Pathogenicity and Efficacy of Beauveria bassiana against the Larval and Adult Stages of Culex pipiens

1. Introduction

Culex pipiens mosquitoes are known to be significant vectors of a number of medically significant pathogens, such as viruses that cause diseases such as West Nile virus and dengue fever [1]. Moreover, they are also important in the spread of Wuchereria bancrofti, the etiological agent of lymphatic filariasis (elephantiasis), a debilitating disease that poses a significant threat to global public health. Lymphatic filariasis is estimated to afflict over 700 million individuals, with around 103 million people at risk of contracting the disease in over 80 countries [2]. Control of vectors is usually considered more practical and efficient than direct control of pathogens; therefore, historically, control of mosquito-borne diseases has focused on reducing mosquito numbers. The most common method of mosquito management has been chemical control, which has mostly been carried out using synthetic insecticides and is still in use today. Nevertheless, the widespread application of these chemicals has caused certain serious environmental issues, i.e., air, water, and soil pollution, thus destroying the ecological balance and worsening environmental quality. Moreover, target mosquito populations have shown an astounding ability to become resilient to these insecticides over time, reducing their effectiveness and constituting a significant challenge to the sustainable management of vectors [3].

Although different plant extracts [4] and insect growth regulators (IGRs) have been used, these methods have failed to suppress the mosquito population entirely, in large part because the insects have adapted and acquired resistance [6]. As a result, increasing interest in finding alternative control measures, especially biological ones, has emerged. Entomopathogenic fungi have been identified as one of these promising biocontrol agents due to their extensive distribution in nature, affordability, and high level of host specificity [7]. They are not problematic to use at higher concentrations because they are safer for humans and the surrounding environment [8].

Among them, one particular species, B. bassiana, is one of the most important entomopathogenic organisms that can infect and cause disease in insects [6]. It is distinguished by extensive distribution, easy recognition, and the capability to create strong spores that can withstand harsh environmental factors. Because of these features, it is able to attain epizootic levels, though its activity is tightly connected to environmental conditions like humidity and temperature [9].

The goal of this study was to isolate pathogenic fungi from wheat-producing buildings and evaluate their effectiveness against the larvae and adults of Cx. pipiens located in several locations. Additionally, this study attempted to determine whether B. bassiana could be used as a safe and eco-friendly biological control agent for mosquitoes (a type of vector).

2. Materials and Methods

2.1. Fungal Isolation and Cultivation

Soil samples were collected from wheat crops in the Al-Diwaniyah province (Al-Shamiyah region) using standard soil sampling protocols [10]. Each sample was randomly selected, and five sites within the soil profile were chosen. Subsamples were collected from the top 10 cm of the soil to obtain a final product of 200 g. Sterile distilled water was used to prepare three serial dilutions (10^-1, 10^-2, and 10^-3). The 10^-2 and 10^-3 dilutions were then inoculated at 0.1 mL onto Petri dishes containing sterile culture media. A sterile Drigalsky spatula was used to evenly spread the dilutions across the surface of the medium.

Two selective media were used: (1) Semi-selective media consisting of glucose (40 g/L), peptone (10 g/L), thiabendazole (0.004 g/L), chloramphenicol (0.5 g/L), crystal violet (0.01 g/L) and agar (15 g/L) in distilled water (pH 6.0); and (2) Oatmeal media consisting of oat flakes (20 g/L), hexadecyltrimethylammonium bromide (CTAB; 0.6 g/L), chloramphenicol (0.5 g/L), and agar (15 g/L) in distilled water (pH 6.0). The plates were incubated at 28 ± 1°C for 5–14 days and examined daily for fungal growth.

2.2. Morphological Features of the Fungal Isolate

Fungal isolates were characterized morphologically by examining macroscopic characteristics of the colony, such as its color (both upper and lower surfaces), type of morphology, texture of surface, margins, and elevation, and by measuring the characteristics of the hyphal and conidiophore structures and the size of the conidia after observing them microscopically [11]. The reproductive structures were observed and measured using an ocular micrometer at both 400× and 1000× magnification and photo-documented using a Carl Zeiss Optical Microscope (Model 467065-9902-18VA).

