Fabrication of green nanoinsecticides from agri-waste of corn silk and its larvicidal and antibiofilm properties

2.1 Collection of CS and methanol extraction

Zea mays L. silk was procured from agricultural firms of Udumalaipettai, which is located at Tiruppur District, Tamil Nadu. It was validated by Taxonomist of Botanical Survey of India, whose reference number was BSI/SRC/5/23/2019/Tech./4 and deposited at Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu, India. Double distilled water (DDH₂O) was used to clean the silk threads which was further dried away from the sunlight at 28 ± 2°C. Two hundred and fifty grams of silk threads were extracted for a time span of 72 h using absolute methanol by employing Soxhlet apparatus. A 20% yield was obtained, which was maintained at 4°C for future use.

2.2 Production and characterization of CS-fabricated AgNPs

AgNPs were prepared according to Murugan et al. [20] with minor changes following Sasidharan et al. [21]. In order to prepare the nanoparticles, 10 mL of CS extract was allowed to react with 90 mL of 1 mM AgNO₃ for a time span of four hours.

The absorption curve of AgNPs was obtained by UV-Vis spectrophotometry (UV-3600 spectrometer, Shimadzu, Japan). Transmission electron microscopy was done by TEM-JEOL model instrument 1200 EX, Japan, operating at 10 kV and energy dispersive X-ray spectroscopy by EDX-JEOL mode 6390, Japan. FTIR was performed using a Perkin–Elmer spectrum 2000 FTIR, USA. XRD results were obtained using Phillips PW1830 equipment [22].

2.3 GC–MS analysis of CS extract

GC–MS analysis has been acquired on a Clarus 500 Perkin–Elmer gas chromatograph with a Turbo mass gold-Perkin–Elmer detector. The column dimensions were 30 m × 0.25 mm × 0.25 µm with the 5% diphenyl/95% dimethyl poly siloxane stationary phase column. For GC–MS detection, an electron ionization system with an ionization energy of 70 eV was used. Helium gas (99.99%) was used as the carrier gas at a constant flow rate of 1 mL·min⁻¹ and an injection volume of 3 mL was employed (split ratio of 10:1). Injector temperature was 250°C; ion-source temperature was 280°C. The oven temperature was programed from 110°C (isothermal for 2 min), with an increase of 10°C‧min⁻¹ to 200°C (no hold), then from 5°C‧min⁻¹ to 280°C, ending with a 9 min isothermal at 280°C. Mass spectra were taken at 70 eV, a scan interval of 0.5 s and fragments from 45 to 450 Da. Total GC running time was 36 min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Software adopted to handle mass spectra and chromatograms was a Turbomass version (5.2.0.2.14).

2.4 Larvicidal/pupicidal experiments in the laboratory

Each stage of larval instar and pupae of mosquitoes was reared in a sustainable way as described by Suresh et al. [23]. Twenty-five larvae of each instar and pupae were treated with 250 mL solutions containing 249 mL of deionized H₂O and 1 mL of required concentration of CS methanolic extract (100–500 ppm) or CS-AgNPs (2–10 ppm) according to a procedure outlined by Sujitha et al. [24]. Five replicates were used for each experiment and deionized water without extract/nanoformulation was used as control; the percentage of death was calculated after 24 h of exposure.

2.5 Experiments on predation in the laboratory

Mettur Dam located in the Salem District of Tamil Nadu in India was the site for collection of Guppy fish (Poecilia reticulata). Acclimatization of the Guppy fishes was done in the laboratory prior to any experiment. Four hundred numbers of Ae. aegypti larvae (I–1V instar) were placed with one guppy fish adult in a 2 L glass tank filled with deionized water plus 1 mL of the desired concentration of the CS extract silk-mediated AgNPs. All the experiments were conducted in five replicates. Control was deionized water only. The experimental tanks were checked every 24 h at daytime and nighttime and the number of preys eaten by guppy fish was calculated. After each checking, the predated mosquito instars were replaced with new ones. Predatory efficiency was determined using the following formula:

2.6 Assessing larvicidal activity in the field

Six different outdoor water bodies were used to study the larvicidal activities of bio-fabricated AgNPs and the CS extract. After 24-, 48-, and 72-h exposure, the larval density calculations were done by following the procedure of Panneerselvam et al. [25]. Lethal toxicity was assessed on third and fourth instar larvae of mosquito. Six replicates were performed maintaining 27 ± 2°C and 80% relative humidity as previously done by Panneerselvam et al. [25]. Ten times the Lethal Concentration₅₀ was used in accordance to Murugan et al. [26]. On the basis of surface area (0.25 m²) and volume (250 L), the quantity of insecticide to be used was ascertained. The following formula was used to calculate the percentage of larval density reduction:

where C is the total number of mosquitoes in the control and T is the total number of mosquitoes in the treatment.

