Novel development of Lecanicillium lecanii-based granules as a platform against malarial vector Anopheles culicifacies

Mosquitoes are infectious vectors for a wide range of pathogens and parasites thereby transmitting several diseases including malaria, dengue, Zika, Japanese encephalitis and chikungunya which pose a major public health concern. Mostly synthetic insecticides are usually applied as a primary control strategy to manage vector-borne diseases. However excessive and non-judicious usage of such chemically derived insecticides has led to serious environmental and health issues owing to their biomagnification ability and increased toxicity towards non-target organisms. In this context, many such bioactive compounds originating from entomopathogenic microbes serve as an alternative strategy and environmentally benign tool for vector control. In the present paper, the entomopathogenic fungus, Lecanicillium lecanii (LL) was processed to make the granules. Developed 4% LL granules have been characterized using the technique of Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The developed formulation was also subjected to an accelerated temperature study at 40 °C and was found to be stable for 3 months. Further, GCMS of the L. lecanii was also performed to screen the potential biomolecules present. The developed formulation was found to be lethal against Anopheles culicifacies with an LC50 value of 11.836 µg/mL. The findings from SEM and histopathology also substantiated the mortality effects. Further, the SEM EDX (energy dispersive X-ray) studies revealed that the treated larvae have lower nitrogen content which is correlated to a lower level of chitin whereas the control ones has higher chitin content and healthy membranes. The developed LL granule formulation exhibited high toxicity against Anopheles mosquitoes. The granule formulations can be used as an effective biocontrol strategy against malaria-causing mosquitoes.


Introduction
Vector-borne diseases (VBDs) have contributed remarkably to the global burden of diseases, accounting for around 17% of all infectious diseases. Among the VBDs, malaria has been regarded as a major cause of morbidity and mortality, particularly in sub-Saharan Africa (SSA), with approximately half the world's population predicted to be at the risk (Wilson et al. 2020). India is endemic to several mosquito-borne diseases, including malaria, dengue, chikungunya, Japanese encephalitis, and lymphatic filariasis (Ragavendran et al. 2017b;Benelli and Duggan 2018;Dye-Braumuller et al. 2022;Sogan et al. 2023) and contributes to 4% of the malaria burden globally however it has been a significant contributor (87%) to the total number of malaria cases in Southeast Asia (Ghosh and Rahi 2019). Around 0.16 million cases of malaria and 90 deaths were reported by the National Vector Borne Disease Control Programme (NVBDCP) in 2021.
Recent advancements in the health sector have led to the discovery of many effective therapeutic drugs in the global market. Although these drugs have been able to control malaria to some extent. However, the incidence of parasite resistance to antimalarial drugs has shifted the control strategy towards vector control, which is an indispensable tool that plays a notable role in reducing the incidences of VBDs (White 2004;Sogan et al. 2018). Chemical insecticides are usually applied as a primary control strategy to manage vector-borne diseases. Consequently, the non-judicious usage of synthetic insecticides has led to various environmental and health issues, along with persistent residues and toxicity towards non-target organisms and escalating issues of insecticidal resistance in the vector population (Benelli 2015;Mittal and Subbarao 2003).
In the present scenario, bioactive compounds of biological origin from bacteria, fungi, plants, and entomopathogenic microbes have the potential to be used as alternatives to chemical insecticides (Nathan et al. 2004;Benelli et al. 2012;Vivekanandhan 2018;Kumar et al. 2021;Sogan et al. 2023). The fungal based products are biodegradable and exhibit high toxicity against mosquitoes with relatively lesser toxicity towards non-target organisms. These characteristic features make them ideal and eco-friendly tools which can be exploited for mosquito vector control (Kirschbaum 1985;Shaalan et al. 2005a, b;Kumar et al. 2021). Mycelial extracts of various fungi have been reported for their larvicidal, cellulolytic, and cytotoxic activities (Valentin Bhimba et al. 2011;Zhang et al. 2013;Fahd 2018).
The entomopathogenic fungi (EPF) are disease-causing organisms in insects and are particularly specific to sap-sucking insects and mosquitoes (Mnyone et al. 2009;Thiyagarajan et al. 2014). Lecanicillium lecanii (synonym Verticillium lecanii (Zimm.) Viegas, thereon L. lecanii (Zimm.) Zare & W. Gams or LL). L. lecanii is reported to be a very predominant and important entomophagous hyphomycetes that occur on a wide range of hosts such as aphids, thrips and mites along with orders including Diptera, Homoptera, Hymenoptera, Lepidoptera in all the climatic regions. Other important substrates for L. lecanii are rusts and other fungi (Alavo 2015).
Lecanicillium lecanii is an EPF which infects its hosts (invertebrates) through the external cuticle. The infection involves three phases, firstly, the adhesion and germination of the fungal spores on the host cuticle. Secondly, the penetration of the insect integument by a germ tube, and finally the development of the fungus inside the insect body, which eventually results in the death of the infected host and cover them with spores and hyphae under favourable humid conditions (Quinlan 1988;Wang et al. 2021).
Fungal spores are hydrophobic in nature and tend to form clumps, thereby reducing the area that is effectively covered. As a result, large amounts of fungal spores are required. Moreover, dry unformulated fungal spores are more exposed to UV radiation and high temperatures, which may have negative effects on spore persistence and germination rate (Bhukhari et al. 2011). Mostly, microbial products are available as powdered formulations, which are difficult to handle and dose (Gola et al. 2019). Therefore, an ideal formulation can aid in the easy handling and application of bioinsecticides and increases its efficacy by improving an area of contact with the host and protecting the active bioagent from environmental factors (Goettel et al. 2010). The anopheline larvae have surface-feeding behavior therefore any formulation intended to infect them should spread the fungal spore/active over the water surface (Merritt et al. 1992;Ramoska et al. 1981). In this context, we developed a low-cost granulated formulation of L. lecanii and evaluated its efficacy against anopheline larvae.

