A study on the larvicidal and adulticidal potential of Cladostepus spongiosus macroalgae and green-fabricated silver nanoparticles against mosquito vectors

: Mosquito vectors in the present universe cause a major problem due to the transmission of pathogens with high morbidity. The present research aimed to explore the larvicidal and adulticidal toxicity of the Cladostepus spon-giosus extract and its fabricated AgNPs on key mosquito vectors. The synthesized AgNPs were con ﬁ rmed by UV-Vis spectroscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectrophotometry, and X-ray di ﬀ raction analysis. In the mosquitocidal assay, the C. spongiosus extract has shown good larvicidal mortality against Aedes aegypti (88.9%), Anopheles stephensi (84.1%), and Culex. quinquefasciatus (81.6%). Further, adulticidal mortality percentages were 78.8%, 75.4%, and 67.6% against An. stephensi, Ae. Aegypti, and Cx. quinquefasciatus at 1,000 ppm. AgNPs revealed larvicidal mortality percentages of 94.8% against An. ste-phensi , 92.8% against Ae. Aegypti , and 90.6% against Cx. quinquefasciatus ; the adulticidal potential was also revealed to be higher against An. stephensi (89.4%) followed by Ae. aegypti (86.8%) and Cx. quinquefasciatus (83.2%). Comparing the results achieved from the C. spongiosus extract and its derived AgNPs, promising activity was attained against key mosquito vectors at a minimal dose of 70 ppm of AgNPs. Thus, C. spongiosus-mediated AgNPs can be an alternative tool in controlling key mosquito vectors


Introduction
Mosquitoes are the arthropod vectors for diseases like malaria, dengue, and filariasis, which cause significant global mortality and morbidity with increased insecticide resistance [1,2]. World Health Organization reports showed 247 million cases of malaria in 2021 and 619,000 deaths [3]; it is a primordial disease caused by Plasmodium parasites and spread through the bites of infected female Anopheles mosquitoes [4,5]. Dengue is a viral infection mainly transmitted by Aedes aegypti and Ae. albopictus [6], and it has been estimated that 100-400 million infections occur per year [7]. Also, the Aedes mosquito vector carries the pathogens, which cause yellow fever, dengue, zika fever, and encephalitis [8]. The most common clinical manifestations observed are fever, rash, eye pain, arthralgias, myalgias, and haemorrhage [9]. The Culex species are the vector of encephalitis and West Nile fever in tropical and subtropical climatic conditions [10]. It is opportunistic and feeds on humans and animals [11]. The life stages of the mosquito are usually targeted using diverse conventional and microbial agents [12]. However, these agents have adverse effects on human health and the environment [13,14]. Therefore, recently green sources have been implemented to augment the control of mosquito vectors, with special reference to botanical mosquitocidal. Botanical is endowed with good photochemical potential, which has promising activities against insect vectors [15] and non-target effects against other beneficial organisms [12,[16][17][18]. Besides terrestrial, oceans are known to have magnificent diversities of living organisms covering 70% of the total surface area of the Earth. Algae are photosynthetic organisms and include a wide range of species with the extreme habitat of microalgae to seaweeds [19]. Algae are rich in antioxidants due to the presence of ascorbic acid, reduced glutathione, phenols, and flavonoids [20][21][22]; they have been reported as insecticidal, repellent, and oviposition deterrence on insects [21,23,24].
Cladostephus spongiosus (Hudson) is a marine brown alga, ranging 2-3 mm in width, and appears narrow, tough, and wiry in nature. Alves et al. [25] highlighted that the algae exhibit a number of biological activities including anti-microbial, anti-cancer, anti-inflammatory, and antiprotozoal. Insecticides synthesized from natural products, such as metal and metal oxide nanomaterials of herbal origin, could have a prominent role in the existing situation. Nanomaterials of varied sizes have gained broad applications based on definite particulate dispersions with a size range of 1-100 nm [26,27]. Silver nanoparticles (AgNPs) possess potential microbial, anticancer, and mosquitocidal properties [28][29][30].
Nanoparticles are smaller in their structure with definite shape and are more specific in their action; hence, they have gained significance in the research fraternity and many other diverse fields [26,27]. The AgNPs synthesized have more potential against microbes, cancer, mosquitoes, and anticancer [28,31]. Green synthesized nanoparticles from plants are gaining significance owing to their simplicity and the rapid rate of synthesis of nanoparticles of diverse morphologies and eco-friendliness [32]. The present study aimed to explore the bio-fabrication of macroalgae-mediated AgNPs and the produced nanomaterials were categorized through various biophysical techniques. Further, the larvicidal and adulticidal potentials of the C. spongiosus extract and C. spongiosus-synthesized AgNPs against key mosquito vectors, An. stephensi, A. aegypti, and Cu. quenifascienses, are studied.

