Introduction

Mosquitoes, belonging to the order Diptera and family Culicidae, are common insects found worldwide, except in Antarctica (Hawkes and Hopkins 2022). As of January 2024, 113 genera and 3726 species of mosquitoes have been recorded globally (Harbach 2024). Fortunately, less than 10% of mosquito species spread human diseases (Yee 2022), and among them, the three genera (Anopheles, Aedes and Culex) comprise significant mosquito vectors of different diseases (Tandina et al. 2018). Anopheles mosquitoes are vectors for malaria (Sinden 2007; Ross 2024), a potentially fatal disease caused by Plasmodium. Culex mosquitoes are vectors for lymphatic filariasis (Manson-bahr 1962; Haynes 2001), a chronic inflammatory disease caused by filarial worms. Aedes mosquitoes transmit more than 26 arboviruses and filarial worms to various animals, including humans (Kraemer et al. 2015; Tippelt et al. 2020). Climate, vegetation, and human activities are key determinants of the distribution of these mosquitoes (Sukupayo et al. 2024). The genus Aedes comprises around 970 species (Harbach 2024), including the medically important Aedes aegypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1895). These two Aedes mosquito species are effective carriers of lethal disease-causing agents such as the chikungunya virus, dengue virus, yellow fever virus, zika virus, and others, resulting in a considerable negative impact on human health and significant economic losses worldwide (Mordecai et al. 2020). Among different arboviral diseases transmitted by Aedes mosquitoes, dengue fever is the most significant mosquito-borne viral disease worldwide. This group of mosquitoes caused millions of disease cases with thousands of deaths in 2022 (ECDC 2023). Aedes aegypti and Aedes albopictus mosquitoes are the principle vectors responsible for transmitting dengue disease in numerous urban areas across Southeast Asia (Smith 1956; Mohamad and Zuharah 2014). These vectors are known to breed in containers, whether natural or artificial, found in and around human settlements (Naish et al. 2014; Zuharah and Sumayyah 2019). A. aegypti is native to the African continent, whereas A. albopictus is native to the Southeast Asian forest (Russell et al. 1969; Tedjou et al. 2020). A. aegypti mainly breeds indoors and outdoors, while A. albopictus typically breeds outdoors, although it is well adapted to the peridomestic environment (Ebi and Nealon 2016). Both species are well adapted to human habitation for oviposition and larval development in natural and artificial water bodies (Naish et al. 2014). Although both feed from morning until dusk, A. albopictus also bites at night, indicating its role in transmitting diseases throughout the day and night (Higa 2011). Since dengue vaccines are still being developed, the most effective method currently available to prevent this disease remains controlling the mosquito vector (Mohamad and Zuharah 2014). Various pesticides have been used for mosquito control. However, this approach presents challenges such as rising environmental pollution, chemical and labor costs and public reluctance to have chemicals in their domestic water containers (Focks 2007). Additionally, mosquito vectors have resisted several pesticides (WHO 2012). Consequently, the rising threat of Aedes-borne diseases necessitates urgently exploring alternative vector control methods specifically targeting these mosquito species.

In this context, the use of biological control agents, such as mosquitoes from the genus Toxorhynchites (Diptera), is one of the most promising alternatives to chemical methods in controlling container-breeding mosquitoes like Aedes. Within the diverse realm of mosquito species, a unique paradox emerges in the form of Toxorhynchites. These mosquitoes are the largest in the world, often referred to as "elephant mosquitoes" or "mosquito eaters" because of their size and long trunk-like proboscis (Focks 2007). The genus Toxorhynchites comprises approximately 100 species distributed across four subgenera (Harbach 2024) and demonstrates an impressive global distribution (Tyagi et al. 2015). They are found across various continents, including in countries such as Indonesia, India, and Thailand in Asia, and Tanzania in Africa. In North America, these mosquitoes can be found in locations such as Florida, USA and Mexico (Tyagi et al. 2015). Notably, species like Toxorhynchites splendens exhibit an even wider distribution, extending to countries like Bangladesh, Nepal, Myanmar, and Sri Lanka (Tyagi et al. 2015).