Test organism and substrate requirements
Lecanicillium lecanii (MTCC 956) was procured from the Centre for Rural and Development Technology (CRDT), Indian Institute of Technology (IIT) Delhi. The culture was maintained on PDA (potato dextrose agar) slants and was stored at 4 °C. The serial dilution method was used to count spores (CFU/ml) and it was found to have 1.2 × 10 8 CFU/ml.

Characterization of the Lecanicillium lecanii through GCMS
The GCMS spectrum of L. lecanii extract was performed in the electron ionization (EI) mode at 70 eV using GCMS (Shimadzu QP2010). LL extract was prepared by grinding ~ 0.1 g of fungus biomass using a pestle and mortar and further the ground fungal biomass was subjected to solvent (methanol) extraction using the rotatory shaker and the extract was filtered through Whatman no 1 paper. Around 1.0 µL extract of fungus was injected into a 30 m capillary column (0.25 mm i.d × 0.25 µm film). The initial oven temperature was programmed at 70 °C (hold time 5 min), which was increased at the rate of 10 °C/min to 300 °C (hold time 5 min) and the sample injection temperature was 270 °C, with a split ratio of 10. Various constituents were identified based on the retention indices and by comparing their mass spectral fragmentation patterns matching against the commercial library mass spectra of the National Institute of Standards and Technology (NIST-14) and Wiley-8.

Formulation development and physical characterization of granules and stability studies
For the preparation of L. lecanii granules or myco-granules, the fungal biomass (LL as active ingredient, 4 g) was ground using a pestle and mortar followed by the absorption of LL on the optimized amount of the silica (5 g). Thereafter, xanthan gum (3 g) was added to the mixture and finally, the clay (88 g) was added as filler. The prepared granules were passed through the mesh having the pore size of 105 µm to obtain the granules of the desired size and again kept for drying at room temperature for 2 h to obtain the mycogranules. The moisture content of the granules obtained after drying was nearly 2%.

