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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Unveiling the Entomopathogenic Potential of Beetle-associated Fungi for Culex Management in South-West Algeria

N
Nouha Halima Bendekhis1,*
0009-0009-0595-1669
A
Ali Boulanouar2
0000-0002-2464-2337
L
Lakhdar Mebarki2
0000-0003-2960-1996
1Division of Laboratory for Valorization of Vegetal Resource and Food Security in Semi-arid Areas University Tahri Mohamed Bechar, Bechar, Algeria.
2Division of University, Tahri Mohamed Bechar, Bechar, Algeria.

ABSTRACT

Background: The escalating threat of mosquito-borne diseases demands innovative control strategies. This study explored the potential of beetle-associated fungi as biological control agents against Culex mosquito larvae in southwestern Algeria.

Methods: Fungal strains were isolated from beetle carcasses collected from pesticide-free regions of Beni Abbès and Béchar using dilution plating techniques. Based on metabolic profiles, four strains were selected for bioassays: Three Aspergillus species and one Penicillium species. Culex larvae were exposed to fungal concentration of 106 conidia/ml in sterile rearing water. Larval mortality was monitored daily over seven days.

Result: The results demonstrated significant larval mortality across all fungal strains. Aspergillus species showed faster mortality rates within three to five days, while Penicillium sp took seven days to show complete death. The LT50 values also confirm the rapid action of Aspergillus strains with Aspergillus sp 1 showing the most potent activity (LT50=1.25 days). On the other hand, Penicillium sp was slower with an LT50 value of 3 days, these findings highlight the power EPF associated with beetles to be an effective tool for mosquito management in the region.

INTRODUCTION

Entomopathogenic fungi (EPFs) have become a promising biological control agents for managing insect pests. Their broad host range, endophytic capabilities and plant growth-promoting properties, the EPFs offer a number one environmentally friendly alternative to chemical insecticides (Panwar and Szczepaniec, 2024). Despite the ongoing danger posed by mosquito-borne diseases (Bursali et al., 2024), that requires the creation of effective control strategies, the potential of EPFs in areas such as southwest Algeria remains poorly investigated (Zimowska and Krol, 2019). Due to the possibility of mosquitoes’ resistance to the chemical pesticides EPFs can be a useful biological alternative (Pratibha et al., 2025; Ramirez et al., 2023).
       
However, the unique environmental conditions in southwest Algeria such as climate, local mosquito species and the availability of suitable fungal strains, influence the efficacy and sustainability of the EPFs (Li et al., 2024). A lot of studies have shown that EPFs are so effective against a variety of mosquito species, however nothing is known about the entomopathogenic capacities of beetle associated fungi in controlling mosquito vectors.
       
This study evaluates the entomopathogenic capability of beetle-associated fungi against mosquitoes in southwest Algeria. By investigating this innovative approach, we seek to contribute to the creation of sustainable and region-specific mosquito control strategies.

MATERIALS AND METHODS

This study was conducted in laboratory for valorization of vegetal resource and food security in semi-arid areas at university Tahri Mohamed Bechar, Algeria during December 2021-March 2024.
 
Isolation
 
Fungal strains were isolated from beetle carcasses, that were collected in pesticide-free regions of Beni Abbes (Igli) and Bachar (Wakda). Using a dilution plating method, we aseptically homogenized 5 g of each carcass in 45 ml of sterile distilled water. Decimal dilutions of the resulting suspension were then inoculated onto chitinous Potato Dextrose Agar (PDA) for fungal cultivation. The isolated fungi were purified and identified using classic methods (Abdel-Raheem, 2019; Erick de Jesús de Luna-Santillana et al., 2020).
 
Insect collection
 
Culex mosquito larvae were collected from Igli during April to July in both 2022 and 2023. Larvae were transported in containers containing the original wastewater while maintaining optimal temperature conditions. To ensure continuous feeding, algae and other organisms were included in the containers. Water parameters, specifically pH (5-8) and temperature (25±2°C) were monitored throughout.
 
Bioassay
 
We selected four fungal strains based on their metabolic profiles observed on agar plates. Then, a standardized concentration of 106 conidia/ml was added into 50 ml of sterile rearing water containing 10 third-instar mosquito larvae. A drop of Tween 80 was added to each treatment to ensure homogeneous spore distribution. We included control groups containing only sterile rearing water and Tween 80. All setups were maintained in the dark at 25°C± 2°C. Larval mortality was recorded daily for seven days (Jaber et al., 2016). The experiment was replicated three times. Lethal Time 50 (LT50) values were calculated using Microsoft Excel.

