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Table of Contents
SHORT RESEARCH COMMUNICATION
Year : 2018  |  Volume : 55  |  Issue : 2  |  Page : 159-164

Nontoxic water soluble nanocarbons prevent respiration of mosquito larvae, causing anoxia


1 Department of Chemistry, Nanoscience and Synthetic Leaf Laboratory at Downing Hall, Center for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology (IIEST), Shibpur, Howrah, India
2 Nanoscience and Synthetic Leaf Laboratory at Downing Hall, Center for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology (IIEST), Shibpur, Howrah, India

Date of Submission23-Aug-2017
Date of Acceptance19-Mar-2018
Date of Web Publication1-Oct-2018

Correspondence Address:
Sabyasachi Sarkar
Nanoscience and Synthetic Leaf Laboratory at Downing Hall, Center for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology (IIEST), Shibpur, Botanic Garden, Howrah–711 103
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.242564

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  Abstract 


Keywords: Anoxia; biocompatible; mosquito larvicide; nontoxic, water soluble nanocarbon


How to cite this article:
Ghosh S, Das GS, Majumdar R, Sarkar S. Nontoxic water soluble nanocarbons prevent respiration of mosquito larvae, causing anoxia. J Vector Borne Dis 2018;55:159-64

How to cite this URL:
Ghosh S, Das GS, Majumdar R, Sarkar S. Nontoxic water soluble nanocarbons prevent respiration of mosquito larvae, causing anoxia. J Vector Borne Dis [serial online] 2018 [cited 2018 Oct 21];55:159-64. Available from: http://www.jvbd.org/text.asp?2018/55/2/159/242564



The anthropophilic nature of Aedes aegypti mosquito[1],[2] over other mosquitoes is a serious concern, as it largely affects human population living in the heavily congested cities across the world[3],[4]. The female Ae. aegypti mosquitoes that spread the dengue, chikungunya and Zika viruses, generally bite during day time, when people are at work. Aedes aegypti mosquitoes lay eggs in artificial containers, plastic or earthen bowls and similar objects partially filled with water around road side in cities and town. Generally, in urban lifestyle decorative flower pots, vases and other appliances (filled with water) are used in living rooms or in office places, wherein the water remains unchanged for days. Such unchanged water is enough for mosquito eggs to hatch under tropical environment. During rainy session bowls, cups, tyres, barrels, and puddles, etc. nearby the residence area in the cities augment mosquito nursery.

For mosquito control, the larva stage might be an attractive target[5], because mosquito larvae are restricted in water in limited places[6],[7]. Mosquito larvae control methods include biological control using predators or parasites[8],[9]; chemical control using insecticide[10],[11] environmental management by eliminating the breeding sites[12],[13] and physical control using mechanical means[14]. Mosquito is unlikely to develop resistance to the physical control[15]. The old but effective physical control method uses surface oil film that creates a barrier for aerial oxygen to reach larvae inside water pool and affects its respiration[16],[17]. When any oil is sprayed in water resource which contain mosquito larva, the applied oil quickly diffuses over the surface creating a film which prevent the passage of air to the water pool that prevents respiration of larvae, causing death. The highly volatile petroleum oils paralyze the larvae by their lethal effect, causing death in minutes, whereas the nonvolatile viscous oils gradually accumulate in their respiratory system and suffocate them to death after several hours. But the disadvantage of such control methods is that it completely separates out aerial oxygen in contact with the water which not only block the air supply to mosquito larva, but also to the other creatures and aerobic microorganisms present in water. All aquatic plant systems also get adversely affected by such oil mess. Further, highly inflammable petroleum oil carries fire hazard and bad odour. The rate of evaporation of the used oil controls the frequency of its repeat use and this will add its polluting effect in the environment. From operational point of view, the application of chemical insecticides using thermal vapourization technique is a house-hold process to repel or kill mosquito. However, such release of neurotoxin to dispel mosquito in low dosage continuously for days and months might have serious implications on the human nerve systems[18]. Furthermore, the long-term use of chemical insecticides, has led to development of immunity in the mosquitoes against them, resulting their usage in higher doses, that cause even more health hazards in infants and old people.

