Evaluation of synergistic effect of entomopathogenic fungi Beauveria bassiana and Lecanicillium lecacii on the mosquito Culex quinquefaciatus

Aditya Shankar Kataki; Francesco Baldini; Anjana Singha Naorem

doi:10.1371/journal.pone.0308707

Abstract

Vector-borne diseases resulted into several cases of human morbidity and mortality over the years and among them is filariasis, caused by the mosquito Culex quinquefasciatus. Developing novel strategies for mosquito control without jeopardizing the environmental conditions has always been a topic of discussion and research. Integrated Vector Management (IVM) emphasizes a comprehensive approach and use of a range of strategies for vector control. Recent research evaluated the use of two entomopathogenic fungi; Beauveria bassiana and Lecanicillium lecanii in IVM, which can serve as potential organic insecticide for mosquito population control. However, their combined efficacy has not yet been evaluated against mosquito control in prior research and a gap of knowledge is still existing. So, this research was an attempt to bridge up the knowledge gap by (1) Assessing the combined efficacy of Beauveria bassiana and Lecanicillium lecanii on Culex quinquefasciatus (2) To investigate the sub-lethal concentration (LC₅₀) of the combined fungal concentration and (3) To examine the post-mortem effects caused by the combined fungal concentration under Scanning Electron Microscope (SEM). The larval pathogenicity assay was performed on 4^th instar C. quinquefasciatus larvae. Individual processed fungal solution of B. bassiana and L. lecanii were procured and to test the combined efficacy, the two solutions were mixed in equal proportions. To evaluate the sub-lethal concentration (LC₅₀), different concentrations of the combined fungal solution were prepared by serial dilations. The mortality was recorded after 24 hours for each concentration. Upon treatment and evaluation, The LC₅₀ values of B. bassiana and L. lecanii were 0.25 x 10⁴ spores/ml and 0.12 x 10⁴ spores/ml respectively and the combined fungal concentration was 0.06 x 10³ spores/ml. This clearly indicated that the combined efficacy of the fungi is more significant. Further, SEM analysis revealed morphological deformities and extensive body perforations upon combined fungal treatment. These findings suggested that combining the two fungi can be a more effective way in controlling the population of Culex quinquefasciatus.

Citation: Kataki AS, Baldini F, Naorem AS (2024) Evaluation of synergistic effect of entomopathogenic fungi Beauveria bassiana and Lecanicillium lecacii on the mosquito Culex quinquefaciatus. PLoS ONE 19(9): e0308707. https://doi.org/10.1371/journal.pone.0308707

Editor: Rachid Bouharroud, National Institute of Agricultural Research - INRA, MOROCCO

Received: March 19, 2024; Accepted: July 29, 2024; Published: September 6, 2024

Copyright: © 2024 Kataki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript.

Funding: Aditya Shankar Kataki was supported by Assam Science and Technology Council (ASTEC), Guwahati, Assam, India. Francesco Baldini was supported by the Academy Medical Science Springboard Award (ref: SBF007\100094).

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

1.1 Mosquito and their health and economic burden

Vectors are living organisms that are responsible for transferring infectious pathogens between humans or from animals to humans [1] and the diseases that are transmitted by potential vectors (eg: mosquitoes, fleas, cockroaches, tsetse flies, ticks, bugs etc.) are considered as vector-borne diseases [2]. Among vectors, mosquito borne diseases (MBD’s) are considered responsible for around 700 million cases of human morbidity and around a million of mortality every year around the globe [2]. Aedes, Anopheles, and Culex are recognized as most important mosquito vectors, responsible for the causing the deadly diseases dengue, malaria, filariasis, West Nile fever, yellow fever, Zika fever and Japanese encephalitis [1, 3]. According to prior research and survey, there are 219 million cases of malaria every year globally and around 3.9 billion are at risk contacting dengue in over 129 countries [1]. Chikungunya and Zika has led to an average yearly loss of over 106,000 and 44,000 disability-adjusted life years (DALYs) worldwide, respectively, between 2010 and 2019 [4, 5]. In addition, there has been significant economic losses too due to mosquito borne diseases (MBD’s) worldwide. As per reports, direct global cost of malaria has been estimated to be around $12 billion per year [6] and the estimated global cost of dengue is US $306 billion between 2020–2050 [7]. Similarly, mosquitoes of the family Culicidae is distributed worldwide comprising about 3500 species is responsible for high burden nuisance and spread of diseases in any urban cities [8, 9]. The species Culex quinquefasciatus is considered as the principal vector of bancroftian filariasis and a potential vector of the disease Dirofilaria immitis [10, 11]. This mosquito species has also been found to be a potential vector of several other arboviruses like avian pox, Rift Valley fever virus and West Nile virus and protozoa like Plasmodium relictum that causes bird malaria [12]. However, despite their high nuisance in urban society [8] and being a potential vector of diseases, they remain less studied compared to anophelines or aedes.

1.2 Current control methods and drawbacks

Currently various chemical, biological and mechanical methods are implemented to control the mosquito population and mosquito-borne diseases resulting in increase of resistance in them gradually [13–15]. The chemical methods mainly include the use of long-lasting insecticide treated nets (LLINs), indoor residual spraying (IRS) and use of mosquito repellants among others [13] Biological methods mainly include genetic modifications like sterile insect technique (SIT) [13, 16], use of fungi from genera Beauveria, Metarhizium, Lagenidium etc. [17, 18], use of fish, protozoans and bacterial agents. Mechanical control methods mainly involve the use of traps with chemical attractants like CO₂, NH₃, lactic acid etc that attract female mosquitoes along with eave tubes and attractive sugar baits [19, 20].

However, the chemical insecticides like DDT, pyrethroid, deltamethrin, organochlorine etc are found to be associated with many neurological and immunological disorders in human beings and are also carcinogenic leading to formation of tumors [21]. Additionally, chemical insecticides can affect natural predators, parasitoids and other organisms that assist in natural pest control resulting in harm to a broad variety of insects outside their intended target [22]. The ecosystem, including plant health, soil quality and other organisms that depend on these insects for a variety of ecological services, may be negatively impacted by loss of diversity due to the use of insecticides [22]. Moreover, target pests may become resistant to a particular insecticide after an extended period of exposure making it less effective [15, 23]. Thus, finding alternatives to these chemical insecticides has always been a topic of discussion and research among scientists.

1.3 Entomopathogenic fungi- A biological control agent for mosquito control

In search of alternatives, microbial biopesticides have being found to have undergone a great momentum around the globe [22]. Several studies and experiments have demonstrated the potential use of entomopathogenic fungi for controlling mosquito vectors without jeopardizing the nature [24, 25]. It has been well cited in previous research experiments that entomopathogens can contribute to reducing selective pressure for pesticide resistance development in pest populations [26, 27]. Beauveria bassiana and Lecaniciilium lecanii has been found to play a key role in management and elimination of forestry, veterinary and agricultural pests [22, 28] Particularly in honeybee research, experimental investigations employing entomopathogenic fungi have yielded great success [29]. Thus, the potential use of such entomopathogens to control mosquito population and restrict the spread of mosquito-borne disease has largely driven the research and investigation on fungal-mosquito interactions over more than a century [17, 30–32]. Mosquito larvae are exposed to fungi when they are exposed to plan detritus, within the water column and at the surface of water [30]. Adult mosquitoes are exposed to fungi in both indoor and outdoor environments when they rest, blood feed, mate and oviposit [30]. It has been well cited in prior research and experiments that the fungal infection reduces the life span of insecticide resistant mosquitoes [32–34] and thus entomopathogenic fungi can be used as a synergy in various insecticides or alone in IVM strategies [32].

1.4 Mode of action of entomopathogenic fungi

The main feature that makes these entomopathogenic fungi unique is their infectivity through direct contact and penetration [29] B. bassiana and L. lecanii successfully penetrate the cuticle of the insect and then reach the haemocoel to overcome the hosts innate immune defense response [30]. The conidia or spores germinate on the cuticle of the insect host, penetrate through the cuticle and spread in the hemolymph finally resulting the death of the host [35]. Upon penetration, it employs a combination of biochemical and mechanical mechanisms to infiltrate the host integument and reach the haemocoel [21]. When mycelium reaches a nutrient-rich environment, it switches to a specialized yeast-like cell phenotype known as hyphal bodies or blastophores [36]. and start to exploit the nutrient rich environment of the insect blood which ultimately results in colonizing tissues and release of toxic metabolites. It eventually degrades the free amino acids in the hemolymph and inhibits several key metabolic enzymes, including glutathione S- transferases (GST), carboxylesterase (CarE), and cytochrome P450 (CYP450) [18, 29, 32, 35–37] that ultimately results in death of the host upon fungal infection by mummification and mycosis [29].

