Study on the Characterization of Coelomic Fluid of Eudrilus Eugeniae and Its Effect on Plants and Animal Cells in the In-Vitro Culture System

Study on the characterization of coelomic fluid of Eudrilus eugeniae and its effect on plants and animal cells in the in-vitro culture system

Thesis submitted to Bharathidasan University, Tiruchirappalli for the award of the degree of DOCTOR OF PHILOSOPHY IN BOTANY

Submitted by A. VEERAMANI

Under the guidance of Dr. R. RAVIKUMAR Ph.D., Assistant Professor PG and Research Department of Botany Jamal Mohamed College (Autonomous) Tiruchirappalli - 620 020.

November 2010

DECLARATION

I do hereby declare that this work has been originally carried out by me under the guidance and supervision of Dr. R. Ravikumar, Assistant Professor, P.G and Research Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli and this work has not been submitted elsewhere for any other degree, diploma or other similar titles.

Place: Tiruchirappalli Date:

(A. Veeramani)

POST GRADUATE AND RESEARCH DEPARTMENT OF BOTANY JAMAL MOHAMED COLLEGE (Autonomous) TIRUCHIRAPPALLI-620 020.

Dr. R. RAVIKUMAR Ph.D.,

CERTIFICATE

Study on the characterization of coelomic

Thisfluid is of to Eudrilus certify that eugeniae this thesis and itsentitled effect on plants and animal cells in the In-vitro culture system submitted by Mr. A. VEERAMANI, for the degree of Doctor of Philosophy in Botany, to the Bharathidasan University is based on the results of studies carried out by him under my guidance and supervision. This thesis or any part thereof has not been submitted elsewhere for any other degree.

Place: Tiruchirappalli

Date:

R. Ravikumar

Office: 0431-2331235 Extn. 302 Mobile: 9787486255 E-Mail: [email protected] Residence: Plot No.63, Sriram Nagar, Kattur, Tiruchirappalli-620 019.

Acknowledgement

I owe a great many thanks to a great many people who helped and supported me during my Ph.D work.

My deepest thanks to Dr. R. Ravikumar, my research supervisor and also Assistant Professor in the Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India, for guiding and correcting various documents of mine with attention and care. He has taken pains to go through the project and make necessary correction as and when needed.

I am much indebted to Dr. V. Nandhagopal, Associate Professor, Department of Botany, National College (Autonomous), Tiruchirappalli, Tamil Nadu, India, for his valuable advice as a Doctoral Committee member.

I express my thanks to the Management of Jamal Mohamed College and Dr. M. Sheik Mohamed, Principal, for extending support and the facilities to carry out part of my research work in the Departments of Botany and Biotechnology.

I extend my sincere thanks to Dr. S. Ahamed John, Head, PG and Research Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India for permitting me to undertake my research programme in the Department. I would like to express my thanks to the members of the faculty of the PG and Research Department of Botany for giving me valuable suggestions and support in course of my work.

My deep sense of gratitude to Dr. M.M. Shahul Hameed, Head, Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, Tamil Nadu, India for extending the facilities of the Department to perform in vitro studies in Plant and Animal cells that remains a vital part of my research work.

I am grateful to the Principal, Head and members of the faculty of the Department of Botany, Arignar Anna Govt. Arts College, Namakkal, for their constant encouragement all through my Ph.D work. I wish to specially thank my friend and colleague Dr. M. Rajasekarapandian, Assistant professor, Department of Zoology, in every possible way for the encouragement that he showed upon me.

I gratefully acknowledge Mr. S. Senthil Kumar and Mr. M.S. Mohamed Jaabir, Assistant Professors, Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, for their timely help and interpretation of the results.

My family deserves special mention for their inseparable support and prayers. Though I need not thank them, I record my sincere gratitude for the care and support of my wife, son, daughter and my son-in-law in this occasion.

It is a pleasure to pay tribute also to all those friends and fellow-scholars in the Department of Biotechnology for various helps rendered to me. I am proud to record that I had several opportunities to work with exceptionally hard working scholars like them.

Finally, I would like to thank everybody who was important to the successful realization of thesis, as well as expressing my apology that I could not mention personally one by one.

A. Veeramani

S. No. CONTENTS Page No.

1. INTRODUCTION 01

2. REVIEW OF LITERATURE 09

CHAPTER I: Evaluation of different collection methods for coelomic fluid 3. 36 from Eudrilus eugeniae and its biochemical characterization

4. CHAPTER II: Antimicrobial activity of the coelomic fluid of E .eugeniae 64

CHAPTER III: Investigation of plant growth promoting property of the 5. coelomic fluid in plant tissue culture system 79

CHAPTER IV: Evaluation of anticancer property of coelomic fluid of 6. 104 Eudrilus eugeniae in SiHa cell line

CHAPTER V: To study the impact of textile effluent on the fecundity and 7. 118 population of Eudrilus eugeniae

8. SUMMARY 133

10. REFERENCES

11. PUBLICATIONS

“Earthworms-The intestines of the soil"

– Aristotle.

"The plow is one of the most ancient and most valuable of man's inventions; but long before he existed, the land was in fact regularly plowed and still continues to be thus plowed by earthworms. It may be doubted whether there are many other animals which have played so important a part in the history of the world, as have these lowly organized creatures. Without the work of this humble creature, who knows, nothing of the benefits he confers upon mankind, agriculture, as we know it, would be very difficult, if not wholly impossible"

–Charles Darwin.

These are the words, which reveal the importance of Earthworms. It is well known that the earthworms have the ability to support the growth of plants and they can increase the fertility of the soil. There are about 3920 named species of earthworm so far reported worldwide. In

India, so far, 509 species, referable to 67 genera and 10 families, have been reported (Kale,

1991). Earthworms play an important role in agro-ecosystem like enhancing decomposition, humus formation, nutrient cycling and soil structural development (Kladiviko et al., 1986). The practice of vermiculture is at least a century old but it is now being revived worldwide with diverse ecological objectives such as waste management, soil detoxification and regeneration and sustainable agriculture. Earthworms act in the soil as aerators, grinders, crushers, chemical degraders and biological stimulators. They secrete enzymes, proteases, lipases, amylases, cellulases and chitinases which bring about rapid biochemical conversion of the cellulosic and

1 the proteinaceous materials in the variety of organic wastes which originate from homes, gardens, dairies and farms. Recent works have elucidated some of the mechanisms by which earthworms enhance soil aggregation. Ingested aggregates are broken up in liquid slurry that mixes soil with organic material and binding agents. The defecated casts become stable after drying and also earthworms initiate the formation of stable soil aggregates in mining soils.

Ecology of earthworms

Earthworms are burrowing animals and form tunnels by literally eating their way through the soil. The distribution of earthworms in soil depends on factors like soil moisture, availability of organic matter and pH of the soil. They occur in diverse habitats specially those which are dark and moist. Organic materials like humus, cattle dung and kitchen wastes are highly attractive sites for some species. Earthworms are generally absent or rare in soil with a very coarse texture, in soil and high clay content, or soil with pH < 4 (Gunathilagraj, 1996).

Earthworms are very sensitive to touch, light and dryness. Water-logging in the soil can cause them to come to the surface. Worms can tolerate a temperature range between 5ºC to 29ºC. A temperature of 20ºC to 25ºC and moisture of 50–60 percent is optimum for earthworm function

(Hand, 1988).

Biology of earthworms

Earthworms are long, narrow, cylindrical, bilaterally symmetrical, segmented animals without bones. The body is dark brown, glistening and covered with delicate cuticle. They weigh about 700–1400 mg after 10 weeks. They have a muscular gizzard which finely grinds the food

(fresh and decaying plant debris, living or dead larvae and small animals, and bacteria and

2 protozoa mixed with earth) to a size of 2–4 microns. The gut of the earthworm is inhabited by millions of decomposer micro-organisms. They are bisexual animals and cross-fertilization occurs as a rule. Copulation may last for about an hour, the worms then separate. Later the clitellum of each worm eject cocoon where sperms enter to fertilize the eggs. Up to 3 cocoons per worm per week are produced. From each cocoon about 10–12 tiny worms emerge.

Earthworms continue to grow throughout their life and the number of segments continuously proliferates from a „growing zone‟ just in front of the anus. Earthworms contain 70–80 percent high quality lysine rich protein on a dry weight basis. They can be useful as animal feed. Usually the life span of an earthworm is about 3 to 7 years depending upon the type of species and the ecological situation.

Eudrilus eugeniae

Eudrilus eugeniae is an earthworm species indigenous in Africa but it has been bred extensively in USA, Canada, Europe and Asia for the fish bait market, where it is commonly called as the African Night-crawler. E. eugeniae is a large worm appearing brown and red to dark violet like animal flesh. Their length ranges from 3.2 to 14 cm, and 5 to 8 mm in diameter.

It grows faster and better than other species. Life span of this worm ranges from 1 to 3 years. It grows rapidly and reasonably prolific. Under optimum conditions it would be ideal for animal feed protein production. However there has been relatively little work on the biology and ecology of this species. The African night crawler Eudrilus eugeniae is used extensively in commercial vermin culture especially in India. Increased attention is also being given to this species as a possible waste decomposer and as a protein source.

Coelomic fluid of earthworm

A fluid within the coelom of earthworm is known as the coelomic fluid and this fluid is collected by stimulating them in different methods like mild electric shock, puncturing of coelomic cavity and worm water shock method. The coelomic fluid functions as a hydrostatic skeleton and also serves as the circulatory medium. The fluid contains cytolytic, agglutinating and/or antibacterial components, which are involved in the immune systems. Presumably the function of this system is to destroy membranes of foreign cell, a mechanism that causes cell death by cytosol release, and is attributed to the coelomycetes, which secrete humoral effectors into the coelomic fluid. Coelomic fluid is also reported for having anticancer activity. The high concentration of coelomic fluid exhibited toxic effect on HeLa cells, causing the cell lysis and break down into pieces. Antibacterial activity of coelomic fluid is reported to be selective. The coelomic fluid from earthworm is known to contain immunoactive cells and molecules involved in immune defense. Earthworm coelomic fluid is found to contain molecules that bind anti IgA and anti IgG. Elucidation of the earthworm binding site on anti IgG and anti IgA could make earthworm coelomic fluid a valuable reagent in immunological, chemical and biological research.

Vermiculture biotechnology promises to usher in the „Second green revolution‟ by completely replacing the destructive agro-chemicals which did more harm than good to both the farmers and their farmland. Earthworms restore and improve soil fertility and significantly boost crop productivity. Earthworms excreta (vermicast) is a nutritive „organic fertilizer‟ rich in humus, NPK, micronutrients, beneficial soil microbes - „nitrogen-fixing and phosphate solubilizing bacteria‟ and „actinomycetes‟ and growth hormones „auxins‟, „gibberellins‟ and

„cytokinins‟. Both earthworms and its vermicast and body liquid (vermiwash) are scientifically proving as both „growth promoters and protectors‟ for crop plants. In the experiments with corn and wheat crops, tomato and egg-plants it displayed excellent growth performances in terms of height of plants, colour and texture of leaves, appearance of flowers and fruits, seed ears etc, as compared to chemical fertilizers and the conventional compost. There is also less incidences of

„pest and disease attack‟ and „reduced demand of water‟ for irrigation in plants grown on vermicompost. Presence of live earthworms in soil also makes significant difference in flower and fruit formation in vegetable crops. Biomass of earthworms, a byproduct of Vermiculture

Biotechnology (VBT) is rich in „high quality protein‟ and source of nutritive feed materials for fishery, poultry and dairy industries and also for human consumption (Rajiv et al., 2010).

Vermiculture and environmental management

Vermiculture is practiced for the mass production of earthworms with the multiple objectives of waste management, soil fertility and detoxification and vermicompost production for sustainable agriculture. The practice was started in the middle of 20th century and the first serious experiments were established in Holland in 1970, and subsequently in England, and

Canada. Later vermiculture practices were followed in USA, Italy, Philippines, Thailand, China,

Korea, Japan, Brazil, France, Australia and Israel (Edward, 1988). Collie (1978) and Hartenstein and Bisesi (1989) have reported on the management of sewage sludge and effluents from intensively housed livestock by vermiculture in USA. Vermiculture is being practiced and propagated on a large scale in Australia as a part of the „Urban agriculture development program‟ which utilizes the urban wastes. Australia‟s „Worm grower association‟ is the largest in world with more than 1200 members.

India has yet to appreciate the full importance of vermiculture despite the potential for the production of 400 million tonnes of vermicompost annually from waste degradation (Sinha,

1996). Senapati (1992) has stressed the importance of vermiculture for the management of all cellulosic wastes in India. Gunathilagraj and Ramesh (1996) and Gunathilagaraj and

Ravignanam (1996) reported respectively about management of coir and sericultural wastes by earthworms in India. Kale et al. (1993), Seenappa and Kale (1993) and Seenappa et al. (1995) have each advocated vermicomposting and management on aspects of sugar factory waste, solid wastes from the aromatic oil industries, and distillery wastes in India. In 1998, the Government of India announced exemption from tax liability to all those institutions, organizations, and individuals in India practicing vermiculture on a commercial scale. Vermicomposting plants are operating in Pune and Bangalore with 100 tonnes day−1 capacity (Sinha, 1996). Chennai,

Mumbai, Indore, Jaipur and several other Indian cities are also setting up vermiculture farms.

Earthworms in general are highly resistant to many pesticides and have been reported to concentrate the pesticides and heavy metals in their tissues. They also inhibit the soil borne pathogens and work as a detoxifying agent for polluted soil (Davis, 1971; Ireland, 1983). These properties of earthworms can be utilized for effluent treatment and heavy metal and pesticides removal from industrial and agricultural wastes. Earthworms are important „secondary decomposers‟ and vermicomposting in nature is an ongoing process if the natural population of earthworms are undisturbed. Vermiculture engineers the growth of beneficial nitrogen fixing and decomposer bacteria and actinomycetes fungus in the degraded waste (vermicompost). India has voracious waste eater tropical species of earthworms. The warm and moist climatic conditions of

India are also favorable for earthworm rapid biodegradation action. An earthworm promotes the

6 growth of „beneficial decomposer bacteria‟ in waste biomass and acts as an aerator, grinder, crusher, chemical degrader and a biological stimulator. Given the optimum conditions of temperature and moisture, earthworms eat the organic component of the waste biomass, which is finely ground into small particles in their gizzard and passed on to the intestine for enzymatic actions.

The worms secrete enzymes; proteases, lipases, amylases, cellulases and chitinases in their gizzard and intestine which bring about rapid biochemical conversion of the cellulosic and the proteinaceous materials in the organic wastes (Hand, 1988). The gizzard and the intestine work as a „bioreactor.‟ Only 5–10 percent of the chemically digested and ingested material is absorbed into the body and the rest is excreted out in the form of fine mucus coated granular aggregates called „vermicastings‟ which are rich in nitrates, phosphates and potash. Earthworm participation enhances natural biodegradation and decomposition of wastes from 60 to 80 percent

(given optimum temperature and moisture) thus significantly reducing the composting time by several weeks. The process of decomposition is odour-free because earthworms release coelomic fluids in the decaying waste biomass which have antibacterial properties and kill pathogens

(Pierre et al., 1982). Earthworms also create aerobic conditions in the waste materials, inhibiting the action of anaerobic micro-organisms which release foul-smelling hydrogen sulfide and mercaptans. Eisenia fetida, E. andrei, Eudrilus eugeniae, Lumbricus rubellus and Perionyx excavatus are major waste eater and biodegrader earthworm species. They are used worldwide for waste degradation and are found to be very successful functionaries for the ecological management of organic municipal wastes (Edwards, 1988). E. eugeniae and P. excavatus are believed to be the more versatile waste managers (Graff, 1981; Kale et al., 1982).

Though earthworms are popular among the farmers to scientists for their plant growth promoting activities to anticancer activities, there are not enough studies available that has recorded the molecular basis of these findings. Hence, it was proposed to characterize the coelomic fluid and its various biological activities such as anti microbial and anti-cancer besides its obvious plant growth promoting property. Being a soil dweller and a known decomposer, a study has been conducted to evaluate the impact of textile effluent discharge the population of the earthworm. Eudrilus eugeniae species of earthworm was chosen for this study and the objectives were

1. Evaluation of different collection methods for coelomic fluid from Eudrilus eugeniae and

its biochemical characterization.

2. Evaluation of the antimicrobial activity of the coelomic fluid on selected pathogenic

strains.

3. To investigate the plant growth promoting property of the coelomic fluid in plant tissue

culture system.

4. To evaluate the anticancer potential of coelomic fluid of Eudrilus eugeniae in SiHa cell

line

5. To study the impact of textile effluent on the fecundity and population of Eudrilus

eugeniae.

In 1881, Charles Darwin published his last book "The formation of vegetable mould through the action of worms with observation on their habits" shortly before his death. The book drew attention to the great importance of earthworms in the breakdown of dead plant material and the release of essential nutrients from it. However, only in last few decades, potential of earthworms for breaking down organic waste has been explored in depth and many large scale vermicomposting facilities have been developed all over the world with varying success.

Earthworms

The earthworm derives its name from the fact that it burrows and eats its way into the earth. Earthworms have been on the earth for over 20 million years. There are 3920 species of earthworms distributed throughout the world. Aquatic worms are called as microdrilli and terrestrial earthworms are known as megadrilli. In India, there are about 509 species of earthworms, belonging to 67 genera. Besides these, more than 20 species from other countries have been introduced into India. These are known as 'peregrines'. Earthworm occur in diverse habitats, organic materials like manures, litter, compost etc are highly attractive for earthworms but they are also found in very hydrophilic environment close to both fresh and brackish water, some species can survive under snow (Sharma et al., 2005).

Classification of earthworm

Kingdom: Animalia, Phylum: Annelida, Class: Oligochaeta, Order: Opisthopora, Family:

Lumbricidae, Genus: A large number of genera have been described in literature, Species: A large number of species under each genus have been described in literature. Earthworms have also been classified on the basis of their ecological niche (Bouche, 1977) and feeding behaviour

(Lee, 1985) [Fig. 2.1]. A brief discussion of different ecological groups of the earthworms is given below:

E. eugeniae

P. excavatus

Fig. 2.1: Classification of earthworms based on ecological groups and niche

Epigeic species

These species live above the mineral soil surface typically in the litter layers and plant debris and feed on them. These are phytophagous. Most of the species have insignificant role in humus formation and are not good for use in field conditions for soil reclamation. They have high reproductive rate and high cocoon production rate. However, their life span is relatively short. They show high metabolic activity and hence are particularly useful for vermicomposting.

Examples are Eisenia fetida, Eisenia andrei, Eudrilus eugeniae, Perionyx excavates and

Drawida modesta.

Endogeic species

These species inhabit mineral soil beneath the top soil surface generally forming horizontal tunnels to the soil surface. They feed on soil more or less enriched with organic matter. They are probably important in improvement of soil texture and structure (pedogenesis) and are not much beneficial in organic matter decomposition and recycling of plant nutrients.

Their reproduction rate is moderate and they have shorter life span. Example is Octochaetona thurstoni.

Anecic species

These are surface feeding earthworms that construct and live in permanent burrows in the mineral soil layers but come to the surface to feed on organic matter, mostly plant litter, and pull it into their burrows. They are important in burying surface litter. They are great help in incorporation of organic matter into the soil, and distribution and cycling of plant nutrients, and also in improvement of soil structure and texture (pedogenesis). These species have low cocoon production rate and limited reproductive capacity, but their life span is longer. Examples are

Lampito mauritti, Lumbricus terrestris and Octochaetona serrata. A summary of characteristics used by Bouche to distinguish the earthworms on the basis of ecological niche is given in

Table A (Gajalakshmi and Abbasi, 2004a).

Classification of earthworms based on feeding behavior

According to their feeding habits, earthworms are classified into detritivorous and geophagus (Lee, 1985). Detritivores feed at or near the soil surface mainly on plant litter or dead roots and other plant debris in the organic matter rich surface soil or on mammalian dung. These include epigeic and anecic forms. These are also called as humus formers. Geophagus feed deeper beneath the surface, ingesting large quantities of organically rich soil. These include endogeic forms. These are also called as humus feeders. For the purpose of vermicomposting of different organic wastes, generally epigeic species of earthworms are used widely in India

(Ismail, 2005). It is generally known that the epigeic species Eudrilus eugeniae, Perionyx excavates and Eisenia fetida have a potential as waste decomposers. In order to utilize these species successfully in vermicomposting and vermiculture all aspects of their biology and physical requirements must be known. The life-cycle of each of the three species are now well documented after intensive studies under controlled conditions. Venter and Reinecke (1988) presented studies on Eisenia fetida, Reinecke et al. (1992) on Eudrilus eugeniae, and Hallatt et al., (1990) on Perionyx excavatus. From a comparison of the lifecycle it is evident that all three species are prolific breeders, maintaining a high reproduction rate under favourable conditions of temperature, moisture and food availability.

Food and feeding habits of earthworms

Earthworms exhibit a high degree of niche diversity (Table A). Surface dwellers largely feed upon leaf litter on soil surface. Burrow formers swallow soil and derive nutrition from it.

The quantity and quality of food available in an ecosystem determines population size, composition and diversity of earthworm community. In general, daily ingestion of feed varies

12 from 100 to 300 mg/g of worm body weight. According to one estimate, an earthworm can consume 8 to 20 g dung/year. So at a population density of 1,20,000 adults/ha, dung consumption would be 17.20 tones/ha/year (Bhatnagar and Palta, 1996). In a temperate deciduous forest, annual leaf fall of approximately three tones/ha/year will be consumed just in threemonths (Satchell, 1983). These estimates thus amply indicate that earthworms are important in soil biota mixing and incorporating organic matter into soil. Some earthworms are able to selectively digest certain microorganisms (Dash et al., 1984).

Characteristics Epigeic Endogeics Anecics

Body Size Small Large Moderate

Burrowing habit Reduced Developed Strongly developed Longitudinal No Little Developed contraction Hooked chetae Absent Absent Present

Sensitivity to light Low Strong Moderate

Mobility Rapid Slow Moderate

Skin moistening Developed Feeble Developed Dorsal and Pigmentation Homochromic Absent Anterior Fecundity High Low Moderate

Maturation Rapid Slow Moderate

Respiration High Feeble Moderate Survival under adverse as cocoons By quiescence True diapause conditions

Table A: Summary of characteristics to classify the earthworms on the basis of ecological niche (Gajalakshmi and Abbasi, 2004a)

Biology of earthworms

Earthworms are long, thread-like, elongated cylindrical, soft bodied worms with uniform ring like structures all along the length of their body. Earthworms vary greatly in size. In India some peregrine species like Microscotex phosphoreus (Duges) are only 20 mm long while some endemic geophagous worms such as Drawida grandus (Bourus) may reach up to one meter in length. The colour of earthworms generally ranges from a brownish- black tinge to purple with some exceptions. Generally the dorsal side of the worms is darker while ventral side is paler.

Their bodies are segmented which are arranged linearly and outwardly highlighted by circular grooves which are called annuli.

The number of segments varies from 80-100 or more. The first segment into which the mouth opens is called as peristomium. On the dorsal surface of the peristomium is a lobe like structure called prostomium which overhangs the mouth. The last segment is called the anal segment and it has a perforation for the anus at the hind end. At the sides of the body on the ventral surface of each segment are four pairs of short, stubby brittles or setae. The setae provide action for movement and also enable the worms to cling to their burrows when predators try to pull them out. Earthworms possess both male and female gonads. At maturity, it develops swollen region behind the anterior which is called as clitellum. It deposits its eggs in a cocoon without the free larval stage.

Cocoon production starts at the age of 6 weeks and continues till the end of 6 months.

Under favorable conditions, one pair of earthworms can produce 100 cocoons in 6 weeks to 6 months (Ismail, 1997). Cocoon is a translucent, small, spherical protective capsule in which

14 earthworms lay their eggs. The shape, size, colour and number of cocoons vary from species to species. The incubation period of a cocoon is roughly about 3-5 weeks, in temperate worms it ranges between 3-30 weeks and in tropical worms within 1-8 weeks. Quality of organic waste is one of the factors determining the onset and rate of reproduction (Garg et al., 2005). Epigeic earthworms swallow large quantities of decaying animal waste and plant litter. The quantity of food taken by a worm varies from 100 to 300 mg g-1 body weight day -1 (Edwards and Lofty,

1972). While the worm is feeding, the buccal chamber is everted and the food is drawn into the mouth by the sucking action of the muscular pharynx. The gizzard serves to break the food into fine particles which is then sent into the intestine where gastric juices act on the ingested food to digest proteins, fats and carbohydrates. The excreta is egested through the anus as castings

(Ismail, 2005).

