Evaluation of bioactivity of plant extracts against pathogens in shrimp

Aquaculture is the fastest-rising food sector in the world, and is considered a high-protein source to meet the food demand as the natural resources are overexploited. Shrimp farming is one of the most productive aquaculture sectors in the world, especially in the Asia-Pacific region, with export earnings amounting to billions of US dollars per year (Ganjoor, 2015). The black tiger shrimp (Penaeus monodon) and the white leg shrimp (Litopenaeus vannamei) dominate the production, which accounts for approximately 90 % of the total shrimp production in the world (FAO, 2014). Nowadays, intensification in shrimp farming has resulted in disease outbreaks which have been considered a major constraint to shrimp production, causing great economic losses in the affected shrimp farms due to the massive mortality (Kumar et al., 2014; Moss et al., 2012). It had been estimated that 60% of the shrimp losses were due to viral pathogens and a further 20% were caused by bacterial pathogens (Stentiford et al., 2012). Major viral pathogens of shrimp include White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), Yellow Head Virus (YHV), Taura Syndrome Virus (TSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) (Flegel, 2006; Sivasankar et al., 2015). Among the viruses, the WSSV is extremely virulent and lethal, highly prevalent, and currently causes the most adverse effects for the global penaeid shrimp aquaculture industry (Walker and Mohan, 2009), leading to massive mortality (80 to 100%) within 5-10 days (Chou et al., 1995). Besides viral diseases, bacterial diseases also have a devastating effect on affected shrimp farms and have gained more attention due to the presence of Vibrio species, which are associated with high mortality in shrimp hatcheries and farms (Saulnier, 2000). Some Vibrio species reported for causing disease outbreaks throughout the world are V. parahaemolyticus, V. harveyi, V. alginolyticus, V. vulnificus, V. fluvialis, V. anguillarum, V. penaeicida, V. campbelli, V. cholera, V. splendidus etc. (Lightner, 1992; Anderson et al., 1988; Song et al., 1993; Lee et al., 1996; Takahashi et al., 1985a; Costa et al., 1998). Among those bacteria, Vibrio parahaemolyticus is considered the devastating causative agent for Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS), which has an acute mortality in shrimp (Penaeus monodon) within 35 days approximately after stocking in grow-out ponds (Joshi et al., 2014; Tran et al., 2013). Besides these diseases, larval stages of shrimp are most commonly affected by the fungi Lagenidium callinectes and Serolpidium spp. (Karunasagar et al., 2004). Due to the continuous and increasing use of antibiotics in aquaculture, there is an increasing danger of developing antibiotic-resistant pathogenic bacteria in shrimp, which makes antibiotic treatment in humans ineffective (Madhuri et al., 2012; Zanetti et al., 2001; Yin et al., 2008). Medicinal plants are used as an alternative agent to treat infectious disease, and to minimize side effects associated with synthetic antimicrobials. Medicinal plants have the potential for being effective herbal drugs against fish - and other aquaculture pathogens, because plant extracts often have minimal side effects, contain secondary metabolites, are inexpensive, easily biodegradable, and extracts can be easily prepared (Madhuri et al., 2012, Tekwu et al., 2013). To reduce the risk of disease, the development of new antimicrobial agents for the treatment of shrimp infection is of increasing interest. In the last few years, a number of studies have been conducted to verify the effectiveness of natural antibacterial compounds (mainly plant extracts) against bacterial infections of aquatic animals (Kumar et al., 2006; Chou et al., 2010; Caruana et al., 2012; Chang et al., 2013; Talpur, 2014; Sivagnanavelmurugan et al., 2015; Acar et al., 2015; Dhayanithi et al., 2015; Thanigaivel et al., 2015; Srinivasan et al., 2017; Mohtar et al., 2016; Hoque et al., 2011; Nguyen et al., 2018). There are some medicinal plants such as A. marmelos, L. camara, C. dactylon, M. charantia, P. amarus, P. guajava etc. exhibiting antiviral activity against WSSV, IPNV, YHV in the form of different extracts (Direkbusarakom et al., 1997; Balasubramanian et al., 2007; Citarasu et al., 2006; Balasubramanian et al., 2008b). For this Ph.D. thesis, we will evaluate the antimicrobial compounds of natural plant extracts against the aforementioned pathogens in Penaeus monodon both in vitro (Vibrio species, fungi) and in vivo (V. parahaemolyticus, V. harveyi, WSSV). At the beginning of the study, based on an extensive literature survey, plants will be selected based on ethnomedicinal evidence and tested against specific pathogens to identify the active plants. From the top three/four plants which have high activity against selected pathogens, we will then identify the bioactive compounds through Bioassay-guided fractionation. The plants will also be used for cytotoxicity test and in vivo experiments against known pathogen-challenged shrimp in the lab of Fisheries and Marine Resource Technology Discipline of Khulna University, Bangladesh. Structure-activity relationship and synergy studies will also be performed for the bioactive compounds of the selected plants. It is expected that several bioactive compounds will be discovered from the plants, which have potential activity against specific pathogens in shrimp to lower the mortality rate during the culture stage and also in the hatchery. Such result will have a high impact of lowering the disease outbreak in shrimp culture in Bangladesh as well as elsewhere around the world.