification approaches using nucleases such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and clustered regularly interspaced palindromic repeats (CRISPR/Cas) [6]. The early development of these tools has allowed researchers to explore gene drive technologies for population control, replacement as well as pathogen blocking [7].

Black flies

Out of nearly 2000 species of blackflies, approximately 30 of the genus Simulium are identified as vectors of human diseases. Black flies are major vectors of nematodes Onchocerca volvulus that cause onchocerciasis or river blindness. Estimated 220 million people are at risk of onchocerciasis, and approximately 1.15 million people have lost their sight [8]. Vector control in black flies has been mainly targeted towards the larval breeding areas with insecticides. The larvicide Bacillus thuringiensis israelensis (Bti) is currently the only effective treatment with more than 90% larval lethality that has been used for over 40 years [9]. While the blackfly genome project is still underway (NIH White Paper), a mitochondrial genome has been published [10]. Genetic methods for the control of black flies are currently absent.

Sand flies

Phlebotomine sand flies belong to the Phlebotomus/Lutzomyia genus, with nearly 1000 identified species. Sand flies can transmit pathogens causing leishmaniasis, Carrion’s disease, sand fly fever, meningitis, and encephalitis [11]. Leishmaniasis is caused by protozoan flagellates and accounts for over 60,000 yearly deaths making it one of the deadliest neglected tropical diseases [12]. Prevention strategies include insecticides topical repellents, and clothing or barriers such as nets. Bacillus sphaericus bacteria carried by adult flies to larval habitats in burrows have also been used as larvicides [13]. CRISPR-Cas9 was used to knockout relish, an essential gene in the immune deficiency pathway of Phlebotomus papatasi which led to increased susceptibility to bacterial infection followed by lower survivability. The flies also showed higher numbers of parasites when infected with Leishmania major indicating the relationship between the immune response and vector competence of the sand fly [14, 15]. This suggests the possibility for limiting Leishmania spread through strengthening the immune system of the fly, however, overexpression studies are yet to be performed.

Tsetse flies

Tsetse flies (Glossina sp.) carry the trypanosoma protozoan parasites and the causative agents of trypanosomiasis, also known as sleeping sickness. Older, effective measures for control of tsetse flies have been environmental: elimination of reservoir hosts, clearing of woodlands, and periodic burning to prevent brush growth [16]. Another method that has been used to successfully eradicate tsetse flies in Zanzibar relied on the introduction of large numbers of sterilized male flies into a wild population [17]. Currently, deltamethrin-treated screens are being used to limit the population density of flies [18]. The Tsetse genome was sequenced in 2014 and several transcriptomes are available for the identification of target genes [19]. Core promoter architecture was shown using a computational approach [20], however, genetic transformation is yet to be established. Tsetse goes through adenotrophic viviparity, or “gland-fed, live birth”. Females produce a single egg at a time, which passes into the uterus, is fertilized there, and then grows into a maggot within the mother until 3rd instar. This unique biology of tsetse flies makes it difficult to inject transgenic constructs. To bypass this limitation, scientists are using the paratransgenic approach, where they use intralarval microinjection to introduce a maternally inherited mutant bacterial endosymbiont Sodalis glossinidius producing anti-trypanosoma nanobodies [21]. Strategies such as Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control (described later in Genetic Control Section) that bypass embryo injection might be useful for tsetse fly transformation.

Triatomine bugs

Rhodnius prolixus, the kissing bug, transmits protozoa, Trypanosoma cruzi, the causative agent of Chagas disease. The current control methods focus on spraying pyrethroid insecticides and preventing transmission of contaminated donated blood. Although the disease had been virtually eradicated in Central America, the risks of reintroduction and re-infestations remain, requiring constant surveillance and vector control. Recently, ReMOT Control was successfully used for gene editing in this vector [22]. The availability of genome, transcriptomes, and cell lines along with the gene-editing by ReMOT Control makes R. prolixus a suitable candidate for the future development of genetic vector control strategies.

Fleas

Historically, flea-borne diseases are among the most important medical diseases of humans. Plague and murine typhus have been known for centuries while the new flea-transmitted pathogens, like Rickettsia felis and Bartonella henselae were identified in the past 30 years. Flea-borne infections are emerging or re-emerging throughout the world and control methods are needed. The genome of cat fleas has been sequenced; however, the genetic tools are still lacking in this vector (Table 1) [23].

