Swift synthesis of zinc oxide nanoparticles using unripe fruit extract of Pergularia daemia: An enhanced and eco-friendly control agent against Zika virus vector Aedes aegypti.

In this study Pergularia daemia unripe fruits were used to synthesize zinc oxide nanoparticles (Pd-ZnONPs). UV-vis Spectroscopy detected the production of ZnONPs. XRD, FTIR, SEM, and TEM studies were used to characterize the synthesized Pd-ZnONPs. Aedes aegypti (Ae. aegypti) third instar larvae were analyzed to diverse concentrations of Pd-unripe fruit extract and Pd-ZnONPs for 24 hours to assess the larvicidal effect. Mortality was also detected in Ae. aegypti larvae under laboratory conditions, with corresponding LC50 and LC90 values of 11.11 and 21.20 µg/ml respectively. As a result of this study, the levels of total proteins, esterases, acetylcholine esterase, and phosphatase enzymes in the third instar larvae of Ae. aegypti were significantly lower than the control. These findings suggest that Pd-ZnONPs could be used to suppress mosquito larval populations.

Immunological and antibiofilm property of haemocyanin purified from grooved tiger shrimp (Penaeus semisulcatus): An in vitro and in silico approach.

Haemocyanin (Hc) is a non-specific innate immune protein present in the haemolymph of arthropods and molluscs. In the current study, we characterized the structural and immunological properties of Hc from grooved tiger shrimp, Penaeus semisulcatus. Hc was isolated from the haemolymph of P. semisulcatus by gel filtration column chromatography using Sephadex G-100. High-performance liquid chromatography of the purified Hc emerged as a single peak through a retention time of 3.3 min demonstrating the homogeneity nature of the protein. X-ray diffraction analysis revealed a distinct peak at 31.7° indicating the crystalline character of the purified Hc. Circular dichroism spectra of the purified Hc displayed negative ellipticity bands close to 225 nm and 208 nm representing ß-sheet secondary structure. The purified Hc agglutinated sheep RBCs, yeast Saccharomyces cerevisiae and fungal Candida albicans. In addition, the purified Hc displayed antibacterial activity against Gram-positive bacteria (Bacillus thuringiensis and Bacillus pumilis) and Gram-negative bacteria (Vibrio parahaemolyticus and Vibrio alginolyticus) with a minimal inhibitory concentration of 50 µg/ml. Antibiofilm activity revealed the potential of purified Hc to inhibit biofilm formation of both Gram-positive and Gram-negative bacterial pathogens. Furthermore, live/dead staining of biofilms demonstrated the reduced viability of bacterial cells after exposure to the purified Hc. In silico molecular modeling was carried out using the sequence of Hc from SwissProt and molecular docking was performed with the cell surface components found in Gram-positive and Gram-negative bacteria. Overall our study demonstrates the involvement of Hc in the native immune reaction of P. semisulcatus by eliciting pathogen recognition. Thus, Hc could enhance disease resistance against pathogenic infection in shrimp aquaculture.

Facile synthesis of haemocyanin-capped zinc oxide nanoparticles: Effect on growth performance, digestive-enzyme activity, and immune responses of Penaeus semisulcatus.

ZnO nanoparticles (NPs) synthesized using haemocyanin (Hc-ZnONPs) purified from haemolymph of Penaeus semisulcatus were characterized using various techniques. HR-TEM and SEM microscopy indicated Hc-ZnONPs had a typical size of 20-50 nm and were spherical. The objective of current investigation was to assess the effects of dietary supplementation of Hc-ZnONPs on the development and activity of digestive and metabolic enzymes, as well as the antioxidant levels in P. semisulcatus. Trial basal diets were supplemented with Hc-ZnONPs at rates of 0, 10, 20, 40, 60, and 80 mg kg-1 (dry feed weight) and were fed to P. semisulcatus for 30 d. For 60 mg kg-1 Hc-ZnONPs-supplemented feed, significantly (P < 0.05) enhanced endurance, development, and activity of the digestive enzyme were observed. The enzymatic antioxidants and metabolic enzymes activities in the muscle exhibited no significant changes when 10-60 mg kg-1 Hc-ZnONPs-supplemented feed was fed to P. semisulcatus. Conversely, feeding the P. semisulcatus with 80 mg kg-1 Hc-ZnONPs produced a harmful outcome, with significant increase in the enzymatic antioxidants and metabolic enzymes. Consequently, 80 mg kg-1 Hc-ZnONPs was identified as lethal to P. semisulcatus. Hence, it is proposed that the diet of P. semisulcatus can be supplemented with up to 60 mg kg-1 Hc-ZnONPs for improving the endurance, development and immunity.

