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

2. ABSTRACT:

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3. 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.

4. RESEARCH DESIGN:

A. INTRODUCTION:

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 Eunapius. carteri.
(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 in human research but also in 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 spineless animals belong to phylum, “the pore bearers” (Porifera), serve as most primitive multicellular animals, existing for millions of year ago. They frequently produce bioactive compounds as compared to other living microorganisms. Because sponges cannot move and lack physical defenses, they are highly susceptible to marine predators such as fish, turtles, and invertebrates. Thus, it is not surprising that sponges have developed a wide suite of defensive chemicals to deter predators, (Anjum, 2016) microbial infections, biofouling, and overgrowth by other sessile organisms (M. F. Mehbub, 2018).

While natural products have traditionally been harvested from terrestrial sources, from sponges and their associates, approximately 5,300 different natural compounds are known (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 microflora. 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). Eunapius carteri (Porifera: Demospongiae: Spongillidae a common variety of freshwater sponge) is distributed in seasonal ponds and lakes.

F. METHODOLOGY:

G. TIME FRAME:

5. REFERENCES

1) Anjum, K. A. (2016). Marine Sponges as a Drug Treasure . Biomolecules & Therapeutics, 24(4), 347–362. doi:http://doi.org/10.4062/biomolther.2016.067
2) Bibi, F. &. (2016). Bacteria from marine sponges: A source of new drugs. Current Drug Metabolism, 17. doi:10.2174/1389200217666161013090610.
3) G. Annie Selva Sonia, A. L. (2008). Antibacterial Activity of Marine Sponge Extracts against. The Israeli Journal of Aquaculture , 60(3), 172-176. Retrieved from https://pdfs.semanticscholar.org/c894/5e6c27890a0b5b9e7e8b10261e989940f760.pdf
4) M. F. Mehbub, J. E. (2018). A controlled aquarium system and approach to study the role of sponge-bacteria interactions using Aplysilla rosea and Vibrio natriegens. Nature. doi:10.1038/s41598-018-30295-y
5) Margey Tadessea, B. G. (2008). Screening for antibacterial and antifungal activities in marine benthic invertebrates from northern Norway. Journal of Invertebrate Pathology , 99(3), 286-93. doi:http://dx.doi.org/10.1016/j.jip.2008.06.009
6) Matthew C. Fisher, D. A. (2012). Emerging fungal threats to animal, plant and ecosystem health. Nature, 484, 186–194. doi:10.1038/nature10947
7) Prabha Devi, R. S. (2013). Antifungal Potential of Marine Sponge Extract against Plant and Fish. Oceanography, 1(3), 112. doi:http://dx.doi.org/10.4172/2332-2632.1000112
8) Roberta J. Melander, H.-b. L. (2016). Marine sponge alkaloids as a source of anti-bacterial adjuvants. Bioorg Med Chem Lett. , 26(24), 5863–5866. doi: doi:10.1016/j.bmcl.2016.11.018
9) Soumalya Mukherjee, A. S. (2016). Immunomodulatory effects of temperature and pH of water in an Indian freshwater sponge. Journal of Thermal Biology, 59, 1-12. doi:https://doi.org/10.1016/J.JTHERBIO.2016.04.005
10) University, F. A. (2018). Deep-sea marine sponges may hold key to antibiotic drug resistance. ScienceDaily. Retrieved from www.sciencedaily.com/releases/2018/06/180619123013.htm
USF, U. o. (2016, May). Scientists discover Antarctic sponge extract can help kill MRSA: New findings may provide opportunity for developing new drugs to fight dangerous bacteria currently highly resistant to treatment. ScienceDaily. Retrieved from www.sciencedaily.com/releases/2016/05/160518094706.htm

