Archive \ Volume.12 2021 Issue 3

Larvicidal Effects of Carbon Nanotubes Loaded with Selected Marine 'Sponges' Extracts

 

Naser Ahmed Alkenani1*, Mona Ali Basabreen1, Lamia Ahmed Shaala2-4, Majed Ahmed Alshaeri1, Jazem Abdullah Mahyoub1, Ihaan Ullah1, Khalid Mohammed Algamdi1, Diaa Tohamy Youssef5,6

 

1Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia. 2Natural Products Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia. 3Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia. 4Suez Canal University Hospital, Suez Canal University, Ismailia 41522, Egypt. 5Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia. 6Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt.


Abstract

Recently, the use of eco-friendly and biodegradable insecticides has gained great attention. The present study was concerned to evaluate the larvicidal potential of the extracts of the Red Sea sponges Xestospongia testudinaria and Amphimedon chloros and biogenic carbon nanotubes (CNTs) against Aedes aegypti (Diptera: Culicidae). The third instar larvae of Ae. aegypti was used to test the insecticidal activity of the methanolic extract of X. testudinaria and A. chloros. The results showed that the tested concentrations (62.5, 125, 250, and 500 ppm) of both extracts possess high and moderate larvicidal effects after 48 h of exposure. The methanolic extract of A. chloros with CNTs showed 96 % (LC50 = 15.569 ppm) mortality after 24 h of exposure. While the A. chloros, extract without CNTs, the larval mortality was 99 % (LC50 = 65.77 ppm) after 48 h of exposure. These results suggested that the synthesized biogenic CNTs can be used as an ideal eco-friendly approach for controlling A. aegypti.

Keywords: Red-Sea sponges, Organic extracts, Larvicidal activities, Biodegradable


INTRODUCTION

Mosquitoes (Diptera Culicidae) are the most critical group in blood-sucking arthropods [1]. Mosquitoes not only create a nuisance to humans but also transmit serious diseases [2]. They belong to three prominent families, e.g., Anophelinae, Culicidae, Toxorhyncitinae, which have been further categorized into approximately 3700 species. The hooked beak can recognize the adult mosquito not to penetrate the skin to have a meal of blood. It feeds only on flower nectar [3, 4].

Most genera Anopheles, Culex, and Aedes, transmit different types of infectious diseases, e.g., Japanese Encephalitis, Dengue fever, Yellow fever, Malaria, Filariasis, etc., causing large scale of deaths each year worldwide [1, 5]. Arthropods transmit dengue, a serious viral disease, that occurs worldwide. Therefore, a high diffusion is observed of Dengue Hemorrhagic Fever (DHF) in Asia and Pacific countries [6]. This condition triggers an acute illness that can kill patients more rapidly than Acquired Immunodeficiency Syndrome, an immune disease (AIDS).

Chemical vector control application is a conventional approach; however, it has environmental and human dangers [7]. In recent years, repeated use of synthetic insecticides for mosquito control has weakened natural biological control mechanisms and resulted in resurgences in mosquitoes' population [8]. Repeated use of chemical pesticides led to the development of resistance by mosquitos against such pesticides [9]. It is necessary to find alternatives to control mosquitoes [10]. It is a big challenge to monitor mosquitoes without the effect of producing larvicide-resistant insects successfully. The World Health Organization (WHO) is also promoting the future use of several insecticides with various action modes. This can be accomplished either by using a mixture of insecticides, by cycling through multiple growing seasons, or by a combination of both [11].

Natural products from plant and marine animals showed promising effects to control mosquitoes [9]. Pesticides from plant origin are eco-friendly, readily biodegradable, and non-toxic to animals [12].

Marine organisms are considered an essential source for numerous bioactive compounds and secondary metabolites to combat these environmental challenges [9, 13]. Secondary metabolites produced by marine invertebrates like sponges displayed interesting insecticidal activities [9].

Carbon nanotubes exist in two different forms, including the Single-Walled Nanotubes (SWNT) and nanotubes with several walls (MWNT) [14]. The nanoparticles can be synthesized by using fungi, which render the nanoparticles more biocompatible. Therefore, bacteria, yeast, and fungi are potentially useful in preparing metal nanoparticles [14].