RESULTS AND DISCUSSION

Isolation

The isolation study revealed that Aspergillus species (Fig 1 a, b and c) constituted the dominant fungal flora, comprising 75% of the isolated chitinolytic fungi, while Penicillium species (Fig 1d) accounted for the remaining 25%. Three distinct Aspergillus species and one Penicillium species were selected for bioassay experiments. Fig 1 illustrates the macroscopic morphological variations, including texture and colour, among these strains. Additionally, microscopic characteristics were observed using microculture techniques.

Fig 1: Pictures of some fungi isolated for use in the bioassay, depict the macroscopic and microscopic morphology of the isolated fungi.


 
Bioassay results
 
Experimental conditions were standardised by exposing 10 third-instar mosquito larvae to a consistent concentration of each fungal strain under controlled temperature, light and pH parameters. All fungal strains induced significant mosquito mortality, although with varying temporal dynamics. Strains Aspergillus sp 1, Aspergillus sp 2 and Aspergillus sp 3 exhibited rapid mortality rates, reaching 100% within five, four and three-days post-infection, respectively (Fig 2). In contrast, Penicillium sp demonstrated a slower progression, achieving complete mortality by the seventh day.

Fig 2: Mortality rates of Culex mosquito larvae exposed to Aspergillus and Penicillium species.


       
Strain Aspergillus sp 1 consistently induced high mortality rates, with a rapid escalation from 40% on day one to 100% by day five. Aspergillus sp 2 also demonstrated potent larvicidal activity, reaching 100% mortality by day four. Similar to Aspergillus sp 1, the strain Aspergillus sp 3 rapidly induced mortality, achieving 100% within three days. Conversely, Penicillium sp exhibited a more gradual mortality curve, culminating in complete larval death by day seven. Aspergillus strains exhibited a broad spectrum of pathogenicity, affecting both larval and adult stages of Culex mosquitoes. While all strains induced mortality in larval stages, primarily targeting the gut and siphon, as evidenced in Fig 3, their impact on adults was also pronounced. Adult mosquitoes exposed to these fungi displayed fungal accumulation in various body segments, leading to mortality shortly post-metamorphosis. In some cases, only the exoskeleton remained.

Fig 3: Fungal effects on Culex mosquitoes and larvae.


 
Lethal time 50
 
The LT50 values provide a quantitative measure of treatment efficacy and speed of action. The strain Aspergillus sp 1 exhibited the shortest LT50 of approximately 1.25 days (Fig 4), demonstrating the most rapid and potent insecticidal activity. Aspergillus sp 3 followed with an LT50 of roughly 1.4 days, while the strain Aspergillus sp 2 displayed a slightly longer LT50 of 1.67 days. In contrast, Penicillium sp demonstrated the longest LT50 of 3 days, indicating a slower onset of action and reduced efficacy compared to the other strains.

Fig 4: Lethal time 50 of Culex mosquitoes population exposed.


       
This study aimed to evaluate the potential of entomopathogenic fungi isolated from scarab cadavers as biocontrol agents against Culex mosquitoes. Dominant fungal isolates recovered from scarab cadavers in southwestern Algeria were Aspergillus species, corroborating previous findings on the prevalence of Aspergillus in Coleoptera and from arthropod Penicillium and Cladosporium (Jaber et al., 2016). Aspergillus and Penicillium species are common within the microbiome of scarab cadavers and it’s compatible with their terrestrial habitat, especially in arid settings (Alfiky, 2022). According to Gebremariam (2021) high temperatures, ultraviolet rays, low humidity and sandy soils are all features that encouraged the proliferation of these fungi. The ubiquitous nature of Aspergillus species explains its dominance in our study, aligning with the work of Tekaia et al. (2005). 
       
All entomopathogenic fungi employed in this study showed a chitinolytic activity and achieved 100% mortality against Culex mosquitoes. This is explained by their natural entomopathogenicity and ability to degrade chitin which is a vital component of insect cuticles.
       
The observed variations in mortality rates among fungal treatments might be caused by the differences in vegetative growth rates, though Parveen and jeyarani (2023) research results confirmed that the optimum temperature for entomopathogenic fungi growth and toxicity is 25°C to 30°C. The slow and gradual increase in mortality caused by Penicillium sp reaching 100% by day seven (Fig 2) indicates a delayed release of endotoxins and delay the onset of death (Sun et al., 2002). Additionally, the thicker cuticle and decreased food intake of later larval stages may have reduced spore penetration, further influencing mortality rates (Lord and Fukuda, 1990; Apperson et al., 1992).
       