To overcome the ill effects of these control strategies a new method, i.e. use of nanocarbons in water pots and other hot-spots is presented in this study. Once added in water, being non-destructive, it remains confined in container for years that may not require much replenishment. This odourless water soluble nano carbon is environment friendly and non-toxic to E. coli, zebra fish and other living species[19]; and has been shown to promote plant growth[20]. It showed no toxicity when used in control doses in imaging the full life cycle of either Drosophila melanogaster[21] or imaging Anopheles, Culex and Aedes species of mosquito[22]. These nanocarbons have been also reported to have negligible cytotoxicity. However, when used at slightly elevated concentration like 3 ppm, it displays a unique property by getting deposited in the sticky part of the spiracle and over the air tube siphon of mosquito larvae. And within four weeks it chokes the respiration of Ae. aegypti mosquito (observed in Culex and Anopheles spp also, but data not shown) larvae causing anoxia.

While imaging the entire life cycle of mosquito using fluorescent water soluble nanocarbon particles (wsCNP), it was observed that exceeding 3 ppm concentration of wsCNP arrests the growth of larva for four weeks and after this period their exposure became fatal. Hence, it was speculated that the wsCNP might be physiologically affecting the biochemical events involved in the larval growth.

Interestingly, the larvae treated under the exposure of wsCNP when transferred mid-way (after 2 or 3 weeks of exposure) to fresh water could be revived fully (with ability to grow further). In tropical environment, the transformation from larval to pupal stage requires just four to five days, but in the presence of 3 ppm level of wsCNP, the larval stage gets prolonged to four weeks and they do not even attend the pupal stage. The larvae stopped growing in this period, skipping moulting processes compared with the control. The larval stages demand a lot of food for growth. Even in the presence of sufficient food the long exposure to < 3 ppm level of wsCNP compels them to starve due to breathing problem.

For the study, Aedes aegypti larvae were reared after collecting them from a shallow pool near neem pond at IIESTS, Howarh, India. They were reared in a small aquarium in a temperature conditioned room at 25 °C, 80% relative humidity and 16:8 h light/dark photo period of standard laboratory conditions. Few IV instar larvae of Ae. aegypti mosquito were reared overnight separately in another aquarium in water containing soluble carbon nano particle (wsCNP) of concentration, 3 μg/ml, prepared as described earlier[17],[18],[19],[20]. Controls and treated larvae were imaged with optical fluorescence (placed in slide/ cover glass) and SEM (dehydrated in ethanol overnight and frozen under liquid nitrogen, fixed with carbon tape). The optical microscopic images were taken by Leica DM 2500 with fluorescence attachment. For SEM, frozen samples were coated in a sputter coater for 120 sec and then the images were taken by Hitachi S-3400N SEM machine.