Enhanced antibacterial activity of hemocyanin purified from Portunus pelagicus hemolymph combined with silver nanoparticles - Intracellular uptake and mode of action.

Recently, biogenic nanoparticles have been considered promising candidates for manufacturing antibacterial nanodrugs. Here, we synthesized AgNPs using the crab-borne antibacterial agent hemocyanin and assessed the antibacterial action against several pathogenic bacteria. In this study, the crustacean immune protein hemocyanin (Pp-Hc, 78 kDa) purified from Portunus pelagicus hemolymph was used to fabricate silver nanoparticles. Characterization of hemocyanin-fabricated AgNPs (Pp-Hc AgNPs) were achieved using ultraviolet-visible spectrophotometer, X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), high-resolution-transmission electron microscopy (HR-TEM), and energy-dispersive X-ray spectroscopy. The antibacterial efficacy of AgNO3,Pp-Hc and Pp-Hc AgNPs was compared by growth inhibition, antibiofilm and live and dead analyses. Based on the results, Pp-Hc AgNPs was more efficient than Pp-Hc and AgNO3 against pathogenic bacteria. Mechanistic analysis revealed membrane damage and reactive oxygen species (ROS) generation, suggesting that Pp-Hc and Pp-Hc AgNPs rely to similar modes of action. Intracellular protein molecules and nucleic acid leakage confirmed that Pp-Hc AgNPs increase membrane permeability, leading to cell death. Based on our results, capping of the exterior surface of nanoparticles with antimicrobial crab-borne peptides, such as Pp-Hc, improves their functions as potential agents against bacterial diseases, which may be useful in clinical applications.

Searching for crab-borne antimicrobial peptides: Crustin from Portunus pelagicus triggers biofilm inhibition and immune responses of Artemia salina against GFP tagged Vibrio parahaemolyticus Dahv2.

Marine organisms represent a huge source of novel compounds for the development of effective antimicrobial drugs. The present study focus on the purification of the antimicrobial peptide crustin from the haemolymph of the blue swimmer crab, Portunus pelagicus, by blue Sepharose CL-6B matrix assisted affinity column chromatography. Crustin showed a single band with a molecular mass of 17 kDa in SDS-PAGE analysis. The XRD analysis exhibited peaks at 32° and 45° while a distinct peak with a retention time of 1.8 min resulted in high performance liquid chromatography (HPLC) pointing out the crystalline nature and purity of crustin, respectively. Crustin purified from P. pelagicus (Pp-Cru) showed immunological activities, triggering encapsulation, phagocytosis on Sepharose beads and yeast (Saccharomyces cerevisiae) respectively. Furthermore, encapsulation of GFP tagged V. parahaemolyticus in Artemia salina and challenging study were assessed under CLSM and the potential of Pp-Cru was examined in vivo. In addition, the growth reduction and biofilm inhibition potential of Pp-Cru on Staphylococcus aureus, Enterococcus faecalis (Gram- positive bacteria) and Pseudomonas aeruginosa, Escherichia coli (Gram-negative bacteria) was evidenced by inverted and confocal laser scanning microscopic analysis, revealing that 100 µg/ml of Pp-Cru can disrupt the biofilm matrix thereby the thickness of biofilm was significantly reduced. Overall, the present investigation might provide a sensitive platform to realize the significant function of Pp-Cru in crustacean immune mechanism as well as its potential to bacterial growth inhibitor. The functional properties of purified Pp-Cru antimicrobial peptide may lead to a superior understanding of innate immune response in P. pelagicus species, which suggest the promising application for drug development in aquaculture.

Sargassum wightii-synthesized ZnO nanoparticles - from antibacterial and insecticidal activity to immunostimulatory effects on the green tiger shrimp Penaeus semisulcatus.