This study aimed to investigate the effect of the extracts of two Red Sea sponges, X. testudinaria and A. chloros, against Ae. aegypti, as well as the analysis of resistance extent in the strain and to evaluate the effect of the interaction of Multi-Walled Carbon Nanotubes (MWCNTs) and marine animal extracts against the larvae of mosquitoes Ae. aegypti. 

MATERIALS AND METHODS

Collection of Red Sea Sponges

The Red Sea sponges, X. testudinaria, and A. chloros (Figure 1) were collected from the Saudi Red Sea by Hands using SCUBA diving at different depths (-12-25 meters). The samples were kindly identified by Dr. Rob van Soest, The Naturalis, The Netherlands. Samples were kept frozen at -20 until organic extracts were prepared.

Mosquitos' Sampling

The mosquitoes, Ae. Aegypti, were collected from Jeddah and were reared in Mosquito Research Unit, King Abdulaziz University. The Xestospongia testudinaria and Amphimedon chloros were collected from the Red Sea coast in Saudi Arabia at depths from 15 to 25 meters by scuba divers. All samples were taken from the substratum and transferred to the laboratory of King Fahd Medical Research Center, Abdulaziz University. After that, it was freeze-dried to minimize problems with foaming and emulsions.

 

a)

b)

Figure 1. The Red Se sponges X. testudinaria (left) and A. chloros (right).

Preparation of the Crude Extracts of the Sponges

The samples were freeze-dried before extraction. The freeze-dried sponges were extracted with methanol solvent (absolute%) (2 × 800 mL). The extracts were dried under reduced pressure. The dried sample was resuspended insolvent.

Biosynthesis of Carbon Nanotubes Using Biological Extracts

An MWCNT (Sigma Aldrich, USA) stock solution was prepared according to the manufacturer guideline, using 0.02% Suwannee River Natural Organic Matter (SRNOM) as a dispersant in an ultrasonic bath (Decon FS300) for 2 hours. SWCNTs were synthesized using inductivity coupled plasma mass spectrometry (ICP-MS) and characterized using SEM, TEM, Raman spectroscopy, DLS, and zeta potential. Carbon nanotubes were synthesized in collaboration with the Environmental Protection and Sustainability Department at KAU.

Larvicidal Bioassay

Larvae of third instars were used for larvicidal bioassay. The stock solution was prepared by dissolving 1 g of the crude extracts into 99 mL of distilled water. The extract was kept within the refrigerator (3 °C) in dark glass containers until experiments were conducted. Twenty larvae of the 3rd instars of A. aegypti were tested, and five replications were used for each concentration. Four concentrations, including 62.5, 125, 250, and 500 ppm, along with a standard control, were used against larvae. The 'larvae's mortality rate was recorded after extract use, and larval mortality was calculated in each concentration.

Preparation of the Extracts with Carbon Nanotubes

One gram of each of the extracts of X. testudinaria or A. chloros were added, separately, to 1 mL of carbon nanotubes and 98 mL distilled water in a flask, and the mixtures were kept at room temperature for 24 h until the color changed.

Statistical Analysis

Data were expressed as a mean ± SD (1971). The statistics of mortality was carried out. In addition to 25%, 50%, 75%, 80%, 90%, and 95% of the test material's mortalities, the corresponding concentration Probit (Ldp line) of the 3rd instar larvae were estimated.

RESULTS AND DISCUSSION

Bioassay of Carbon Nanotubes

   Despite advances in medical research, mosquitoes are responsible for transmitting life-threatening pathogens in nearly all tropical and subtropical countries. The use of pharmacological control agents is, therefore, important. The results (Table 1) revealed that the active series of A. chloros extract concentrations were 62.5-500 ppm and the mortality rate of larval of A. aegypti mosquito of this concentration between 51-99 %, respectively. The results also show the toxicity line of A. chloros that needed to kill 50% of treated larvae after 48 h of this extract was 65.77 ppm (Figures 2 and 3). While A. chloros was recorded, the confidence limit in the lower and upper of the LC50 value was 52.1635 and 77.8909 ppm. However, the concentrations of A. chloros that needed to kill 90% of Ae. Aegypti 3rd instar larvae were 209.7424, and the confidence limit in both lower and upper of LC90 value was 176.1857 and 267.217 ppm, respectively. The value of chi was 0.724.

