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

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

Ramachandran Ishwarya, Baskaralingam Vaseeharan, Suganya Subbaiah, Abdul Khudus Nazar, Marimuthu Govindarajan, Naiyf S. Alharbi, Shine Kadaikunnan, Jamal M. Khaled, Mohammed N. Al-anbr PII: DOI: Reference:

S1011-1344(18)30351-8 doi:10.1016/j.jphotobiol.2018.04.049 JPB 11232

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

31 March 2018 19 April 2018 30 April 2018

Please cite this article as: Ramachandran Ishwarya, Baskaralingam Vaseeharan, Suganya Subbaiah, Abdul Khudus Nazar, Marimuthu Govindarajan, Naiyf S. Alharbi, Shine Kadaikunnan, Jamal M. Khaled, Mohammed N. Al-anbr , Sargassum wightii-synthesized ZnO nanoparticles – from antibacterial and insecticidal activity to immunostimulatory effects on the green tiger shrimp Penaeus semisulcatus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jpb(2018), doi:10.1016/j.jphotobiol.2018.04.049

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

Ramachandran Ishwarya1 , Baskaralingam Vaseeharan*, Suganya subbaiah1 , Abdul Khudus

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Nazar2 , Marimuthu Govindarajan3,4 , Naiyf S. Alharbi5 , Shine Kadaikunnan5 , Jamal M. Khaled5 ,

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1

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Mohammed N. Al-anbr5

Nanobiosciences and Nanopharmacology Division, Biomaterials and Biotechnology in Animal

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Health Lab, Department of Animal Health and Management, Alagappa University, Science

2

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Block 6th floor, Burma colony, Karaikudi 630004, Tamil Nadu, India Mandapam Regional Centre, Central Marine Fisheries Research Institute, Mandapam, Tamil

Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology,

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3

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Nadu, India

Annamalai University, Annamalainagar 608 002, Tamil Nadu, India Department of Zoology, Government College for Women, Kumbakonam 612 001, Tamil Nadu,

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5

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India

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh

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11451, Saudi Arabia

* Corresponding Author. BaskaralingamVaseeharan. Tel: +91 4565 225682. Fax: +91 4565 225202. E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract The green synthesis of metal nanoparticles using phytochemical from marine seaweeds is a fastgrowing research field in nanotechnology. Here, the Bio synthesis of Zinc nanoparticles was achieved via the hot water extracts of Sargassum wightii. The hot water extract prepared from S.

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wightii (H Sw) and the ZnO NPs were distinguished by UV-visible and FTIR spectroscopy, SEM

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and XRD. Then, the nanomaterial both were evaluated for antibiofilm activity towards aquatic

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pathogens. The nanoparticles’ immunostimulating potential on green tiger prawns, Penaeus semisulcatus was studied through immersion and dietary administration. Shrimp immune

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parameters (i.e., total hemocytes count (THC), respiratory bursts (RBs), phenoloxidase (PO) and

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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

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activity against Gram-positive B. subtilis, S. aureus and Gram-negative (S. sonnei, P.

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aeruginosa) pathogens. It was accomplished that the H Sw ZnO NPs able to be used as the bacteriostat and immunostimulants for prawn through immersion and administration to enhance

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immunity of green tiger shrimp. Furthermore, the toxicity effects of H Sw ZnO NPs were 100%

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at 24 h in the 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 (LC 50 49.22;

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LC90 86.96 mg/ml), if compared to the H Sw.

Keywords: crustaceans; antibiofilm activity; dengue; larvicides; microbial pathogens; Zika virus

ACCEPTED MANUSCRIPT 1. Introduction Invertebrates like crustaceans encloses a native immune system, characterized by lack of immunoglobulin and memory, although rely on innate immunity to protect themselves from all intruding pathogens and other outside issues that frequently intimidate their life. Haemocytes

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take part in the significant responsibility in the host immune reaction and performing functions

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like, formation of nodules, cytotoxicity mediation, phagocytosis and encapsulation [1]. The

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Penaeus semisulcatus is an Indo-Pacific species spread across the global ledge in the Eastern Mediterranean Sea, West Pacific Ocean, Indian Ocean and is one of the majority significant

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profitable species in this part of the world [2]. This shrimp supports the main beneficial and

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commercially essential fisheries in crustacean aquaculture. One of the most important restraints in shrimp farming is infection caused via contagious pathogens along with virus and bacteria are

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extremely significant and dependable for cruel commercial loss in aquaculture industry universal

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[3]. On the way to evade the economic losses, antibiotics are regularly used in aquaculture farming, to avoid or care for disease eruptions, and the normal administration because

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supplements in shrimp food or in baths and vaccinations is a regularly used approach as

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prophylactics, therapeutics, or growth supporters [4]. Furthermore, the constant application of composites like antibiotics has been related with the expansion of drug-resistant bacteria together

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interior and exterior of aquaculture amenities [5]. Therefore, aquaculture disease management must focus on lifelong and eco friendly methods. In recent times, rising awareness is being compensated to utilize the plant compounds for infection management in aquaculture as another to chemical treatment [6]. Plant products have been described to motivate hunger and support increasing the weight, to act as Immunostimulants rising the immunocompetency and infection

ACCEPTED MANUSCRIPT resistance through improving together specific and non-specific defense mechanisms in crustacean aquaculture [7]. In fish and crustaceans, the immune stimulatory properties of immunostimulants such as polysaccharides, chitosan and glucan have been broadly reported in the review by Sakai et al.