2.7 Anti-biofilm assay

Biofilm was formed by pouring 100 μL of trypticase soy broth with 1% glucose solutions into plates maintained at 35 ± 2°C for 24 h. About 0.9% saline solution was used to remove the unattached cells followed by staining with 2% crystal violet. Furthermore, 90 μL of trypticase soy broth and 10 μL of CS-fabricated AgNPs were added to all the parallel well biofilms. All the experiments were conducted in three sets. To elucidate biofilm inhibition potential, 100 μL of Staphylococcus aureus or Staphylococcus epidermidis in trypticase soy broth was followed by 24-h of incubation. Planktonic cells were discarded after washing. Biofilm was obtained according to the procedure outlined by Mu et al. [27] and Stefanovic and Hegde [28].

2.8 Extraction of EPS

Filtration of supernatant was accomplished using 0.22 μm nitrocellulose membrane followed by treatment with chilled absolute ethyl alcohol and incubation for 12 h at 4°C to obtain EPS as a precipitate [29]. EPS was quantified according to Dubois et al. [30].

2.9 Statistical analysis

Data were analyzed using SPSS software v. 16.0. Larval toxicity data and biofilm inhibition data were ascertained by two-way ANOVA with two factors (i.e., concentration and mosquito life stages/bacterial strains), followed by Tukey’s HSD test (P = 0.05). Mosquito mortality data were determined by Probit analysis [31]. Two-way ANOVA was adopted to analyze mosquito larval population data from field bioassay with two factors (i.e., mosquito treatment and the elapsed time from the treatment) followed by Tukey’s HSD test.

Guppy fish predation data were determined via JMP 7 using a weighted generalized linear model with two fixed factors:

where y is the vector of the observations (i.e., the number of consumed preys), X is the incidence matrix, ß is the vector of fixed effects (i.e., the mosquitocidal treatment and targeted instar), and ε is the vector of random residual effect. A probability level of P < 0.05 was used for the significance of difference between values.

3.1 Chemical profiling of Z. mays (GC-MS)

The GC-MS analysis of Z. mays extracts show the presence of 14 compounds as shown in Figure 1 and Table 1. For instance, Abirami et al. [32] reported that the presence of 1-(+) ascorbic acid 2, n-hexadecanoic acid in the ethanol extract of CS. Comparably, the fatty acid peak profile of Z. mays was noticed in the chromatogram [33]. Furthermore, 9,12-octadecadienoic acid, methyl ester, and 9-octdecenoic acid methyl ester were observed in Casimiroa edulis La Llave and lex leaf extract with significant insecticidal and antifeedant properties on Spodoptera littoralis [34].

Figure 1

GC-MS spectrum of the Z. mays silk extract.

3.2 CS-AgNP synthesis and characterization

CS-fabricated AgNPs changed the color from pale yellow to dark brown at 4 h of photo period. An extreme absorption peak for AgNPs was noticed at 450 nm (Figure 2). The broadening of the peak indicated that the particles are polydispersed. Similarly, Rajkumar et al. [35] noticed that Z. mays-mediated AgNPs exhibited a sharp absorption peak at 420 nm. Also, Hema et al. [36] observed the maximum UV-Vis absorption peak of Z. mays-decorated AgNPs between 410 and 450 nm.

Figure 2

UV-Vis spectrum of Z. mays silk-fabricated AgNPs.

CS-AgNPs were either spherical or cubic in structure, with a mean size ranging between 10 and 50 nm (Figure 3). Similarly, Patra and Baek [37] noticed that the Zea mays L. silky hairs-fabricated AgNPs displayed spherical in shape and the size ranging from 100 to 450 nm. Furthermore, Eren and Baran [38] observed that the Zea mays L. leaves extract-based AgNPs exhibit the size less than 100 nm and Zea mays L. (corn flour) synthesized nanosilver particles showed monodispersed spherical in shape. The EDX analysis of CS-AgNPs revealed strong peaks of silver (42.4%) confirming the high purity of AgNPs (Figure 4), with the optical absorption peak at 3 keV due to surface plasmon resonance [25,35]. Also, the EDX analysis exhibited the peak of oxygen (O), indicating that CS-AgNPs stabilized by the presence of organic compounds in the Z. mays extract. This result was well matched with Chen et al. [39] who have reported results on the corn straw-synthesized AgNPs.

Figure 3

TEM image of Z. mays silk-fabricated AgNPs.

Figure 4

EDX spectrum of Z. mays silk-fabricated AgNPs.