Adult Toxorhynchites mosquitoes do not feed on blood. Instead, they only feed on nectar from flowers or fruits using their sizeable and downwardly curved proboscis (Collins and Blackwell 2000; Focks 2007), making them harmless to humans. Unlike other blood-hungry females, Toxorhynchites mosquitoes pack a protein punch from their larval stage, allowing them to lay eggs without needing a single blood meal (Watts and Smith 1978; Mercer et al. 2005).

Interestingly, the larvae are dynamic predators, preying on various aquatic invertebrates, including mosquito larvae (Padgett and Focks 1981; Focks 2007; Millado and Sumalde 2018), making them natural regulators of mosquito populations. The Toxorhynchites spp. larvae are ambush predators, meaning that they wait quietly for prey to come to them (Zuharah et al. 2015). These voracious hunters do not actively search, but once the prey comes close, they unleash their powerful mandibles in a swift strike (Zuharah et al. 2015; Vinogradov et al. 2022). The entire process happens quickly, with the captured prey consumed within a few minutes (Steffan and Evenhuis 1981; Zuharah et al. 2015). This ambush-hunting behavior makes Toxorhynchites larvae natural regulator of mosquito populations, consequently helping to control the spread of numerous mosquito-borne diseases (Focks 2007; Schiller et al. 2019; Vinogradov et al. 2022).

Toxorhynchites mosquitoes show a distinct preference for laying their eggs in a wide variety of both artificial and natural containers, similar to their prey, Aedes aegypti and A. albopictus (Trpis 1972; Nyamah et al. 2011). Moreover, almost any container holding water can serve as a breeding site, especially if it is partly shaded (Trpis 1972; Donald et al. 2020). Turbid water in both immature stages and predator–prey interactions efficiently attracts female T. splendens for egg deposition (Phasomkusolsil et al. 2022). Surprisingly, bacterial by-products of Toxorhynchites feeding attract Aedes mosquitoes, drawing them to containers containing Toxorhynchites larvae (Albeny-Simões et al. 2014). These factors highlight the significance of Toxorhynchites mosquitoes in controlling Aedes larvae. However, there is a lack of clarity regarding how the type of container, water depth, and water volume affect the reproductive behavior of Toxorhynchites. Therefore, this research aims to investigate how container type, water depth, volume, and even diurnal variations impact the predatory behavior of Toxorhynchites against Aedes larvae. Understanding these influences is crucial for optimizing the utilization of naturally occurring Toxorhynchites spp. as biological control agents. The experiment could potentially lead to widen our understanding on Toxorhynchites spp. and their predatory behavior in different environmental settings. Such information is needed to control mosquitoes as well as reductions in mosquito-borne diseases, particularly in low and middle-income countries such as Nepal, where cost-effective measures are essential for disease prevention.

Materials and methods

Collection and rearing of larvae

In July 2023, predatory larvae of Toxorhynchites spp. and prey larvae of Aedes spp. were collected using a dipping and pipetting methods from discarded tires near Tamakoshi Bazar (27° 37′ 2.82′′ N, 86° 04′ 37.23′′ E, 875 m asl) along Lamosangu-Ramechhap Highway in Dolakha, Nepal. In addition to the larvae collected in Dolakha, more Aedes larvae were collected from Sipadol (27° 39′ 8.01′′ N, 85° 26′ 33.00′′ E, 1380 m asl), Bhaktapur. All collected larvae were kept in separate plastic bottles and brought to the Nepal Academy of Science and Technology laboratory, Lalitpur, Nepal. In the laboratory, the collected fourth-stage larvae of Toxorhynchites spp. were placed in a glass container (beaker) and left for 24 h to acclimatize them to the new laboratory conditions. Aedes larvae were also introduced into the containers as their food source.

