Mahima Yadav,Nisha Dahiya,Neelam Sehrawat
Mosquito gene targeted RNAi studies for vector control
RNA interference,Vector control,Anopheles,Aedes,Culex,Delivery methods
Abstract Vector-borne diseases are serious public health concern. Mosquito is one of the major vectors responsible for the transmission of a number of diseases like malaria, Zika, chikungunya, dengue, West Nile fever, Japanese encephalitis, St. Louis encephalitis, and yellow fever. Various strategies have been used for mosquito control, but the breeding potential of mosquitoes is such tremendous that most of the strategies failed to control the mosquito population. In 2020, outbreaks of dengue, yellow fever, and Japanese encephalitis have occurred worldwide. Continuous insecticide use resulted in strong resistance and disturbed the ecosystem. RNA interference is one of the strategies opted for mosquito control. There are a number of mosquito genes whose inhibition affected mosquito survival and reproduction. Such kind of genes could be used as bioinsecticides for vector control without disturbing the natural ecosystem. Several studies have targeted mosquito genes at different developmental stages by the RNAi mechanism and result in vector control. In the present review, we included RNAi studies conducted for vector control by targeting mosquito genes at different developmental stages using different delivery methods. The review could help the researcher to find out novel genes of mosquitoes for vector control. Supplementary Information The online version contains supplementary material available at 10.1007/s10142-023-01072-6.
Mosquito gene targeted RNAi studies for vector control

Vector-borne diseases are serious public health concern. Mosquito is one of the major vectors responsible for the transmission of a number of diseases like malaria, Zika, chikungunya, dengue, West Nile fever, Japanese encephalitis, St. Louis encephalitis, and yellow fever. Various strategies have been used for mosquito control, but the breeding potential of mosquitoes is such tremendous that most of the strategies failed to control the mosquito population. In 2020, outbreaks of dengue, yellow fever, and Japanese encephalitis have occurred worldwide. Continuous insecticide use resulted in strong resistance and disturbed the ecosystem. RNA interference is one of the strategies opted for mosquito control. There are a number of mosquito genes whose inhibition affected mosquito survival and reproduction. Such kind of genes could be used as bioinsecticides for vector control without disturbing the natural ecosystem. Several studies have targeted mosquito genes at different developmental stages by the RNAi mechanism and result in vector control. In the present review, we included RNAi studies conducted for vector control by targeting mosquito genes at different developmental stages using different delivery methods. The review could help the researcher to find out novel genes of mosquitoes for vector control.

The online version contains supplementary material available at 10.1007/s10142-023-01072-6.