Collection of plant materials and extraction
The alga Cladostepus spongiosus ((Hudson) C. Agardh, 1817) was collected from Haql (29°20025.400 N, 34°56053.5100 E), in the Tabuk region, Saudi Arabia, during morning hours and identified by a plant taxonomist at the Department of Biology, Tabuk University, Saudi Arabia. The algae harvested were washed in running tap water twice or thrice and air-dried in shade at room temperature (25 ± 2°C). The dried algae were chopped into fine pieces using an electric blender (Torrington, CT, USA) and used for further extraction and experimental procedures. C. spongiosus was extracted using Soxhlet apparatus with ethanol for 48 h at 76-78°C. About 200 g of fine-powdered algae was dissolved in 750 mL of ethanol, and the concentrated crude extract obtained was further evaporated in a rotary evaporator at 40-60°C to remove excess solvent and stored at 2-8°C for further activities.

Preparation of the standard solution
The standard solution was prepared by adding 1 mL of the extract to 98.5 mL of deionized water and 0.5 mL of Triton-×100 as an emulsifying agent. This gives a concentration of 1,000 ppm, which can be diluted for further experiments.

Synthesis of C. spongiosus-based AgNPs and characterization
About 1 mL of the C. spongiosus ethanol extract was added to 1 mL of silver nitrate (AgNO 3 ) 10 −3 , 97.5 mL of distilled water, and 0.5 mL of Triton-X100. The above solution was further incubated at room temperature until the colour changed to grey, which indicated the synthesis of nanoparticles [33]. The nanoparticles synthesized were subjected to Fourier transform infrared spectrophotometry (FTIR), energy-dispersive X-ray (EDX) spectroscopy, and transmission electron microscopy (TEM).

Mosquito culture
Mosquitoes of Indian strains Ae. aegypti, An. stephensi, and Cx. quinquefasciatus were cultured and maintained in the entomology laboratory, at Tabuk University, Saudi Arabia, for more than 6 years without exposure to insecticides.
Cultures were maintained at 27 ± 2°C and 75-85% relative humidity under a 14:10 L/D photoperiod. Larvae were fed with Brewer's yeast and dog biscuits, and algae were collected at 3:1:1. The pupae that emerged were transferred to a cup of tap water and replaced in a cage (dimension: 50 cm × 50 cm × 50 cm) for adult emergence. Adults were fed with 10% sucrose solution, and further larvae emerging from the eggs were used for further experimental purposes.

Larvicidal bioassays
Bioassays were performed for 24 instars of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus at varied concentrations: 300, 450, 600, and 750 ppm of the crude extract of C. spongiosus and 10, 25, 40, 55, and 70 ppm of C. spongiosus-fabricated AgNPs. No food was provided throughout our experiments and the mortality rate was observed after 24 h. The experiments were repeated five times and each concentration had a control. The mean mortality was determined using Abbott's [34] formula:

= ×
Percentage of mortality Number of dead larvae Number of larvae introduced 100. (1)

Adulticidal bioassay
Different concentrations of the ethanolic algal extract at 200, 400, 600, 800, and 1,000 ppm and 10, 25, 40, 55, and 70 ppm of C. spongiosus green-fabricated nanoparticles were tested for adulticidal activity. Different concentrations of extracts were impregnated to Whatman no. 1 filter paper (12 cm × 15 cm); ethanol was used as a control. The impregnated paper was air-dried for 5 min and kept in an exposure tube. Twenty adult mosquitoes, 2-5 days old, starved for blood meal were introduced into the holding tube for 1 h. The tested mosquitoes were blown back to the holding tube after exposure. The tubes were accomplished with a 10% glucose solution, which was soaked in cotton and was used as feed for the surviving mosquitoes. At the end of 24 h, the number of dead mosquitoes was recorded to determine their mortality.

