Toxicity of Ulva lactuca and green fabricated silver nanoparticles against mosquito vectors and their impact on the genomic DNA of the dengue vector Aedes aegypti

Abstract Marine seaweeds are known to have a potential role against microbial and pesticidal activities. Ulva lactuca, a green macroalgae extract analysed through gas chromatography mass spectrometry reveals 31 compounds. Resistance of mosquito vectors to synthetic insecticides remains a major problem. Discovering and applying natural agents to act against disease vectors is challenging. The activities of the extract and nano‐fabricated green synthesised silver nanoparticles were checked for use against Aedes aegypti and Culex pipiens. The crude extract and synthesised silver nanoparticles exhibited a notable larvicidal effect, and very effective inhibition of pupal and adult emergence. Inhibition of adult emergence of Ae.aegypti was 97.7% and in Cu.pipiens, it was 93.3%. Our genotypic study of Deoxyribonucleic acid from treated larvae utilising random primers MA‐09, MA‐12 and MA‐26 revealed damaged nucleotide sequences when compared with the controls. The antimicrobial activity of both the extract and green synthesised nanomaterials showed prominent activity against pathogenic drug resistant bacteria. Our results contribute to further development of eco‐friendly insecticides with lower cost of preparation. This could further contribute to further research helping future generations to be free from these deadly disease‐causing vectors and pathogenic microbes.


| INTRODUCTION
Mosquitoes (Diptera: Culicidae) are the major vector for most of the deadly transmitted diseases in the world [1,2] affecting more than 700,000,000 people per year throughout the world [3]. Mosquitoes have long slender body with long legs and piercing mouthparts. Anopheles, Aedes, and Culex are the three most common mosquito vector genera. They are biting Dipterans that suck blood from humans and other animals while acting as carriers of several diseases. Most of these diseases, such as malaria, filariasis, dengue, chikungunya, yellow fever, Zika virus, etc., are commonly spread throughout the human populations [4]. The resurgences of these diseases are attributed to multiple breeding sites in addition to resistance among mosquito populations due to repeated usage of chemical insecticides increasing resistance among mosquito population as well as causing serious environmental pollution [5]. Thus, an alternative approach is needed to fight against mosquito populations.
Seaweeds have wide range of biological applications from nutrition to therapeutic applications in pharmaceutical industries, etc. Ulva lactuca is a macroalgae and an edible seaweed found abundantly in the marine environment. This species is found practically everywhere in shallow waters, including estuaries, throughout the World Ocean [6,7]. It has wide acceptance in society because it is biodegradable, ecofriendly and cost effective. Reports have also suggested that seaweeds have potential insecticidal activity [8,9]. Study of the bioactive compounds present in seaweed can contribute to our knowledge of their mode of action in combating mosquito This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. larvae [9]. U. lactuca has long been used for the treatment of diseases in Chinese medicines [10] and is also a rich source of ash, proteins, lipids, fibre and carbohydrates [11].
Aedes aegypti Linn. (Diptera: Culicidae) is the major vector of dengue, haemorrhagic fever, yellow fever and chikungunya [12]. Two thirds of the world's population harbours this dengue vector [13], including a total of more than 128 countries [14]. Culex pipiens L. is a commonly occurring mosquito in urban and semi-urban areas. It acts as a primary enzootic vector of West Nile in birds and acts as the vector for transmission of this virus to humans [15]. It was found to be highly resistant against most chemical insecticides such as temephos and fenthion [16].
In addition to our direct use of botanical extracts as insecticides, green biosynthesis of nanomaterials plays a vital role for increasing effectiveness [17]. As a plant-based synthesis, it is eco-friendly and does not contain or generate any hazardous substances [18]. The phytoconstituents present in these natural insecticides were proven to have mosquitocidal and insecticidal properties [19]. As Ag has long been used widely for biomedical and veterinary purposes, it has demonstrated no harmful effects against humans or the environment.
In this research, an ecofriendly nano-based insecticide was synthesised from Ulva lactuca. The bio-efficacies of both the U. lactuca extract and green nano-synthesised silver nanoparticles were studied against Ae. aegypti and Cu. pipiens mosquito vectors. Measures of larvicidal, pupal emergence, adult emergence and extent of inhibition were conducted. Genotoxicity of green synthesised nanoparticles on Deoxyribonucleic acid (DNA) was determined by random amplified polymorphic DNA (RAPD) analysis through agarose gel electrophoresis. Antimicrobial activity the of U. lactuca extract and fabricated AgNPs toxicity against pathogenic strains were studied and their efficacy was analysed.

