Nature's Solution to Aedes Vectors: Toxorhynchites as a Biocontrol Agent

This review summarizes the predatory potential of Toxorhynchites mosquitoes as biological control agents for Aedes vectors. A single larva can consume hundreds of mosquito larvae during its development, with preference for larger prey and higher consumption rates at higher prey densities. Studies suggest Toxorhynchites can significantly reduce Aedes populations. Beyond direct predation, they may indirectly influence prey behavior and adult mosquito lifespan. Despite the demonstrably positive effects of Toxorhynchites species as biocontrol agents, there are acknowledged limitations that require further investigation. These limitations include potential variations in effectiveness across diverse habitats and mosquito developmental stages. Additionally, long-term ecological sustainability and potential ramifications warrant further research. Future efforts should prioritize optimizing rearing and release strategies to enhance effectiveness. Exploring the potential for combined control methods with other biocontrol agents or traditional methods is also crucial. Finally, investigating the influence of environmental factors on predation rates can further refine and optimize the application of Toxorhynchites in mosquito control programs.


Introduction
Mosquitoes are notorious vectors for a multitude of diseases worldwide, as the genera Aedes, Anopheles, and Culex are important vectors of mosquito-borne diseases [1].Among them, Aedes spp.are vectors for many arboviruses.Te two major species, Aedes aegypti (Linnaeus 1762) and Aedes albopictus (Skuse 1894), are related to emerging or reemerging infectious diseases resulting serious public health concerns [2].Aedes mosquitoes are diurnal, target both animals and humans for blood meals, and have earned notoriety for their capacity to transmit over 20 viruses and flarial worms, including those with severe implications to human health [3].Two species Aedes aegypti and Aedes albopictus were originated from the African continent and Southeast Asian forests, respectively.Tey are profcient vectors of chikungunya virus, dengue virus, yellow fever virus, and zika virus, inficting signifcant health repercussions and economic losses worldwide.
Dengue fever, the most prevalent mosquito-borne disease, has seen a tenfold increase in reported cases globally between 2000 and 2019 [4].Tis translates to more than 3.9 billion people in over 129 countries being afected, with over 40,000 deaths every year [5].While Asia carries about 70% of world's disease burden, the Americas, Southeast Asia, and the Western Pacifc are hardest hit.Te situation worsens as dengue fever creeps into new areas like Europe, while existing regions experience intense regular outbreaks [6].According to the WHO, the Americas experienced the most cases in 2023 with over 4 million reported, highlighting this growing threat.Southeast Asia remains a major hotspot with countries like Tailand and Vietnam experiencing high numbers of casualties [4].Africa also faces a rising burden with outbreaks in 15 countries.Similarly, the Eastern Mediterranean region is experiencing an increase, with nine countries regularly facing outbreaks.While Europe is not considered endemic, a few countries like Italy, France, and Spain did report outbreaks in 2023 [4].Tus, this rise in mosquito-borne diseases is of global concern.Chikungunya, another Aedes-borne disease, saw major outbreaks in East Africa, the Indian Ocean (2005)(2006), and the Americas and Oceania (2013-2014) [7][8][9].Zika also caused major outbreaks in the Americas (2015-2016) [9].Moreover, yellow fever has reemerged with recent epidemics in sub-Saharan Africa and Brazil [10].
Te primary strategy to minimize the risk of mosquitoborne infections involves reducing mosquito populations [11].Te emergence of insecticide resistance and the detrimental efects of conventional insecticides on the environment have fueled a growing interest in biological control methods for mosquito vector management [12].Various biocontrol agents from the insect orders Diptera, Odonata, Coleoptera, and Hemiptera have been explored globally to control mosquito populations [13].From the outset, scientists have recognized the value of utilizing the natural predator-prey relationships within the environment for efective mosquito control.Tese natural predators including juvenile fsh, Odonata larvae (dragonfy and damselfy nymphs), and mosquito larvae ofer a sustainable and environmentally friendly approach to control mosquito [14][15][16].A fascinating paradox exists among these predators: the Toxorhynchites mosquito.Tese giants of the mosquito world, nicknamed "elephant mosquitoes" or "mosquito eaters," possess a proboscis specifcally adapted for nectar, not blood.Toxorhynchites mosquitoes, primarily tropical dwellers found in the lush forests of equatorial and tropical regions, have a distribution that generally falls within a band between 35 °north and 35 °south latitude [17].However, there are a few exceptions.Some species, like Toxorhynchites rutilus, has adapted to the temperate zones of the Northern Hemisphere [18] with a wider range within North America, stretching from Mexico all the way to the Atlantic coast [19,20].While a few species have ventured beyond the tropics, Southeast Asia remains a hotspot for Toxorhynchites diversity, boasting as many as 24 documented species [21].
Te larvae of Toxorhynchites mosquitoes hold immense potential for mosquito control [22,23].Tese larvae are exceptional predators throughout their development, particularly adept at preying on mosquito larvae of public health signifcance, such as Aedes aegypti (L.), Aedes albopictus (Skuse), and Culex quinquefasciatus [24,25].Interestingly, Toxorhynchites often lay eggs in the same containers favored by Aedes aegypti and Aedes albopictus [26,27].A single Toxorhynchites larva can devour a staggering number of prey larvae-up to several thousand during their whole larva stage [22].Teir predatory prowess extends beyond direct consumption.Toxorhynchites larvae exhibit a fascinating behavior known as compulsive killing, where they kill mosquito larvae but leave them uneaten, especially before pupation [28].Additionally, they can indirectly infuence prey development, further hindering their population growth [29].Consequently, Toxorhynchites larvae represent a promising model for Aedes mosquito control.Terefore, the aim of the current review is to analyze the role of Toxorhynchites larvae in the control of Aedes mosquito around the world.