Characterization of the granules
For friability: The LL granules were subjected to shaking for 5 min and thereafter were sieved with a sieve having pore size of 212 μm to remove the fine powder. Afterwards, the sample of the residual coarse granules LL (20.0 g) were placed in an Erweka friabilator (Heusenstamm, Germany) rotating at speed of 15-20 rpm for 10 min, to calculate the friability of the residual coarse granules. The percentage of dust formed was determined and taken as an index of friability. The test was done in triplicate.
Carr's Index (CI): Carr's index provides information about the flowability of granules (Shah et al. 2008). The CI was determined using the equation below: where ρB is bulk and ρT is the tap density of granules. Bulk density was measured in terms of the volume and weight of the LL granules. For bulk density, LL granules were weighed first before being measured, and they were then directly poured into a 100 mL measuring cylinder without being tapped. For tap density the cylinders were filled with granules and tapped until there was no longer any change in the volume mark of the cylinder (Thapa et al. 2019). A CI score below 10 suggests excellent flowability, whereas one above 25 indicates poor flowability.
Granule strength: A Texture Analyser (TA XT plus, Stable MicroSystems, UK) equipped with a 5 kg load cell was used to study the granule strength. Using a 9 mm diameter stainless steel probe, each dry LL granule was crushed. A significant number of granules (at least 30 granules) were examined in each experiment to provide statistically valid results.
Weight variation test: 20.0 g of granules were selected randomly and weighed individually. Then the average weight of 20.0 g of granules was determined. The % weight variation was calculated as follows: The acceptable range of weight variation is ± 5% from the average weight (Lachman et al. 1991).
Swelling investigation for the granules: Using a compound microscope, we examined the surface morphology of the granules at various time intervals in an aqueous environment to access the swelling behaviour of the LL granules.

Accelerated temperature study
The compatibility between L. lecanii and other ingredients used in the present formulation was ascertained by Fourier transform infrared spectroscopy (FTIR) (Bruker Optics (Germany-made) Tensor 27 model). The diffused reflectance mode with a resolution of 0.4 cm −1 was used to scan the observations over the range of 400 cm −1 to 4000 cm −1 . For the accelerated temperature study, the developed LL granules were stored at room temperature (25 °C) and higher temperatures (40 °C) for three months and their stability was determined through CFU(Colony Forming Unit) count. For CFU, 1.0 g of the formulation was suspended in 100.0 mL distilled water. Further the serial dilution approach, was followed where LL suspension has been serially diluted, and was plated (spread plate method) on sterile petri-plates having potato dextrose agar media and incubated at 30 ℃ for 3 days. The number of CFUs produced were counted.

X-ray diffraction studies (XRD)
XRD was performed using an X-ray diffractometer (XRD-Rigaku-ultima IV, Japan) with Cu radiation generated at 40 mA. The diffractometer was operated between 60 and 150 kV. Samples were scanned in the range of 2θ values from 10 to 500.

Culture of Anopheles culicifacies larvae and doseresponse bioassay
The National Malaria Research Institute's insectary was used to raise A. culicifacies larvae at a constant temperature of 25 ± 2 °C with a relative humidity of 75 ± 2% and a photoperiod of 14:10 (Light/Dark cycles). Dog biscuits and powdered yeast (in a ratio of 3:1) were provided as larval food (3:1 ratio). Using a dropper, the pupae were removed from the enamel trays and placed into plastic containers (12 × 12 cm) filled with 500 mL of water. For adult emergence, the pupae were kept in plastic jars in a 90 × 90 × 90cm 3 mosquito cage.
For the dose-response bioassay, various concentrations of LL granules were prepared using double distilled water (2.5.0, 5.0, 10.0, 20.0, 40.0, and 80.0 µg/mL). Around twenty 3rd instar larvae of A. culicifacies were exposed to 200.0 mL of various test concentrations. The negative control (blank granules) was also tested. All the test containers were kept at room temperature and were covered with mosquito nets. During the experimental period, no food was given to the larvae. All the experiments were performed in triplicates. The mortality (using Abbott's formula) and survival rate was determined after 24 h of the exposure period (Abbott 1925, WHO 2005. The LC 50 and LC 90 values of exposed larvae were calculated using probit analysis (Finney 1971).