| Seaweed collection and extraction
The seaweed Ulva lactuca was collected from the Red Sea coast of Haql (29°20 0 25.4″N, 34°56 0 53.51″E), Tabuk region, Saudi Arabia, in the early mornings. They were cleaned with distilled water, shade dried at an average temperature of 27°C and 77% relative humidity and powdered. About 10 gm of the powdered sample was aqueous extracted using a soxhlet apparatus using ethanol at a boiling point of 78°C for 8 h and concentrated with a rotary vacuum evaporator at 40°C. The yield extracted was stored at 4°C for further assays.

| Gas chromatography mass spectrometry analysis of U. lactuca extract
The aqueous extract of U. lactuca was subjected to gas chromatography mass spectrometry (GC-MS) analysis. One ml of the sample was dissolved in high-performance liquid chromatography grade methanol and an active fraction was subjected to GC and MS JEOL GC mate equipped with a secondary electron multiplier. JEOL GCMATE II GC-MS, an Agilent Technologies 6890N Network GC system for gas chromatography, was used. The column (HP5) contained fused silica 50 � 0.25 mm I.D and the run time was 20 min. Column temperature was programed at 110°C with a 10°C/min rise to 230°C, and 250°C was chosen for for injector temperature. Helium (99.99%) was the carrier gas, and the split ratio was 5:4. The sample (1 μL) was evaporated in a splitless injector at 280°C . The compounds were identified by gas chromatography coupled with mass spectrometry with source at 270°C and an electron impact mode at 70 eV. The molecular weight, molecular formula, and structure of the compounds of the tested materials were ascertained by interpretation on the mass spectrum of GC-MS using the database of the National Institute Standard and Technology.

| Synthesis and characterisation of U. lactuca-fabricated Ag nanoparticles
About 100 ml of the Ulva lactuca extract was mixed with freshly prepared 1 mM AgNO 3 solution. A yellowish colour indicated the formation of U.lactuca-fabricated AgNPs. The synthesised green based AgNPs were further characterised by Uv-Vis, transmission electron microscopy (TEM), energydispersive X-ray spectroscopy (EDAX), Zeta potential and Xray diffraction (XRD) (Phillips PW1830, 40 kV, 30 mA with CuKα radiation).

| Mosquito culture
Saudi Arabian strains of Ae.aegypti and Cu. pipiens mosquitoes were cultured in an entomology laboratory at Tabuk University, Saudi Arabia, for more than 6 years without exposure to insecticides. Vectors were maintained at 27 � 2 • C and 75%-85% relative humidity under a 14:10 L/D photoperiod. Brewer's yeast and dog biscuits were used as diet for larvae.
Pupae obtained from the culture were moved to a small container with tap water and placed inside the screened cages of (50 � 50 � 50 cm) for emergence of adults. The adults that emerged were deprived of sugar for 12 hours and housed with a mouse placed in resting cages overnight for blood feeding by females on the fifth day. Adults were maintained and the larvae that emerged from the eggs were used for our experiments.

| Larvicidal bioassays
An aqueous extract was prepared, and 1 gm of crude extract was dissolved in 100 ml of distilled water. Then it was used for different ppms. Bioassays were performed for fourth instars of Ae. aegypti and Cu. pipiens at different concentrations of the crude extract, with U. lactuca at 100, 300, 500, 700 and 900 ppm and 50, 100, 150, 200 and 250 ppm of U. lactuca fabricated AgNPs.

