Vector Control: Insights Arising from the Post-Genomics Findings on Insects’ Reproductive Biology

Abstract

The high prevalence of neglected vector-borne diseases, such as Chagas disease and dengue fever, imposes enormous health and financial burdens in developing countries. Historically, and still, to this day, the main effective methods to manage those diseases rely on vector population control. Although early efforts in understanding vector-specific biology resulted in important advancements in the development of strategies for the management of vector-borne diseases, studies regarding the complex physiology of local vector species were weakened by the expanding use of insecticide-based tools, which were, at the time, proven simpler and effective. The rising threat of insecticide resistance and climate change (which can expand endemic areas) has reemphasized the need to rely on thorough species-specific vector biology. One approach to controlling vector populations is to disrupt molecular processes or antagonize the metabolic targets required to produce viable eggs. Here, we discuss new findings arising from post-genomics molecular studies on vector reproductive biology and discuss their potential for the elaboration of new effective vector control interventions.

1. Introduction

1.1 Vector-borne diseases

Vector-borne diseases remain among the deadliest and most prevalent infectious diseases worldwide. Mosquito-transmitted arboviral diseases inflict an enormous burden in tropical areas of the world. A previous study estimated that almost 400 million people are infected by the dengue virus (DENV) every year [1]. While tremendous progress on vector control has been made over the twentieth and twenty-first centuries, we are now facing a critical moment. Record-breaking numbers of dengue cases were detected in the Americas in 2019 [2]. This is aggravated by the fact that multiple serotypes circulate in these regions, maximizing the risk of hemorrhagic fever and other severe complications. Other arboviruses are also of concern. For example, the Zika virus (ZIKV), transmitted by Aedes aegypti, rapidly spread in the Americas in 2014–2015, where it was linked with a surge of newborn malformations, including microcephaly [3, 4], inflicting a lifelong impact on children, their parents, and the health public system. Other viruses, like Mayaro (MAYV), chikungunya (CHKV), and yellow fever (YFV), continue to circulate and periodically reemerge. In addition to the arboviruses, vector-borne parasitic diseases, such as Chagas disease, are also medically important. The triatomine bug Rhodnius prolixus is a primary vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected disease endemic to Central and South America. Chagas disease remains the main cause of death related to neglected infectious diseases in the Americas. Currently, Chagas affects approximately 8 million people and migration among endemic and non-endemic regions has expanded its occurrence to approximately 350,000 infected carriers around the globe [5].

Overall, these diseases not only result in a high number of deaths and hospitalizations but also generate a huge economic impact due to the disability of people during their learning and working ages [6, 7]. While most restricted to tropical areas, where vectors meet the perfect conditions for mating and reproduction, models of climate change predict an expansion of the global areas suitable for vector reproduction. Under these models, areas of Europe and America have already seen an increase in the suitability of vector populations and this trend is going to accelerate in the following decades [8, 9]. This is of special concern as these pathogens meet an immunologically naive local population lacking any previous exposure to such diseases.

2. Reproduction as a target for vector control

The interference in insect vector natural populations has remained one of the key strategies for the control of vector-borne diseases. For the past 50 years, vector control policies have relied on the utilization of insecticide-based tools. With the rising of resistance spreading across populations, a major threat to the ongoing success of control programs has been acknowledged [10].

Although insects are the largest and most diverse group of animals on the planet, most species are regarded as species with a high reproductive capacity. Females can generally produce a large number of eggs in a short period, and the high rates of embryo viability boost their natural populations. Blood-feeding (hematophagy) is necessary for most human disease vectors to obtain the energy and nutrients required for efficient oogenesis, enabling the abovementioned high rates of oviposition [11]. Within a vector reproductive cycle, the overall process of converting protein from the blood meal into yolk protein precursors (YPPs), as well as coordinating their delivery to developing oocytes is the most complex stage of reproduction and requires the coordination of intricate metabolic and neuroendocrine pathways in the adult female. As a result, a comprehensive understanding of the complexity of egg production is the most promising approach to designing safe tools for interference in vector reproduction (Figure 1).

