Mosquitoes transmit pathogens that cause human diseases such as malaria, dengue fever, chikungunya, yellow fever, Zika fever, and filariasis. Biotechnological approaches using microorganisms have a significant potential to control mosquito populations and reduce their vector competence, making them alternatives to synthetic insecticides. Ongoing research has identified many microorganisms that can be used effectively to control mosquito populations and disease transmission. However, the successful implementation of these newly proposed approaches requires a thorough understanding of the multipronged microorganism–mosquito–pathogen–environment interactions. Although much has been achieved in discovering new entomopathogenic microorganisms, antipathogen compounds, and their mechanisms of action, only a few have been turned into viable products for mosquito control. There is a discrepancy between the number of microorganisms with the potential for the development of new insecticides and/or antipathogen products and the actual available products, highlighting the need for investments in the intersection of basic research and biotechnology.
1. Introduction
Microorganisms constitute a large group of genetically diverse biological entities found in a wide range of terrestrial and aquatic habitats, playing crucial roles in the balance of ecosystems
[1][2][3]. Advances in microbiology, molecular biology, and genomics enabled the biotechnological exploration of microbes, allowing the discovery and production of antibiotics
[4][5], foods
[6][7], alcoholic beverages
[8], bioremediators
[9][10], fertilizers
[11], and biopesticides
[12][13]. The microorganisms associated with mosquitoes have drawn special attention to their potential applications in public health (
Figure 1)
[14][15][16].
Figure 1. Microorganisms and their applications for controlling vector populations and disease transmission. Microorganisms are sources of molecules with insecticide (biopesticides) antipathogen (biopharmaceuticals) activities. Interactions of environmental and symbiotic fungi and bacteria with mosquitoes and their microbiota may affect mosquito and pathogen survival, having implications for vector control and disease transmission. Research that elucidates these interactions is crucial because it underpins the development of novel biotechnological products aimed at effective vector control and reducing disease transmission. Created with
BioRender.com (accessed on 5 August 2023).
Associations between mosquitoes and their microbiota have gained significant attention in scientific research due to their impact on vector competence
[17][18][19][20][21][22]. In the following, w
e discuss ways these associations can influence vector competence
are discussed. Understanding these interactions is essential for developing effective vector-borne disease control strategies and reducing their impact on public health.
2. Symbiotic Bacteria and Their Potential against Infectious Agents
The mosquito microbiota influences host development, nutrition, reproduction, and immune responses to invading organisms
[23][24][25][26]. While the composition of the mosquito microbiota is largely defined by the environment in which they live
[27][28][29], resident bacteria can modulate the development and replication of parasites and viruses within their vectors
[30][31][32][33][34][35][36][37]. Although this modulation can enhance or reduce the survival and replication of pathogens within mosquitoes, those mosquito–microbiota interactions that negatively affect pathogens offer possibilities to control arthropod-borne diseases.
For example, the Gram-negative bacteria,
Escherichia coli H243,
E. coli HB101,
Pseudomonas aeruginosa, and
Ewingella americana inhibit the formation of
Plasmodium falciparum oocysts, in
Anopheles stephensi (Liston, 1901)
[38].
Enterobacter sp. (
Esp_Z) isolated from the intestine of
Anopheles gambiae (Giles, 1902) inhibited the development of malaria parasites when reintroduced into this same vector species
[39][40]. The formation of oocysts of
Plasmodium berghei was affected by the presence of
Serratia marcescens-HB3 in
An. stephensi [41]. In
An. gambiae,
Escherichia coli,
S.
marcescens, and
Pseudomonas stutzeri reduced the prevalence and intensity of
P. falciparum infection
[42]. The
Serratia Y1 strain exerts inhibitory activity on
P. berghei ookinetes by activation of the Toll immune pathway in
An. stephensi [43].
Serratia ureilytica (Su_YN1) produces an antimalarial lipase (AmLip) that inhibits the formation of
P. falciparum oocysts in
An. stephensi and
An. gambiae [44].
Asaia SF2.1 also inhibits
Plasmodium development in anophelines
[45].