| Developmental studies
The larvae treated with different concentrations of the extract and U.lactuca fabricated AgNPs were observed for any developmental changes in their further stages. The larvae were observed, and measurements were taken. Rates of pupation, adult emergence and inhibition of emergence were calculated.
The mortality rate observed was noted after 24 h and the Inhibitory concentration (IC 50 and IC 90 ) was calculated by using Probit analysis (Minitab ® 17). The experiment was repeated for five replicates and the percentage of mortality was calculated by using Abbott's [20] formula.

| DNA extraction from Ae. aegypti
DNA was extracted from Ae. aegypti larvae through an extraction kit obtained from DNeasy Blood & Tissue Kits (Qiagen). The larval tissue was chopped, and 10 mg of tissue placed in 1.5 ml centrifuge tubes to which we added 180 µL of ATL buffer. To this was added 20 μL of proteinase K. This was then incubated at 56°C in a shaking water bath until the tissue was lysed. The mixture was vortexed for 15 s, adding 200 μL of AL buffer to the sample. After vortexing, we added 200 μL of ethanol (96%-100%). This mixture was transferred to aDNeasy Mini spin column and centrifuged at ≥6000 x g (8000 rpm) for 1 min. We then discarded the flow through and added 500 μL AW1buffer and centrifuged for 1 min at ≥6000 x g (8000 rpm). Again the flow through was discarded, and we added 500 μL Buffer AW2 and centrifuged for 3 min at 20,000 x g (14,000 rpm). We placed the column in new centrifuge tube and added 200 μL of AE buffer, incubated it at room temperature for 1 min and then centrifuged for 1 min at ≥ 6000 x g (8000 rpm). The obtained elute contained the concentrated DNA.

| Randomly amplified polymorphic DNA-PCR
The genomic DNA extracted from Ae. aegypti was amplified using random primers for further studies. The Polymerase Chain Reaction (PCR) mixtures were prepared with a final volume of 15 μL, the master mix containing 1x PCR buffer 1.

| Agarose gel electrophoresis
The amplified DNA fragments of Ae. aegypti through PCR were analysed by agarose gel electrophoresis based on the procedure followed by Lalli et al. [21]. Agarose gel was prepared with 1.5% Tris-acetate-EDTA (TAE) buffer in which amplified DNA fragments through RAPD were mixed with bromophenol blue dye at a 5: 1 ratio. This was loaded carefully into the electrophoresis chamber wells along with a standard DNA marker containing TAE buffer (4.84 g Tris base, pH 8.0; 0.5 M EDTA/1 L) and finally loaded onto the agarose gel (1.5% gel stained with 10 μg/ml of ethidium bromide). The gel containing samples was connected to the power supply (120 V) for 45 min and the DNA bands were observed under the UV transilluminator to determine the profiles of the treated insects.

| Antibacterial activity
The antibacterial activities of U. lactuca and the fabricated AgNPs were determined by using the agar disc diffusion method on Muller Hinton agar medium. The bacterial strains were cultured initially in a nutrient broth prior to use.

| Statistical analysis
The mortality numbers obtained from our experiment were subjected to analysis of variance (ANOVA of arcsine, logarithmic and square root transformed percentages). Significant differences between treated and control groups were analysed by Tukey's multiple range test (significance at p < 0.05) using the Minitab ® 17 programme. Probit analysis with a dependability interval of 95% using the Minitab ® 17 programme was used to find the lethal concentrations required to kill 50% (LC 50 ) of larvae in 24 h.