On the other hand, the collected third and second-stage larvae of Toxorhynchites spp. were reared in a single beaker to facilitate their transformation into fourth-stage larvae. Aedes larvae served as their primary food source during this period. After the emergence of fourth-stage larvae, the experiment was repeated for each larva as mentioned above.

Impact of container types, water volumes, and depth on Toxorhynchites spp. predation

We investigated how different container types and variations in water volumes and depths could influence predation behavior within controlled laboratory conditions. This is because the Aedes spp. are container-breeders and the current data of predatory behavior of Toxorhynchites spp. on the former species in new environment can be translated for prevention strategies. The laboratory environment was maintained at a temperature of 25 ± 2 °C. Three different types of containers were selected: glass tumblers (14.5 cm height × 6.5 cm diameter), glass bowls (7 cm height × 16 cm diameter), and plastic bottles (13.5 cm height × 9.5 cm diameter). Each container type was filled with specific volumes of dechlorinated water, resulting in varying water depths. For the glass tumblers, 100 ml, 200 ml, and 300 ml with corresponding water depths of 4.0 cm, 8.0 cm, and 12.0 cm, respectively, were used. The glass bowls were filled with 150 ml and 300 ml of water. The water depths were 1.0 cm and 2.0 cm, respectively. Similarly, the plastic bottles were filled with 150 ml and 300 ml of water, with depths of 1.5 cm and 3.0 cm, respectively. Thus, the experiment included three container types, four different water volumes (100 ml, 150 ml, 200 ml, and 300 ml), and seven different water depths (1.0 cm, 1.5 cm, 2.0 cm, 3.0 cm, 4.0 cm, 8.0 cm, and 12.0 cm). The acclimatized fourth-stage Toxorhynchites larvae were individually introduced to each container alongside 50 Aedes larvae as prey to initiate the experiment formally. After 24 h, we observed and recorded the number of prey consumed by the predator. We carefully noted two key points: First, the predation rate, which refers to the number of prey killed and consumed by Toxorhynchites spp. within a specific time period (e.g., per day). It reflects the rate of actual prey consumption by the predator. Second, the predatory impact, which is the total number of prey killed by Toxorhynchites spp. within a specific time period, regardless of whether they were consumed or not. The predatory impact captures the broader effect of the predator on the prey population, including potential "compulsive killing" behavior observed before pupation. We conducted four replicates of the experiment. The experiment included a single control treatment without a predator. This control group utilized a glass tumbler filled with 200 ml of water at a depth of 8 cm. Both the control and experimental groups were given 10 mg of finely powdered fish food to feed the prey larvae.

Impact of day and night time on Toxorhynchites spp. predation efficiency

To evaluate the predation efficiency of Toxorhynchites spp. during both daytime and nighttime, eight experimental setups, described in the previous section were prepared and replicated over three consecutive days and nights. The study focused on distinguishing diurnal and nocturnal predation behaviors, with data collection occurring from 6 am to 6 pm representing daytime observations and from 6 pm to 6 am, representing nighttime observations. The glass windows were left uncovered during both day and night to allow natural light exposure, and no artificial light was provided at night to maintain a realistic nocturnal environment. Including eight replicates ensures a robust statistical foundation for assessing predation efficiency during these distinct temporal periods.

Statistical analysis

The effects of container type, water depth, and water volume on the predation rate and predatory impact of Toxorhynchites spp. preying on Aedes spp. were analyzed separately using generalized linear models (GLM) with Poisson distribution of errors and a log link function. Additionally, Mann–Whitney U test was employed to assess day-night differences in predation rates and predatory impacts. All analyses were performed with SPSS version 20.0, and figures were created using GraphPad Prism Version 5.00.

