Potential larvicidal bioefficacy of copper sulfide-based hybrid nanoemulsions of eucalyptus oil against Aedes aegypti (Linnaeus)

Abstract

Background & objectives: 

Nanotechnology, an emerging field, has acquired considerable attention for the control of vectors. The present study aimed to synthesize, characterize copper sulfide- and eucalyptus oil-based hybrid nanoemulsions and investigate their larvicidal potential against Aedes aegypti by studying larvicidal bioassay, morphological aberrations, histopathological alterations, biochemical analysis and evaluation of risk assessment in non-target organisms.

Methods: 

Hybrid nanoemulsions were prepared by mixing aqueous copper sulfide nanoparticles (CuSNPs) with non-polar eucalyptus oil in five ratios (1:1, 1:2, 1:3, 1:4 and 1:5) by sonication, screened and characterized using Transmission electron microscopy (TEM). Larvicidal activity was recorded and toxicity values were calculated by log-probit method. Morphological, histological and biochemical changes were examined in Aedes aegypti larvae after treatment. Nanohybrids were also tested under simulated conditions and against non-target organism.

Results: 

The nanohybrid ratio of 1:5 was found to be stable after thermodynamic stability tests. TEM studies revealed average size of 90±7.90 nm with globular shape. LC₅₀ and LC₉₀ toxicity values of prepared CuSNPs were calculated out to be 5.00 and 5.81ppm after 24 hours treatment. Effective concentration of prepared nanohybrid (6.5ppm) tested under simulated conditions showed maximum larvicidal mortality after 48 hours of exposure. No toxicity towards the Mesocyclops spp. was observed after treatment of these nanohybrids even up to 21 days.

Interpretation & conclusion: 

Copper sulfide based hybrid nanoemulsions were found to show efficient larvicidal property which can be used for the formulation of ecofriendly bio-larvicide against Aedes aegypti.

INTRODUCTION

Aedes aegypti (Linnaeus) is the primary carrier of deadly mosquito-borne diseases that cause dengue, dengue hemorrhagic fever, yellow fever and this vector is widely spread over large areas of the tropics and subtropics. Ae. aegypti is adapted to urban sites and prefers clean water containers for egg laying and further development[¹]. Dengue is endemic in 129 countries and Western Pacific, South-Eastern Asia, Americas are seriously affected regions[²]. At present, no effective vaccine is available for dengue so the only way to reduce the incidence of this disease is by mosquito control, which is frequently dependent on insecticide application[³]. Chemical measures implemented were effective initially, but these have failed as their constant use has resulted in resistance among mosquitoes, insect outbreak, environmental pollution and undesirable effects on non-target organisms[⁴]. Plant extracts with insecticidal, larvicidal and repellent properties have been tried in the recent past for the control of various insect pests and vectors and is well documented from many parts of India[⁵]. In addition, plants extracts, essential oils and isolated compounds, like flavonoids, alkaloids and terpenoids were identified as larvicides and mosquito repellents[⁶]. Eucalyptus is amongst the most significant plants belonging to family Myrtaceae possessing more than 700 species. Eucalyptus globulus oil has shown eco-friendly nature along with significant larvicidal and repellent actions against mosquito vectors[⁷]. Application of nanotechnology is quite effective against mosquitoes by formation of nanoparticles from plant extracts which is a biodegradable and clean process. Herbal oils are mixed with different nanoparticles to manage various mosquito species and is a fast-growing field of research[⁸]. Many metal nanoparticles have been reported with different biological properties[⁹] which if used in combination with essential oil nanoemulsions may prove to be effective in mosquito control strategies.

Sulfide analogues of transition metals are hypothe sized as one of the most detoxified forms of metals[¹⁰]. Various transition metals applied in different forms get their ultimate fate by sulfidation in the environment for their detoxification. The eminent bioefficacy and detoxified nature of metal sulfide nanoparticles are drawing the attention of scientists for their use against variety of environmental menaces. Copper sulfide (CuS) is one of the most detoxified forms of copper[¹⁰] which is natural, thermally stable and water insoluble[¹¹] but still retains the biopotential of copper. It is relatively safe and non-toxic to humans[¹¹]. Till date no study has been carried out on the synthesis of water dispersed CuS nanoparticles along with eucalyptus oil nanoemulsion and evaluation of their larvicidal potential. Therefore, the present study was planned to develop hybrid nanoemulsion (eucalyptus oil nanoparticles + copper sulfide nanoparticles) for evaluation of its potential as a safe larvicide against Aedes aegypti.