Lice

Body lice and head lice are the most common ectoparasites of humans. Many infectious diseases such as typhus, relapsing fever, and trench fever are caused by the pathogens, Rickettsia prowazekii, Borrelia recurrentis, and Bartonella quintana, respectively, are transmitted by the body lice, Pediculus humanus corporis. These diseases remain a major public health concern in populations living in poor hygiene conditions because of war, social disruption, severe poverty, or gaps in public health management. The vectorial capacity of head lice is still a matter of debate [24]. The body louse genome has been sequenced; however, molecular tools, are still lacking in this vector [25].

Non-insect vectors

Ticks

Ticks from the genus Ixodes are of medical and veterinary importance as they transmit causative agents of Lyme disease, ehrlichiosis, anaplasmosis, tularemia, babesiosis, and Rocky Mountain Spotted Fever, to mention a few. Lyme disease alone results in nearly 480,000 cases yearly in the United States [26]. Approaches for tick control include habitat modification, such as applying acaricides through spraying or spot treatments. Additionally, removing the low-growing vegetation and brush limits the tick survival and human contact of ticks [27]. The first tick genome assembly was completed in 2016 for I. scapularis [28] followed by other Ixodes spp.. Recently the genetic toolkit has been expanded and the first embryo injection protocol was developed [29]. These advances allowed for the first CRISPR/Cas9-mediated knock-out [30]. Regulatory elements have been identified in ticks (see Promoter section); however, functional spatial/temporal and inducible promoters are still lacking. Although not yet published (preprint available) [31], functional inducible endogenous and 3xP3 promoters have been identified and tested in I. scapularis cell line.

Arthropod promoters

According to the central dogma of molecular biology, genetic information is encoded in DNA, which is transcribed into RNA and translated into proteins, influencing the phenotypical and functional characteristics of cells, tissues, organs, and organ systems. RNA polymerase II (RNAPII) is the enzyme responsible for the transcription of protein-coding genes and certain noncoding RNA genes. RNAPII recognizes specific regions of DNA surrounding the transcription initiation site (TSS) to assemble the pre-initiation complex and initiate transcription. This region, referred to as the core promoter is a crucial regulator of gene expression level and can be characterized by various elements. In arthropods, common DNA motifs of the core promoter such as TATA box, initiator (Inr), and downstream promoter element (DPE) have been identified using D. melanogaster as the model organism and were summarized by Haberle and Lenhard [32]. These elements are not universal, being found only in a fraction of core promoters and absent in many others, making promoter identification difficult and often a species-specific endeavor [33].

Genetic control of arthropod vectors requires functional promoters that are either ubiquitous, tissue-specific, or inducible. Therefore, it is essential to establish endogenous (from the same species) as well as heterologous (from a different species) and synthetic promoters that may function across multiple organisms. Identification of endogenous promoters is especially difficult in organisms with incomplete or large, repetitive genomes such as ticks. Similarly, heterologous promoters are excellent when functional; however, promoters derived from distant species may not always work [34, 35]. To overcome this challenge modern techniques such as assay for transposase-accessible chromatin using sequencing (ATAC-seq) are being deployed to identify promoters on a genome-scale as discussed later in this review [36]. Currently, thanks to the decades of research, genetic manipulation tools available for mosquitoes are more developed than for any other arthropod vector. This offers a great opportunity to choose the best approach and adapt it to other, less studied vectors. While the elegant review of Rojas and James [37] discussed mosquito promoters, we have restated the available mosquito and in some cases D. melanogaster promoters to compare with other vectors.

Ubiquitous promoters

Ubiquitous promoters are highly conserved. They show strong activity in various cells and tissues across developmental stages. These promoters can be constitutive or inducible. Constitutive ubiquitous promoters are commonly housekeeping genes, while inducible promoters are genes expressed under specific conditions. An advantage of ubiquitous promoters is the ease of transgene expression and the pattern that can be mapped in various tissues and lifecycles. Actin 5C, polyubiquitin, ubiquitin, heat shock protein (HSP) 30 and 70, and U6 are examples of ubiquitous promoters that have been successfully used in arthropod research.