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and Ringo et al. [8,9]. The consequence of plant mediated products on native and adaptive

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immune reaction and to avoid and manage crustacean’s diseases was evaluated by Harikrishnan

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et al.[10]. Seaweeds are used as nourish for fish and further marine animals and as well as the basis of elevated value agar, food for human being expenditure, and pharmaceutical components

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[11]. Many studies focused on the management of hot water extracts from different seaweeds

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recover the shrimp’s immune action and resistance against diseases. Hot-water extracts from numerous marine seaweeds like, Porphyra yezoensis, Gloiopeltis furcata, Gracilaria

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tenuistipitata, Gelidium amansii, Gloiopeltis complanata, Hypenea charoides, Chondrus

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ocellatus, Undaria pinnatifida, Sargassum autunnale, Lessonia nigrescens and Sargassum duplicatum.

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Mosquitoes act as key vectors for the transmission of dengue, malaria, yellow fever, Zika

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virus and other important infectious diseases of public health relevance [12 -18]. The current revival of these diseases is owing to superior number of breeding spaces in nowadays dispose of

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the world. Further, the random use of synthetic insecticides are well recognized for their speedy action except a major disadvantage in their application is, they are non-selective and might be dangerous to non-target species [19-22]. All these limitations on the usage of artificial pesticides have motivated researches for an environmentally safe, degradable and target specific insecticides against mosquitoes [23-32]. Moderately a lot of research has been performed in higher plants on their purely active material with toxic and repellent properties against arthropod

ACCEPTED MANUSCRIPT vectors [33-36] and only some attentions were focused on the larvicidal properties of the marine seaweeds [37-40]. Recently, the increase of efficient green chemistry methods for synthesis of nanoparticles has become a focus of researchers. The nanoparticles are used in therapeutic, agricultural, and pharmaceutical industries has acquired a huge deal of interest, with a focus on

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growth of more suitable methods using green biotechnology tools for production of eco-friendly,

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nontoxic and environmentally kind nanoparticles [41,42].

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Sargassum wightii is most significant varieties existing to the group Sargassum and broad array of bioactive compounds have been described as this seaweed [43]. This seaweed

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extensively dispersed on the southern coastline of Tamil Nadu, India and used for aquatic animal

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nourish, food elements and nourishment. It is a perfect goal for examine the occurrence of biomolecules for different biomedical and potential sources of an anti-bacterial agent and

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Immunostimulants. Therefore, the current study was designed to investigate the immune

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response of green tiger shrimp P. semisulcatus treatment with the hot-water extract of S. wightii and H Sw - ZnO NPs. The shrimp that were absorbed in seawater and fed diets encompassing

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hot-water extract of S. wightii and H Sw - ZnO NPs were observed for immune parameters.

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Ae. Aegypti.

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Furthermore, the synthesized H Sw - ZnO NPs found to produce a high larvicidal activity against

2. Materials and methods 2.1 Collection and Preparation of hot water extract of S. wightii Fresh S. wightii were collected from intertidal rocky seashore area in Mandapam, Tamilnadu, India. Seaweeds were rinsed thoroughly with d H2 O and dehydrated at 37 °C and then lyophilised and powdered. Fine seaweed powder (10 g) were weighed and shifted into of

ACCEPTED MANUSCRIPT conical flask and added into 50 ml of d H2 O then heated for 20 min. After, seaweed extract was filtered through Whatman No.1 filter paper and stocked in refrigerator at 4 °C for additional use.

2.2 Bacterial strains

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The bacterial activity of H Sw and H Sw ZnO NPs against both Gram positive Bacillus

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subtilis (KT763078), Staphylococcus aureus (MTCC) and Gram negative Shigella sonnei

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(K7769531), Pseudomonas aeruginosa (HQ693274) were used. Bacteria were grown in a test

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tube containing Nutrient broth and maintained in a shaker for 24 hrs at 37 °C.

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2.3 Biosynthesis of zinc nanoparticles

The warm water extract of S. wightii (5 ml) was added to the 1 mM zinc acetate 95 ml of

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aqueous solution. Then the reaction solution was placed under magnetic stirrer at 3-4 h for 70 °C

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with constant stirring. After, the solution was centrifuged at 4000 rpm for 10 min the white pellet

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was collected and washed by distilled water.

