TOPIC OF RESEARCH

TOPIC OF RESEARCH:
“Immuno-modulatory compound from fresh water sponge Spongilla spp. helps fishes to fight against bacterial and fungal infections”
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.
OUTLINE:
Bioactive compounds from fresh water sponges Spongilla spp. and its antimicrobial activities.

Extraction of bioactive compound from Spongilla spp. fresh water sponge.

Purification of biological active compound and forming crude compound.

Isolation of bacterial pathogens, Aeromonas. salmonicida, Flavobacterium. branchiophilum, Pseudomonas spp. or Vibrio spp from infected fishes.

Isolation of fungal pathogens, stains Exophiala. spp., Aphanomyces. Invadans and Saprolegnia spp. from fungal infected fishes.

Checking of biological active crude compound against three different bacterial- fish pathogens.

Checking of biological activity of crude compound against three different fungal- fish pathogens.

Minimum inhibitor concentration against those bacterial or fungal fish pathogens against which crude compound shows its resistance.

RESEARCH DESIGN:

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 CITATION Pra13 l 1033 (Prabha Devi, 2013).

Sponges produces many forms of biological active compounds which possess antimicrobial activities. Researchers isolated many compounds from marine sponges as immuno-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 which 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.
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?
HYPOTHESIS:
Marine sponges contains variety of bioactive compounds which having immune-modulatory 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?
Aeromonas. salmonicida causes Furunculosis forms boil-like lesions or may also appear dark in coloration at the base of their fins.

Flavobacterium. Branchiophilum causes gill disease, leading to gill proliferation in fishes.

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?
Exophiala. spp are ubiquitous yeast and are distinct olive to black-brown color lesions on fishes.

Saprolegnia.spp freshwater specie of fungi which causes cottony/woolly, white growth on the skin, or gills, or on fish eggs.

Aphanomyces. Invadans causes deep ulcerative orange to red lesions on skin scrapes.

OBJECTIVE OF RESEARCH:
Considering the importance of ecofriendly compounds having antimicrobial activities as synthetic drugs produces harm full effects on both environment and health. As a green health management, popularity of drugs drive from natural sources is important.

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.

LITERATURE REVIEW:
Sponges are animals which consider one of the most primitive animal on earth, existing from millions of years by surviving major mass extents. They are belonging to phylum Porifera containing pores although their body. They mainly marine sponges are being studied not only because for its evolutionary and ecologically importance, but also due to their ability to produce bioactive compounds CITATION Swa16 l 1033 (Swapnil Gaikwad, 2016). As they cannot move lack definite tissue organization and physical defenses they are highly susceptible predators such as fishes. Thus, it is not surprising that sponges have developed a wide suite of defensive chemicals to deter predators CITATION Anj16 l 1033 (Anjum, 2016), bio-fouling, microbial infections, and overgrowth by other sessile organisms CITATION Moh18 l 1033 (M. F. Mehbub, 2018).

Natural products have conventionally been collected from terrestrial bases, while from sponges and their associates produces approximately 5,300 identified different natural compounds CITATION Bib16 l 1033 (Bibi, 2016). Now these compound are starting isolate from aqua origin. A major causative reason to this development is the fact that modern technology has gain access at ease to the great biodiversity of life present under the water CITATION Mar08 l 1033 (Margey Tadessea, 2008).
Oceans are greatest primitive, vital and exceptional form of life on the globe. It provides a huge range of living organisms inhabiting diverse micro and macro flora. Nowadays, water resources are widely studied because of numerous reasons. One of the main motive is to identifying new species as it covers more than 70% of sphere surface which containing 36 living known phyla of both fauna and flora CITATION Bib16 l 1033 (Bibi, 2016).

Compounds drive from sponges contains anticancer, anti-inflammatory, antiviral, antibacterial, anticoagulant CITATION Rob16 l 1033 (Roberta J. Melander, 2016), anti-tumor CITATION Mar08 l 1033 (Margey Tadessea, 2008), anti-fungal CITATION Pra13 l 1033 (Prabha Devi, 2013), anti-protozoal, anti-diabetic, anti-malarial, anti-platelet, anti-leukemic, anti- tuberculosis, cytotoxic CITATION GAn08 l 1033 (G. Annie Selva Sonia, 2008) and immune-modulatory activities CITATION Sou16 l 1033 (Soumalya Mukherjee, 2016). Considering their scope of anti-microbial activity against fish pathogenic, Sponge extracts are prime candidates as sources of bioactive metabolites CITATION GAn08 l 1033 (G. Annie Selva Sonia, 2008)The discovery of penicillin in the mid-20th century modernized the treatment of deadly disease. Since then, antimicrobial agents have saved the lives and improve the suffering of billions of individuals. Now multi-resistant bacteria threaten to cause new epidemics CITATION Bib16 l 1033 (Bibi, 2016).
Proof advocate that development of resistance to any new antimicrobial treatment drug is inevitable CITATION Pra13 l 1033 (Prabha Devi, 2013). So these growing resistance has made necessary for search for novel antibiotics for human along with agricultural and aqua-cultural purposes. In the water atmosphere nutrients are in diluted form and diversity of organisms present in it are high and their competition for living space leads to development of defense strategies including formation of antimicrobial compound within their body. Still many of them infected and died off but possibly adversative effects on aqua-culture ecology and human well-being produces by usage of synthetic drugs, has lead in the limitations of commercial chemicals and antimicrobial agents in the aquatic environment CITATION GAn08 l 1033 (G. Annie Selva Sonia, 2008).