Composting, Vermicomposting and Vermiculture

Composting is bioconversion of organic matter by heterotrophic microorganisms

(bacteria, fungi, actinomycetes and protozoa) into humus-like material called compost. The process occurs naturally provided the fight organisms, moisture, aerobic conditions, feed material and nutrients are available for microbial growth. By controlling these factors the composting process can occur at a much faster rate.

Vermicomposting is the process by which worms are used to convert organic materials

(usually wastes) into a humus-like material known as vermicompost. The goal is to process the material as quickly and efficiently as possible. Vermiculture is the culture of earthworms. The goal is to continually increase the number of worms in order to obtain a sustainable harvest. The

15 worms are either used to expand a vermicomposting operation or sold to customers who use them for the same or other purposes. If the goal is to produce vermicompost then we want to have maximum worm population density all of the time. If the goal is to produce worms then we keep the population density low enough so that reproductive rates are optimized.

Principle of vermicomposting

Certain species of earthworms can ingest organic waste rapidly and fragment them into much fine particles by passing them through gizzard. The earthworms maintain aerobic conditions in the vermicomposting process, ingest solids and convert a portion of it to earthworm biomass and respiration products and egest peat like material termed as worm castings (Loehr et al., 1985). Castings are much more fragmented, porous and microbially active than parent material (Edwards, 1988a; Edwards, 1998b; Edwards and Bohlen, 1996) due to humification and increased decomposition. The earthworms derive their nourishment from the microorganisms involved in the waste decomposition; and organic waste to be decomposed. The earthworms and the microorganisms act symbiotically to accelerate and enhance the decomposition of the organic waste. The composition of the worm casting depends on the parent material. During this process, important plant nutrients such as nitrogen, potassium, phosphorus, calcium etc. present in the waste are converted through microbial action into forms that are much more soluble and available to plants than those in the parent substrate (Ndegwa and Thompson, 2001). Overall, the vermicomposting process is a result of the combined action of earthworms and microflora living in earthworm intestine and in the organic waste (Albanell et al., 1988).

A revolution is unfolding in vermiculture studies for vermicomposting of diverse organic wastes by vermiculture technology using waste eater earthworms into a nutritive „organic fertilizer‟ and using them for production of „safe food‟, both in quantity and quality without recourse to agro-chemicals. Heavy use of agro-chemicals since the „green-revolution‟ of the

1960‟s boosted food productivity, with the cost of environment and society. It killed the beneficial soil organisms and destroyed their natural fertility, impaired the power of „biological resistance‟ in crops to make them more susceptible to pests and diseases. Chemically grown foods have adversely affected human health. The scientific community all over the world is desperately looking for an „economically viable, socially safe and environmentally sustainable‟ alternative to the agro-chemicals. Vermiculture biotechnology promises to usher in the „Second green revolution‟ by completely replacing the destructive agro-chemicals which did more harm than good to both the farmers and their farmland during the „First green revolution‟ of the 1950 -

60‟s. Earthworms restore and improve soil fertility and boost crop productivity by the use of their excreta - „vermicast‟. They excrete beneficial soil microbes, and secrete polysaccharides, proteins and other nitrogenous compounds into the soil. They promote soil fragmentation and aeration, and bring about „soil turning‟ and dispersion in farmlands.

Worm activity can increase air-soil volume from 8 - 30%. One acre of land can contain up to 3 million earthworms, the activities of which can bring up to 8 - 10 tons of „top soil‟ to the surface every year. Presence of worms improves water penetration in compacted soils by 50%

(Kangmin and Peizhen, 2010; Ghabbour, 1996; Bhat and Kambhata, 1994). A study in India showed that an earthworm population of 0.2 - 1.0 million per hectare of farmlands can be established within a short period of three months. On an average 12 tons/ hectare/year of soil or

17 organic matter is ingested by earthworms, leading to upturning of 18 tons of soil/year, and the world over at this rate it may mean a 2 inches of fertile humus layer over the globe (Bhawalkar and Bhawalkar, 1993; White, 1997). Earthworms have over 600 million years of experience in waste and land management, soil improvement and farm production. No wonder, Charles

Darwin called them as the „unheralded soldiers of mankind and farmer‟s friend working day and night under the soil‟ (Martin, 1976; Satchell, 1983). Importance of earthworms in growth of pomegranate fruit plants was indicated by the ancient Indian scientist Surpala in the 10th Century

A.D. in his epic „Vrikshayurveda‟ (Science of tree growing) (Sadhale, 1996).

The concept of sustainable agriculture

It is not enough to produce „sufficient food‟ to feed the civilization but also to produce a

„high quality of nutritive food‟ which should be „safe‟ (chemical free) and also „protective‟ to human health and to produce it in a sustainable manner to ensure „food security‟ for all, but most for the poor developing countries in the long term. „Food safety and security‟ is a major issue everywhere in the world and this urgently needs a change in strategy of farm production. The new concept of farm production against the destructive „chemical agriculture‟ has been termed as

„sustainable agriculture‟. This is about growing „nutritive and protective foods‟ with the aid of biological based „organic fertilizers‟ without recourse to agro-chemicals. This is thought to be the answer for the „food safety and security‟ for the human society in future. The U.S. National

Research Council (1989) defined sustainable agriculture as „those alternative farming systems and technologies incorporating natural processes, reducing the use of inputs of off-farm sources, ensuring the long term sustainability of current production levels and conserving soil, water, energy and farm biodiversity‟. It is a system of food production which avoids or largely excludes

18 the use of systematically compounded chemical fertilizers and pesticides and use of environmentally friendly organic inputs.

A powerful growth promoter and plant protector

Earthworms vermicompost is a highly nutritive organic fertilizer which is rich in humus, nitrogen (N, 2 - 3%), phosphorus (P, 1.55 - 2.25%), potassium (K, 1.85 - 2.25%), micronutrients, beneficial soil microbes like „nitrogen-fixing bacteria‟ and mycorrhizal fungi. This organic fertilizer was scientifically proved as miracle plant growth promoters (Tiwary et al., 1989; Binet et al., 1998, Chaoui et al., 2003; Guerrero, 2010). Kale and Bano (1986) reports as high as

7.37% of nitrogen and 19.58% of phosphorus as P2O5 in worm‟s vermicast. Furthermore,

Suhane et al. (2007) showed that exchangeable potassium (K) was over 95% higher in vermicompost compared with conventional compost. There are also over 60% higher amounts of calcium (Ca) and magnesium (Mg). Vermicompost has very high porosity, aeration, „drainage‟ and water holding capacity. The more important is that it contains plant-available nutrients and appears to increase and retain the nutrients for longer period of time. Pajon (Undated) rated it as

4 - 7 times more powerful growth promoter than conventional compost. A matter of still greater agronomic significance is that worms and vermicompost increases biological resistance in plants

(due to actinomyctes) and protect them against pest and diseases either by repelling or by suppressing them (Anonymous, 2001; Rodriguez et al., 2000; Edwards and Arancon, 2004).

Many studies have shown that the presence of earthworm and its vermicompost resulted in advantages as explained below.

High levels of bio-available nutrients for plants

Earthworms mineralize the nitrogen (N), phosphorus (P), and all essential organic and inorganic elements in the compost to make it bio-available to plants as nutrients (Buchanan et al., 1988). They recycle N in soil in very short time, ranging from 20 - 200 kg N/ha/year and increase nitrogen contents by over 85% (Patil, 1993). After 28 weeks the soil with living worms contained 75 ppm of nitrate nitrogen (N), compared with the controlled soil which had only 45 ppm (Barley and Jennings, 1959). Worms increase nitrogen levels in soil by adding their metabolic and excretory products (vermicast), mucus, body fluid, enzymes and decaying tissues of dead worms (Dash and Patra, 1979; Whalen et al., 1999). Lee (1985) suggested that the passage of organic matter through the gut of worm results in phosphorus (P) converted to more bio-available forms. This is done by both worm‟s gut enzyme „phosphatases‟ and by the phosphate solubilizing microorganisms in the worm cast (Satchell and Martin, 1984).

High level of beneficial and biologically active soil microorganisms

Among beneficial soil microbes stimulated by earthworms are nitrogen-fixing and phosphate solubilizing bacteria, the actinomycetes and mycorrhizal fungi. Suhane et al. (2007) found that the total bacterial count was more than 1010/gr of vermicompost. It included

Actinomycetes, Azotobacter, Rhizobium, Nitrobacter and Phosphate Solubilizing Bacteria, ranging from 102 - 106 per g of vermicompost.

Humus

Vermicompost contains „humus‟ excreted by worms which makes it markedly different from other organic fertilizers. It takes several years for soil or any organic matter to decompose

20 to form humus while earthworms secrete humus in its excreta. Without humus plants cannot grow and survive. The humic and fulvic acids in humus are essential to plants in four basic ways:

1) Enables plant to extract nutrients from soil; 2) Help dissolve unresolved minerals to make organic matter ready for plants to use; 3) Stimulates root growth; and 4) Helps plants overcome stress. Presence of humus in soil even helps chemical fertilizers to work better (Kangmin, 1998;

Kangmin and Peizhen, 2010). This was also indicated by Tomati et al. (1987) and Canellas et al.

(2002) found that humic acids isolated from vermicompost enhanced root elongation and formation of lateral roots in maize roots. Humus in vermicast extracts „toxins‟, „harmful fungi and bacteria‟ from soil and protects plants.

Plant growth hormones

Edwards and Burrows (1988) and Atiyeh et al. (2000) speculated that the growth responses of plants from vermicompost appeared more like „hormone-induced activity‟ associated with the high levels of nutrients, humic acids and humates in vermicompost.

Researches show that vermicompost use further stimulates plant growth even when plants are already receiving „optimal nutrition‟. It consistently improved seed germination, enhanced seedling growth and development, and increased plant productivity significantly much more than would be possible from the mere conversion of mineral nutrients into plant-available forms.

Neilson (1965), Tomati et al. (1987, 1995) and Suhane et al. (2007) have also reported that vermicompost contained growth promoting hormone „auxins‟, „cytokinins‟ and flowering hormone „gibberellins‟ secreted by earthworms.

Soil enzymes

Vermicompost contain enzymes like amylase, lipase, cellulase and chitinase, which continue to break down organic matter in the soil (to release the nutrients and make it available to the plant roots) even after they have been excreted (Tiwary et al., 1989; Chaoui et al., 2003).

They also increase the levels of some important soil enzymes like dehydrogenase, acid and alkaline phosphatases and urease. Urease play a key role in N2-cycle as it hydrolyses urea and phosphates bioconvert soil phosphorus into bio-available form for plants.

Controlling pest and disease without pesticides

Earthworms are both „plant growth promoter and protector‟. There has been considerable evidence in recent years regarding the ability of earthworms and its vermicompost to protect plants against various pests and diseases either by suppressing or repelling them or by inducing biological resistance in plants to fight them or by killing them through pesticidal action.

Furthermore, the actinomycetes fungus excreted by the earthworms in their vermicast produce chemicals that kill parasitic fungi, such as Pythium and Fusarium (Edward and Arancon, 2004).

Yardim et al. (2006) reported that application of vermicompost reduced the damage by striped

Cucumber beetle (Acalymma vittatum), spotted Cucumber beetle (Diabotrica undecimpunctata) and larval hornworms (Manduca quinquemaculata) on tomatoes in both greenhouse and field experiments. There are several plant protection abilities of earthworms. Recently, Newington et al. (2004) have implicated earthworms as influencing the abundance of above-ground herbivores and their natural enemies (crop pests) which they devour.

Ability to induce biological resistance in plants

Vermicompost contains some antibiotics and actinomycetes which help in increasing the

„power of biological resistance‟ among the crop plants against pest and diseases. Pesticide spray was significantly reduced where earthworms and vermicompost were used in agriculture (Singh,

1993; Suhane et al., 2007).

Ability to repel crop pests

There seems to be strong evidence that worms varmicastings sometimes repel hard- bodied pests (Anonymous, 2001). Edwards and Arancon, (2004) reports statistically significant decrease in arthropods (aphids, buds, mealy bug, spider mite) populations, and subsequent reduction in plant damage, in tomato, pepper, and cabbage trials with 20 and 40% vermicompost additions. George Hahn, doing commercial vermicomposting in California, U.S., claims that his product repels many different insects‟ pests. His explanation is that this is due to production of enzymes „chitinase‟ by worms which breaks down the chitin in the insect‟s exoskelton (Munroe,

2007).

Ability to suppress plant disease

Arancon et al. (2002) reported that vermicompost application suppressed 20 - 40% infection of insect pests that is, aphids (Myzus persicae), mearly bugs (Pseudococcus spp.) and cabbage white caterpillars (Peiris brassicae) on pepper (Capiscum annuum), cabbage (Brassica oleracea) and tomato (Lycopersicum esculentum). Furthermore, Edwards and Arancon (2004) have found that the use of vermicompost in crops inhibited the soil-born fungal diseases. They also found significant suppression of plant-parasitic nematodes in field trials with pepper,

23 tomatoes, strawberries and grapes. The explanation behind this concept is that high levels of agronomically beneficial microbial population in vermicompost protects plants by out-competing plant pathogens for available food resources that is, by starving them and also by blocking their excess to plant roots by occupying all the available sites.

In addition, Edwards and Arancon (2004) also reported the disease suppressing effects of applications of vermicompost, on attacks by fungus Pythium on cucumber, Rhizoctonia on radishes in the greenhouse, by Verticillium on strawberries and by Phomposis and Sphaerotheca fulginae on grapes in the field. In all these experiments vermicompost applications suppressed the incidence of the disease significantly. They also found that, the ability of pathogen suppression disappeared when the vermicompost was sterilized, convincingly indicating that the biological mechanism of disease suppression involved was „microbial antagonism‟. Meanwhile,

Edwards et al. (2007) reported considerable suppression of root knot nematode (Meloidogyne incognita) and drastic suppression of spotted spider mites (Tetranychus spp.) and aphid

(M. persicae) in tomato plants after application of vermicompost teas (vermiwash liquid). They are serious pests of several crops.

Vermiwash - A growth promoting and plant protecting

The brownish-red liquid which collects in all vermicomposting practices is also

„productive‟ and „protective‟ for farm crops. This liquid partially comes from the body of earthworms (as worm‟s body contain plenty of water) and is rich in amino acids, vitamins, nutrients like nitrogen, potassium, magnesium, zinc, calcium, iron and copper and some growth hormones like „auxins‟, „cytokinins‟. It also contains plenty of nitrogen-fixing and phosphate

24 solubilising bacteria (nitrosomonas, nitrobacter and actinomycetes). Vermiwash has great

„growth promoting‟ as well as „pest killing‟ properties. Buckerfield and Webster (1998) reported that weekly application of vermiwash increased radish yield by 7.3%. Thangavel et al. (2003) also observed that both growth and yield of paddy increased with the application of vermiwash and vermicast extracts.

Farmers from Bihar in North India reported growth promoting and pesticidal properties of this liquid. They used it on brinjal and tomato with excellent results. The plants were healthy and bore bigger fruits with unique shine over it. Spray of vermiwash effectively controlled all incidences of pests and diseases significantly reduced the use of chemical pesticides and insecticides on vegetable crops and the products were significantly different from others with high market value (Suhane et al., 2007; Sinha et al., 2009). George et al. (2007) studied the use of vermiwash for the management of „Thrips‟ (Scirtothrips dorsalis) and „Mites‟

(Polyphagotarsonemus latus) on chilli amended with vermicompost to evaluate its efficacy against thrips and mites. Vermiwash was used in three different dilutions e.g. 1:1, 1:2 and 1:4 by mixing with water both as „seedling dip‟ treatment and „foliar spray‟. Six rounds of vermiwash sprays were taken up at 15 days interval commencing at two weeks after transplanting.

Among the various treatments, application of vermicompost at the rate of 0.5 ton/ha with

6 sprays of vermiwash at 1:1 dilution showed significantly lower incidence of thrips and mites attack. The treatment resulted in very low mean population of thrips and mites. In addition, the application of vermicompost gave a highest yield (2.98 quintal/ha). Giraddi (2003) also reported significantly lower pest population in chilli applied with vermiwash (soil drench 30 days after

25 transplanting, and foliar spray at 60 and 75 days after transplanting) as compared to untreated crops. Suthar (2010 a) has reported hormone like substances in vermiwash. He studied its impact on seed germination, roots and shoots length in Cyamopsis tertagonoloba and compared with urea solution (0.05%). Maximum germination was 90% on 50% vermiwash as compared to

61.7% in urea solution. Maximum root and shoot length was 8.65 and 12.42 cm on 100% vermiwash as compared to 5.87 and 7.73 cm on urea. The seedlings with 100% vermiwash foliar spray showed the maximum level of total protein and soluble sugars in their tissues.

Studies on the role of Vermiculture Biotechnology (VBT)

There have been several reports that earthworms and their excretory products (vermicast) can induce excellent plant growth and enhance crop production. Baker et al. (1997) found that the earthworms (Aporrectodea trapezoids) increased growth of wheat crops (Triticum aestivum) by 39%, grain yield by 35%, lifted protein value of the grain by 12% and also resisted crop diseases as compared to the control. Baker et al. (2006) also reported that in Parana, Brazil invasion of earthworms significantly altered soil structure and water holding capacity. The grain yields of wheat and soybean increased by 47 and 51%, respectively, Palanisamy (1996) reported that earthworms and its vermicast improve the growth and yield of wheat by more than 40%.

Bhatia et al. (2000), Sharma (2001) and Suthar (2010a, 2010b) also reported better yield and growth in wheat crops applied with vermicompost in soil. Kale et al. (1992) who studied on the agronomic impacts of vermicompost on rice crops (Oryza sativa) reported that greater population of nitrogen fixers, actinomycetes and mycorrhizal fungi inducing better nutrient uptake by crops and better growth. Jeyabal and Kuppuswamy (2001) studied the impact of vermicompost on rice- legume cropping system in India.

They showed that the integrated application of vermicompost, chemical fertilizer and biofertilizers (Azospirillum and Phosphorbacteria) increased rice yield by 15.9% over chemical fertilizer used alone. Guerrero and Guerrero (2008) also reported good response of upland rice crops grown on vermicompost. Buckerfield and Webster (1998) found that worm worked waste

(vermicompost) boosted grape yield by two-fold as compared to chemical fertilizers. Treated vines with vermicompost produced 23% more grapes due to 18% increase in bunch numbers.

Furthermore, a study on grapes carried out on „eroded wastelands‟ in Sangli district of

Maharashtra, India, treated with vermicasting at the rate of 5 tons/ha showed that the grape harvest was normal with improvement in quality, taste and shelf life. The soil analysis showed that within one year pH came down from 8.3 - 6.9 and the value of potash increased from 62.5 -

800 kg/ha. There was also marked improvement in the nutritional quality of the grape fruits

(Sinha et al., 2009).

Arancon et al. (2004) studied the agronomic impacts of vermicompost and inorganic

(chemical) fertilizers on strawberries (Fragaria ananasa) when applied separately and also in combination. Significantly, the „yield‟ of marketable strawberries and the „weight‟ of the „largest fruit‟ was 35% greater on plants grown on vermicompost as compared to inorganic fertilizers in

220 days after transplanting. Also, there were 36% more „runners‟ and 40% more „flowers‟ on plants grown on vermicompost. Also, farm soils applied with vermicompost had significantly greater „microbial biomass‟ than the one applied with inorganic fertilizers. Singh et al. (2008) also reported that vermicompost increased the yield of strawberries by Sinha et al. 32.7% and also drastically reduced the incidence of physiological disorders like albinism (16.1 - 4.5%), fruit malformations (11.5 - 4%), grey mould (10.4 - 2.1%) and diseases like Botrytis rot. By

27 suppressing the nutrient related disorders, vermicompost application increased the yield and quality of marketable strawberry fruits up to 58.6%.

Webster (2005) studied the agronomic impact of vermicompost on cherries and found that, it increased yield of „cherries‟ for three years after „single application‟ inferring that the use of vermicompost in soil builds up fertility and restore its vitality for long time and its further use can be reduced to a minimum after some years of application in farms. Studies on the production of important vegetable crops like tomato (Lycopersicum esculentus), eggplant (Solanum melangena) and okra (Abelmoschus esculentus) have yielded very good results (Guerrero and

Guerrero, 2006; Gupta et al., 2008; Sinha et al., 2009). Agarwal et al. (2010) studied growth impacts of earthworms (with feed materials), vermicompost, cow dung compost and chemical fertilizers on okra (A. esculentus). Worms and vermicompost promoted excellent growth in the vegetable crop with more flowers and fruits development. But the most significant observation was drastically less incidence of „Yellow Vein Mosaic‟, „Color Rot‟ and „Powdery Mildew‟ diseases in worm and vermicompost applied plants. Meena et al. (2007) studied the growth impacts of organic manure (containing earthworm casts) on garden pea (Pisum sativum) and compared with chemical fertilizers. It produced higher green pod plants, higher green grain weight per plant, higher percentage of protein content and carbohydrates and higher green pod yield (24.8 - 91%) as compared to chemical fertilizer.

Baker et al. (2006) reported a study of earthworms on soil properties and herbage production in a mined field micro-plot experiment in Ireland. The presence of earthworms had little effect on herbage production in the first year. But total herbage yield was 25% greater in the

28 second year and 49% greater in the third year in plots receiving annual topdressing of cattle slurry with earthworms compared to similarly-treated plots with cattle slurry but without earthworms. The conclusion drawn from such study is that earthworms in soil are paramount in plant productivity. In the first year, it took the worm to restore and condition the mined soil. By second year, enough nutritive „vermicast‟ got accumulated in soil and improved soil fertility which promoted higher herbage yield (25 %). In the third year, the worm population in soil increased significantly leading to higher excretion of vermicast, higher soil fertility and higher plant production (49%). In a bucket experiment they found that the cumulative herbage yields over a period of 20 months was 89% higher in buckets with earthworms added with cattle manure as compared to those without earthworms but only with cattle manure, and only 19% higher in buckets receiving exclusive chemical fertilizers.

Ansari (2008) studied the production of potato (Solanum tuberosum) by application of vermicompost in a reclaimed sodic (alkaline) soil in India. With good potato growth, the sodicity

(ESP) of the soil was also reduced from initial 96.74 - 73.68 in just about 12 weeks. The average available nitrogen (N) content of the soil increased from initial 336.00 - 829.33 kg/ha. Sinha et al. (2009) reported that farmers at Phaltan in Satara district of Maharashtra, India, applied live earthworms to their sugarcane crop grown on saline soils irrigated by saline ground water. The yield was 125ton/ha of sugarcane and there was marked improvement in soil chemistry. Within a year, there was 37% more nitrogen, 66% more phosphates and 10% more potash. The chloride content was less by 46%. Earthworms and its vermicompost works like „miracle growth promoter‟ and is nutritionally superior to the conventional compost and chemical fertilizers.

Reduced incidence of „pest and disease attack‟, and „better taste of organic food products

29 especially „fruits and vegetables‟ grown with vermiculture are matter of great socioeconomic and environmental significance (Hand, 1988; Lee, 2003). Presence of earthworms in soil particularly makes a big difference in growth of flowering and fruit crops and significantly aid in fruit development. The 18% increase in yield of wheat crops over chemical fertilizers in their farm studies made in India has great economic and agronomic significance. Use of vermicompost over the years build up the soil‟s physical, chemical and biological properties restoring its natural fertility.

Subsequently, reduced amount of vermicompost is required to maintain productivity.

VBT will truly bring in „economic prosperity‟ for the farmers, „ecological security‟ for the farms and „food security‟ for the people. With the growing global popularity of „organic foods‟ which became a US $ 6.5 billion business every year by 2000, there will be great demand for earthworms and vermicompost in future (Sinha et al., 2010b). The „natural control of crop pests‟ influenced by earthworms seems particularly fruitful research area to be pursued. More study is required to develop the potential of „vermiwash‟ as a sustainable, non-toxic and environmentally friendly alternative to the „chemical pesticides‟. Earthworms are justifying the beliefs and fulfilling the dreams of Charles Darwin who called earthworms as „friends of farmers‟ and that of Anatoly Igonin of Russia who said „Earthworms create soil and improve soil‟s fertility and provides critical biosphere‟s functions: disinfecting, neutralizing, protective and productive‟.