Figure 1.

Targets for intervention within the reproductive cycle of vectors. After digestion, adult females are able to lay a large number of highly viable eggs, thus contributing to the increase and maintenance of vector natural populations. The complex physiology process of transforming the contents of the blood meal into mature fertilized eggs requires intricate coordination to accomplish vitellogenesis, delivery of the yolk to the oocytes (yolk uptake), eggshell biogenesis (choriogenesis), and fertilization (mating habits). Interference in any of those stages directly impairs vectors' egg production capacity and embryo viability, rendering drastically reduced reproduction rates.

The molecular physiology of oogenesis is highly conserved within the different insect vectors [11, 12, 13]. In brief, oogenesis is triggered by signals from nutritional status and the blood meal. The levels of the sesquiterpene juvenile hormone (JH) [14], secreted by the corpora allata in the brain, increase over the early periods of insect maturity triggering changes in the fat body that become sensitive to the ovary-producing steroid hormone ecdysone [15]. After the blood meal, the brain stops JH synthesis and releases the ovarian ecdysiotropic hormone, signaling to the ovaries to produce ecdysone. In the fat body, ecdysone is hydroxylated to 20-hydroxyecdysone (20E) and binds to the 20E receptor EcR/USP to trigger vitellogenesis, that is, the production of the YPPs (yolk protein precursors). YPPs are secreted to the hemolymph and delivered to the developing oocytes in the ovaries via receptor-mediated endocytosis. Apart from the huge metabolic challenge of transforming the blood meal into a large number of eggs, the maximum capacity of egg production is also dependent on successful mating, fertilization, and proper conditions for embryo development [12, 16, 17, 18, 19, 20, 21, 22, 23].

3. Potential targets for intervention within vectors reproduction

3.1 The molecular mechanisms of vitellogenesis and oogenesis

The conversion of protein from the blood meal into YPPs for eggs biogenesis is a vital component of the reproductive cycle and understanding how this process is regulated is necessary to design safe, specific, and effective ways to block reproduction in vectors. Mosquito 20E has been shown to play multiple roles in Anopheles.Apart from regulating monogamy in Anopheles, the male-transferred 20E was shown to be important in maintaining sperm viability over the female lifetime through induction of the heme peroxidase 15 (HPX15) [24]. Accordingly, HPX15 knockdown was shown to dramatically increase the ratio of infertile eggs. Upon insemination, the male 20E further interacts with the female Mating-Induced Stimulator of Oogenesis (MISO) and induces an increase in fecundity by increasing the expression of LP and oocyte numbers [25]. Finally, 20E was also shown to be necessary for Anopheles egg-laying [26].

On that note, several genes that are somehow essential for oogenesis and generate unviable embryos have been identified and functionally tested in R. prolixus. The orthologue of Bicaudal C (BicC), a gene originally identified in D. melanogaster involved in embryonic patterning has shown to be maternally expressed and essential for the arrangement of the follicle cells [27]. The control of iron and heme homeostasis is particularly critical for hematophagous insects, especially for the strictly hematophagous triatomines, such R. prolixus. In this model, the silencing of multiple iron-related genes, namely, ferritin, iron responsive protein 1 (IRP1), heme oxygenase (HO), and heme exporter feline leukemia virus C receptor (FLVCR), impairs oogenesis and embryo viability [28, 29].

The role of receptor-mediated endocytosis in yolk uptake has been investigated in oocytes of many insect species. The internalization of yolk proteins through the presence of a specialized endocytic cortex in the oocytes, which includes prominent microvilli, coated pits, coated vesicles, and endosomes have been shown in several species, including Aedes [30, 31, 32, 33, 34, 35, 36, 37, 38] However, the regulations encompassing the recruitment of the endocytic machinery to specific sites of the oocyte cortex and the signals that govern the oocyte endocytic pathways and endosomal maturation are yet to be addressed. In R. prolixus, ATG6/Beclin1 class-III PI3K complexes I and II were shown to be essential for YPP uptake. Insects silenced for the genes present in both complexes produce yolk-deficient eggs generating unviable embryos due to the lack of generated phosphatidylinositol-3P (PI3P) to recruit the endocytic machinery in vitellogenic oocytes [39, 40].