Aspergillus species, on the other hand, show greater larvicidal activity against Culex, achieving total mortality within 3-5 days, indicating their potential for practical applications. These results align with previous studies showing that Aspergillus niger has a powerful larvicidal (Abideen et al., 2021; Rai et al., 2023) and poricidal properties, especially when paired with silver nanoparticles (Awad et al., 2022). Aspergillus parasiticus has also strong entomopathogenic properties (Abrar et al., 2022). Other studies have demonstrated that Aspergillus germination is effective against various mosquito species and its metabolism has proven to be effective (Ragavendran and Natarajan, 2015; Ragavendran et al., 2018; Balumahendhiran et al., 2019).
       
Aspergillus species isolated from scarab, often considered environmental contaminants especially soil (Beemrote et al., 2024), have shown unexpected potential as entomopathogens. They were as pathogenic as Beauveria bassiana against Aedes and Culex mosquitoes. (Jaber et al., 2016). Mosquitoes’ mortality caused by EPF is a complex process influenced by multiple factors. Host characteristics, including species, age and population density, significantly impact disease progression and mortality rates in addition to fungal virulence. Consequently, attributing mortality solely to fungal dosage is an oversimplification (Batta, 2005; Mantzoukas et al., 2019; Mantzoukas and Grammatikopoulos, 2020; Mantzoukas et al., 2022).
       
EPF spores infect insects with both hard and soft exoskeletons (Sharma et al., 2023) and function as midgut toxins just like Nerium oleander leaf extracts (Boulkenafet et al., 2023). Fig 3 illustrates spore accumulation in siphons, guts and adult mosquito articulations. Primarily ingested through the mouth, spores readily infect mosquito larvae when applied to the water surface (Bukhari et al., 2010). Once inside the host, spores obstruct feeding structures, colonize internal tissues and release toxins, causing damage to larval and mosquito guts as depicted in Fig 3 (Hegedus and Khachatourians, 1995).
       
Studies have confirmed that mechanical obstruction of tracheal trunks and larval siphons by fungi is a primary factor contributing to larval mortality (Daniel et al., 2017; Amobonye et al., 2020). The interplay between insects and their associated microbial communities, both on the cuticle and within the gut, significantly impacts the efficacy of entomopathogenic fungi. Mosquitoes harbouring gut microbiota exhibit accelerated mortality rates when exposed to the studied fungi compared to their microbiota-depleted counterparts (Liu et al., 2023). Notably, the presence of Wolbachia, an endosymbiotic bacterium commonly found in Culex mosquitoes, does not provide a protective advantage against fungal infections (Ramirez et al., 2021). The conclusions drawn from these studies align closely with our own research results.
       
Larval immune systems face a multifaceted challenge in combating EPF infections, potentially reducing the likelihood of developing resistance to these biological control agents (Mulla et al., 2003). While studies indicate increased antibacterial defences during metamorphosis, antifungal immunity remains relatively unchanged (Kokoska et al., 2005; Meylaers et al., 2007) which explains the mortality of adult mosquitoes (Fig 3). Studies like Shoukat et al., (2020) confirm that prolonged exposure to EPFs can compromise immune function and other physiological processes, as evidenced by continued mortality rates, melanisation responses in larval siphons and tearing of midguts.
       
Beyond their efficacy against mosquitoes, the EPFs employed in this study also exhibit potential for controlling scarab populations. Research indicates that EPF strains adapted to specific environments demonstrate enhanced effectiveness against local pests (Liu et al., 2021). However, this localised adaptability requires careful evaluation of the wider pest control implications. Although using a single fungal pathogen to target multiple insect pests has benefits, it also raises the risk of non-target organism mortality, demanding careful administration methods (Ortiz-Urquiza et al., 2015).
       
This study also revealed notable differences in the efficacy of different entomopathogenic fungi against Culex mosquitoes, as determined by LT50 values. These findings highlight the complex interplay between fungal species, mosquito hosts and environmental factors influencing disease progression and mortality rates.
       
As observed with strains Aspergillus sp 1, Aspergillus sp 2 and Aspergillus sp 3, rapid mortality rates suggest powerful insecticidal properties. As opposed to Penicillium sp that showed a slower killing rate, implying a potentially less virulent or less adaptable isolate.
       
