The widespread use of synthetic pesticides has led to many problems, including the emergence of resistant organisms, environmental degradation and harm to human health. Therefore, new microbial pesticides that are safe for human health and the environment are urgently needed. In this study, rhamnolipid biosurfactant produced by Enterobacter cloacae SJ2 was used to evaluate toxicity to mosquito (Culex quinquefasciatus) and termite (Odontotermes obesus) larvae. The results showed that there was a dose-dependent mortality rate between treatments. The LC50 (50% lethal concentration) value at 48 hours for termite and mosquito larval biosurfactants was determined using a nonlinear regression curve fitting method. The results showed that the 48-hour LC50 values (95% confidence interval) of larvicidal and antitermite activity of the biosurfactant were 26.49 mg/L (range 25.40 to 27.57) and 33.43 mg/L (range 31.09 to 35.68) respectively. According to histopathological examination, treatment with biosurfactants caused severe damage to organelle tissues of larvae and termites. The results of this study indicate that the microbial biosurfactant produced by Enterobacter cloacae SJ2 is an excellent and potentially effective tool for Cx control. quinquefasciatus and O. obesus.
Tropical countries experience a large number of mosquito-borne diseases1. The relevance of mosquito-borne diseases is widespread. More than 400,000 people die from malaria each year, and some major cities are experiencing epidemics of serious diseases such as dengue, yellow fever, chikungunya and Zika.2 Vector-borne diseases are associated with one in six infections worldwide, with mosquitoes causing the most significant cases3 ,4. Culex, Anopheles and Aedes are the three mosquito genera most commonly associated with disease transmission5. The prevalence of dengue fever, an infection transmitted by the Aedes aegypti mosquito, has increased over the past decade and poses a significant public health threat4,7,8. According to the World Health Organization (WHO), more than 40% of the world’s population is at risk of dengue fever, with 50–100 million new cases occurring annually in more than 100 countries9,10,11. Dengue fever has become a major public health problem as its incidence has increased worldwide12,13,14. Anopheles gambiae, commonly known as the African Anopheles mosquito, is the most important vector of human malaria in tropical and subtropical regions15. West Nile virus, St. Louis encephalitis, Japanese encephalitis, and viral infections of horses and birds are transmitted by Culex mosquitoes, often called common house mosquitoes. In addition, they are also carriers of bacterial and parasitic diseases16. There are more than 3,000 species of termites in the world, and they have been around for more than 150 million years17. Most pests live in the soil and feed on wood and wood products containing cellulose. The Indian termite Odontotermes obesus is an important pest that causes severe damage to important crops and plantation trees18. In agricultural areas, termite infestations at various stages can cause enormous economic damage to various crops, tree species and building materials. Termites can also cause human health problems19.
The issue of resistance from microorganisms and pests in today’s pharmaceutical and agricultural fields is complex20,21. Therefore, both companies should look for new cost-effective antimicrobials and safe biopesticides. Synthetic pesticides are now available and have been shown to be infectious and repel non-target beneficial insects22. In recent years, research on biosurfactants has expanded due to their application in various industries. Biosurfactants are very useful and vital in agriculture, soil remediation, petroleum extraction, bacteria and insect removal, and food processing23,24. Biosurfactants or microbial surfactants are biosurfactant chemicals produced by microorganisms such as bacteria, yeasts and fungi in coastal habitats and oil-contaminated areas25,26. Chemically derived surfactants and biosurfactants are two types that are obtained directly from the natural environment27. Various biosurfactants are obtained from marine habitats28,29. Therefore, scientists are looking for new technologies for the production of biosurfactants based on natural bacteria30,31. Advances in such research demonstrate the importance of these biological compounds for environmental protection32. Bacillus, Pseudomonas, Rhodococcus, Alcaligenes, Corynebacterium and these bacterial genera are well-studied representatives23,33.
There are many types of biosurfactants with a wide range of applications34. A significant advantage of these compounds is that some of them have antibacterial, larvicidal and insecticidal activity. This means that they can be used in the agricultural, chemical, pharmaceutical and cosmetic industries35,36,37,38. Because biosurfactants are generally biodegradable and environmentally beneficial, they are used in integrated pest management programs to protect crops39. Thus, basic knowledge has been obtained about the larvicidal and antitermite activity of microbial biosurfactants produced by Enterobacter cloacae SJ2. We examined mortality and histological changes when exposed to different concentrations of rhamnolipid biosurfactants. In addition, we evaluated the widely used Quantitative Structure-Activity (QSAR) computer program Ecological Structure-Activity (ECOSAR) to determine acute toxicity for microalgae, daphnia, and fish.