MATERIAL & METHODS

Preparation of hybrid nanoemulsions (nanohybrids)

Stock aqueous solution of copper sulfide nanoparticles (CuSNPs) was prepared using sonochemical irradiation method[¹²]. The pure form of eucalyptus oil (Eucalyptus globulus Labillardiere) was obtained from Loba Chemie Private Limited, Mumbai, India. The aqueous solution of prepared CuSNPs was mixed with non-polar eucalyptus oil nanoemulsion while sonication along with drop of Tween 20 to set metal sulfide decorated non polar eucalyptus oil hybrid nanoemulsions. Lipophilic non polar fractions of CuSNPs and eucalyptus oil nanoemulsions were mixed in 1:1, 1:2, 1:3, 1:4 and 1:5 ratios.

Screening of nanohybrids

For screening of stable hybrid nanoemulsion, these were first visually observed for optical transparency, appearance and phase separation and then were analysed for thermodynamic stability stress tests[¹³]. For this, prepared hybrid nanoemulsions were centrifuged at 3000rpm for 30 min and observed for phase separation. The nanoemulsion which was found to be the stable one during this process was further analyzed for heating-cooling cycle by keeping stable hybrid namoemulsion at 40°C and 4°C, alternatively for 48 h at each temperature and repeated three times.

Morphological characterization

The morphology and size of hybrid nanoemulsions were recorded in Hitachi Transmission electron microscope Hi 7650 (TEM) at an accelerated voltage of 80kV by casting a drop of hybrid nanoemulsion which was negatively stained with phosphotungstic acid and placed on a 200-mesh carbon coated copper grid in Electron Microscopy and Nanotechnology (EMN) Laboratory, PAU, Ludhiana. TEM micrographs were acquired using TEM with a tungsten source.

Larvicidal bioassay

Water samples were collected from different peri-domestic water collections sites from urban areas of Ludhiana district in Punjab, using plastic dippers, from June to September 2019. Ae. aegypti larvae were identified on the basis of their morphological features by using standard keys[¹⁴]. The bioassay method was carried out according to World Health Organization (WHO) procedure[¹⁵]. Nanohybrids were tested at 7.5, 7.0, 6.5, 6.0 and 5.5 ppm diluting most stable and effectively coated nanohybrid(s) using de-chlorinated water for getting effective concentration of prepared nanohybrids. For each concentration, treatment sets were established in triplicate containing twenty larvae at 4^th instar stage. A vehicle-control with Polyvinylpyrrolidone (PVP, a surfactant) and a control set with dechlorinated water were also run simultaneously in triplicate. All mosquito larvae were adequately fed with dog biscuits and yeast powder mixed in 3:1 ratio (2mg/100ml). Larval mortality was recorded after 3, 6, 12, 24 and 48 h of treatment. The control mortalities were corrected by using Abbott’s formula[¹⁶].

The concentration of nanohybrids with highest mortality within lesser time duration out of tested concentrations was calculated and marked as the effective concentration.

Morphological observations

Morphological changes in nanohybrids treated larvae were observed with help of Scanning electron microscopy (SEM) and compared with control larvae. This study was carried out in the EMN Laboratory, Punjab Agriculture University. For SEM studies, treated and control larvae were fixed in 2.5% glutaraldehyde, washed in 0.1M sodium cacodylate buffer and secondarily fixed with 1% osmium tetroxide. Larvae were further washed with 0.1M sodium cacodylate buffer and dehydrated in different grades of ethyl alcohol. Larvae were dried in vacuum desiccators, spurted with 30 nm of gold mounted on aluminium stub. Samples were viewed under Scanning electron microscope (SEM-S3400N).

Histological observations

Treated and control larvae were collected and washed properly in 0.9% saline solution. Then larvae were fixed in 10% neutral buffered formalin (NBF), processed, sectioned and stained using a standard histological procedure[¹⁷].

Biochemical analysis

The larvae treated with effective larvicidal concentration of copper-based hybrid nanoemulsion were processed for the determination of total proteins18 and activity of digestive enzymes i.e., α-amylase19, protease[²⁰] and lipase[²¹].