2.3. Establishment of Permanent Cultures of Cx. pipiens

Larvae at various developmental stages were collected from drainage sites in Al-Diwaniyah province using a long-handled scoop, transported to the laboratory in plastic containers, and reared in dechlorinated water. Larvae were fed a standardized diet consisting of wheat, corn, protein, and rice (1:1:1:0.25 ratio; 2 g per container). Species identification was confirmed at the Natural History Museum, University of Baghdad, using standard taxonomic keys. Figure 1 shows the sample collection, laboratory cultivation, and morphological features of Cx. pipiens larvae.

2.4. Effect on Mortality Percentages of the Four Larval Instars

Each of the four larval instar stages was selected at random, with 40 larvae per concentration of the fungal suspension being tested. Larvae were distributed among four containers, each containing 100 mL of a fungal concentration, with the fourth containing sterile distilled water (control treatment). After exposure, the treated larvae were carefully transferred with a fine brush into 250 mL beakers containing sterile distilled water mixed with 10 mg of larval diet. The beakers were incubated at 28°C under a 14-hour photoperiod. Mortality percentages were recorded at 24 and 72 hours post-treatment [12].

2.5. Effect on Mortality Percentages of Adult Cx. pipiens

A sufficient quantity of pupae was gathered from the stock culture and placed individually into 10-ml tubes, with the openings filled with cotton until an adult emerged. One-liter glass beakers were arranged, each containing a small dish with a cotton pad wetted in a 10% sugar solution. All beakers were sprayed with 5 ml of the corresponding fungal suspension using a manual sprayer at a distance of about 15 cm; the control treatment was sprayed with sterile distilled water. Then ten newly emerged adults (male and female separately) were placed into the treated beakers with the help of an aspirator. Each concentration (including the control) was replicated three times in the experiment. The incubation temperature was 28°C, and mortality rates were recorded after 24 and 72 hours [13].

2.6. Laboratory Pathogenicity Assays of Fungal Species

Pathogenicity tests were conducted to assess the effects of various doses of B. bassiana under ambient laboratory conditions (mean temperature 28°C, relative humidity 53%). In each assay, a random number of insects (15 adults) were placed in sterile plastic containers.

The fungal inoculum was prepared by inoculating 14-day-old cultures grown on Potato Dextrose Agar (PDA) with 5 mL of sterile distilled water in 9-cm Petri dishes. Tween-80 was added as a surfactant. Conidia were harvested using a sterile glass rod, and the filtrate was homogenized on a magnetic stirrer for 10 minutes. The mixture was then filtered through a glass funnel lined with sterile gauze, and 5 mL of distilled water was added to the suspension to maximize conidial recovery. The 10 mL filtrate was transferred to a glass flask, and the resulting suspension was labeled the stock suspension [10].

An aliquot of 1 mL was examined using an improved Neubauer hemocytometer to determine the conidial concentration. The number of conidia in the four corner large squares was counted to obtain the mean number of conidia per square. This average was then multiplied by 1×10⁴ to obtain the number of conidia per milliliter. The following dilution formula was used to obtain the desired experimental concentrations [14]:

Concentrations were prepared at 3 × 10⁵, 3 × 10⁶, and 3 × 10⁷ spore/ml.

2.7. Statistical Analysis

The experiments were conducted following a two- or three-factor model using CRD analysis. Mortality ratios were corrected using Abbott's equation. The corrected percentages were converted to angular values using the Arcsine Transform to ensure normal distribution. Statistical analysis was carried out to assess differences between treatments via LSD analysis at a significance level of probability ≤ 0.05 [15]. Mortality percentages were corrected using the method of Abbott [16].

3. Results

3.1. Morphological Characterization of B. bassiana

Fungal isolates belonging to B. bassiana were selected for this study. In macroscopic examination, the colonies exhibited a white to creamy, powdery appearance with irregular margins (Figure 2). Microscopic examination revealed well-defined reproductive structures and conidia with typical morphology, size, and pigmentation. Septate hyphae and conidiogenous cells, measuring 5.4–8.7 µm in length and 2.0–2.8 µm in width (SD: 0.6–1.0 and 0.1–0.7 µm, respectively), were used to characterize the isolates. These cells had a wide basal area and a small apical extension (rachis), on which many conidia were produced in a typical sympodial pattern. The conidia were hyaline, nonporous, and spherical or sub-spherical, with a mean diameter of 1.7–2.3 µm (SD: 0.5–0.6 µm).