On the other hand, A. chloros extract with carbon nanotubes showed significant concentrations against 3rd instars larvae of Ae. aegypti mosquito at 62.5-500 ppm, respectively, and the mortality rate of the 3rd larval instars of Ae. aegypti mosquito of this concentration was between 75-96, respectively (Tables 1 and 3). Therefore, the toxicity line of A. chloros with CNTs that needed to kill 50% of treated larvae after 24 h of this extract was 15.569 ppm (Figures 2 and 3). The value of the lower and upper confidence limit of LC50 was 2.751 and 32.3797 ppm. However, LC90 value was 408.121, and in a consecutive range, the LC90 limit was 173.8269 ppm.  In sum, the value of chi was 0.0878, which means the toxicity line of A. chloros with CNTs was more effective and toxic without CNTs (Table 1). The results in Tables 2 and 3 showed the significant concentrations of X. testudinaria extracts were 62.5-500 ppm and the mortality rate of the 3rd larval instar of A. aegypti mosquito of this concentration between 41-98 ppm, respectively. Our results also showed the toxicity line of X. testudinaria that needed to kill 50% of treated larvae after 7 days of this extract was 95.6729 ppm (Figure 2). While X. testudinaria was recorded, the confidence limit in lower and upper of LC50 value was 78.843 and 111.8294 ppm, and the concentrations of X. testudinaria were needed to kill 90% of Ae. Aegypti 3rd larvae were 375.6465 ppm, and the confidence limit in both lower and upper of LC90 values were 308.6802 and 490.1225 ppm, respectively. The value of chi was 3.4612 (Table 2).

On the other hand, X.  testudinaria extract with CNTs shows significant concentrations against 3rd instars larvae of Ae. aegypti mosquito and was 62.5-500 ppm, respectively, and the mortality rate of the 3rd larval instars of Ae. aegypti mosquito of this concentration between 7-97%, respectively (Tables 2 and 3). Therefore, as shown in Figure 2, the toxicity line of X. testudinaria with CNTs needed to kill 50% of treated larvae after 72 h of this extract was 158.3125 ppm. However, the values ranged from 143.6556 to 174.2906 ppm in both lower and upper confidence limits of the LC50. But in a consecutive 298.1747 and 406.3433 ppm the value was both lower and upper LC90 limit value.  Finally, the value of chi was 1.7042 (Table 2).

 

 

Table 1. Results of the Larvicidal Activities of A. chloros Extract with and without CNTs against Ae. aegypti Larvae

Extract Tested

Conc.  (ppm)

Larval

Mortality (%)

Mean* ± SD

LC

Con

ppm

Confidence

Limit

Lower – Upper

Slope

Chi**

Amphimedon chloros

62.5

51d ±1.15

25

35.7236

46.0915-24.2809

2.5446

0.7240

125

74C ±1.17

50

65.77

52.1635 -77.8909

250

94b ±1.05

75

121.0879

104.7328-140.8442

500

99a ±0.57

90

209.7424

267.217-176.1857

Control

3e ± 0.01

95

291.3802

402.573-234.2076

Amphimedon chloros with CNTs

62.5

75d ±1.12

25

3.6822

11.5333-0.2296

1.0772

0.0878

125

84C±0.577

50

15.569

2.751-32.3797

250

90b ±1.14

75

65.8287

31.2113-96.0083

500

96a ±0.48

90

240.9911

408.1217-173.8269

Control

2e ± 0.03

95

523.9155

1424.4649-330.8671

 

 

Table 2. Results of the Larvicidal Activities of X. testudinaria Extract with and without CNTs against Ae. Aegypti Larvae

Extract Tested

Conc.  (ppm)

Larval

Mortality (%)