Anti-cancer activity

Earthworm has been recorded with a long history. Five hundred years ago, Shizhen Li compiled the famous medical book Compendium of Material, in which the earthworm (Earth

30 dragon) was recorded as a drug prescribed for antipyretic and diuretic purposes in the form of dried powder in clinic. Now the remedy is still used in the folk. In the end of 19th century,

Fredericq [1878] discovered one enzyme secreted from the alimentary tract of earthworm. Then several proteases were separated from the earthworm in 1920 (Keilin, 1920). They could dissolve casein, gelatin, and albumin. This was the preliminary research about the earthworm proteases. Large-scale research about earthworm protease began in 1980. Mihara et al. [1983] isolated a group of proteases with fibrinolytic activity from the earthworm Lumbricus rubellus.

Subsequently different purification methods were applied to isolate the enzymes, including gel filtration, affinity chromatography, ion exchanging chromatography, and high-pressure liquid chromatography (HPLC). More proteases have been obtained from different species, such as lumbrokinase (Mihara et. al., 1983), earthworm-tissue plasminogen activator (Wu and Fan,

1986), earthworm plasminogen activator (Yang and Ru, 1997; Yang et al., 1998a; 1998b; 1998c;

1998d; 1998e) component A of EFE (EFEa) (Tang et al., 2000; Tang et al., 2002), and biologically active glycolipoprotein complex (G-90) [Popovic et al., 2001; Popovic et al., 1998;

Hrzenjak et al., 1998a; 1998b; Hrzenjak et al., 1992; Grdisa et al., 2001).

Clinical application and medical research- the earthworm protease as a fibrinolytic agent

The formation of thrombus in the blood causes many devastating diseases such as stroke and myocardial infarction. Several enzymes have been used as the thrombolytic agents including urokinase (UK), streptokinase, recombinant tissue-type plasminogen activator, staphylokinase, and recombinant prourokinase (Verstraete M. 2000; Zhao J. and Li D. 2002). These agents are administered via intravenous injection generally. Some of them are effective, but they also have some limitations such as fast clearance, lack of resistance to reocclusion, bleeding complications,

31 and other adverse effects (Verstraete M. 2000). The earthworm protease functions in the fibrinolysis and plasminogen activation, distinct from those enzymes (UK, tissue-type plasminogen activator, etc.) (Kasai et al., 1985; Madison et al., 1995; Kim et al., 1993).

Therefore they have been used to treat the thrombosis. The proteases during oral experiments both in animals and clinics show significant fibrinolytic efficacy. A distinct amelioration is observed in the treatment of blood high-viscosity syndrome and thrombocytosis (Cong et al.,

2000). In addition, the proteases are stable during a long-term storage at room temperature

(Nakajima et al., 2000), in the form of oral capsule. Earthworm is easily raised, which renders the isozymes into a relatively inexpensive thrombolytic agent. So far, the earthworm proteases have been used as an orally administered fibrinolytic agent to prevent and treat clotting diseases, such as myocardial infarction and cerebral thrombus (Jin et al., 2000).

Antitumor

Cancer has a reputation of being an incurable disease. Although some methods such as surgery, chemotherapy, radiation therapy, and immunotherapy are available, they are far from reaching the goal of complete removal of the cancer cells without damage to the rest of the body.

It is demonstrated that the earthworm crude extract has the ability to kill the cancer cells directly in vitro (Zhang and Wang 1987; Zeng et al., 1995) and inhibit the occurrence and development of tumor in vivo (Wang et al., 1986). Furthermore, it has been proved that the earthworm proteases enhance the curative effects by both radiation therapy and chemotherapy (Zhang et al.,

1991; Zhang et al., 1992). The most malignant tumors secrete urokinase-type plasminogen activator (u-PA). In order to inhibit the hyperactivity of the u-PA, inhibitors of plasminogen activators are synthesized by the surrounding cells for tissue protection, resulting in a high

32 concentration of fibrin locally. The glycolipoprotein mixture (G-90) was isolated from the homogenate of E. fetida (Popovic et al. 1998; Hrzenjak et al., 1998a; 1998b; Grdisa et al., 2001), which is assayed in a euglobulinic test applied to fibrin clot from blood plasma of patients who suffered from malignant tumors. The effect of G-90 on the fibrinolysis rate is related to not only its concentration, but also to histological type where the malignant tumors invade. The blood with the fibrin clots derived from the dogs with cardiopathies and the dogs with malignant tumors was examined for the time of coagulation and fibrinolysis by adding different substances including G-90. The clotting time in the presence of G-90 shows dogs with malignant tumors healthy dogs with cardiopathies (Popovic et al., 2001).

Recently, a glycosylated component is separated from the earthworm E. fetida by Xie and coworkers (Xie et al., 2003), which has relations with apoptosis of tumor cells. It is highly homologous to LrP-I-1 and LrP-I-2. It is identified to be a plasmin and also a plasminogen activator. From the results of the phase-contrast microscopy observation of apoptotic cells and the localization of fluorescent antibodies in cell nucleus, the antitumor activity is observed. The earthworm protease possesses obvious anti-tumor activity in the hepatoma cells. The proliferation of the hepatoma cell treated with the proteases is inhibited in proportion to the concentration of the proteases. The growth of tumor xenograft in nude mice is significantly suppressed after being fed with the earthworm protease for four weeks. At the same time, it has been found that the earthworm protease can induce apoptosis of hepatoma cells and down- regulated the expression of matrix metal protease-2. As described above (Chen et al., 2007), the earthworm protease is a potential candidate for treating some kind of tumors (Rong et al., 2010).

Earthworm mediated bioremediation

Bioaugmentation of soil with xenobiotic-degrading micro-organisms is generally hindered by the poor transport and dispersal of soil inoculants (Elsas and Heijnen, 1990), and has been criticized as an ineffective strategy for treating contaminated soils (Goldstein et al., 1985).

In situ bioremediation is also limited by the supply of suitable electron acceptors (Harding, 1997;

Margesin et al., 2000). While anaerobic conditions can lead to removal of halogenated substituted xenobiotics, such as the PCB‟s, textile dyes etc (Wiegel and Wu, 2000), their mineralization is exclusively aerobic (Furukawa, 1982; Robinson and Lenn, 1994).

Consequently, in oxygen limited sites, such as the soil subsurface, mineralization of toxic and other synthetic dyes (xenobiotics) can be limited without manual mixing of the contaminated soil

(McDermott et al., 1989) or the introduction of oxygen from forced air, pure oxygen or hydrogen peroxide (Alexander, 1999; Harkness et al., 1993).

In nature, the movement of soil animals and earthworms can enhance the transport and distribution of bacteria. Earthworms have been shown to improve the dispersal of soil inoculants through bioturbation (Daane et al., 1997; Doube et al., 1994; Hampson and Coombes, 1989;

Hutchinson and Kamel, 1956; Singer et al., 1999; Stephens et al., 1994; Thorpe et al., 1996), and transport of the microbial inoculant into the burrows via bypass Flow (Bouma et al., 1982;

Edwards et al., 1992; Ehlers, 1975; Farenhorst et al., 2000; Lee, 1985; Madsen and Alexander,

1982; Pivetz and Steenhuis, 1995). Earthworm activity and burrowing also has been shown to increase soil aeration (Kretzschmar, 1978; Kretzschmar, 1987; Lee, 1985; Schack-Kirchner and

Hildebrand, 1998). Through their mucilaginous secretions, earthworms `prime' the soil, thereby increasing microbial activity and mineral nutrient availability (Wolters, 2000).

Despite the abundance of evidence suggesting earthworms could contribute significantly to improving in situ xenobiotic remediation through mixing, aeration, and improved soil fertility, there has been surprisingly little research on their use in a bioremediation strategy and especially the textile bioremediation.

Vermi‟ stands for earthworm, which are regarded as „Farmer‟s friend‟ from the time immemorial. Earthworms are known for their beneficial role in the soil system and form a major component of the soil ecosystem. They have been efficiently ploughing the land for millions of years and are also known as biological indicators of soil fertility (Batt and Steiner, 1992). They support adequate growth of bacteria, fungi, and actinomycetes and protozoan, which are essential for sustaining a healthy soil (Desetir, 1991). They act on organic debris and accelerate the decomposition process in natural manure known as “vermicomposting”.

Vermiculture is a mixed culture containing soil bacteria mixed and an effective strain of earthworms (NIIR Board, 2008). Earthworm has efficiency to consume all types of organic rich waste material including vegetable waste, industrial and other organic waste. Vermicroposting refers to the production of plant nutrient rich excreta of worms.

Earthworms play a vital role in plant growth. It is a quite possible to effect quick change over for sustainable agriculture by harnessing brand new vermicompost technology to the soil.

An earthworm‟s body consists of a series of cylindrical segments, and earthworms move by changing the dimensions of each segment (Gray and Lissmann 1938). A spacious fluid-filled cavity, the coelom, runs the length of the earthworm, providing the animal with support. This fluid is called as the „Coelomic fluid‟. The coelom acts as a hydrostatic skeleton, transferring the forces generated by the muscles to the animal‟s environment (Alexander 1983). Coelomic fluid does not flow from one segment to the next because an earthworm‟s coelom is divided internally by walls called septa. A segment can be regarded as a cylinder of constant volume, because the coelomic fluid cannot be compressed, and experiments by Newell (1950) demonstrated that

36 coelomic fluid does not flow from one segment to the next. A decrease in segment length must be accompanied by an increase in segment circumference, and vice versa. When circular muscles contract, the segment lengthens and axial pressure is produced. When longitudinal muscles contract, the segment shortens and radial pressure is produced. In normal movement the activity of the earthworm‟s muscles is co-ordinated to give waves of segment elongation and contraction which pass from the anterior to the posterior. Earthworms use axial pressure to thrust a segment forwards. Radial pressure is used to widen the burrow, and it also plays a role in enabling the earthworm to grip the burrow walls. The thickness of the circular muscles led Chapman (1950) to propose that radial pressure was the more important pressure for a burrowing earthworm.

Chapman suggested that earthworms burrowed primarily by using radial pressure to widen existing crevices in the soil, and he called this concept “crevice burrowing”. Radial pressures have been found to be higher than axial ones in burrowing earthworms (McKenzie and Dexter

1988a, 1988b; Keudel and Schrader 1999), as Chapman (1950) predicted. Earthworms can ingest soil to create crevices, and then widen the crevices using radial pressure (Kemper et al. 1988). In addition to widening a burrow, earthworms may use radial pressure to reduce stresses in the soil around the anterior of the body.

In recent times, the commercial vermin culturists have started promoting a product called vermiwash. This vermiwash would have enzymes, secretions of earthworms which would stimulate the growth and yield of crops and even develop resistance in crops receiving this spray.

Such a preparation would certainly have the soluble plant nutrients apart from some organic acids and mucus of earthworms and microbes (Shivsubramanian and Ganeshkumar, 2004). But so far there are no experimental evidences or standardized method for collecting the coelomic

37 fluid – the so called „Vermiwash‟. Neither is there any report on the physicochemical properties of this fluid nor any characterization recorded so far in its original concentrated form.

The present study was carried out to evaluate the composition of coelomic fluid by considering different collection methods. The fluid was then subjected to physical and biochemical characterization. For this, following objectives were set.

Standardization of the procedure for collecting coelomic fluid from E. eugeniae

Determination of the biochemical constituents of coelomic fluid.

Characterization of proteins present in the coelomic fluid by SDS PAGE.

Characterization of protease activity of the coelomic fluid by gelatin diffusion assay.

Characterization of endogenous metabolite profile of Coelomic fluid.

Materials and methods

Collection of earthworms, treatment and their maintenance

Earthworm, Eudrilus eugeniae were collected from the Periyar Research Organization for

Bio-Technique & Eco-System (PROBE), Periyar Maniyammai University, Vallam, Tanjore Dist,

Tamil Nadu, South India. Organic waste served as a medium of growth for the worms in the sieved garden soil and cow dung mixed in the ratio of 2:1. After mixing subsequent amount of water, the worms were allowed to be contained in the worm pit of 8‟x2‟ dimension. The optimum temperature was maintained constantly by spreading some banana leaves over the culture basket. Viability of the worms, cocoon production, hatchlings and weight of the worms were carefully monitored periodically. These earthworms served as the source for the collection of coelomic fluid and subsequent analysis.

Collection of coelomic fluid (vermiwash)

Different methods for the collection of the Coelomic fluid was carried out as explained below.

Warm water method

Exactly, 15 g of worms were taken from the culture pit and washed in running distilled water. The worms were then placed on the filter paper to remove excess water droplets and dropped into 25 ml of water in a beaker. The temperature of the water was maintained at 40 C for 30 minutes. The water was then collected back and centrifuged at 5000 rpm for 10 minutes to sediment the larger particles and other debris. This served as the equivalent of the commercially available fluid called „Vermiwash‟. The supernatant was carefully removed and filter sterilized

39 through 0.2µM syringe filter. The filtrate was carefully collected in a sterile microfuge tube and stored in aliquots at -20 C for further analyses (Figure 1.3a).

Heat Shock Method

Exactly, 15 g of the worms were taken from the culture pit and washed in distilled water.

The worms were then dried on a filter paper and carefully placed into the nylon mesh rolled into a „cone‟ shape to fit into the glass funnel. The glass funnel was held in a burette clamp on a titration stand. The set up is shown in the figure 1.3d. A bag of hot water (45 - 50 C) was kept over the funnel such that the worms could feel the rise in the temperature due to the hot water above. The coelomic fluid was made to release through the dorsal pores on the body due to „heat- shock‟ and was collected in a sterile clean dry test tube. The collected coelomic fluid was then centrifuged at 5000 rpm for 10 minutes to deposit the debris and the clear supernatant was sterilized through 0.2µM syringe filter into a clean dry sterile microfuge. The filtrate was stored in aliquots at -20 C for subsequent use and analyses.

Electric shock method

Exactly, 15 grams of worms were taken from the culture pit and washed in running distilled water. The worms were then placed on the filter paper to remove excess water droplets and were taken in a nylon mesh rolled to fit inside a glass funnel. The nylon mesh was given connection to the 5 Volt electric shock using an eliminator (step-down transformer). The worms were kept under shock like this continuously for 30 minutes and then the fluid was collected. The collected fluid was centrifuged at 5000rpm for 10 minutes to sediment larger particles and other debris. The supernatant was carefully removed and filter sterilized through 0.2 µm (pore size)

40 syringe filter. The filtrate was stored in aliquots at -20 C for subsequent use and analyses (1.3b

& c).

Cold shock method

Exactly 15 grams of the worms were taken from the culture pit and washed in distilled water. The worms were then dried on a filter paper and carefully placed into the nylon mesh rolled into a „cone‟ shape to fit into the glass funnel. The glass funnel is held in a burette clamp on a titration stand. The set up is shown in the figure 1.3d. A bag of ice was kept over the funnel such that the worms could feel the drop in the temperature due to the ice above it. The coelomic fluid was made to release through the dorsal pores on the body due to the „cold-shock‟ and was collected in a sterile clean dry test tube. The collected coelomic fluid was then centrifuged at

5000rpm for 10 minutes to deposit the debris and the clear supernatant was sterilized through

0.2µM syringe filter into a clean dry sterile microfuge. The filtrate was stored in aliquots at -

200C for further use and analyses.

Biochemical analysis of coelomic fluid

The coelomic fluid was then subjected to biochemical estimations such as of total sugar, reducing sugar, cholesterol, triglyceride, urea, uric acid, free amino acids, total protein and enzyme activities viz. Glutamate Oxaloacetate Transaminase, Glutamate Pyruvate transaminase and alkaline phosphatase.

Biochemical estimations

Estimation of carbohydrates (Total Sugar)

For estimation of carbohydrates, coelomic fluid both concentrated and diluted fluid was analyzed for the presence of carbohydrates. The total sugars in the samples were estimated by the phenol sulphuric acid method.

Reagents

Phenol reagent- 5 gm of redistilled phenol was dissolved in 95 ml of distilled water.

Method

Known aliquots of sample were made up to 1ml with distilled water and to this 0.5 ml of phenol reagent was added and mixed well. Then 5ml of 96 % sulphuric acid was added and placed in a water bath at 30degree Celsius for 20 minutes and the absorbance was read at 490nm.

The amount of total sugars in the sample was estimated by comparing the results with a standard glucose curve.

Estimation of reducing sugar (by DNS Method)

Principle

Reducing sugars convert dinitrosalicylate under alkaline condition (sodium potassium tartarate) to amino – nitrosalicylate which is an orange – yellowish compound that has an absorption maximum at 540 nm.

Reagents required:

Standard maltose solution

100 mg of maltose is dissolved in distilled water and made up to 100 ml in standard flask.

DNS reagent

About 1 g of 3, 5 – Dinitrosalicylate, 30 g of Sodium potassium Tartarate and 1.6 g of

KOH were dissolved in distilled water and made up to 100 ml. The reagent needed to be prepared fresh before use.

Procedure

In a series of clean and dry test tubes marked as S1 to S5, 0.2 to 1.0 ml were taken. To the tubes marked T1 and T2, 0.2 and 0.5 ml were taken and all the tubes were made up to 1 ml with distilled water. Tube containing 1.0 of distilled water alone was taken as blank. All the tubes were added with 0.5 ml of DNS reagent. The tubes were incubated in boiling water bath for 10 minutes until orange color developed. After cooling, 4 ml of water was added to all the tubes uniformly and their absorbance was measured at 520 nm.

Estimation of free amino acids (by Ninhydrin Method)

Principle

Ninhydrin also chemically known as triketohydrindene hydrate reacts with amino acids to give a purple colored complex (Ruhemann‟s purple) with an absorption maximum at 570 nm.

However, imino acids such as praline and hydroxyproline yield a yellow color with an absorption maximum at 440 nm.

Ninhydrin oxidizes the amino acid to aldehyde, releasing carbondioxide and ammonia.

During the course of the reaction, ninhydrin gets reduced to hydridantin. The hydridantin formed condenses with ninhydrin in the presence of ammonia to yield a purple colored complex called

Ruhemann‟s purple. Primary amines also react with ninhydrin, however, there is no liberation of carbondioxide.

Reagents required

Buffer

Around 0.5 M acetate buffer was prepared for pH 5.5

Standard amino acid solution (10 m/ml)

Exactly 100 mg of the standard amino acid was dissolved in 100 ml of distilled water.

Ninhydrin reagent

Exactly 2.0 g of ninhydrin was dissolved in 30 ml of acetone and 20 ml of 0.5 M acetate buffer was also added. The pH was set to 5.5. the solution was prepared freshly before use.

Procedure

Clean dry test tubes marked S1 to S5 were pipetted with 10 to 50 l of the standard amino acid solution. In the „Test‟ tubes, 10 and 20 l of the sample was taken. All the tubes were made up to the volume of 4 ml with distilled water and added with 1 ml of freshly prepared ninhydrin reagent. The tubes were covered with aluminium foil and incubated in boiling water

44 bath for 10 minutes. the tubes were all cooled to room temperature and 1 ml of 50 % ethanol was added to each tube. The tubes were allowed to stand for 5 minutes in room temperature and then read at 550 nm.

Estimation of proteins (by Lowry et al., 1951)

Proteins are the macromolecules (polypeptides) derived from the amino acid chains.

Their structure and function is determined by the amino acid sequences. Proteins are amphoteric in nature i.e. they have free amino acid and carboxyl groups (NH and COOH). They are present in the protoplasm as ions and exhibit electrical charges.

Reagents

Solution A-

About 2gm of sodium hydroxide and 10gm sodium carbonate (anhydrous) in 500ml water.

Solution B-

1ml of 1.35% potassium sodium tartarate and 0.1ml of 5.5% copper sulphate solution.

Solution C-

About 50ml of solution A and 1ml of solution B were mixed just before use.

Method

Known aliquots of the samples were made up to 1ml using distilled water. 5ml of solution C was added and mixed and allowed to stand for 10 minutes for colour development.

The colour developed was read at 660 nm after 30 minutes, against reagent blank. A standard graph was drawn by taking concentration of protein in X axis and optical density (OD) on Y axis. The amount of protein present in the sample can be calculated from the graph.

Estimations of other parameters and enzyme activities

Levels of cholesterol, triglyceride, urea, uric acid, and enzyme activity of Pyruvate

Transaminase & oxaloacetate transaminase and alkaline phosphatase were analyzed from Dr.

Arunagiri Diagnostic lab, Thillainagar, Tiruchirappalli, Tamil Nadu, India.

Sodiumn Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed with the coelomic fluid of Eudrilus eugeniae to characterize proteins present in the coelomic fluid. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) involves the separation of proteins based on their size. By heating the sample under denaturing and reducing conditions, proteins become unfolded and coated with SDS detergent molecules, acquiring a high net negative charge that is proportional to the length of the polypeptide chain.

When loaded onto a gel matrix and placed in an electric field, the negatively charged protein molecules migrate towards the positively charged electrode and are separated by a molecular sieving effect. After visualization by a protein specific staining technique, the size of a protein can be estimated by comparison of its migration distance with that of a standard of known molecular weight.

Sample preparation

The coelomic fluid collected was centrifuged at 5000rpm for 10 minutes to deposit the debris and the clear supernatant was filter sterilized through 0.2µ syringe filter into a clean dry sterile microfuge.

Protein isolation by acetone wash method

Coelomic fluid sample was taken in a 5ml polypropylene tube and four times the volume of ice-cold acetone was added to the sample. Tubes were vortexed for 10 seconds and then incubated for sixty minutes at -20o. Then the sample was centrifuged at 10,000 rpm for 10 minutes at 4oC. The supernatant was decanted and the tubes were incubated at room temperature till acetone evaporated completely (approximately for 2 hours). After the addition of 200-400µl of sample loading dye, the sample was kept in water bath for 10 minutes.

Sample application

The protein of the coelomic fluid was processed by acetone precipitation method and loaded into the well for SDS-PAGE separation. However, to understand the effect of different sample processing methods, crude sample of the fluid (after centrifugation to remove cellular debris), dialysate of the crude coelomic fluid and dialysate of the acetone precipitated protein were also loaded onto to separate wells and subjected to electrophoretic separation.

Staining of separated proteins

At the end of electrophoresis, gel was removed and stained with coomasie blue until the clear lanes were appeared.

Determination of molecular mass

Coelomic fluid sample from Eudrilus eugeniae was run on SDS-PAGE with concurrent run of standard protein markers. The molecular mass of the coelomic fluid proteins were visualized on BioRad Gel Documentation System and analyzed using Quantity One Software™.

Gelatin Diffusion Assay to determine the protease activity of the coelomic fluid of

Eudrilus eugeniae. (Kauschke et al., 1997).

Principle

Proteases are the enzymes that catalyze the splitting of proteins into smaller peptide fractions and amino acids by a process known as proteolysis. Gelatin is the denatured product of collagen, and is an abundant protein. A special agarose gel plate was made for this study.

Presence of protease activity will show zone of digestion as lighter area around the well and the area of diffusion can be visualized by precipitating the undigested proteins.

Sample preparation

The coelomic fluid obtained by the cold shock method, was centrifuged at 5000 rpm for

10 minutes to deposit the debris and the clear supernatant was sterilized through 0.2µ syringe filter into a clean dry sterile microfuge and it was used as the source of proteinase.

Reagents

1. Gelatin agarose medium

0.1M Tris-HCL - 0.6g/50ml

Gelatin - 0.25g/50ml

Agarose - 0.75g/50ml

2. Protein precipitating solution

12N HCL - 8.75ml/20ml

HgC12 - 15g/5ml

Dis.water - 80ml

Method

Modified Maskel and Di Capua's (1988) method of invitro gelatin lysis assay was employed to find the protease activity in the coelomic fluid of Eudrilus eugeniae. Briefly, a solution of 0.5% gelatin and 1.5% agarose was prepared in 0.1 M Tris-HCl, pH 7:0 (Tris).

Exactly 5ml of gelatin-agarose gel was poured onto a rectangular dish of 8 x 4 cms. Wells of 6 mm were cut into solid agarose and filled with 10µl, 20µl, and 30µl of coelomic fluid respectively. After 4 hr of incubation at 37°C, the gelatin-agarose gel was precipitated with 5 ml of solution containing 15g of HgC12 and 20ml 12N HC1 in 80ml distilled water. The diameter of the clear circle around each well was measured. The diameter of the circle was proportional to the protease activity of the coelomic fluid.