Semi-field bioassay
Semi-field bioassay is predominant in understanding the efficacy of the LL in complex environmental conditions. Therefore, the toxicity of LL granules was also evaluated under semi-field conditions (during August) against the field-collected larval stages (Delhi -NCR) following the previously described method by Lee and Ahn (2013) with some modifications. For each selected test concentration (2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 µg/mL) of dose-response bioassay three buckets of 5 L capacity were used. These buckets were provided with 2 L of the test concentration of LL granules. Around 200 3rd instar larvae (field-collected larvae) were released into these buckets. A nylon mesh screen was used to cover these buckets to avoid any predators and egglaying by any other insect species. These buckets were then placed in the shade under natural environmental conditions (Delhi-NCR). The negative control (Blank granules) was also tested. Mortality was recorded after 24 h. The moribund larvae were also considered dead. The LC 50 and LC 90 values were calculated using probit analysis (Finney 1971).

Histopathology analysis of the treated larvae
To observe the histopathological impact of granules on the third instar larvae of A. culicifacies, histopathological investigations were performed. Larvae were treated with lethal concentration (LC 50 values). A setup containing distilled water served as a control group. Post 24 h of treatment the larvae were removed from the treated solution and were stored in a buffered formalin reagent (pH 7.2). The LL-treated larvae were then passed through graded ethanol series followed by embedding in paraffin wax (Al-Mekhlafi 2018). A thin longitudinal section (LS) of the tissue was stained with Hematoxylin and Eosin (Hi-Media labs) and was finally cut using a microtome (Leica, Germany) and mounted on a glass slide. The LS of the midgut region was examined under the microscope.

Scanning electron microscopy (SEM) and scanning electron microscopy -energy dispersive X-ray analysis (SEM EDX)
For SEM analysis, larvae (treated as well as control) were fixed with 2.0% glutaraldehyde and left overnight at 4 °C. Samples were washed with PBS buffer after overnight incubation. Samples were gold-coated for SEM and carbon coated for EDX. The SEM and EDX observations were made under the following analytical condition: EHT = 20.0 kV and Signal A = SE1 (ZEISS EVO 50).

Results and discussion
The findings of the present research study have revealed that entomophagous fungi are important microorganisms that can be exploited to produce new, bio-control agents and bioformulations that can be used as a tool in our arsenal in the fight against mosquito vector-borne diseases (Baskar et al. 2020). The developed LL granules have fulfilled all the physical parameters criteria and were found to be stable. Further LL granules have been characterized using the technique of SEM and FTIR.

GCMS based metabolic profiling of fungal extract
The chemical profile of L. lecanii mycelia extract was done by GCMS analysis. In the database of the National Institute Standard and Technology (NIST), active principles with their retention time (Rt), molecular formula, molecular weight, and percentage (area %) was used for the interpretation of Mass spectra data. GCMS of the fungal extract revealed the major components as Octadec-9-enoic acid (32.63) trans, trans-9,12-Octadecadienoic acid propyl ester (22.04), Hexadecanoic acid, butyl ester (20.90), while octadecanoic acid, butyl ester (6.87), eicosanoic acid, butyl ester (0.41) Docosanoic acid, butyl ester (0.30) Squalene (1.64) were present in minor concentrations (Table 1). Similarly, the GC-MS analysis of the hexane extract of Trichoderma sp. and its partitions showed linoleic as the main component followed by oleic, palmitic and stearic acids. Methyl linoleate and methyl oleate were present as minor components (Kaushik et al. 2020). The bioactive compounds from the Trichoderma isolate EFI 679 were fatty acids and their methylated derivatives, present in the Hexane extract in free form and as part of the triglyceride, exhibiting aphid antifeedant effects (Kaushik et al. 2020).

