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.

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 3^rd 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 3^rd 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 3^rd 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 3^rd instars larvae of Ae. aegypti mosquito at 62.5-500 ppm, respectively, and the mortality rate of the 3^rd 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 3^rd 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 3^rd 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 3^rd instars larvae of Ae. aegypti mosquito and was 62.5-500 ppm, respectively, and the mortality rate of the 3^rd 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).