1H-NMR Analysis

Sample preparation for 1H-NMR

Colomic fluid centrifuged at 12,000 rpm for 20 min. about 450 µl of the fluid was then mixed with 200 µl of 0.2 M phosphate buffer solution (pH 7.4) and 100 µl of D2O.

NMR spectroscopy

Characterization of endogenous metabolite profile

1H NMR spectra were obtained at 300 K using a Bruker Avance DRX600 spectrometer

(Bruker, Coventry, UK), operating at 600.22 MHz and equipped with a 5 mm broad-band inverse probe. One dimensional spectra were acquired using a standard pulse sequence for water suppression, with an additional T1 relaxation delay of 2s. About 4096 free induction decays

(FIDs) were collected with a spectral width of 12,019 Hz into 64 K data points. An exponential function equivalent to a line-broadening of 0.3 Hz was applied prior to Fourier transformation.

Results

Collection of earthworms and harvesting of coelomic fluid

Earthworm, Eudrilus eugeniae were collected from the Periyar Research Organization for

Bio-Technique & Eco-System (PROBE), Periyar Maniyammai University, Vallam, Tanjore Dist,

Tamil Nadu, South India (Figure 1.1 (a,b,c & d). Organic waste served as a medium of growth for the worms in the sieved garden soil and cow dung mixed in the ratio of 2:1. The mixture was allowed to dry under sun-light for 10 hours. After mixing subsequent amount of water, the worms were allowed to be contained in suitable cement tanks of 1 ft depth and 2 ft diameter

(Figure 1.2a, b, c, d & e). Besides, earthworms were reared also in large bunds of 5-6 ft in length with sufficient amounts of soil and cow dung mixed together for a height of 30cms. To maintain the earthworms in bunds and the plastic trays, dry leaves and litters were used as a coverage over which water was sprinkled as often as required to maintain the moisture. Once the coelomic fluids were collected for various experimentation purposes, the worms were left in separate plastic trays for atleast 14 days before the next collection.

Collection methods

Four different methods for the collection of coelomic fluid were carried out namely warm water method, electric shock, cold shock and Heat shock method. In warm water method, worms

(15 grams) were kept immersed under 25 ml of warm water (450C) and the whole water was used in place of the coelomic fluid, therefore the volume of collection was 25±3 ml in 30 minutes (Figure 1.3a). In the electric shock method, the known quantity of earthworms (15 grams) was subjected to mild electric shock (5 Volt) for 30 minutes (Figure 1.3b & c). This accounted for 0.2 ±0.17 ml. In cold shock method, the same quantity of earthworms were

51 subjected to cold-shock using ice packing and the fluid was collected in a clean dry test tube.

Cold shock method produced 1.5 ml of fluid collection in 30 minutes (Figure 1.3d). Similar to this, in the hot shock method, ice-cold pack was replaced with hot water bag (55-60ºC) with a different set of worms (Figure 1.3d). This heat shock method yielded 0.5 ±0.25 ml of coelomic fluid. All these fluids were subsequently employed for further experimentation. Other methods of fluid collection was abandoned due to enormous water content as in warm water method and mortality in electric shock method (Table 1.1).

Consequence of different collection methods

There was marked influence on the earthworms after different collection methods. The worms that underwent electric shock method and heat shock method were almost dead. This clearly indicated that the conditions for collection were of extremely stressful for the worms. As worms almost died, the possibility of using electric shock method and heat shock method did seem to be viable. In case of warm water method, worms were alive and active. However, there was no increase in the volume of the water showing mere dilution of the fluid if there had been any secretion indeed. The worms that were subjected to cold-shock method were alive and active though it secreted comparatively larger volume (1.5 ml) of fluid than the other methods (Table

1.2). The fluid collected appeared clearly brown in colour without any marked debris that was seen in the electric and heat shock method. As the worms were indifferent after the collection, they were left in a separate basket of cow-dung-soil mixture for subsequent collection after a time period of two weeks.

Effect of the cold shock collection method on the survivability of the earthworms and subsequent collection

Cold shock method of fluid collection was found to be the safest method of coelomic fluid. This method of placing worms under the ice did not seem to be harmful as seen by the survivability of the worms after every time of collection. It was observed by growing the worms separately in plastic tray after the collection of coelomic fluid. The growth of the worms were found to be normal even after three rounds of fluid collection within a time period of one month.

There was no significant reduction in the volume of fluid collection in subsequent collections.

(Table 1.2).

Biochemical estimations

As the fluid collected by electric shock method was too low and that of warm water method was too diluted, they were not chosen for further studies including biochemical estimations. Total sugar, reducing sugar, protein and free amino acid contents were analyzed in the coelomic fluids collected by the cold-shock and heat shock methods. The concentrations of total sugar, protein and amino acids were almost same in the fluids collected by the above said two different methods. However, heat shock method of fluid collection was stressful to the worms leading to the death of the worms within few hours. As the fluid collection was also significantly lower by 70% than that of the cold shock method. Therefore, subsequent biochemical estimations such as cholesterol, triglycerides, urea, uric acid, glutamate, oxaloacetate transaminase, pyruvate transaminase and alkaline phosphatase were carried out in the fluid collected by cold-shock method. Results of the analysis are tabulated (Table 1.3).

Characterization of coelomic fluid proteins by SDS-PAGE method

The protein characterization of the coelomic fluid of the earthworm species Eudrilus eugeniae was performed by the technique Sodium-dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE). The coelomic fluid of E. eugeniae and the standard marker was loaded in two separate lanes to mark the difference in their protein. The molecular mass of those proteins was determined by comparing with relative mobility of the molecular mass of protein markers using the Quantity One Software from BioRad (USA).

Lane 1 was loaded with BSA and revealed two bands of proteins of molecular weight 66 and 45 KDa. Lanes 2 and 6 were loaded with acetone precipitated crude coelomic fluid. This revealed 5 characteristic protein bands of molecular weights 58, 47, 30, 20 and 16 KDa. Lane 3 was loaded with the dialysate of the crude coelomic fluid and revealed two peptide bands of 13 and 9 KDa. Lanes 4 & 5 revealed a single protein band of 29 KDa when loaded with the dialysate of the acetone precipitate fraction of the coelomic fluid. The PAGE is presented in

Figure 1.4.

Demonstration of protease activity by Gelatin Diffusion Assay

Protease activity was clearly visible from the precipitation of gelatin present in the

Agarose medium. The digestion of gelatin confirmed the proteolytic activity of the coelomic fluids of Eudrilus eugeniae. Clear zones were observed after 4 hrs of incubation of coelomic fluid at 370C on the wells of gelatin-agarose plates. The proteolytic reaction of coelomic fluid increased rapidly during the first hour of incubation at 370C at pH 7 and the maximum diameter of lysis was obtained at the 10th hour of incubation. The diameter of the zone also increased with

54 the increase in the concentration of the coelomic fluid/well. The zones were 1.5 cms and 0.8 cms in diameter for 20 µl and 40 µl respectively. The diameter of zone around each well was clearly viewed by precipitating the undigested gelatin. This study showed that the coelomic fluid of

Eudrilus eugeniae have proteolytic activity (Figure 1.5). The zone of lysis was directly proportional to its protease activity due to the increase in the volume of fluid charged inside the gel. The result is presented in Table 1.4.

NMR spectroscopic result

The one-dimensional 1H NMR spectrum shows a number of both broad and sharp resonances. The broad peaks arise from proteins still present in the coelomic fluid (despite an initial centrifugation of the coelomic fluid prior to analysis), and these have not been characterized using NMR spectroscopy. Superimposed on these broad lines are sharp peaks arising from small molecule metabolites. Many of the peaks can be assigned by inspection based upon comparison with tabulated literature values (Lindon et al., 1999; Nicholson et al., 1995) and these are labeled directly on the spectrum (Fig. 1.6).

It is apparent that the small molecule composition of the coelomic fluid is dominated by organic acids. The largest single resonance observed in the one-dimensional 1H NMR spectrum is that of succinate at N 2.41. The next most prominent resonances in the spectrum are also from organic acids: malate and acetate. Several other organic acids can be observed within the one- dimensional 1H NMR spectrum. Other compound classes are also present: alanine was the only free amino acid to be visible in the one-dimensional spectrum, the nucleotide nicotinamide mononucleotide (NMN) was observed; the nucleoside adenosine was also present, at a much

55 lower concentration than NMN. Finally, myo-inositol, glycerol, and trimethylamine-N-oxide

(TMAO) were detected. Assignment of the resonances to NMN was made by comparison with the aromatic region resonances, in both chemical shift and multiplicity terms, to those of N- methyl nicotinamide (Lindon et al., 1999). In this spectrum, however, an additional doublet at N

6.55 is observed and has been assigned to the anomeric proton of the ribose group. The integrated area of the resonance at N 6.55 is highly correlated with the area of each of the different aromatic resonances (at N 9.31, 9.10, 8.95, and 8.21) across many different spectra. In addition, the N 6.55 signals shows coupling to a triplet at N 4.95, which is in turn coupled to the resonance at N 4.37. These data are consistent with the signals that would be expected of NMN.

The signals could also arise from the closely related compound nicotinic acid mononucleotide.

Biochemical estimations

Total Sugar, reducing sugar, protein and free amino acid contents were analyzed in the coelomic fluids collected by the cold-shock method and tabulated as below.

Collection Methods

Among the collection methods adopted, cold-shock method of fluid collection was found to be the best due to biochemical estimations that revealed the presence of sugars, proteins and amino acids. In electric shock method, the fluid was collected, however; the worms almost died or came out of the funnel due to intolerance.

DISCUSSION

Earthworms are protostomian animals endowed with a true coelom of mesenchymal origin. The coelomic cavity is filled with coelomic fluid containing free wandering coelomocytes derived from the mesenchymal lining of the cavity. The coelomic cavity is metameric and the segments are separated by transversal septa. Regulated transport of the coelomic fluid and coelomocytes between neighboring segments is ensured by channels comprised of sphincters within the septa. Each segment of the coelomic cavity is opened to the outer environment by paired nephridia and by one dorsal pore through which soluble metabolites and corpuscular material, respectively, can be excreted or expelled (Bilej et al., 2000; Roch, 1979; Weidong et al., 2003).

The coelomic fluid is generally secreted by the earthworm for maintaining the moisture and to help in its physiological activities including respiration and burrowing. However, the animal was facilitated to secrete this fluid by giving external stimuli like rise or drop in temperature or by applying external voltage. There are a number of reports for the collection of coelomic fluid by applying electric stimulation (Hideshi et al., 2004; Weidong et al., 2003).

Likewise, without any standardized procedure for collection, the fluid is being collected along with enormous quantity of water and called as Vermiwash (Zambare et al., 2008;

Abdullah, 2008).

However, there is no mention about the viability of the worms in these reports after the collection procedure. As a facilitator for fluid collection, temperature was chosen in this study

57 and therefore, worms were kept under high (55 - 60ºC) and low temperatures (10 - 15ºC) using water/ice bag. This method of fluid collection is termed cold-shock or heat shock method.

Though fluid is collected, cold-shock method of collection produced higher volume than the heat shock method. This method of fluid collection is a novel method and has not been cited anywhere else in articles so far. This method of collecting fluid using low temperature has been coined as „cold shock method‟.

There are several advantages of this cold-shock method as discussed here. Compared to the vermiwash, coelomic fluid is concentrated and can be collected within an hour involving no skilled labour. It does not require any apparatus such as the step-down transformer / Power eliminator. Most important of all, worms are un-harmed and show normal activities even after the collection process unlike in the other method involving the electric shock. Therefore, worms are employed for the process again for atleast three rounds within a month. Whereas, in electric shock method, worms almost die. Being concentrated, the coelomic fluid can be stored, packaged for transportation owing to its application or diluted at the user-end.

Several bioactive proteins have been found in the coelomic fluid of earthworms. These proteins exhibit a variety of antibacterial (Lassegues et al., 1981; Valembois et al.,1982), hemolytic (Andrews and Kukulinsky, 1975; Du Pasquier and Duprat, 1968; Roch, 1979; Roch et al., 1981), cytotoxic (Kauschke and Mohrig, 1987), hemagglutinating (Roch et al., 1984;

Valembois et al., 1984, 1986), and proteolytic (Tuckova et al., 1986; Valembois et al., 1973) activities, and the biological and chemical nature of the compounds responsible for such activities has been studied extensively, for more than 2 decades (Bilej, 1994; Roch, 1979;

Valembois et al., 1986). Some of the active proteins have been isolated and their biological and chemical characteristics have been successively demonstrated.

In the present work, there had been five different protein bands that appeared. Among these, two proteins of 58 and 47 KDa was seen. This is in consistent with the earlier findings

(Lassegues et al., 1997; Milochau et al., 1997). In their study, two proteins, of 40 and 45 kDa, designated as fetidins exhibited hemolytic and antibacterial activity. These were isolated initially from the coelomic fluid of E. fetida. Subsequently, three proteins with hemolytic activity, designated H1, H2, and H3 and having respective molecular masses of 46, 43, and 40 kDa, were isolated from the coelomic fluid of E. fetida (Eue et al., 1998). H1 andH2 have been reported to have exclusively hemolytic activity, while H3 has both lytic and agglutinating activities. The chemical structures of these compounds have not yet been determined.

In addition to the compounds mentioned above, proteolytic enzymes from coelomic fluid have been characterized by Mohrig et al. (1989), Roch et al. (1991), Leipner et al. (1993), and

Kauschke et al. (1997). A serine protease inhibitor was purified and characterized by Roch et al.

(1998). Lysozyme-like compounds with the potential ability to cleave mucopeptides in bacterial cell walls were identified in the coelomic fluid of E. fetida by Cotuk and Dales (1984a,b) and, subsequently, Lassalle et al. (1988) purified lysozyme with a molecular mass of 20 kDa from the coelomic fluid of E. fetida and determined its amino acid composition. Similar finding is also seen in the present work. When the acetone precipitated crude coelomic fluid was electrophoresced, 20 KDa protein was obtained.

Ito et al. (1999) purified a lysozyme of 13 kDa from E. foetida and obtained a partial amino acid sequence. This 13 KDa protein was present in the dialyzed crude coelomic fluid. It is likely that at least two classes of bacteriolytic factors must be involved in the humoral defenses of annelids: antibacterial compounds, such as fetidins, and lysozyme-like enzymes (Cotuk and

Dales, 1984b; Hirigoyenberry et al., 1990). Wojdani et al. (1984) demonstrated an activity that stimulated mitosis of cultured mouse and human lymphocytes in the coelomic fluid of

Lumbricus terrestris, and Hanusova et al. (1999) found a factor in E. fetida that had a mitogenic effect on murine splenocytes.

CF of earthworms expresses naturally a strong proteolytic activity, which is even increased after challenging but displayed individually differences. A simple agar diffusion assay incorporating gelatin as substrate demonstrated characteristics of coelomic fluid proteases in E. euginiae. Proteases are generally involved in immune mechanisms by activation of enzyme cascades [complement activation, clotting reaction, pro-PO-cascade], degrading of foreign material and inducing apoptosis. In invertebrates serine proteases are especially important for immune functions and interactions of humoral and cellular reactions as well, demonstrated for the clotting reaction and phenoloxidase activation (Johansson and Soderhall, 1996; Muta and

Iwanaga, 1996; Roch et al., 1998; Seki et al., 1994). Proteases are reported to range between 24 to 33 KDa. (Cho et al., 2004; Wang et al., 2003; Ajlan and Bailey, 2000). The SDS-PAGE analysis reveal protein bands of 20 and 29 KDa representing the serine group of proteases.

Inhibition assays classified the majority of CF protease as serine proteases (Kauschke et al., 1997; Mohrig et al., 1989). Most likely CF proteases are regulated by inhibitors to prevent

60 autolysis. However only one potential protease inhibitor, classified as serine protease inhibitor has been isolated from the CF of E. fetida so far (Roch et al., 1998). Protease pattern and levels of protease activity as well might be considered as promising biomarker candidates, suitable in monitoring physiological challenging environmental conditions in earthworms, easy to analyse.

Importance of earthworm proteases is realized now-a-days. It is applicable in both experiment and production, such as medical usage (fibrinolytic agent, Tissue type plasminogen activator), environmental protection and nutritional protection (Rong et al., 2009).

Many studies have investigated the composition of the coelomic fuid of earthworms at an enzymatic level, and this has demonstrated the presence of haemolytic, proteolytic, and cytotoxic enzymes that are active against foreign cells and peptides (e.g. (Kauschke et al., 1997; Bilej et al., 1995; Yamaji et al., 1998; Eue et al., 1998). However, there is currently little knowledge of the metabolite complement of the coelomic fuid of earthworms.

1H-NMR is well suited as a technique for the analysis of metabolites in complex biological matrices, and is thus a useful probe of physiological parameters. A wide range of small organic molecules can be characterised and quantifed using this technique (Cooper et al.,

2002), and the approach is particularly useful when, as in this case, there is no prior knowledge of the potential analytes. Only minimal sample preparation is required, and no further sample derivatisation or separation is required (Nicholson et al., 1983; Nicholson and Wilson, 1989;

Lindon et al., 1999). Consequently, there is neither preselection of target analytes nor loss of components caused by sample preparation. Analysis of bio- fuids, particularly urine and blood

61 plasma, has been shown to be successful in detecting metabolic changes induced by xenobiotics in mammalian systems (Nicholson et al., 1983; Nicholson and Wilson, 1989; Griffin et al.,

2000). The same approach has also successfully been applied to the analysis of earthworms using a tissue homogenate prepared from the entire earthworm (Gibb et al., 1997; Warne et al., 1999).

This use of the whole worm homogenate possesses the obvious disadvantage that a metabolic change resulting in the relative concentration or depletion of endogenous biochemicals in a specific tissue or fluid may be obscured in the whole-worm homogenate. A secondary disadvantage is that the extra procedural steps of homogenisation and extraction are required compared to biofluid analysis. This increases sample preparation time and may discriminate against certain target groups of analytes. As a consequence, the coelomic fluid has been subjected to NMR analysis to record various detectable biochemicals present in it. 1H-NMR study of the coelomic fluid has revealed the presence of many different components. Many of these include most common metabolites of fumarate, malonate, malate, succinate and glycerol.

This study demonstrates the advantages of using a nonselective technique such as 1H-

NMR, which reports on the entire metabolite complement of a biofluid. Succinate, malonate and acetate concentrations in coelomic fluid have been demonstrated to be a potential combination biomarker, although none of these were a prior selected as analytes, nor even known to be likely components of the coelomic fluid of E. eugeniae.

The application of multivariate data analysis techniques to the biochemical profiles of the entire small-molecule complement of biological samples, as detected by 1H-NMR, has been described as `metabonomics' (Robertson et al., 2000; Nicholson et al., 2000). Metabonomics is

62 likely to be useful in understanding the ejects of changes in gene expression, in conjunction with the parallel techniques of genomics and proteomics (functional genomics) (Lindon et al., 2000).

The metabonomic approach has previously (Gibb et al., 1997; Warne et al., 1999) and in this study been demonstrated to be an appropriate technique for earthworm biochemical analysis. MS spectroscopy is also a valuable tool for detecting new potential biomarker compounds. The analysis and interpretation of endogenous biomarkers is likely to be of great value in the assessment of ecological risks to terrestrial invertebrates, as it can provide an `early warning' of toxicity, as well as providing information on the integrated ejects of cumulative exposures to complex mixtures that cannot be gained from chemical analysis of pollutant residues (Depledge et al., 1994; Scott-Forsmand et al., 2000). Morgan et al. (1999) stressed the importance of combining information from biomarker studies at multiple levels of organization (e.g. a general toxic response together with ejects at the genetic and protein level), ideally with mechanistic connections between levels. MS spectroscopy- based metabonomics is well suited to providing multivariate biomarker data at a biomolecular toxicity level, which could then be combined with higher-level (for example, nucleic acid or protein level) toxicity data. The rapid nature of metabonomic analysis would enable testing to discover biomarker compounds that are relevant to different hierarchies of response, namely, general toxic insult and stress, ejects of different chemical classes, and specific toxic responses to individual chemicals, together with a mechanistic understanding of the process.

The earthworm is one of the typical saprozoic organisms, living in the environment replete with microorganisms some of which may be a threat to their existence. To survive in such an environment, they have developed efficient immuno-defense mechanisms against invading microorganisms. Immunity in invertebrates, like immunity in vertebrates, involves both humoral and cellular mechanisms. From studies on insects, it was established that many antibacterial peptides have been successfully extracted.

Earthworms have been living with the aid of their defense system since the early phases of evolution, although they always face the invasion of pathogen microorganisms in their environments (Lassegues et al., 1981; Engelmann et al., 2004). The studies which have been continued for about 50 years showed that earthworms have humoral and cellular immunity mechanisms (Kauschke et al., 1997; Beschin et al., 1998; Hanusova et al., 1999; Bilej et al.,

2001; Field et al., 2004). It has been found that coelomic fluid of the earthworms contains more than 40 proteins and exhibits several biological activities as follows: cytolytic, proteolytic, antimicrobial, hemolytic, hemagglutinating, tumorolytic, mitogenic activities (Cotuk and Dales

1984; Lange et al., 1997; Lange et al., 1999; Cooper and Roch 2003).

These investigations with earthworms have usually intensified with Eisenia fetida,

Lumbricus terrestris and Dendrobaena venata (Dales and Kalac 1992; Milochau et al., 1997;

Eue et al., 1998; Furlong et al., 2002; Kalac et al., 2002; Koening et al., 2003). The hemolytic action of coelomic fluid of E. fetida was first defined by Du Pasquier and Duprat (1968a). They demonstrated that hemolytic factor was active against sheep red blood cell and various other vertebrate erythrocytes (Du Pasquier and Duprat 1968b). Lassegues et al. (1981) found that this

64 hemolytic factor also inhibited the growth of different bacterial species which were isolated from nature. They showed that these bacteria antigens were common with sheep red cells. The coelomic fluid of Eisenia fetida andrei was demonstrated to possess an antimicrobial activity against Aeromonas hydrophila and Bacillus megaterium which are known as earthworm pathogens (Valembois et al., 1982; Pan et al., 2003). Afterwards, Milochau et al. (1997) obtained two proteins, named Fetidins, from dialyzed coelomic fluid of earthworms and confirmed that this antibacterial activity was due to fetidins. Cho et al. (1998) found that

Lumbricus rubellus also has two antibacterial agents named Lumbricin 1 and Lumbricin 2.

Recently, two types of antibacterial factors which include lysozyme-like molecules with hemolytic activity as well as a pattern recognition protein named coelomic cytolytic factor (CCF) have been identified in Eisenia fetida earthworms (Kohlerova et al. 2004). Bruhn et al. (2006) stated that lysenin which was a different protein of Eisenia fetida and lysenin-like proteins had several cytolytic activities which exerted hemolytic, antibacterial and membrane-permeabilizing properties.

The function of polysaccharides in vivo not only provides energy and participates in body structure, but also has various biological functions, including anti-hemagglutination, reducing blood fats, anti-viral, anti-tumor, anti-radiation and enhancing immunity of organisms

(Hwan et al., 1996; Xie, 2001; Zhang et al., 1998; Zhang et al., 2003). Polysaccharides also participate in various cell activities and connect with vital movements in vivo. The glycoprotein and lipopolysaccharides play an important role in cell recognition and secretion as well as in protein modification and transfer process. The earthworm is one of the traditional Chinese medicinal components. Nowadays, the development of earthworm medical value concentrates on

65 fibrinolytic enzymes and antibacterial proteins, but there has been no report on earthworm polysaccharide research. Agglutinins have been found in skin secretions, coelomic fluid and earthworm tissues. Agglutinins are one of nature‟s glycoproteins.

All the antimicrobial reports of the earthworm is attributed to its fluid secretion called coelomic fluid. The fluid contains cytolytic, agglutinating and/or antibacterial components, which are involved in the immune systems (Vetvicka et al., 1994). Presumably the function of this system is to destroy membranes of foreign cell, a mechanism that causes cell death by cytosol release, and is attributed to the coelomycetes, which secrete humoral effectors into the coelomic fluid. Based on this, the antimicrobial activity was studied in the coelomic fluid.

Materials and methods

Earthworm: Eudrilus eugeniae

The worms used in this study were Eudrilus eugeniae. The earthworms were collected from the Agriculture Wing, Department of Biotechnology, Periyar Maniyammai University,

Vallam, Tamil Nadu, India.