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2.4 Characterization of biosynthesized hot water extracts of S. wightii and H Sw - ZnO NPs The synthesized H Sw extract and HSw - ZnO NPs were characterized for their structure

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and morphology by XRD and SEM analysis. In XRD analysis, the powdered samples were recorded by Powder X- Ray Diffractometer (X’ Pert Pro- PAnalytic) Shimazhu cooperation, Nakagyo, ku, Japan and SEM images of the samples were taken by FE-SEM (Quanta FEG 250).The spectral studies of H Sw extract and HSw - ZnO NPs was carried out by determining the optical density (OD) via scanning UV-Vis spectrophotometer worked at a resolution of 1 nm

ACCEPTED MANUSCRIPT between 280 to 800 nm and the chemical structure and functional groups was observed by using FTIR spectrometer (Thermo scientific Nicolet – ISS) USA.

2.5 Experimental design

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Green tiger shrimp Penaeus semisulcatus were harvested from the CMFRI, Mandapam,

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Tamilnadu, India and the shrimps were reared at the Department of Animal Health and

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Management laboratory, Alagappa University, Karaikudi. Shrimp were located in fiberglass tanks for two weeks prior to the experiment. Throughout the acclimation time, the shrimp were

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fed twice every day by commercial shrimp diet. There were two analysis, an immersion and

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dietary administration for immune parameters studies carried out for three triplicates. The immune parameters like THC, PO, RB and SOD activity, test and control groups included 8

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shrimps every one. The shrimp ranged as of 9.5 g to 15.4 g, average 12.5 ± 3.2 g through not

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significant range variation amongst the treatments for the immersion and dietary administration

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analysis.

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2.6 The immune parameters of P. semisulcatus that received hot-water extract of S. wightii and H Sw - ZnO NPs

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The immune parameters of P. semisulcatus that received hot-water extract of S. wightii and H Sw - ZnO NPs experiments followed the method by Fu et al. (44). In brief, there are 4 concentrations (control, 200, 400 & 600 mg l-1 ) and three exposure times (1, 3 & 5 h) are used in the immersion test. Every treatment set was immersed in twenty liters of seawater having hot-water extract of S. wightii and H Sw - ZnO NPs at 200, 400 & 600 mg l-1 , respectively. Four

ACCEPTED MANUSCRIPT treatments (control, 0.5, 1.0 & 2.0 g kg-1 ) with four sampling period (1, 3, 5 & 7 days) were used for the diet administration studies.

2.7 Measurements of immune parameters

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2.7.1 Total haemocytes count

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Hot-water extract and H Sw - ZnO NPs treated shrimps were sampled after 1, 3 and 5 h

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in the immersion test and fed diet having hot-water extract and H Sw - ZnO NPs after 1, 3, 5 and 7 days were used for the assays. Haemolymph (200 µl) was separately collected from the

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ventral sinus of all shrimp via 2-ml sterilized syringe and placed in a tube having anticoagulant

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solution. The haemolymph (one drop) was positioned in a haemocytometer to calculate total

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haemocyte count, by an inverted phase-contrast microscope (Nikon Eclipse TS 100, Japan).

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2.7.2 Phenoloxidase activity

Phenoloxidase activity was performed through the method of Hernandez-Lopez et al. [45]

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with slight modifications. The haemolymph was centrifuge at 800 x g at 4 °C for 15 min. Then,

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centrifugation, the pellet washed quietly in 1 ml cacodylate-citrate buffer and centrifuged another time. 100 µl of aliquot was incubate for 15 min at 27 °C among trypsin (50 µl) and L-DOPA

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were added, then pursued cacodylate buffer (800 µl) in 10 min later. The PO activity was calculated the OD at 490 nm by spectrophotometer (Model U-1800, Shimadzu) and uttered as dopachrome creation in fifty µl of haemolymph.

2.7.3 Respiratory burst (RB) activity

ACCEPTED MANUSCRIPT The RB of haemocytes was measured by the following method of Su et al. [46] with minor changes. In brief, 100 µl of haemolymph was kept in triplicate in a micro titre plate (96well plate) before layered with poly-lysine solution (800 µl) added to develop cell adhesion. The OD at 630 nm was calculated in triplicate by microplate reader. RB was uttered as NBT-

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reduction for each ten µl haemolymph.

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2.7.4 Superoxide dismutase activity

The SOD activity of haemocytes was measured by the following procedure of Liu et al.

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[47]. Briefly, the diluted haemolymph (1000 µl) was centrifuged at 900 x g and 4 °C for 15 min.

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1 unit of Superoxide dismutase was definite as the quantity needed inhibiting the rate of xanthine

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decrease via 50% and exact activity was uttered as SOD unit ml-1 .

