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1. TOPIC OF RESEARCH:
“Immuno-modulatory compound from fresh water sponge Spongilla helps fishes to fight against bacterial and fungal infections”

2. ABSTRACT:

Development of new antimicrobial drugs is vital, as current finding suggests that resistance producing against microbial drugs is inevitable. As synthetic drugs produces high health and environment risks, natural compound containing immune-modulatory activity isolated from different organisms gaining popularity as they are ecofriendly. Sponges produces variety of bioactive compounds, most among other organisms. However majority of the study focus compounds isolated from marine sponges, freshwater sponges mainly ignore as far. This study screens, extracts bioactive compound isolated from freshwater sponge Spongilla spp. and purify it by help of high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) respectively forming crude compound.
Antimicrobial activity of crude compound against 3 bacterial fish pathogen strains Aeromonas. salmonicida, Flavobacterium. branchiophilum, Pseudomonas spp. or Vibrio spp. and 3fungal fish pathogen stains Exophiala. spp., Aphanomyces. Invadans and Saprolegnia spp. and their minimal inhibitory concentration (MIC) of the compound is evaluate. Its finding may help in designing new antimicrobial drugs against common fish pathogens in aquaculture.

Keywords: Freshwater sponge; Antimicrobial activity; High performance liquid chromatography; Thin layer chromatography; Minimum inhibitory concentration; Aquaculture.

OUTLINE:

? Bioactive compounds from fresh water sponges and its antimicrobial activities.

• Extraction of bioactive compound from fresh water sponge.
• Purification of bioactive compound and forming crude compound.
• Isolation of bacterial pathogens from infected fishes.
• Isolation of fungal pathogens from fungal infected fishes.
• Antibacterial activity of crude compound against fish pathogens.
• Antifungal activity of crude compound against fish pathogens.
• Minimum inhibitor concentration of crude compound against fish pathogens.

3. RESEARCH DESIGN:

A. INTRODUCTION:

Resistance against disease increasing day by day which lead to increase the necessity of new drugs discoveries. New researches suggests that new drugs isolates from natural sources are so much important for green heath management and to minimizing the toxicological as well as environmental risks produces by synthetic antimicrobial drugs (Prabha Devi, 2013).

Sponges produces many forms of biological active compounds which possess antimicrobial activities. Researchers isolated many compounds from marine sponges as imuno-modulatory compound but identification from freshwater sponge are still lacking.

Extraction of immune-modulatory compound from natural sources i.e. from fresh water sponge and testing its biological activity against fish pathogens may help in discovery of new drug tip its significance in field of biotechnology. Isolation from natural sources may leads lowering the contamination, environment risks and may also lead in reduction of food insecurity as the drug for aquaculture minimize the ratio of fishes to get infected and die. World’s large population is depending on fishes as their food, either develop country like Japan or poor African countries.

B. RESEARCH QUESTION:

• Replacement of exciting antibacterial and antifungal drugs with more effective and natural ones has become main question to address.
• Is fresh water sponges help freshwater fishes to increases its immunity to fight against different bacterial and fungal infections?

C. HYPOTHESIS:

Marine sponges contains variety of bioactive compounds which having immunomodulatory activities. Fresh water sponges may also contain these type of bioactive compounds having antibacterial and antifungal activities which may help fresh water fishes to fight against infections and aid them in increasing their immunity.
Aims to find antibacterial and antifungal activities from fresh water sponges.

? SUPPLEMENTARY QUESTIONS:

• From where we can isolate bioactive compound?
From fresh water sponge Spongilla. Spp
(Porifera: Demospongiae: Spongillidae a common variety of freshwater sponge).

• Why fresh water sponges?
Marine sponges are known to produces many metabolites processing various bioactive activities, some are also went for clinical trials. Whereas fresh sponges is less studied group having relatively limited information.

• Will fresh Sponges help more than marine sponges?
Technical problems associated with cultivation and harvesting large amounts of marine sponges. Whereas Fresh water sponges are easy to cultivate and grow and harvest artificially as we can grow them in aquariums.

• What type of bacteria infect fishes? And what disease they cause?
1) Aeromonas. salmonicida causes Furunculosis forms boil-like lesions or may also appear dark in coloration at the base of their fins.
2) Flavobacterium. Branchiophilum causes gill disease, leading to gill proliferation in fishes.
3) Pseudomonas.spp and vibrio.spp can cause frayed fins with hemorrhaging at the base of the fin, sores, lethargy, and swelling of the belly.