Our results exceeded those of previous studies highlighting the efficacy of certain fungal species, such as Beauveria bassiana, which had LT50 values from 2 to 5 days against Culex pipiens larvae and 3.68 days against Aedes albopictus larvae (Kirsch et al., 2022; Istabraq et al., 2023; Waheeb, 2023; Renuka et al., 2023). And Metarhizium anisopliae, that had LT50 values from 3.2 to 4.7 days against Culex genera (Choi et al., 2020).
       
In contrast, the LT50 of entomopathogenic fungi against Aedes aegypti ranged from 6.4 to 16.3 days, with Aspergillus tamarii scored the highest virulence and Trichoderma euskadiense the lowest (Aguilar-Durán et al., 2023). Another study highlighted that the LT50 values were approximately 2 days for Aedes aegypti and 2.5 days for Culex quinquefasciatus, underscoring the effectiveness of Aspergillus tamarii extracts as larvicides for these species (Baskar et al., 2020).
               
However, the observed variations in LT50 values across different mosquito species emphasise the need for a targeted approach to fungal-based mosquito control, considering the specific ecological and epidemiological context. The differential susceptibility of mosquito species to entomopathogenic fungi may be attributed to a combination of factors, including variations in cuticle composition, immune response and behavioural patterns. Further research is warranted to elucidate the underlying mechanisms contributing to these observed differences.

CONCLUSION

Overall, the Aspergillus species have shown to be highly effective against not only Culex instar but also adults, especially Aspergillus 1 which showed the lowest LT50 value proving its rapid action compared to the others. Whereas Penicillium showed the slowest and less effective insecticidal activity. Future research should focus on characterising the specific bioactive compounds responsible for its insecticidal activity and investigate the environmental stability.

ACKNOWLEDGEMENT

This research was conducted independently, without any external funding.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All experiments involving mosquitoes were conducted following institutional and national guidelines for the care and use of invertebrates in research. As mosquitoes are invertebrates, no specific ethical approval was required under current animal welfare regulations.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Unveiling the Entomopathogenic Potential of Beetle-associated Fungi for Culex Management in South-West Algeria

    N
    Nouha Halima Bendekhis1,*
    0009-0009-0595-1669
    A
    Ali Boulanouar2
    0000-0002-2464-2337
    L
    Lakhdar Mebarki2
    0000-0003-2960-1996
    1Division of Laboratory for Valorization of Vegetal Resource and Food Security in Semi-arid Areas University Tahri Mohamed Bechar, Bechar, Algeria.
    2Division of University, Tahri Mohamed Bechar, Bechar, Algeria.

    ABSTRACT

    Background: The escalating threat of mosquito-borne diseases demands innovative control strategies. This study explored the potential of beetle-associated fungi as biological control agents against Culex mosquito larvae in southwestern Algeria.

    Methods: Fungal strains were isolated from beetle carcasses collected from pesticide-free regions of Beni Abbès and Béchar using dilution plating techniques. Based on metabolic profiles, four strains were selected for bioassays: Three Aspergillus species and one Penicillium species. Culex larvae were exposed to fungal concentration of 106 conidia/ml in sterile rearing water. Larval mortality was monitored daily over seven days.

    Result: The results demonstrated significant larval mortality across all fungal strains. Aspergillus species showed faster mortality rates within three to five days, while Penicillium sp took seven days to show complete death. The LT50 values also confirm the rapid action of Aspergillus strains with Aspergillus sp 1 showing the most potent activity (LT50=1.25 days). On the other hand, Penicillium sp was slower with an LT50 value of 3 days, these findings highlight the power EPF associated with beetles to be an effective tool for mosquito management in the region.

    INTRODUCTION

    Entomopathogenic fungi (EPFs) have become a promising biological control agents for managing insect pests. Their broad host range, endophytic capabilities and plant growth-promoting properties, the EPFs offer a number one environmentally friendly alternative to chemical insecticides (Panwar and Szczepaniec, 2024). Despite the ongoing danger posed by mosquito-borne diseases (Bursali et al., 2024), that requires the creation of effective control strategies, the potential of EPFs in areas such as southwest Algeria remains poorly investigated (Zimowska and Krol, 2019). Due to the possibility of mosquitoes’ resistance to the chemical pesticides EPFs can be a useful biological alternative (Pratibha et al., 2025; Ramirez et al., 2023).
           