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2.8 Anti biofilm activity of hot-water extract of S. wightii and H Sw - ZnO NPs 1 mL of the bacterial culture were distributed into the 24-well micro titre plate with glass

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pieces (13 X 13 mm) along with presence and absence hot-water extract of S. wightii and H Sw -

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ZnO NPs (50 and 100 mg mL-1 ). The plates were incubating the room temperature at 12 h in flat condition. After, the glass pieces were washed by 0.01M phosphate buffer saline (PBS) and 0.4%

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crystal violet dye was used to stain the glass pieces and showed below light microscope at 40X magnification. Also for CLSM analysis, glass pieces in the nonexistence and existence of hotwater extract of S. wightii and H Sw - ZnO NPs the 0.4% of acridine orange is used to stain the glass pieces for 1min.

2.9 Bacterial viability assay

ACCEPTED MANUSCRIPT A live/dead Bac Light bacterial viability assay was carried out using kit L-7012, Invitrogen. At first 1.0 × 105 CFU/mL the overnight cultures of bacterial strains were inoculated to every well of a 24-well plate. The 24-well plate were treated with hot-water extract of S. wightii and H Sw - ZnO NPs at 100 and 50 µg/mL and incubated at 37 °C for 1 h. Then the cells

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were centrifuge at 5000 x g for 15 min and the pellet was rinsed with PBS buffer. After the cells

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were stained with two different dye SYTO ® 9 (3 mM) and propidium iodide (20 mM), at 37 °C

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in the dark for 20 min and the treated cells were observed by CLSM microscopy.

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2.10 Mosquito larvicidal efficacy

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Early third instars of Ae. aegypti were reared following the method by Govindarajan et al. [16]. Larvicidal bioassays testing hot water extract, Zinc acetate and H Sw - ZnO NPs toxicity

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against Ae. aegypti were carried out. For every assay, 20 larvae were introduced into 250 mL of

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water, the control was set up with dechlorinated tap water. Different concentrations (50, 100, 150, 200 and 250 µg/ml) of hot water extract and H Sw - ZnO NPs (20, 40, 60, 80 and 100

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µg/ml) were tested. Mortality was evaluated following 24 h of exposure, every assay was

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performed in five replicates.

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2.10.1 Histological and microscopy studies For histopathalogical analysis, Ae. aegypti larvae treated with hot water extract and H Sw - ZnO NPs were fixed and mounted in paraffin blocks. Larval tissue was sliced with 8 µm thickness using glass knives in a rotary microtome. The sections were mounted on glass slides and stained with haemotoxylin and eosin. After that, larval tissues and the NP accumulation was observed using a stereomicroscope (Nikon SMZ 745T Japan).

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3. Results 3.1 Characterization of H Sw-ZnONPs

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3.1.1 UV–visible spectroscopy

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The formation of ZnO NPs was established from the visual consideration. The hot water

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extract of S. wightii without zinc acetate has no color alteration. The absorbance spectra of S. wightii hot water extract at different wave length range from 250- 400 nm and the ZnO NPs

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synthesized from hot water extract of S. wightii were executed after 24 at 150 °C. H Sw - ZnO

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NPs shows strong UV absorption spectra shows peak absorbance at 327 nm elucidates the

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fundamental band gap absorption of ZnO crystals (Fig 1a).

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3.1.2 X-ray diffraction (XRD)

The XRD pattern exposes the formation of H Sw - ZnO NPs, which aspects to the

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crystalline nature of nanoparticles (Fig 1b). The representative XRD model of hot water extract

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of S. wightii in the array of 20 – 80° at a scanning step of 0.01. Several Bragg reflections with 2θ values of 27.8° and 45.0° observed matching to (111) and (222) planes respectively. A number

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of Bragg reflections with 2θ values of 31.6°, 45.4°, 56.3°and 75.2° are observed consequent to (100),(102),(110),(220) planes (JCPDS card No 77- 2414).

3.1.3 Functional groups being involved in H Sw-ZnO NPs synthesis FTIR spectral analysis was carried out to recognize the potential biomolecules in the S. wightii hot water extract. FTIR spectra of dried hot-water extract of S. wightii and H Sw - ZnO

ACCEPTED MANUSCRIPT NPs are shown in Fig. 1c. In S. wightii hot water extract peaks are observed at 444, 1028, 1659, 2344 and 3393 cm-1 respectively. After reaction with Zinc acetate, the peaks are shifted to a superior wave number side, such as 705, 830, 1039, 1713, 1789, 2532, 2924, 3418 and 3584 cm1. The broad absorption band is observed in 3584 and 3418cm-1 can be consigning to H- bonded

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in alcohol or phenol groups. The peaks at 2924 cm-1 can be allotted to –C-H alkene groups. The

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strong peak observed in 1789 and 1713cm-1 allotted to C=O stretching in aromatic groups. The

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peaks between 2352 and 2287 cm-1 equivalent to the O-H alcohol group. The peaks at 1538 and 830 cm-1 are consequent to Zn-O stretching and deformation vibration, correspondingly. FTIR

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spectra visibly specify O– H stretching which is more dependable for the formation of H Sw -

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3.1.4 Scanning electron microscopy

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ZnO NPs since it is greatly shifted.