The green synthesis of metal nanoparticles using phytochemical from marine seaweeds is a fast-growing research field in nanotechnology. Here, the biosynthesis of zinc oxide nanoparticles was achieved using the hot water extract of Sargassum wightii. The hot water extract prepared from S. wightii (H Sw) and ZnO NPs were studied by UV-visible and FTIR spectroscopy, SEM and XRD. Then, both products were evaluated for antibiofilm activity towards aquatic pathogens. The nanoparticles' immunostimulating potential on green tiger prawns, Penaeus semisulcatus was studied through immersion and dietary administration. Shrimp immune parameters (i.e., total hemocytes count (THC), respiratory bursts (RBs), phenoloxidase (PO) and superoxide dismutase (SOD) activity) were significantly affected by exposure or ingestion of ZnO nanoparticles. In addition, the hot water extract and ZnO nanoparticles had high antibiofilm activity against Gram-positive (B. subtilis, S. aureus) and Gram-negative (S. sonnei, P. aeruginosa) microbial pathogens. It was accomplished that the ZnO nanoparticles can be used as the bacteriostatic and immunostimulant agents through immersion and dietary administration enhancing immunity of green tiger shrimp. Furthermore, the toxicity effects of ZnO nanoparticles were 100% at 24 h on Aedes aegypti 3 rd instar larvae at the concentration of 100 µg/mL and the greatest efficacy was accomplished by H Sw ZnO NPs against the Ae. aegypti after 24 h (LC50 49.22; LC90 86.96 mg/mL), if compared to the seaweed extract alone. Morphological and histological damages triggered by nanoexposure were investigated.

Identification, characterization and immune response of prophenoloxidase from the blue swimmer crab Portunus pelagicus and its antibiofilm activity.

Prophenoloxidase is a conserved Cu-containing enzyme acting as a major defense molecule in the immune response of crustaceans. In the present research, we purified prophenoloxidase from the haemolymph of Portunus pelagicus (Pp-proPO) by Blue Sepharose CL-6B chromatography. Pp-proPO exhibited only one band with molecular weight of 75kDa on SDS-PAGE. The purified Pp-proPO was characterized through X-ray diffraction (XRD) and high-performance liquid chromatography (HPLC). Pp-proPO showed phagocytic activity on the yeast Saccharomyces cerevisiae as well as encapsulation on sepharose CL-6B beads associated with CM sepharose and beads of sodium alginate. Pp-proPO also led to strong agglutination on human erythrocytes. Furthermore, Pp-proPO showed magnified PO activity when altered with activated particles acting as pathogen combined molecular patterns (PAMPs), metal ions or other chemicals. Pp-proPO showed relevant antibiofilm activity on Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. Overall, the above results allowed us to claim that Pp-proPO play a key role in immune defense mechanisms of P. pelagicus crabs, in particular towards microbial pathogens; notably we added basic information to the functional characterization of Pp-proPO, as well as to understand its immunological role in crustaceans defense systems.

Swift fabrication of Ag nanostructures using a colloidal solution of Holostemma ada-kodien (Apocynaceae) - Antibiofilm potential, insecticidal activity against mosquitoes and non-target impact on water bugs.

Recent research in entomology and parasitology focused on the efficacy of green fabricated nanomaterials as novel insecticides. In this study, we synthesized poly-dispersed and stable silver nanoparticles (AgNPs) using the leaf extract of Holostemma ada-kodien. The nanostructures were characterized by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray, and X-ray diffraction analysis. The efficacy of H. ada-kodien leaf extract and AgNPs in vector control was evaluated against the mosquitoes Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus, which act as major vectors of important parasitic and arboviral diseases. AgNPs showed higher toxicity if compared to the H. ada-kodien leaf aqueous extract, LC50 towards larvae of A. stephensi, A. aegypti, and C. quinquefasciatus were 12.18, 13.30, and 14.70 µg/mL, respectively. When the AgNPs were tested on non-target water bugs, Diplonychus indicus, the LC50 value was 623.48 µg/mL. Furthermore, 100 µl/mL of AgNPs achieved significant antimicrobial activity against Bacillus pumilus, Enterococcus faecalis, Pseudomonas aeruginosa, Proteus vulgaris, and Candida albicans. Light and confocal laser scanning microscopy highlighted a major impact of the H. ada-kodien-synthesized AgNPs on the external topography and architecture of microbial biofilms, both on Gram-positive and Gram-negative bacteria. Overall, this study sheds light on the insecticidal and antibiofilm potential of H. ada-kodien-synthesized AgNPs, a potential green resource for the rapid synthesis of polydispersed and highly stable AgNPs.

Facile green synthesis of zinc oxide nanoparticles using Ulva lactuca seaweed extract and evaluation of their photocatalytic, antibiofilm and insecticidal activity.