Methods
We conducted a comprehensive search for articles in Science Citation Index (SCI)-indexed journals, focusing on Aedes mosquito control and the potential of Toxorhynchites as a biocontrol agent.Utilizing PubMed and Research4Life, we searched using the keywords "Aedes control" and "Toxorhynchites" to capture relevant strategies and the specifc role of Toxorhynchites.Te initial search yielded 269 articles.To maintain high scientifc rigor and align with the review's objectives, we applied specifc inclusion/exclusion criteria related to Aedes control and Toxorhynchites' biocontrol role.Tis resulted in a fnal selection of 42 articles for detailed analysis (Figure 1).To deeply understand existing research, a standardized data extraction method was employed, collecting information on study design, methodology, key fndings, and limitations from each article (Supplementary Table 1).

Exclusion Criteria
(i) Studies focused on Toxorhynchites for purposes other than Aedes mosquito control (e.g., their biology, taxonomy, and distribution).(ii) Studies only mentioned Toxorhynchites in passing without any data on their use in biocontrol.(iii) Studies published in journals that are not SCIindexed.(iv) Studies published in languages other than English (unless translations were available).13

Toxorhynchites minimus
Sri Lanka [46] 4 Journal of Tropical Medicine recruited to the fght against mosquitoes, demonstrating the genus's promising role in biocontrol solutions.

Toxorhynchites Record from Nepal.
Only one species of Toxorhynchites (Tx.splendens) has been documented in Nepal.Toxorhynchites splendens was frst recorded in 1956 by Peter and Dewar [64].It has been documented in Sunsari, Rupandehi, Jogikuti, Sindhuli, and Khuntpani in Nepal, as reported by Darsie and Pradhan in 1990 [65].It has also been reported from the Banke district by Darsie et al. in 1996 [43].However, there have been no published records of Toxorhynchites in Nepal after 1996.

Promising Species for Biocontrol.
Several Toxorhynchites species show high potential as biological control agents for mosquito populations, particularly Aedes aegypti, and the vector of dengue hemorrhagic fever.Teir key strengths lie in their predatory efciency and eco-friendly nature.Many Toxorhynchites larvae are voracious predators, consuming hundreds of mosquito larvae per individual throughout their lifespan [44,45].Field trials documented reductions in Aedes populations by up to 83% after Toxorhynchites introductions [46].Unlike chemical insecticides, Toxorhynchites pose minimal risk to nontarget organisms and the environment [36,47].Tis makes them particularly attractive for areas with environmental, biodiversity, and other concerns.