Despite studies and research on their effectiveness to use as an organic insecticide and synergically with other insecticides for mosquito population control [38, 39], the combined efficacy of the two entomopathogenic fungi B. bassiana and L. lecacii has not yet been explored in previous research and experiments and thud needs to be studied and understand. This can indeed serve as an effective IVM strategy to prevent the emergence of insecticide resistance in mosquitoes. Furthermore, till date, no research has been done on assessing the morphological deformities and abnormalities that might have resulted upon combined fungal treatment of B. bassiana and L. lecacii on mosquitoes. Thus, this research was an attempt to bridge up existing gap of knowledge. The hypotheses of the research were (1) Combining the two entomopathogenic fungi B. bassiana and L. lecanii will produce better efficacy results than their individual treatments (2) The sub-lethal concentration (LC₅₀) of the combined fungal solution will be less than their individual solutions.

To test the hypotheses, the research addresses mainly three objectives: (i) evaluating the synergistic effect of B. bassiana and L. lecanii and comparing them with individual effect (ii) assessing the sub-lethal concentration (LC₅₀) of the combined fungal formulation and individual treatments against C. quinquefasciatus (iii) examining the post-mortem effects and morphological deformities on fungal infected larvae under SEM and compare it with the untreated larvae.

2. Materials and methods

2.1 Institutional Ethical Clearance (IEC)

The present work involved the use of mosquitoes but not any human. So, Institutional Ethical Clearance certificate of approval was obtained from Institutional Biosafety Committee (IBSC) of Cotton University, Guwahati, Assam; India with reference no–CU/IBSC/2023/21 dated on 9^th March 2023.

2.2 Collection sites of Culex quinquefasciatus larvae samples

The 4^th instar larval samples of C. quinquefasciatus were collected during the pre-monsoon period (March- April’23) from different locations in Guwahati Metropolitan city, Kamrup (M) district, Assam (26.1158° N, 91.7086° E) as shown in [Fig 1]. The selection of the sample collection sites was based on the factor that despite high population density of C. quiquefasciatus [9] and residents in these urban areas, no relevant survey or experimental study on mosquitoes till date were conducted.

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Fig 1. Sentinel-2 data was downloaded from USGS Earth Explorer portal (https://earthexplorer.usgs.gov/).

Three bands-Band 8 (Near Infrared), Band 4 (Red) and Band 3 (Green) were used to prepare the False Colour Composite (FCC) Image. This was performed using Composite Band Tool in Arc GIS 10.2 software. In FCC image, vegetation appears in red, water bodies are shown in dark blue, and built-up areas are depicted in light blue. Barren land, rock, outcrops, and sandbars are represented in shades ranging from light blue to grey.

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2.3 Experiment location

The entire experiment was carried out in the laboratory of Zoology department, Cotton University, Guwahati, Assam, India. The laboratory condition was at temperature 25°C to 37°C and humidity 70% to 80% with a 12-hour day/night cycles.

2.4 Larvae collection and laboratory rearing

The mosquito larvae from different sites were collected using a larval collection dipper and scoopers as per standard protocols [40, 41]. Upon collection, the mosquito larvae were transferred in small containers covered with lids for identification before rearing. The larvae were identified using the books entitled “The Ecology of Malaria mosquitoes” by Charlwood JD and “The biology of mosquitoes” by A.N Clements [42, 43]. Upon identification, only larvae of C. quinquefasciatus were selected and others were discarded. The larvae were transferred to trays of 5” x 7” in dimension for larval bioassay test. The mosquito larvae were fed with dog biscuits and millet powder and yeast powder in 3:3:1 ratio as done in prior research [21] during the experiment.

2.5 Preparation of different concentrations of B. bassiana fungal solution

Processed fungal solution of B. bassiana with concentration 2 x 10⁸ spores/ml was procured from the S.S. Biotech brand of Guwahati, Assam, India. This was considered as stock concentration for B. bassiana fungal formulation. The viability of the spores was determined by culture on nutrient agar and counting the colonies formed. Further, serial dilations were performed upon the stock concentration in 1:10 dilution factor by following serial dilution protocol [44] and different concentrations of B. bassiana fungal formulation were prepared to evaluate the sub-lethal concentration (LC₅₀) against C. quinquefasciatus larvae. Upon preparation they were mixed in the larval pans. The different fungal concentrations of B. bassiana prepared were 2.5 x 10⁶ spores/ml, 1.5 x 10⁴ spores/ml, 1.25 x 10⁴ spores/ml, 0.52 x 10⁴ spores/ml, 0.25 x 10³ spores/ml and 0.12 x 10³ spores/ml. The counting of the spores was carried out using hemocytometer.

2.6 Preparation of different concentrations of L. lecanii fungal solution

Processed fungal solution of L. lecanii with concentration 1 x 10⁹ spores/ml was procured from the S.S. Biotech brand of Guwahati, Assam, India. This was considered as stock concentration for L. lecanii fungal formulation and similarly the viability of the spores was determined by culture on nutrient agar and counting the colonies formed. Further, serial dilations were performed serial dilations were performed upon the stock concentration in 1:10 dilution factor by following serial dilution protocol [44] and different concentrations of L. lecanii were prepared to evaluate the sub-lethal concentration (LC₅₀) against C. quinquefasciatus larvae. Similarly, upon preparation, they were mixed in the larval pans. The different fungal concentrations of L. lecanii prepared were 1x 10⁵ spores/ml, 0.5 x 10⁵ spores/ml, 0.25 x 10⁴ spores/ml, 0.12 x 10⁴ spores/ml, 0.06 x10⁴ spores/ml and 0.03 x 10⁴ spores/ml. The counting of the spores was carried out using hemocytometer.

2.7 Preparation of combined fungal solution of B. bassiana and L. lecanii

A stock solution of 100 ml, combining two entomopathogenic fungi B. bassiana and L. lecanii was prepared by adding 50 ml each of the individual fungal solution. The concentration of the stock combined fungal solution was 6 x 10¹⁰ spores/ml and was kept in a 250 ml conical flask covered with aluminum foil to avoid contamination at 27°C room temperature. To evaluate the sub-lethal concentration (LC₅₀) the combined fungal solution, serial dilations were performed similarly upon the stock concentration in 1:10 dilution factor by following serial dilution protocol [44]. After performing serial dilations, the combined fungal concentration procured were 0.5 x 10⁴ spores/ml, 0.25 x 10⁴ spores/ml, 0.12 x 10⁴ spores/ml, 0.06 x 10³ spores/ml, 0.03 x 10³ spores/ml and 0.02 x 10³ spores/ml.

2.8 Larval pathogenicity bioassay

The larval pathogenicity bioassay was carried out using standard mosquito larval bioassay protocols [45] for the individual fungal solution of B. bassiana, L. lecanii and for the prepared combined fungal solution. The experiment was conducted by exposing larvae in conical flask of 250 ml. Three conical flasks for each; Control (distilled water), B. bassiana fungal solution, L. lecanii fungal solution and combined fungal solution was taken. The three conical flasks were considered as three replicates for each solution. Around twenty larvae were transferred and exposed in each replicate. Upon exposure and observation, larval mortality was noted down after 24 hours as mentioned by Standard protocols for larval bioassay and done in other studies too [45–47]. The mortality was corrected using Abbott’s formulae of corrected mortality [48, 49].

2.9 Preparation of larvae sample for SEM analysis

After treatment with the fungal solution the larvae specimens were fixed with 2.5% glutaraldehyde for 24 hours followed by dehydration in a series of acetone solutions (30%, 50%, 70%,90%) for 10 mins in each set and finally in 100% for 5 mins. Then the larval sample was air dried, and sputter coated with a 45nm gold coated and was observed under SEM (Sigma VP) [50]. Observations were mainly made on the head and thoracic regions of control and treated larvae, the abdominal segments and in the terminal region of larvae showing respiratory siphon for comparison studies.

2.10 Statistical analysis

Statistical analysis was performed in RStudio software (version 2024.04.2+764) and the packages ‘ggeffects’ ‘ggplot2’ ‘lme4’ ‘Matrix’ and ‘lmerTest’ using generalized linear mixed-effects model (GLMM) to investigate the significance of several explanatory variables ‘fungi’, ‘concentration’ and their interaction ‘fungi * concentration’ on the response variable ‘mortality’ caused by individual and combined fungal solution. The model used binary distribution and the final simplified model was selected based on AIC. The final model was plotted using the “ggplot2” package of the RStudio software. Additionally, Abbotts formulae was calculated by = (% test mortality -% control mortality/ 100—control mortality x 100) [49] and probit analysis was done using tables to estimate the probits and fitting the relationship by eye.