3.2 Choriogenesis as an emerging target for safe interventions

The final checkpoint of oogenesis, before fertilization, is the triggering of the choriogenesis program, in which the multiple layers of the chorion are secreted by the follicle cells that envelop the developing oocytes. Remarkably, while the chorion’s primary protective function is conserved in insects, its general composition and structure have evolved in a highly species-specific manner, giving rise to a wide range of morphologies and functional adaptations. The main chorion proteins in insects have been identified in models, such as the silk moth Bombyx mori, the fruit fly D. melanogaster, and the mosquito Aedes Aegypti, and revealed to be broadly unrelated to their counterparts in each of these species [41, 42]. Proteins that are conserved in a wide variety of organisms are not ideal target molecules as vector control agents because of deleterious effects on non-target organisms, such as vertebrates, pollinating agricultural insects, and beneficial predators. As a result, studies on the molecular biology of the chorion biogenesis in insect vectors are biotechnology strategic as they are likely to unravel safe molecular targets that are at the same time essential for reproduction and highly specific to one species.

The A. aegypti eggshell is composed of different structural proteins, enzymes, odorant-binding proteins, as well as many uncharacterized proteins of unknown function. Melanization proteins and enzymes of the eggshell have been identified [43, 44, 45, 46, 47, 48, 49], and proteomics studies have been performed [42]. Isoe and colleagues [50] designed in silico analyses to identify mosquito-specific genes that are essential for successful embryo development. After systematic RNAi functional screening of over 40 selected genes, the authors identified a chorion-related protein named eggshell organizing factor 1 (EOF1), which is essential for eggshell biogenesis and embryo development. The EOF1 sequence includes an F-box functional motif, which is characterized by the interaction with the SKP1 protein in the SCF E3 ubiquitin ligase complex [51]. Although its exact function in the eggshell has not been elucidated, such findings are very promising in terms of designing safe strategies for vector control.

In R. prolixus, some aspects of the chorion ultrastructure and permeability properties were previously explored [52, 53, 54] and the identification of the specific chorion proteins Rp30 and Rp45, the latter associated with antifungal activity, was also described [55]. In this model, the cell biology of the follicle cells (FCs), the tissue that synthesizes and secretes the chorion components, has been explored. Early transcriptome analysis showed that the FCs are committed to transcription, translation, and vesicular traffic [56]. Accordingly, electron microscopy evidenced the FC’s typical secretory cell morphology with a high content of vesiculated rough endoplasmic reticulum [57, 58]. Systemic RNAi experiments targeting the autophagy-related genes ULK1/ATG1, the autophagy-dedicated E2-conjugating enzyme ATG3 [57, 59], and E1-activating and E2-conjugating ubiquitin enzymes [60] generated particular phenotypes of chorion malformations due to alterations in the general protein homeostasis of the FCs during choriogenesis, resulting in extremely lower rates of embryo viability. Taken together, the data points to a high degree of complexity in the chorion biogenesis program in R. prolixus, rendering the process extremely sensitive to changes in proteostasis of the FCs, and, thus, an interesting target for slight but effective interventions.

Resistance to desiccation is another potential intervention target. Although mosquito eggs are laid in water, they are susceptible to dehydration in the first hours of development. Thus, this property directly affects mosquito reproduction. In A. Aegypti, the serosal cuticle secretion (an inner layer of the chorion secreted during embryogenesis) coincides with an increase in dry resistance and the presence of chitin as one of the serosal cuticle components has been detected [61, 62, 63]. In R. prolixus, chitin was detected in the ovaries and the embryonic cuticle [64, 65]. Additionally, exposure to lufenuron (a chitin synthesis inhibitor) and chitin synthase RNAi experiments reduced oviposition and embryo viability [66]. Therefore, the synthesis and deposition of chitin or chitin-like components in the eggshells are also promising targets for reproduction interventions.