Mean* ± SD

LC

Con

ppm

Confidence

Limit

Lower – Upper

Slope

Chi**

Xestospongia testudinaria

62.5

41d ±1.73

25

46.5774

33.5163 - 58.9324

2.1576

3.4612

125

55C ±1.15

50

95.6729

78.843   - 111.8294

250

81b ±1.15

75

196.5184

170.1258- 231.3436

500

98a ±1.17

90

375.6465

308.6802- 490.1225

Control

3e ± 0.12

95

553.554

432.8351- 782.416

Xestospongia testudinaria with CNTs

62.5

7d ±1.15

25

105.6292

117.6326-92.8279

3.8382

1.7042

125

41C ±0.67

50

158.3125

143.6556-174.2906

250

77b ±1.15

75

237.2719

213.6052-268.766

500

97a ±1.21

90

341.5209

406.3433-298.1747

Control

3e ± 0.31

95

424.6879

522.9458-362.2603

 

 

This study in agreement with many scientific studies shows that sponges play an essential role in controlling mosquitoes that transmit many diseases to humans. Investigation of the ethanol extracts of marine sponges Topsentia ophiraphidites, Amphimedon compressa, Ircinia campana, Agelas sventres, and Svenzea zeai showed that the extract of Amphimedon compressa was more effective against Ae. Aegypti [15]. The methanol extract of the marine sponge Cliona celata (Grantand) showed the highest larvicidal activity against Ae. aegypti and Culex quinquefasciatus larvae [8]. The methanol extract of the sponge Acanthella elongate displayed, among other marine sponge species, the highest larvicidal activity against larvae of Culex sp. [16].

The statistical analysis of the extract of the sponge Amphimedon chloros with CNTs showed high effectiveness against the Ae. aegypti mosquitoes in their larval stage and RR was 4.224 times more effective than Amphimedon chloros extract without CNTs (Table 3).

a)

b)

c)

d)

e)

e)

Figure 2. The morphological deformities on Ae. aegypti mosquito larvae were treated using A. chloros and X. testudinaria extracts with CNTs and without CNTs. (a) Adult incompletely emerged and shown the body attached in the pupa exuviae. (b) The intermediate stage between pupa and adult (Pupa winged). (c) Segments of larval body contraction. (d)  Albino pupa. (e) Pigmentation and neck elongation. (f)  Evident elongation of the neck region & Cells explosion.

 

Table 3. Susceptibility of 3rd Instar of A. aegypti Larvae to the Extracts of A. chloros and X. testudinaria with and without CNTs followed by Continuous Exposure to the Extracts

Marine invertebrate extracts

Effect concentration

(ppm)

Larval mortality (%)

Statistical parameters

LC50

RR

Slope

A. chloros with CNTs

62.5-500

75-96

15.569

1

1.077

A. chloros

62.5-500

51-99

65.77

4.224

2.545

X. testudinaria

62.5-500

41-98

95.673

6.145

2.158

X. testudinaria with CNTs

62.5-500

7-97

158.312

10.168

3.838

CONCLUSION

Mosquitoes are an important group in blood-sucking arthropods. Despite advances in medical investigations, mosquitoes transmit various life-threatening pathogens in almost all tropical and subtropical regions. Recently, the use of biodegradable and eco-friendly insecticides has received much attention. This study evaluated the effect of the extracts of two Red Sea sponges, X. testudinaria and A. chloros, against Ae. aegypti, and analyzed the resistance extent in the strain. Moreover, the effect of the interaction of Multi-Walled Carbon Nanotubes (MWCNTs) and marine animal extracts against the larvae of mosquitoes Ae. aegypti was evaluated. In line with other studies, this study showed that sponges play a crucial role in controlling mosquitoes. The statistical analysis exhibited high effectiveness against Ae. aegypti mosquitoes in their larval stage and RR was 4.224 times more effective than Amphimedon chloros extract without CNTs.

ACKNOWLEDGMENTS: We would like to thank Dr. Rob van Soest for the identification of the 'sponges' specmens. Our thanks also to the staff of the EPS laboratory for helping in the preparation of carbon nanotubes. We appreciate all the staff members at the research station of dengue fever, Department of Biological Sciences, College of Science at King Abdulaziz University, Jeddah, and King Fahd Medical Research Center support during this study.

CONFLICT OF INTEREST: None

FINANCIAL SUPPORT: None

ETHICS STATEMENT: None

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