Actin 5C is a strong ubiquitous promoter of Drosophila melanogaster [38]. Its activity in the yellow fever mosquito, Aedes aegypti, was reported back in 2000 by driving the expression of green fluorescent protein (GFP) [39] and more recently, to express Cas9 in the Southern house mosquito, Culex quinquefasciatus, [40]. In ticks, actin putative promoter from the Asian long-horned tick, Haemaphysalis longicornis, has been identified and used to drive the expression of luciferase in two cell lines, a cattle tick, Rhiphicephalus (Boophilus) microplus, BME26, and a black-legged tick, Ixodes scapularis, ISE6, cell line [41].

Ubiquitin (UB) and polyubiquitin (PUB) are amongst the most conserved proteins. They are found in almost all cellular tissues in eukaryotic organisms and are involved in cellular proteolysis and gene transcription regulation through histone conjugation. Because of the high level of conservation, UB and PUB promoters are commonly used to establish transgenesis in non-model organisms and have been successfully used in arthropod vectors such as mosquitoes, C. quinquefasciatus and Ae. aegypti and a plant pest, the Caribbean fruit fly, Anastrepha suspensa [42, 43].

Heat shock protein genes code for protein chaperones that prevent cytotoxic protein aggregation. HSP70 promoter in D. melanogaster (DmHSP70) is very well characterized and is induced by stress such as chemicals, anoxia, and heat. DmHSP70 promoter has also been used heterologously in Aedes and Anopheles mosquitoes, A. suspensa, and ticks cell line, ISE6 [31,44, 45]. Aedes aegypti endogenous HSP70 promoter is active in all life stages and all tissues in response to heat shock and can be used as an efficient inducible promoter [46]. Similarly, endogenous HSP70 from tick, Ixodes scapularis, was recently shown to be inducible in ISE6 cell line [31].

With the advent of CRISPR/Cas editing, the need for RNAP III promoters capable of transcribing guide RNA (gRNA) greatly increased. U6 is the most common RNAP III promoter used to drive small hairpin (shRNA) expression. It has been utilized in mosquitoes [43] and is a crucial component of gene drive systems in insects [47]. The endogenous U6 promoters were recently identified in the New World screwworm fly, Cochliomyia hominivorax, although not a disease vector, it is an obligate parasite and a major pest of livestock. Based on the number of site-specific mutations in the target gene in G1 larvae, one out of 7 U6 promoters showed the highest activity and was proposed to use in CRISPR/Cas9-based genetic systems [48]. Outside the three insects: Drosophila (D. melanogaster, D. suzukii [49]), mosquitoes (Ae. aegypti and C. quinquefasciatus), and the screwworm fly [48], U6 promoters have not been identified and provide an important area of research for other disease vectors.

Spatial/temporal promoters

Some promoters exhibit exclusive spatial control, influencing gene expression in specific tissues such as the nervous system, salivary glands, midgut, fat body, and germline. Meanwhile, others operate temporally, regulating gene expression during developmental stages or physiological changes like feeding. Spatial-temporal promoters coordinate the timing and location of gene activation (as reviewed by Rojas and James [50]).

Brain/nervous system

The expression of a wide variety of genes is regulated in response to increased neuronal excitation and activation of cellular signaling pathways [], that regulate transcription, synaptic function, and metabolism in all organisms, including insects [51]. These genes can be classified into immediate-early genes (IEGs) and delayed-early genes (DEGs) depending on the temporal expression. Some IEGs encode transcription factors whose protein products, in turn, regulate the expression of DEGs. The promoter regions of IEGs have been used in D. melanogaster in combination with optogenetic tools to address the functional importance of behaviorally relevant circuits [52]. However, none of these have been utilized in insect vectors.

Three genes encoding proteins involved in the maintenance and structure of chemical synapses: neuronal Synaptobrevin (nSyb), Synaptotagmin1 (Syt1), bruchpilot (brp) together with embryonic lethal abnormal vision (elav) – a gene encoding the neuron-specific mRNA splicing proteins have been used to generate the most popular pan-neuronal drivers available in D. melanogaster. Brp and Syt1 have been shown to be neuron-specific in Ae. aegypti via CRISPR/Cas9 mediated knock-ins. However, promoter regions are yet to be identified [53].