Toxorhynchites splendens. Toxorhynchites splendens
larvae are known to be efcient predators, consuming a signifcant number of prey larvae daily.Studies show impressive predation rates in the laboratory.For example, single third-instar larvae in West Bengal, India, have been observed to consume over 50 Ae.albopictus larvae daily [44].Similar results were observed in Okinawa, Japan, where research recorded that fourth-instar Tx. splendens larvae could consume as many as 55 Ae. albopictus larvae daily, while third-instar larvae exhibited a lower predation rate, consuming around 20 larvae per day [45].Another study in Sri Lanka demonstrated that third-and fourth-stage larvae of Tx. splendens could devour one Ae.albopictus larvae in about 30 minutes [46].Even with some studies showing lower predation rates [48], the efectiveness of Tx. splendens translates encouragingly from laboratory to real-world settings.Semifeld experiments showed signifcant reductions in Ae. albopictus larvae [44].Field trials documented up to 83% decline in Aedes populations following Tx.splendens introductions [46].Additionally, a negative correlation between Tx. splendens and Ae.albopictus larvae in ovitraps was observed [26].Even monthly adult releases efectively reduced target mosquito broods in containers [49].However, factors such as container type and prey density (ofering 10 to 50 prey individuals per predator) infuenced Tx. splendens predation rates, with horizontal containers with wide openings being more suitable [48].
Additionally, experiments explored Tx. splendens' feeding activity in the presence of alternative food, reaffrming its role as a mosquito larvae predator.Tey exhibit a clear preference for consuming mosquito larvae, and their consumption is inversely proportional to the search area and directly proportional to prey density (the number of prey given) [44,50].Interestingly, fourth-instar Tx. splendens larvae might also kill prey without consuming them [45].Researchers also investigated Tx. splendens' behavior to optimize control programs, revealing hunting preferences for Aedes aegypti over Aedes albopictus, with predation increasing with prey density [51].Tx. splendens larvae prefer Aedes larvae of the respective stages [45,51], with a preference for Ae.aegypti larvae over Ae. albopictus and Anopheles sinensis due to more active movements [47].However, Tx. splendens larvae exhibited a slightly lower predation ability for Ae.aegypti compared to Culex quinquefasciatus (10.6 vs. 12 larvae/day) [50].
Research suggests Tx. splendens larvae may even kill prey without consuming them.While this highlights their effectiveness, the long-term ecological implications need thorough investigation.Furthermore, complete eradication of target mosquito populations might not be achievable solely with Tx. splendens releases [46].Integrated control programs combining Tx. splendens with other methods may be necessary.However, potential ecological impacts and limitations on complete eradication necessitate a cautious approach with Tx. splendens as a biocontrol agent.

Toxorhynchites amboinensis.
Laboratory studies reported that a single predator larva can consume more than 230 prey larvae on average throughout its larval stage at lower prey density (20 Ae. aegypti larvae/200 ml water/ Toxorhynchites larva).Interestingly, it moves more than 350 times at higher prey density (Ae.aegypti larvae/200 ml water/Toxorhynchites larva) [50] regardless of the prey species ofered (Ae.aegypti, Ae. albopictus, and Cx.quinquefasciatus).Tese fndings highlight Tx. amboinensis as a potential predator for a variety of mosquito larvae, such as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus.Te fourth-instar larvae were found to be the most predacious, with compulsive predation observed during the late stage of this instar [30], confrming its predatory efcacy and pinpointing the fourth-instar larvae as the most efective stage.Interestingly, these studies also revealed a preference for mosquito larvae by Tx. amboinensis, with higher predation rates at higher prey densities.It suggests Tx. amboinensis can be a valuable component of an integrated mosquito control strategy, potentially ofering both environmental and health benefts by reducing reliance on chemical insecticides due to its preference for mosquito larvae and increased predation at higher prey densities.
Field trials investigated Tx. amboinensis as a promising biological agent for mosquito control.Studies in New Orleans [31] demonstrated signifcant reductions (up to 45%) in Ae. aegypti densities following weekly releases of Tx. amboinensis larvae.Tis predator's efectiveness was further amplifed when combined with reduced insecticide use.When integrated with Malathion, mosquito control reached up to 96% compared to 29% with Malathion alone [32].Interestingly, increasing the number of Tx. amboinensis Journal of Tropical Medicine released did not yield additional control, suggesting researchers could develop optimized release strategies [31].However, the success rate of Aedes spp.control by Tx. amboinensis depends greatly on the type of habitat, as the mean overall reduction of Aedes by the introduction of Tx. amboinensis was recorded as 22% in tins and 63% in tires over a 10-month period in Wailoku Village and Yanuca Island [33].Habitat suitability assessments were therefore essential for determining the feasibility of Tx. amboinensis as a control agent in specifc locations.Overall, Tx. amboinensis demonstrated promise as a biological control agent for vector mosquito species.Its efectiveness as a predator, potential for integration with reduced insecticide use, and environmentally friendly approach make it a valuable candidate for further development.However, successful implementation requires careful consideration of release strategies, habitat suitability, and ecosystem dynamics.Future research should focus on optimizing these aspects to maximize the potential of Tx. amboinensis for sustainable mosquito control.