3.2. Larval Stage Susceptibility to B. bassiana

Table 1 illustrates the effect of different B. bassiana conidial concentrations on the mortality of Cx. pipiens larval instars. The LC₅₀ for the first instar was 5.241×10⁵ and 2.935×10⁶ spores/ml after 24 and 72 hours, respectively. For the second instar, the values were 1.845×10⁶ and 5.988×10⁶ spores/ml; for the third instar, 1.868×10⁶ and 6.696×10⁶ spores/ml; and for the fourth instar, 4.45×10⁶ and 8.913×10⁶ spores/ml, at the same intervals. No mortality was observed in the control group, indicating a direct correlation between concentration and mortality rates. Furthermore, a positive relationship was evident between exposure duration and mortality across all four instars.

3.3. Adult Susceptibility and Sex-Specific Responses

Table 2 shows the impact of B. bassiana on adult Cx. pipiens. The LC₅₀ values for males were 1.245×10⁷ and 4.314×10⁶ spores/ml, while for females they were 1.456×10⁷ and 6.159×10⁶ spores/ml, after 24 and 72 hours, respectively. The data support a direct correlation between conidial concentration, exposure time, and mortality rates. Statistical comparison showed significant differences in percent adult mortality according to concentration and sex, with males showing higher resistance compared to females; this is because higher concentrations result in higher mortality, as there are more germinating spores that attack the host and affect its immune system.

4. Discussion

Insects can have varying levels of effective immune defenses against fungal pathogens and thus will have different levels of susceptibility to those pathogens; but the variability of these defenses is not distributed consistently among insect populations. The immune system can develop sufficient defenses at low conidial loads; however, there is a significant reduction in the immune system's ability to protect against pathogenic fungi at high conidial loads. The sexually dimorphic nature of insect immune systems considerably complicates our understanding of the role of fungal biocontrol agents [17,18].

Male and female Cx. pipiens use very different immunological strategies that are representative of their unique ecological roles and physiological limitations [18]. Male mosquitoes employ what would be considered a constitutive immune strategy, with a high level of basal expression of immune-related genes (i.e., sustained immune defenses) even when not exposed to pathogens. This constant state of immunological readiness allows male mosquitoes to mount a highly rapid and complete immune response to fungal pathogens upon exposure. This is due, in large part, to the fact that the male mosquito's diet consists exclusively of plant sugars and floral nectar, which provide the nutritional requirements for sustaining immune system function. On the other hand, female Cx. pipiens have developed a tolerance-based immunological strategy. Following blood meals, they experience profound physiological and hormonal fluctuations that substantially modulate their immune competency [19]. Rather than attempting to clear pathogens completely — an energetically expensive proposition — females maintain a moderate immune response that permits coexistence with certain fungal loads. This evolutionary trade-off prioritizes reproductive success, allowing females to conserve energetic resources for the demanding processes of oogenesis and oviposition rather than committing them to futile pathogen-elimination efforts.

An understanding of the mechanistic basis of fungal pathogenesis is fundamental to understanding the promise of entomopathogenic fungi as biological control agents. The infection process occurs through direct contact in both adult males and females [20]. Fungal spores are applied to an insect's surface, initiating an invasive cascade. The fungus penetrates the insect's protective cuticle to systematically colonize different tissues in the hemocoel while evading host immune responses. Entomopathogenic fungi have multiple routes for invasion, including directly breaching the cuticle, accessing the digestive tract, penetrating through the respiratory spiracles, or entering through existing wounds [21]. The fungus secretes powerful degrading enzymes (proteases, lipases, and chitinases) to dismantle the structural components of the insect exoskeleton, allowing it to penetrate [21,22]. After successful colonization of the host, fungal hyphae spread throughout the host and essentially consume the entire insect. Finally, the production of conidiophores and fruiting bodies marks the completion of the infection process and leads to the insect's death. In addition, some entomopathogenic fungi are able to persist in the environment in the absence of a host, extending their potential impact beyond direct contact situations [22].