Collection of coelomic fluid by cold shock method

In this method approximately 15 grams of worms are taken from the culture pit and washed with sterile distilled water. The worms are then dried on a filter paper and placed in a nylon mesh rolled into a „cone‟ shape to fit into the glass funnel. The funnel is held in a burette clamp on a titration stand. The set-up is shown in the Figure 1.3d. A bag of ice that fits over the funnel was placed above the worms such that the worms could feel the drop in temperature due to the ice pack above. The coelomic fluid was made to release through the dorsal pores of its body due to the drop in temperature surrounding it. The fluid was collected in a clean sterile dry test tube that was fit to the end of the funnel. The collection was carried out for 30 minutes and the worms were quickly released into a separate worm–culture pit for relaxation. The collected coelomic fluid was then centrifuged at 5000rpm for 10 minutes to deposit the debris and the clear straw-colored supernatant was then filter sterilized through 0.2 m syringe filter into a clean, dry, sterile microfuge in a Laminar Air flow chamber and stored under -20ºC. This collection method is described as Cold Shock method in this chapter.

Collection of pathogenic bacteria and phytopathogenic fungi

Pathogenic organisms were selected based on pathogenesis and drug resistance. They were obtained from the Department of Microbiology, Doctors Diagnostics, Thillainagar,

Tiruchirappalli, Tamil Nadu, India. The pathogenic bacteria tested were Salmonella typhi,

Salmonella paratyphi, Enterococcus foecalis, Aeromonas liquifaciens, Klebsiella pneumonia,

Pseudomonas aeruginosa, Serratia marscecens, Escherichia coli, Streptococcus pyogenes and

Staphylococcus aureus. The phytopathogenic fungi were Rhizatonia solani, Trichoderma viride,

Alternaria solani, Sclerotium rolfsii, Fusarium sp., Colletrotrichum sp., and Aspergillus sp., and were procured from the Department of Plant Pathology, Tamil Nadu Agricultural University,

Melur, Madurai.

Methods of antimicrobial susceptibility testing

Preparation of dried filter paper discs

Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter, which are placed in a Petri dish and sterilized in a hot air oven. The discs were loaded with 20, 40, 60, 80 and 100 µl per disc.

Preparation of inoculum

Stock cultures were maintained at 40C on slope of Nutrient agar. Active cultures for experiments were prepared by transferring a loop-ful of cells from stock culture from a test tube of Mulluer Hinton Broth (MHB) and were incubated without agitation for 24 hours, 370 C. The cultures were diluted with fresh Mueller Hintor Broth to achieve optical densities corresponding to that it Colony Forming Units per ml (CFUs).

Agar diffusion method

Antimicrobial sensitivity was done by the Kirby-Bauer disc diffusion method. The principles of determining the effectivity of a noxious agent to a bacterium were well enumerated at the turn of the century. The discovery of antibiotics made these tests (or their modification) too cumbersome for the large numbers of tests necessary to be put up as a routine. The ditch plate method of agar diffusion used by Alexander Fleming was the fore-runner of a variety of agar diffusion methods devised by workers in this field. The Oxford Group used these methods initially to assay the antibiotic contained in blood by allowing the antibiotics to diffuse out of reservoirs in the medium in containers placed on the surface.

With the introduction of a variety of antimicrobials it became necessary to perform the antimicrobial susceptibility test as a routine. For this, the antimicrobial contained in a reservoir was allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms. Even now a variety of antimicrobial containing reservoirs are used but the antimicrobial impregnated absorbent paper disc is by far the commonest type used. The disc diffusion method of AST is the most practical method and is still the method of choice for the average laboratory. Automation may force the method out of the diagnostic laboratory but in this country as well as in the smaller laboratories of even advanced countries, it will certainly be the most commonly carried out microbiological test for many years to come. It is, therefore, imperative that microbiologists understand the principles of the test well and keep updating the information as and when necessary. All techniques involve either diffusion of antimicrobial agent in agar or dilution of antibiotic in agar or broth. Even automated techniques are variations of the above methods.

Method

The invitro anti-microbial activity was screened by using Mueller Hinton Agar (MHA) obtained from Himedia, Mumbai.

The MHA plates were prepared by pouring 15 ml of molten media into sterile Petri plates.

The Plates were allowed to solidify for 5 minutes.

About 0.1 % inoculum suspension was swabbed uniformly and the inoculum was allowed to dry for 5 minutes.

The different concentrations of extracts (1.5, 2.0 and 2.5 mg/disc) were loaded on 6mm sterile discs.

The loaded disc was placed on the surface of the medium and the compound was allowed to diffuse for 5 minutes.

The plates were kept for incubation at 370 C for 24 hours.

At the end of incubation, inhibition zones formed around the discs were measured with transparent ruler in mm.

The studies were performed in triplicates.

Determination of hemolytic activity (Elif et al., 2008)

Hemolytic activity was carried out with rabbit, sheep and human red blood cells. After the cells were washed three times by centrifugation (2000 rpm, 4ºC and 15 minutes) with phosphate buffered salt (PBS) solution, erythrocytes were suspended to a final concentration of

2%. Coelomic fluids were diluted serially two-fold in Holtfreter‟s earthworm saline solution.

Coelomic fluid suspensions and erythrocyte suspensions were mixed (1:1, v/v) and incubated 2

70 hours at 25ºC. After the incubation period, all aliquots were collected and centrifuged (2000 rpm,

4ºC and 10 minutes) for the separation of free hemoglobin from cell fragment. Following the centrifugation step, supernatants were collected and hemolyse quantities were determined spectrophotometrically at 405 nm. In all spectrophotometric measurements, coelomic fluid suspensions were used as a blank, whereas 0.003% saponin suspension in sterile distilled water and the mixture of Holtfreter‟s earthworm saline solution:erythrocyte suspensions [1:1 (v/v) ratio] were used as positive and negative controls, respectively. Percentage of hemolysis was calculated by using the following formula:

(Absorbance of sample) / (absorbance of the total hemolysis)] x 100 = % hemolysis.

Total hemolysis was obtained by adding saponin. Hemolytic activity was also carried out with blood agar diffusion method. In this method, 6 mm diameter wells were cut from an agarose gel which contains 1% erythrocytes, and then wells were filled with 20 µl of the same suspensions as spectrophotometric hemolyse assay (dilution assay). After the incubation at 25ºC for 24 hours, diameters of hemolyse zones were measured.

Reagents required: 100% Holtfreter's solution NaCl 3.5 g

NaHCO3 0.2 g KCl 0.05 g

MgSO4 stock solution 333 µl

CaCl2 stock solution 333 µl dH20 1 liter pH was set between 7 and 7.5.

50% Holtfreter's solution

NaCl 1.75 g

NaHCO3 0.1 g KCl 0.025 g

MgSO4 stock solution 335 µl

CaCl2 stock solution 335 µl dH20 1 liter pH was set between 7 and 7.5.

MgSO4 stock solution: 300 g of MgSO4 in 500 ml deionized water.

CaCl2 stock solution: 150 g of CaCl2 in 500 ml deionized water.

RESULTS

Antimicrobial activity of the coelomic fluid

The zone of inhibition was recorded for various concentrations such as 20, 40, 60, 80 and

100 µl/disc. There were no zones of inhibition for the concentrations of 20 and 40µl/disc, but there was a moderate zone of inhibition at 60µl/disc against Klebsiella pneumoniae. The coelomic fluid demonstrated a maximum of 11mm zone of inhibition against the latter, at a concentration of 100µl/disc, followed by 10 mm at 80 µl/disc concentration. Salmonella paratyphi and Salmonella typhi were susceptible to the fluid demonstrating 10 and 8 mm zones of inhibition at 100µl/disc concentration respectively. Similar pattern was observed in S. paratyphi and S. typhi at 80µl/disc concentration with 8 mm and 7 mm of zones of inhibition.

There was moderate zones of inhibition at 100µl/disc concentrations in Streptococus pyogenes and Psuedomonas aeruginosa with 6 mm of zone of inhibition. The fluid was ineffective against the other bacterial and fungal phytopathogens (Table 2.1 & 2.2; Figures 2.1). Among the fungi, coelomic fluid demonstrated a moderate zone of inhibition at 100µl/disc concentration.

Hemolytic activity

To evaluate the hemolytic activity of coelomic fluids, human erythrocytes were used.

Coelomic fluid exhibited hemolytic activity to a moderate level. There were clear zones of hemolysis seen on the blood-agar plates. Streptococcus sp. was also simultaneously screened for demonstrating the hemolytic activity as a positive control. The hemolysis observed on the agar plate is shown in the figure 2.1 (H&I).

Discussion

1984; Lange et al., 1997; Lange et al., 1999; Cooper and Roch 2003). Investigations with earthworms have usually intensified with Eisenia fetida, Lumbricus terrestris and Dendrobaena venata (Dales and Kalac 1992; Milochau et al., 1997; Eue et al., 1998; Furlong et al., 2002;

Kalac et al., 2002; Koenig et al., 2003). The hemolytic action of coelomic fluid of E. fetida was first defined by Du Pasquier and Duprat. They demonstrated that hemolytic factor was active against sheep red blood cell and various other vertebrate erythrocytes (Du Pasquier and Duprat

1968). Lassegues et al., (1981) found that this hemolytic factor also inhibited the growth of different bacterial species which were isolated from nature.

However, there are no earlier reports on such activities in the coelomic fluid of Eudrilus eugeniae and the present work established a record in this aspect. Antimicrobial peptides are considered as a universal host – defense tool of earthworms against microbial infection. In the course of evolution, earthworms have developed efficient defense mechanisms against microbes they ingested during feeding or uptaken into the body from the environment after injury (Bilej et

74 al., 2000; Cooper and Roch, 2004; Dhainaut and Scaps, 2001; Jarosz and Glinski, 1997). The concentration of naturally occurring bacteria is usually in the order of 6 x105 CFU/ ml of coelomic fluid or 0.9 x105 CFU per worm of an average size (Dales and Kalac, 1992). Kohlerova et al. (2004) found that injecting approximately 107 CFU bacteria (10 times more than the number of naturally occurring bacteria) to earthworms did not affect the viability of Eisenia fetida but was sufficient to activate the defense mechanisms of earthworms (Kohlerova et al.,

2004). Coelomic fluid (CF) of earthworms contains a 42-kDa coelomic cytolytic factor (CCF)

(Bilej et al., 1995) that recognizes a broad range of bacterial species.

The binding of CCF to cell wall components of Gram-negative bacteria (O-antigen of lipopolysaccharide) via lectin-like interactions can activate the prophenoloxidase (proPO) cascade involving serine proteases and fetidins (Bilej et al., 2001; Salzet et al., 2006). As a major defense pathway against bacterial pathogens, such signaling pathway then leads to the production of melanin, amain component with the antimicrobial and cytotoxic activities

(Cerenius and Soderhall, 2004). However, these reports comply to the natural flora of the soil.

When clinical isolates were tested for susceptibility to antimicrobial effect of the constituents of coelomic fluid, there was no significant finding as expected.

The coelomic fluid of annelids exerts numerous biological activities. Among the factors involved in earthworm humoral immunity, particular attention has been devoted to cytolytic components secreted by coelomocytes into the coelomic cavity. The cytolytic activity of the coelomic fluid was generally demonstrated on vertebrate erythrocytes, and the resulting effect was referred to as haemolysis. The majority of the haemolysins identified so far show

75 haemagglutination activity and, more interestingly, a spectrum of antibacterial and/or bacteriostatic activities against pathogenic soil bacteria (Roch 1979; Valembois et al. 1982,

1986; Roch et al. 1991). Therefore, the biological relevance of the E. fetida cytolytic and/or agglutinating system consists in part in growth inhibition of the worm's potential pathogens that live in manure and possess antigen(s) common with red blood cells. Reports on antibacterial peptides in the coelomic fluid of E. eugeniae are probably due to such antibacterial peptides and these have been called as fetidins, lysenins etc. beside the well known lysozyme like proteins.

These proteins have been also been detected in the SDS-PAGE as described in the chapter I.

Lysozyme is a ubiquitous enzyme hydrolyzing 1,4--D-linked glycosidic bond of the peptidoglycan in the bacterial cell wall and thus efficiently protecting the host against Gram- positive bacteria infections. Lysozyme activity was evidenced in coelomocyte extracts and to lesser extent in the coelomic fluid of E. fetida earthworms (Cotuk and Dales 1984). More recently, the active protein was isolated as a 13-kDa protein, characterized, and partially sequenced (Ito et al. 1999). The N-terminal sequence of E. fetida lysozyme was reported to reveal considerable homology with lysozyme from mollusks, echinoderms, and the nematode

Caenorhabditis elegans, while the homology to other known types of lysozymes was negligible.

This suggests that earthworm enzyme belongs to a distinct family of invertebrate lysozymes. The reason behind insignificant antibacterial activity of coelomic fluid is probably because all the tested isolates were Gram negative. Moreover, isolates tested in the experiment are multidrug resistant strains and have been obtained from a clinical pathology laboratory. However, a possible contribution of the cytolytic molecules to the destruction of membranes from self- transformed cells cannot be ruled out.

Several antitumor and antibacterial were found by many researchers (Lassegues et al.,

1997; Yamaji et al., 2003; Yamaji et al., 1998; Lange et al., 1999). Roch et al. (1991) and

Milochau et al. (1997) both found proteins with molecular weights of 45 kDa and 40 kDa possessing several activities, such as antibacterial, hemolysis, and hemoagglutination from E. fetida (Milochau et al., 1997; Roch et al., 1981). In the present study as well, a protein band of

47KDa was obtained in E.eugeniae. One small peptide was isolated from the earthworm coelomic fluid with a molecular weight of 510.3 Da and antibacterial activity (Yanqin et al.,

2004).

There were many investigations on the presence of antimicrobial substances in annelids since the first bacteriolytic molecules were identified as lysozyme-like molecules, active only against Gram-positive bacteria (Jollwes and Zuili, 1960; Schubert, and Messner, 1971;

Lassègues, et al., 1989). The coelomic fluid of the earthworm, Eisenia fetida andrei

(Oligochaeta, Lumbricidae) was demonstrated to possess an antimicrobial activity directed against earthworm pathogenic bacteria, namely; Gram-negative Aeromonas hydrophila and

Gram positive Bacillus megaterium by Valembois et al (1982). Later, Milochau et al. (1997) purified two proteins, named fetidins, from dialysed coelomic fluid of the earthworm and confirmed that the antimicrobial activity existing in the coelomic fluid was essentially due to fetidins. Nevertheless, the present attempt to test the presence of any antimicrobial activity against the pathogenic bacteria and fungi for the cause of finding the evidence to this attribute if any. In the present study, against 10 bacteria and 7 fungi, there was no significant inhibitory zone formation except in Salmonella typhi, Salmonella paratyphi and Klebsiella pneumonia being gram negative bacteria. Besides, the bacterial strains were procured from the clinical diagnostic

77 laboratory and already proved to be resistant for most of the existing antimicrobial drugs in the market. In this regard, the coelomic fluid of Eudrilus eugeniae can prove effective for further investigations.

The term „Sustainable Development‟ was coined by Bruntdland Commisssion Report

„Our Common Future‟ in 1982 which redefined the concept of human development as the development (both social & economic) to- „meet the „needs‟ (but not the „greed‟) of the present generation without compromising with the abilities of the future generations to meet their own needs and that should improve the total quality of all life (human beings, plants and animals) on

Earth now and in the future too, while maintaining the social and ecological integrity (natural and man-made ecosystems) of the earth upon which all life depends and which can provide good quality of life to all the people born on Earth, while protecting their basic life-support systems

(air, water, soil, flora and fauna) and also safely disposing all the wastes generated by them‟

(UNDP, 1994; UNEP/GEMS, 1992; UNEP, 1998). The scientific community all over the world is desperately looking for an „economically viable, socially safe & environmentally sustainable‟ alternative to the destructive „chemical agriculture‟ which would not only „maintain‟ but also

„enhance‟ farm production per hectare of available land as the farmlands all over the world is shrinking in the wake of rapid urbanization. Then, it is not enough to produce „sufficient food‟ to feed the civilization (which was the primary objectives of chemical based green revolution) but also to produce a „high quality of food‟ which should be „safe‟ (chemical free) and also

„protective‟ to human health (good combination of macro and micro nutrients and vitamins) and do it in a sustainable manner to ensure „food security‟ for all, but most for them in the poor Third

World nations in the long term. „Food Safety & Security‟ is a major issue everywhere in the world. This will amount to embarking on a „Second Green Revolution‟ and this time by „Organic

Farming‟ practices completely giving up the use of agro-chemicals (Bettolo and Marini, 1987;

Bradley and Peggy, 2000; FAO, 2001; FAO, 2004; Horrigan et al., 2002; Sinha and Rajiv K,

2008).

The new concept of farm production against the destructive „Chemical Agriculture‟ has been termed as „Sustainable Agriculture‟. This is about growing „nutritive and protective foods‟ with the aid of biological based „organic fertilizers‟ without recourse to agro-chemicals. This is thought to be the answer for the „food safety and security‟ for the human society in future. The

U.S. National Research Council (1989) defined sustainable agriculture as „those alternative farming systems and technologies incorporating natural processes, reducing the use of inputs of off-farm sources, ensuring the long term sustainability of current production levels and conserving soil, water, energy and farm biodiversity‟. It is a system of food production which avoids or largely excludes the use of systematically compounded chemical fertilizers and pesticides and use of environmentally friendly organic inputs. To the maximum extent feasible, organic farming systems rely upon crop rotations, crop residues, animal manures, legumes and green manures to maintain soil productivity and to supply plant nutrients. It emphasizes on both preventive and curative methods of pest control such as the use of pest resistant cultivars, biocontrol agents and cultural methods of pest-control.

In the US, the top 25% of sustainable agriculture farmers practicing „organic farming‟ now have better gross margins and better yields than the top 25% of their counterparts still practicing chemical agriculture (Anonymous, 1980). Swedish farmers are practicing the

„Cleanest Agriculture‟ in world now since 1972. They have developed an alternative system of agriculture based upon the vision of „kretslopp‟--„agriculture which aims to be in harmony with the cycle of nature‟ and therefore, highly sustainable. They have drastically cut the use of pesticides, herbicides and fungicides by 70 per cent since 1985 (UNEP/GEMS, 1992; UNEP,

1996).

Vermicompost (metabolic products of earthworms feeding on organic wastes) is proving to be highly nutritive „organic fertilizer‟ and a „miracle growth promoter‟ rich in NPK (nitrogen

2-3%, potassium 1.85-2.25% and phosphorus 1.55-2.25%), micronutrients, beneficial soil microbes and also contain „plant growth hormones & enzymes‟. Evidences are accumulating all over the world including the present study that the earthworms and their coelomic fluids can do the miracle. They can „build up soil‟, „restore soil fertility‟, „sustain farm production‟ and also deliver „safe food‟ for the civilization.

Most of the reports and investigations are available with the vermicompost or with vermiwash spray to the direct plants in the field or in the soil if in a contained environment.

There is no direct report of invitro studies for the plant growth promoting factors present in the coelomic fluid of these earthworms. To scientifically validate this observation, coelomic fluid was supplemented in the MS medium in place of vitamins and hormones; and the plant was allowed to grow by standard plant tissue culture method with MS media as the basal medium.

Nigella sativa L.

Nigella sativa is an annual flowering plant belongs to the family Ranunculaceae, native to southwest Asia. It grows to 20–30 cm (7.9–12 in) tall, with finely divided, linear (but not thread-like) leaves. The flowers are delicate, and usually colored pale blue and white, with 5–10 petals. The fruit is a large and inflated capsule composed of 3–7 united follicles, each containing numerous seeds. The seed is used as a spice. Nigella sativa has been used for medicinal purposes for centuries, both as a herb and pressed into oil, in Asia, Middle East, and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach and intestinal health, kidney and liver function, circulatory and immune system support, as analgesic, anti--inflammatory, anti--allergic, anti-oxidants, anti-cancer, anti-viral and for general well-being. In Islam, it is regarded as one of the greatest forms of healing medicine available.

Prophet Mohammed once stated that the black seed can heal every disease except death.

Black cumin oil contains nigellone, which protects guinea pigs from histamine-induced bronchial spasms (Mohammad and Batool, 2002) (perhaps explaining its use to relieve the symptoms of asthma, bronchitis, and coughing). The presence of an anti--tumor sterol, beta- sitosterol, lends credence to its traditional use to treat abscesses and tumors of the abdomen, eyes, and liver. (Sharaf, 1990). Nigella sativa oil has been reported to be effective in treating opioid dependence (Aqel, 1993) Nigella sativa also has been reported to reduce calculi formation in rats' kidneys (Reiter and Brandt, 1985).

Tinospora cordifolia Miers.,

Tinospora cordifolia, also called Guduchi is an herbaceous vine of the family

Menispermaceae indigenous to the tropical areas of India, Myanmar and Sri Lanka. The plant is a glabrous climbing shrub found throughout India, typically growing in deciduous and dry forests. The leaves are heart shaped. The succulent bark is creamy white to grey in color, with deep clefts spotted with lenticels. It puts out long, slender aerial roots, often growing on mango or neem trees (Wagner and Hildebert, 1999). Flowers are yellow, growing in lax racemes from nodes on old wood. Fruits are drupes, turning red when ripe (Warrier et al., 1996). Tinospora cordifolia and similar species like Tinospora crispa and Tinospora rumphii Boerl are used in

Ayurvedic and Jamu herbal medicine as a hepatoprotectant, protecting the liver from damage that may occur following exposure to toxins, as well as in Thailand, Philippines. Recent research has demonstrated that a combination of T. cordifolia extract and turmeric extract is effective in preventing the hepatotoxicity which is otherwise produced as a side effect of conventional pharmaceutical treatments for tuberculosis using drugs such as isoniazid and rifampicin

(Adhvaryu et al., 2008).

Clitoria ternatea L.

Clitoria ternatea is a plant species belonging to the Fabaceae family. It is a perennial herbaceous plant. Its leaves are elliptic and obtuse. It grows as a vine or creeper, doing well in moist neutral soil. The most striking feature about this plant are its vivid deep blue flowers. They are solitary, with light yellow markings. They are about 4 cm long by 3 cm wide. There are some varieties that yield white flowers. The fruits are 5 - 7 cm long, flat pods with 6 to 10 seeds in each pod. They are edible when tender. It is grown as an ornamental plant and as a revegetation

83 species (e.g., in coal mines in Australia), requiring little care when cultivated. Its roots fix nitrogen and therefore this plant is also used to improve soil quality.

In Southeast Asia the flowers are used to colour food. In Malay cooking, an aqueous extract is used to colour glutinous rice for kuih tekan (also known as pulut tai tai in

Peranakan/Nyonya cooking) and in nonya chang. In Thailand, a syrupy blue drink is made called nam dok anchan In Burma the flowers are used as food, often they are dipped in batter and fried.

In animal tests the methanolic extract of Clitoria ternatea roots demonstrated nootropic, anxiolytic, anti-depressant, anti-convulsant and anti--stress activity (Jain, (2003). The active constituent(s) include tannins, resins, starch, taraxerol & taraxerone. Clitoria ternatea root extracts are capable of curing whooping cough if taken orally. The extract from the white- flowered plant can cure goiter. Its roots are used in ayurveda Indian medicine (Aparajite, 1981).

Andrographis paniculata Nees.,

Andrographis paniculata is a herbaceous plant in the family Acanthaceae, native to India and Sri Lanka. It is widely cultivated in southern Asia, where it is used to treat infections and some diseases, often being used before anti-biotics were created. Mostly the leaves and roots were used for medicinal purposes. Since ancient times, A. paniculata is used in traditional

Siddha and Ayurvedic systems of medicine as well as in tribal medicine in India and some other countries for multiple clinical applications. The therapeutic value of Kalmegh is due to its mechanism of action which is perhaps by enzyme induction. The plant extract exhibits anti- typhoid and anti-fungal activities. Kalmegh is also reported to possess anti-hepatotoxic, antibiotic, anti-malarial, anti-hepatitic, anti-thrombogenic, anti-inflammatory, anti--snake venom,

84 and anti-pyretic properties to mention a few, besides its general use as an immunostimulant agent. A recent study conducted at Bastyr University, confirms the anti-HIV activity of andrographolide.