• What type of fungus infect fishes? And what disease they cause?
1) Exophiala. spp are ubiquitous yeast and are distinct olive to black-brown color lesions on fishes.
2) Saprolegnia.spp freshwater specie of fungi which causes cottony/woolly, white growth on the skin, or gills, or on fish eggs.
3) Aphanomyces. Invadans causes deep ulcerative orange to red lesions on skin scrapes.

D. OBJECTIVE OF RESEARCH:
Considering the importance of green health management in aquaculture and concern about the contamination, toxicological and environmental risks posed by synthetic drugs has led to an increase in the popularity of developing natural products as a source of ecofriendly compounds possessing antimicrobial activities.
In addition, the evolving resistance of microorganisms to existing antibiotics is becoming major issue not only for humans but also for aquaculture. This threat is increasing day by day causing immense economic losses resulting in food insecurity. Hence, replacement of existing antibiotics with more operational and safer ones has become an important subject to discuss.

E. LITERATURE REVIEW:
Sponges are animals which consider one of the most primitive animal on earth, existing from millions of years by surviving major mass extinct. They are belonging to phylum porifera containing pores although their body. They produces variety of bioactive compound as they cannot move lacking physical defenses, highly susceptible predators such as fishes. Thus, it is not surprising that sponges have developed a wide suite of defensive chemicals to deter predators (Anjum, 2016), biofouling, microbial infections, and overgrowth by other sessile organisms (M. F. Mehbub, 2018).

Previously natural products have traditionally been harvested from terrestrial sources, while from sponges and their associates produces approximately 5,300 different natural known compounds (Bibi, 2016). A major contributing factor to this development is the fact that modern technology has made it easier to gain access to the great biodiversity of life found in the oceans (Margey Tadessea, 2008).

Oceans are most primitive, important and unique form of life on the earth. It provides a huge diversity of living organisms inhabiting diverse micro flora. The marine resources are widely studied nowadays because of numerous reasons. One of the reason is, the oceans cover more than 70% of planet surface and among 36 living phyla known yet, 34 of them are found in marine environments with more than 300000 known species of fauna and flora (Bibi, 2016).

Compounds isolated from sponges contains anticancer, anti-inflammatory, antiviral, antibacterial, anticoagulant (Roberta J. Melander, 2016), antitumour (Margey Tadessea, 2008), antifungal (Prabha Devi, 2013), cytotoxic, antidiabetic, antimalarial, antiplatelet, antiprotozoal, antileukemic, anti- tuberculosis, (G. Annie Selva Sonia, 2008) and immunomodulatory activities (Soumalya Mukherjee, 2016). Considering their scope of antibiotic activity against fish pathogenic bacteria, marine sponge extracts are prime candidates as sources of bioactive metabolites (G. Annie Selva Sonia, 2008)

The discovery of penicillin in the mid-twentieth century revolutionized the treatment of infectious disease. Since then, antimicrobial agents have saved the lives and eased the suffering of millions of people. Multi-resistant bacteria threaten to cause new epidemics (Bibi, 2016).

Evidence suggest that development of resistance to any new antimicrobial agents is inevitable (Prabha Devi, 2013). So the evolving resistance has made necessary a search for new antibiotics for human as well as aqua cultural purposes. In the aquatic environment, competition for space and nutrients leads to evolution of antimicrobial defense strategies. This, along with possibly adverse effects on the ecosystem and human health problems, has resulted in restrictions on the use of commercial antibiotics and chemicals in the aquatic environment (G. Annie Selva Sonia, 2008).

Emerging infectious diseases (EIDs) caused by fungi are increasingly recognized as presenting a worldwide threat to food security. This is not a new problem and fungi have long been known to constitute a widespread threat to plant species. However, pathogenic fungi (mycoses) have not been widely recognized as posing major threats to animal health. This perception is changing rapidly owing to the recent occurrence of several high-profile declines in wildlife caused by the emergence of previously unknown fungi (Matthew C. Fisher, 2012). For more than two decades worldwide and fungal infections are amongst the common diseases in hatchery and aquaculture systems leading to the demise of fish population resulting in great economic loss (Prabha Devi, 2013).