Physical methods for the characterization of LL granules
Various fungal biomass amounts (in percentage) were used for the preparation of myco-granules as an active ingredient; however, it was observed that 4 g of fungal biomass was found to be better than other amounts of biomass (data not shown). LL granules have a diameter of 7 mm (Fig. 1). The LL granules were characterized for various parameters such as dimension, hardness, weight, friability (extent of dust produced by the tablet) bulk density and Carr index. The calculated values of hardness and friability were 2.5 kg/cm 2 and 2.4%, respectively (Table 2). Hardness and friability are two important physical parameters that define the mechanical strength of the granules (Jensen 2011). These tests are performed by manufacturers to ensure that the granules would tolerate the rigors of handling, packaging, and transportation process, and would maintain the desired properties during the storage period. The values of these parameters for stable granules have been laid as ideal friability (less than 5%), and ideal hardness (more than 1 kg/m 2 ) Hence, the results (Table 2) suggested good stability of the formulated LL granules during storage and transportation.

Swelling study for the granules
The morphology of LL granules was observed by accessing the swelling behaviour and changes that occurred in the matrix ( Fig. 2A). Initially, at 0 min the surface of the granules was more relaxed and had space in between. However, with subsequent increases in time, the granules become more compact at 5 min, 10 min, and 15 min. (Fig. 2B-D). This change in the compactness of granules indicates that granules absorb water and absorbed water entraps across interstitial spaces, which led to the swelling of the granules with increae in time. The change in morphology and compactness of granules with increasing time indicated that granules might have diffusion mechanisms responsible for sustaining the release of the active (Siddique et al. 2010).

Accelerated temperature study
FTIR spectroscopy is an important technique to study the functional groups and various interactions between the different components of any developed formulation. Here, we scanned both the LL and developed LL granules at the 400 to 4000 cm −1 range (Fig. 3A, B). The peak at 2990-4000 cm −1 range is mainly due to water absorption. The FTIR spectra of L. lecanii have revealed absorbance at 3284 cm −1 , 1636.36 cm −1 , 1411.94 cm −1 , 1258.56 cm −1 , and 1088.07 cm −1 . The band at 3248 cm −1 may be due to polymeric hydroxyl compounds (Lingegowda et al. 2012). The band at 1411.94 cm -1 is due to C-O/C-H bending. Absorbance bands at around 1088cm −1 are due to C-O-C stretch of the polysaccharides group. Absorption at 1258.56 cm −1 is attributed mainly due to the alkyl ketonic group. Absorption at 1636 cm −1 is probably due to the amide group of the proteins (Naumann 2009;Salman et al. 2010Salman et al. , 2012Huleihel et al. 2018).
FTIR of the developed formulation also exhibited a prominent peak at 1636 cm −1 which depicts the incorporation of L. lecanii as the main active ingredient in the present formulation. It also reiterates that other additives used in the present formulation are compatible with the active ingredient used. LL granules were also assayed for their CFU (colony forming unit) count after storage at the accelerated temperature of 40 °C for three months. The results revealed that there was a slight change in the CFU from 1.2 × 10 8 to 1 × 10 8 .

XRD profile of the fungus and developed LL granules
L. lecanii shows an amorphous peak at a 2 theta value of 26.12. however XRD results of L. lecanii entrapped granules showed the absence of amorphous peaks of L. lecanii (Fig. 4A, B). This further confirms the entrapment of L. lecanii into granules and the transition of L. lecanii from amorphous form to crystalline form. The LL granules are composed of clay and other additives where fungus represents just 4% of the total composition, a major component is dominated by the clay, followed by the xanthan gum, and silica which is crystalline, therefore, the developed matrix is crystalline.

