INTRODUCTION:

Probiotics have been utilized by humans for health advantages and for processing a variety of dietary products.Probiotic benefits comprise: resistance to microbial infectious, reducing serum cholesterol concentration^1,2, and use of exopolysaccharides from probiotic as an anti-colon cancer ³. In addition to being used in materials, health care, and medicine, nanoparticles are used in every facet of daily life.Silver nanoparticles (AgNPs) biologically synthesized is less toxic than AgNPs synthesized using chemical method⁴. Copper nanoparticles (CuNPs) offer a wide range of uses as antibacterial due to their high reactivity and ability to combine with other materials.

Numerous studies have established CuNPs superior antibacterial efficacy against antibiotics resistance bacterial species⁵. The production of nanoparticles involves a wide variety of bioresources, including plants, plant products, bacteria, yeast, algae and viruses. Both multicellular and unicellular organisms can create inorganic nanoparticles either extracellularly or intracellularly ⁶. Also, actinomycetes strains were used for AgNPs synthesis and recorded antimicrobial activity against pathogen⁷. Furthermore, silver nanoparticles were impregnation on bacterial cellulose (BC) membranes and the BC-Ag composite exhibited highly active antimicrobial agent^8,9.

In general, biosynthesized AgNPs and CuNPs are more specialized andsafer, particularly in Nano form^10,11,12. Culex antennatus Becker play a major role in prevalence of Rift Valley Fever virus in Nile Delta of Egypt^12,13.14. For many decades, larvae of mosquito species usually targeted using imitative chemical insecticides as last resortto eliminate the rapid spread of diseases by vector control ^15,16. However, application of these chemical insecticides has many obstacles, such as mosquito resistance to chemical insecticides and negative impact of these insecticides on environment and non-target organisms^17,18,19. Application of nanoparticles in mosquito control consider one of the modern promising methods since they are cheap and does not require using of extremely toxic chemicals in their preparation. In the recent years, anincreasing number of nanoparticles proved its activity against different mosquito species at very low concentrations^20,21,22.

The aim of our research, synthesis and characterization of nanoparticles (Ag and Cu) from eco-friendly bacteria and determine the correlation of nanoparticles against C. antennatus third larval instar, as well as reproductive potential of resulted females.

MATERIALS AND METHODS:

Culture of Probiotic: Lactobacillus reuteri NRRL B-14171 was chosen from the culture collection of the Agricultural Research Service (house research arm of the U.S. Department of Agriculture).The strain was sub-cultured in 10 ml of De Man, Rogasa and Sharpe broth (MRS) broth (Fluka No. 93780) and incubated aerobically at 30 °C for 24 h²³.

Biosynthesis of nanoparticles:

Silver nanoparticles biosynthesis: Centrifugation was used to separate the bacterial biomass for 10 minutes at 5000 rpmand AgNO₃. 1mM solution was added to cell free supernatant of Lactobacillus reuteri NRRL B-14171 then, incubated overnight at room temperature. Color changes in the solution were used to determine the synthesis of AgNPs²⁴.

Copper nanoparticles biosynthesis: Lactobacillus reuteri NRRL B-14171 was cultured in MRS broth. After centrifugation, the broth culture pH was then adjusted at 6 using 1M NaOH.CuSO4 was dissolved in an aqueous solution, and the mixture was then incubated at 37°C overnight.At the bottom of the flask, the color shifted from yellow to a dark greenish, signifying the production of copper nanoparticles.The nanoparticles were centrifugation and dried at 40°C in an oven for 4 hours after being rinsed with deionized water.

Characterization of Nanoparticles:

UV–Vis spectrophotometry:

(T80+UV/VIS Spectrometer, PG Instrument Ltd., UK) was measured to determine the absorbance of Ag and CuNPs over an absorbance range of 300 to 800 nm for both NPs until no more absorbance changes were detected.