Many structurally diverse marine sponge secondary metabolites have been shown to exhibit antibiotic activities against several Gram-positive bacteria including Streptomyces. pyogenes, Staphylococcus.aureus and Bacillus. subtilis. However many of these natural products are in active against Gram-negative bacteria (Roberta J. Melander, 2016).
In most cases development and production of sponge derived drugs is hindered by environmental concerns and technical problems associated with harvesting large amounts of sponges. But now presence of sustainable source of sponge-derived drug candidates could be generated by establishing a symbiont culture or by transferring its biosynthetic genes into culturable bacteria (Anjum, 2016). There are a few examples of marine derived compounds which have successfully reached the market as therapeutic drugs (Margey Tadessea, 2008).

Multi drug resistant Staphylococcus. aureus (MRSA) formerly particularly problematic in places such as hospitals and nursing homes, is now found in commonly-used places. Scientists have isolated an extract from a sponge found in Antarctica, tested it on MRSA biofilm and found that it eliminate more than 98 percent of MRSA cells. The highly-resistant MRSA infection (USF, 2016). Several strains were identified for their potent antifungal activity, and for both antifungal and antibacterial activities (University, 2018).

Benthic marine invertebrates (Sponges) were found to be a promising source of novel bioactive compounds against human and fish pathogenic bacteria and fungi (Margey Tadessea, 2008). Freshwater poriferans are relatively a less studied group with limited scientific information (Soumalya Mukherjee, 2016). Spongilla. Spp (Porifera: Demospongiae: Spongillidae a common variety of freshwater sponge) is distributed in seasonal ponds and lakes.

F. METHODOLOGY:

Collection and Laboratory Acclimation of Spongilla. Spp:
Protocol is design according to (Margey Tadessea, 2008) and (Prabha Devi, 2013) Spongilla. Spp shallow water sponge will manually collect from the water bodies. Sponges will carefully remove from jetty pylons with a scraper, kept wrap in plastic bags, and will immediately transport to the laboratory. Associated macro organisms (mainly algae and polychaetes) will remove from the biological material before lyophilisation. Samples of Spongilla. Spp will then identify by Polymerase chain reaction (PCR), pool, lyophilize and separately frozen at -20 o C.

Extraction of Bioactive Compound from Sponge:
Extraction protocol as describe by (Margey Tadessea, 2008), (Prabha Devi, 2013) and (G. Annie Selva Sonia, 2008) frozen sponge sample will thaw and extract exhaustively with acetone (Prabha Devi, 2013) or extract thrice with distilled methanol and the pooled organic solution made from each species will filter by suction through a Buchner funnel line with Whatman No. 1 filter paper. (G. Annie Selva Sonia, 2008). Solvent will remove by rotary evaporator. The free aqueous extract thus obtain will transferred into a separating flask and fractionate sequentially using Diethyl Ether (DE) follow by Butanol (Bu) to obtain the DE-fraction and the Bu fractions respectively (Margey Tadessea, 2008). The crude extracts will now screen for antibacterial and antifungal activity.

Pathogens Collection:
Pathogen collection as describe by (Prabha Devi, 2013) Fish pathogens will isolate from infected fish. Isolation will carry out using standard techniques. Briefly, one gram wet weight of the fish sample from the infected region will rinse thrice in sterile fresh water and homogenize it by using a sterile mortar and pestle in 5ml sterile freshwater.

For Bacterial Pathogens:
Serial dilutions (up to 4 dilutions) will make and spread plated on Luria agar (M. F. Mehbub, 2018) as standardize growth media due to the simplicity and accessibility of its formulation. Plates will incubate at 26°C for 2-3 days. The isolates will repeatedly sub-cultured until pure bacterial isolates will obtain and then store on Luria broth until use.