3. Results

3.1 Larval mortality upon treatment with B. bassiana fungal solution

Upon treatment with different concentrations of B. bassiana fungal solution, it was observed that the sub-lethal concentration (LC₅₀) for it was around 0.52 x 10⁴ spores/ml (Table 1). Larvae that were exposed in concentration 2.5 x 10⁶ spores/ml was giving mean morality of 90% and thus it was considered as LC₉₀ concentration.

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Table 1. Larval mortality observed post 24 hours of treatment with B. bassiana fungal solution.

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3.2 Larval mortality upon treatment with L. lecanii fungal solution

Upon treatment with different concentrations of L. lecanii fungal solution, it was observed that the sub-lethal concentration (LC₅₀) for it was around 0.12 x 10⁴ spores/ml (Table 2). Larvae that were exposed in concentration 1 x 10⁵ spores/ml was giving mean morality of around 87–90% and thus it was considered as LC₉₀ concentration.

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Table 2. Larval mortality observed post 24 hours of treatment with L. lecanii fungal solution.

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3.3 Larval mortality upon treatment with different concentrations of combined fungal solution of B. bassiana and L. lecanii

After treatment with different concentrations combined fungal formulation of B. bassiana and L. lecanii, it was observed that the sub-lethal concentration (LC₅₀) for it was around 0.06 x 10³ spores/ml (Table 3). Larvae that were exposed in concentration above 1 x 10⁵ spores/ml was giving mean morality of around 90–95% and thus it was considered as LC₉₀ concentration.

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Table 3. Larval mortality observed post 24 hours of treatment with combined fungal solution of B. bassiana and L. lecanii.

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Upon performing statistical analysis, it was found that the response variable “mortality” was significantly associated an interaction between fungal infection (individual species and combined) and their concentration with chisq (χ²) = 16.579, Df = 2 and Pr(>Chisq) = 0.0002511 ***.

Thus, the sublethal (LC₅₀) concentration of individual fungal solutions of B. bassiana and L. lecanii were 0.52 x 10⁴ spores/ml and 0.12 x 10⁴ spores/ml respectively. However, when combined, the LC₅₀ value was 0.06 x 10³ spores/ml indicating better efficacy and synergic effect against C. quinquefasciatus mosquito species as shown by graphical representation in [Fig 2].

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Fig 2. Percentage mortality of C. quinquefasciatus larvae at different concentrations (log transformed) of individual and combined fungal solution.

The X- axis of the graph represents the “Log concentration” and the Y-axis represents the “Percentage Mortality”. From the graph the sigmoid curve for combined effect produces 50% (LC₅₀) mortality in C. quinquefasciatus at much lesser concentration than the two individual fungi.

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3.4 Depositional of fungal spores in the larvae upon combined fungal treatment at different concentrations

Upon application of the combined fungal solution of B. bassiana and L. lecanii in different concentrations to the C. quinquefasciatus larvae, different degrees of deposition of fungal spores were observed under stereo microscope at magnification 40X for each concentration [Fig 3(A)–3(H)]. When carefully observed, the larvae kept in control (distilled water) had no deposition of fungal spores in any region of the body. Additionally, the alimentary canal along with the cuticle also seemed to be intact and complete with no distortion and breakage [Fig 3(A)]. On the contrary, larvae exposed in higher concentrations of combined fungal solution were found to have extensive deposition of fungal spores in them [Fig 3(B) and 3(C)]. These larvae were observed to had completely distorted thorax and alimentary canal with deposition of fungal spores. As the concentrations were gradually decreased, the deposition of fungal spores was also observed to get reduce and the alimentary canal and cuticle were intact [Fig 3(D)–3(F)]. However, the thorax and head regions were found to be swollen up with deposition of fungal spores. Finally, at the lowest concentration, distortion was evidently less, and the alimentary canal and cuticle was found to be completely intact, with minimal deposition of fungal spores in the head and thoracic region of the larvae [Fig 3(G) and 3(H)]. A closer view of the abdominal segment (AB) of treated larvae with combined effect of the entomopathogenic fungi B. bassiana and L. lecannii showing deposition of fungal spores has been captured under stereo microscope and is shown in [Fig 4].

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Fig 3. Images of larvae of C. quinquefasciatus under stereo microscope at magnification 40X to show deposition of fungal spores upon treatment with different concentrations of combined fungal solution of B. bassiana and L. lecanii and compare it with the control untreated larvae.

[a] control (distilled water) with no fungal spores (NFS) [b] 5 x 10⁷ spores/ml and [c] 2.85 x 10⁶ spores/ml with high fungal spores (HFS) [d]1.25 x 10⁵ spores/ml [e] 1x 10⁵ spores/ml and [f] 0.25 x 10⁴ spores/ml with moderate fungal spores (MFS) and [g] 0.0625 x 10³ spores/ml and [h] 0.31 x 10³ spores/ml with low fungal spores (LFS).

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Fig 4. A closer view of the abdominal segment (AB) of treated larvae with combined effect of the entomopathogenic fungi B. bassiana and L. lecannii showing deposition of fungal spores.

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3.5 Observations under Scanning Electron Microscope (SEM)

Upon treatment of larvae of C. quinquefasciatus with the sub-lethal concentration (LC₅₀) combined fungal solution of B. bassiana and L. lecanii, mosquito larvae were visualized under the Scanning Electron Microscope (SEM) to observe the extent of morphological deformities caused. The (Fig 5), represents the pictures of control untreated larvae (left) and the treated larvae (right). Upon our observation and careful analysis, it was found that the control larvae have a smooth cuticle surface [Fig 5(A)] whereas treated larvae [Fig 5(B)] had varied level of shrinkage and ruptured cuticle throughout the body.

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Fig 5.

Scanning Electron Microscope (SEM) images of C. quinquefasciatus larvae of control (left) and combined fungal treated larvae (right) showing deformity and fungal hyphae (FH). Full view of control (a) and treated larvae (b). Abdominal segments (AB) of control (c) and treated (d and g) larvae. The terminal region (TR) of larvae showing respiratory siphon (RS) of control (e) and treated (f and h) larvae.

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Additionally, when the cuticle of both the control and treated larvae was analyzed more closely, a complete distortion and aberration of the larval abdominal cuticle was observed [Fig 5(D) and 5(G)] in the treated larvae compared to the control larvae [Fig 5(C)]. The terminal segments were found to be completely shrunken in the treated larvae [Fig 5(F) and 5(H)] which was not actually observed in the control untreated larvae [Fig 5(E)]. Further, abnormalities observed in the treated larvae include breakage in the larval epithelium and bloated thoracic regions.

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4. Discussion

The rising concern of insecticide resistance gradually over the years in mosquitoes around the globe has led researchers to think about other sustainable alternatives for mosquito population control [15, 51–53]. As a result, the use of entomopathogenic fungi alone and synergically with other insecticides has been mentioned in many prior experiments and research work as alternatives to artificial chemical insecticides for mosquito population control and other agricultural pests too [17, 25, 27, 29, 32, 35, 37, 38, 54, 55]. Also, the matter of fact that these entomopathogenic fungi are safe with minimal risks to humans, animals and the environment, makes them a better alternative to the existing chemical artificial insecticides like DDT, Permethrin, Deltamethrin, Organochlorines, Organophosphates etc for mosquito population control [56–58].

However, previous studies didn’t evaluate the combined efficacy of the two entomopathogenic fungi B. bassiana and L. lecanii against mosquito population control. In our research we combined the two fungal solutions of B. bassiana and L. lecanii and found that by combining the fungal solution, it becomes more virulent than the individual fungi against the mosquito species C. quinquefasciatus. The viability of spores was determined by culture on nutrient agar and counting the colonies formed. The LC₅₀ values for B. bassiana and L. lecanii were 0.52 x 10⁴ spores/ml and 0.12 x 10⁴ spores/ml respectively and when they were combined in equal proportions, the LC₅₀ value was 0.06 x 10³ spores/ml against C. quinquefasciatus upon exposure for 24 hours. Thus, according to our results and observation, the sub-lethal concentration (LC₅₀) of the combined entomopathogenic fungi was ten times more virulent than the individual fungal solutions indicating better efficacy in population control of mosquito species C. quinquefasciatus.

In addition, the SEM analysis of the treated larvae with combined fungal solution revealed shrinkage and deformities in the head, thorax and abdomen of the mosquito. Hence, it established the fact that, when the two entomopathogenic fungi were combined at such low concentration, they were able to cause fungal infection and deformities in the larvae leading them to death by mycosis and mummification.

The sub-lethal concentration (LC₅₀) value is defined as the concentration of a chemical that kills 50% of the test animals exposed to the concentration for a set period [59]. This is the concentration that is taken into consideration while making any commercial insecticide alone or mixing with other insecticides because animal or insect toxicity studies do not necessarily extrapolate to humans [60, 61]. Thus, it is important to know what the sub-lethal (LC50) concentration or dose of any new insecticide or fungicide is while developing them before using them commercially against insects and pests in large scale so that it doesn’t lead to human infection and diseases.