Toxorhynchites moctezuma.
A dose-dependent efect, with more Tx.moctezuma larvae leading to a sharper decline in Ae. aegypti emergence, was documented in the experiment conducted at the Caribbean Epidemiology Centre in Spain.One or two Tx.moctezuma larvae could halt adult Ae. aegypti emergence for a week, and fve or ten larvae could prevent emergence for up to 16 weeks [37].Additionally, sustained releases of Tx. moctezuma larvae for fve months resulted in lower mosquito indices in released villages relative to unreleased ones [38], suggesting long-term efectiveness.Tx. moctezuma larvae exhibit minimal cannibalism, but their fourth-instar stage displays compulsive killing behavior [37].Tese contrasting traits present both challenges and opportunities for their use as biocontrol agents.Te successful suppression of Ae. aegypti populations through systematic releases of Tx. moctezuma larvae on Union Island [39] further bolstered the case for this biological control method.However, a decline in Tx. moctezuma's efectiveness over time was observed [36], highlighting the need for research on maintaining long-term control.Most studies focused on single-release events or short-term trials [37,39].Terefore, large-scale feld studies were needed to assess long-term efcacy and ecological impact.Additionally, cost-efective rearing and release strategies were not addressed in the presented studies.Overall, the evidence strongly supports the potential of Tx. moctezuma larvae as a powerful biological control agent for Ae.aegypti.However, further research is necessary to address limitations like diminishing control over time, develop practical rearing and release methods, and conduct a thorough assessment of potential ecological impacts before widespread implementation.[40,66].A single larva could consume or kill nearly 50 mosquito immatures daily, exceeding 300 during development [40].Another research documented as many as 400 prey larvae during their larval development and also demonstrated killing prey without consuming it [40,66], which could signifcantly reduce potential adult Ae. aegypti emergence.Te fourth-instar Tx. rutilus were more likely to kill pupae than the larvae of the same age.Interestingly, they completely ignored the frst instar altogether [40].Tis preference for larger prey size suggests a potential limitation for Tx.rutilus as a biocontrol agent-while powerful; it might not be equally efective against all mosquito life stages.Additionally, studies documented cannibalism occurring in confned spaces with limited resources [66].Understanding and mitigating these behaviors are crucial for optimizing the efectiveness and sustainability of Tx. rutilus-based interventions.However, natural populations of Tx. rutilus were often insufcient for mosquito control, necessitating the rearing and release of additional adults.Tis approach increases complexity and cost.Additionally, the efectiveness of Tx. rutilus releases relied on precise timing, requiring releases before mosquito populations surged [41].Adult dependence on nectar sources added another layer of complexity [41].Availability of suitable nectar sources can limit their efectiveness in certain areas.While such programs ofer potential advantages like dispersal and positive public perception, successful implementation requires careful planning, monitoring, and adaptation to local conditions.A study investigating the combined efects of pyriproxyfen (an insecticide) and Tx.rutilus on Ae. aegypti found that the combined approach signifcantly inhibited adult emergence compared to using either method alone [42].However, further research with larger sample sizes and diverse feld conditions is necessary to confrm these fndings and assess the long-term efcacy and ecological implications of this combined approach.Overall, Tx. rutilus showed promise as a biological control agent, but its limitations necessitate a multifaceted approach.Future research can refne rearing and release strategies, alongside exploring complementary control methods, to maximize efectiveness of Tx. rutilus in managing Ae.aegypti populations.