Results

Predation rate and predatory impact

The fourth instar larvae of Toxorhynchites spp. were each fed 50 Aedes spp. larvae, and their consumption was recorded over 24 h for four days. A total of 40 observations were collected. The descriptive analysis of the observed data yielded a mean predation rate (± SE) of an individual Toxorhynchites spp. larva of 23.29 ± 1.73. Similarly, the mean predatory impact within 24 h of a single Toxorhynchites spp. was 25.18 ± 1.77. Furthermore, no mortality was observed in the control group, and Aedes mosquitoes proceeded to complete development into adulthood in the lack of the predatory Toxorhynchites spp.

Effect of container types on the predation rate and predatory impact of Toxorhynchites spp.

In the experiment, a total of 28 containers were used, including 12 glass tumblers, eight glass bowls, and eight plastic bottles. Predation rate and predatory impact were calculated for 24 h. The mean predation rate of Toxorhynchites spp. on Aedes species was highest (26.25 ± 3.10) in glass bowls compared to other containers, but difference was not statistically significant (χ2 = 2.47, df = 2, p = 0.29) (Fig. 1a). A similar result was found for predation impact (28.13 ± 3.18) in glass bowls, which was also not statistically significant (χ2 = 2.25, df = 2, p = 0.33) (Fig. 1b).

Fig. 1
figure 1

Predation rates (mean ± SE) (a) and predatory impact (mean ± SE) (b) of Toxorhynchites spp. preying on Aedes spp. in different container types (dots indicate observed data)

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Effect of water depth on the predation rate and predatory impact of Toxorhynchites spp.

In the experiment seven different water depths, ranging from 1 to 12 cm, were used to investigate the impact on the predation rate and predatory impact of Toxorhynchites larvae on Aedes larvae within 24 h. The mean predation rate was the highest (26.75 ± 5.66) in a water depth of 1 cm compared to others without any statistical significance (χ2 = 2.94, df = 6, p = 0.82) (Fig. 2a). In addition, the mean predatory impact was highest and equal at water depths of 1 cm (29.50 ± 5.39) and 4 cm (29.50 ± 3.52). However, data were insignificant (χ2 = 3.77, df = 6, p = 0.71) (Fig. 2b).

Fig. 2
figure 2

Predation rates (mean ± SE) (a) and predatory impacts (mean ± SE) (b) of Toxorhynchites spp. on Aedes spp. in different water depths (dots indicate observed data)

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Impact of water volumes on the predation rate and predatory impact of Toxorhynchites spp.

In this study, four water volumes, ranging from 100 to 300 ml, were used to assess the impacts of Toxorhynchites larvae predation on Aedes larvae within 24 h. The mean predation rate was highest in the 100 ml water volume (25 ± 3.76) compared to other three volumes. Nevertheless, there was no statistical significance (χ2 = 0.45, df = 3, p = 0.93) (Fig. 3a). Similarly, the mean predatory impact was also highest (29.50 ± 3.52) in the 100 ml water volume compared to others, without any statistical significance (χ2 = 1.53, df = 3, p = 0.68) (Fig. 3b).

Fig. 3
figure 3

Predation rates (mean ± SE) (a) and predatory impact (mean ± SE) (b) of Toxorhynchites spp. on Aedes larvae in different water volumes (dots indicate observed data)

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Predation efficiency at daytime and nighttime

Among the eight setups that ran continuously for three days and nights, the mean predation rate and predatory impact were assessed for those periods. The mean predation rate during the daytime was slightly higher than at night (12.38 ± 1.18 vs. 10.13 ± 0.94) without any statistical significance (p = 0.20) (Fig. 4a). Similarly, the mean predatory impact during the day was slightly higher than at night (13.13 ± 1.24 vs. 10.63 ± 0.90) without any significant difference (p = 0.15) (Fig. 4b).

Fig. 4
figure 4

Predation rates (mean ± SE) (a) and predatory impact (mean ± SE) (b) for day and night treatments (dots indicate observed data)

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Discussion

The occurrence of Toxorhynchites splendens in Nepal has been previously documented based on morphological characteristics (Peters and Dewar 1956; Darsie et al. 1996). Consistent with these earlier finding, our morphological examination suggested the current specimens as T. splendens. However, we have designated them as Toxorhynchites spp. because polymerase chain reaction and sequencing analysis are still currently underway.