The sub-lethal concentration (LC₅₀) evaluated from the current research for the combined fungal solution of B. bassiana and L. lecanii against C. quinquefasciatus mosquito species, can be used as a reference for producing other organic sustainable novel insecticides. The combined fungal solution can be either applied alone or synergically with other insecticides as done with different group of fungi in prior research [38, 39] for controlling different vectors responsible for causing diseases in human and wildlife. Keeping in mind the morphological deformities shown in the experiment through SEM analysis, the concentration can either be increased or decreased for other insects or species of mosquito in different parts of the world where the resistance status of them varies accordingly. This will ensure that their population is below the threshold level and consequently people contaminate with less vector-borne diseases. Apart from that, this combined fungal formulation of B. bassiana and L. lecanii can be used in local sewages and stagnant water bodies too which are considered as potential breeding sites and habitats for mosquitoes. These will ensure the mosquitoes contaminate themselves with the fungal infection in their larval stage itself and not reach the pupal stage of development, ultimately decreasing the adult population of the mosquitoes in the community.

However, more research is required to understand and determine whether it can be used as aerosols to eradicate other harmful pests and insects.

Since, in recent studies and research, the use of entomopathogenic fungi has drawn a lot of attention [17, 18, 27, 37, 55], researchers and toxicologists may use this information as a starting point to investigate the effects of the fungal solution on different organisms or to compare the toxicity of different substances. This will help them in understanding and establishing safe exposure limits along with developing appropriate sustainable mitigation strategies for vector control in society.

5. Conclusion

The present study was an attempt to show the combined efficacy of the two entomopathogenic fungi B. bassiana and L. lecanii against C. quinquefasciatus population control. The research addressed significantly the fact that when the fungi are combined, they indeed show better efficacy than the two individual fungi in controlling population of C. quiquefasciatus. In addition, it can be used as a reference in developing alternative organic insecticides for tackling the developing insecticide resistance in mosquitoes around the globe to control their population and spread of vector-borne disease. Thus, the study will be instrumental for producing other insecticides and controlling potential vectors. Moreover, by determining the lethal concentration further for aquatic organisms or invertebrates, it will help evaluate the risk posed by the fungal solution to them. This information thus, will indeed aid in future decision making and strategies in IVM and sustainable vector control in society.

Acknowledgments

The authors would like to thank the Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam, India for their support in performing SEM analysis. Moreover, we would also like to thank the Zoology department of Cotton University, Guwahati, Assam, India and School of Biodiversity, One Health and Veterinary Medicine (SBHOVM) of the University of Glasgow, United Kingdom. Also, the first author would like to thank Dr Nameirakpam Nirjanta Devi; Biotechnology department, Cotton University, Guwahati, Assam, India, for her valuable suggestions and insights.