Among fungi used as biological control agents, B. bassiana has become one of the most successful. It has a very broad host range and shows significant effectiveness in controlling insects. Studies show that this fungus can infect approximately 200 different types of insects, with about half of them belonging to two specific groups (Lepidoptera and Coleoptera) [23]. Like many other pathogenic fungi, B. bassiana infects via contact through the growth of conidia on the cuticle of an insect. When B. bassiana's conidia land on a suitable insect and the environment is conducive to germination, these conidia begin developing into new fungal mycelium by extending their germ tube from the conidia, at the site of contact with the insect's cuticle. Through the extension of the germ tube, B. bassiana secretes specific degradative enzymes that degrade the cuticle components of the insect, starting with protein, then chitin, and finally lipids. The unique appeal of B. bassiana for use in integrated pest management systems is that it is selective for host insects while posing little risk to non-targets. Additionally, B. bassiana can be produced in large quantities at low cost and is relatively easy to prepare for commercial application [24].

Numerous studies have thoroughly documented the effectiveness of B. bassiana in combatting a wide variety of insect pests. The fungus produced 58–91% mortality rates in Aphis craccivora (cowpea aphid) seven days post-treatment [25], due to the secretion of fungal chitinase/lipase enzymes and a variety of toxins that permit the fungus to penetrate deeply into the insect's body [26]. One of the most noteworthy indications of B. bassiana's effectiveness as a disease-vector pathogen is in mosquito populations, especially Anopheles gambiae. A study on the effect of B. bassiana on adult Cx. pipiens determined an LT₅₀ (median lethal time) of 3.5 days, while another study on the effect of Metarhizium anisopliae found a similar LT₅₀ of 3.49 days [20]. Larval Cx. pipiens also appear to be quite susceptible; ingestion of fungal conidia by larvae leads to toxic metabolite production that causes septicemia and death [27,28]. Due to its effectiveness against both adult and larval forms, B. bassiana presents significant promise as a tool for vector control.

The broad-spectrum potential of B. bassiana is further evidenced by its activity against numerous economically important pest species. In studies examining Aphis fabae (black bean aphid), a 100% culture filtrate concentration of B. bassiana produced mortality rates of 52.17% in nymphs and 54.10% in adults [18], indicating its utility as a versatile biological control agent. Against Spodoptera frugiperda (fall armyworm), B. bassiana achieved substantial mortality rates, though specific efficacy varied depending on multiple biological and environmental factors. These variations in mortality rates across different target insects may be attributed to differences in spore germination rates, growth-factor availability, enzyme production efficiency, spore adhesion strength, and various other circumstances that collectively support fungal survival and proliferation [29]. This variability underscores the importance of optimizing application conditions and spore concentrations for specific target pests.

B. bassiana showed concentration-dependent efficacy in laboratory studies. Research on Tribolium castaneum identified a positive correlation between mortality rates and spore concentrations; the highest concentration tested, 17×10⁸ spores/ml, provided 93.33% mortality of adults by 10 days post-treatment [30]. This relationship illustrates the importance of spore concentration for successful infection, corresponding to previous findings on the performance of the insect immune system at different conidial loads. By integrating knowledge of sexual dimorphism in insect immune systems with evidence of B. bassiana efficacy, researchers can develop improved biocontrol options. Given the unique immune responses of male and female insects, researchers may develop more targeted applications based on sex-related vulnerability differences. Combined with B. bassiana's selectivity towards specific insect hosts, low cost, broad-spectrum activity, and substantial environmental persistence, researchers can increasingly rely on this fungus as part of a sustainable, integrated pest management program. Further research on interactions between host immune mechanisms and virulence factors will enable greater use of entomopathogenic fungi as precision biological control agents against agricultural pests and disease vectors.

5. Conclusion

Findings from this investigation have provided much insight into the association of entomopathogenic fungi with mosquito larvae within Al-Diwaniyah City. Specifically, the study concluded that B. bassiana is a very effective means of biologically controlling Cx. pipiens, particularly within its early larval stages.

As such, B. bassiana serves as an environmentally friendly alternative to chemical insecticides and demonstrates that conidial concentration and exposure duration are related to the level of mortality in treated mosquitoes. The results of this research also indicate varying degrees of susceptibility among different instar stages of larval mosquitoes, as well as death via mechanical and enzymatic mechanisms when the fungus is present on these species. Therefore, in order to successfully control Cx. pipiens, the use of B. bassiana provides a much more environmentally safe alternative than chemical pesticides and could improve sustainable agriculture for wheat production by offering an eco-friendly option for controlling mosquito populations, while enabling the use of only the most virulent strains of B. bassiana for large-scale implementation.

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