Salivary glands

During blood-feeding, arthropod vectors secrete saliva into the host. If the vector is infected, saliva will likely contain pathogens. Therefore, the salivary gland serves as a crucial tissue for pathogen transmission, and identifying salivary gland-specific promoters is essential for pathogen transmission studies. Maltase-like I (MALI) is expressed in salivary glands, encoding the α-glucosidase enzyme that breaks down starch into glucose. Another closely associated salivary gland promoter is Apyrase (APY), whose genes encode 5’-nucleotidases inhibiting ADP-induced platelet recruitment and facilitating hematophagy at the feeding site. APY is preferentially expressed in female mosquitoes. MALI and APY promoters, fused to the luciferase (luc) reporter gene have been utilized in Ae. aegypti to drive expression in a developmental, sex-specific, and tissue-specific manner [54]. Anopheline Anti-Platelet Protein (AAPP) is a component of mosquito saliva. It is important for proper blood uptake because it inhibits platelet aggregation by binding collagen during feeding. AAPP promoter has been used in Ae. aegypti to drive the expression of an anti-dengue effector gene (Mnp) and shown reduced viral infection and replication in the salivary glands of infected female mosquitoes [55].

Gut

Pathogens are first acquired during feeding on an infected vertebrate host. The midgut is the first tissue that a pathogen encounters when ingested by vectors during a blood meal taken on an infected host. Therefore, gut-specific promoters are good candidates for research aimed at vector-pathogen interactions. Carboxy peptidase (CP) is a protease expressed in the midgut of Ae. aegypti and A. gambiae and it is strongly induced by a blood meal. CP promoter has been successfully used for transgene expression in a spatial-temporal manner [56]. Another mosquito bloodmeal-inducible gene is G12. It has a peak expression at 24 hours post-bloodmeal in the malaria vector, A. stephensi and G12 promoter shows strong and gut-specific activity. It has been proposed as a good candidate to drive targeted transgene expression coinciding spatially and temporally with pre-sporogonic stages of Plasmodium parasites in the mosquito gut [57].

Fat body

The fat body is a relatively large tissue distributed throughout the body of arthropods. As the central storage tissue for nutrients and energy reserves, the fat body plays crucial roles in development, metamorphosis, immunity, and reproduction. Large amounts of storage proteins, lipoproteins, and vitellogenin are synthesized in the fat body, many of which are released and accumulated in a sex- and stage-specific manner in the hemolymph [58]. Vitellogenin is a gene responsible for encoding the primary precursor of egg yolk proteins. The promoter of this gene has been employed to drive sex, tissue, and stage-specific transgene expression in mosquito species such as A. stephensi, C. tarsalis, and Ae. aegypti [50].

Germline

Germline-specific promoters are a subset of very well-conserved tissue-specific promoters that drive the expression of genes in ovaries or testes. The germline promoters are required for gene drive strategies, particularly the ones that utilize engineered site-specific homing endonucleases [59]. When expression of an endonuclease such as Cas9 is activated in the germline (male and/or female), a double-stranded cut on the target site on the wild-type chromosome followed by homology-directed DNA repair results in the proportion of offspring that receive the drive transgene will be higher than the expected Mendelian 50%. This strategy has been used for Anopheles and Culex mosquitoes using germline-specific promoter vasa for direct expression of Cas9 [60].

In D. melanogaster, vasa, an ATP-dependent RNA helicase, is a crucial factor in the development of primordial germ cells (PGCs) in embryonic gonads and germline during oogenesis [61]. Additionally, vasa is essential for the efficient translation and activation of another important germline gene, Nanos [62]. Nanos serves as a transcriptional regulator involved in the abdominal patterning of embryos, the migration of PGCs, and the development of stem cells [62]. Active vasa promoter has been identified and used for the expression of fluorescent markers in A. gambiae [63] while Nanos promoter has been used to express mariner MosI transposase in Ae. aegypti [64] and luciferase in A. stephensi [65]. Notably, nanos promoter has also been employed in creating a CRISPR-Cas9-based next-generation gene drive system in the malaria vector mosquito A. gambiae [66].

A testis-specific β2-tubulin promoter has been utilized to drive testis-specific marker expression in A. gambiae [67] and for population control in germline-specific drive systems to induce sterility and control malaria transmission by the mosquito A. stephensi [68]. In A. gambiae, the doublesex (dsx) promoter permits sex separation due to its selective expression pattern in male larvae at early developmental stages with the use of large particle flow cytometry. Early sex sorting is beneficial for Sterile Insect Technology (SIT), especially for the release of sterile males for vector control [69].

The female-specific indirect flight muscle promoter from the Ae. aegypti Actin-4 gene has been used in the release of insects carrying a dominant lethal (RIDL) system, which leads to the expression of a synthetic tetracycline-repressible transactivator (tTA) in a stage-, tissue-, and sex-specific manner resulting in female-specific RIDL strains for population suppression of Ae. aegypti mosquitoes [70].