Andrographolide, the chief constituent extracted from the leaves of the plant, is a bitter water-soluble lactone exhibiting protective effects in carbon tetrachloride induced hepatotoxicity in rats. Its LD50 in male mice was 11.46gm/kg, ip. This bitter principle was isolated in pure form by Calabrese et al. (2000). Such other activities as liver protection under various experimental conditions of treatment with galactosamine, paracetamol etc. are also attributed to

Andrographolide. The hepatoprotective action of andrographolide is related to activity of certain metabolic enzymes. Andrographis paniculata plant extract is known to possess a variety of pharmacological activities. Andrographolide, the major constituent of the extract, is implicated in its pharmacological activity. A study has been conducted on the cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells. Andrographolide treatment inhibited the in vitro proliferation of different tumor cell lines, representing various types of cancers.

Aristolochia bracteata Retz.,

A small glabrous shrub occurring in the Sahel zone of the Region from Mali to Nigeria, and in Tropical East Africa, Arabia and India. An infusion of the dried leaves, sometimes with the dried root added, is used in Nigeria by Hausa and Fula as an anti--helminthic, a use that is also known in India. The freshly bruised leaves are mixed with castor-oil and used in Nigeria topically on pimples (Burkill, 1985). In India the plant is used to treat scabies (Burkill, 1985),

85 and in the Ogaden of Ethiopia on leg-itch (Burkill, 1985).The root is bitter. Roots mixed with lime-juice are taken for snake-bite, scorpion-stings, etc., in Nigeria (Burkill, 1985). East of Lake

Chad also the root is applied to scorpion-sting (Burkill, 1985).The flowers are sometimes worn in Nigeria as a juju or charm against snake-bite and scorpion-stings (Burkill, 1985).The fresh root yields two acidic crystalline compounds, one with bright yellow needles, m.p. 275–7°C, is identical with aristolochic acid, the other has orange yellow needles, m.p. 240–52°C. The seeds also contain the same two substances and also a greenish-brown non-drying fixed oil (Burkill,

1985). An unnamed alkaloid is reported present in the root and stem of Indian material (Burkill,

1985).The vernacular names and uses of this species are probably similar to those of A. albida with which it is doubtless inter-changeable.

Medicinal plants occupy a distinct place in the life of human, right from the primitive till today (Latha and Pari, 2003). Use of plants as a source of medicine has been inherited and is an important component of health care system in India (Seth et al., 2004). Aristolochia bracteolata is used in traditional medicine as a gastric stimulant and in the treatment of cancer, lung inflammation, dysentery and snake bites (Negi et al., 2003). This plant belongs to the family

Aristolochiaceae and known as kidmar. It has insecticidal properties. Its roots and leaves are bitter and anti-helmintic, and are medicinally important. Almost every part of the plant have medicinal usage. Identifying bioactive compounds and establishing their health effects are active areas of scientific enquiry (Etherton et al., 2004).

Materials and methods

Collection of coelomic fluid by cold shock method

In this method approximately 40 grams of worms are taken from the culture pit and washed with sterile distilled water. The worms are then dried on a filter paper and placed in a nylon mesh rolled into a „cone‟ shape to fit into the glass funnel. The funnel is held in a burette clamp on a titration stand. The set-up is shown in the figure 1.3d. A bag of ice that fits over the funnel was placed above the worms such that the worms could feel the drop in temperature due to the ice pack above. The coelomic fluid was made to release through the dorsal pores of its body due to the drop in temperature surrounding it. The fluid was collected in a clean sterile dry test tube that was fit to the end of the funnel. The collection was carried out for 30 minutes and the worms were quickly released into a separate worm–culture pit for relaxation. The collected coelomic fluid was then centrifuged at 5000rpm for 10 minutes to deposit the debris and the clear straw-coloured supernatant was then filter sterilized through 0.2 syringe filter into a clean, dry, sterile microfuge in a Laminar Air flow chamber and stored under -200C.

In vitro propagation using supplements of coelomic fluid in place of vitamins and hormones

The effect of the coelomic fluid was studied in vitro with the explants from Nigella sativa, Tinospora cordifolia, Clitoria ternatea, Andrographis paniculata and Aristolochia bracteata.

Sterilization of explants

For the surface sterilization, the explants were first washed in running water. Then the shoots were cut into 5 cm and washed with distilled water. Shoots were then washed with active

Tween 20 and then in distilled water Fungicide treatment was done by treating with 0.1%

Bavistin for 15 minutes. Then the explants were washed thoroughly with distilled water for 2-3 times to remove the fungicide. Bactericidal treatment is done with sodium Hypochloride for 3-4 minutes. Then the explants were again washed with sterile water for 3-4 times to remove sodium hypochloride. Then the explants were again treated with 0.1% mercuric chloride for 3-4 minutes.

Finally, the explants were washed with sterilized water for 3-4 times and then trimmed on both sides. Trimmed explants were then inoculated into the MS media. MS media was prepared as per normal procedure using standard stock solutions for macro nutrients, micro nutrients and vitamins. Plants were grown in MS media for rooting, shooting and callus induction with respective hormones and were taken as control while the test medium was prepared with supplements of coelomic fluid in place of macro nutrients, micro nutrients, vitamins and hormones.

Coelomic fluid supplementation

Control was taken as per normal procedure using appropriate volumes of macronutrients, micro nutrients, vitamins and hormones and were kept for observations. On the other hand, coelomic fluid was supplemented in place of vitamins and hormones at different concentrations such as 5ml/L, 10ml/L and 20 ml/L. These „Test‟-culture medium were also kept for observation for rooting, shooting and callus induction.

Preparation of stock solutions

The preparation of MS medium by this method is based on four-concentrated stock solution, which are prepared and kept in the refrigerator.

Stock solution 1: Macronutrients (10× concentration sufficient to make 10L of MS media). The following chemicals were dissolved in 1 L of distilled water

NH4NO3 - 16.5g

KNO3 - 19.0g

CaCl2. 2H2O - 4.4g

MgSO4. 7H2O - 3.7g

KH2PO4 - 1.7g

Stock solution 2: Micronutrients (100 x concentration sufficient for 10L) The following chemicals were added to 1 L of distilled water.

MnSO4. 4 H2O - 2230mg

ZnSO4. 4 H2O - 860 mg

H3BO3 - 620 mg KI - 8.30mg

Na2MoO4. 2H2O - 2.50mg

CuSO4. 5H2O - 2.50mg

CoCl2. 6H2O - 2.50 mg

Stock solution 3: Vitamins (100 x concentration sufficient for 10 L) Following vitamins were mixed in 100ml of distilled water. Glycine - 20mg Nicotinic acid - 5mg Pyridoxine HCl - 5mg Thiamine Hcl - 1mg Myo-inositol - 1000mg

Stock solution 4: Fe-EDTA (100 x concentration sufficient for 10 L) Following chemical salts were added to 100ml of distilled water.

FeSO4. 7H2O - 278mg

Na2EDTA. 2H2O - 373mg EDTA was first dissolved prior to iron sulphate in dark bottle at 40C.

Preparation of MS medium from stock solutions

The stock solution of the MS basal medium was prepared as suggested above and aliquot of the frozen stock solution was thawed at room temperature just before use. To make 1 liter of the medium, about 500ml of distilled water was added to a clean Erlen-meyer flask (2 liter) with

100ml of stock solution-1 and 10 ml of stock solution-2, stock solution-3 and stock solution-4 while stirring with a magnetic stirrer. Approximately, 30g of sucrose was mixed with little water and made into a solution. This solution was added slowly to prevent clumping of the sucrose in the volume and adjusted to pH 5.8. Agar (8 g/l) was added slowly while shaking and the medium was boiled to dissolve the agar and sterilized at 1210C at 15 lbs for 15 minutes. For the use of plant growth regulators, BAP was used in the concentration of 1 to 1.5 mg/Litre.

Culturing of explants

1. The sterilized explants were transferred to sterile tissue paper and blotted.

2. The edges were cut into pieces containing node and were cultured on MS basal medium which was prepared as per treatment to determine appropriate coelomic fluid combination for optimal growth.

Tests for plant growth promoting property of coelomic fluid

The effect of coelomic fluid was tested separately and in combination with vitamins and hormones while having MS media as the basal medium. Altogether there were four groups such as mentioned in the (Table 3.1)

S. No. Name of the Test Group Description (with Abbreviation)

1. Control Group (C) MS + Vitamins + Hormones

2. Test Group I (T1) MS + Hormones + Coelomic fluid

3. Test Group II (T2) MS + Vitamins + Coelomic fluid

4. Test Group III (T3) MS + Coelomic fluid

Table 3.1: Showing the test groups of the study

Results

Demonstration of growth promoting property of the coelomic fluids

Plant tissue culture experiments with coelomic fluid of Eudrilus eugeniae demonstrated the ability of the coelomic fluid to promote shoot induction in Nigella sativa, Tinospora cordifolia, Clitoria ternatea, Andrographis paniculata and Aristolochia bracteata.

Plant tissue culture medium supplemented with coelomic fluid in place of vitamins and hormones showed sprouting of shoots and callus at comparatively shorter time period than the

MS medium and hormone.

Nigella sativa

Under standard illumination and temperature maintained in the plant tissue culture laboratory, the control group of Nigella sativa explants produced callus formation (+++) after a period of 20 days. Height of the control plant was around 0.6 cm with rudimentary leaves. In case of Test Group I (T1), the height of the plant significantly increased to 2.2 cms with 3 leaves and two multiple shoots in the presence of 2% of coelomic fluid. There was no callus formation when compared to the Control Group I. In the T1 group, 0.5% supplementation of coelomic fluid produced a moderate level of callus formation. In Test Group II (T2), with 1 % and 2% of coelomic fluid when used in place of hormones (MS media + Vitamins + CF), produced a highly significant growth of 11.7 and 10.9 cms in height; 14 and 16 number of leaves; 14 and 13 multiple shoots respectively. However, the combination of CF with vitamins in MS media was not productive for callus formation. In this Test Group, there was no growth observed from the explants that were under 0.5 % concentration of coelomic fluid. In the Test Group III (T3), MS

92 media supplemented with Coelomic fluid produced 11 leaves in the plant with 12.2 cm height and 11 shoots. Similar to the T1 and T2 group of plants, callus formation was absent. There was no growth observed from the explants when treated with 2% of coelomic fluid. (Table 3.2,

Figure 3.1)

Tinospora cordifolia

After 20 days of incubation under standard illumination and temperature, the growth- promoting characteristics of the coelomic fluid for Tinospora cordifolia was recorded. In the

Control Group (C), there are no leaves and multiple shoots and the plant grew to a height of 0.8 cm. In the case of coelomic fluid supplemented treatment, the T1 tube containing 400µl of the fluid showed best results with 2 leaves and 2.1 cms of shoot length in comparison to T1 control

(Table 3.3; Figure 3.4). However, caulogenesis was observed in the T1 control unlike the other tubes in the T1 category. In T2 treatment, 400µl of coelomic fluid with vitamins were equally good with T2 control. In the T2 tube with 200µl of coelomic fluid, there was no significant increase in the growth characteristics in comparison to the T2 control. Nevertheless, there was callus formation at 200µl of coelomic fluid in the T2 group. In the T3 treatment, 200µl of coelomic fluid with no supplement of vitamins and hormones resulted in better growth performance with 5 leaves, 2 multiple shoots and a shoot length of 2.2 cms. This is a significant finding when compared to the T3 control and Control Group (C) as well (Table 3.3; Figure 3.2).

In Test Group II and III (T2 and T3), there was no significant growth observed in 0.5 % of coelomic fluid treatment.

Clitoria ternatea

Under the standard illumination and temperature, the explants were maintained in the plant tissue culture laboratory and observed for growth parameters after 20 days‟ time period. In the Control Group, the plants that were in MS media and BAP, grew to a height of 1.5 cms with a single leaf in a single shoot. There was no callus formation seen in any of this control group of tubes. In Test Group (T1), with 0.5 and 2% of coelomic fluid in MS media and hormone (BAP), the plant grew to a height of 2.7 and 2.3 cm respectively. This was a significant growth when compared to the control. There was no difference in the no. of leaves and shoots when compared to the control group. However, there were two shoots produced under 2% concentration. Test

Group II (T2), demonstrated maximum height, higher no. of leaves and significant no. of multiple shoots. In particular, 0.5% and 1 % concentration of the coelomic fluid in MS media containing vitamins produced plants with 3.3 and 3.1 cm height;2 and 3 no. of leaves; 3 and 2 multiple shoots respectively. However, there was no callus formation in any of the groups including the control. Growth parameters of the plants in the Test Group III (T3) treated with coelomic fluid alone in MS basal medium, demonstrated a similar trend as observed in T2. In 1 and 2 % concentration of the coelomic fluid, 2.9 cm and 2.0 cm height was produced with 4 and

2 leaves. There were 2 multiple shoots seen in these two concentrations. However, there was no callus formation observed in any of the treatment groups including the control. There was no growth observed in T3 control which was MS media alone. Similarly, there was no growth observed in the control of T1 group (MS media + Hormone alone) as well. (Table 3.4: Figure

3.3).

Andrographis paniculata

After a 20 days of incubation under standard illumination and temperature in the plant tissue culture laboratory, in Andrographis paniculata, there were two no. of leaves, in a single shoot that grew to a height of 1.8 cms in Control Group (C). In Test Group I (T1), there were significant increase in the no. of leaves, multiple shoots and height of the plant among the three different concentrations of coelomic fluid tested in comparison to the Control Group (C) . In Test

Group I (T1), the tube that had 400µl of coelomic fluid with BAP (1mg/ml) in MS basal media showed a significant increase in the no. of leaves (7 numbers), height of plant (2.6 cm) and multiple shoots (2 numbers) (Table 3.5; Figure 3.4). In Test Group II (T2), the tube with 100µl of coelomic fluid along with vitamin supplement in MS basal medium demonstrated a significant increase in the no. of leaves (7 leaves), height of the plant (2 cms) and multiple shoots (2 numbers). In Test Group III (T3), the tube with 400µl of coelomic fluid only with MS as the basal medium has demonstrated best result with a significant 8 number of leaves, 3 multiple shoots that grew to a height of 3 cms which is a significant record in comparison to the Control

Group.

Aristolochia bracteata

After 20 days of maintenance of the explant/plant under standard plant tissue culture laboratory operations, T3 group showed a significant result with respect to height, no. of leaves and multiple shoot formation when compared to the other treatment groups and control. In particular, with 1 % and 2 % concentration of coelomic fluid, the maximum height of the plant was 3.2 and 2.9 cm with 6 and 5 no. of leaves under single shoot. There was callus formation observed in the explant that was under 0.5 % of coelomic fluid in T3.T1 Group of plants

95 demonstrated growth to a height of 1.8 and 1.2 cm with 4 and 5 leaves from a single shoot in 1 and 0.5 % concentrations of coelomic fluid in MS media containing hormone (BAP). In the

Treatment Group II (T2), the coelomic fluid when present along with MS media containing vitamins, produced 2 and 1.3 cm of plant shoot with 5 and 1 leaves respectively. This treatment group also produced 2 multiple shoot at 1 % concentration of coelomic fluid. In the test groups

(T1 and T2), there was no sign of any growth observed from the explants when treated with 2% of coelomic fluid along with MS media replacing vitamins and hormones respectively. (Table

3.6 Figure 3.5).

Discussion

Chemical agriculture triggered by widespread use of agro-chemicals in the wake of

„green revolution‟ of the 1950s-60s came as a „mixed-blessing‟ rather a „curse in disguise‟ for mankind. It dramatically increased the „quantity‟ of the food produced but severely decreased its

„nutritional quality‟ and also the „soil fertility‟ over the years. The soil has become addict and increasingly greater amount of chemical fertilizers are needed every year to maintain the soil fertility and food productivity at the same levels. Increased use of agro-chemicals have virtually resulted into „biological droughts‟ (severe decline in beneficial soil microbes and earthworms which help to renew the natural fertility of soil) in soils in the regions of green revolution in world where heavy use of agro-chemicals were made. Higher uses of agro-chemicals also demands high use of water for irrigation putting severe stress on ground and surface waters. The scientific community all over the world is desperately looking for an „economically viable, socially safe & environmentally sustainable‟ alternative to the destructive „chemical agriculture‟ which would not only „maintain‟ but also „enhance‟ farm production per hectare of available land as the farmlands all over the world is shrinking in the wake of rapid urbanization. Then, it is not enough to produce „sufficient food‟ to feed the civilization (which was the primary objectives of chemical based green revolution) but also to produce a „high quality of food‟ which should be

„safe‟ (chemical free) and also „protective‟ to human health (good combination of macro and micro nutrients and vitamins) and do it in a sustainable manner to ensure „food security‟ for all, but most for them in the poor third world nations in the long term. „Food safety & security‟ is a major issue everywhere in the world. This will amount to embarking on a „second green revolution‟ and this time by „organic farming‟ practices completely giving up the use of agro-

97 chemicals (Bettolo and Marini, 1987; Bradley and Peggy, 2000; FAO, 2001; FAO, 2004;

Horrigan et al., 2002; Sinha and Rajiv K, 2008).

Sustainable agriculture is a process of learning new and innovative methods developed by both farmers and the farm scientist and also learning from the traditional knowledge and practices of the farmers and implementing what were good in them and also relevant in present times. Vermiculture was practiced by traditional and ancient farmers with enormous benefits accruing for them and their farmlands. There is need to revive this „traditional concept‟ through modern scientific knowledge - „Vermiculture revolution‟. Sir Charles Darwin called the earthworms as „farmer‟s friends‟. There is great wisdom in this statement of the great visionary scientist who advocated to use the earthworms, the „nature‟s gift‟ in farm production.

Earthworms are an important organism in the soil doing great service for mankind for millions of years now. It combines immense social, economic and environmental values together which is now being realized and recognized. A newer branch of biotechnology called

„Vermiculture technology‟ is emerging by the use of earthworms to solve various environmental problems from waste management to land (soil) improvement. Sir Charles Darwin, the great visionary biological scientist highlighted about its role in „soil improvement and farm production‟ long time ago and traditional farming community was also practicing vermiculture in their farms. Unfortunately, very little attention was given to it by post-Darwin biological scientists and the modern agricultural scientists and also the farming community of world who saw „agrochemicals‟ as a technological boon to produce more food in shorter time.

A revolution is unfolding in vermiculture studies (rearing of useful earthworms species) for multiple uses in sustainable waste management and sustainable agriculture (Bhawalkar, , 1995;

Fraser-Quick, 2002; Martin, 1976; Satchell, 1983). Earthworms have over 600 million years of experience in waste & land management, soil improvement & farm production. No wonder, Sir

Charles Darwin called them as the „unheralded soldiers of mankind and farmer‟s friend working day and night under the soil‟ (Rajiv et al., 2009c).

Reports are plenty to describe the effect of earthworms on vermicomposting and organic farming. However there had been no studies undertaken with reference to their direct influence on plant growth promotion in vitro. In this aspect, the present study is a unique approach trying to fill the lacuna in this area. Coelomic fluid is the fluid that exists within the coelom of the animal and the soil is usually kept moist by fluid secretion in part throughout its outer surface.

This formed the basis for taking coelomic fluid as the target of study.

In Nigella sativa, there is a marked increase in the height of the plant (1 % CF with MS

Media + vitamins) producing 14 shoots from single nodal explant. In Tinospora cordifolia,

Coelomic fluid has demonstrated its efficiency at 1 % supplementation level in MS media alone producing 5 leaves, 2.8 cm of plant growth and two shoots from the nodal explant. This is clearly significant and the highest among the other respective group of controls. Similarly, in

Aristolochia bracteata, 1 % of coelomic fluid supplementation alone has produced best results in comparison to the control. Andrographis paniculata also demonstrated significant increase in the number of leaves per explant with higher number of shoots at 2 % concentration of coelomic fluid separately in the MS media. Very much similar to the plants mentioned above,

Clitorea ternatea also produced marked increase in the height of the shoot and number of leaves when used in the absence of both hormones and vitamins in MS media composition. All observations made in these five plants clearly indicated a fact that, coelomic fluid is capable of inducing shoot, faster growth of the shoot, bearing higher number of leaves and formation of multiple shoot in the explant. It is reported that coelomic fluid is rich in amino acids, vitamins, nutrients like nitrogen, potassium, magnesium, zinc, calcium, iron, and copper and some growth hormones like „auxins‟ and „cytokinins‟. It also contains plenty of nitrogen fixing and phosphate solubilizing bacteria (nitrosomonas, nitrobacter and actinomycetes) (Rajiv et al., 2009, a, b, c, d and e) . However, the role of nitrogen and phosphate solubilizing bacteria had been eliminated by filter sterilization process in the present study when supplementing the MS media further adding evidence to the possible role of the „mysterious metabolites‟ that is present in the coelomic fluid.

The present study is a clear indication and an evidence for the role of earthworm and its secretion called coelomic fluid for producing better growth parameters in the crop that is cultivated. Many no. of studies have been carried out with a diluted form of the earthworm secretion called vermiwash. Vermiwash is commercially available and been tested for growth promoting properties when used as a foliar spray. When used it on brinjal and tomato with excellent results. The plants were healthy and bore bigger fruits with unique shine over it. Spray of vermiwash effectively controlled all incidences of pests and diseases, significantly reduced the use of chemical pesticides and insecticides on vegetable crops and the products were significantly different from others with high market value. These farmers are using

100 vermicompost and vermiwash in all their crops since last 4 years completely giving up the use of chemical fertilizers & pesticides.

Composts are aerobically decomposed products of organic wastes such as the cattle dung and animal droppings, farm and forest wastes and the municipal solid wastes (MSW). Bombatkar

(Bombatkar and Vasanthrao, 1996) called them as „miracle‟ for plant growth. They supply balanced nutrients to plant roots and stimulate growth; increase organic matter content of the soil including the „humic substances‟ that affect nutrient accumulation and promote root growth

(Canellas et al., 2000; Siminis et al., 1998). They in fact improve the total physical and chemical properties of the soil. They also add useful micro-organisms to the soil and provide food for the existing soil micro-organisms and thus increase their biological properties and capacity of self- renewal of soil fertility (Ouédraogo et al., 2001; Shiralipour et al., 1992). Beside these, a number of reports on vermicomposts provide us insight into the use of these humble animals as plant growth promoters. It cannot be denied that these vermicomposts are not without the essential secretions of this coelomic fluid.

For conservation and large scale production of the important species, tissue culture seems to be reliable alternative. It is a faster method of asexual reproduction in comparison to propagation through seeds. Most of the plants raised through seeds are highly heterozygous and shows great variation in growth, habit, and yield and may have to be discarded because of poor quality of products for their commercial release. Likewise majority of plants propagated by vegetative means contains systemic bacteria, fungi, viruses which also affect the yield and quality of the product. Some medicinal plants are not amenable to multiplication of desired

101 cultivars. In recent years, tissue culture has emerged as a promising technique to obtain pure and elite population under in vitro conditions. The importance of plant cell and tissue culture in plant science is vast and varied. Plant transformation and gene cloning are becoming important for improvement via genetic engineering. However, the development of an efficient reproducible tissue culture regeneration protocol is the first step in utilizing the power and potential of this new technology. In this technique, synthetic plant growth regulators have been used since its practice began and often involve enormous period of time for the standardization of the protocol.

Any advent that reduce the time period or improve the growth characteristics would have commercial application for the exploitation.

Micropropagation is being used extensively for rapid clonal propagation of many fruit, nuts and ornamental trees (Zimmerman, 1985; Hammerschlag, 1986; Hansman and Y de Novoa,

1986), since it enables rapid propagation and hastens the availability of new cultivars

(Hammerschlag, 1980; Zimmerman, 1981). Using this method, a million-fold increase per year in the rate of clonal multiplication over conventional methods is possible (Murashige, 1974). A micropropagation method could assist in building up the clonal stock of elite genotypes by enabling rapid multiplication after their selection in a breeding programme.

Use of coelomic fluid in plant tissue culture technique especially as a micropropagation technique is a novel approach where in the present study opens up a new avenue demonstrating the efficiency of the fluid to act as natural plant growth promoter especially in shoot induction and multiple shoot formation. Shoot induction and multiple shoot formation within a shorter

102 period of time using coelomic fluid is a breakthrough technique for those who practice plant tissue culture on commercial scale and for research.

Though earthworms are known to be „farmers‟ friend‟ since ages with innumerable reports on vermicompost and its use to increase the growth and yield of the crop, there has not be any study undertaken to test and screen the biochemical constituents from the perspective as a

„plant growth regulator‟ especially in an isolated system as described in this chapter. The present study is an eye-opener in this regard and it is now well established that coelomic fluid is of immense potential to explore further to make the fruits of its power be available globally.