Infectious diseases spread by vectors account for 17% of all communicable diseases and cause 7,00,000 deaths per year worldwide (World Health Organization 2020a). Globally, cases of mosquito-borne disease have been increasing, and after the COVID-19 pandemic, mosquito-borne diseases have been resurging from the disease-eradicated region (Franklinos et al. 2019; Ong et al. 2022). In 2021, 247 million cases of malaria were reported globally (WHO report 2022k), and recent outbreak of dengue, yellow fever, chikungunya, and Zika poses a serious threat in many areas (Rana et al. 2021; Bagcchi 2023; Islam et al. 2022; Cortes-Escamilla et al. 2022; Tuells et al. 2022; Vairo et al. 2019; Pielnaa et al. 2020). It is expected that by 2050, about half of the population of the world would be at risk of arbovirus transmission (Kraemer et al. 2019). Vector-borne disease can arise from parasites, viruses, or bacteria, and mosquito acts as a host for a number of disease-causing pathogens like West Nile virus, Zika virus, DENV virus, Flavivirus, CHIKV, Plasmodium, and Wuchereria bancrofti that pose a serious threat to human health. Mosquitoes belonging to genera Aedes, Anopheles, and Culex play an important role in the transmission of vector-borne disease. Aedes and Anopheles are the primary vectors for the viral pathogen and parasites. Culex is the vector for both the parasite and viruses (Sultana et al. 2020). The mosquito-borne diseases, host, causing pathogen, symptoms, outbreaks, and vaccine have been mentioned in the supplementary file. Although the mosquito population has been reduced to some extent by the use of insecticide-treated nets, indoor residual spraying, mosquito repellant, sterilant, insecticide, targeting of mosquito breeding habitat, and sound traps, these methods did not appear to be very successful (Benelli et al. 2016). Due to the lack of effective drugs and vaccines against most vector-borne diseases, the emergence of resistance in mosquitoes against insecticides like DDT, pyrethroids, carbamates, organophosphates, and organochlorines and the destruction of the ecosystem via reiteration use of insecticides made humans compelled to use another alternative approach for vector control (Sultana et al. 2020; Airs & Bartholomay 2017). There is a requirement to use another environment-friendly approach for controlling the mosquito population as it is the most effective way to combat life-threatening vector-borne diseases (Mysore et al. 2021). RNA interference is an innovative in vivo approach in which the effect of mRNA transcript is reduced through posttranscriptional modification on the basis of sequence complementarity with double-stranded RNA (Airs & Bartholomay 2017; Balakrishna Pillai et al. 2017). The pathway required two core proteins, Dicer protein (endonuclease) which recognizes dsRNA and generates small RNAs and Argonaut protein that takes generated RNAs and screens complementary target mRNA. Target mRNA is degraded or its translation is inhibited during the process known as posttranslational gene silencing (Azimzadeh Jamalkandi et al. 2014). Fire et al. (1998) discovered this phenomenon in the nematode Caenorhabditis elegans via the introduction of dsRNA into the body cavity to manipulate gene expression resulting in sequence-specific gene silencing (Reis 2017). In the field of mosquito genomics, RNAi is one of the best methods being used to suppress the effect of mosquito endogenous genes and genes encoding for pathogens in vivo. RNAi provided new insight into fundamental research to disrupt the physiology of the mosquito life cycle by suppressing the gene associated with fecundity, behavior pattern, survival, and vector status so that burden of mosquito-borne disease on the human population could be reduced. As this technology is more popular in agriculture in terms of managing insect pests, it is interesting to consider the implementation of RNAi in mosquito control as a bioinsecticide (Airs & Bartholomay 2017). CRISPR/Cas is the more advance tool that could be used to explore genome editing (Li et al. 2022). A number of mosquito genes have been targeted for vector control by RNAi mechanism. Two reviews have briefly explained the application of RNAi for controlling the mosquito population. Balakrishna Pillai et al. (2017) elucidated the implementation of RNAi for understanding vector-pathogen interaction by using various delivery methods targeted at different developmental stages, insecticide resistance genes of vector, and Munawar et al. (2020) explained different methods used for targeting larva genes to overcome the mosquito burden. In the present review, we have focused on the genes targeted at different developmental stages whose inhibition by RNAi is associated with direct mortality of individuals, physical or physiological abnormalities, vectoral capacity, and pesticide susceptibility. We tried to avoid the gene targeted to understand mosquito biology by RNAi in terms of their specific function as explained in Balakrishna Pillai et al. (2017) review. Our main goal is to find out novel mosquito candidate genes that can be used as bioinsecticides in the near future without depleting natural resources in order to overcome the burden of pesticide resistance in mosquitoes over a period of time. We also did not include the mosquito gene on which the RNAi study had been taken, but their inhibition effect did not affect mosquito survival.

Articles for this review were searched through the database PubMed, Scopus, and ScienceDirect and from the reference list of relevant articles. The WHO site was used for collecting information regarding mosquito-borne disease, disease-causing pathogens, symptoms, outbreaks, and vaccine availability (https://www.who.int/). Research Gate was also used for some articles which were not in full text on PubMed. Search term “RNAi based strategy for mosquito control” was used while using the PubMed, Scopus, and ScienceDirect databases. Title and abstract of the research and review articles were screened individually. Research articles and reviews relevant to the topic were included in the study. Last literature search was performed on 30 January 2023.

Figure 1 illustrates the results of the search. A total of 1526 articles were identified with the search term “RNAi based strategy for mosquito control” using PubMed, Scopus, and ScienceDirect databases. A total of 55 articles have been included in the study after reading the full text. The RNAi studies targeted mosquito gene candidate results in the mortality, abnormality, susceptibility towards insecticide, and affect fecundity of mosquitoes have been discussed in the present review. Mosquito gene-based RNAi studies that did not provide the significant result or low mortality results have been excluded.