In the domain of non-insect vector promoters, particularly in ticks, the utilization of heterologous promoters from insects has generally proven ineffective in driving expression [71]. Efforts have been undertaken to identify tick-specific promoters; this has been comprehensively reviewed by Pham et al. [71]. Several inducible promoters, including those derived from subolesin (an immune gene from the akirin family) [72], a ferritin gene promoter, and an HSP70 promoter, have been of interest in ticks[71].

Heterologous and synthetic promoters

While not always as active or efficient as endogenous promoters, heterologous and synthetic promoters prove very useful in arthropod research, particularly in organisms in which transgenic methods are not well established. Synthetic promoters, such as CAG, are artificial constructs not naturally expressed in organisms. CAG promoter comprises the first exon and intron of the chicken β-actin gene, the cytomegalovirus immediate early (CMV-IE) promoter, and the 3’ acceptor splice of the rabbit β-globin gene. CAG has been utilized to drive transgene expression in cell lines of the cattle tick R. (B.) microplus and in the embryos of the black-legged tick, Ixodes scapularis [30]. Another well-established synthetic promoter is 3xP3. It is constituted by three repeats of Pax6 transcriptional activator binding sites and the HSP70 TATA box. Based on the evolutionary conserved role of Pax6 in metazoan eye development it is assumed that the 3xP3 promoter will be functional in most arthropod vectors with eyes [71]. Its activity has been confirmed in several insects including mosquito vectors [50]. Outside mosquitoes, 3xP3 has been shown to work in several non-vector insects however, it has not been tested in any other insect disease vector. An I. scapularis specific 3xP3 (I. scapularis HSP70 core promoter instead of D. melanogaster HSP70 core promoter) was recently shown to be functional and heat-inducible in this tick species [31].

The baculovirus immediate-early gene 1 (IE1) promoters from the nuclear polyhedrosis viruses of the alfalfa looper, Autographa californica (AcMNPV) and the silkmoth, Bombyx mori (BmNPV), have found application in transgenesis across various arthropods [73]. Their expression can be augmented by insect hormones like ecdysone or juvenile hormone analogs (JHA). A fusion of AcMNPV IE1 promoter and homologous region 5 (HR5) enhancer from the same virus was used to drive the expression of enhanced GFP in the malaria vector, A. gambiae [73]. IE1 promoter has also been used in CRISPR-Cas9-based knock-ins in C. quinquefasciatus [74] and in the development of population suppression strains of A. stephensi [75]. Another baculovirus promoter utilized in arthropod transgenics is the AcMNPV polyhedrin promoter. In ticks, it has been used to drive the expression of Haemaphysalis longicornis chitinase (CHT1) in Spodographa frugiperda Sf9 cells and in unfed larvae and adult ticks [76].

Approaches for promoter identifications

Promoters play a crucial role in gene expression. Historically TSS and motifs like TATA box were used in in computational prediction tools, however their performance was limited [77]. Several ubiquitous promoters and that of baculovirus origin were identified in D. melanogaster and then translated directly to mosquitoes [78]. It took decades of work to identify functional promoters for Drosophila and mosquitoes; however, lessons learned in this process, as well as technological development that allows high throughput screening of genomes can greatly shorten the time required for identifying promoters in other vectors.

Many sequencing technologies have been developed in recent years that allow genome-wide identification of promoter regions. A popular approach for discovering promoters is to identify TSS using High-Throughput Sequencing (HTS). In cap analysis of gene expression (CAGE) biotinylation of the 7-methylguanosine cap of RNAPII transcripts is used to select the capped transcripts and fully sequence the 5’-end of the transcripts [79]. Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE) relies on long paired reads for RNAPII transcript enrichment. It has been used for the discovery of TSS in Drosophila [80] and for promoter selection in non-model organisms such as the flatworm, Macrostomum lignano [81]. Rapid amplification of cDNA ends (RACE) uses gene-specific primers for known exons to obtain the 5’ end of a transcript from cDNA. Even though RACE has lower throughput, it is routinely utilized in arthropod research [82–84].

Chromatin Immunoprecipitation Sequencing (ChIP-seq) [39] and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) [85] allow researchers to identify active promoters by isolating protein-DNA complexes and using HTS to map TSS and associated regulatory regions.