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The SEM images of H Sw and H Sw - ZnO NPs were recorded and are shown in figure 2. The H Sw seems to be further widespread surface area, though the definite surface area of the

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seaweed biomass cannot be calculated as their small specific surface area (Fig. 2a &b).

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Moreover, by contrasting SEM pictures in Fig. 2c &d with that in Fig. 2a &b, found that the morphology of H Sw surface has scarcely altered still after exposing Zinc nanoparticles. Also,

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the distinct alter to the surface morphology on hot-water extract of S. wightii was observed after adsorption of metals. According to the H Sw - ZnO NPs, the superior overstated SEM image obviously reveals the incidence of spherical and uniform ZnO NPs with size range among 40 and 50 nm (Fig. 2c). The lower magnified image illustrates the occurrence of coarse ZnO nanostructures with approximate morphology, which are made up of mixture of numerous spherical ZnO NPs (Fig. 2d).

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3.2 The immune parameters of P. semisulcatus that received H Sw and H Sw - ZnO NPs The THC of P. semisulcatus treated with H Sw at 400 and 600 mg l -1 was considerably superior to the control following 5 h in immersion methods (Fig.3a) and treated in H Sw -ZnO

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NPs at 200 and 400 mg l -1 was considerably superior to control shrimp after 3 and 5 h in

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immersion method (Fig. 3b). In addition, the THC of P. semisulcatus treated with hot-water

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extract at 2.0 g kg-1 was appreciably superior to the P. semisulcatus fed diets method following 5 days and 1.0 g kg -1 was considerably superior to the P. semisulcatus that fed diets control

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following 7 days (Fig. 3c). H Sw -ZnO NPs at 2.0 g kg-1 was considerably superior to shrimp fed

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diets control after 3 days and diet containing H Sw -ZnO NPs at 1.0 g kg -1 was considerably superior to the P. semisulcatus that fed diets the control after 5 and 7 days in fed diet method

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(Fig. 3d).

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The phenoloxidase activity of P. semisulcatus treated with H Sw at 400 and 600 mg l -1 was considerably superior to 200 mg l

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and that control following 5 h in immersion method

was considerably superior to 200 mg l -1 , and that control following 1, 3 and 5 h (Fig.

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-1

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(Fig. 4a).The effect of immersed H Sw -ZnO NPs, the PO activity of shrimp at 400 and 600 mg l

4b).The Phenoloxidase activity of P. semisulcatus fed diets having superior to and H Sw -ZnO

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NPs at 0.5, 1.0 & 2.0 g kg -1 was appreciably superior to P. semisulcatus fed diets control following 5-7 and 3-7 days respectively (Fig. 4c & d). Respiratory burst of P. semisulcatus were immersed in H Sw on 600 mg l -1 was considerably superior to the control P. semisulcatus after 3 and 5 h (Fig. 5a). The effects of immersed H Sw -ZnO NPs, the respiratory burst activity of shrimp 200, 400 and 600 mg l

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was

considerably superior to the control following 1, 3 and 5 h (Fig. 5b). The RB of P. semisulcatus

ACCEPTED MANUSCRIPT fed diets having H Sw at 1.0 and 2.0 g kg -1 was considerably superior to the control diet after 5 and 7 days (Fig. 5c). RB of P. semisulcatus fed diets having H Sw at 0.5, 1.0 and 2.0 g kg -1 was considerably superior to shrimp that control diet following 3, 5 and 7 days(Fig. 5d). In Superoxide dismutase activity there is no o significant difference was observed for P.

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semisulcatus between four diets at H Sw -ZnO NPs the beginning and after 1-5 days. Conversely,

Microscopic observations of hot-water extract of S. wightii and H Sw - ZnO NPs

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3.3

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superior to control shrimp after 7 days (Fig. 6a & b).

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Superoxide dismutase activity of shrimp fed diets having 0.5, 1.0 and 2.0 g kg-1 was considerably

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The light and CLSM images provided visual observations of hot-water extract of S. wightii and H Sw - ZnO NPs biofilm formed Gram positive and negative bacteria was apparent

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on exposure to (Fig. 7&8). Further, dense and compressed biofilm structure was showed in

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untreated sample. The H Sw - ZnO NPs efficiently reduce biofilm formation in the test bacterial pathogens lacking kill the cells while contrasted to the hot-water extract of S. wightii. Moreover,

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CLSM results obviously exposed that the architecture of the biofilms was fewer thick and thinner

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when contrasted with that of untreated control (Fig. 7). The microscopic images prove that the H Sw - ZnO NPs achieves entire dispersal of biofilm architecture by loosening the microcolonies in

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the treated sample (Fig. 7&8).