| RESULTS
The results obtained from the GC-MS analysis of aqueous extracted U. lactuca show the presence of 31 compounds as shown in Figure 1 Table 1). The aqueous extract of U. lactuca incubated with AgNO 3 showed a yellowish colour which indicates the formation of U. lactucafabricated AgNPs which was confirmed by UV-vis spectroscopy ( Figure 2) and the absorption maxima of U. lactuca-synthesised AgNPs was at 453 nm. The particle size of the synthesised particles ranging 20-50nm is shown in Figure 3. The elemental composition of U. lactuca-synthesised AgNPs show copper and carbon by EDAX spectroscopy in Figure 4. The XRD profile of U. lactuca-F I G U R E 2 UV-Vis spectrum of AgNPs synthesised by the Ulva lactuca extract

F I G U R E 3 TEM of AgNPs synthesised using the U. lactuca extract
150 -AZIZ fabricated AgNPs shows prominent peaks. The characteristic study of green synthesised U. lactuca-fabricated AgNPs shows a definitive absorption peak at near 8 keV, which is shown in Figure 5. The zeta potential values of U. lactuca-synthesised AgNPs were found to have a negative zeta potential and confirm the stability developed formulation ( Figure 6). The U. lactuca extract against strains of Ae. aegypti and Cu.pipiens showed variation in their activity when exposed at 100, 300, 500, 700 and 900 ppm. The larvae show a dosedependent mortality with significant differences in their results. The highest mortality of 60.3% was noted in Ae. aegypti with 42.1% in Cu. pipiens at 900 ppm concentration. The pupae emergence from the treated larvae showed higher percentage of 90% at 100 ppm concentration in Cu. pipiens and 85% in Ae. aegypti. Adult emergence was reduced, and the treated larvae showed 2.3% reduction in Ae.aegypti and 5.1% reduction in Cu. pipiens at a higher concentration of 900 ppm. Thus, the adult inhibition percentage is higher at higher concentrations; in Ae. aegypti it was 98% and 95% in Cu. pipiens. Results are shown in Figures 1-10 The impact of U.lactuca extract against Ae. aegypti larvae exposed to LC 50