Larvicidal efficacy of the LL granules
Entomopathogenic fungi have been documented to have anti-larvicidal activity and may serve as an instrumental tool in the control of mosquito-borne diseases (Charnley In the present study, the toxicity of L. lecanii granules was noted against A. culicifacies larval instars (III) ( Table 3). At 80 μg/ml concentration of LL showed 100% mortality with an LC 50 value of 11.836 µg/mL. The treated larvae with LL were observed to have a dark colour, thorax and their abdominal segments (1st-5th) were predominately damaged). The untreated A. culicifacies showed a normal appearance with well-developed eye, head, thorax, and abdomen segments of the whole body. Similar findings have been reported previously by Raghuvendra et al. (2017). Entomopathogenic fungi have been demonstrated to have good larvicidal efficacy (Thiyagarajan et al. 2014;Raghuvendra et al. 2017;Baskar et al. 2020). The entomogenous fungal pathogenicity begins with the adhesion of the spores onto the insect's outer cuticle, and then spores germinate and penetrate the cuticle entering the host. Once the fungus penetrates it produces toxic secondary metabolites that overcome the insect immune system (Vivekanandhan et al. 2020). However, in response to the fungal infection, the host may activate both cellular and humoral immune responses which include phagocytosis, encapsulation, nodulation, melanization, and expression of antimicrobial peptides. (Dimopoulos et al. 2003;Wang et al. 2021).
A previous study reported that Paecilomyces fungal exhibited excellent mortality against larvae and was found to be safer on non-target organisms, (Vyas et al. 2007). Likewise, Abutaha et al. (2015) reported that an endophytic fungus Cochliobolus spicifer had great mortality effects against

Efficacy of the LL granules under the semi-field condition
The toxicity of LL granules was also evaluated under semifield conditions against the field-collected larval stages (Delhi -NCR) and compared with the results observed in previous bioassays conducted with laboratory-reared mosquito larvae (Table 4). Although LL granules resulted in higher LC 50 34.752 µg/mL and LC 90 184.710 µg/mL values which may be due to the prevailing natural environmental condition. Present findings revealed that LL was found to be equally effective against both laboratory-reared and field collected larvae under semi-field conditions, Thus, it is safe to conclude that LL holds much promise for the development of alternative larvicides that can be effective even against the field-collected mosquito populations and it may help in the control of vector population at the community level.

Histopathological analysis
Histopathological analysis was performed to get insight into the mode of action of the developed LL granules. It revealed edema, swelling, and the prolonged elongation of epithelial cells in the midgut region of the treated  mosquito larvae. While in contrast, untreated larvae exhibited the normal midgut epithelium with a single layer of digestive cells, well-developed brush border, cell membrane, and cytoplasmic regions (Fig. 5A, B). Histological alterations observed in the present research study are in accordance with previous reports by Farida et al. (2018) who observed that the entomopathogenic fungus, Beauveria bassiana when treated with 4th instar larvae of C. pipiens showed many histological alterations and malformations inside tissues, especially the cuticle, adipose cells, and midgut region as compared to untreated larvae. Interestingly, Ragavendran et al. (2017b) reported that the midgut cells of A. aegypti and C. quinquefasciatus (4th instar larvae) had shown enlargement in the gut lumen, decreased intercellular contents, and degeneration of nuclei, after treatment with Penicillium daleae mycelium metabolites (Raghuvendra et al. 2017a, b). Similarly, Abutaha et al. (2015) reported larvae treated with Aspergillus sydowi showed disruption of the peritrophic membrane, cytoplasmic vacuolization, and deformities of cellular microvilli.