The bioactivity of semiconductor nanocomplexes has been poorly studied in the field of pesticide science. In this research, the synthesis of zinc nanoparticles was accomplished through new effortless green chemistry process, using the Ulva lactuca seaweed extract as a reducing and capping agent. The production of U. lactuca-fabricated ZnO nanoparticles (Ul-ZnO Nps) was characterized by powder X-ray diffraction (XRD), UV-visible, Fourier transform infrared (FTIR) spectroscopy, selected area electron diffraction (SAED) analysis and transmission electron microscopy (TEM). The U. lactuca-fabricated ZnO NPs were tested for their photodegradative action against organic dyes, as well as for antibiofilm and larvicidal activities. The UV visible absorbance spectrum of Ul-ZnO NPs exhibited the absorbance band at 325nm and TEM highlighted average crystallite sizes of nanoparticles of 10-50nm. Methylene blue (MB) dye was efficiently corrupted under sunlight in presence of Ul-ZnO NPs. Excellent bactericidal activity was shown by the Ul-ZnO Nps on Gram positive (Bacillus licheniformis and Bacillus pumilis) and Gram negative (Escherichia coliand Proteus vulgaris) bacteria. High antibiofilm potential was noted under both dark and sunlight conditions. The impact of a single treatment with Ul-ZnO NPs on biofilm architecture was also analyzed by confocal laser scanning microscopy (CLSM) on both Gram positive and Gram negative bacteria. Moreover, Ul-ZnO NPs led to 100% mortality of Aedes aegypti fourth instar larvae at the concentration of 50µg/ml within 24h. The effects of ZnO nanoparticle-based treatment on mosquito larval morphology and histology were monitored. Overall, based on our results, we believe that the synthesis of multifunctional Ul-ZnO Nps using widely available seaweed products can be promoted as a potential eco-friendly option to chemical methods currently used for nanosynthesis of antimicrobials and insecticides.

Eco-friendly fabrication of Ag nanostructures using the seed extract of Pedalium murex, an ancient Indian medicinal plant: Histopathological effects on the Zika virus vector Aedes aegypti and inhibition of biofilm-forming pathogenic bacteria.

The control of Zika virus mosquito vectors and well as the development of drugs in the fight against biofilm-forming microbial pathogens, are timely and important challenges in current bionanoscience. Here we focused on the eco-friendly fabrication of Ag nanostructures using the seed extract of Pedalium murex, an ancient Indian medicinal plant. Initial confirmation of Ag nanoparticles (AgNPs) production was showed by a color change from transparent to dark brown. The UV-Visible spectrum (476nm), X-ray diffraction peaks (101, 200, 220 and 311) and Fourier transform infrared spectroscopy shed light on the production of green-capped AgNPs. Morphological structure analysis using HR-TEM showed that the AgNPs were mostly hexagonal in shape with rough edges, and a size of 20-30nm. The larvicidal potential of P. murex seed extract and AgNPs fabricated using the P. murex seed extract (Pm-AgNPs) was tested on fourth instar mosquito larvae of the Zika virus vector Aedes aegypti. Maximum efficacy was achieved by Pm-AgNPs against Ae. aegypti after 24h (LC50 34.88; LC90 64.56mg/ml), if compared to the P. murex seed extract. Histopathological analyses showed severe damages to the hindgut and larval muscles in NPs-treated Ae. aegypti larvae. The sub-MIC concentrations of Pm-AgNPs exhibited significant anti-biofilm activity against Gram positive (Enterococcus faecalis, Staphylococcus aureus) and Gram negative (Shigella sonnei, Pseudomonas aeruginosa) bacterial pathogens, as showed by EPS and MTP assays. Light and CLSM microscopic studies highlighted a significant impact of P. murex seed extract and Pm-synthesized AgNPs on the surface topography and architecture of bacterial biofilm, both in Gram positive and Gram negative species. Overall, results reported here contribute to the development of reliable large-scale protocols for the green fabrication of effective mosquito larvicides and biofilm inhibitors.

Toxicity of herbal extracts used in ethno-veterinary medicine and green-encapsulated ZnO nanoparticles against Aedes aegypti and microbial pathogens.