Altogether, and combined with the above-mentioned high degree of species-specificity of the chorion proteins, choriogenesis has the potential to emerge as the foremost target for the generation of new and environmentally safe strategies to achieve vector control.

3.3 Molecular neuroendocrine control of egg production

Major advancements have been achieved in the understanding of the neurohormonal control of egg production. In R. prolixus, a detailed model depicting the control of oogenesis, ovulation, and oviposition has been designed and elegantly reviewed by Lange and colleagues [13]. Post-genomics has allowed the identification and functional characterization of dozens of reproduction-related neuropeptide receptor families, processing enzymes, and neurochemicals. Historically, R. prolixus has been an important model, wherein the basics of insect physiology have been determined [67, 68, 69, 70]. Interestingly, the integration of the post-genomics findings with the smartly designed early physiology experiments has allowed the elucidation and depiction of many aspects of the global endocrinal integration in this vector.

3.4 Crosstalk between reproduction and immunity

A relationship between reproductive potential and immune status has been long established. Collectively, these studies suggest a tradeoff between immune activation and egg production, reflected by the identification of follicular atresia and other cell death pathways [71, 72]. Recent advances are now highlighting the role of nutrient-sensing pathways and vector immunometabolism [73]. Future studies will provide further insight into how signaling pathways, such as TOR and Insulin pathways, well-known vitellogenic and immune regulators, coordinate energetic balance during infection. Interestingly, previous work using natural combinations of vector-parasite has suggested that coevolution might have minimized the impact of infection [74], possibly by the fact that immune tolerance can induce a less-energetic costly immune response.

Rerouting of yolk components can be used as a nutritional factor for parasite development. Mosquito LP has been incorporated into Plasmodium oocyst as a lipid source [75]. While parasite development was accelerated by LP delivery, it did not induce any detectable reproductive cost [76]. Interestingly, mosquito lipids influenced not only total parasite numbers, but also Plasmodium sporozoite virulence upon transmission to vertebrate hosts [77]. Similarly, VG is a key component for Plasmodium survival. An interplay between YPPs and immune response has been demonstrated. Both LP and VG were shown to reduce the efficiency of the binding of the major parasite-killing TEP1 [78], increasing parasite survival following mosquito infection.

3.5 Interventions on mosquito mating and insemination

Mosquitoes are thought to use a set of sonorous, visual, and chemical cues to identify and attract their partners. While the manipulation of such signals used to guide mosquitoes is an interesting target to prevent mosquito mating, the molecular identity of its components, such as sex pheromones and their odorant-binding receptors are scant. In that sense, both Anopheles and Aedes mosquitoes can adopt a swarming behavior during mating. Aggregation pheromones have been identified in Anopheles [79]. Such compounds can be used to manipulate mating behavior in wild vector populations and are a likely target of vector control strategies. More recently, genes regulating cuticular hydrocarbon productions and the circadian cycle have been described to be coordinated with light and temperature to guide swarming in Anopheles [80]. Aggregation pheromones have also been described in A. aegypti (Fawaz et al., 2014). Interestingly, Aedes swarming does not require swarming before mate and Aedes mosquitoes have been shown to mate in pairs throughout the day [81].

Upon mating, male sperm is transferred to a spermatheca (one in Anopheles, two in Aedes) where it is stored for the lifetime of the female mosquito. The role of odorant receptors in activating spermatozoa flagella has been previously shown [82]. While several candidate agonists were shown to activate flagellar beating, its physiological ligand remains to be further defined. Upon insemination, females are thought to mate once in their lifetime. In most Anopheles species, this is enforced by the formation of a mating plug that forms a barrier to prevent further female insemination [83]. The mating plug is composed of seminal secretions produced by the male accessory glands [84], and 20-hydroxyecdysone (20E) embedded is thought to play a signaling role in inducing monogamy in the female [26]. While a mating plug is not formed in Aedes, a physical barrier is temporarily formed by components of the male sperm produced at the male accessory gland [85]. Later, bioactive proteins collectively known as matrone can modulate female behavior at the neuronal levels and induce monogamy [86, 87]. Nevertheless, the exact molecular composition of matrone remains to be defined. A further understanding of the molecular basis for male-induced monogamy is of great importance, as it could potentially identify chemicals that could be used (e.g., in aerosols) to prevent virgin female mating.