The feeding strategy Toxorhynchites spp. play a significant role in efficiently managing prey populations. Their feeding behavior is commonly described as "opportunistic" because these predatory larvae do not actively hunt for prey. Instead, they lay in wait and ambush potential prey that meets their mandibles (Zuharah et al. 2015). Research suggests that a single Toxorhynchites larva can consume a significant number of other mosquitoes, even hundreds, while it is growing (Mohamad and Zuharah 2014; Digma et al. 2019; Vinogradov et al. 2022). Toxorhynchites species like T. amboinensis, T. rutilus septentrionalis, and T. splendens have a behavior called "prepupal compulsive killing" (Steffan and Evenhuis 1981; Russo 1986; Vinogradov et al. 2022). This behavior involves their larvae getting rid of potential prey before it turns into pupae, even though they do not eat it.

Our study found a mean predation of 25.18 (± 9.35) Aedes larvae consumed by a single Toxorhynchites larva within 24 h. The highest number of prey consumed by a single larva in our experiment was 45. This exceeds the previously reported mean predation of 17.68 with a maximum consumption of 23 larvae per 24 h (Digma et al. 2019). However, the average predation observed in our experiment is lower than the mean predation of 51.67 (± 2.52) with a maximum consumption of 54 larvae reported in a study from India (Malla et al. 2023).

The type of container was previously thought to impact predation (Padgett and Focks 1981; Mohamad and Zuharah 2014), but our study found no significant differences in container types. Toxorhynchites larvae are opportunistic predators. When they encounter mosquito larvae in a given container, they are likely to feed on them. This opportunistic behavior means that the container type is less important than the presence of prey.

While some studies have suggested that the volume of the search area affects the mean number of prey larvae consumed by T. splendens larvae (Malla et al. 2023), our findings contradict this, as we have observed that the volume of water does not have a significant effect on predation. This is consistent with earlier research findings (Dominic and Das 1998; Mohamad and Zuharah 2014). These results indicate that Toxorhynchites spp. can effectively control Aedes larvae regardless of water volume, making them suitable for dry and rainy seasons.

Additionally, Toxorhynchites spp. displayed consistent diurnal and nocturnal predation potentialities, aligning with previous findings from Banjarbaru, Indonesia (Muhamat et al. 2022). This implies that their efficiency in regulating prey populations is not influenced by environmental light intensity, making them suitable for daytime and nighttime control. While no mortality was observed in the control treatment, a limitation of this study is the absence of control groups for each combination of water container type, depth, and volume. A full factorial design with separate control groups for each condition would have strengthened the isolation of the predator’s specific effects on prey mortality.

When considering the use of Toxorhynchites spp. as a biocontrol agent for Aedes species, it is important to recognize some limitations. These challenges include limitation of natural container breeding sites, difficulties in captive rearing, and the time-consuming nature of evaluating predation efficiency in natural habitats (Donald et al. 2020). Additionally, there is a risk of disease spread associated with Toxorhynchites spp., as they sometimes stockpile their prey (Frank et al. 1984). This highlights the need for detailed studies to confirm their predation characteristics and ensure their effectiveness as a control method.

The ongoing fight against Aedes-borne diseases demands innovative and sustainable control strategies. Our study demonstrated that Toxorhynchites spp. are effective biological control agents for Aedes mosquitoes. Their predation behavior remained consistent across different container types, water depths, and volumes. This adaptability suggests they can thrive in various aquatic environments throughout both dry and rainy seasons. Furthermore, consistent predation under both daylight and nighttime conditions suggests minimal impact from environmental factors like light, making them effective predators throughout the day and night. This approach offers a valuable alternative considering the lack of effective vaccines for Aedes-borne diseases like dengue, chikungunya, and zika.