    However, the unique environmental conditions in southwest Algeria such as climate, local mosquito species and the availability of suitable fungal strains, influence the efficacy and sustainability of the EPFs (Li et al., 2024). A lot of studies have shown that EPFs are so effective against a variety of mosquito species, however nothing is known about the entomopathogenic capacities of beetle associated fungi in controlling mosquito vectors.
           
    This study evaluates the entomopathogenic capability of beetle-associated fungi against mosquitoes in southwest Algeria. By investigating this innovative approach, we seek to contribute to the creation of sustainable and region-specific mosquito control strategies.

    MATERIALS AND METHODS

    This study was conducted in laboratory for valorization of vegetal resource and food security in semi-arid areas at university Tahri Mohamed Bechar, Algeria during December 2021-March 2024.
     
    Isolation
     
    Fungal strains were isolated from beetle carcasses, that were collected in pesticide-free regions of Beni Abbes (Igli) and Bachar (Wakda). Using a dilution plating method, we aseptically homogenized 5 g of each carcass in 45 ml of sterile distilled water. Decimal dilutions of the resulting suspension were then inoculated onto chitinous Potato Dextrose Agar (PDA) for fungal cultivation. The isolated fungi were purified and identified using classic methods (Abdel-Raheem, 2019; Erick de Jesús de Luna-Santillana et al., 2020).
     
    Insect collection
     
    Culex mosquito larvae were collected from Igli during April to July in both 2022 and 2023. Larvae were transported in containers containing the original wastewater while maintaining optimal temperature conditions. To ensure continuous feeding, algae and other organisms were included in the containers. Water parameters, specifically pH (5-8) and temperature (25±2°C) were monitored throughout.
     
    Bioassay
     
    We selected four fungal strains based on their metabolic profiles observed on agar plates. Then, a standardized concentration of 106 conidia/ml was added into 50 ml of sterile rearing water containing 10 third-instar mosquito larvae. A drop of Tween 80 was added to each treatment to ensure homogeneous spore distribution. We included control groups containing only sterile rearing water and Tween 80. All setups were maintained in the dark at 25°C± 2°C. Larval mortality was recorded daily for seven days (Jaber et al., 2016). The experiment was replicated three times. Lethal Time 50 (LT50) values were calculated using Microsoft Excel.

    RESULTS AND DISCUSSION

    Isolation

    The isolation study revealed that Aspergillus species (Fig 1 a, b and c) constituted the dominant fungal flora, comprising 75% of the isolated chitinolytic fungi, while Penicillium species (Fig 1d) accounted for the remaining 25%. Three distinct Aspergillus species and one Penicillium species were selected for bioassay experiments. Fig 1 illustrates the macroscopic morphological variations, including texture and colour, among these strains. Additionally, microscopic characteristics were observed using microculture techniques.

    Fig 1: Pictures of some fungi isolated for use in the bioassay, depict the macroscopic and microscopic morphology of the isolated fungi.


     
    Bioassay results
     
    Experimental conditions were standardised by exposing 10 third-instar mosquito larvae to a consistent concentration of each fungal strain under controlled temperature, light and pH parameters. All fungal strains induced significant mosquito mortality, although with varying temporal dynamics. Strains Aspergillus sp 1, Aspergillus sp 2 and Aspergillus sp 3 exhibited rapid mortality rates, reaching 100% within five, four and three-days post-infection, respectively (Fig 2). In contrast, Penicillium sp demonstrated a slower progression, achieving complete mortality by the seventh day.

    Fig 2: Mortality rates of Culex mosquito larvae exposed to Aspergillus and Penicillium species.


           
    Strain Aspergillus sp 1 consistently induced high mortality rates, with a rapid escalation from 40% on day one to 100% by day five. Aspergillus sp 2 also demonstrated potent larvicidal activity, reaching 100% mortality by day four. Similar to Aspergillus sp 1, the strain Aspergillus sp 3 rapidly induced mortality, achieving 100% within three days. Conversely, Penicillium sp exhibited a more gradual mortality curve, culminating in complete larval death by day seven. Aspergillus strains exhibited a broad spectrum of pathogenicity, affecting both larval and adult stages of Culex mosquitoes. While all strains induced mortality in larval stages, primarily targeting the gut and siphon, as evidenced in Fig 3, their impact on adults was also pronounced. Adult mosquitoes exposed to these fungi displayed fungal accumulation in various body segments, leading to mortality shortly post-metamorphosis. In some cases, only the exoskeleton remained.