There are a few RNAi studies have been done on the eggs of mosquitoes for vector control. Meleshkevitch et al. (2013) performed a silencing experiment in Ae. aegypti by using Na+ methionine transporter 5 (NAT5) via the soaking method. NAT5 is involved in the transport of L-Meth under the influence of Na+, but the silencing effect was neutralized after the late larva stage and significant mortality of larvae was observed during ecdysis; the emergence of adults from pupae was also suppressed as well.

The larval stage is one of the most important developmental stages of mosquitoes, at which genes targeted by the RNAi mechanism provided fruitful results in the larvae control strategy. Different methods have been used for the transfer of ds RNA of the gene of interest in mosquito larvae via microorganisms like bacteria (E. coli), fungi (Saccharomyces spp.), and algae (Chlamydomonas, Chlorella) and by various technical approaches like soaking, microinjection, and nanoparticles (Taracena et al. 2019; Mysore et al. 2017; Hapairai et al. 2020; Van Ekert et al. 2014; Khalil et al. 2021). The inhibition of gene expression is usually done by real-time PCR. With the deep analysis of the implementation of all methods on mosquito mortality, the yeast delivery system was found to be the most efficient in carrying the highest mortality of larvae (Mysore et al. 2021, Mysore et al. 2019a, b; Mysore et al. 2017). It might be due to the complete knockout of ds RNA, which is the biggest challenge of RNAi to tackle. Studies conducted on the inhibition effect of larvae-targeted genes on mortality by RNAi mechanism are mentioned in Table 1, and Fig. 2 shows the list of larva gene targets according to the category mentioned in the text.

Ataxin 2-binding protein (Mysore et al. 2021), dopamine 1 (Hapairai et al. 2020), leukocyte receptor complex 0.51 (Mysore et al. 2017), synaptotagmin, semaphorin (Mysore et al. 2019a, b), offtrack (Mysore et al. 2017), shaker (Mysore et al. 2020) gene-targeted via yeast, and hr3 gene via algal delivery system affected the highest larvae survival. The microbial delivery method also gave a good result in offtrack and leukocyte receptor complex suppression and carrying high mortality of An. gambiae larvae (Mysore et al. 2017). Juvenile acid methyl transferase (Van Ekert et al. 2014) inhibition delayed pupation and eclosion rate as well as carried 80% mortality of Ae. aegypti larvae by fungal delivery method. A no. of scientists have worked on chitin synthetase 1 and 2 or chitin synthetase A and B in different mosquitoes by using multiple delivery systems (Khalil et al. 2021; Lopez et al. 2019; Zhang et al. 2015, 2010; Singh et al. 2013). Among them, chitin synthetase A and B silencing carried out the highest ~ 80% mortality of larvae by the soaking method in Ae. aegypti (Lopez et al. 2019).

Vacuolar adenosine triphosphatase (V-ATPase), 3-hydroxy kynurenine transaminase (3-HKT), voltage-gated Na+ channel (V-ATPase), inhibitor of apoptosis (IAP1), dopamine 1, β-tubulin, steroid receptor coactivator (SRC), and chitin synthetase enzyme inhibition carried out a medium range of mortality of ~ 50–70% among larvae by various delivery methods (Khalil et al. 2021; Kumar et al. 2013; Bona et al. 2016; Hapairai et al. 2020; Singh et al. 2013; Das et al. 2015). Vacuolar sorting protein (SNF7) and steroid receptor coactivator (SRC) inhibition affected high mortality of larvae by quantum dot nanoparticle than only chitosan nanoparticle. Chitin synthetase 1, heat shock protein gene, and 3,4-dihydroxyphenylacetaldehyde synthetase gene inhibition results in three times higher mortality of larvae through soaking and nanoparticle method, respectively (Singh et al. 2013; Chen et al. 2019).

Sex-specific genes like testis genes, gas and doublesex gene silencing with the soaking method results in the emergence of a maximum no. of sterile male progeny. Doublesex gene inhibition affected half of larvae survival, and females that emerged from ds-treated larvae failed to develop and reproduce (Whyard et al. 2015).