SEM and SEM EDX studies of the LL-treated mosquitoes
Scanning electron microscopy (SEM) of A. culicifacies larvae treated with the LC 50 value of the LL granules, was performed to get insight into the mode of action. The images from SEM have substantiated the mode of action of the LL granules which is through penetration inside the cuticle, once the entomopathogenic fungal spores adhere to the insect's outer cuticle, it germinates, and penetrates the whole body of the host.  matrix of the gut epithelium and trachea of the arthropods including insects (Zhu et al. 2007). The rigidity and structural integrity to the mosquito larvae is provided by chitin which is essential for their survival.
For this particular study, we have used C:N values as an indirect measure of the relative amounts of chitin among treated and controlled mosquito larvae. Based on the previous reports we have presumed that the C:N ratios of invertebrates reflect relative amounts of chitin. The range of %C (41.1%) and %N (5.3%) values reported for structural chitin in other invertebrates from Yen et al. 2009 and % C (26.2) and % N (4.0%) values reported by Abdulkarim et al. (2013) would translate to similar C:N ratios. In the treated sample the C:N is 2.26 while in the control sample the C:N is 3.05. (Fig. 7A, B). Higher C:N ratio reveals that the chitin content of the control is higher as compared with the chitin content of the treated mosquito larvae. This may be due to the various enzymatic activity of the LL fungi which degrades the chitin of the cell wall so that the fungal spores or hyphae can penetrate deep inside the mosquito larvae for further sporulation which eventually results in the mortality of the mosquito larvae.

Efficacy of the LL granules over unformulated fungal spores
Granules are usually made either by coating a fine powder onto a substrate, e.g., sand, using a sticker such as PVP solution or by solvent impregnation onto an absorbent carrier. (Knowles 2008). However, the granules generally can be coated with resins or polymers to control the rate of effectiveness of active ingredients. Coating with a polymer such as xanthum gum ensures the controlled relapses of the action slowly and steadily and by maintaining the desired concentration of the action for a longer duration.
Xanthan gum is a high molecular weight heteropolysaccharide gum and has been widely used in oral and topical formulations, cosmetics, and food (Wade and Weller 1994;Santos et al. 2004). It has been documented to facilitate the slow release of drugs from hydrophilic matrix formulations (Lu et al. 1991;Talukdar and Plaizier-Vercammen 1993;Ingani and Moes 1988).
Lecanicillium lecanii is generally susceptible to UV light, temperature, and environmental conditions, thus granular formulation will slowly release the active ingredient to the surface which will ensure the slow and steady supply of the formulation's active ingredient, thereby ultimately killing the larvae. This will also increase its shelf life compared to non-formulated fungus. Moreover, the clay present in the granular formulation protects it from the harmful effect of UV light on account of its UV    (Chavan and Kadam 2010). Therefore, humidity and UV exposure must be considered important factors when formulating L. lecanii-based formulation. The use of wetting agents also helps to blend, and maintain humidity (Bobin 1999;Candau 1999). The binders also play an important role in making granules. The LL granule developed in the present research study is an economical alternative to synthetic insecticides and will also address the issue of insecticide resistance.

Conclusion
Despite the growing predominance of different types of biopesticides in the market and the huge potential to be used as an effective vector control agent, so far only 4% of the biopesticides are registered. In the present research study, we have developed an effective granular formulation using the fungus, L. lecanii and evaluated its efficacy against the vector, Anopheles culicifacies. Developed granules have shown good efficacy towards the target organisms this is particularly important as this will help in minimizing toxicity towards the non-target organisms due to their specific actions as compared with synthetic insecticides. Moreover, fungus-based products will also address the issue of insecticide resistance as they are very specific in their mode of action. The efficacy shown by LL granules will complement the existing tools in the vector control toolbox for malaria vectors. The outcome of the present study suggests that LL granules are more potent, selective, biodegradable, and natural mosquito larvicidal agents and can be used as tools for developing eco-friendly larvicides. However, non-target organisms may be included for comparison in future studies.