    Fig 3: Fungal effects on Culex mosquitoes and larvae.


     
    Lethal time 50
     
    The LT50 values provide a quantitative measure of treatment efficacy and speed of action. The strain Aspergillus sp 1 exhibited the shortest LT50 of approximately 1.25 days (Fig 4), demonstrating the most rapid and potent insecticidal activity. Aspergillus sp 3 followed with an LT50 of roughly 1.4 days, while the strain Aspergillus sp 2 displayed a slightly longer LT50 of 1.67 days. In contrast, Penicillium sp demonstrated the longest LT50 of 3 days, indicating a slower onset of action and reduced efficacy compared to the other strains.

    Fig 4: Lethal time 50 of Culex mosquitoes population exposed.


           
    This study aimed to evaluate the potential of entomopathogenic fungi isolated from scarab cadavers as biocontrol agents against Culex mosquitoes. Dominant fungal isolates recovered from scarab cadavers in southwestern Algeria were Aspergillus species, corroborating previous findings on the prevalence of Aspergillus in Coleoptera and from arthropod Penicillium and Cladosporium (Jaber et al., 2016). Aspergillus and Penicillium species are common within the microbiome of scarab cadavers and it’s compatible with their terrestrial habitat, especially in arid settings (Alfiky, 2022). According to Gebremariam (2021) high temperatures, ultraviolet rays, low humidity and sandy soils are all features that encouraged the proliferation of these fungi. The ubiquitous nature of Aspergillus species explains its dominance in our study, aligning with the work of Tekaia et al. (2005). 
           
    All entomopathogenic fungi employed in this study showed a chitinolytic activity and achieved 100% mortality against Culex mosquitoes. This is explained by their natural entomopathogenicity and ability to degrade chitin which is a vital component of insect cuticles.
           
    The observed variations in mortality rates among fungal treatments might be caused by the differences in vegetative growth rates, though Parveen and jeyarani (2023) research results confirmed that the optimum temperature for entomopathogenic fungi growth and toxicity is 25°C to 30°C. The slow and gradual increase in mortality caused by Penicillium sp reaching 100% by day seven (Fig 2) indicates a delayed release of endotoxins and delay the onset of death (Sun et al., 2002). Additionally, the thicker cuticle and decreased food intake of later larval stages may have reduced spore penetration, further influencing mortality rates (Lord and Fukuda, 1990; Apperson et al., 1992).
           
    Aspergillus     species, on the other hand, show greater larvicidal activity against Culex, achieving total mortality within 3-5 days, indicating their potential for practical applications. These results align with previous studies showing that Aspergillus niger has a powerful larvicidal (Abideen et al., 2021; Rai et al., 2023) and poricidal properties, especially when paired with silver nanoparticles (Awad et al., 2022). Aspergillus parasiticus has also strong entomopathogenic properties (Abrar et al., 2022). Other studies have demonstrated that Aspergillus germination is effective against various mosquito species and its metabolism has proven to be effective (Ragavendran and Natarajan, 2015; Ragavendran et al., 2018; Balumahendhiran et al., 2019).
           
    Aspergillus     species isolated from scarab, often considered environmental contaminants especially soil (Beemrote et al., 2024), have shown unexpected potential as entomopathogens. They were as pathogenic as Beauveria bassiana against Aedes and Culex mosquitoes. (Jaber et al., 2016). Mosquitoes’ mortality caused by EPF is a complex process influenced by multiple factors. Host characteristics, including species, age and population density, significantly impact disease progression and mortality rates in addition to fungal virulence. Consequently, attributing mortality solely to fungal dosage is an oversimplification (Batta, 2005; Mantzoukas et al., 2019; Mantzoukas and Grammatikopoulos, 2020; Mantzoukas et al., 2022).
           
    EPF spores infect insects with both hard and soft exoskeletons (Sharma et al., 2023) and function as midgut toxins just like Nerium oleander leaf extracts (Boulkenafet et al., 2023). Fig 3 illustrates spore accumulation in siphons, guts and adult mosquito articulations. Primarily ingested through the mouth, spores readily infect mosquito larvae when applied to the water surface (Bukhari et al., 2010). Once inside the host, spores obstruct feeding structures, colonize internal tissues and release toxins, causing damage to larval and mosquito guts as depicted in Fig 3 (Hegedus and Khachatourians, 1995).
           