References

1. Vector-borne diseases [Internet]. [cited 2024 Jun 28]. Available from: https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases
2. Chilakam N, Lakshminarayanan V, Keremutt S, Rajendran A, Thunga G, Poojari PG, et al. Economic Burden of Mosquito-Borne Diseases in Low- and Middle-Income Countries: Protocol for a Systematic Review. JMIR Res Protoc [Internet]. 2023 Jan 1 [cited 2024 Jun 28];12(1). Available from: /pmc/articles/PMC10750235/ pmid:38079215
- View Article
- PubMed/NCBI
- Google Scholar
3. Caraballo H, King K. Emergency department management of mosquito-borne illness: malaria, dengue, and West Nile virus. Emerg Med Pract [Internet]. 2014 May 1 [cited 2024 Jun 28];16(5):1–23; quiz 23. Available from: https://europepmc.org/article/med/25207355 pmid:25207355
- View Article
- PubMed/NCBI
- Google Scholar
4. Puntasecca CJ, King CH, Labeaud AD. Measuring the global burden of chikungunya and Zika viruses: A systematic review. PLoS Negl Trop Dis [Internet]. 2021 Mar 1 [cited 2024 Jun 29];15(3):e0009055. Available from: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0009055 pmid:33661908
- View Article
- PubMed/NCBI
- Google Scholar
5. Aryaprema VS, Steck MR, Peper ST, Xue R De, Qualls WA. A systematic review of published literature on mosquito control action thresholds across the world. PLoS Negl Trop Dis [Internet]. 2023 Mar 1 [cited 2024 Jun 29];17(3):e0011173. Available from: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0011173 pmid:36867651
- View Article
- PubMed/NCBI
- Google Scholar
6. Andrade M V., Noronha K, Diniz BPC, Guedes G, Carvalho LR, Silva VA, et al. The economic burden of malaria: a systematic review. Malar J [Internet]. 2022 Dec 1 [cited 2024 Jun 29];21(1):1–10. Available from: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-022-04303-6
- View Article
- Google Scholar
7. Chen S, Cao Z, Jiao L, Chen W, Prettner K, Kuhn M, et al. The Global Economic Burden of Dengue in 2020–2050: Estimates and Projections for 141 Countries and Territories. [cited 2024 Jun 29]; Available from: https://papers.ssrn.com/abstract=4691773
- View Article
- Google Scholar
8. Nchoutpouen E, Talipouo A, Djiappi-Tchamen B, Djamouko-Djonkam L, Kopya E, Ngadjeu CS, et al. Culex species diversity, susceptibility to insecticides and role as potential vector of Lymphatic filariasis in the city of Yaoundé, Cameroon. PLoS Negl Trop Dis [Internet]. 2019 [cited 2024 Jun 28];13(4). Available from: /pmc/articles/PMC6464241/
- View Article
- Google Scholar
9. CK J., Alak ChD, Kaushik M. Mosquito vector survey in Guwahati city of Assam, India. J Entomol Nematol. 2014 Mar 31;6(3):42–50.
- View Article
- Google Scholar
10. Belkin JN. MOSQUITO STUDIES (DIPTERA, CULICIDAE). VII. THE CULICIDAE OF NEW ZEALAND. 1968;
11. Lai CH, Tung KC, Ooi HK, Wang JS. Competence of Aedes albopictus and Culex quinquefasciatus as vector of Dirofilaria immitis after blood meal with different microfilarial density. Vet Parasitol [Internet]. 2000 Jun 27 [cited 2024 Jun 28];90(3):231–7. Available from: https://pubmed.ncbi.nlm.nih.gov/10842003/ pmid:10842003
- View Article
- PubMed/NCBI
- Google Scholar
12. Bhattacharya S, Basu P, Sajal Bhattacharya C. The Southern House Mosquito, Culex quinquefasciatus: profile of a smart vector. J Entomol Zool Stud [Internet]. 2016 [cited 2024 Jun 28];4(2):73–81. Available from: https://www.researchgate.net/publication/330579096
- View Article
- Google Scholar
13. Okumu F, Moore S. Combining indoor residual spraying and insecticide-treated nets for malaria control in Africa: A review of possible outcomes and an outline of suggestions for the future. Malar J [Internet]. 2011 Jul 28 [cited 2024 Jun 29];10(1):1–13. Available from: https://link.springer.com/articles/10.1186/1475-2875-10-208
- View Article
- Google Scholar
14. Onen H, Luzala MM, Kigozi S, Sikumbili RM, Muanga CJK, Zola EN, et al. Mosquito-Borne Diseases and Their Control Strategies: An Overview Focused on Green Synthesized Plant-Based Metallic Nanoparticles. Insects [Internet]. 2023 Mar 1 [cited 2024 Jun 29];14(3):221. Available from: https://www.mdpi.com/2075-4450/14/3/221/htm pmid:36975906
- View Article
- PubMed/NCBI
- Google Scholar
15. Liu N. Insecticide resistance in mosquitoes: Impact, mechanisms, and research directions. Vol. 60, Annual Review of Entomology. Annual Reviews Inc.; 2015. p. 537–59.
16. Wilke ABB, Marrelli MT. Genetic control of mosquitoes: Population suppression strategies. Rev Inst Med Trop Sao Paulo. 2012 Sep;54(5):287–92. pmid:22983293
- View Article
- PubMed/NCBI
- Google Scholar
17. Kanzok SM, Jacobs-Lorena M. Entomopathogenic fungi as biological insecticides to control malaria. Trends Parasitol [Internet]. 2006 [cited 2024 Jun 29];22(2):49–51. Available from: https://pubmed.ncbi.nlm.nih.gov/16377249/ pmid:16377249
- View Article
- PubMed/NCBI
- Google Scholar
18. Litwin A, Nowak M, Różalska S. Entomopathogenic fungi: unconventional applications. Vol. 19, Reviews in Environmental Science and Biotechnology. Springer; 2020. p. 23–42.
19. Sternberg ED, Ng’Habi KR, Lyimo IN, Kessy ST, Farenhorst M, Thomas MB, et al. Eave tubes for malaria control in Africa: Initial development and semi-field evaluations in Tanzania Lucy Tusting, Jo Lines. Malar J [Internet]. 2016 Sep 1 [cited 2024 Jun 29];15(1):1–11. Available from: https://link.springer.com/articles/10.1186/s12936-016-1499-8
- View Article
- Google Scholar
20. Dormont L, Mulatier M, Carrasco D, Cohuet A. Mosquito Attractants. Journal of Chemical Ecology 2021 47:4 [Internet]. 2021 Mar 16 [cited 2024 Jun 29];47(4):351–93. Available from: https://link.springer.com/article/10.1007/s10886-021-01261-2 pmid:33725235
- View Article
- PubMed/NCBI
- Google Scholar
21. Vivekanandhan P, Kavitha T, Karthi S, Senthil-Nathan S, Shivakumar MS. Toxicity of beauveria bassiana-28 mycelial extracts on larvae of culex quinquefasciatus mosquito (Diptera: Culicidae). Int J Environ Res Public Health. 2018 Mar 3;15(3). pmid:29510502
- View Article
- PubMed/NCBI
- Google Scholar
22. Mascarin GM, Jaronski ST. The production and uses of Beauveria bassiana as a microbial insecticide. World J Microbiol Biotechnol [Internet]. 2016 Nov 1 [cited 2024 Jun 28];32(11). Available from: https://pubmed.ncbi.nlm.nih.gov/27628337/ pmid:27628337
- View Article
- PubMed/NCBI
- Google Scholar
23. Platt N, Kwiatkowska RM, Irving H, Diabaté A, Dabire R, Wondji CS. Target-site resistance mutations (kdr and RDL), but not metabolic resistance, negatively impact male mating competiveness in the malaria vector Anopheles gambiae. Heredity (Edinb) [Internet]. 2015 Sep 14 [cited 2024 Jun 25];115(3):243. Available from: /pmc/articles/PMC4519523/
- View Article
- Google Scholar
24. Environmental and Microbial Relationships. Environmental and Microbial Relationships. 2007;
- View Article
- Google Scholar
25. Accoti A, Engdahl CS, Dimopoulos G. Discovery of Novel Entomopathogenic Fungi for Mosquito-Borne Disease Control. Frontiers in Fungal Biology [Internet]. 2021 Jul 27 [cited 2024 Jun 28]; 2:637234. Available from: www.frontiersin.org pmid:37744144
- View Article
- PubMed/NCBI
- Google Scholar
26. Farenhorst M, Mouatcho JC, Kikankie CK, Brooke BD, Hunt RH, Thomas MB, et al. Fungal infection counters insecticide resistance in African malaria mosquitoes. Proc Natl Acad Sci U S A [Internet]. 2009 Oct 13 [cited 2024 Jun 28];106(41):17443–7. Available from: https://www.pnas.org/doi/abs/10.1073/pnas.0908530106 pmid:19805146
- View Article
- PubMed/NCBI
- Google Scholar
27. Farenhorst M, Knols BGJ, Thomas MB, Howard AFV, Takken W, Rowland M, et al. Synergy in Efficacy of Fungal Entomopathogens and Permethrin against West African Insecticide-Resistant Anopheles gambiae Mosquitoes. PLoS One [Internet]. 2010 [cited 2024 Jun 28];5(8):e12081. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012081 pmid:20711409
- View Article
- PubMed/NCBI
- Google Scholar
28. BARCANU E, AGAPIE O, GHERASE I, TĂNASE B, VÎNĂTORU C. Biological Control Using Microorganisms Metarhizium anisopliae, Beauveria bassiana and Lecanicillium lecanii Against Tuta absoluta (Meyrick). Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca Horticulture. 2022 Oct 24;78(2):164–8.
- View Article
- Google Scholar
29. Bava R, Castagna F, Piras C, Musolino V, Lupia C, Palma E, et al. Entomopathogenic Fungi for Pests and Predators Control in Beekeeping. Veterinary Sciences 2022, Vol 9, Page 95 [Internet]. 2022 Feb 21 [cited 2024 Jun 28];9(2):95. Available from: https://www.mdpi.com/2306-7381/9/2/95/htm pmid:35202348
- View Article
- PubMed/NCBI
- Google Scholar
30. Tawidian P, Rhodes VL, Michel K. Mosquito-fungus interactions and antifungal immunity. Insect Biochem Mol Biol. 2019 Aug 1;111. pmid:31265904
- View Article
- PubMed/NCBI
- Google Scholar
31. Scholte EJ, Knols BGJ, Takken W. Autodissemination of the entomopathogenic fungus Metarhizium anisopliae amongst adults of the malaria vector Anopheles gambiae s.s. Malar J [Internet]. 2004 Nov 28 [cited 2024 Jun 29];3. Available from: https://pubmed.ncbi.nlm.nih.gov/15566626/ pmid:15566626
- View Article
- PubMed/NCBI
- Google Scholar
32. Renuka S, Vani H C, Alex E. Entomopathogenic Fungi as a Potential Management Tool for the Control of Urban Malaria Vector, Anopheles stephensi (Diptera: Culicidae). Journal of Fungi 2023, Vol 9, Page 223 [Internet]. 2023 Feb 8 [cited 2024 Jun 28];9(2):223. Available from: https://www.mdpi.com/2309-608X/9/2/223/htm pmid:36836337
- View Article
- PubMed/NCBI
- Google Scholar
33. Blanford S, Chan BHK, Jenkins N, Sim D, Turner RJ, Read AF, et al. Fungal pathogen reduces potential for malaria transmission. Science [Internet]. 2005 Jun 10 [cited 2024 Jun 29];308(5728):1638–41. Available from: https://pubmed.ncbi.nlm.nih.gov/15947189/ pmid:15947189
- View Article
- PubMed/NCBI
- Google Scholar
34. Scholte EJ, Takken W, Knols BGJ. Infection of adult Aedes aegypti and Ae. albopictus mosquitoes with the entomopathogenic fungus Metarhizium anisopliae. Acta Trop. 2007 Jun 1;102(3):151–8. pmid:17544354
- View Article
- PubMed/NCBI
- Google Scholar
35. Islam W, Adnan M, Shabbir A, Naveed H, Abubakar YS, Qasim M, et al. Insect-fungal-interactions: A detailed review on entomopathogenic fungi pathogenicity to combat insect pests. Microb Pathog. 2021 Oct 1;159. pmid:34352375
- View Article
- PubMed/NCBI
- Google Scholar
36. Ma X, Ge Q, Taha RH, Chen K, Yuan Y. Beauveria bassiana Ribotoxin (BbRib) Induces Silkworm Cell Apoptosis via Activating Ros Stress Response. Processes 2021, Vol 9, Page 1470 [Internet]. 2021 Aug 23 [cited 2024 Jun 29];9(8):1470. Available from: https://www.mdpi.com/2227-9717/9/8/1470/htm
- View Article
- Google Scholar
37. Upadhyay V, Rai D, Rana M, Mehra P, Pandey AK. Verticillium lecani (Zimm.): A potential entomopathogenic fungus. International Journal of Agriculture, Environment and Biotechnology. 2014;7(4):719.
- View Article
- Google Scholar
38. Thakur N, Tomar P, Sharma S, Kaur S, Sharma S, Yadav AN, et al. Synergistic effect of entomopathogens against Spodoptera litura (Fabricius) under laboratory and greenhouse conditions. Egypt J Biol Pest Control. 2022 Dec 1;32(1).
- View Article
- Google Scholar
39. Ahmed MAI, Matsumura F. Synergistic actions of formamidine insecticides on the activity of pyrethroids and neonicotinoids against aedes aegypti (Diptera: Culicidae). J Med Entomol. 2012 Nov;49(6):1405–10. pmid:23270169
- View Article
- PubMed/NCBI
- Google Scholar
40. Standard Operating Procedure for conducting larval and pupal surveys for Anopheles. 2022;
41. Guidelines for laboratory and field testing of mosquito larvicides [Internet]. [cited 2024 Jun 29]. Available from: https://www.who.int/publications/i/item/WHO-CDS-WHOPES-GCDPP-2005.13
42. Clements AN, Clements AN. The biology of mosquitoes. A. N. Clements. 1992 [cited 2024 Jun 29]; Available from: https://books.google.com/books/about/Biology_of_Mosquitoes_Development_Nutrit.html?id=dLjwAAAAMAAJ
- View Article
- Google Scholar
43. Charlwood JD. The ecology of malaria vectors [Internet]. [cited 2024 Jun 30]. 284 p. Available from: https://www.routledge.com/The-Ecology-of-Malaria-Vectors/Charlwood/p/book/9780367248482
- View Article
- Google Scholar
44. Serial Dilution Protocol [Internet]. [cited 2024 Jun 30]. Available from: https://bpsbioscience.com/serial-dilution-protocol
45. Wang Y, Li T, Liu N. Mosquito Larval Bioassays. Cold Spring Harb Protoc [Internet]. 2023 Jul 1 [cited 2024 Jun 30];2023(7):pdb. prot108040. Available from: http://cshprotocols.cshlp.org/content/2023/7/pdb.prot108040.full pmid:36882293
- View Article
- PubMed/NCBI
- Google Scholar
46. Standard WHO protocol of larval toxicity assay—Google Search [Internet]. [cited 2024 Jun 26]. Available from: https://www.google.co.in/search?q=Standard+WHO+protocol+of+larval+toxicity+assay
47. LC50 calculations help predict toxicity—Responsible Seafood Advocate [Internet]. [cited 2024 Jul 1]. Available from: https://www.globalseafood.org/advocate/lc50-calculations-help-predict-toxicity/
48. Campbell BE, Miller DM. A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars. J Vis Exp [Internet]. 2017 Mar 15 [cited 2024 Jun 30];2017(121):55092. Available from: /pmc/articles/PMC5409339/ pmid:28362364
- View Article
- PubMed/NCBI
- Google Scholar
49. Abbott WS. A METHOD OF COMPUTING THE EFFECTIVENESS OF AN INSECTICIDEl. Vol. 3, JoURNAL oF THE Arunnrclu Moseurro Coxrnol AssocrATroN Vot.
- View Article
- Google Scholar
50. Yu KX. Rapid Method for Scanning Electron Microscopy of the Larvae of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) [Internet]. Available from: https://www.researchgate.net/publication/282291458
51. Munywoki DN, Kokwaro ED, Mwangangi JM, Muturi EJ, Mbogo CM. Insecticide resistance status in Anopheles gambiae (s.l.) in coastal Kenya. Parasit Vectors. 2021 Dec 1;14(1). pmid:33879244
- View Article
- PubMed/NCBI
- Google Scholar
52. Mzilahowa T, Chiumia M, Mbewe RB, Uzalili VT, Luka-Banda M, Kutengule A, et al. Increasing insecticide resistance in Anopheles funestus and Anopheles arabiensis in Malawi, 2011–2015. Malar J [Internet]. 2016 Nov 22 [cited 2024 Jun 23];15(1):1–15. Available from: /pmc/articles/PMC5120501/ pmid:27876046
- View Article
- PubMed/NCBI
- Google Scholar
53. Alout H, Roche B, Dabiré RK, Cohuet A. Consequences of insecticide resistance on malaria transmission. PLoS Pathog [Internet]. 2017 Sep 1 [cited 2024 Jun 25];13(9). Available from: /pmc/articles/PMC5589250/ pmid:28880906
- View Article
- PubMed/NCBI
- Google Scholar
54. Dey A. Beauveria Bassiana: Entomopathogenic Fungi as Biopesticide Reported from Cachar, Southern Assam. Journal of Agriculture and Allied Sciences e-RRJAAS |. 2022;11.
- View Article
- Google Scholar
55. Scholte EJ, Knols BGJ, Samson RA, Takken W. Entomopathogenic fungi for mosquito control: A review. Journal of Insect Science [Internet]. 2004 Jun 23 [cited 2024 Jul 1];4. Available from: /pmc/articles/PMC528879/ pmid:15861235
- View Article
- PubMed/NCBI
- Google Scholar
56. Sharma A, Sharma S, Yadav PK. Entomopathogenic fungi and their relevance in sustainable agriculture: A review. Cogent Food Agric. 2023;9(1).
- View Article
- Google Scholar
57. Zimmermann G. Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Sci Technol. 2007;17(6):553–96.
- View Article
- Google Scholar
58. Zimmermann G. Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Sci Technol [Internet]. 2007 [cited 2024 Jul 1];17(9):879–920. Available from: https://www.tandfonline.com/doi/abs/10.1080/09583150701593963
- View Article
- Google Scholar
59. CCOHS: What is a LD₅₀ and LC₅₀? [Internet]. [cited 2024 Jul 1]. Available from: https://www.ccohs.ca/oshanswers/chemicals/ld50.html
60. Lane TR, Harris J, Urbina F, Ekins S. Comparing LD50/LC50Machine Learning Models for Multiple Species. ACS Chemical Health and Safety. 2023 Mar 27;30(2):83–97. pmid:37457397
- View Article
- PubMed/NCBI
- Google Scholar
61. The MSDS HyperGlossary: LC-50, 50% Lethal Concentration [Internet]. [cited 2024 Jul 1]. Available from: http://www.ilpi.com/msds/ref/lc50.html#