| DISCUSSION
Development of eco-friendly natural pesticides against insects has a major role in public health and environment protection. Seaweeds that can be obtained in enormous quantities and having non-harmful properties, can be used widely for their biological activity [1]. Gas chromatography mass spectrometry analysis of aqueous extracted U. lactuca shows the presence of 31 compounds. Similar ethanolic extracts of U. lactuca revealed 17 compounds [22]. Most of the compounds identified from U. lactuca have medicinal properties previously identified by researchers [23]. And controlling vector population by green synthesised AgNPs is now an emerging technique [24]. The nanoparticle synthesised from U. lactuca extract is evident by the colour changes; this may likely be due to surface plasmon resonance oscillation of the nanoparticles. An absorption peak for seaweed-fabricated AgNPs is noticed at 453 nm. Comparably, Sargassum muticum-synthesised AgNPs show a maximum absorption peak at 400 nm [25]. TEM analysis reveals a particle size of 20-50 nm for this synthesised nanoparticle. Earlier researchers [25][26][27] consider particles of 1-100 nm and solid particles of 10-1000 nm [24] to be nanoparticles.
The U. lactuca-fabricated AgNPs show positive anti-vector results when compared with crude extract. This was evident from the observed vector mortality percentages of treated strains. This is due to lower particle size and easier penetration of target insect larvae, thus providing more advantages than conventional insecticides. The plant-mediated and fabricated nanoparticles are synthesised in a single-step; therefore, there is no requirement of high pressure, high temperature or more toxic chemicals, etc. [28].
The toxicity of U. lactuca crude extract towards strains shows highest mortality against Ae. aegypti when compared with Cu. pipiens. This suggests Ae. aegypti are more susceptible to the extract. Pupal emergence and adult emergence from the treated larvae were also observed to be lower. Inhibition percentage of this strain was greatest at Ae. aegypti (98%) when compared with Cu. pipiens. Similar to our research, Suresh et al. [29] observed the aqueous leaf extract of S. maritima against Ae. aegypti. The active compounds which were present in the green extracts were more active against disease vectors, binding to target sites and providing better results. This suggests that aqueous extracts are toxic towards larvae in the same way Gracilaria firma extracts are toxic against Ae. aegypti at an earlier stage [30].
An inhibitory concentration of U. lactuca against Ae. aegypti was obtained at a lower concentration when compared with Cu. pipiens. Fabricated AgNPs show their highest mortality in Cu.pipiens (65.5%) and pupal emergence was also lower in Cu.pipiens, but adult emergence was lower in Ae. aegypti when compared with Cu. pipiens. This implies a decrease in reduction of adult emergence which contributes towards an environment free from Ae. aegypti. Even though our extract showed reduced mortality against Ae. aegypti, adult emergence and emergence inhibition was observed to be higher. Studies focussed on plant fabricated nanoparticles against mosquito larvae are highly toxic at low concentrations [31].
The green-fabricated AgNPs showed the highest percentage of mortality at the minimum concentration of 250 ppm in Cu. pipiens when compared with Ae. aegypti. Thus, a small quantity of the synthesised nanoparticle has a F I G U R E 1 2 RAPD analysis of DNA isolated from Ae. aegypti amplified with random primers. One-Control, 2-LC 50 , 3-LC 90 (a) (b) (c) F I G U R E 1 3 Antimicrobial activity of Ulva lactuca extract and its mediated AgNPs AZIZ capability to bind to the insect cuticle, penetrate inside the cells, and destroy further functions of the cells by protein synthesis, enzymatic activity, etc. Artemisia-synthesised AgNPs exhibited similar results when exposed to mosquito vectors [32].
The RAPD-PCR analysis of treated larvae revealed a higher number of bands in the control larvae when compared with results obtained from LC 50 and LC 90 larvae. This clearly indicates that the random primers were unable to find their complementary sequences for amplification. The larvae treated by synthesised AgNPs might have denatured the DNA of the larvae and this could delete the base pairs, or cause nversion, mutation, or other DNA damages so that the DNA was not able to recognise random primers to amplify. In any case, the DNA was damaged by the treatment concentrations in the larvae. Similar study made on microbes from the extract of Capparis spinosa showed a genotoxic effect which was confirmed through RAPD-PCR [33].
Antimicrobial activity of the U. lactuca extract and synthesised AgNPs shows potential against pathogenic microbes such as KP, PA, Staphylococcus aureus and MRSA. Our study results revealed synthesised that nanoparticles from U. lactuca show good activity measured by their zone of inhibition by the disc diffusion method, in comparison with crude extract of U. lactuca. Similarly antimicrobial activity of greensynthesised AgNPs against gram positive Staphylococcus aureus and gram-negative Escherichia coli, PA and Enterobacter spp. tthrough the disc diffusion method implies good activity [34]. This implies that green-synthesised nanoparticles are more active against microbial strains than are crude extracts. It is evident from earlier research that AgNPssynthesised nanoparticles from Areca catechu exhibited potential activity against antibiotic-resistant bacteria [35]. The compound phlorotannin from brown seaweeds also exhibits good antiviral, antibacterial, antifungal, and larvicidal activities [36].
Thus, seaweeds possess good insecticidal properties, and this activity increases when fabricated to form AgNPs. Researchers found seaweeds are more active against insects than terrestrial plant-derived bio-insecticides [8]. These synthesised AgNPs have a potential to control Ae. aegypti and Cu. pipiens, thus acting as a promising tool to control and eradicate vector borne diseases.

| CONCLUSION
Insecticides of chemical origin are highly toxic towards pests but often persist inside the environment and remain toxic for future generations. This has adverse effects on biological systems and creates abnormal ecological changes. Plant-based fabricated nanoparticles are an eco-friendly intervention to address some of the present problems with chemical pesticides. AgNP-based nano-formulations are proven to be effective in our study compared to aqueous extracts. This research contributes towards possible use of herbal nanopesticides against certain deadly vector-borne diseases. Further research needs to focus on the mode of action, stability, toxicity and non-target effects of these agents against beneficial insects.