Several gene expression inhibition elevated the susceptibility of mosquitoes towards insecticides as the insecticide resistance in mosquitoes has been increased with continuous exposure. Abcg4 transporter (Negri et al. 2019) and voltage-gated sodium channel (Bona et al. 2016) increased the susceptibility of larvae towards pyrethroid and p glycoprotein (Figueira-Mansur et al. 2013) susceptibility towards temephos by the soaking method. Chitin synthase 1 (Zhang et al. 2015, 2010) and chitin synthase 2 (Zhang et al. 2010) inhibition increases sensitivity towards diflubenzene and calcofluor white or dithiothretol, respectively, by the nanoparticle method.

Pupa genes are a good target for controlling the mosquito population. Knockdown at the pupa stage can persist during developmental interval time between pupae and can extend up to the adult stage (Regna et al. 2016). Most RNAi studies conducted to target pupa genes hindered the eclosion process or carried physiological abnormalities in the adults that emerged from treated pupae. Prophenoloxidase III inhibition in Zika virus potential vector Ar. subalbatus allowed survival of only 3–4% of emerged adults with malformed wings and legs (Tsao et al. 2009). Laccase2 and tyrosine hydroxylase inhibition in malarial vector An. sinensis inhibited the eclosion process and affected cuticles and survival rate as well (Du et al. 2017; Qiao et al. 2016). Semaphorin 1a and cecropin B gene inhibition carried physiological abnormalities like defective antenna lobes and the inability of adults to detach from pupae resulting in the death of individuals (Mysore et al. 2013; Liu et al. 2017). Testis genes boule (bol), fuzzy onions (fzo), growth arrest specific protein 8 (gas8), no-hitter (nht), and zero population growth (zpg) inhibition results in fecundity reduction and the progeny produced from mating of adults (emerged adults from testis gene ds RNA-treated pupae) found to be less viable (Whyard et al. 2015). Table 2 shows the list of pupa genes targeted by the RNAi mechanism.

A number of RNAi studies focused on adult stage genes in order to manage vector population. Adult stage is easy to handle to carry delivery of RNA as comparison to egg and pupa stage. A number of methods had been used to deliver RNA to inhibit the target gene expression. Most of gene targeted at adult stage affected reproductive capacity in terms of egg production and some genes inhibition affected survival leading to mortality of individuals.

Ataxin 2-binding protein (Mysore et al. 2021) and dopamine 1 receptor (Hapairai et al. 2020) gene silencing carried high mortality among different vectors including An. gambiae, Cx. quinquefasciatus, Ae. albopictus, and Ae. aegypti via yeast attractive targeted sugar bait delivery method. Coatomer protein 1 (Isoe et al. 2011) and stearoyl Co-A desaturase (Ferdous et al. 2021) gene expression inhibition led to high mortality of adult mosquitoes through microinjection delivery method. Ataxin 2-binding protein (Mysore et al. 2021), ecdysone receptor (Maharaj et al. 2022), and akirin (Letinić et al. 2020) gene suppression executed an effective mortality in adult mosquito microinjection delivery method. Epsilon glutathione transferase (GST) gene inhibition increased the susceptibility of mosquitoes towards insecticide deltamethrin (Lumjuan et al. 2011). In Ae. aegypti, 3,4-dihydroxyphenylacetaldehyde synthetase (Chen et al. 2019) gene inhibition and shaker gene (Mysore et al. 2020) inhibition by microinjection cause more than 50% mortality in different vectors including Ae. aegypti, Ae. albopictus, An. gambiae, and Cx. quinquefasciatus.

A few studies executed silencing of male mosquito gene and mating of these specific gene-deficient mosquitoes with virgin females affected fecundity rate adversely. Male sex-specific gene heme peroxidase 12-deficient mosquitoes mating with female results in reduction of fecundity capacity by ~ 50% (Kumari et al. 2022). Ammonia transporter is a crucial factor for sperm viability and fertility; silencing also affected fecundity rate of female mosquitoes (Durant & Donini 2020).