Infectious diseases by bacteria are well known but now it caused by fungi are progressively documented as presenting a worldwide hazard to food security. This is not a fresh problem, food insecurity because of fungi have been long known as it is also great threat to fruits, vegetables and widespread plant species. However, fungi have not been usually known as posing great threats to animal health. This perception is altering rapidly due to the recent event of several high-profile failures in wildlife caused by the occurrence of unknown fungi CITATION Mat12 l 1033 (Matthew C. Fisher, 2012). For more than two eras fungal infections are among the common infections in aqua systems leading to the decease of fish population causing excessive economic loss CITATION Pra13 l 1033 (Prabha Devi, 2013).
Benthic marine Sponges were found to be a source of unique bioactive secondary metabolites against human and fish pathogens CITATION Mar08 l 1033 (Margey Tadessea, 2008). Many of these diverse bioactive metabolites have been exhibit antibiotic activities against several Gram-positive bacteria, however many of these metabolites are inactive against Gram-negative bacteria CITATION Rob16 l 1033 (Roberta J. Melander, 2016).
Previously development and production of sponges associated metabolites are hindered because of environmental concerns as harvesting large quantity of sponges from the oceans adversely effected overall aquatic ecosystem. But now due to presence of modern technology like recombinant DNA technology, it’s become easier to transfer genes which produces bioactive metabolites into cultural bacteria and cultivate from them. CITATION Anj16 l 1033 (Anjum, 2016). There are a few therapeutic drugs which are drive from marine compounds and now successfully reached for clinical trials CITATION Mar08 l 1033 (Margey Tadessea, 2008).
Several strains of marine sponges were identified for their potent antifungal activity, antibacterial activities CITATION Flo18 l 1033 (University, 2018). New research also extracted marine sponge from Antarctica and tested it on multi drug resistant Staphylococcus. aureus biofilm which eliminate approximately 98% of cells. MRSA which is previously present only in areas such as hospitals, but now starts found in commonly-used places caused serious epidemic CITATION Uni16 l 1033 (USF, 2016).
Freshwater invertebrates i.e. sponges are neglected so far, relatively a less studied group with limited scientific information CITATION Sou16 l 1033 (Soumalya Mukherjee, 2016). As Most of world restricted to study marine sponge as they are abundantly present and have more diversity as compared to their freshwater SpongesCITATION Swa16 l 1033 (Swapnil Gaikwad, 2016). Spongilla. Spp (Porifera: Demospongiae: Spongillidae a common variety of freshwater sponge) is distributed in seasonal ponds and lakes.

METHODOLOGY:
Collection and Laboratory Acclimation of Spongilla. Spp:
Protocol is design according to CITATION Mar08 l 1033 (Margey Tadessea, 2008) and CITATION Pra13 l 1033 (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 CITATION Mar08 l 1033 (Margey Tadessea, 2008), CITATION Pra13 l 1033 (Prabha Devi, 2013) and CITATION GAn08 l 1033 (G. Annie Selva Sonia, 2008) frozen sponge sample will thaw and extract exhaustively with acetone CITATION Pra13 l 1033 (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. CITATION GAn08 l 1033 (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 CITATION Mar08 l 1033 (Margey Tadessea, 2008). The crude extracts will now screen for antibacterial and antifungal activity.
Pathogens Collection:
Pathogen collection as describe by CITATION Pra13 l 1033 (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 CITATION Moh18 l 1033 (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. CITATION Pra13 l 1033 (Prabha Devi, 2013)Metabolite Purification High Performance Liquid Chromatography (HPLC):
Protocol is set as per CITATION Moh18 l 1033 (M. F. Mehbub, 2018) and CITATION Pra13 l 1033 (Prabha Devi, 2013) to assess sponge metabolite profiles, and HPLC analyses will perform as per protocol defined by CITATION Moh18 l 1033 (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 CITATION Moh18 l 1033 (M. F. Mehbub, 2018) and CITATION Pra13 l 1033 (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.

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 spp. collection. As we have to collect three Bacterial and tree fungal pathogens, it can be done within 3 months after Spongilla spp. identification.
After identification of Spongilla spp. 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.