[ref1] 1. Vector-borne diseases [Internet]. [cited 2024 Jun 28]. Available from: https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases

[ref2] 2. Chilakam N, Lakshminarayanan V, Keremutt S, Rajendran A, Thunga G, Poojari PG, et al. Economic Burden of Mosquito-Borne Diseases in Low- and Middle-Income Countries: Protocol for a Systematic Review. JMIR Res Protoc [Internet]. 2023 Jan 1 [cited 2024 Jun 28];12(1). Available from: /pmc/articles/PMC10750235/ pmid:38079215
View Article
PubMed/NCBI
Google Scholar

[3] View Article

[4] PubMed/NCBI

[5] Google Scholar

[ref3] 3. Caraballo H, King K. Emergency department management of mosquito-borne illness: malaria, dengue, and West Nile virus. Emerg Med Pract [Internet]. 2014 May 1 [cited 2024 Jun 28];16(5):1–23; quiz 23. Available from: https://europepmc.org/article/med/25207355 pmid:25207355
View Article
PubMed/NCBI
Google Scholar

[7] View Article

[8] PubMed/NCBI

[9] Google Scholar

[ref4] 4. Puntasecca CJ, King CH, Labeaud AD. Measuring the global burden of chikungunya and Zika viruses: A systematic review. PLoS Negl Trop Dis [Internet]. 2021 Mar 1 [cited 2024 Jun 29];15(3):e0009055. Available from: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0009055 pmid:33661908
View Article
PubMed/NCBI
Google Scholar

[11] View Article

[12] PubMed/NCBI

[13] Google Scholar

[ref5] 5. Aryaprema VS, Steck MR, Peper ST, Xue R De, Qualls WA. A systematic review of published literature on mosquito control action thresholds across the world. PLoS Negl Trop Dis [Internet]. 2023 Mar 1 [cited 2024 Jun 29];17(3):e0011173. Available from: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0011173 pmid:36867651
View Article
PubMed/NCBI
Google Scholar

[15] View Article

[16] PubMed/NCBI

[17] Google Scholar

[ref6] 6. Andrade M V., Noronha K, Diniz BPC, Guedes G, Carvalho LR, Silva VA, et al. The economic burden of malaria: a systematic review. Malar J [Internet]. 2022 Dec 1 [cited 2024 Jun 29];21(1):1–10. Available from: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-022-04303-6
View Article
Google Scholar

[19] View Article

[20] Google Scholar

[ref7] 7. Chen S, Cao Z, Jiao L, Chen W, Prettner K, Kuhn M, et al. The Global Economic Burden of Dengue in 2020–2050: Estimates and Projections for 141 Countries and Territories. [cited 2024 Jun 29]; Available from: https://papers.ssrn.com/abstract=4691773
View Article
Google Scholar

[22] View Article

[23] Google Scholar

[ref8] 8. Nchoutpouen E, Talipouo A, Djiappi-Tchamen B, Djamouko-Djonkam L, Kopya E, Ngadjeu CS, et al. Culex species diversity, susceptibility to insecticides and role as potential vector of Lymphatic filariasis in the city of Yaoundé, Cameroon. PLoS Negl Trop Dis [Internet]. 2019 [cited 2024 Jun 28];13(4). Available from: /pmc/articles/PMC6464241/
View Article
Google Scholar

[25] View Article

[26] Google Scholar

[ref9] 9. CK J., Alak ChD, Kaushik M. Mosquito vector survey in Guwahati city of Assam, India. J Entomol Nematol. 2014 Mar 31;6(3):42–50.
View Article
Google Scholar

[28] View Article

[29] Google Scholar

[ref10] 10. Belkin JN. MOSQUITO STUDIES (DIPTERA, CULICIDAE). VII. THE CULICIDAE OF NEW ZEALAND. 1968;