3.6 Sterile insect technique (SIT)

General models from the middle of the twentieth century had already predicted the potential of releasing sterile male releases to suppress insect populations [88]. This approach known as the sterile insect technique (SIT) has been originally accomplished by irradiation of mosquitoes. Sterile animals have been released, mostly by preliminary investigations, in several locations around the world with varied success, as previously discussed [89]. While claims of reduced competitive rates of irradiated mosquitoes are still a matter of debate [90], the biggest issues facing SIT seem to rely on the scalability and sustainability of such efforts [89]. An alternative approach has been the release of transgenic-induced sterile mosquitoes. Still, the logistic challenges of such practices remain a major challenge for field implementations in large geographic areas, with wild-type mosquito populations rapidly returning after release interruption [91]. At present, the development of efficient and flexible gene drive techniques, such as CRISPR/Cas9, remains a promising approach to the development of efficient cost-effective SIT implementations independent of continuous mosquito release [92].

3.7 Wolbachia-induced cytoplasmic incompatibility

The utilization of Wolbachia-induced cytoplasmic incompatibility remains one of the most promising alternatives for insecticide-independent strategies of vector control. Wolbachia is an arthropod-specific bacteria that establish a systemic infection and can be vertically transmitted by infecting the host oocytes [93]. Several strains of Wolbachia are known to induce a phenomenon known as cytoplasmic incompatibility (CI) where the progeny from infected males and uninfected females are turned nonviable [94]. While recent reports have identified populations of A. aegypti carrying native Wolbachia infections [95], these seem to be deviations from a general rule where Wolbachia strains are not naturally able to infect A. aegypti. Nevertheless, Wolbachia strains have previously been adapted to infect A. aegypti by trans-infection in the lab, and CI has been shown to manifest in this model. In that sense, the release of CI-carrying Wolbachia-infected males has been proposed as a strategy to suppress Aedes populations, and field trials have been implemented [96, 97]. The molecular mechanisms mediating cytoplasmic incompatibility started to be elucidated and two key genes linked with the prophage WO have been identified [98, 99]. Transgenesis of such genes would provide alternatives to induce CI in the absence of Wolbachia infections [100]. Such strategies would be beneficial for the cases where stable Wolbachia trans-infections have not been achieved, as is the case of many anophelines.

4. Concluding remarks

The control of vector populations has shrunk the map of many vector-borne diseases [10, 101], but new strategies will need to be developed to continue this process. Although the fundamental biology behind oocyte development is known and mostly conserved, its molecular mechanisms are still to be explored. The recent completion of multiple genome sequencing projects will allow comparative genomics studies that not only increase our knowledge about reproductive processes but also facilitate the identification of novel species-specific targets for vector control. Research directed to understanding how this process is regulated and being able to manipulate the female’s capacity to produce so many viable eggs will lead to safe and effective ways to block reproduction in blood-feeding insects. To accomplish this, there is an urgent need to integrate the post-genomics findings with the species-specific vectors’ physiology. Such tactics are the safest path to unravel evidence-based information and design customized tools to manage vector populations in different endemic areas.

Acknowledgments

This work was funded by the following grants. Fundação Carlos Chagas Filho De Amparo À Pesquisa Do Estado Do Rio De Janeiro (FAPERJ) (JCNE E-26/2031802017; http://www.faperj.br/) to I.R.; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (INCT-EM 16/2014; http://cnpq.br/) to I.R.; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (https://www.gov.br/capes/pt-br) to I.R. The authors thank Vinicius Torrão for the excellent technical assistance with the illustration design.

Conflict of interest

The funders had no role in study design, data collection, analysis, and decision to publish, or preparation of the manuscript.