Doaa Ibrahim ^1* Ioan Pet ² Reham G A Anter ³ Abdelwahab A Abdelwarith ⁴ Mona Rahman ³ Basant Shafik ⁵ Elsayed Younis ⁴ Asmaa Basiony ⁶ Shimaa Ahmed ⁷ Aya S M Metwally ⁸ Heba A Tolba ⁹ Mirela Ahmadi ² Asmaa T. Y. Kishawy ¹⁰

• ¹ Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
• ² Department of Biotechnology, Faculty of Bioengineering of Animals Resources, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, Romania, Timisoara, Romania
• ³ Department of Parasitology, Faculty of Veterinary Medicine, Zagazig University, Zagazig city, 44511, Egypt, Zagazig, Al Sharqia, Egypt
• ⁴ Department of Zoology, College of Science, King Saudi University, Riyadh 11451, Saudi Arabia, Riyadh, Saudi Arabia
• ⁵ Department of Animal Wealth Development, Animal and Poultry Production, Faculty of Veterinary Medicine, Benha University, Toukh city P.O. Box 13736, Qalyubia, Egypt, Benha, Egypt
• ⁶ Infection control unit, Zagazig University Hospitals, Zagazig University, 44511, Egypt, Zagazig, Egypt
• ⁷ Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh 13736, Egypt, Benha, Egypt
• ⁸ Department of Pharmacology, Faculty of Veterinary Medicine, Aswan University, Aswan, Egypt, Zagazig, Al Sharqia, Egypt
• ⁹ Department of Fish Health and Management, Central Laboratory of Aquaculture Research (CLAR), AboHamad, Egypt, Agriculture Research Center (ARC), Egypt, Zagazig, Egypt
• ¹⁰ Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig, Egypt, egy, Egypt

Imperative to identify novel natural sources from marine environment with unique immunomodulatory and antioxidant efficacy is a point of interest in intensive fish farming. In this sense, marine sponge extract derived from Smenospongia (SS-extract) was tested for its potential antiinflammatory, antioxidant and antimicrobial activities during the concurrent infection with Trichodina sp and Flavobacterium columnare in Nile tilapia. A total of 625 Nile tilapia fingerlings were distributed into five groups: control group (with no additives) and four groups fed control diet fortified with SS-extract at the level of (50, 100, 150 and 200mg/kg) for 60 days. The parasitic challenge with Trichodina sp was done at the end point of feeding trial and at 7 days post infection (dpi), fish were coinfected by F. columnare. Significant improvement in Nile tilapia growth related parameters was detected in groups received SS-extract at the levels of 150 and 200 mg/kg. Scavenging ability for free radicals (ABTS and DPPH) was maximized with higher SS-extract supplemental levels. Simultaneously, the antioxidant defense of Nile tilapia fed 150 and 200 mg/kg was augmented with a contrary decline in oxidative associated indicators (reactive oxygen species: ROS, hydrogen peroxide: H2O2, and Malondialdehyde: MDA). Feeding on higher concentrations of SS-Extract potentiated the immune response of Nile tilapia before infection and even after coinfection which parallel with subsiding the exaggerated inflammatory response after concurrent infection. Concurrently, mRNA expression of autophagy-encountered genes (Atg5 and 12, LC3-II and BCLN1) showed their highest expression in 200 mg/kg diet with lowest expression of mTOR gene. The infection intensity and rate and mucous score were displayed their lowest levels in 200 mg/kg SS-extract at 20 dpi. Interestingly, higher levels of SS-extract triggered the antimicrobial peptides (upregulation of Hepcidin and βdefensin-1). In contrast excessive expression of endoplasmic reticulum genes (atf4, JAK1, PERK and eif2α) in control infected group was downregulated in 200 mg/kg SS-extract. The quantification of F. columnare indicated that the columnaris coinfection severity displayed its lowest rate in 200 mg/kg SS-Extract supplemental group. Collectively, strategic siting based on fortification Nile tilapia's diet with SS-extract was elucidated to reinforce its immune and antioxidant defense during the concurrent infection.