Virus replication in their vectors is also regulated by the mosquito microbiota. Bacteria of the genera
Proteus,
Paenibacillus, and
Chromobacterium inhibited the replication of dengue virus serotype 2 (DENV-2) when administered to mosquitoes
[46][47]. Some of the mechanisms by which symbiotic bacteria can hamper pathogen development have been elucidated and can be exploited to inhibit the spread of infectious agents by mosquitoes (
Figure 2).
Figure 2. Biotechnological potential of mosquito symbiotic bacteria against infectious agents. (
A), secretion of toxic substances that either kill or arrest the development and replication of viruses and parasites. (
B), formation of physical barriers through large population accumulation or rearrangements of molecules secreted into the midgut lumen, preventing the passage of parasites to organs essential for their successful development. (
C), activation of the mosquito immune system, which not only reduces the load of symbiotic bacteria but also leads to the elimination of invading parasites through the secretion of toxic molecules, preventing their propagation in the mosquito’s body. (
D), competition with infectious agents for space and nutrients can have dire consequences for these pathogens as they must compete with a vastly larger population of symbiotic bacteria in the mosquito’s midgut lumen. This results in limited resources for the pathogens, ultimately leading to their decreased survival and replication within the mosquito. (
E), paratransgenesis involves populating vector insects with genetically engineered symbiotic microorganisms that effectively hinder the development of parasites through synthesizing and secreting antipathogen molecules. Created with
BioRender.com (accessed on 5 August 2023).
3. Wolbachia-Based Strategy for Controlling Mosquito-Borne Viruses: Mechanisms, Efficacy, and Implications
The
wMel and
wAlbB strains of
Wolbachia pipientis, an intracellular bacterium, inhibit dengue, CHIKUNGUNYA, and Zika virus replication within mosquito cells
[48][49][50][51][52]. However, another
Wolbachia strain,
wPip, does not inhibit virus infection in
Ae.
aegypti [53] and the mechanism by which
Wolbachia interferes with virus replication has not been fully elucidated. Current hypotheses include competition between
Wolbachia and the virus for physical space within mosquito cells and metabolite resources
[54][55] and
Wolbachia-induced modulation of the host’s immune system and immune priming. Immune priming entails sensitizing or preparing the mosquito’s immune system for a faster and more efficient response to a specific pathogen, such as a virus, upon subsequent exposure
[56][57].
Despite the lack of a complete understanding of the mechanism or mechanisms involved in
Wolbachia-associated modulation of viral suppression, the
Wolbachia-carrying mosquito-based strategy has been deployed as a public health intervention to control dengue transmission (The World Mosquito Program
https://www.worldmosquitoprogram.org/) (accessed on 15 February 2023). A randomized study carried out in the city of Yogyakarta, Indonesia, compared the areas where
Ae. aegypti carrying
Wolbachia was released with areas without
Wolbachia. The results revealed a 77% lower incidence of dengue cases, in the
Wolbachia-treated area
[58]. Another study conducted in the city of Niterói, Rio de Janeiro, Brazil, reported a 69% reduction in dengue, 56% in chikungunya, and a 37% reduction in Zika incidence three years after the beginning of the release of
Ae. aegypti with
Wolbachia [59].
Although these results bring optimism regarding the use of
Wolbachia for the control of dengue transmission, these bacteria can have variable effects on mosquito-borne viruses. For example, the
Wolbachia strain
wMel strongly blocked Mayaro virus (MAYV) infections in
Ae. aegypti, but another strain,
wAlbB, did not influence MAYV infection in this same vector.
Aedes aegypti infected with
wAlbB and
wMel showed enhanced Sindbis virus infection rates
[60]. The variable effects of
Wolbachia on vector competence bring into question the safety of the current release of
Wolbachia-infected mosquitoes. Furthermore, the potential impact of these bacteria on biodiversity has not been thoroughly investigated
[61][62], and the risk of the emergence of DENV variants that escape virus-specific inhibition in
Wolbachia-infected mosquitoes
[63][64], underscores the importance of further research on interactions between
Wolbachia, mosquitoes, viruses, and other organisms.