1.1 History
Electric Vehicles
Electric vehicles, as the name suggests, run at least partially on electricity. Instead of fossil fuel-driven internal combustion engines, these vehicles are powered by electric motors for propulsion. The electric motor, in turn, derives energy from rechargeable batteries, solar panels or fuel cells.
Historically, electric cars have been around for more than a century. Interestingly, the first known electric car was built in Aberdeen, Scotland way back in 1837. Exhibited at the Royal Scottish Society of Arts Exhibition in 1841, the vehicle, weighing seven tons, could carry a load of six tons at speed of around four miles per hour over a distance of one and a half miles.
Its arrival coincided with the growing status of electricity as one of the preferred methods for vehicular propulsion. Additionally, with the invention of rechargeable batteries in 1859, innovations around EVs slowly, but steadily, started emerging. Incidentally, towards the end of the 19th century, battery-powered electric cabs started plying on the streets of London and New York.
The first known electric car was built in Aberdeen, Scotland way back in 1837.
In London, for instance, Walter C. Bersey built a fleet of electric taxis, called “hummingbirds”, which became operational in 1897. Around the same time, New York-based company, Samuel’s Electric Carriage and Wagon Company, designed around 62 electric cabs.
Despite its early popularity, however, electric vehicles witnessed a decline globally in the first half of the 20th century. Lack of proper charging infrastructure and simultaneous improvements in road infrastructure resulted in the dwindling popularity of EVs. At the same time, with the advancements of the automobile industry, car owners were increasingly looking for vehicles with greater range and speed than electric cars.
However, by the 1960s, EVs once again started garnering the interest of automakers. In 1959, for instance, the American Motor Corporation entered into a joint research agreement with Sonotone Corporation to develop an electric car powered by a “self-charging” battery.
In the decades since then, numerous electric car concepts have been showcased around the globe, including the Scottish Aviation Scamp (1965), the Electrovair (1966), the Electron (1977). Tracing the history of electric vehicles, we found that the first modern version of the electric car, as we know it today, was built in the early 2000s.
In 2004, Elon Musk-founded Tesla Motors started working on the Tesla Roadster, which was the first highway-legal all-electric car running on lithium-ion batteries. Over the years, most carmakers have jumped on the EV bandwagon, with Tesla, Ford, Nissan, Hyundai, Toyota and others leading the race.

Hybrid Vehicles

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it was none other than Dr. Ferdinand Porsche who built the first car to combine an internal-combustion engine with electric motors. The car, which was constructed in 1898, featured a gasoline engine that was used to power a generator that fed four electric motors, one per wheel hub. The car’s range was 40 miles.
By 1905, however, Henry Ford had begun mass-producing inexpensive cars with gasoline engines, hammering the first nails into the coffin of the early hybrid models.
By 1905, however, Henry Ford had begun mass-producing inexpensive cars with gasoline engines, hammering the first nails into the coffin of the early hybrid models.
Commonly considered to be the company that popularized hybrids, Toyota had its first hybrid prototype on the road in 1976. Two decades later, the first Prius was introduced to the Japanese market in 1997, the same year that Audi introduced the Audi Duo, a hybrid based on the A4 Avant, to the European market. Though Audi and Toyota mass-marketed the first modern gas/electric hybrids in Europe and Asia, it was Honda that brought hybrid technology to Americans with the introduction of the 1999 Insight. A year later, the Toyota Prius went on sale in the U.S.

1.2 Overview of electric and hybrid vehicle

Electric vehicles touted as the future of mobility, are fitted with onboard batteries which, unlike conventional fuel tanks, can be charged using electricity. These batteries, in turn, store and use the energy needed to power a set of electric motors, which ultimately propels the car forward.
Because an electric car is devoid of clutch, gearbox and even an exhaust pipe, it is significantly quieter and offers a smoother ride than conventional gasoline-driven vehicles. When fully charged, a standard EV is capable of covering somewhere between 150 km to 170 km before it needs to be recharged.