In this study, the antitermite activity (toxicity) of purified biosurfactants at various concentrations ranging from 30 to 50 mg/ml (at 5 mg/ml intervals) was tested against Indian termites, O. obesus and fourth species )Evaluate. Larvae of instar Cx. Larvae of mosquitoes quinquefasciatus. Biosurfactant LC50 concentrations over 48 hours against O. obesus and Cx. C. solanacearum. Mosquito larvae were identified using a nonlinear regression curve fitting method. The results showed that termite mortality increased with increasing biosurfactant concentration. The results showed that the biosurfactant had larvicidal activity (Figure 1) and anti-termite activity (Figure 2), with 48-hour LC50 values (95% CI) of 26.49 mg/L (25.40 to 27.57 ) and 33.43 mg/l (Fig. 31.09 to 35.68), respectively (Table 1). In terms of acute toxicity (48 hours), the biosurfactant is classified as “harmful” to the tested organisms. The biosurfactant produced in this study showed excellent larvicidal activity with 100% mortality within 24-48 hours of exposure.
Calculate the LC50 value for larvicidal activity. Nonlinear regression curve fitting (solid line) and 95% confidence interval (shaded area) for relative mortality (%).
Calculate the LC50 value for anti-termite activity. Nonlinear regression curve fitting (solid line) and 95% confidence interval (shaded area) for relative mortality (%).
At the end of the experiment, morphological changes and anomalies were observed under the microscope. Morphological changes were observed in the control and treated groups at 40x magnification. As shown in Figure 3, growth impairment occurred in the majority of larvae treated with biosurfactants. Figure 3a shows a normal Cx. quinquefasciatus, Figure 3b shows an anomalous Cx. Causes five nematode larvae.
Effect of sublethal (LC50) doses of biosurfactants on the development of Culex quinquefasciatus larvae. Light microscopy image (a) of a normal Cx at 40× magnification. quinquefasciatus (b) Abnormal Cx. Causes five nematode larvae.
In the present study, histological examination of treated larvae (Fig. 4) and termites (Fig. 5) revealed several abnormalities, including reduction in abdominal area and damage to muscles, epithelial layers and skin. midgut. Histology revealed the mechanism of inhibitory activity of the biosurfactant used in this study.
Histopathology of normal untreated 4th instar Cx larvae. quinquefasciatus larvae (control: (a,b)) and treated with biosurfactant (treatment: (c,d)). Arrows indicate treated intestinal epithelium (epi), nuclei (n), and muscle (mu). Bar = 50 µm.
Histopathology of normal untreated O. obesus (control: (a,b)) and biosurfactant treated (treatment: (c,d)). Arrows indicate intestinal epithelium (epi) and muscle (mu), respectively. Bar = 50 µm.
In this study, ECOSAR was used to predict the acute toxicity of rhamnolipid biosurfactant products to primary producers (green algae), primary consumers (water fleas) and secondary consumers (fish). This program uses sophisticated quantitative structure-activity compound models to evaluate toxicity based on molecular structure. The model uses structure-activity (SAR) software to calculate the acute and long-term toxicity of substances to aquatic species. Specifically, Table 2 summarizes the estimated mean lethal concentrations (LC50) and mean effective concentrations (EC50) for several species. Suspected toxicity was categorized into four levels using the Globally Harmonized System of Classification and Labeling of Chemicals (Table 3).
Control of vector-borne diseases, especially strains of mosquitoes and Aedes mosquitoes. Egyptians, now difficult work 40,41,42,43,44,45,46. Although some chemically available pesticides, such as pyrethroids and organophosphates, are somewhat beneficial, they pose significant risks to human health, including diabetes, reproductive disorders, neurological disorders, cancer, and respiratory diseases. Moreover, over time, these insects can become resistant to them13,43,48. Thus, effective and environmentally friendly biological control measures will become a more popular method of mosquito control49,50. Benelli51 suggested that early control of mosquito vectors would be more effective in urban areas, but they did not recommend the use of larvicides in rural areas52. Tom et al 53 also suggested that controlling mosquitoes at their immature stages would be a safe and simple strategy because they are more sensitive to control agents 54 .