Toxorhynchites brevipalpis.
A detailed analysis conducted at the Department of Biology, University of Notre Dame, Indiana, revealed that predation rates of Tx. brevipalpis on Ae. aegypti larvae varied with temperature, with the highest predation (n � 358) observed at 30-32 °C during larval development [27].Interestingly, Tx. brevipalpis also killed prey larvae nearing pupation without consumption [27].Similarly, combining Metarhizium brunneum fungus with Tx. brevipalpis larvae resulted in signifcantly lower Ae.aegypti larval survival rates than using either approach alone [35].However, generalizing these laboratory fndings to feld applications requires careful consideration of ecological complexities and practicalities.Future research should prioritize feld trials to validate the efcacy and sustainability of combined control strategies.

Toxorhynchites violaceus.
Experiments performed in the laboratory to fnd the survival rate of fourth-instar larvae of Ae. aegypti in the presence of fourth-instar larvae of Tx. violaceus showed that although the initial survival rate was 98% in 24 hours, it decreased subsequently, reaching 0% by 192 hours [53].Tis suggests a signifcant increase in the predatory potential of Tx. violaceus fourth-instar larvae for consuming Ae. aegypti larvae, ultimately leading to the elimination of all Ae.aegypti larvae.While this laboratory experiment was promising, the real-world efectiveness of Tx. violaceus remained uncertain.Furthermore, feld testing is imperative as natural environments introduce complexities that laboratory settings cannot fully replicate.
3.3.7.Toxorhynchites towadensis.Tx. towadensis exhibited a curious behavior of killing prey without consuming them at high prey densities.While the study ofers valuable insights, its generalizability is limited by the artifcial setting.Real-world environments present greater complexities in prey availability, habitat structure, and potential interactions with other species.Future research should explore how these factors interact with prey density to provide a more holistic understanding of Tx. towadensis' predatory behavior in natural settings.Moreover, the study solely focused on prey density.Investigating the infuence of prey type and environmental conditions on Tx. towadensis' behavior and development would provide a more comprehensive picture of its potential role in mosquito population control strategies [52].
3.3.8.Toxorhynchites theobaldi.Aedes aegypti mosquitoes, instead of avoiding predators, preferred oviposition sites with evidence of Tx. theobaldi predation (including dead conspecifc larvae) due to the increased bacterial abundance by Tx. theobaldi feeding activity [54].Te study sheds light on the intricate web of indirect efects within predator-prey interactions, where predation can infuence prey behavior through alterations in the microbial community.Tis suggests a potential double beneft for employing Tx. theobaldi as a biocontrol agent: direct reduction of prey populations and the inadvertent attraction of egg-laying mosquitoes to areas where their ofspring are more susceptible to predation [54].However, limitations exist.Te laboratory setting might not fully capture the complexities infuencing oviposition choices in natural environments.Additionally, the study focused solely on bacterial cues.Future research should explore the specifc bacterial strains involved and how they interact with other environmental factors infuencing mosquito behavior.

Combined Study of Diferent
Species.An investigation of feeding strategies in fve Toxorhynchites mosquito species (Tx.amboinensis, Tx. rutilus, Tx. theobaldi, Tx. brevipalpis, and Tx.splendens) found no signifcant diference in the average number of strikes needed for a successful capture across all species.However, Tx. amboinensis (37 prey/day) and Tx.brevipalpis (35 prey/day) captured prey at a significantly higher rate than Tx.splendens (27 prey/day), Tx. theobaldi (25 prey/day), and Tx.rutilus (19 prey/day) [34].Interestingly, Tx. theobaldi exhibited a higher daily consumption rate (18.9 ± 0.97 prey/day) than other predators, even killing prey beyond their immediate needs before pupation [34].Tese fndings suggest potential variations in feeding strategies among Toxorhynchites species.Future research should delve deeper, investigating factors like predatory activity, handling time, and prey type to provide a more comprehensive understanding of these species' feeding strategies.Furthermore, examining if these patterns hold true across all developmental stages, from larvae to adult, is crucial for a complete picture.