Statistical analysis
The observed mortality from our experiment was exposed to the analysis of variance (ANOVA of arcsin, logarithmic, and square root transformed percentages). The significant differences obtained between treated and control groups were evaluated by Tukey's multiple range test (significance at P < 0.05) using the Minitab ® 17 program.

Results
The nanofabricated AgNPs synthesized from C. spongiosus were characterized using FTIR, spectrophotometry, and they showed significant peaks.  Figure 1 and Table 1. EDX analysis of C. spongiosus-mediated green-fabricated AgNPs showed strong signals of silver atoms at 3 keV. This implies the synthesis of AgNPs, as shown in Figure 2. The signals apart from silver, such as carbon and copper, are due to the TEM grid.
UV-visible spectrophotometric analysis of synthesized nanoparticles showed an absorption peak at 341 nm, which indicated the synthesis of AgNPs. The results obtained are shown in Figure 3.
The TEM results also implied the synthesis of nanoparticles. The shape and size of the nanoparticles synthesized were 20 and 50 nm, as shown in Figure 4.
X-ray diffraction (XRD) analysis of the synthesized nanoparticles showed the corresponding peaks of (111), (200), (220), and (311) planes, confirming that the material synthesized was nanoparticles. The results obtained are shown in Figure 5.
The larvicidal activity of the ethanolic crude extract of C. spongiosus against treated mosquito vectors such as Ae. aegypti, An. stephensi, and Cx. quinquefasciatus showed promising activity, which was evident by its mortality percentage. Larvae treated at concentrations of 150, 300, 450, 600, and 750 ppm showed good mortality in a dosedependent manner. The mortality percentage was higher at higher concentrations and least at lower concentrations. At 150 ppm, the mortality percentages were 24.2% against Cx. quinquefasciatus, 26.7% against Ae. Aegypti, and 29.5% against An. stephensi. At a higher concentration of 750 ppm, the mortality percentages were 81.6% in Cx. quinquefasciatus, 84.1% in Ae. aegypti, and 88.9% in An. stephensi. This implies that the extract has exceeded its potential to control mosquito larvae vectors. The results obtained are shown in Figure 6.
The larvicidal activity of green-fabricated C. spongiosus-mediated AgNPs against mosquito vectors showed good activity. Larvae treated at different concentrations of 10, 25, 40, 55, and 70 ppm showed good potential to control     Figure 7. The adulticidal activity of the ethanolic extract of C. spongiosus showed good activity against the treated vectors. The adulticidal potential was implied by its mortality    Figure 8.