A number of adult targeted gene silencing affected ovary size, ovarian follicle length, oocyst maturation, oocyst number, egg development, and fecundity rate and reduced reproductive output carried out by mosquitoes. Juvenile acid methyl transferase (Van Ekert et al. 2014), ras homolog enriched in brain GTPase (Roy & Raikhel 2011), coatomer protein 1 (Isoe et al. 2011), ribosomal proteins S6 and S26 (Estep et al. 2016), AeSigP-66,427 (Pascini et al. 2020), hr38 (Dong et al. 2018), autophagic genes (Bryant & Raikhel 2011), transferrin (Rani et al. 2022), trehalase (Tevatiya et al. 2020), target of rapamycin kinase (Hansen et al., 2005), protein tyrosine phosphatase homolog (Moretti et al. 2014), and kruppel homolog 1 (Ojani et al. 2018) gene silencing decreased the ability to reproduce and provided rewarding result to control mosquito population. Inward rectifier potassium (Raphemot et al. 2014) and frizzled 2 (Weng & Shiao 2015) gene silencing by microinjection interfered with egg production in An. gambiae and Ae. aegypti, respectively. In Ae. aegypti, regulator of ribosome synthesis, ribosome protein large subunit 32, and methoprene-tolerant (Wang et al. 2017) gene inhibition by microinjection results in a reduction in follicle length and egg number. The silencing effect of the mentioned gene is listed in Table 3, and Fig. 3 is showing the adult stage targeted genes by RNAi.

Mosquito-borne diseases are posing a major threat to mankind for many decades. The only way to tackle these diseases is to use an effective vaccine. But there are a number of mosquito-borne disease like chikungunya, elephantiasis, and Zika virus for which no vaccine is available and some developed vaccines have lower efficacy like RTS/01 for malaria. In future time, mosquito could be a potential vector for other perilous diseases. In this situation, mosquito control approach is the only route to overcome this extreme burden. A number of approaches have been used for controlling the mosquito population, but the population of mosquitoes has been growing despite the adoption of a variety of treatments due to their rapid rate of reproduction. Consistent use of chemical insecticide disturbed the air, water, and soil environment and also made mosquito to evolve with greater extent to counteract the effect of insecticides. At present time, RNAi has emerged as a novel strategy in the field of functional genomics study of organisms and has been used for mosquito control by targeting a particular mosquito gene. Inhibition of the mosquito gene, which is essential to its survival, may result in mosquito death. A number of researchers used RNAi method to inhibit mosquito gene expression to find out the outcomes of gene inhibition. In the present review, we explained different studies conducted on RNAi-mediated silencing effect on vector survival by targeting mosquito genes of different developmental stages via different delivery methods. With literature finding, we concluded that larva genes ataxin 2-binding protein, dopamine 1, leukocyte receptor complex 0.51, synaptotagmin, semaphorin, offtrack, hr3, juvenile acid methyl transferase, chitin synthetase A and B, and shaker gene silencing targeted by different methods provided the most effective results as shown in Table 1. At the larval stage, shaker, synaptotagmin, and semaphorin gene inhibition has also been applied in semifield conditions and provided fruitful results in larva control. Most of the pupa stage targeted genes provided effective results in terms of abnormalities, generated due to gene inhibition assay. Prophenoloxidase III and cecropin B gene suppression at the pupal stage is found to be more efficient by microinjection method and adult stage targeted gene ataxin 2-binding protein, dopamine 1 receptor, coatomer protein 1, and stearoyl co-A desaturase gene silencing carried the highest mortality in adults. With deep analysis of all result findings, it was concluded that yeast delivery method is the most efficient method in carrying the highest mortality among different mosquito species, i.e., yeast delivery method is capable of complete inhibition of the target gene. This review is providing the information all about the mosquito gene target that can be used for further bioinsecticide for mosquito control without depleting the natural ecosystem. We need to find the most efficient delivery method so that complete inhibition of gene could carry out maximum mortality of gene of interest for vector control so that vector-borne disease could be controlled. No doubt, stability and large-scale production at lower cost are the major challenges in the implementation of RNAi in field conditions. In vivo strategies could solve the problem of cost and yield. It is regarded that using bacterial expression system could solve the dsRNA cost and large-scale production. Further studies are required in this area to implement RNAi for vector control in the field.

Below is the link to the electronic supplementary material.Supplementary file1 (PDF 333 KB)