[ref11] 11. Lai CH, Tung KC, Ooi HK, Wang JS. Competence of Aedes albopictus and Culex quinquefasciatus as vector of Dirofilaria immitis after blood meal with different microfilarial density. Vet Parasitol [Internet]. 2000 Jun 27 [cited 2024 Jun 28];90(3):231–7. Available from: https://pubmed.ncbi.nlm.nih.gov/10842003/ pmid:10842003
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref12] 12. Bhattacharya S, Basu P, Sajal Bhattacharya C. The Southern House Mosquito, Culex quinquefasciatus: profile of a smart vector. J Entomol Zool Stud [Internet]. 2016 [cited 2024 Jun 28];4(2):73–81. Available from: https://www.researchgate.net/publication/330579096
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref13] 13. Okumu F, Moore S. Combining indoor residual spraying and insecticide-treated nets for malaria control in Africa: A review of possible outcomes and an outline of suggestions for the future. Malar J [Internet]. 2011 Jul 28 [cited 2024 Jun 29];10(1):1–13. Available from: https://link.springer.com/articles/10.1186/1475-2875-10-208
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref14] 14. Onen H, Luzala MM, Kigozi S, Sikumbili RM, Muanga CJK, Zola EN, et al. Mosquito-Borne Diseases and Their Control Strategies: An Overview Focused on Green Synthesized Plant-Based Metallic Nanoparticles. Insects [Internet]. 2023 Mar 1 [cited 2024 Jun 29];14(3):221. Available from: https://www.mdpi.com/2075-4450/14/3/221/htm pmid:36975906
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref15] 15. Liu N. Insecticide resistance in mosquitoes: Impact, mechanisms, and research directions. Vol. 60, Annual Review of Entomology. Annual Reviews Inc.; 2015. p. 537–59.

[ref16] 16. Wilke ABB, Marrelli MT. Genetic control of mosquitoes: Population suppression strategies. Rev Inst Med Trop Sao Paulo. 2012 Sep;54(5):287–92. pmid:22983293
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref17] 17. Kanzok SM, Jacobs-Lorena M. Entomopathogenic fungi as biological insecticides to control malaria. Trends Parasitol [Internet]. 2006 [cited 2024 Jun 29];22(2):49–51. Available from: https://pubmed.ncbi.nlm.nih.gov/16377249/ pmid:16377249
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref18] 18. Litwin A, Nowak M, Różalska S. Entomopathogenic fungi: unconventional applications. Vol. 19, Reviews in Environmental Science and Biotechnology. Springer; 2020. p. 23–42.

[ref19] 19. Sternberg ED, Ng’Habi KR, Lyimo IN, Kessy ST, Farenhorst M, Thomas MB, et al. Eave tubes for malaria control in Africa: Initial development and semi-field evaluations in Tanzania Lucy Tusting, Jo Lines. Malar J [Internet]. 2016 Sep 1 [cited 2024 Jun 29];15(1):1–11. Available from: https://link.springer.com/articles/10.1186/s12936-016-1499-8
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref20] 20. Dormont L, Mulatier M, Carrasco D, Cohuet A. Mosquito Attractants. Journal of Chemical Ecology 2021 47:4 [Internet]. 2021 Mar 16 [cited 2024 Jun 29];47(4):351–93. Available from: https://link.springer.com/article/10.1007/s10886-021-01261-2 pmid:33725235
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref21] 21. Vivekanandhan P, Kavitha T, Karthi S, Senthil-Nathan S, Shivakumar MS. Toxicity of beauveria bassiana-28 mycelial extracts on larvae of culex quinquefasciatus mosquito (Diptera: Culicidae). Int J Environ Res Public Health. 2018 Mar 3;15(3). pmid:29510502
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref22] 22. Mascarin GM, Jaronski ST. The production and uses of Beauveria bassiana as a microbial insecticide. World J Microbiol Biotechnol [Internet]. 2016 Nov 1 [cited 2024 Jun 28];32(11). Available from: https://pubmed.ncbi.nlm.nih.gov/27628337/ pmid:27628337
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref23] 23. Platt N, Kwiatkowska RM, Irving H, Diabaté A, Dabire R, Wondji CS. Target-site resistance mutations (kdr and RDL), but not metabolic resistance, negatively impact male mating competiveness in the malaria vector Anopheles gambiae. Heredity (Edinb) [Internet]. 2015 Sep 14 [cited 2024 Jun 25];115(3):243. Available from: /pmc/articles/PMC4519523/
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref24] 24. Environmental and Microbial Relationships. Environmental and Microbial Relationships. 2007;
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref25] 25. Accoti A, Engdahl CS, Dimopoulos G. Discovery of Novel Entomopathogenic Fungi for Mosquito-Borne Disease Control. Frontiers in Fungal Biology [Internet]. 2021 Jul 27 [cited 2024 Jun 28]; 2:637234. Available from: www.frontiersin.org pmid:37744144
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref26] 26. Farenhorst M, Mouatcho JC, Kikankie CK, Brooke BD, Hunt RH, Thomas MB, et al. Fungal infection counters insecticide resistance in African malaria mosquitoes. Proc Natl Acad Sci U S A [Internet]. 2009 Oct 13 [cited 2024 Jun 28];106(41):17443–7. Available from: https://www.pnas.org/doi/abs/10.1073/pnas.0908530106 pmid:19805146
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref27] 27. Farenhorst M, Knols BGJ, Thomas MB, Howard AFV, Takken W, Rowland M, et al. Synergy in Efficacy of Fungal Entomopathogens and Permethrin against West African Insecticide-Resistant Anopheles gambiae Mosquitoes. PLoS One [Internet]. 2010 [cited 2024 Jun 28];5(8):e12081. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012081 pmid:20711409
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref28] 28. BARCANU E, AGAPIE O, GHERASE I, TĂNASE B, VÎNĂTORU C. Biological Control Using Microorganisms Metarhizium anisopliae, Beauveria bassiana and Lecanicillium lecanii Against Tuta absoluta (Meyrick). Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca Horticulture. 2022 Oct 24;78(2):164–8.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref29] 29. Bava R, Castagna F, Piras C, Musolino V, Lupia C, Palma E, et al. Entomopathogenic Fungi for Pests and Predators Control in Beekeeping. Veterinary Sciences 2022, Vol 9, Page 95 [Internet]. 2022 Feb 21 [cited 2024 Jun 28];9(2):95. Available from: https://www.mdpi.com/2306-7381/9/2/95/htm pmid:35202348
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref30] 30. Tawidian P, Rhodes VL, Michel K. Mosquito-fungus interactions and antifungal immunity. Insect Biochem Mol Biol. 2019 Aug 1;111. pmid:31265904
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref31] 31. Scholte EJ, Knols BGJ, Takken W. Autodissemination of the entomopathogenic fungus Metarhizium anisopliae amongst adults of the malaria vector Anopheles gambiae s.s. Malar J [Internet]. 2004 Nov 28 [cited 2024 Jun 29];3. Available from: https://pubmed.ncbi.nlm.nih.gov/15566626/ pmid:15566626
View Article
PubMed/NCBI
Google Scholar

[100] View Article

[101] PubMed/NCBI

[102] Google Scholar

[ref32] 32. Renuka S, Vani H C, Alex E. Entomopathogenic Fungi as a Potential Management Tool for the Control of Urban Malaria Vector, Anopheles stephensi (Diptera: Culicidae). Journal of Fungi 2023, Vol 9, Page 223 [Internet]. 2023 Feb 8 [cited 2024 Jun 28];9(2):223. Available from: https://www.mdpi.com/2309-608X/9/2/223/htm pmid:36836337
View Article
PubMed/NCBI
Google Scholar

[104] View Article

[105] PubMed/NCBI

[106] Google Scholar

[ref33] 33. Blanford S, Chan BHK, Jenkins N, Sim D, Turner RJ, Read AF, et al. Fungal pathogen reduces potential for malaria transmission. Science [Internet]. 2005 Jun 10 [cited 2024 Jun 29];308(5728):1638–41. Available from: https://pubmed.ncbi.nlm.nih.gov/15947189/ pmid:15947189
View Article
PubMed/NCBI
Google Scholar

[108] View Article

[109] PubMed/NCBI

[110] Google Scholar

[ref34] 34. Scholte EJ, Takken W, Knols BGJ. Infection of adult Aedes aegypti and Ae. albopictus mosquitoes with the entomopathogenic fungus Metarhizium anisopliae. Acta Trop. 2007 Jun 1;102(3):151–8. pmid:17544354
View Article
PubMed/NCBI
Google Scholar

[112] View Article

[113] PubMed/NCBI

[114] Google Scholar

[ref35] 35. Islam W, Adnan M, Shabbir A, Naveed H, Abubakar YS, Qasim M, et al. Insect-fungal-interactions: A detailed review on entomopathogenic fungi pathogenicity to combat insect pests. Microb Pathog. 2021 Oct 1;159. pmid:34352375
View Article
PubMed/NCBI
Google Scholar