4. Symbiotic Microorganisms and Paratransgenesis
Paratransgenesis involves the colonization of vector insects with genetically engineered symbiotic microorganisms that are effective in inhibiting parasite development
[65][66][67][68]. Ideal symbionts for effective paratransgenesis are easily manipulated genetically, colonize mosquitoes efficiently, spread into mosquito populations (vertical and horizontal transmission), and are efficient in inhibiting pathogen development in mosquitoes
[69]. Proof-of-principle experiments performed primarily with bacteria demonstrated that genetically modified microorganisms expressing antipathogen molecules are capable of interfering with or blocking the development of malaria parasites in mosquitoes
[70][71][72]. Among the mosquito symbiotic bacteria, strains of
Asaia,
Pantoea,
Serratia,
Pseudomonas, and
Thorsellia have been evaluated as candidates for paratransgenesis
[73][74][75][76][77].
The fungus
Metarhizium anisopliae has been genetically transformed to express anti-
Plasmodium proteins. Mosquitoes treated with transgenic
M. anisopliae had 71–98% fewer sporozoites present in their salivary glands
[78]. Scorpine, one of the molecules expressed by transgenic
M. anisopliae, also affects negatively dengue virus replication, expanding the application of genetically transformed fungi to control arbovirus transmission
[79].
Densovirus, a small DNA virus (4–6 kb) belonging to the Parvoviridae family (Densovirinae subfamily), infects arthropods such as mosquitoes and is maintained in natural populations through both horizontal and vertical transmission from infected adults to larvae. With one of the smallest known viral genomes, Densovirus serves as a valuable molecular tool. Its compact genome can be inserted into an infectious plasmid, which can then be used to express antiparasitic genes, both in cell cultures and in live mosquitoes
[80][81][82][83].
The discovery of mosquito symbiotic bacteria
[71][84][85][86], viruses
[80][81][82][83], and fungi
[87] is an active area of research. Advances toward deploying paratransgenesis as a tool for blocking pathogen transmission by mosquitoes also include the identification of antipathogen effector peptides and mechanisms of cellular secretion
[66][71]. In bacteria, one efficient way of secreting effector molecules from the bacterial cytoplasm into the lumen of the mosquito intestine was engineered using
Escherichia coli’s hemolysin-A secretion system. This process involves the export of proteins, such as hemolysin HlyA, through a specific mechanism that includes an export signal at the C-terminal end of the protein, along with the membrane proteins HlyB, HlyD, and the outer membrane protein TolC
[88]. The use of bacteria for paratransgenesis is the most advanced alternative compared with viruses and fungi applications. However, concerns about the safety of releasing engineered bacteria into the environment and the potential unforeseen consequences still require attention when contemplating field tests for paratransgenesis.
Self-limiting paratransgenesis
[69] has been suggested as an alternative for initial field trials. This approach proposes the utilization of transient expression of antipathogen compounds from a plasmid that is gradually lost, reverting bacteria to their original wild type. Risk assessment still needs to be carried out and laws and regulations need to be created and enacted before paratransgenesis can be tested in field conditions. However, the processes by which genetically modified microorganisms (GMs) can be spread in nature and how they should act to inhibit the development of target parasites in mosquitoes have already been envisaged. This is illustrated in
Figure 3, which presents the paratransgenesis process as a multifaceted approach to combating mosquito-borne diseases.
Figure 3. Strategies for dissemination of GM microorganisms in the wild and their continual circulation among mosquitoes. (
A) Male and female mosquitoes are fed in the laboratory with a sucrose solution containing the GM microorganisms and then released into the wild to mate with other wild mosquitoes. The spread of GM microorganisms can also occur through the provision of sucrose baits in the field enriched with GM microorganisms. This enables the GM microorganisms to be transmitted forward, allowing them to spread throughout the wild mosquito population and aiding in reducing vector-borne disease transmission. (
B) Release of GM microorganisms into natural larval breeding sites. The GM microorganisms are ingested by the larvae and remain associated with them until adulthood. If mosquitoes become infected with a parasite that is a target of the effector molecules produced by the GM microorganisms, these molecules will interfere with the development of the target pathogen, thereby preventing its transmission. (
C) Persistence of GM microorganisms in mosquitoes for generations through vertical and horizontal transmission. Vertical transmission occurs from parents to offspring, while horizontal transmission takes place between mosquitoes during mating or sharing of breeding sites. The presence of GM microorganisms can continue to impact mosquito populations for an extended period. Created with
BioRender.com (accessed on 5 August 2023).