Enhancers are distal regulatory DNA sequences that positively affect the expression of a gene and are a useful tool in genetic manipulation. Enhancer trapping is a common method for identifying enhancers. It involves inserting a reporter gene, such as GFP (Green Fluorescent Protein), into the genome randomly or near potential regulatory regions. Reporter assays then assess the activity of nearby genomic regions, allowing for the identification of active promoters and enhancers. Other methods for enhancer identification include computational approaches [86].

Although this is not a comprehensive list of technologies utilized by researchers for promoter identification, it provides a glimpse into the available methods. The sequencing technologies are favorable because they can be applied for non-model organisms and even in organisms that lack a well-assembled genome.

In addition to the sequencing technologies, CRISPR-based screening technologies, such as CRISPR-Cas9 and CRISPRi (CRISPR interference), enable large-scale functional genomic screens to identify regulatory elements, including promoters. They can facilitate the identification of functional promoters by simultaneously testing thousands of targets by using pools of gRNAs to direct Cas9 to either knock-out or knock-down chosen DNA sites [87]. These technologies are more suitable for vectors with established cell lines.

Machine learning and artificial intelligence (AI)

With the advances in next-generation sequencing and data availability, more focus is being put on computational approaches to identify TSS [20, 36]. Genome-wide bioinformatic analysis provides a deeper understanding of core promoter structure [33], and recent developments in artificial intelligence and new machine learning algorithms can use the abundance of data to identify regulatory sequences [88], greatly reducing the time and cost for discovering new species-specific promoters.

Genetic methods for generating transgenic arthropods

Access to transgenic methods is crucial for research on any organism, particularly for arthropod vectors. Throughout the decades many methods for genetic manipulation have been developed with the last ten years being the steady rise in the dominance of CRISPR/Cas-mediated genome engineering. However, other methods such as transposons, ZFNs, and TALENs remain valuable.

Transgenic approaches using transposases

Transposable elements were some of the first methods used for genetic manipulation. Starting with P-elements in D. melanogaster [89] various transposon-based systems such as piggyBac, hobo, Hermes, Minos, and Mos have been developed and used in different species including mosquito vectors [90] and have been reviewed previously [37].

Genome editing using targeted approaches

Targeted genome editing is highly desirable for reverse genetic studies and developing genetic control systems. ZFNs, TALENs, and CRISPR/Cas are being actively researched in the context of arthropod vectors. ZFNs consist of a DNA binding domain made of three to six individual zinc finger repeats that recognize the specific sequence on the genome and a FokI nuclease domain that cleaves the specific site of DNA [91]. They have been successfully used to create knockouts in Ae. Aegypti. However, the complexity of ZFNs coupled with difficulties in the design process makes this approach less appealing to non-model researchers [6]. TALENS are nucleases that can be designed to introduce a double-stranded break in DNA with very high specificity and minimal off-target effect. This approach has been successfully deployed in Ae. Aegypti [92]. Similar to ZFN, the prohibitive cost of developing specific endonucleases resulted in the low adaptability of this approach in non-model organisms.

CRISPR-Cas9 and other CRISPR systems are widely known for their simplicity and reliability. The system works by introducing double-stranded breaks at a specific target site in the DNA and can be designed to universally target virtually any genomic sequence. By utilizing non-homologous end-joining or homologous recombination mechanisms of the cells CRISPR-based systems offer easy access to both knock-out and knock-in experiments, which are crucial for gene editing and developing gene drives. So far it has been widely adapted to modify the genomes of mosquitoes [43, 66] and its utility has been shown in ticks [30], sand flies [14], and triatomine bugs [21].

Current approaches for arthropod gene editing require microinjection of materials into embryos. However, many insect species, especially those whose reproduction systems preclude access to early embryos for injection such as tsetse fly, require alternative methods for genome editing. Several alternative methods that bypass the requirement of embryo injection have been developed; the first among these was ReMOT Control [93]. In this method, peptide ligands derived from yolk protein precursors are fused to Cas9 protein, and the complex of the engineered Cas9 and single-guide RNAs (sgRNAs) is injected into female adults to introduce mutations in developing oocytes. ReMOT-mediated targeted mutagenesis has been successfully used in several arthropod disease vectors including mosquitoes: Ae. aegypti [93]. A. stephensi [94] , Cx. pipiens [95], R. prolixus [21], and ticks [30]. Other methods such as DIPA-CRISPR [96] and BAPC/SYNCAS [97] are based on similar concepts but do not use a ligand for receptor-mediated endocytosis.