Dengue and chikungunya are arboviral diseases mainly vectored by the mosquito Aedes aegypti. Presently, there is no treatment for these viral diseases and their prevention is still based on vector control measures. Nanopesticides fabricated using herbal extracts as reducing and capping agents currently represent an excellent platform for pest control. In this scenario, the present study assessed the acute toxicity of seven plants employed in ethno-veterinary medicine of southern India, as well as the green synthesis of zinc oxide nanoparticles, on third-instar larvae of A. aegypti. Larvae were exposed to extracts of the seven plants obtained with solvents of different polarity (acetone, ethanol, petroleum ether, and water) for 24 h. Maximum efficacy was observed for Lobelia leschenaultiana leaf extracts prepared using all the four solvent extracts (LC50 = 22.83, 28.12, 32.61, and 36.85 mg/L, respectively). Therefore, this plant species was used for the synthesis and stabilization of ZnO nanoparticles based on its maximum efficacy against third-instar larvae of A. aegypti. L. leschenaultiana-encapsulated ZnO nanoparticles showed 100% mortality when tested at 10 mg/L, the LC50 was extremely low,  1.57 mg/L. Zinc acetate achieved only 65.33% when tested at 60 mg/L, with a LC50 of 51.62 mg/L. Additionally, ZnO nanoparticles inhibited growth of Pseudomonas aeruginosa, Proteus vulgaris, Shigella sonnei, and Vibrio parahaemolyticus and also inhibited biofilm formation on selected microbila pathogens, showing impact on EPS production and hydrophobicity. Overall, our results suggest that L. leschenaultiana-fabricated ZnO nanoparticles have a significant potential to control A. aegypti mosquitoes and Gram-negative bacterial pathogens.

Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties.

The present study reveals purification and characterization of immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus (Pp-Lec). The Pp-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 155 kDa in SDS-PAGE. The surface morphology of purified Pp-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 3.3 min was appeared in high performance liquid chromatography (HPLC) and X-ray diffraction (XRD) analysis expresses a single peak at 31.5° which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Pp-Lec exhibits encapsulation activity against sepharose beads and yeast agglutination activity against Saccharomyces cerevisiae. Moreover, the purified Pp-Lec has the ability to agglutinates with the human erythrocytes among tested and which was observed by light microscopy. In addition, purified Pp-Lec showed the broad spectrum of antibacterial activity against Gram-positive Bacillus pumulis, Bacillus thuringiensis, Enterococcus faecalis and Gram negative Citrobacter amalonaticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter murliniae, Citrobacter freundii, Morganella morganii. Antibiofilm potential of purified Pp-Lec against selective Gram-negative bacteria showed the disruption of biofilm architecture at the concentration of 50 µg ml-1.

Haemolytic and antibiofilm properties of haemocyanin purified from the haemolymph of Indian white shrimp Fenneropenaeus indicus.

Haemocyanin (Hc) is an important non-specific immune macromolecule present in the haemolymph of both mollusks and crustaceans. In the present study, Hc was purified from the haemolymph of Indian white shrimp Fenneropenaeus indicus by gel filtration chromatography and it exhibits a single band with a molecular weight of 74 kDa on SDS-PAGE. The X-ray diffraction (XRD) and High performance liquid chromatography (HPLC) result of purified Hc express single peak at 31.5° be a sign of crystalline nature and appear as a single peak with a retention time of 5.6 min signify the homogeneity nature of the protein respectively. The purified Hc exhibited haemolytic activity against chicken erythrocytes. The haemolytic activity of purified Hc in optimum conditions observed to be pH 6.0, temperature 40 °C, in the presence of calcium. As well purified Hc exhibited the antibiofilm activity against both Gram positive and Gram negative bacteria. Moreover, the haemolysis can be inhibited to different degrees by osmoprotectants of diverse molecular masses, signifying that it follows a colloid-osmotic mechanism. This study conclude that purified Hc from F. indicus remarkably possess haemolytic and antibiofilm activity.

Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity.

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tiger shrimp, Penaeus semisulcatus by gel fermentation chromatography using blue Sepharose matrix. The two purified prophenoloxidase macromolecules are of about 76 and 75 kDa determined through SDS-PAGE and named as Penaeus semisulcatus prophenoloxidase I (PSproPO I) and Penaeus semisulcatus prophenoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography (HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre against human erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activity against yeast Saccharomyces cerevisiae and encapsulation activity against Sepharose CL 6B beads compared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I and PSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogen associated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containing PSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria. The above results concluded that no significant differences were found between the purified PSproPO I and PSproPO II immune indices and functions. This study might provide a sensitive platform to understand more about the critical roles of PSproPO I and PSproPO II in crustacean immune system.