Figure 5 reveals the XRD pattern of Z. mays-synthesized AgNPs. The XRD results of Z. mays-synthesized AgNPs were corresponding with JCPDS card number (04–0783). The XRD patterns of CS-AgNPs exhibited strong Bragg reflections at 38.47, 44.09, 64.56, and 77.37 corresponding to the planes (111), (200), (220), and (311) (Figure 5), which well matched with the reports of Hema et al. [36], who observed that flower-shaped AgNPs from Z. mays had strongest diffraction peaks at 38.6° and 64°, while Rajkumar et al. [35] noticed diffraction peaks at 38.2, 44.42, 64.4, 76.7, and 81.45, which indicates the crystalline nature of AgNPs from Z. mays.

Figure 5

XRD pattern of Z. mays silk-fabricated AgNPs.

The FTIR spectra of AgNPs synthesized from Z. mays are shown in Figure 6. The synthesized AgNP spectrum reveals various characteristic peaks at 3,350, 2,974, 1,738, 1,661, and 1,273 cm⁻¹, respectively. A peak at 3,350 cm⁻¹ was assigned to N−H stretching of the amine group and an observed peak at 1,661 cm⁻¹ was attributed to –C═C−H stretching of alkenes. The absorption peaks at 1,378 and 2,974 cm⁻¹ of AgNPs are relative to the presence of alkynes, carboxylic acids, and phenolic group [36]. The peak at 1,738 cm⁻¹ corresponds to the carbonyl stretching vibration, while the peak at 1,660 cm⁻¹ can be attributed to C═C stretching vibration. The peak at 1,273 cm⁻¹ corresponds to the C–O stretching vibration. The slight shifting of various functional groups in the AgNPs from Z. mays displayed the progress of the reduction processes with capping and stabilization [40].

Figure 6

FTIR spectrum of Z. mays silk-fabricated AgNPs.

The zeta potential value of Z. mays-fabricated AgNPs was measured as −14.3.86 (Figure 7). The synthesized AgNPs were found to have negative zeta potential, confirming the stability developed formulation [41]. Comparably, Erdogan et al. [42] observed that the zeta potential value of AgNPs from Cynara scolymus L. was about −32.3 ± 0.8 mV. The negative value might be due to the capping action of active metabolites present in the Z. mays extract.

Figure 7

Zeta potential of Z. mays silk-fabricated AgNPs.

In addition to this, the typical size of Z. mays-synthesized AgNPs was examined by DLS analysis (Figure 8) and determined an average particle size of 104 nm. Comparably, Murugan et al. [3] noted that the Cymbopogon citrates-synthesized Au nanoparticles had an average particle size of about 20 nm. Furthermore, Dwivedi and Gopal [43] observed that Chenopodium album L.-fabricated Ag and Au nanomaterials were stable under a wide pH range due to their high zeta potential.

Figure 8

DLS spectrum of Z. mays silk-fabricated AgNPs.

3.3 Bio toxicity effect of CS extract and CS-AgNPs on Ae. aegypti

CS extract exhibited significant toxicity on dengue vector, Ae. aegypti, and the acquired LC₅₀ values ranged from 231.67 (I instar) to 447.89 μg·mL⁻¹ (pupae) (Table 2). Moreover, CS-AgNPs were more toxic on Ae. aegypti when compared with CS extracts. Indeed, their LC₅₀ values ranged from 2.35 (I instar) to 6.24 μg·mL⁻¹ (pupae) (Table 3). To our knowledge, no reports on the larvicidal activity of CS extract and CS-fabricated AgNPs against Ae. aegypti have been reported. The current results were compared with other reports of plant extracts, for example, Valoniopsis pachynema (G. Martens) Børgesen extract showed high toxicity against An. stephensi with LC₅₀ values ranging from 216.560 (larva I) to 359.868 μg·mL⁻¹ (pupa), while for An. sundaicus they ranged from 188.871 to 311.860 μg·mL⁻¹ [24]. Recently, Alshehri et al. [44] stated that Heritiera fomes Buch.-Ham leaf extract showed toxicity on An. stephensi and Ae. aegypti, in a dose-dependent manner.