Binary systems for expression control

While not a genome editing system, the binary GAL4 -UAS system is a well-established tool for the modification of gene expression in a controlled spatiotemporal manner in D. melanogaster [41]. The system utilizes the transgenic lines expressing the yeast transcription activator GAL4 and crosses them with Gal responsive upstream activation sequence (UAS) enhancer bound to the gene of interest. This allows the study of the expression of cloned genes separately from their respective enhancers in specific tissues or cell types. The system’s advantage lies in driving ectopic expression, reducing the likelihood of developmental defects or lethality. The system has been successfully used in mosquitoes [98]. Similar to Gal4-UAS system, the binary Q-system was originally developed in D. melanogaster [99]. It utilizes two genes from the qa cluster of the fungus, Neurospora crassa, and consists of the transcriptional activator, QF, the enhancer, QUAS, and the repressor, QS. Q system works in a variety of organisms including mosquitoes[35]. GAL4/UAS and Q systems are independent binary systems and can be used simultaneously to express several reporters in different subsets of cells. These bipartite gene expression systems offer fine spatial and temporal control of gene expression and are crucial for understanding gene function as well as for enhancertrapping. but their application in other disease vectors has not yet been described.

Concluding remarks

Vector-borne diseases continue to exert a significant burden on humans around the world and are not limited to rural or underdeveloped regions. With the advancements in science, genetic methods of control are becoming more effective and feasible to implement offering long-lasting solutions to population control of mosquito vectors as well as pathogens they transmit. While translating Drosophila genetic research to mosquitoes took much longer due to technical hurdles, new technologies such as CRISPR-Cas9 now may allow rapid translation of ideas to non-model disease vectors (see Outstanding Questions). However, adopting these methods still requires in-depth knowledge of the biology of vectors, primarily in the context of transgene expression. Unfortunately, synthetic and heterologous promoters do not always exhibit the expected level of activity making the identification of endogenous promoters an extremely important part in adapting genetic methods of control to less studied arthropod vectors. The literature review highlights the need for the identification of promoters in disease vectors for developing genetic control methods. Currently, endogenous promoters are available for mosquitoes, tsetse flies, and ticks. However, the spatial/temporal and germline promoters are still lacking in most vectors. The CRISPR/Cas9 technology has now provided an opportunity to develop genetic tools for all organisms including disease vectors. Therefore, the research focus on ancillary tool development such as promoter identification is necessary. Additionally, RNPIII-based promoters such as U6 need to be identified for the expression of guide RNAs for CRISPR-based editing.

Outstanding Questions.

• How do we make the genetic modification toolkit currently available for mosquitoes accessible for other disease vectors?

• How can machine learning models identify general promoters for arthropod vectors?

• Can machine learning and artificial intelligence tools be trained for promoter identification?

In spite of a growing number of examples of identification of endogenous promoters, this approach has been somewhat limited in part due to challenges with genome assemblies and better annotation. The progress in artificial intelligence and machine learning might aid in identifying specific promoters (see Outstanding Questions). Therefore, future directions in the field of vector-borne diseases will need to explore species-specific methods to express genes of interest. With this in mind, we propose that more emphasis should be put on sequencing and assembling high-quality genomes of arthropod vectors. Based on our experience in developing transgenic techniques in flatworms [80] and ticks [30] we view this as the first step in utilizing the full potential offered by decades of research done on mosquitoes and D. melanogaster. In addition, advances in next-generation sequencing and computational methods made genome assembly an affordable endeavor available for most research facilities.

Highlights.

• Genetic toolkit is now extended to disease vectors.

• For genetic control methods such as CRISPR/Cas-based editing and gene drives, promoters with robust and tissue-specific expression are needed.

• Availability of genomes and machine learning technologies provide an opportunity to identify promoters in non-model vectors.

Glossary

ATAC-seq

assay for transposase-accessible chromatin using sequencing, method for determining regulatory regions of the genome.

CRISPR/Cas

clustered regularly interspaced palindromic repeats Cas associated nuclease system, precise genome editing tool.

Receptor-Mediated Ovary Transduction of Cargo (ReMOT)

method of delivering Cas9-gRNA complex into the developing oocytes.

Vector-borne disease (VBD)

illnesses caused by parasites, viruses and bacteria that are transmitted by organisms such as arthropods.