Larvicidal bioassay under simulated conditions

Pond water (5 liters), soil, dried leaves and algae were taken in plastic tubs to provide simulated conditions and effective nanohybrid concentration having maximum larval mortality was tested. Then, hundred 4^th instar larvae were introduced in treated, control and vehicle-control sets in triplicate. Mortality was recorded after 3, 6, 12, 24 and 48 h.

Toxicity testing against non-target organism

Water was collected from small pond having natural enemies, predators and different crustaceans along with Aedes larvae using a zooplankton net. Non-target organism was identified as cyclopoid copepods, Mesocyclops spp. on the basis of morphological characters[²²]. Twenty Mesocyclops spp. were exposed to effective concentration of nanohybrids in small plastic containers. Population counts and mortality was recorded after 12, 24 and 48 h initially and then at weekly intervals up to 21 days.

Statistical analysis

Data was statistically analyzed by comparing the larval mortality in treated sets with that of control sets by using ANOVA (Duncan multiple range test) on SPSS software version 16. LC₅₀and LC₉₀ values were calculated by log probit method[²³].

Ethical statement: Not applicable

RESULTS

Screening and characterization of nanohybrids

Hybrid nanoemulsion having CuSNPs and oil @ 1:5 was found to be the most stable after screening as per the parameters shown in Table 1. Transmission Electron Microscopy (TEM) showed globular shape of droplets with their droplet size 90±7.90nm [Figure 1].

Figure 1: Transmission electron micrograph of stable CuS-based hybrid nanoemulsion of eucalyptus oil (1:5).

Table 1: Parameters used for testing prepared eucalyptus oil and copper sulfide-based hybrid nanoemulsions

Larvicidal bioassay

Among the tested five different concentrations of the most stable copper-based hybrid nanoemulsion, 100% mortality was observed within 24 h after treatment with 6.5ppm [Table 2 & Figure 2] and this was found to be the most effective concentration out of the tested concentrations, as it resulted in 100% killing of Ae. aegypti larva before their conversion to next developmental stage i.e., pupae. Toxicity values in terms of LC₅₀and LC₉₀were calculated to be 5.00 and 5.81ppm respectively after 24 h (Table 2).

Figure 2: Maximum mortality of 4^th instar Aedes aegypti larvae after exposing to different concentrations of CuS-based nanohybrid.

Table 2: Effect of different concentrations of stable (1:5) copper-based hybrid nanoemulsion of eucalyptus oil on mortality of 4^th instar Aedes aegypti larvae

Morphological observations

Copper-based hybrid nanoemulsion treated larvae showed overall shrinkage of the body as compared to control larvae which were having well developed appearance with distinguished head, thorax and abdominal region. Control larvae were showing smooth head surface with clearly visible antennae, eye region and mouth brushes [Figure 3]A while treated larvae had shrunken head without antenna [Figure 3]B. Abdominal region of treated larvae showed outward bulging with numerous wrinkles [Figure 3]D & [Figure 3]E while in control larvae abdominal segments were very clear and bearing comb scales with distinct larger median spine [Figure 3]C. Siphon region of control larvae was found to have has smooth surface with spiracular valves and pectan spines [Figure 3]F while treatment resulted in contraction of siphon and complete disruption of anal papillae [Figure 3]G & [Figure 3]H.

Figure 3: Scanning electron micrographs showing morphology of 4^th instar Aedes aegypti larvae A. Control head region; B. Treated head region; C. Control abdominal segments; D. & E. Treated abdominal segments; F. Control terminal region; G & H Treated terminal region with copper-sul fide based hybrid nanoemulsion.

Histological observations

The head area of control larvae was found to have a distinct pair of imaginary eyes (E), an imaginary pair of antennae bud (IBA), a pair of brush inner retractor muscle (IRB) and brush outer retractor muscle (ORB) and brain with a pair of optic lobes (OL) were also clearly visible as shown in Figure 4A. The treatment of larvae with CuS-based hybrid nanoemulsion led to disintegration of brain and other related structures [Figure 4]B. Imaginary buds were found to be intact in control larvae [Figure 4]C. However, treated larva demonstrated cracks and complete disorganization of these buds [Figure 4]D. Foregut has gastric caeca in thorax region showing cylindrical epithelial cells (EC), vesicles (V), nucleus (N), peritrophic membrane (PM), basement membrane (BM) and muscle fibers (MF) with well-developed microvilli (MV) in control larvae [Figure 4]E. Diversification, vacuolization, disorganization of cells was observed in treated larvae [Figure 4]F. A clear food channel showing lumen (L), muscle fibers (MF) was visible throughout entire abdomen [Figure 4]G in control larvae, but layers of epithelial cells were disintegrated in treated larvae [Figure 4]H.