103

Earthworm has been recognized in oriental medicine as anti-inflammatory, analgesic and antipyretic agent (Noda et al., 1992). It shows anticancer effect by preventing excess glucose uptake (Nagasawa et al., 1991). It is also implicated in hemostasis by acting either as a fibrinolytic or anti-coagulatory agent, which results in the facilitation of blood circulation (Wang et al., 1989). The earthworm, therefore has been suspected to contain proteases which specifically dissolve the fibrin clots or anticoagulants which selectively interfere with the intrinsic pathway of the blood coagulation cascade (Mann et al., 1990; Davie et al., 1991);

Leipner et al., 1993; Kim et al., 1995; Woo et al., 1996).

Earthworms have been widely used in traditional Chinese medicine for thousands of years. However, it is only during the past few decades, with the development of biochemical technologies, that research on the pharmaceutical effects of earthworms has been initiated.

Fibrinolytic enzymes were first isolated from earthworms in 1980‟s, (Mihara et al., 1983; Lu et al., 1988; Lin et al., 2000) and, since that time, the medical value of earthworms has been given much more consideration. It was found that earthworm extracts could significantly diminish the coagulation of platelets and promote the dissolution of thrombi in the blood. Its therapeutic and preventive effects for thrombosis-related disease have been confirmed clinically. In fact, earthworm extracts had been clinically made into medications for the treatment of thrombosis- related disease in China. Recently earthworm extract was found to have an anti-tumor effect (Xie et al., 2003; Hu et al., 2002; Yuan et al., 2004; Lin and Zhou, 2002; Zhao et al., 2002).

Apoptosis is a fundamental physiological process in mammals in which cells die by activating an intrinsic suicide mechanism. Defects in apoptotic signaling pathways play critical

104 roles in a multiplicity of pathophysiological status including cancer. The highly regulated systematic nature of apoptosis lends itself to distinct morphological and biochemical criteria including selective, and tightly controlled activation of proteolytic cascades that results in an ordered disassembly of cells. In anticancer research, the focus is on those bioactive compounds of natural origin that are known for toxic effects. Based on the reports of antimicrobial activity of the coelomic fluid and few citations of its anticancer activity against HeLa cell line, experiments were conducted to validate if there are any cytotoxic activity present in the coelomic fluids that could be a potent lead molecule in search for inducing apoptosis.

105

Materials and methods

Earthworm – Eudrilus eugeniae

Worms used in this study were Eudrilus eugeniae. The earthworms were collected from the Periyar Research Organisation for Bio-Technique & Eco-System (PROBE), Periyar

Maniyammai University, Vallam, Thanjavur, Tamil Nadu, India.

Collection of coelomic fluid by cold facile method

In this method approximately 40 grams of worms were taken from the culture pit and washed with sterile distilled water. The worms are then dried on a filter paper and placed in a nylon mesh rolled into a „cone‟ shape to fit into the glass funnel. The funnel is held in a burette clamp on a titration stand. The set-up is shown in the figure 1.3d. A bag of ice that fits over the funnel was placed above the worms such that the worms could feel the drop in temperature due to the ice pack above. The coelomic fluid was made to release through the dorsal pores of its body due to the drop in temperature surrounding it. The fluid was collected in a clean sterile dry test tube that was fit to the end of the funnel. The collection was carried out for 30 minutes and the worms were quickly released into a separate worm–culture pit for relaxation. The collected coelomic fluid was then centrifuged at 5000 rpm for 10 minutes to deposit the debris and the clear straw-colored supernatant was then filter sterilized through 0.2 m syringe filter into a clean, dry, sterile microfuge in a Laminar Air flow chamber and stored under -200C.

Screening Anti-cancer activity in SiHa Cell line

The coelomic fluid was subjected to anticancer activity in SiHa cell line under different dilution as described below. SiHa cell line was procured from NCCS, Pune. The cells were

106 grown in RPMI 1640 medium supplemented with 10% Fetal Calf serum; with antibiotics streptomycin at a concentration of 100 microgram per milliliter and Penicillin at a concentration of 100 units per milliliter and incubated at 37oC with 5% carbon dioxide.

MTT assay

It is a sensitive, quantitative and reliable colorimetric assay that measures the viability, proliferation and activity of cells. This assay is based on capacity of mitochondrial dehydrogenase enzyme in living cells, which convert the yellow water-soluble substrate

3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazoliumbromide (MTT) into a dark blue formazan product that is insoluble in water. The amount of formazan produced is directly proportional to the cell number in a range of cell lines. The results are consistent with those obtained from [3H] thymidine uptake assay. The MTT assay is more useful in detection of cells that are not dividing but are still active. Therefore it can be used to distinguish between proliferation and cell activation. The technique permits the processing of a large number of samples with a high degree of precision using a multi-well scanning spectrophotometer (Micro ELISA reader).

Reagents

MTT stain; Hydrochloric acid; Propan-2-ol

Procedure

MTT stock solution of 5mg/ml (sigma, St. Lowis) in phosphate buffered saline (PBS) at pH 7.4

was prepared and filters through a sterile filter to remove the debris or the insoluble residues.

To 100 l of the suspension or cells in microtitre well 10 l of MTT stain was added.

107

The mixture incubated in a humidified incubator at 37 c for 3 hours.

Exactly 100 l of 0.04M hydrochloric acid in propan-2-ol was added to each well and mixed

thoroughly to dissolve blue formazan crystal.

The plate was read on Micro-ELISA reader at a wavelength of 570nanometer and a reference

wavelength of 630nanometer.

Cell viability test

The most common method for counting the viable cell was Haemocytometer- Nauber counting chamber method. The use of viability stain Trypan blue was ensuring the quantitative analysis of culture. It was the stain that can cross the membrane of dead or nonviable cells and become swollen, larger and appear dark blue in color. Viable cells were stay small, round and refractile and appear as colorless cells.

Reagent

About 0.4 gram Trypan blue in 100 ml physiological saline.

Procedure

The Haemocytometer and cover slip was thoroughly washed and dried.

The cell suspension was gently mixed and the Trypan blue solution was added in drops

depending on the concentration of cells.

The sample was drawn in Pasture pipette and gently placed on the counting chamber.

The chamber was then focused under light microscope in low power for observing the cells.

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The number of cells in one millimeter square area was counted until at least 200 unstained cells

was observed.

The both the stained and unstained cells was counted.

Cell culturing

Protocol for sub culturing

Required

RPMI 1640 medium

Phosphate buffered saline

Trypsin – EDTA

Procedure

The flask was carefully examined under inverted microscope, if any contaminant were grown.

Then the flask was transferred to the Class III inoculation chamber and the old medium was

aseptically discarded.

Then flask was washed with PBS to remove the excess medium in the flask.

Exactly 6 ml of Trypsin- EDTA solution was added to the flask and keep as such without

disturbances for 2-6 minutes. During that time monolayer cells were separated as individual

cells.

6 ml of sterilized medium was added to the flask to stop the action of Trypsin- EDTA solution.

Transfer the mixture to screw cap tubes aseptically for centrifugation. Cells were settled down.

The supernatant was discarded and then add 1ml of sterilized medium and vortexed.

Count the viable cells using Haemocytometer.

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The sample was added to new flasks to that add 4 to 5 ml of sterilized medium to the flask and

incubated at 370 C with 5% carbon dioxide.

Assessment of cell morphology

Reagent

Acridine orange/ethidium bromide staining solution

One part of 100µg/ml Acridine orange in PBS and part of 100µg/ml ethidium bromide in

PBS.

Hoechst 33258; Stock solution

Exactly 1 mg of the dye was added to 1ml of PBS buffer.

Working solution

About 5µl of the stock solution was taken and the volume was made up to 500µl using distilled water.

Method

SiHa cells were grown in 6 well plates (5×103 cells /well) for hr. the cells were then incubated with the IC50 concentration of the methanolic extacts for 24 and 48 hr. the medium was discarded and the cells were washed with PBS. The cells were then trypsinized and placed on a glass slide and stained with Acridine Orange and Ethidium Bromide (AOEB) or Hoechst

33258 stain. The cells then viewed in an epifluoresent microscope (Olympus, Japan).

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DNA agarose gel electrophoresis assay

Cells treated with different concentrations of coelomic fluid (4µl/ml and 5µl/ml concentration), were harvested and washed three times with ice-cold phosphate buffered saline

(PBS) (pH 7.2) and analyzed . After centrifugation for 15 minutes at 10,000rpm, the cells were subjected to DNA isolation using Mammalian DNA isolation kit (Himedia, Mumbai, India) using the spin column technique. The DNA was then separated by Agarose Gel Electrophoresis

(Yanqin et al., 2005) and viewed under UV illumination.

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RESULTS

Anti-proliferative assay in SiHa Cell Line

MTT assay

The cytotoxicity of the coelomic fluid cultured on SiHa human cervical cancer cells by exposing cells to 2µ/ml, 4µl/ml, 5µl/ml, 6µl/ml and 7µl/ml from the stock concentrations for 24 hrs. The reduced MTT-formazan was dissolved in DMSO and the absorbance was read in 96- well-plate reader. The graphs were plotted as % inhibition (absorbance at y-axis) against the concentration of the drug (x-axis). The IC50 concentration was determined as the drug concentration that is required to reduce the half of the cells from the total population. The IC50 value in respect of the on the SiHa cell growth 5µl/ml concentration. Apoptosis screening was carried out with sublethal dosages fixed at 4µl/ml and 5µl/ml concentration. Lower dose range of the extracts is sufficient to kill the cells at 24 hrs .

Morphological changes in target cells after the treatment with coelomic fluid

The morphological changes in cell lines were visualized by phase contrast microscopic observation. The views of the cells clearly indicated morphological changes in the coelomic fluid treated groups when compared to that of the control. According to these views, cell damage resembled necrosis; shrunken cells were observed, swollen nuclei and preserved nucleoli. It is believed that the observation is due to the coelomic fluid, which presumably or partly derived from coelomocytes that caused cell death; however, results do not distinguish between necrosis and apoptosis very clearly.

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Staining with Acridine Orange-Ethdium Bromide (AOEB) and Hoechst 33258

Acridine orange (AO) – ethidium bromide (EB) and hoechst staining were done for the

SiHa cells treated with IC50 concentration of the coelomic fluid for 24hrs. The control cells appeared green and blue respectively. Photographic evidence of AOEB staining of treated cells was indicative of apoptosis. Therefore, coelomic fluid of E. eugeniae concluded to be significant for further apoptotic studies. However, the coelomic fluid has demonstrated suggestive findings for further investigation due to necrotic lesions seen at higher concentration well. Cells stained with both the stains revealed cytoplasmic blebbing, presence of apoptotic bodies, marginalization of chromatin and innumerable micronuclei in cells treated with the sub-lethal dosage levels of coelomic fluid (Figure 4.2). These cytological changes indicated that the cells were committed to specific mode of cell death, probably apoptosis.

Agarose gel electrophoresis

Agarose gel electrophoresis results of DNA extracted from untreated and treated cells by coelomic fluid are shown in Figure 4.3. The DNA from untreated cells was unfragmented with one band (E), addition of 4µl/ml of coelomic fluid to SiHa cells showed a smear pattern (B and

C) indicating DNA ladder probably due to apoptosis.

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Discussion

In recent years, increased attention has been focused on the evolution of the invertebrate immune system. There is specific interest in effector molecules in antimicrobial and cytotoxic responses. Biologically active molecules are present since early phases of evolution onwards. In fact, invertebrates, vertebrates and plants utilize analogous immunodefense molecules for millions of years (Vizioli and Salzet, 2002; Nappi and Ottaviani , 2000). The coelomic fluid of earthworms exhibits different biological functions, including bacteriostatic, proteolytic, cytolytic

(hemolytic) and mitogenic activities (Cooper et al., 2002). There is a factor known as EFAF

(Eisenia fetida andrei factor), named fetidin, which exhibits antibacterial and hemolytic activities (Milochau et al., 1997). It is well known that certain molecules from the coelomic fluid inhibit bacterial growth and lyse different mammalian red blood cells (Mohrig et al., 1996;

Lange et al., 1997; Eue et al., 1998). This literature reveals the existence of these immune molecules in the coelomic fluid and presumes that earthworm effector leukocytes (e.g. coelomocytes) can participate in the production of these humoral factors. Coelomocytes share activities of innate cellular immune systems including phagocytosis, wound healing, graft rejection, nodule formation and encapsulation.

Earlier, special interest had been taken in detecting highly conserved molecules including cell surface antigens, cytokines, respiratory burst enzymes and hormones in the invertebrate immune-competent cells (Engelmann et al., 2002). According to the literature, coelomocytes can be divided into three main populations by light microscopic cytology: hyaline and granular amoebocytes, and chloragocytes; however, two cell populations (large coelomocytes and small coelomocytes) were detected by the first flow cytometric analysis (Cossarizza et al., 1996).

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Recent flow cytometric and confocal microscopic studies showed three well-separated cell populations, expressing different cell surface and intracellular markers (Engelmann et al., 2004;

Engelmann et al., 2002a,b, Engelmann et al., 2010). Further recent studies identified cellular cytotoxicity related to earthworm coelomocytes (Quagliano et al., 1996. Published data showed cellular cytotoxic effects of coelomocytes in xenogeneic and allogeneic culture of earthworm coelomocytes as well as coelomocyte cytotoxicity against mammalian tumor cell lines (Cooper et al., 1995; Suzuki and Cooper, 1995). In this chapter, the cytotoxicity of the coelomic fluid of the earthworm E. eugeniae was focussed.

Earthworm coelomic fluid contains biologically active molecules and leucocytes that participate in phagocytosis encapsulation and granulomas. Several proteins and peptides with anti-tumor and anti-bacterial activities have been reported in coelomic fluids (Cossarizza et al.,

1996; Quagliano et al., 1996; Cooper et al., 1995; Suzuki and Cooper, 1995; Eyambe et al.,

1991). This research has investigated the antitumor activity of coelomic fluid of the earthworm

E. eugeniae and have analyzed the mechanisms of the antitumor activity of coelomic fluid using in vitro methods.

Earthworm, Dilong, is a cold, slightly salty traditional Chinese natural product. Li

Shizhen illustrated 40 diseases in the book Bencao Gangmu, in which earthworms act as a main component. In clinics, earthworm is used to treat chronic bronchitis, bronchial asthma, psychosis, digestive tract ulcer, peptic ulcer, epidemic parotitis, herpes zoster, urticaria, burn, scald, bladder calculi, urinating obstacle, and cancer (Vizioli and Salzet, 2002; Nappi AJ,

Ottaviani, 2000; Cooper et al., 2002). Recently more and more researches focus on the main

115 component investigation with antitumor activity from different parts of the earthworm. Several components with antitumor activity were found from the whole body tissue of earthworm including G-90, a mixture of different molecules with molecular weights of 33 kDa, 40 kDa, 42 kDa, 45 kDa and 60 kDa [4-7-8]. The necrotic and apoptotic activity of the coelomic fluid of E. eugeniae must be related to the components contained within the coelomic fluid. Several antitumor and antibacterial were found by many researchers (Cossarizza et al., 1996, Cooper et al., 1995; Suzuki and Cooper, 1995; Eyambe et al., 1991).

The coelomic fluid of earthworms contains cytolytic and hemagglutinating molecules, which can be released from various coelomocytes into the coelomic fluid besides several metabolites in itself. In this chapter, the effects of coelomic fluid and coelomocyte lysates were measured on standard mammalian tumor cell line – SiHa. In vivo experiments correlate with the results presented in this in vitro study; coelomic fluid exerted lethal effects against a wide spectrum of vertebrate animal cells but not on those of invertebrates (Kobayashi et al., 2001.

Some coelomocyte types (e.g. chloragocytes) contain cytotoxic effector molecules and these cytotoxic effects were remarkable after using native coelomic fluid; however, similar effects but with lower intensity were shown after treatment with CCLs (Kobayashi et al., 2001) .

In the present study, coelomic fluid showed a dose dependent effect on HeLa cell detected with MTT assays, AO/EB, Hoechst 33258 assay and DNA agarose gel electrophoresis.

These results which show that high concentration of coelomic fluid induce cell necrosis were similar to those reported by Engelmann, which stated that cell-free coelomic fluid significantly decreased ratios of living cells compared to controls in a dose dependent manner (Raftos and

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Hutchinson, 1995) and Eue et al. (1998), which summarized that Eisenia foetida has a toxic effect on a variety of cell types, such as chicken fibroblasts. Different from report by Nasi et al.

(2002) that coelomic fluid could not lead to cell apoptosis and Linnert and Kauschke (2005) that coelomic fluid of earthworm did not cause Caki-1 apoptotic, the present results demonstrated that 4µl/ml coelomocyte-free coelomic fluid could induce SiHa cell apoptosis in a dose and time dependent manner similar with the former study on oligo peptide from earthworm coelomic fluid

(Yanqin et al., 2005).

The necrotic and apoptotic activities of coelomic fluid of earthworms must be related to components contained within the coelomic fluid. These results and other similar ones that use natural products from animals (Cooper et al., 1995; Akporiaye and Kudalore, 1989; Urdiales et al., 1996) might help to settle a basic question for the pharmacological development of earthworm coelomic fluid in the future as an effective ancient medicine. Overall, it can be concluded from the present study that according to the MTT assay, AO/EB double staining and

DNA agarose gel electrophoresis assays, a mass concentration of coelomic fluid of earthworm,

4µl/ml, can kill SiHa cells by cell necrosis and lysis. Coelomic fluid induced SiHa cell apoptosis in a dose and time dependent manner. From the present, it is proposed that earthworm coelomic fluid may be a future candidate for treating tumors in clinic.

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Textile industries consume large amount of water (60-4001/kg of fabric) and chemicals for wet processing (AEPA, 1998). The chemical reagents used in textile sector are diverse in chemical composition ranging from inorganic to organic. The inputs of wide range of chemicals, which, if not incorporated in the final products (fabric), become waste and turn out to be part of water ecology. Generally, textile effluent is colored, varying in hydraulic flow rate, having high; pH, temperature, biological oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS) and total suspended solids (TSS) (Buckley, 1992; Banat et al., 1996;

Ghoreishi and Haghighi, 2003).

Color is imparted to textile effluents because of various dyes and pigments used. Many dyes are visible in water at concentrations as low as 1 mg/l. textile wastewaters, typically with dye content in the range of 10-200mg/l are therefore highly colored. In addition to this, various salts and chemicals are major sources of heavy metals in wastewater (Wagner 1993). Sediments, suspended and dissolved solids are important repositories for toxic heavy metals and dyes

(Tamburlini, et al., 2002; Chapman et al., 1982) causing rapid depletion of dissolved oxygen leading to oxygen sag in the receiving water (Ademoroti et al., 1992).

The key environmental issues associated with textile manufacture are; water use, treatment and disposal of aqueous effluent. Textile effluents are mostly discharged after minimal or no pretreatment into the adjoining water channels, streams and estuaries (Macaskie and Dean,

1984; Niu et al., 1993). There is a growing emphasis on biological remediation associated with their cost effective and long lasting nature. Presently, textile belt of Tamil Nadu as popularly called had been draining their effluents into the streams and waste lands that were once

118 cultivable until strict rules and regulations were passed to confine their effluents. Since then, the industries have been treating their effluents either by chemical treatment or using biological agents such as bacterium or fungi. However, the large quantities of water ultimately reach the ecosystem even after careful recycling and purification processes as being done recently. Since earthworms are major components in the cultivable lands where the textile industry polluted waters drain into, a study has been undertaken to record the impact of these textile dye industry effluents (raw and chemical or biologically treated) on the population of these worms. Eudrilus eugeniae was employed for the purpose.

Eudrilus eugeniae is an earthworm species indigenous in Africa but it has been bred extensively in the USA, Canada, Europe and Asia for the fish bait market, where it is commonly called the African night crawler. E. eugeniae is a large worm that grows extremely rapidly and is reasonably prolific and, under optimum conditions it would be ideal for animal feed protein production; however there has been relatively little work on the biology and ecology of this species (Madge 1969; Neuhauser et al., 1979; Viljoen and Reinecke 1989, 1992; Reinecke et al.,

1992; Reinecke and Viljoen 1993). In particular, there are no citations regarding the impact of the textile industrial effluent on the population of this species.

In the present study an attempt has been made to manage the textile effluent by vermicomposting technique using an exotic earthworm, Eudrilus eugeniae. The role of earthworm as a decomposer is known since Darwin. Rajesh Banu et al. (2001) and Oboh et al.

(2007) reported that earthworm Eudrilus eugeniae shows the potential to manage paper mill

119 sludge successfully. The end product of vermicomposting is rich in essential micro and macronutrients along with microorganism in a very simple form (Logakanthi et al., 2000;

Parthsarathi et al., 2007). Adding cast not only improves the soil structure and fertility but also leads to improvement in overall plant growth and thus increases their yield too (Kavian and

Ghatnekar, 1991; Kavian et al., 1998). The experiments were carried out for a period of 8 weeks to assess the impact of the effluent on the earthworm. For this, the growth, reproduction and population dynamics of E. eugeniae was studied in the presence of textile effluent. The study summarizes its potential for development as a putative agent for bioremediation of textile industrial polluted soil.

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Materials and Methods

Collection of Raw, chemically and biologically treated textile dye effluent

Textile dye effluents were collected from the United Bleachers Limited at Mettupalayam separately as raw effluent, chemically treated effluent and biologically treated effluent. These were brought to the lab and used for the restoration of moisture content in the earthworm medium as per the treatment regime.

Collection of earthworms, treatment and their maintenance

Earthworm, Eudrilus eugeniae were collected from the Periyar Research Organization for

Bio-Technique & Eco-System (PROBE), Periyar Maniyammai University, Vallam, Tanjore Dist,

Tamil Nadu, South India. Organic waste served as a medium of growth for the worms in the sieved garden soil and cow dung mixed in the ratio of 2:1. The mixture was allowed to dry under sun-light for 10 hours. After mixing subsequent amount of water, the worms were allowed to be contained in suitable perforated plastic containers with the soil (figure 5.1). The study was carried out in four groups such as the Control (Group I) receiving only tap water, Group II: receiving raw effluent, Group III receiving the chemically treated effluent and Group IV receiving the biologically treated effluent. The effluents were given for the first ten days followed by the tap water for all the groups in order to accommodate study in a small volume of soil taken in the culture-basket. The study was prolonged for a period of 8 weeks with a changeover of only 30% of medium (wt/wt) at the end of 4 weeks. Care was taken to rear the worms with 82% of moisture in the immediate surroundings. The optimum temperature was maintained constantly by spreading some banana leaves over the culture basket. Viability of the worms, cocoon production, hatchlings and weight of the worms were recorded for every seven

121 days from day 1. At the time of recording, the worms were taken out gently in a cotton mesh and washed with a gentle flow of water. For this, the soil from each basket was carefully placed on a clean white surface and the worms, cocoons and hatchlings were collected carefully and weighed or counted as appropriate.

Collection of raw, chemically and biologically treated textile dye effluent

Earthworm culture medium

The organic waste served as a medium for the growth of earthworms containing sieved garden soil and cow dung was mixed well in the radio of 2:1. The mixture was allowed to dry under the sun light for ten hours. After mixing subsequent amount of water, earthworms were allowed to inhabit the medium as per the treatment category.

Culturing of earthworms

Three earthworms each having similar weights and length was placed in a specially designed perforated plastic basket of 10 cm diameter× 8 cm deep having 300 g of earthworm culture medium as prepared above. Three replicates for each treatment were kept at room temperature.

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Treatment Groups

The medium was provided with appropriate effluent/water as per the treatment groups given below. As given in the table, the treatment was provided with appropriate effluents / water for the first ten days and from the eleventh day onwards, all the treatment group received only tap water for a period of 8 weeks.

Treatment Description of the Volume of No. of. days Group Treatment treatment/day of treatment Number 1. Sterilized water 15 ml 10 2. Raw effluent 15 ml 10 Chemically Treated 3. 15 ml 10 Effluent Biologically Treated 4. 15 ml 10 Effluent

Table 5.1: Treatment Groups for the study of growth and Fecundity in Eudrilus eugeniae.

Maintenance

Earthworms were maintained with more than 80% of moisture by sprinkling the water daily. The optimum temperature was maintained constantly by spreading some banana leaves over the culture medium. At regular intervals, as the cattle solids were consumed by the earthworms, 100 g of the medium was removed and replaced with 100 g fresh solid wastes.