    Studies have confirmed that mechanical obstruction of tracheal trunks and larval siphons by fungi is a primary factor contributing to larval mortality (Daniel et al., 2017; Amobonye et al., 2020). The interplay between insects and their associated microbial communities, both on the cuticle and within the gut, significantly impacts the efficacy of entomopathogenic fungi. Mosquitoes harbouring gut microbiota exhibit accelerated mortality rates when exposed to the studied fungi compared to their microbiota-depleted counterparts (Liu et al., 2023). Notably, the presence of Wolbachia, an endosymbiotic bacterium commonly found in Culex mosquitoes, does not provide a protective advantage against fungal infections (Ramirez et al., 2021). The conclusions drawn from these studies align closely with our own research results.
           
    Larval immune systems face a multifaceted challenge in combating EPF infections, potentially reducing the likelihood of developing resistance to these biological control agents (Mulla et al., 2003). While studies indicate increased antibacterial defences during metamorphosis, antifungal immunity remains relatively unchanged (Kokoska et al., 2005; Meylaers et al., 2007) which explains the mortality of adult mosquitoes (Fig 3). Studies like Shoukat et al., (2020) confirm that prolonged exposure to EPFs can compromise immune function and other physiological processes, as evidenced by continued mortality rates, melanisation responses in larval siphons and tearing of midguts.
           
    Beyond their efficacy against mosquitoes, the EPFs employed in this study also exhibit potential for controlling scarab populations. Research indicates that EPF strains adapted to specific environments demonstrate enhanced effectiveness against local pests (Liu et al., 2021). However, this localised adaptability requires careful evaluation of the wider pest control implications. Although using a single fungal pathogen to target multiple insect pests has benefits, it also raises the risk of non-target organism mortality, demanding careful administration methods (Ortiz-Urquiza et al., 2015).
           
    This study also revealed notable differences in the efficacy of different entomopathogenic fungi against Culex mosquitoes, as determined by LT50 values. These findings highlight the complex interplay between fungal species, mosquito hosts and environmental factors influencing disease progression and mortality rates.
           
    As observed with strains Aspergillus sp 1, Aspergillus sp 2 and Aspergillus sp 3, rapid mortality rates suggest powerful insecticidal properties. As opposed to Penicillium sp that showed a slower killing rate, implying a potentially less virulent or less adaptable isolate.
           
    Our results exceeded those of previous studies highlighting the efficacy of certain fungal species, such as Beauveria bassiana, which had LT50 values from 2 to 5 days against Culex pipiens larvae and 3.68 days against Aedes albopictus larvae (Kirsch et al., 2022; Istabraq et al., 2023; Waheeb, 2023; Renuka et al., 2023). And Metarhizium anisopliae, that had LT50 values from 3.2 to 4.7 days against Culex genera (Choi et al., 2020).
           
    In contrast, the LT50 of entomopathogenic fungi against Aedes aegypti ranged from 6.4 to 16.3 days, with Aspergillus tamarii scored the highest virulence and Trichoderma euskadiense the lowest (Aguilar-Durán et al., 2023). Another study highlighted that the LT50 values were approximately 2 days for Aedes aegypti and 2.5 days for Culex quinquefasciatus, underscoring the effectiveness of Aspergillus tamarii extracts as larvicides for these species (Baskar et al., 2020).
                   
    However, the observed variations in LT50 values across different mosquito species emphasise the need for a targeted approach to fungal-based mosquito control, considering the specific ecological and epidemiological context. The differential susceptibility of mosquito species to entomopathogenic fungi may be attributed to a combination of factors, including variations in cuticle composition, immune response and behavioural patterns. Further research is warranted to elucidate the underlying mechanisms contributing to these observed differences.

    CONCLUSION

    Overall, the Aspergillus species have shown to be highly effective against not only Culex instar but also adults, especially Aspergillus 1 which showed the lowest LT50 value proving its rapid action compared to the others. Whereas Penicillium showed the slowest and less effective insecticidal activity. Future research should focus on characterising the specific bioactive compounds responsible for its insecticidal activity and investigate the environmental stability.

    ACKNOWLEDGEMENT

    This research was conducted independently, without any external funding.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All experiments involving mosquitoes were conducted following institutional and national guidelines for the care and use of invertebrates in research. As mosquitoes are invertebrates, no specific ethical approval was required under current animal welfare regulations.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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