Figure 4: Longitudinal sections of 4^th instar Aedes aegypti larvae: A. Head region of control larva showing imaginai eyes (E), imaginai bud of antenna (IBA), inner retractor muscle of brush (IRB), outer retractor muscle of brush (ORB) and optic lobes (OL) (100X); B. Copper sulfide based nanohybrid treated larva showing disintegration of head region (100X); C. Head highlighting the region of imaginal bud of antennae (IBA) of control larva showing intact IBA (400X); D. Copper sulfide based nanohybrid treated larva showing cracks and disorganization in IBA (400X); E. Thorax highlighting the gastric caeca (GC) of control larva having epithelial cells (EC), nucleus (N), peritrophic membrane (PM), basement-membrane (BM), muscle fibres (MF), microvilli.

The midgut epithelium of control larva consisted of a single layer of digestive cells with well-developed microvilli (MV) [Figure 5]A. Lysis of epithelial cells, degenerating microvilli was seen in treated larvae [Figure 5]B. The anterior midgut of control larva showed numerous well-developed fat body (FB) tissues [Figure 5]C. However, complete disappearance of fat bodies was observed at different areas of mid gut of treated larvae [Figure 5]D. Hindgut region of control larvae was found to have intact epithelium layer with food in lumen [Figure 5]E while complete distortion was observed in treated larvae [Figure 5]F.

Figure 5: Longitudinal sections of 4^th instar Aedes aegypti larvae (400X) A. Epithelium layer of midgut of control larva showing cells having peritrophic membrane (PM), basement membrane (BM) and microvilli (MV); B. Copper sulfide based nanohybrid treated larva showing lysis of epithelial cells; C. Midgut region highlighting fat bodies (FB) of control larva showing deposition of FB; D. Copper sulfide based nanohybrid treated larva showing disappearance of FB at various areas; E. Terminal region highlighting hindgut of control larva showing intact epithelial layer and food in gut lumen (FD); F. Copper sulfide based nanohybrid treated larva showing complete distortion of epithelium (EP).

Biochemical analysis

While studying the various biochemical parameters, a significant reduction was observed in protein content and specific activity of α-amylase and protease activity of treated larvae, while lipase activity got increased in treated larvae as compared to control (Table 3).

Table 3: Effect of effective concentration of copper sulfide-based hybrid nanoemulsion on the different biochemical parameters of 4^th instar larvae of Aedes aegypti

Larvicidal bioassay under simulation

Under simulated conditions, more than 90% larval mortality was recorded after 48h of exposure with effective copper sulfide-based hybrid nanoemulsion (6.5ppm) as shown in Table 4, indicating that these nanohybrids can be applied in the field conditions.

Table 4: Larvicidal potential of effective concentration of copper sulfide-based hybrid nanoemulsions against Aedes aegypti under simulated conditions

Toxicity testing against non-target organism

When effective copper sulfide-based hybrid nanoemulsion @ 6.5ppm was exposed to the tested non-target organism i.e., Mesocyclops spp. (n= 20), no mortality was observed upto 21 days of treatment and 100% survival was found. Even, no morphological changes were recorded in Mesocyclops spp. after this treatment.

DISCUSSION

Eucalyptus globulus nanoemulsion against Ae. aegypti larvae revealed that increasing concentration caused mortality in less time[²⁴] which was similar to our experimental results of larvicidal bioassay. Nanoemulsion based on Baccharis reticulate EO was found to have D-limonene as its major component and when tested at 25% it resulted in killing of Ae. aegypti larvae[⁶]. Eucalyptus globulus and many other essential oils have shown ovicidal activity and repellency to oviposition of Aedes aegypti[²⁵]. Testing of copper and zinc nanoparticles along with Azadirachta indica leaf extract revealed that monometallic forms of nanoparticles i.e., copper and zinc alone showed less larvicidal efficacy in comparison to copper-zinc bimetallic nanoparticles against Culex quinquefasciatus[²⁶].