For Fungal Pathogens:
Serial dilutions (up to 4 dilutions) will make and spread plated on Sabourauds dextrose agar (SDA, Hi Media) containing 50 mgml-1 of antibiotic chloramphenicol to inhibit bacterial growth. Plates will incubate at 26°C for 2-3 days. The isolates will repeatedly sub-cultured until pure fungal isolates will obtain and then store on SDA slants until use. (Prabha Devi, 2013)

Metabolite Purification High Performance Liquid Chromatography (HPLC):
Protocol is set as per (M. F. Mehbub, 2018) and (Prabha Devi, 2013) to assess sponge metabolite profiles, and HPLC analyses will perform as per protocol defined by (M. F. Mehbub, 2018). At constant flow rate, 100 mg of freeze-dried sponge tissue was extracted three times, powdered sponge tissue was transferred to a new tube and dissolved with 1 ml methanol in an ultrasonic tank for 5 min with high energy setting, centrifuged and the pellet retained after transferring the supernatant.
The pellet was extracted twice and the combined crude extracts and finally dissolved with 1 ml methanol. This crude extract was filtered through a 13 mm 0.2 µm Syringe Filter and added to a 2ml tube with glass insert. Then, 50 µl of this filtered solution was injected into the HPLC system described above. The peaks will observe at 200 to 800nm wavelength range.

Thin Layer Chromatography (TLC):
This step is design according to protocol design by (M. F. Mehbub, 2018) and (Prabha Devi, 2013) to further elaborate the nature of the metabolites produced, we used TLC. A slurry of the Diethyl Ether (DE) fraction in silica gel was prepared by dissolving the crude extract in minimum quantity of DE and dried under nitrogen. This dry slurry was loaded onto a silica gel glass column and initially eluted with hexane followed by increasing concentration of diethyl ether in hexane. Next elution was performed using chloroform followed by increasing concentration of methanol in chloroform and finally eluted with methanol. Like fractions were combined on the basis of TLC and the combined fractions were subjected to bioassay screening against pathogenic bacteria and fungi. Separation on TLC may be detected under a UV lamp at 254 or 366 nm wavelength range.

Biological Activity:
Antimicrobial activities for the crude fractions and the pure compound against fish pathogens will determine by agar disc diffusion method. Briefly, paper discs of 6mm diameter will impregnate with 25 µg of the crude extract and 10 µg of the pure compound dissolve in diethyl ether. The zone will then measure in millimeter and scored as (– no activity; + mild activity; ++ moderate activity; +++ Significant activity; and ++++ strong activity). Positive and negative control will also use.
For bacterial pathogens discs will place on Mueller Hinton Agar (MHA) plates possessing a lawn of the different strains to be test. The cultures will incubate for 24 hours at 37°C and for fungal pathogens discs will place on Potato Dextrose Agar (PDA) plates possessing a lawn of the different strains to be test. The cultures will incubate for 48 hours at 27°C to obtain maximum growth in the culture media so as to visualize the clear zone of growth inhibition around each discs. Experiment repeat thrice to know the reproducibility of results

Minimum Inhibitory Concentration (MIC):
Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of the pure compound that inhibits visible growth of the microorganism around the disc. MIC values of crude compound against test pathogens will determine according to the Kirby and Bauer disc diffusion method. The discs load with the compound will prepare in the same way as described above. Each of the pathogen will inoculated in different plate.
For antibacterial MIC inoculum of 250µl solution will spread over each MHA agar plate surface and incubate it for 24h for 37°C and for antifungal MIC inoculum of 250µl solution of each fungi pathogen spread over the surface on potato dextrose agar and plates will incubate for 48-72 h at 28°C. An array of the discs containing different concentrations (µg/ ml) of the compound will place on the plates to determine the MIC values of the compound. Dried discs will use as negative control discs and standard drug will use as positive control. MIC will determine in triplicate.

G. TIME FRAME:

• Collection and laboratory acclimation of Spongilla. spp and its identification through PCR can take up to 3 months according to availability of reagents.

• Bacterial and fungal fish pathogens collection can be start along with Spongilla collection. As we have to collect three Bacterial and tree fungal pathogens, it can be done within 3 months after Spongilla identification.

• After identification of Spongilla, we have to wait until pathogens collection. After collection we will thaw and then extract bioactive immune-modulatory compound. Extraction of bioactive compound from sponge can be done within two week.

• Metabolite Purification by HPLC and TLC both can be done within 3 months, forming pure crude compound.

• Antibacterial and antifungal biological activity of crude compound done in systematic repeat manner in order to get authentic results. This step can be done in 3 month.

• To find out minimum inhibitory concentration of crude compound only for those against which it give antimicrobial activity, will be done in a month.

• Thesis writing can be done within 3 month after complete whole research work.
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