Biosurfactant production by a potent strain (Enterobacter cloacae SJ2) showed consistent and promising efficacy. Our previous study reported that Enterobacter cloacae SJ2 optimizes biosurfactant production using physicochemical parameters26. According to their study, the optimal conditions for biosurfactant production by a potential E. cloacae isolate were incubation for 36 hours, agitation at 150 rpm, pH 7.5, 37 °C, salinity 1 ppt, 2% glucose as carbon source, 1 % yeast. the extract was used as a nitrogen source to obtain 2.61 g/L biosurfactant. In addition, the biosurfactants were characterized using TLC, FTIR and MALDI-TOF-MS. This confirmed that rhamnolipid is a biosurfactant. Glycolipid biosurfactants are the most intensively studied class of other types of biosurfactants55. They consist of carbohydrate and lipid parts, mainly fatty acid chains. Among glycolipids, the main representatives are rhamnolipid and sophorolipid56. Rhamnolipids contain two rhamnose moieties linked to mono‐ or di‐β‐hydroxydecanoic acid 57 . The use of rhamnolipids in the medical and pharmaceutical industries is well established 58 , in addition to their recent use as pesticides 59 .
The interaction of the biosurfactant with the hydrophobic region of the respiratory siphon allows water to pass through its stomatal cavity, thereby increasing the contact of the larvae with the aquatic environment. The presence of biosurfactants also affects the trachea, the length of which is close to the surface, which makes it easier for the larvae to crawl to the surface and breathe. As a result, the surface tension of water decreases. Since the larvae cannot attach to the surface of the water, they fall to the bottom of the tank, disrupting hydrostatic pressure, resulting in excessive energy expenditure and death by drowning38,60. Similar results were obtained by Ghribi61, where a biosurfactant produced by Bacillus subtilis exhibited larvicidal activity against Ephestia kuehniella. Similarly, the larvicidal activity of Cx. Das and Mukherjee23 also assessed the effect of cyclic lipopeptides on quinquefasciatus larvae.
The results of this study concern the larvicidal activity of rhamnolipid biosurfactants against Cx. Killing of quinquefasciatus mosquitoes is consistent with previously published results. For example, surfactin-based biosurfactants produced by various bacteria of the genus Bacillus are used. and Pseudomonas spp. Some early reports64,65,66 reported larval-killing activity of lipopeptide biosurfactants from Bacillus subtilis23. Deepali et al. 63 found that rhamnolipid biosurfactant isolated from Stenotropomonas maltophilia had potent larvicidal activity at a concentration of 10 mg/L. Silva et al. 67 reported the larvicidal activity of rhamnolipid biosurfactant against Ae at a concentration of 1 g/L. Aedes aegypti. Kanakdande et al. 68 reported that lipopeptide biosurfactants produced by Bacillus subtilis caused overall mortality in Culex larvae and termites with the lipophilic fraction of Eucalyptus. Similarly, Masendra et al. 69 reported worker ant (Cryptotermes cynocephalus Light.) mortality of 61.7% in the lipophilic n -hexane and EtOAc fractions of E. crude extract.
Parthipan et al 70 reported the insecticidal use of lipopeptide biosurfactants produced by Bacillus subtilis A1 and Pseudomonas stutzeri NA3 against Anopheles Stephensi, a vector of the malaria parasite Plasmodium. They observed that larvae and pupae survived longer, had shorter oviposition periods, were sterile, and had shorter lifespans when treated with different concentrations of biosurfactants. The observed LC50 values of B. subtilis biosurfactant A1 were 3.58, 4.92, 5.37, 7.10 and 7.99 mg/L for different larval states (i.e. larvae I, II, III, IV and stage pupae) respectively. In comparison, biosurfactants for larval stages I-IV and pupal stages of Pseudomonas stutzeri NA3 were 2.61, 3.68, 4.48, 5.55 and 6.99 mg/L, respectively. The delayed phenology of surviving larvae and pupae is thought to be the result of significant physiological and metabolic disturbances caused by insecticide treatments71.