The IV instar larvae were examined using scanning electron microscopy (SEM) and fluorescence optical microscopy [Figure 1],[Figure 2],[Figure 3],[Figure 4]. Three sets of SEM and fluorescence microscopic images exposing siphon area of larva are shown. For fluorescence microscopy, three different light sources were used and under bright field images were made. The SEM images clearly identified the spiracle of the siphon [Figure 1] a,[Figure 1] b,[Figure 1] c and on exposure under water with wsCNP, the sticky oily secretion covering the siphon part[4] facilitate deposition of wsCNP followed by agglomeration [Figure 1] d,[Figure 1] e,[Figure 1] f. On washing with fresh water most of the wsCNP could be removed but a large chunk still remained at the entry point of the spiracle [Figure 1] g,[Figure 1] h,[Figure 1] i. Deposition of wsCNP can be better viewed using fluorescence optical microscopy. The fluorescence images of wsCNP exposed larva [Figure 2] were compared with those treated in fresh water after wsCNP treatment [Figure 3] and both set of images were compared with the control, i.e. unexposed larva [Figure 4]. The difference in fluorescence images of wsCNP treated larva [Figure 2] from untreated [Figure 4] or washed larva [Figure 3] is easily correlated with the survival of Ae. aegypti larva. [Figure 2] shows the optical microscopic images under bright field. In [Figure 2] a,[Figure 2] b,[Figure 2] c very fine detailed parts of the siphon are shown. The deposition of wsCNP over the entire siphon part can be readily seen by fluorescence spectroscopy using three different excitation lines to distinguish these images from auto fluorescence. In all these images the deposition of wsCNP was clearly visible. This was further validated once the wsCNP treated larvae placed in pure water and left overnight, where observed in the optical microscopic images as shown in [Figure 3]. Images of control larvae (without wsCNP exposure) are displayed in [Figure 4]. [Figure 3] and [Figure 4] did not show any perceptible fluorescence; however, [Figure 3] confirmed that on washing with fresh water the deposited wsCNP can be washed away (loss of fluorescence) supporting the observation[19] that discontinuation of wsCNP treatment during four week's exposure by fresh water may help to survive the larvae. The control larval images [Figure 4] further confirm the study results, displaying virtually no auto fluorescence under the experimental condition.
Figure 1: Scanning electron microscopy (SEM) images of Aedes aegypti mosquito larvae showing spiracle of the siphon (arrow marked) under different magnification; (a–c): Control; (d–f): wsCNP treated; and (g–i): wsCNP washed.

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Figure 2: Optical microscope images of Aedes aegypti mosquito larvae siphon after the treatment with wsCNP showing siphon (arrow)—(a–c) under bright field; (d–f) under epifluorescence microscopy at 488 nm; (g–i) at 561 nm; and (j–l) 635 nm.

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Figure 3: Optical microscope images of wsCNP treated Aedes aegypti mosquito larvae siphon after washing with water: (a–c) under bright field; (d–f) under epifluorescence microscopy at 488 nm; (g–i) at 561 nm; and (j–l) at 635 nm showing the diminution of fluorescence due to washing away of the wsCNP (the layers are as described in [Figure 2]).

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Figure 4: Optical microscope images of control Aedes aegypti mosquito larvae siphon: (a–c) under bright field; (d–f) under epifluorescence microscopy at 488 nm; (g–i) at 561 nm; and (j–l) at 635 nm showing pale auto fluorescence (the layers are as described in [Figure 2].) The residual auto fluorescence comparable with wsCNP washed larvae as shown in [Figure 3].

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The oily secretion near the snorkels including the hairy parts[4] facilitate the deposition of wsCNP and as the proof of concept human hair were used as control and treated with wsCNP to show-the deposition, in the bright field images [Figure 5]a. This was distinctly visible and can be distinguished from the auto fluorescence [Figure 5] b,[Figure 5] c,[Figure 5] d.
Figure 5: Optical microscope images of control hair (Top image) and wsCNP treated hair (Bottom image): (a) under bright field; (b) under epifluorescence microscopy at 488 nm; (c) 561 nm; and (d) 635 nm, differentiating auto fluorescence of control (Top image) with wsCNP treated bright fluorescence (Bottom image) under each light.

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Based on these observations it can be concluded that the use of non-toxic, wsCNP is safe to prevent growth of mosquito around house-hold premises. These nano-particles could be economically produced and preserved in the solid form for infinite period of time and can be used as and when required by the people without any help unlike sought to spray chemical insecticides or fogging by specialists.

Conflict of interest

There is no conflict of interest in the publication of this article.

Ethical issues: This study does not involve ethical issues.


  Acknowledgements Top


GS is research associate in DST-SERB project, New Delhi. SS thanks DST-SERB (File No.: EMR/2015/001328), New Delhi, India for financing the project. RM thanks DST-SERB, New Delhi for National Post-Doctoral Fellowship (PDF/2016/000513).



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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