Transmission electron microscopy (TEM):

(Electron probe micro-analyzer JEOL – JXA 840A, Model Japan) images for both nanoparticles provide specialized information on morphological properties, such as shape and size. The samples were prepared by drop coating onto a copper grid that had been coated with carbon. After being vacuum dried, the samples were loaded into a specimen holder. Analysis of the ready grids resulted in the taking of TEM micrographs.

Tested Mosquitoes: Culex antennatus larvae was collected from Sanouras region, Fayoum governorate, Egypt (29^° 24^' 37.527 N, 30^° 52^' 25.075 E) and transferred into Medical Entomology Insectary, Animal house, Department of Zoology, Faculty of Science, Al-Azhar University, Cairofor colonization. Identified C. antennatus larvae reared for five generations using a standard rearing experimental procedure adopted by Hassan et al.²⁶.

Experimental Bioassay: Larvicidal activity of AgNPs and CuNPs were conducted according to procedures described by El-Mehdawyet al.²⁷ and Hassanain et al.²⁸ A 250 ml ofeach tested concentration were prepared using dechlorinated tape water. Then, twenty-five C. antennatus third larval instar were placed in each concentration until pupation. Lethal concentrations (LC₂₅, LC₅₀ and LC₇₅) were calculated and prepared for the next assays. Twenty-five C. antennatus 3^rdlarval instarwere treated with lethal concentrations (LC₂₅, LC₅₀ and LC₇₅) prepared in a volume of 250 ml using dechlorinated tape water. Larvae noted daily until adult emergence. Three sets of each treatment were used.

Females that emerged from treated larvae treated withlethal concentrations were subjected to reproductive potential test according to procedure of Shehata et al.²⁹.

Statistical analysis: To determine LC25, LC50, and LC75, larval mortality percentages were subjected to Probit analysis. One way ANOVA analysis was performed for comparing the differences between the activity of tested nanoparticles using Tucky’s HSD test at 5% probability level. All results recorded as Mean±SD. All data were evaluated using Statistical Package Social Science (SPSS) software version 11.5 (SPSS, 2007).

RESULTS AND DISCUSSION:

Biosynthesis of AgNPs and CuNPs by Lactobacillus reuteri supernatant:

The probiotic Lactobacillus reuteri demonstrated their capability for extracellular biosynthesis of AgNPs and CuNPswhen cell-free supernatant is used. Changing in the color of AgNO₃ and CuSO₄ solutions from yellow to reddish brown and dark greenish, respectively after incubation overnight at room temperature, was indicator for biosynthesis of AgNPs and CuNPs by Lactobacillus reuteri.

Characterization of silver and copper nanoparticles

UV–visible spectrophotometer analysis:

For AgNPs, the reaction mixture's nanoparticles' absorption spectra shows an absorption peak at 410–430 nm(Fig. 1A), the reaction time, demonstrating that the particles are disseminated in the aqueous solution and there is no sign of agglomeration, which confirms the biosynthesis of nanoparticles³⁰. This is thought to be the peak for AgNPs as previously reported by Sikder et al³¹. This proof that nanoparticles exhibit surface Plasmon resonance (SPR) and just one SPR band shows that they are spherical in shape.Furthermore, the findings were consistent with those of another investigation in which Lactobacillus species formed AgNPs with an absorbance of 410 nm^32,33. While proteins could potentially be present based on the peak at 292 nm³⁴. Furthermore, according to reports from other researchers, AgNPs' absorption spectra peak wavelengths range from 391-440 nm.

On the other hand, CuNPs exhibit an absorption peak at 270 nm in the absorption spectrum of the nanoparticles created in the reaction mixture (Fig. 1B).When the cell-free supernatant was added to the CuSO₄ solution, the color changed from yellow to a dark greenish to confirming the reduction to copper nanoparticles. Similar results were reported the absorption spectra peaks for Lactobacillus species CuNPs at 270 nm²⁵.