3.4 Live and dead cell assay The antibacterial efficacy of the hot-water extract of S. wightii as well as the H Sw - ZnO NPs was additionally established by confocal microscopy analysis with Bac Light fluorescent staining (Fig. 8). The green fluorescence point out the survive bacterial cells, whereas the red one

ACCEPTED MANUSCRIPT represent departed bacterial cells. The control samples had a huge amount of survive cells, even as the hot-water extract of S. wightii treated bacterial cells had approximately the same quantities of survive and departed bacterial cells (Fig. 9). However, the H Sw - ZnO NPs treated pathogenic bacteria demonstrated superior amounts of departed bacteria, highlighting the extremely efficient

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bactericidal property of zinc nanoparticles.

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3.5 Mosquito larvicidal activity

The larvicidal activity of different concentrations of hot water extract and H Sw - ZnO

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NPs was tested on third instar larvae of Ae. aegypti mosquitoes, evaluating their efficiency

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following 24 h of exposure. The LC 50 and LC90 along with 95% upper and lower confidence limit values and regression equations for the various tested larvicides are presented in Table 1.

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The hot water extract samples were less toxic over H Sw - ZnO NPs against Ae. aegypti. The

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LC50 and LC90 values of hot water was LC 50 = 130.26; LC90 = 224.69 mg/L and LC 50 and LC90

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values of H Sw - ZnO NPs on larvae of Ae. aegypti were LC50 = 49.22; LC90 = 86.96 mg/L.

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3.5.1 Mosquito histology and microscopy studies The stereomicroscopic observations of third instar larvae of Ae. aegypti treated with hot

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water extract (Fig. 10a) showed the loss of upper hair, lower head hair, antennal hair and lateral hair. In addition, the histological images of Ae. aegypti larvae exposed to hot water extract showed that the larval epithelium was damaged and cells were vacuolated; the hindgut was affected and the epithelium appeared highly vacuolated with damaged intercellular membranes, but still enclosed the cell nuclei (Fig. 10b). Conversely, larvae exposed to H Sw - ZnO NPs,

ACCEPTED MANUSCRIPT rigorous damages to the anal papillae, distorted body and the treated larvae were darkening and shrunken the outer layer along with destructive structure at the stigmal plate on the siphon apex.

4. Discussion

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The hot water extracts of several seaweeds can manipulate the range of antibacterial and

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immunomodulatory properties in aquaculture, and in consideration of the efficiency of

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extraction, a superior competence of extracting secondary bioactive metabolites from seaweeds. Among, the hot water extracts from numerous seaweeds have been broadly considered to enlarge

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the endurance of shellfish against pathogen, and enlarge the non-specific immune system.

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Numerous researches described the use of immunostimulants in marine seaweeds to improve immunity and resistance to pathogens, like the hot water extracts of Gloiopeltis complanata,

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Hypenea charoides and are enlarged immunostimulant activity of common carp Cyprinus carpio,

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and yellowtail Seriola quinqueradiata [48]. Gelidium amansii [44], Gracilaria tenuistipitata [49] and Sargassum duplicatum [50] are improved the immune capacity of white shrimp

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Lithopenaeus vannamei. In the current study, the H Sw and H Sw - ZnO NPs are enlarged the

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THC, PO, RB and SOD activity of P. semisulcatus through immersion and dietary administrations. A previous study reports that the hot water extract of Gracilaria tenuistipitata,

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Gelidium amansii and Sargassum duplicatum is also increased the level of THC, phenoloxidase activity and respiratory burst in the green tiger shrimp P. semisulcatus. To evaluate the cellular immunity in shrimp, the haemogram, radical oxygen intermediates created through post-phagocytic action and PO activity are considered possible markers [51]. In crustaceans, the THC, PO activity, RBs, SOD were used as displays in many studies to evaluate the potential effect of extracts on innate immunity [52-54]. Haemocytes of crustaceans take part

ACCEPTED MANUSCRIPT in significant functions in regulating physiological functions and immune defence system [55]. PO is the active enzyme of the proPO and performs as together appreciation and effectors constituent to encourage cell-to-cell communication and consequently eradicate pathogens [56]. The stimulated PO produced superior cytotoxic quinines can inactivate viral pathogens [57].

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SOD, an antioxidative enzyme, performing significant position in scavenging superoxide anions

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in crustaceans [58]. Reactive oxygen intermediates are released through RBs of phagocytosis,

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which is corresponding to a defence mechanism aligned with microbial infection [59]. Conversely, the extreme accretion of ROIs is particularly toxic to host cells. In a usual

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physiological condition, injurious effect of ROIs are efficiently defused with the antioxidant

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defence method of organisms, which in common encompass enzymes similar to various peroxidases, catalase, superoxide dismutases (SODs) and minute antioxidant molecules [52]. In

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the current study, P. semisulcatus that received H Sw through immersion at 600 mg l-1 following

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5 h, fed diet method at 2.0 g kg-1 following 6 days at rest continued a considerably superior immune parameters representing a persevere effect of the H Sw. However, the THC, PO activity,

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RB, SOD activity of green tiger shrimp that had received the H Sw - ZnO NPs at 200 mg l-1 after

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3h in immersion test and fed diet method at 1.0 g kg-1 after 3 days of treatment. Based on the results the addition of H Sw - ZnO NPs in the diet of P. semisulcatus, improved the immune

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responses by raising its THC, RBs, and PO, SOD activities when compared to the hot water extracts of S. wightii treated P. semisulcatus. As said by the approximates of the Centers for Disease Control (CDC) and the National Institute of Health (NIH), 65%–80% [60,61] of all bacterial infections worldwide are linked with biofilms [62]. The incidence of biofilm- mediated infections, the recognition of new composites by the capability to reduce bacterial colonization and biofilm formation is of critical significance.