[116] View Article

[117] PubMed/NCBI

[118] Google Scholar

[ref36] 36. Ma X, Ge Q, Taha RH, Chen K, Yuan Y. Beauveria bassiana Ribotoxin (BbRib) Induces Silkworm Cell Apoptosis via Activating Ros Stress Response. Processes 2021, Vol 9, Page 1470 [Internet]. 2021 Aug 23 [cited 2024 Jun 29];9(8):1470. Available from: https://www.mdpi.com/2227-9717/9/8/1470/htm
View Article
Google Scholar

[120] View Article

[121] Google Scholar

[ref37] 37. Upadhyay V, Rai D, Rana M, Mehra P, Pandey AK. Verticillium lecani (Zimm.): A potential entomopathogenic fungus. International Journal of Agriculture, Environment and Biotechnology. 2014;7(4):719.
View Article
Google Scholar

[123] View Article

[124] Google Scholar

[ref38] 38. Thakur N, Tomar P, Sharma S, Kaur S, Sharma S, Yadav AN, et al. Synergistic effect of entomopathogens against Spodoptera litura (Fabricius) under laboratory and greenhouse conditions. Egypt J Biol Pest Control. 2022 Dec 1;32(1).
View Article
Google Scholar

[126] View Article

[127] Google Scholar

[ref39] 39. Ahmed MAI, Matsumura F. Synergistic actions of formamidine insecticides on the activity of pyrethroids and neonicotinoids against aedes aegypti (Diptera: Culicidae). J Med Entomol. 2012 Nov;49(6):1405–10. pmid:23270169
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref40] 40. Standard Operating Procedure for conducting larval and pupal surveys for Anopheles. 2022;

[ref41] 41. Guidelines for laboratory and field testing of mosquito larvicides [Internet]. [cited 2024 Jun 29]. Available from: https://www.who.int/publications/i/item/WHO-CDS-WHOPES-GCDPP-2005.13

[ref42] 42. Clements AN, Clements AN. The biology of mosquitoes. A. N. Clements. 1992 [cited 2024 Jun 29]; Available from: https://books.google.com/books/about/Biology_of_Mosquitoes_Development_Nutrit.html?id=dLjwAAAAMAAJ
View Article
Google Scholar

[135] View Article

[136] Google Scholar

[ref43] 43. Charlwood JD. The ecology of malaria vectors [Internet]. [cited 2024 Jun 30]. 284 p. Available from: https://www.routledge.com/The-Ecology-of-Malaria-Vectors/Charlwood/p/book/9780367248482
View Article
Google Scholar

[138] View Article

[139] Google Scholar

[ref44] 44. Serial Dilution Protocol [Internet]. [cited 2024 Jun 30]. Available from: https://bpsbioscience.com/serial-dilution-protocol

[ref45] 45. Wang Y, Li T, Liu N. Mosquito Larval Bioassays. Cold Spring Harb Protoc [Internet]. 2023 Jul 1 [cited 2024 Jun 30];2023(7):pdb. prot108040. Available from: http://cshprotocols.cshlp.org/content/2023/7/pdb.prot108040.full pmid:36882293
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref46] 46. Standard WHO protocol of larval toxicity assay—Google Search [Internet]. [cited 2024 Jun 26]. Available from: https://www.google.co.in/search?q=Standard+WHO+protocol+of+larval+toxicity+assay

[ref47] 47. LC50 calculations help predict toxicity—Responsible Seafood Advocate [Internet]. [cited 2024 Jul 1]. Available from: https://www.globalseafood.org/advocate/lc50-calculations-help-predict-toxicity/

[ref48] 48. Campbell BE, Miller DM. A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars. J Vis Exp [Internet]. 2017 Mar 15 [cited 2024 Jun 30];2017(121):55092. Available from: /pmc/articles/PMC5409339/ pmid:28362364
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref49] 49. Abbott WS. A METHOD OF COMPUTING THE EFFECTIVENESS OF AN INSECTICIDEl. Vol. 3, JoURNAL oF THE Arunnrclu Moseurro Coxrnol AssocrATroN Vot.
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref50] 50. Yu KX. Rapid Method for Scanning Electron Microscopy of the Larvae of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) [Internet]. Available from: https://www.researchgate.net/publication/282291458

[ref51] 51. Munywoki DN, Kokwaro ED, Mwangangi JM, Muturi EJ, Mbogo CM. Insecticide resistance status in Anopheles gambiae (s.l.) in coastal Kenya. Parasit Vectors. 2021 Dec 1;14(1). pmid:33879244
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref52] 52. Mzilahowa T, Chiumia M, Mbewe RB, Uzalili VT, Luka-Banda M, Kutengule A, et al. Increasing insecticide resistance in Anopheles funestus and Anopheles arabiensis in Malawi, 2011–2015. Malar J [Internet]. 2016 Nov 22 [cited 2024 Jun 23];15(1):1–15. Available from: /pmc/articles/PMC5120501/ pmid:27876046
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref53] 53. Alout H, Roche B, Dabiré RK, Cohuet A. Consequences of insecticide resistance on malaria transmission. PLoS Pathog [Internet]. 2017 Sep 1 [cited 2024 Jun 25];13(9). Available from: /pmc/articles/PMC5589250/ pmid:28880906
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

[ref54] 54. Dey A. Beauveria Bassiana: Entomopathogenic Fungi as Biopesticide Reported from Cachar, Southern Assam. Journal of Agriculture and Allied Sciences e-RRJAAS |. 2022;11.
View Article
Google Scholar

[168] View Article

[169] Google Scholar

[ref55] 55. Scholte EJ, Knols BGJ, Samson RA, Takken W. Entomopathogenic fungi for mosquito control: A review. Journal of Insect Science [Internet]. 2004 Jun 23 [cited 2024 Jul 1];4. Available from: /pmc/articles/PMC528879/ pmid:15861235
View Article
PubMed/NCBI
Google Scholar

[171] View Article

[172] PubMed/NCBI

[173] Google Scholar

[ref56] 56. Sharma A, Sharma S, Yadav PK. Entomopathogenic fungi and their relevance in sustainable agriculture: A review. Cogent Food Agric. 2023;9(1).
View Article
Google Scholar

[175] View Article

[176] Google Scholar

[ref57] 57. Zimmermann G. Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Sci Technol. 2007;17(6):553–96.
View Article
Google Scholar

[178] View Article

[179] Google Scholar

[ref58] 58. Zimmermann G. Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Sci Technol [Internet]. 2007 [cited 2024 Jul 1];17(9):879–920. Available from: https://www.tandfonline.com/doi/abs/10.1080/09583150701593963
View Article
Google Scholar

[181] View Article

[182] Google Scholar

[ref59] 59. CCOHS: What is a LD₅₀ and LC₅₀? [Internet]. [cited 2024 Jul 1]. Available from: https://www.ccohs.ca/oshanswers/chemicals/ld50.html

[ref60] 60. Lane TR, Harris J, Urbina F, Ekins S. Comparing LD50/LC50Machine Learning Models for Multiple Species. ACS Chemical Health and Safety. 2023 Mar 27;30(2):83–97. pmid:37457397
View Article
PubMed/NCBI
Google Scholar

[185] View Article

[186] PubMed/NCBI

[187] Google Scholar

[ref61] 61. The MSDS HyperGlossary: LC-50, 50% Lethal Concentration [Internet]. [cited 2024 Jul 1]. Available from: http://www.ilpi.com/msds/ref/lc50.html#

Figures

Abstract

1. Introduction

1.1 Mosquito and their health and economic burden

1.2 Current control methods and drawbacks

1.3 Entomopathogenic fungi- A biological control agent for mosquito control

1.4 Mode of action of entomopathogenic fungi

2. Materials and methods

2.1 Institutional Ethical Clearance (IEC)

2.2 Collection sites of Culex quinquefasciatus larvae samples

2.3 Experiment location

2.4 Larvae collection and laboratory rearing

2.5 Preparation of different concentrations of B. bassiana fungal solution

2.6 Preparation of different concentrations of L. lecanii fungal solution

2.7 Preparation of combined fungal solution of B. bassiana and L. lecanii

2.8 Larval pathogenicity bioassay

2.9 Preparation of larvae sample for SEM analysis

2.10 Statistical analysis

3. Results

3.1 Larval mortality upon treatment with B. bassiana fungal solution

3.2 Larval mortality upon treatment with L. lecanii fungal solution

3.3 Larval mortality upon treatment with different concentrations of combined fungal solution of B. bassiana and L. lecanii

3.4 Depositional of fungal spores in the larvae upon combined fungal treatment at different concentrations

3.5 Observations under Scanning Electron Microscope (SEM)

4. Discussion

5. Conclusion

Acknowledgments

References