During the present study, shrinkage was the main sign seen in all the regions of the hybrid nanoemulsion treated Ae. aegypti larvae; it could be mainly due to dehydration because of the presence of 1,8-cineole, α-Pinene and Globulol which are responsible for larval killing[²⁷]. In An. stephensi larvae, similar degenerative changes and necrosis have also been observed after exposure to crude eucalyptus oil[²⁷]. Treatment of Aedes larvae with Eucalyptus globulus oil nanoemulsion indicated distortions in head and thorax regions through SEM analysis. Other prominent changes recorded during the present study were: mouth brushes were disturbed along with constrictions on the head, totally malformed abdomen, completely damaged anal papillae and similar changes have also been observed in a recent study[²⁴].

Disruptions in various parts of An. stephensi larvae found after treatment with eucalyptus oil can be due to the presence of alkanoids and phenolic compounds. The histopathological changes observed in An. stephensi larvae exposed to E. globulus and A. vera oils[²⁷] were in agreement with the present study. Selenium nanowires (Cr-Se NWs) proved their larvicidal efficacy against mosquito vectors and cause damages in treated larvae including the loss of antennal hairs, breakdown of epithelial layer, significant effect on hindgut and vacuolation of epithelium within cellular membrane damages causing abrupt overall damage to abdomen, thorax and siphon regions as compared to control larvae[⁸]. Thus, such morphological changes in the form of distortion and damages in various body parts of larvae observed due to treatments of essential oils results in larval killing and could be used as a part of mosquito control programme.

In the present study, significantly low level of protein content was observed from the homogenates of treated larvae as compared to control. This decline in protein content of treated larvae can be attributed to one or a combination of factors such as decreased protein synthesis or increased protein breakdown so as to detoxify active ingredients such as 1, 8 cineole, aloesin, and flavonoids, in plant extracts or essential oils ingested by the larvae during water intake. When amylase action is inhibited, organism nutrition is impaired which causes energy shortness. Lipases are enzymes that hydrolyze external associations of fat molecules and have a role in storage and mobilization of lipids in insects. Increased midgut lipase activity may lead to a greater use of exogenous lipids and larvae’s efforts for use from storage lipids. Acetylcholinesterase and mixed-function oxidases activity got decreased by treatment of Petroselinum crispum essential oil against Ae. aegypti[²⁸] which shows that previous data is in line with the present study.

Eucalyptus oil was found more effective against Ae. aegypti larvae at lower concentration as compared to neem oil under simulated field conditions and recommended for its evaluation in coolers so that it can be easily applied in household applications by common public. A. squamosa oil causing 71 and 99 % larval mortality under field and laboratory conditions, respectively against Anopheles[²⁹]. The significantly lower mortality rate of larvae in field conditions as compared to laboratory can be attributed to the exposure of larvicides under sunlight, which might have broken them into nontoxic products. During the present study, laboratory experiments were performed in controlled conditions in BOD at 27±2°C in petri plates and beakers, whereas in case of simulated trials the imitation of field conditions was made with the help of leaves, soil and algae as well as simulated trials were performed under everyday fluctuating temperature conditions in plastic tubs. The reason of higher mortality rate under laboratory can be a proper dispersal of nanoemulsions in water as the experiments were performed in beakers so the larvae got properly exposed to the treated compounds. In case of simulated conditions, leaves, soil or algae may cause hindrance in the complete exposure of copper sulfide hybrid nanoemulsion to the larvae. But overall mortality rate was at par in case of simulated conditions, which suggested that the prepared copper sulfide hybrid nanoemulsion can be implemented as larvicide under field conditions in the future.

For risk assessment studies, similar experiments have been conducted by researchers in which permethrin nanoemulsion was checked for its bioefficacy against different non-target species like freshwater algae, chickpea and zebrafish and mosquitocidal concentration of nanopesticide has been found to be non-toxic30 which was at par with our results.

CONCLUSION

The results of the present study revealed that effective concentration of the prepared copper sulfide hybrid nanoemulsion (6.5ppm) led to efficient killing of Ae. aegypti but at the same this nanohybrid did not cause any harm to the tested aquatic non-target organisms i.e. Mesocyclops spp. treated with the same concentration. So, it is concluded from our findings that these copper sulfide-based hybrid nanoemulsions may act as potential alternative larvicidal agents for the control of Ae. aegypti, being eco-friendly and cost-effectively viable.

Conflict of interest: None

Acknowledgements

The authors are grateful to the Head, Department of Zoology, for providing all the necessary facilities including financial assistance and Department of Science and Technology, Government of India, New Delhi, for providing infrastructural facilities under FIST grant.