ACCEPTED MANUSCRIPT In this regards biodegradable antibiofilm products from the natural origin of together global and oceanic environment. Research in the direction of enhanced sympathetic of prawn defense factors is a potential induction mechanism, influence prawn health, and struggle to disease, other than the information achieved has not up till now lead to some common conclusions. The natural

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immunostimulants are used in fish and shrimp culture to avoid diseases is a capable novel

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development [63-66] which are biodegradable, biocompatible and secure for the surroundings

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and human health. Composites formed by brown algae consist of chief metabolites derived from fucoidans, phlorotannins and fucoxanthins reveals antioxidant, isoprene (complex diterpenoids)

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[67], antifungal, antibiotic, antiviral and anti-cancer activities [68]. In our results proposed that

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the hot water extract of S. wightii and H Sw - ZnO NPs were superior antibiofilm agents against the Gram positive and Gram negative bacteria. LIVE/DEAD staining of both Gram positive and

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negative bacterial biofilms following 24 h challenge with hot water extract of S. wightii and H

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Sw - ZnO NPs observed using CLSM illustrates widespread cell death in mature biofilms treated with H Sw - ZnO NPs verifying the antibiotic nature of the bioactive compounds concerned.

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Marine algae are established to be fundamental source of valuable bioactive material while two

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decades. Numerous studies have verified with biological activities like antimicrobial and larvicidal [69]. Even if a wide number of green fabricated nanoparticles have been tested against

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mosquito larvae [70-72], and this study is based on mosquito larvicidal activities with hot water extract of S. wightii and H Sw - ZnO NPs. However, other members of the Sargassum genus have been reported for their larvicidal activity towards mosquito and the activity reported is more effective compared to the activity in our report. The histopathalogical images showing the effect of exposure to hot water extract of S. wightii and H Sw - ZnO NPs treated larvae were different histological changes like gastric caeca, muscles and siphon was damaged and disorganized. Our

ACCEPTED MANUSCRIPT results concurred with our previous report described that, Ae. aegypti treated with U. lactuca coated ZnO NPs illustrated elevated shrinkage in abdominal region of the treated larvae. A similar trend was reported earlier, the extracts of different seaweeds on Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus larvae [73]. Later, the histological impact of

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Sargassum binderi extract was analyzed on Ae. aegypti larvae showing results in agreement with

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our work [74].

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To conclude, the current research recognized that management of the H Sw and H Sw - ZnO NPs, through immersion, nutritional uptakes improved the immune capacity of green tiger

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shrimp P. semisulcatus by raising THC, PO activity, RB activities. The H Sw - ZnO NPs act as

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an Immunostimulants via immersion method and fed diet method. Furthermore, the hot water extract of S. wightii and H Sw - ZnO NPs also possessed antibiofilm activities against both Gram

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positive and Gram negative biofilm forming bacterial pathogens. In addition, H Sw - ZnO NPs

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are extremely stable and had significant larvicidal activity against A. aegypti. Further efforts are needed to shed light on the mode of action of ZnO nanoparticles on the tested economically

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important invertebrates [75-77], as well as to monitor the fate of this new nanomaterial in the

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environment for potential damage assessment on non-target species [78-79].

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Acknowledgements

The authors would like to extend their grateful thanks to the Alagappa University Research Fellowship (AURF), Karaikudi, India and this work was partly carried by Department of Biotechnology (DBT), New Delhi, India, below the Project grants code: BT/PR7903/AAQ/3/638/2013. The authors extend their appreciation to the Deanship of

ACCEPTED MANUSCRIPT Scientific Research at King Saud University for funding this work through research group No.

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RG-1438- 074.

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Table 1. Larvicidal activity of S. wightii hot water extract and S. wightii-synthesized ZnO nanoparticles against 3rd instar larvae of Aedes aegypti.

(µg/ml)

(%) ± SD

(µg/ml)

(95%

(95%

LCL-

LCL-

UCL)

T

Mortality

(µg/ml)

IP

Concentration

LC90

Hot water

100

31.6±1.4

130.26

224.69

extract of S.