One of the chief features of electric vehicles is that they can be plugged into off-board power sources for charging. Essentially, there are two types of EVs: all-electric vehicles (AEVs) and plug-in hybrid electric vehicles (PHEVs). AEVs, in turn, consist of battery electric vehicles (BEVs) and fuel cell electric Vehicles (FCEVs). Both BEVs and FCEVs are charged from the electrical grid and are also usually capable of generating electricity through regenerative braking.

1.2.3 organic matter
Organic matter is a basic component of soil and it controls physical, chemical and biological properties of the soil (McBride, 1994; Stevenson, 1994). Organic matter is particularly involved in the release and retention of nutrients (i.e Ca, Fe, Ca, Mg, Cu, Mn, K) by cation exchange and adsorption of the potentially toxic organic compound, such as pesticides and industrial wastes (McBride, 1994).
Organic matter is a soil is a commonly and collectively referred to as humic substance and this can be further divided into humic acid, fulvic acid, and humic based on solubility (McBride, 1994). Humic and fulvic acids contribute to the majority of cation binding properties of soils (Tipping, 2005) and dissolved organic carbon (Doc) is the main source of fulvic acids which are known to increase the carrying capacity of soil solutions for strongly organic complexing metals such as Cu (Amery et al., 2007; Ashworth and alloway, 2007); Bolan and Duraisamy, 2003; Cattani et al; 2006; Chaignon et al; 2003; Sauve et al; 1997), and in some instane Ni (Antoniaadias and Alloway, 2002b; ashworth and Alloway, 2004;Doig and Liber, 2007). Metal ions can be complexed by the COO- and CooH-groups resent in both Doc and solid organic matter to form stable complexes (McLaren and Crawford, 1973a; Strevenson, 1994). Conseqently,the amount of organic mtter,in particular DOC increase the opportunity for forming stable organometal complexes (McBride, 1994)
In general these stable soluble organo-metal complexes are considered largely unavailable for plant uptake (Reichman, 2002), and whilst the amount of metal in solution may increase, metal, especially Cu may be less available as a result of complexing with DOC (Amery et al,. 2008;Kalis et al., 2006)
Several studies have shown that the presence of DOC does affect the solubility and bioavailability of Cd (Antoniadis and Alloway, 2002b; Antonladis and Tsadilas, 2007; Gray and McLaren, 2006; Gray et al., 19999a; McLaughlin, 2002; McLaughlin et al., 2006). A study by Antoniadis and Alloway (2002b) found that increased amounts of DOC from biosolids application to land were correlated with an increase in Cd solubilty as well as plant uptake of Cd. In contrast, the case of Cd applied to soils and Cd contained in biosolids, Cd was shown to be less available for plant uptake than the more water-soluble forms, such as Cd salts (McLaughlim et al., 2006). Additionally, a study by Antonladis and Tsadilas (2007), observed that the complexion of Cd by DOC and retention in organic matter were less likely to occur in the presence of Cu, Ni or Zn as Cd does not compete for these sites as well as these metals. Thus while DOC plays an important role in determining the solubility and availability of Cd,it appears to be more influenced by the presence of other cation such as Ca (Antonladies and Tsadilas, 2007; Antonladies et al ., 2008; Gray and McLaren, 2006).
Copper forms strong coordination complexes with organic matter, hence Cu present in soils is relatively immobile, with a large proportion associated with organic matter in a solid phase (Ashworth and Alloway, 2007; Mcbride et al., 1997b; McLaren and crawford, 1973a; Reichman,2002; Stevnson, 1994). In experiments carried out by Button et al. (2005a and 2005b) it was revealed that Cu was sorbed predominantly to soil organic matter and Cu interaction with DOC were shown to influence the sorption-desorption process that primary control Cu behavior in soil (McBride. 1995; McLaren and Crawford, 1973b).Sauve et al. (1997) also confirmed the importance of organic matter in determining Cu solubility and bioavailability with a study on Cu contamination of the range of soils, showing that greater than 90% of Cu was bound organically irrespective of the soil pH.