Wickerhamomyces anomalus strain CCMA 0358 produces a biosurfactant with 100% larvicidal activity against Aedes mosquitoes. aegypti 24-hour interval 38 was higher than reported by Silva et al. A biosurfactant produced from Pseudomonas aeruginosa using sunflower oil as a carbon source has been shown to kill 100% of larvae within 48 hours 67 . Abinaya et al.72 and Pradhan et al.73 also demonstrated the larvicidal or insecticidal effects of surfactants produced by several isolates of the genus Bacillus. A previously published study by Senthil-Nathan et al. found that 100% of mosquito larvae exposed to plant lagoons were likely to die. 74.
Assessing the sublethal effects of insecticides on insect biology is critical for integrated pest management programs because sublethal doses/concentrations do not kill insects but may reduce insect populations in future generations by disrupting biological characteristics10. Siqueira et al 75 observed complete larvicidal activity (100% mortality) of rhamnolipid biosurfactant (300 mg/ml) when tested at various concentrations ranging from 50 to 300 mg/ml. Larval stage of Aedes aegypti strains. They analyzed the effects of time to death and sublethal concentrations on larval survival and swimming activity. In addition, they observed a decrease in swimming speed after 24–48 hours of exposure to sublethal concentrations of biosurfactant (e.g., 50 mg/mL and 100 mg/mL). Poisons that have promising sublethal roles are thought to be more effective in causing multiple damage to exposed pests76.
Histological observations of our results indicate that biosurfactants produced by Enterobacter cloacae SJ2 significantly alter the tissues of mosquito (Cx. quinquefasciatus) and termite (O. obesus) larvae. Similar anomalies were caused by preparations of basil oil in An. gambiaes.s and An. arabica were described by Ochola77. Kamaraj et al.78 also described the same morphological abnormalities in An. Stephanie’s larvae were exposed to gold nanoparticles. Vasantha-Srinivasan et al.79 also reported that shepherd’s purse essential oil severely damaged the chamber and epithelial layers of Aedes albopictus. Aedes aegypti. Raghavendran et al reported that mosquito larvae were treated with 500 mg/ml mycelial extract of a local Penicillium fungus. Ae show severe histological damage. aegypti and Cx. Mortality rate 80. Previously, Abinaya et al. Fourth instar larvae of An were studied. Stephensi and Ae. aegypti found numerous histological changes in Aedes aegypti treated with B. licheniformis exopolysaccharides, including gastric cecum, muscle atrophy, damage and disorganization of nerve cord ganglia72. According to Raghavendran et al., after treatment with P. daleae mycelial extract, the midgut cells of tested mosquitoes (4th instar larvae) showed swelling of the intestinal lumen, a decrease in intercellular contents, and nuclear degeneration81. The same histological changes were observed in mosquito larvae treated with echinacea leaf extract, indicating the insecticidal potential of the treated compounds50.
The use of ECOSAR software has received international recognition82. Current research suggests that the acute toxicity of ECOSAR biosurfactants to microalgae (C. vulgaris), fish and water fleas (D. magna) falls within the “toxicity” category defined by the United Nations83. The ECOSAR ecotoxicity model uses SAR and QSAR to predict acute and long-term toxicity of substances and is often used to predict the toxicity of organic pollutants82,84.
Paraformaldehyde, sodium phosphate buffer (pH 7.4) and all other chemicals used in this study were purchased from HiMedia Laboratories, India.
Biosurfactant production was carried out in 500 mL Erlenmeyer flasks containing 200 mL of sterile Bushnell Haas medium supplemented with 1% crude oil as the sole carbon source. A preculture of Enterobacter cloacae SJ2 (1.4 × 104 CFU/ml) was inoculated and cultured on an orbital shaker at 37°C, 200 rpm for 7 days. After the incubation period, the biosurfactant was extracted by centrifuging the culture medium at 3400×g for 20 min at 4°C and the resulting supernatant was used for screening purposes. The optimization procedures and characterization of biosurfactants were adopted from our earlier study26.
Culex quinquefasciatus larvae were obtained from the Center for Advanced Study in Marine Biology (CAS), Palanchipetai, Tamil Nadu (India). Larvae were reared in plastic containers filled with deionized water at 27 ± 2°C and a photoperiod of 12:12 (light:dark). Mosquito larvae were fed a 10% glucose solution.