150

56.4±0.8

(101.26-

(191.40-

wightii

200

78.8±1.2

250

99.3±1.4

20

21.5±0.6

40

AN

293.11)

34.3±0.8

49.22

86.96

58.6±1.2

(36.01-

(73.10-

82.7±0.8

60.40)

117.40)

CE

PT

80

100.0±0.0

Values are means ± SD of 5 replicates

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(d.f. =4)

y=-

7.863

5960+04188x

n.s.

y=-2.200-

9.020

1.027x

n.s.

M

156.35)

ED

60

100

US

18.2±1.2

nanoparticles

equation

χ2

UCL)

50

ZnO

Regression

CR

Treatment

LC50

d.f. = degrees of freedom n.s. = not significant (α=0.05)

ACCEPTED MANUSCRIPT Fig. 1. (a) UV-Vis optical absorption spectrum of ZnO nanoparticles fabricated using a hot water extract of Sargassum wightii (H Sw - ZnONPs) (left inset: nanoparticles in suspension; right inset: seaweed extract), (b) XRD analysis, and (c) FTIR spectrum. Fig. 2. SEM of the hot water extract of Sargassum wightii (a-b) and ZnO nanoparticles fabricated using the S. wightii extract (H Sw - ZnONPs) (c-d).

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Fig. 3. Total heamocytes count of Penaeus semisulcatus immersed in seawater containing (a) hot water extract of Sargassum wightii and (b) ZnO nanoparticles fabricated using the S. wightii extract at 200, 400 and 600 mg l-1 after 1, 3 and 5 hours. Total heamocytes count of (c) P. semisulcatus that had fed diets containing hot-water extract and (d) H Sw- ZnONPs (d) at 0, 0.5, 1.0 and 2.0 g kg -1 after 1, 3, 5 and 7 days.

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Fig. 4. Phenoloxidase (PO) activity of Penaeus semisulcatus immersed in seawater containing (a) hot water extract of Sargassum wightii and (b) ZnO nanoparticles fabricated using the S. wightii extract at 200, 400 and 600 mg l-1 after 1, 3 and 5 hours. Phenoloxidase (PO) activity of (c) P. semisulcatus that had fed diets containing hot-water extract, and (d) H Sw- ZnONPs (d) at 0, 0.5, 1.0 and 2.0 g kg -1 after 1, 3, 5 and 7 days.

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Fig. 5. Respiratory burst of Penaeus semisulcatus immersed in seawater containing (a) hot water extract of Sargassum wightii and (b) ZnO nanoparticles fabricated using the S. wightii extract at 200, 400 and 600 mg l-1 after 1, 3 and 5 hours. Respiratory burst of (c) P. semisulcatus that had fed diets containing hot-water extract and (d) H Sw- ZnONPs (d) at 0, 0.5, 1.0 and 2.0 g kg -1 after 1, 3, 5 and 7 days.

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Fig. 6. Superoxide dismutase (SOD) activity of Penaeus semisulcatus fed on diets containing (a) hot water extract of Sargassum wightii and (b) ZnO nanoparticles fabricated using the S. wightii extract at 0, 0.5, 1.0 and 2.0 g kg -1 after 1, 3, 5, and 7 days.

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Fig. 7. CLSM of Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Shigella sonnei, Psuedomonas aeruginosa) biofilms treated or not with hot water extract of S. wightii (H Sw Extract) and or S. wightii-fabricated ZnO nanoparticles (H Sw - ZnONPs). Fig. 8. Light microscopy (40X) images of Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Shigella sonnei, Psuedomonas aeruginosa) biofilms treated or not with hot water extract of S. wightii (H Sw Extract) and or S. wightii-fabricated ZnO nanoparticles (H Sw - ZnONPs). Fig.9. Confocal images of live (green) and dead (red) bacterial cells from Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Shigella sonnei, Psuedomonas aeruginosa) bacteria before and after treatment with hot water extract of S. wightii (H Sw Extract) and or S. wightii-fabricated ZnO nanoparticles (H Sw - ZnONPs).

ACCEPTED MANUSCRIPT Fig. 10. Stereomicroscopic images of 3rd instar larvae of Aedes aegypti: negative control, hot water extract of Sargassum wightii (H Sw extract), zinc acetate, and H Sw ZnO NPs (H Sw ZnONPs). Histology of Ae. aegypti 3rd instar larvae: negative control, H Sw extract, zinc acetate, and S. wightii-fabricated ZnO nanoparticles (H Sw – ZnONPs) (b). Red arrows indicate damages

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in head, abdominal, thorax region and siphon.

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Graphical Abstract

ACCEPTED MANUSCRIPT Highlights ZnO nanoparticles were fabricated using the hot water extract of Sargassum wightii (Sw).

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ZnO NPs were characterized by UV-vis, XRD, FTIR and SEM analyses.

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ZnO NPs enhanced immune parameters in green tiger shrimp, Penaeus semisulcatus.

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ZnO NPs nanoparticles showed high antibiofilm activity on Gram-positive and negative

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They were toxic to larvae of Aedes aegypti mosquitoes, which vector Zika virus.

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bacteria.

Graphics Abstract

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