Discussion
The mosquito control program is challenging in the present environment due to its resistance against most of the insecticides. The continuous use of chemical pesticides to control vector-based disease spread has become a global problem against food chain and environmental pollution [35]. Insecticides developed from the natural environment or plant-based origin have good potential with no toxicity against the environment. Several studies are in progress to develop potential insecticides at low cost and with high mortality [35][36][37]. Seaweeds are widely distributed in the universe and can be obtained at lower costs from the seas and oceans [24]. Thus, it can be an alternative source for the present synthetic insecticides [27]. C. spongiosus extracted with ethanol showed promising activities against mosquito vectors. Similarly, earlier research reports reveal that the algae have good larvicidal potential owing to the presence of bioactive compounds [38].
Nanosynthesized green-fabricated nanoparticles have more advantages than crude extracts due to their rapid action at the lowest concentration. Green-fabricated C. spongiosus-mediated AgNPs synthesized via ethanol extraction showed colour changes. Cittararasu et al. [39] revealed that changes in the colour indicated the initial confirmation of nanoparticles, which was compared with the Ag nanoparticles synthesized biologically from Barleria longiflora. The FT-IR spectra of the synthesized nanoparticles showed the presence of S]S disulfide asym., ]CH out of plane, C-H out of plane, C-H out of plane, P-OR ester, S]O sulphate ester, S-H (sharp), and CH 2 , ArO-H. Similarly, Sargassum myriocystum-mediated AgNPs showed the presence of N-H stretch, C]C stretch, 1 C-C (O)-C stretch with esters, and C-C stretch with alkyl halides [40].
EDX analysis revealed the crystalline nature of the particles synthesized. Balaraman et al. [40] revealed the phase formation and crystalline nature of the nanoparticles synthesized from S. myriocystum. The nanoparticles synthesized from C. spongiosus showed Ag ions in the range of 2.7-3 keV. A similar study was reported in which Ag NPs were synthesized from S. siliquosum that shows Ag ions at ∼3 keV [41].
UV-Vis spectrophotometric analysis of the synthesized nanoparticles showed an absorption peak at 341 nm, which indicated the synthesis of AgNPs. Similar to our report, AgNPs prepared on montmorillonite clay in n-hexanol exhibited fluorescence peaks at 341 nm [42].
TEM analysis showed that the synthesized nanoparticles were of size 20 and 50 nm. Reports from earlier findings reveal that particle sizes in the range of 1-100 nm and solid particles in the range of 10-1,000 nm are considered to be nanoparticles [43,44].
The XRD analysis of the synthesized nanoparticles showed corresponding peaks of (111), (200), (220), and (311) planes, confirming that the material synthesized is nanoparticle. Our reports agree with the findings of other researchers, who found similar findings when analysed for marine fabricated AgNPs [43,45,46].
The ethanolic extract of C. spongiosus algae showed promising activities against Cx. quinquefasciatus, Ae. aegypti, and An. stephensi larvae. Similarly, the methanolic extract of brown, green, and red algae tested against Ae. albopictus and Ae. aegypti showed strong larvicidal activity [28]. The ethanolic extract of C. spongiosus treated at concentrations of 150, 300, 600, and 750 ppm showed good mortality against An. stephensi followed by Ae. aegypti and Cx. quinquefasciatus. This implies that the bioactive compounds available in the extract exhibit their potential by binding to their target site [47]. Similarly, the leaf extract of Annona muricata treated against mosquito vectors An. stephensi, Cu. quinquefasciatus, and Ae. aegypti showed good activity when treated at concentrations of 100, 200, 300, 400, and 500 ppm [48].
The adulticidal potential of the ethanolic C. spongiosus extract showed significant activity against all vectors showing maximum activity at 1,000 ppm. The adults showed a maximum of 78.8% followed by 75.4% and 67.6% against An. stephensi, Ae. aegypti, and Cx. Quinquefasciatus, respectively. Similarly, the ethanolic extract of Sargassum polycystum, which was tested against adulticidal potential, showed good activity; when treated at 100, 150, 200, 250, and 300 ppm, it showed significant activity on Ae. aegypti and Cx. quinquefasciatus [49].
The synthesized green-fabricated C. spongiosus-mediated AgNPs which were tested against larvae of An. stephensi, Ae. aegypti, and Cx. quinquefasciatus showed their potential by the mortality percentage. The mortality percentages obtained against the vectors are higher even at lower concentrations. The larvae treated at the lowest concentration of 10 ppm exhibited mortality percentages of 40.6%, 37.4%, and 35.2% against An. stephensi, Ae. aegypti, and Cx. Quinquefasciatus, respectively. Similarly, plant-fabricated nanoparticles show higher mortality against mosquito larvae at lower concentrations [46].
The adulticidal potential of C. spongiosus-mediated AgNPs was good in governing mosquito adults. The treated adults showed maximum percentages of mortality at a concentration of 70 ppm: 89.4% against An. stephensi adults, 86.4% against Ae. Aegypti, and 83.2% against Cx. quinquefasciatus. Likewise, AgNPs fabricated using the Acacia caesia showed promising potential against treated adults at a lower concentration of 45 µg·mL −1 , with a mortality percentage of 100% against An. subpictus, 98.2% against Ae. albopictus, and 95.6% against Cu. tritaeniorhynchus [51].
The percentage of mortality against larvae and adults of An. stephensi, Ae. aegypti, and Cx. quinquefasciatus via C. spongiosus may change from place to place within the same species. This may directly depend on the availability of bioactive compounds in the algae with respect to environmental factors or stress. A similar result was stated by earlier researchers: species of the same division change due to ecological and geographical factors, etc. [52].
Acknowledgements: The author is thankful to the Entomology and Toxicology Laboratory, Faculty of Science, University of Tabuk, Kingdom of Saudi Arabia.
Funding information: The author states no funding involved.
Author contributions: Al Thabiani Aziz performed the entire experimental studies, data compilation, as well as wrote and edited the manuscript.