TEM analysis of nanoparticles:

The size and shape distribution of biologically produced AgNPs and CuNPs were studied using TEM. As showed in (Fig. 1C) silver nanoparticles had diameters ranging from 12 to 35 nm and average sizes of 24 nm, with different shapesbut the majority were spherical. The size and shape of silver nanoparticles have been measured in several investigations using TEM³⁵.The outcome of the TEM investigation is remarkably comparable to one from a previous study in which silver nanoparticles produced by Lactobacillus appeared to have some variation in shape and size, ranging from 2 to 20 nm³⁶.On the other hand, the spherical shape of the CuNPs was visible in the TEM picture (Fig. 1D), which had a 50 nm resolution power and was in the 9–21 nm size range.The presence of extracts on the surface of the Cu particles explained the rectangular patterns created by the nanoparticle overlap.Similar results from another work on CuNPs produced by Lactobacillus casei subsp. casei were obtained from the TEM analysis²⁵.

Fig. 1: (A&B) UV absorption spectrum of biosynthesized AgNPsandCuNPs, (C&D) TEM of AgNPswith 200 nm resolution power andCuNPs with 50 nm resolution power biosynthesized by Lactobacillus reuteri

Larvicidal activity of nanoparticles:

At the highest concentrations (500 and 400 µM), Culex antennatus larval mortality recorded 98.67, 86.67% and 89.33, 76.0% by AgNPs and CuNPs, respectively. Meanwhile, larval mortality percentage decreased to 17.33 and 14.67 at the lowest concentration (100 µM), compared with no larval mortality in control groups (Fig.2).

Fig.2: Activity of tested nanoparticles against Culex antennatus third larval instar. NPs1: Tested Nanoparticles 1; CuNPs: Tested Nanoparticles 2; Means followed by the same letters aren’t statistically significant (P>0.005).

Lethal concentration (LC₅₀) recorded 196.22 and 228.48 µMby AgNPs and CuNPs, respectively (Table 1).

Table 1: Relative effeciency of tested nanoparticles against Culex antennatus third larval instar.

Tested Nanoparticles

LC₂₅

LC₅₀

LC₇₅

χ²

AgNPs

118.30

(111.10-125.50)

196.22

(187.51-204.93)

325.53

(314.52-336.54)

1.11^N.S.

CuNPs

131.98

(126.72-137.24)

228.48

(219.39-237.57)

395.54

(379.31-411.77)

1.64^N.S.

LC: Lethal Concentration; Values between brackets represents 95% Confidence limits (lower CL - upper CL); N.S.; Non-significant (P>0.005).

In addition, lethal concentrations (LC₂₅, LC₅₀ and LC₇₅) of AgNPs and CuNPsaffected C. antennatus both larval and pupal times as compared with untreated groups. Larval time recorded 4.72 days in control group, increased to record 6.08 and 5.42 days bylethal concentration (LC₇₅) of AgNPs and CuNPs, respectively. Also, the highest pupal time (2.38 days) recorded by LC₇₅of AgNPs. All lethal concentrations of CuNPs were insignificantly (P>0.005) affected C. antennatus pupal time, compared with control group(Table 2).The growth index of C. antennatus larvae and pupae recorded 8.61 and 11.39 by LC₇₅of AgNPs and CuNPs, respectively, compared with 15.11 for control congers (Figure 3).

Table 2: Effect of tested nanoparticles on developmental times of Culex antennatus.

Tested Nanoparticles	LC (µM)	Larval time (Days)	Pupal time (Days)	Developmental time (Days)
AgNPs	25	5.67±0.16^c	2.01±0.10^a	7.58±0.24^c
	50	5.71±0.27^c	2.23±0.09^b	7.94±0.36^c
	75	6.08±0.13^d	2.38±0.11^c	8.46±0.23^d
CuNPs	25	5.16±0.56^a	1.88±0.13^a	7.04±0.68^a
	50	5.27±0.06^c	1.98±0.09^a	7.25±0.13^c
	75	5.42±0.06^c	2.04±0.13^a	7.46±0.15^c
Control	---	4.72±0.14^a	1.90±0.12^a	6.62±0.18^a

Means followed by the same letters aren’t statistically significant (P>0.005). See footnote of table 1.