Usefulness of Other Insects Other than Toxorhynchites.
Some other invertebrates such as copepods, dragonfies, and damselfies are also predators of mosquito larvae.Copepods are highly efective predators, capable of signifcantly reducing larval populations, even achieving complete elimination in some cases [67].While copepods primarily target mosquito larvae, they also feed on other aquatic organisms, which help to maintain ecological balance in water bodies [68,69].However, their ability to control mosquito populations might be reduced because they are not exclusively mosquito predators and may consume other organisms [67,70].Additionally, only large copepod species (over 1 mm) can efectively prey on mosquito larvae, limiting the range of species suitable for biocontrol [67].Te impact of copepod introduction on nontarget species and ecosystem dynamics requires careful assessment to avoid unintended consequences [71].Odonata larvae, encompassing dragonfies and damselfies, are voracious mosquito predators, efectively reducing mosquito populations [13].Teir extended larval development makes them ideal biocontrol agents, allowing for continuous mosquito predation [72].However, this long development period can be a drawback.Dragonfy larvae in temporary ponds may not survive extended droughts, hindering control efectiveness [73].Additionally, some Odonata larvae exhibit prey preferences, favoring other aquatic insects over mosquito larvae [74].Te research on copepods, dragonfies, and damselfies suggests they could be useful for mosquito control, but there are also some drawbacks like impact on other organisms and long development times.
Toxorhynchites mosquitoes are a promising choice for biocontrol against mosquitoes compared to Copepoda and Odonata (dragonfies and damselfies).Tey target mosquito larvae specifcally, minimizing disruption to the ecosystem.Additionally, their faster life cycle allows for quicker population control compared to Odonata whose development can be slow.Tese factors make Toxorhynchites a compelling option for eco-friendly mosquito management.Tese studies highlight Toxorhynchites species as mosquito predators [27,33,41,44,48,50].Among Toxorhynchites species, Tx. splendens stands out for its well-documented success in controlling Aedes larvae, though efectiveness might vary against Ae.aegypti.Tx. amboinensis ofers a broader range of mosquito prey but requires study on its ecological impact.Tx. moctezuma shows promise for long-term suppression but may necessitate frequent releases.Tx. rutilus boasts high predation rates but has limitations in target stages and requires careful management to avoid cannibalism.Tx.

Journal of Tropical Medicine
brevipalpis excels in warm climates and might work well with other control methods, but feld data are limited.Tx. violaceus and Tx.towadensis show promise in the laboratory but need real-world testing.While Tx. theobaldi ofers an intriguing indirect control mechanism, its predatory impact is less direct.However, the potential of many Toxorhynchites species remains unstudied, ofering a wealth of potential for future mosquito control research.

Conclusion
Various Toxorhynchites mosquito species exhibit promising potential as biological control agents for mosquito populations, particularly Aedes aegypti, the vector of dengue hemorrhagic fever.Teir key strengths lie in their voracious predatory nature and minimal ecological impact.Species like Tx. splendens, Tx. amboinensis, and Tx.moctezuma have demonstrated signifcant reductions in Aedes populations in both laboratory and feld trials.Based on the available data, among Toxorhynchites species, Tx. splendens stands out for its well-documented success in controlling Aedes larvae and Tx.amboinensis appears to be the most suitable species for controlling Aedes mosquito populations due to its high predation rates, efectiveness against various mosquito larvae, and successful feld trials.However, limitations exist, including potential variations in efectiveness across habitats and developmental stages, as well as the need for further research on long-term sustainability and potential ecological ramifcations.Future research should focus on optimizing rearing and release strategies, exploring combined control methods, and investigating the infuence of environmental factors on predation rates.By addressing these limitations, Toxorhynchites species can become a valuable component of integrated mosquito management programs, ofering a safe and eco-friendly approach to curbing mosquito-borne diseases.
(i) Studies investigated the use of Toxorhynchites larvae or adults for controlling Aedes mosquito populations.(ii) Studies evaluated the efectiveness of Toxorhynchites in reducing Aedes mosquito breeding or biting rates.(iii) Studies conducted in laboratory or feld settings that explored the predatory behavior of Toxorhynchites toward Aedes mosquito larvae.(iv) Studies published in English in the last 51 years (1972 to 2023).

Table 2 :
Global utilization of Toxorhynchites species for biological control.