Culex quinquefasciatus larvae have been found in open and unprotected septic tanks. Use standard classification guidelines to identify and culture larvae in the laboratory85. Larvicidal trials were carried out in accordance with the recommendations of the World Health Organization 86 . SH. Fourth instar larvae of quinquefasciatus were collected in closed tubes in groups of 25 ml and 50 ml with an air gap of two-thirds of their capacity. Biosurfactant (0–50 mg/ml) was added to each tube individually and stored at 25 °C. The control tube used only distilled water (50 ml). Dead larvae were considered to be those that showed no signs of swimming during the incubation period (12–48 hours) 87 . Calculate the percentage of larval mortality using the equation. (1)88.
The family Odontotermitidae includes the Indian termite Odontotermes obesus, found in rotting logs at the Agricultural Campus (Annamalai University, India). Test this biosurfactant (0–50 mg/ml) using normal procedures to determine if it is harmful. After drying in laminar air flow for 30 min, each strip of Whatman paper was coated with biosurfactant at a concentration of 30, 40, or 50 mg/ml. Pre-coated and uncoated paper strips were tested and compared in the center of a Petri dish. Each petri dish contains about thirty active termites O. obesus. Control and test termites were given wet paper as a food source. All plates were kept at room temperature throughout the incubation period. Termites died after 12, 24, 36 and 48 hours89,90. Equation 1 was then used to estimate the percentage of termite mortality at different biosurfactant concentrations. (2).
The samples were kept on ice and packed in microtubes containing 100 ml of 0.1 M sodium phosphate buffer (pH 7.4) and sent to the Central Aquaculture Pathology Laboratory (CAPL) of the Rajiv Gandhi Center for Aquaculture (RGCA). Histology Laboratory, Sirkali, Mayiladuthurai. District, Tamil Nadu, India for further analysis. Samples were immediately fixed in 4% paraformaldehyde at 37°C for 48 hours.
After the fixation phase, the material was washed three times with 0.1 M sodium phosphate buffer (pH 7.4), stepwise dehydrated in ethanol and soaked in LEICA resin for 7 days. The substance is then placed in a plastic mold filled with resin and polymerizer, and then placed in an oven heated to 37°C until the block containing the substance is completely polymerized.
After polymerization, the blocks were cut using a LEICA RM2235 microtome (Rankin Biomedical Corporation 10,399 Enterprise Dr. Davisburg, MI 48,350, USA) to a thickness of 3 mm. The sections are grouped on slides, with six sections per slide. The slides were dried at room temperature, then stained with hematoxylin for 7 min and washed with running water for 4 min. In addition, apply the eosin solution to the skin for 5 minutes and rinse with running water for 5 minutes.
Acute toxicity was predicted using aquatic organisms from different tropical levels: 96-hour fish LC50, 48-hour D. magna LC50, and 96-hour green algae EC50. The toxicity of rhamnolipid biosurfactants to fish and green algae was assessed using ECOSAR software version 2.2 for Windows developed by the US Environmental Protection Agency. (Available online at https://www.epa.gov/tsca-screening-tools/ecological-struct-activity-relationships-ecosar-predictive-model).
All tests for larvicidal and antitermite activity were carried out in triplicate. Nonlinear regression (log of dose response variables) of larval and termite mortality data was performed to calculate median lethal concentration (LC50) with 95% confidence interval, and concentration response curves were generated using Prism® (version 8.0, GraphPad Software) Inc. , USA) 84, 91.
The present study reveals the potential of microbial biosurfactants produced by Enterobacter cloacae SJ2 as mosquito larvicidal and antitermite agents, and this work will contribute to a better understanding of the mechanisms of larvicidal and antitermite action. Histological studies of larvae treated with biosurfactants showed damage to the digestive tract, midgut, cerebral cortex and hyperplasia of intestinal epithelial cells. Results: Toxicological evaluation of the antitermite and larvicidal activity of rhamnolipid biosurfactant produced by Enterobacter cloacae SJ2 revealed that this isolate is a potential biopesticide for the control of vector-borne diseases of mosquitoes (Cx quinquefasciatus) and termites (O. obesus). There is a need to understand the underlying environmental toxicity of biosurfactants and their potential environmental impacts. This study provides a scientific basis for assessing the environmental risk of biosurfactants.
Post time: Apr-09-2024