Fig3: Growth index of Culex antennatus affected by lethal concentrations of tested nanoparticles. See capture of Figure 1.

On the other hand, AgNPs and CuNPssignificantly decreased the mean number of eggs laid by females resulted from treated larvae. At LC₇₅of AgNPs and CuNPs, number of eggs laid by females recorded 39.91 and 52.10 eggs/♀, respectively vs. 104.13 eggs/♀ for the control. Also, non-hatched eggs percentages increased by increasing the concentration of tested nanoparticles. non-hatched eggs recorded 35.20 and 28.64% at LC₂₅of tested AgNPs and CuNPs,respectively, compared with 5.07% for control (Table 3). The highest sterility index (91.87 and 78.21%) attained by LC₇₅of AgNPs and CuNPs, respectively (Figure 4).

Fig 4: Sterility index of Culex antennatus females resulted from larvae treated with tested nanoparticles. See capture of Figure 1.

Tested AgNPs and CuNPsshowed larvicidal activity against Culex antennatus third larval instardepending on concentration used. Generally, AgNPs was more effective against tested larvae than CuNPs. Findings of the present study confirm the previously reported by Kumar et al.³⁷ using silver nanoparticles synthesized by Excoecariaagallocha leaf extract against Aedes aegypti larvae, where LC₅₀ recorded 4.65 mg/L, Kumar et al.³⁸using silver nanoparticles synthesized by Holarrhena antidysenterica extract against A. aegypti and C. quinquefasciatus larvae, where LC₅₀ recorded 5.53 and 9.3 ppm after 72 hours of exposure and El-Mehdawyet al.²⁷using silver nanoparticles synthesized using MHB agar and chitosan against C.pipiens, where LC₅₀ recorded 279.33, 321.33 and 367.37 μM, respectively. In agreement with Arjunanet al.³⁹ and Subramaniam et al.⁴⁰the high activity of tested AgNPs and CuNPscan be attributed to the ability of tested nanoparticles to permeate the larval exoskeleton and reaching inner cells, where they restrict proteins and DNA, inducing changes in their structure and therefore their functions.

Also, increasing sterility index of females resulted from larvae treated with AgNPs and CuNPswas recorded. Effects of AgNPs and CuNPs on reproductivity of C. antennatus females may be due to corporal weakness of resulted females⁴¹, weak metabolic rate⁴²and interference of AgNPs and CuNPs with oogenesis process ⁴³.

CONCLUSION:

Overall, tested of Lactobacillus reuteri AgNPs biosynthesized was more effective against tested larvae, as well as reproductive potential of resulted females than CuNPs. Moreover, more studies on the activity of biosynthesized nanoparticles against other mosquito species are necessary to reduce the spread of diseases transmitted by these species.

CONFLICT OF INTEREST:

The authors state that they have no conflicts of interest.

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Received on 27.10.2022 Modified on 19.11.2022

Research J. Pharm. and Tech 2023; 16(6):2969-2974.

DOI: 10.52711/0974-360X.2023.00490

Tested Nanoparticles	LC (µM)	No. of tested females	Eggs Laid		Non-hatched Eggs
Tested Nanoparticles	LC (µM)	No. of tested females	Total	Mean±SD	Mean±SD	%±SD
AgNPs	25	18	1161	64.50±3.65^d	22.67±2.63^d	35.20±4.06
	50	13	687	52.85±2.19^d	22.69±2.46^d	42.92±4.08
	75	11	439	39.91±2.74^d	31.82±1.89^d	79.85±3.73
CuNPs	25	14	1128	80.57±2.68^c	23.07±3.29^c	28.64±4.04
	50	16	1188	74.25±2.18^d	26.06±2.08^d	35.13±2.93
	75	10	521	52.10±3.90^d	30.60±3.53^d	58.66±4.13
Control	---	15	1562	104.13±4.22^a	5.27±1.58^a	5.07±1.54