3.4 Impact of CS-AgNPs on Poecilia reticulata predation

The predatory activity of P. reticulate fishes was calculated against I-IV instar larvae of A. aegypti. Guppy fish showed a high feeding activity in water containing CS-fabricated AgNPs, indicating lack of intoxication effects on this nontarget species. In standard laboratory conditions, low doses of AgNPs were treated with water where the fishes were introduced, their predatory efficacy were 43.2% (I instar), 74% (II instar), 65.8% (III instar), and 39.5% (IV instar) after 24 h treatment. The predatory efficacy of P. reticulate was 32.2% (I), 52.8% (II), 43.8% (III), and 30.2% (IV instar) (Table 4). Similarly, Saleeza et al. [45] studied the feeding competence of both male and female guppies, P. reticulate on Ae. aegypti, Ae. Albopictus, and Cx. quinquefasciatus, and they reported that female guppies predate more mosquito larvae than male guppies. The predatory rates of female guppies were 121.3, 105.6, and 72.3, and male guppies were 98.6, 73.6, and 47.6 for Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus, respectively. The predatory efficiency of P. reticulate post-treatment with Toddalia asiatica (L.) Lam.-synthesized AgNPs were 89.75% and 74% against II and III instar larvae of Cx. quinquefasciatus [46].

3.5 Field application of CS extract and CS-AgNPs on Ae. aegypti

In the field, the application of CS extracts and AgNPs (10 × LC₅₀) leads to the reduction of larval population into 10.61% and 5.16% after 72 h (Table 5). Similarly, the leaf extract of Catharanthus roseus (L.) G. Don led to An. stephensi larval reduction of 12.26%, 28.80%, and 79.46% after 24, 48, and 72 h post-treatment [47] and the field application of Phyllanthus niruri L. extract (10 × LC₅₀) led to larval density reductions of 39.9%, 69.2%, and 100% after 24, 48, and 72 h, respectively [22]. Concerning AgNPs, Rajaganesh et al. [48] studied Dicranopteris linearis (Burm.fil.) Underw.-synthesized AgNPs (10 × LC₅₀) led to 56.5%, 18.33%, and 100% of reduction after 24, 48, and 72 h, respectively. The bio-toxicity of AgNPs on Ae. aegypti may be due to the tiny size of nanomaterials that can easily penetrate the exoskeleton of insects and even individual cells, producing shedding and other physiological interferences [49].

3.6 Anti-biofilm activity of CS-AgNPs

In this research, we studied the anti-biofilm efficacy of CS-AgNPs against S. aureus and S. epidermidis testing five different concentrations ranging from 25 to 125 μg·mL⁻¹. Results showed a maximum inhibitory effect (90% and 79% inhibition) at 125 μg·mL⁻¹ on S. aureus and S. epidermidis, respectively (Table 6, Figure 9). Similarly, Patra and Baek [37] observed that corn silky hairs-fabricated AgNPs exhibited strong antibacterial effect on five various bacterial pathogens with inhibition zones between 9.23 and 12.81 mm. The results of light microscopy examination revealed alteration in the cell membrane permeability and inhibition of DNA replication when compared with control, and the biofilm thickness was notably reduced after treatment with CS-AgNPs at 125 μg·mL⁻¹ (Figure 9). Interestingly, when treated with CS-AgNPs, a lesser number of scattered cell aggregates in the biofilm have been observed. Overall, these results suggested that high doses of AgNPs increase the oxidative stress leading to bacterial cell detachment [50].

Figure 9

Light microscopic observation (40×) of S. aureus (I) and S. epidermidis (II) adhesion phases on the glass surfaces with different concentration ((a) control, (b) 25 μg·mL⁻¹, (c) 50 μg·mL⁻¹, (d) 75 μg·mL⁻¹, (e) 100 μg·mL⁻¹, (f) 125 μg·mL⁻¹) of Z. mays-synthesized AgNPs against control which is indicative of glass surface with uniformly distributed cells stained with crystal violet.

3.7 Quantification of EPS

Biofilm is formed by adhering complex communities that are encased within extracellular polymeric matrix. The EPS helps to protect the bacterial communities from oxidizing biocides, UV radiation, and antibiotics [51]. Therefore, disintegration of extra polymeric matrix paves way for assassination of biofilm. Herein, we studied the impact of CS-AgNPs on EPS formed by S. aureus and S. epidermidis within the biofilm. From the results, we observed a reduction of EPS formation in both S. aureus and S. epidermidis after treatment with different concentrations of AgNPs (Figure 9). Notably, the reduction of EPS observed at the concentration of 125 μg·mL⁻¹ was of 79.95% and 71.67% S. aureus and S. epidermidis, respectively. The inhibition of biofilm formation and the reduction of EPS synthesis were dose dependent (Table 6). Rajkumari et al. [52] reported the anti-biofilm effect of gold nanoparticles against Pseudomonas aeruginosa and a reduction of EPS formation by 81.29%. Very recently, Alahmdi et al. [53] highlighted that the Clitoria ternatea L.-synthesized ZnO NPs significantly reduced EPS of both E. coli and S. aureus at 25–100 g·L⁻¹, respectively.