Study Parameters

Earthworm fecundity is often expressed in various ways, viz., the rate of cocoon production, hatching success of cocoons and number of offspring‟s emerging from each cocoon.

The success of the composting depends upon the fecundity of the earthworm.

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The impact of effluents on the growth and fecundity of the worms were recorded in terms of weight of the worms, mortality, cocoon production and hatchling in different treatment groups for every seven days and the study was prolonged for a period of 8 weeks.

At the time of recording, the worms were taken out gently in a cotton mesh and washed with a gentle flow of water. For this, the soil from each basket was carefully placed on a clean white surface and the worms, cocoons and hatchlings were collected carefully and weighed or counted as appropriate.

The worms which are cultured in the basket for particular treatment was taken out carefully in a cotton mesh and was washed with the gentle flow of water. Number of viable worms was noted. After removing all the soil particles from the worm‟s body, they were tried with another cotton cloth, than the worms were weighed and the weight was noted. Worms were replaced immediately in the culture medium. It was done at the regular interval of seven days.

Cocoon study

After taking out the worms, the culture medium was spread over the plastic sheet.

Cocoons were carefully visualized and collected on a cotton mesh. The number of cocoons per the individual treatment was counted and noted, than the cocoons were replaced in its own basket for hatchling production.

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Hatchling study

The same procedure was followed as a cocoon study but care should be taken on handling the hatchlings as it was so soft. The hatchlings were taken in a separate medium until whole of the basket has been counted. After the count was finished they were replaced in their own basket.

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Results

Number and weight of the worms, number of hatchlings and cocoons were recorded at an interval of seven days for 8 weeks in all the treatment groups. Viability of the worms were recorded for a period of 8 weeks where, worms in Group II and the Control (Group I) were all viable. Group III and Group IV worms demonstrated mortality to 33% and 100 % respectively at sixth week (Figure 5.2). Absolute mortality observed in Group IV worms is possibly due to the odour, heavy microbial load and / or the metabolites present in the biologically treated effluent.

The mean weight of the worms have reduced significantly (p<0.001) from 3.48±0.92 to about 1.65±0.65 in the control group I. A similar observation was made in the treatment groups

II and III also where there is a reduction in size of worms by 2-3 folds. However, in the biologically treated group (IV), there was high mortality after 5 weeks (Figure 5.3).

In Group IV, in week 5, the weight of the worms reduced to nearly 3 folds when compared to the control which took 8 weeks for similar observation. All the three worms in the biologically treated effluent-soil died in the sixth week itself.

As a measure of the population growth, cocoon production was recorded among all the three treatments in comparison with the control. An average of 7 cocoons were seen in the control group whereas, in the group-II (raw effluent) it was raised to a maximum of 22 in the fifth week and finally were 17 at the end of eight weeks. This is a significant increase when compared to the control group-I (Figure 5.4).

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In the chemically treated effluent-soil, the number of cocoons was comparable to that in the control without any significant change. In contrast to this, there were no cocoon productions seen in the biologically treated-soil group (IV).

Hatchlings were counted every seven days and there were two hatchlings at the end of four weeks in the control group-I which was significantly (p<0.001) different from the (Group II) raw effluent treated-soil which produced 7 cocoons.

Chemically treated and biologically treated-soil groups (III and IV) did not produce hatchlings through cocoons (Figure 5.5). There was significant loss in weight of the worms throughout the experimental period which is probably due to the limited space and soil available in the basket containing it. Chemically treated effluent-soil in group-III demonstrated stress and toxicity due to chemicals to certain levels that caused morbidity and mortality though not to the level of the biologically treated effluent-soil group-IV.

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Discussion

The centrality of earthworms in terrestrial ecotoxicology (Spurgeon et al., 2003) partly reflects their acknowledged status as ecosystem engineers (Lavelle, 1997). These macro invertebrates have highly permeable general body surfaces that render them potentially vulnerable to intoxication by soil pollutants and to invasion by soil-dwelling pathogens.

Consequently, the negative effects of inorganic and organic residues on the structure and function of earthworm immune cells, coelomocytes, have been a subject of considerable research activity (Kurek et al., 2002; Cooper and Roch, 2003; Homa et al., 2003, 2005; Wieczorek-

Olchawa et al., 2003). Whilst these studies have contributed to a better understanding of the evolution of invertebrate immune systems, and have yielded useful cellular biomarkers for application in environmental diagnostics, there are many facets of ontogeny, consanguinity, and functionality of coelomocytes that remain obscure.

A greater proportion (>80%) of the biomass of terrestrial invertebrates is represented by earthworms which play an important role in structuring and increasing the nutrient content of the soil. Therefore, they can be suitable bio-indicators of chemical contamination of the soil in terrestrial ecosystems providing an early warning of deterioration in soil quality (Culy and Berry,

1995; Sorour and Larink, 2001; Bustos-Obregon and Goicochea, 2001). This is important for protecting the health of the natural environment and is of increasing interest in the context of protecting human health (Beeby, 2001) as well as other terrestrial vertebrates which prey upon earthworms (Dell‟Omo et al., 1999). The suitability of earthworms as bioindicators in soil toxicity is largely due to the fact that they ingest large quantity of decomposed litter, manure and other organic matter deposited on soil, helping to convert it into rich topsoil (Reinecke and

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Reinecke, 1999, Sandoval et al., 2001). Moreover, studies have shown that the earthworm skin is a significant route of contaminant uptake (Lord, 1980) and thus investigation of earthworm biomarkers in the ecological risk assessment (ERA) can be helpful (Sanchez-Hernandez, 2006).

They have also been observed to be valuable as index organisms in evaluating the impact of soil- borne contaminants (Ireland, 1979; Callahan et al., 1991; Schaefer, 2001). With the increasing rate of industrialization and urban development, there is no doubt that the environment we live in today is exposed to greater pollution. There is a need therefore to evaluate an off-side test for assessing the impact of textile industry effluent on the activities of the earthworm species

Eudrilus eugeniae.

Under „normal‟ conditions the coelomic cavity of healthy earthworms is inhabited by various soil-derived parasites, such as bacteria, gregarines and fungi (Dales and Kalac., 1992;

Tuckova and Bilej., 1996; Reinhart and Dollahon, 2003; Wieczorek-Olchawa et al., 2003). The populations of extraneous invading organisms are kept in check by the combined activities of coelomocytes and humoral factors. Though, the treatment of textile effluent using microorganism is an eco-friendly and feasible approach, the treated effluent (biologically treated) had an adverse effect in the present study in contrast to the expectation. There was 100% mortality of the worms in the Group IV. This is probably due to two main reasons. First being that the microbial load of the treated effluent. The balance between microbial invaders

(employed in effluent treatment and the soil flora) and the host may be shifted in favour of the invaders either by the elevation of pathogenicity in the source soil or by the impairment of host immunity. This is probably a reason for the observation that there was total mortality of the worms in Group IV. Secondly, mortality might be due to the toxic metabolites. The primary

129 amines that are derived from the microbial decolorization of the dyes, are mutagenic and toxic to the animals, especially the lower vertebrates and the invertebrates (Chung and Stevens, 1993).

The availability of reliable ecotoxicological test methods and risk assessment procedures is paramount for the successful implementation of regulatory guidelines for the classification of polluted soils and chemicals (Van Straalen and Løkke, 1997). Various sensitive endpoints have been identified in earthworm ecotoxicology, such as weight change and reproductive success

(Kokta 1992).

Reproduction is of particular importance in eco-toxicological assessment because of its influence on population dynamics (Spurgeon et al., 1994). Very little comparative data are however available for effects of metals on the reproduction of different earthworms species. The aim of this study was to assess the effects of textile effluent (raw, chemical and biologically treated) on the production and viability of cocoons of Eudrilus eugeniae, a tropical species from

West Africa.

Feeding of earthworm onto a portion of the organic materials in the wastes or undigested/partially digested materials relies on the increased microbial activity and nutrient availability in the soil (Benitez et al., 1999). The indigenous microflora and the change of soil structure caused by earthworm activities create the amenable environment for microbial growth

(Sidhu et al., 2001; Williams et al., 2006). At the same time, earthworm is universally accepted as one of the most suitable representatives of soil animals used for soil contamination surveys

(Gao et al., 2008; Reinecke and Reinecke, 2004); for the capacity of accumulate and concentrate

130 large quantities of inorganic and organic pollutants. At present, the characterization of

Escherichia coli O157:H7 with the ecotoxicological tests has not been reported yet.

The disposal of organic wastes from domestic, agricultural and industrial sources is increasingly causing environmental and economic problems. Vermicomposting, the microbial composting of organic wastes through earthworm activity, has proved to be successful in processing sewage sludges and solids from wastewater (Neuhauser et al., 1988; Domínguez et al., 2000), materials from breweries (Butt, 1993), paper wastes (Butt 1993; Elvira et al. 1996a), urban food and garden residues, animal wastes (Edwards et al. 1985; Edwards 1988; Elvira et al.

1996b; Domínguez and Edwards, 1997), as well as horticultural residues from processed potatoes, dead plants and the brewery and mushroom industries (Edwards 1988). Most of this research has utilized the earthworm species Eisenia fetida and Eisenia andrei, and the potential of both species as a source of protein, to be used for animal feed, has also been reported (Schulz and Graff, 1977; Sabine, 1978; Hartenstein, 1981; Edwards and Niederer, 1988; Edwards, 1988).

Only one other species, Lumbricus terrestris L., has been suggested for this purpose (Fosgate and Babb, 1972).

The conclusions of the present investigation are that Eudrilus eugeniae is a fast-growing and productive earthworm in animal waste that is ideally suited for fast bioremediation of polluted soil. It is more productive in terms of rates of growth than other species and would seem to be a suitable candidate for vermicomposting systems, in regions where the optimal temperature of 25°C is both feasible and economic. Although the large size of E. eugeniae makes it much easier to handle and harvest, than commonly-used species such E. fetida and

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P. excavatus, it is much more sensitive to disturbance and handling and may occasionally migrate from breeding beds. Since it has been grown commercially for fish bait for a long time, it shows that it is comparatively easy to rear.

The results of the present study showed high level of fecundity when compared with the results of other biomanagement studies like paper-sludge and petrochemical-sludge vermicomposting (Rajesh Banu et al., 2001, 2005).

It is necessary to manage huge quantity of textile dye effluent in an ecofriendly manner.

In this context, several physical, chemical and biological methods have been tried to treat solids namely, gravity separation (Singh, 1992), anaerobic digestion (Rajesh Banu et al., 2007), fungal composting (Logakanthi et al., 2006) thermal treatment (Burner, 1997; Binner et al., 2000;

Burton and Ravi Shankar, 2000), pyrolysis (Gayle, 1999; Borup and Middlebrooks, 2000) and stabilization and solidification (Le Grega et al.,1994).

It can be concluded that textile effluent was found to be non-toxic to the earthworm and amenable to vermicomposting. Further studies employing different earthworm species and on the impacts of textile effluent/ effluent polluted soil, would help to evaluate the process and its application may pave way for pilot scale experiment. The extent to which effluent pollution will manifest itself at the population level needs further investigation in order to assess the usefulness of this endpoint in ecotoxicological work.

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C T1 T2 T3

Figure 3.2: Demonstrating the growth promoting property of Coelomic fluid in Tinospora cordifolia C: Control; T1: Coelomic fluid supplemented for Vitamins; T2: Coelomic fluid supplemented for Hormones; T3: Coelomic fluid supplemented for Vitamins and Hormones.

Figure 3.1: Demonstrating the growth promoting property of Coelomic fluid in Nigella sativa C: Control; T1: Coelomic fluid supplemented for Vitamins; T2: Coelomic fluid supplemented for Hormones; T3: Coelomic fluid supplemented for Vitamins and Hormones.

Figure 3.3: Demonstrating the growth promoting property of Coelomic fluid in Clitoria ternatea C: Control; T1: Coelomic fluid supplemented for Vitamins; T2: Coelomic fluid supplemented for Hormones; T3: Coelomic fluid supplemented for Vitamins and Hormones.

Figure 3.4: Demonstrating the growth promoting property of Coelomic fluid in Andrographis paniculata C: Control; T1: Coelomic fluid supplemented for Vitamins; T2: Coelomic fluid supplemented for Hormones; T3: Coelomic fluid supplemented for Vitamins and Hormones.

Figure 3.5: Demonstrating the growth promoting property of Coelomic fluid in Aristolichia bracteata C: Control; T1: Coelomic fluid supplemented for Vitamins; T2: Coelomic fluid supplemented for Hormones; T3: Coelomic fluid supplemented for Vitamins and Hormones.

a b

c d

Figure 1.1: Earthworm rearing pits at Periyar Research Organization for Biotechnique & Eco- System (PROBE), Periyar Maniyammai University, Vallam, Tanjore Dist, Tamil Nadu, South India; (a) Earthworm rearing huts; (b & c) earthworm rearing pits; (d) Vermicompost storage tanks.

a b

C d

Figure 1.2: Preparation of cow-dung and soil mixture for rearing the earthworms; (a & b) Soil mixed and dried under sun light and then sprinkled with water for thorough mixing; (c) worms reared in cement tanks filled with cow-dung-soil mixture and (d) earthworms periodically checked for its alive status; (e) earthworms cleaned and kept on filter paper prior to coelomic fluid extaction.

a b

c d

Figure 1.3: Coelomic fluid collection by various methods. (a) Warm water method; (b & c) Electric shock method; (d) Cold-shock and heat–shock method.

Figure 4.2: Microphotography of SiHa cell lines treated with the coelomic fluid. ABC – SiHa cell line under Phase Contrast Microscope (Control); DEF – SiHa cell line under AOEB staining (Fluorescent microscope); and GHI – SiHa cell line under Hoescht 33258 staining (Fluorescent microscope); ADG – Control group; B,E,H, - SiHa cell line after 24 hrs of treatment; C,F, I – SiHa cell line after 48 hrs of treatment of coelomic fluid at 5 µl/ml.

Figure 4.1: Morphology of SiHa cell line under Bright Field View (A & D) and Phase Contrast View (B,C and E); A & B – SiHa cell line under confluency before trypsinization at 10X; C – SiHa cell line under confluency before trypsinization at 40X; D & E – SiHa cell line after trypsinization at 10X.

A B

C D

Figure 5.1: Earthworms (Eudrilus eugeniae) reared in baskets for the study on growth and fecundity under the effluence of effluent (Raw, chemical and biologically treated effluents) in comparison to the water as control. (A & B – perforated basket containing soil-cowdung mixture; C & D – treatments).

4.5

4 Control(Group-1) 3.5 3 Raw effluent(Group-2) 2.5 Chemically Treated 2 Effluent(Group-3) Biologically Treated Worms weight 1.5 Effluent(Group-4) 1 0.5 0 1 2 3 4 5 6 7 8 Weeks

Figure 5.2: Graph showing the weight of worms under the treatment of raw, chemically treated, and biologically treated effluents in comparison to the tap water (Control).

3 Control(Group-1)

2.5

2 Raw effluent(Group-2)

1.5

1 Chemically Treated 0.5 Effluent(Group-3) No.of.Viable Worms No.of.Viable

0 Biologically Treated 1 2 3 4 Effluent(Group-4) 5 6 7 8 Weeks

Figure 5.3: Graph showing the number of viable worms under the treatment of raw, chemically treated, and biologically treated effluents in comparison to the tap water (Control).

20 Control(Group-1)

15 Raw effluent(Group-2)

10 Chemically Treated Effluent(Group-3)

No.of.Cocoons Biologically Treated 5 Effluent(Group-4)

Weeks

Figure 5.4: Graph showing number cocoons formed under the treatment of raw, chemically treated, and biologically treated effluents in comparison to the tap water (Control).

7 Control(Group-1) 6

5 Raw effluent(Group-2)

4 Chemically Treated Effluent(Group-3) 3

No.of.Hatchlings Biologically Treated 2 Effluent(Group-4)

0 1 2 3 4 5 6 7 8 Weeks

Figure 5.5: Graph showing number cocoons formed under the treatment of raw, chemically treated, and biologically treated effluents in comparison to the tap water (Control).

A B C

D E F

G H I

Figure 2.1: Plates showing antimicrobial activity and hemolytic activity of the coelomic fluid. A- S. aureus; B – S. typhi; C – E. coli; D - P. aeruginosa; E - K. pneumoniae; F – Bacillus sp.; G – E. foecalis; H & I – Hymolysis of blood agar plate by coelomic fluid.

Lane 1 2 3

Figure 4.3: DNA agarose gel electrophoresis under UV trans-illumination showing the ladder formation as streaks in lanes 2 and 3 in comparison to the control DNA on lane 1.

Figure 1.4: SDS PAGE of coelomic fluid; Lane 1: BSA; Lane 2 & 6: Crude C.F Acetone ppt. ptn. (58, 47, 30, 20 & 16 KDa); Lane 3: Dialyzed Crude C.F (13 & 9 KDa); Lane 4 & 5: Centrifuged C.F Acetone ppt. after dialysis (29 KDa).

Figure 1.5: Protease activity demonstrated by the coelomic fluid; Conc. of coelomic fluid: U1 & U2 – 40 µl ; U3 & U4 –20 µl

Cold Shock Heat Shock Warm Water Electric Shock Method Method Method Method Wt. of Worms 15 ± 0.25 15 ± 1.1 15 ± 1.2 15 ± 0.98 (gm) Volume of Fluid 0.5 ± 0.1 1.5 ± 0.1 0.5 ± 0.01 25 ± 0.9 (ml) Time (Minutes) 30 30 30 30 Worms almost Worms are alive Worms almost Worms are alive died or came out and active; die; Remarks and active; Fluid is of the funnel; low Concentrated concentrated highly Diluted volume of fluid fluid fluid collection

Table 1.1: Showing the results for fluid collection methods

I Collection on Day-10 II Collection on Day -20 III Collection on Day -30 Wt. of Worms (gm) 15 ± 1.5 15 ± 0.75 15 ± 1.02 Volume of Fluid (ml) 1.3 ± 0.1 1.28 ± 0.21 1.33 ± 0.15 Time (Minutes) 30 30 30 Worms are alive and Worms are alive and Worms are alive and Remarks active; Concentrated active; Concentrated active; Concentrated fluid fluid fluid

Table 1.2: Volumes of coelomic fluid collected and the status of the worms after collection

S. No. Compound Concentration 1. Total Sugar 720± 17 µg/ml 2. Reducing Sugar 282 ± 11 µg/ml 3. Protein 3600 ± 129 g/ml 4. Amino Acids 1400 ± 98 µg/ml 5. Cholesterol 30 ± 1.5 mg/dl 6. Triclyceride 30 ±2.1 mg/dl 7. Urea 2.1 ± 0.9 mg/dl 8. Uric acid 1.3 ± 0.21 mg/dl 9. Glutamate Oxaloacetate Transaminase 22 ± 1.3 IU/l 10. Glutamate Pyruvate Transaminase 30± 1.5 IU /l 11. Alkaline phosphatase 30 ±2.1 IU /l

Table 1.3: Showing the biochemical results of coelomic fluid (mg/dl - milligrams/deciliter: IU/l - international units/liter)

Volume of coelomic fluid Diameter of the circle 10 µl - 20 µl 0.8 ± 0.l cm 30 µl 1.5 ± 0.1 cm

Table. 1.4 Showing the diameter of the lysis circle formed by the coelomic fluid of Eudrilus eugeniae.

Conc. No. of No. of Height of the Callus Explant Treatment Coelomic Multiple Result Leaves Plant (cm) Formation Fluid Shoots

MS+BAP - 0.6 ± 0.18 - +++ + (Control)

Control - 0.3 ± 0.09 - + + CF in place of 0.5% - 0.8 ± 0.17 - ++ + Vitamins 1% - 0.5 ± 0.23 - + + (T1) 2% 3.3 ± 0.41 2.2 ± 0.30 2.3 ± 0.5 - + Control 3.4 ± 0.19 2.2 ± 0.41 2.1 ± 0.6 - + CF in 0.5% - - - - - place of 14.3 ± 1% 11.7 ± 0.19 14.2 ± 0.72 - + Nigella sativa Hormones 0.61 (T ) 2 16.2 ± 2% 10.9 ± 0.31 13.1 ± 0.31 - + 0.18 CF in Control 9.3 ± 0.52 4.5 ± 0.52 8.4 ± 0.17 - + place of 0.5% 1.1 ± 0.18 0.6 ± 0.31 - - + Hormones 11.3 ± & 1% 12.2 ± 0.51 11.3 ± 0.52 - + Vitamins 0.41 (T3) 2% - - - - -

Table 3.2: Nigella sativa under different concentrations of coelomic fluid of Eudrilus eugeniae

Conc. of Height of No. of No. of Callus Explant Treatment Coelomic Plant Multiple Result Leaves Formation Fluid (cm) Shoots

MS+BAP 0.8 ± 4 ± 0.5 - - + (Control) 0.03

0.5 ± Control - - + + 0.23 1.2 ± CF in place 0.5% - - - + of Vitamins 0.32 (T1) 1% - - - - - 2.5 ± 2.1 ± 2% - - + 0.21 0.21 2.5 ± Control 2.5 - - + 0.32 CF in place 0.5% - - - - - of 1.2 ± Hormones 1% 0.8 ± 0.3 - + +

Tinospora cordifolia 0.51 (T2) 2.5 ± 1.3 ± 2% 2.8 ± 0.2 - + 0.16 0.61 1.3 ± Control 0.5 ± 0.3 - - + CF in place 0.08 of 0.5% - 1 ± 0.23 - - + Hormones 5.3 ± 2.2 ± 2.4 ± 1% - + & Vitamins 0.42 0.41 0.19 (T3) 3.2 ± 1.3 ± 2% - - + 0.18 0.18

Table 3.3: Tinospora cordifolia under different concentrations of coelomic fluid of Eudrilus eugeniae

Conc. of No. of No. of Height of Callus Explant Treatment coelomic multiple Result leaves the plant formation fluid shoots

MS + BAP 1.4±0.21 1.5±0.41 1.3±0.52 - + (control) Control - - - - - CF in the place of 0.5% 1.3±0.32 2.7±0.28 1.2±0.41 - + vitamins 1.0% - - - - - (T1) 2.0% 1.4±0.18 2.3±0.9 2.1±0.23 - + Control 2.45±0.27 2.9±0.42 1.7±0.15 - + CF in the place of 0.5% 2.52±0.61 3.3±0.51 3.4±0.31 - + Hormones 1.0% 3.2±0.41 3.1±0.71 2.1±0.23 - + Clitorea ternatea ternatea Clitorea (T2) 2.0% - - - - - Control - - - - - CF in the place of 0.5% 2.1±0.32 1±0.50 1.3±0.61 - + Hormones 2.4 ± 1.0% 4.45±0.17 2.9±0.23 - + and vitamins 0.250 (T3) 2.0% 2.4±0.31 2±1 2.3±0.21 - +

Table 3.4: Clitoria ternatea under different concentrations of coelomic fluid of

Eudrilus eugeniae

Conc. of No. of No. of Height of Callus Explant Treatment coelomic multiple Result leaves the plant formation fluid shoots

MS + BAP 2.4±0.71 1.8±0.31 1.3±0.23 - + (control)

Control 1.1±0.42 0.8±0.28 - - + CF in the place of 0.5% 3.3±0.51 1.9±0.31 3.1±0.42 - + vitamins 1.0% 2.1±0.22 1.9±0.42 2.3±0.31 - + (T1) 2.0% 7.3±0.57 2.6±0.32 2.5±0.52 - + Control 5±1 1.3±0.27 1.1±0.27 - + CF in the place of 0.5% 7.3±0.56 2.2±0.42 2.1±0.18 - + Hormones 1.0% 6.1±0.71 1.9±0.67 2.3±0.42 - +

Andrographis paniculata (T2) 2.0% - - - - - CF in the Control 1.2±0.20 0.7±0.29 - - + place of Hormones 0.5% - - - - _ and 1.0% 2.3±0.40 2.3±0.17 2.4±0.09 - + vitamins (T3) 2.0% 8.1±0.61 3.1±0.22 3±0.21 - +

Table 3.5: Andrographis paniculata under different concentrations of coelomic fluid of

Eudrilus eugeniae

Conc. of No. of No. of Height of Callus Explant Treatment coelomic multiple Result leaves the plant formation fluid shoots

MS + BAP 2.3±0.15 1.3±0.27 - - + (control)

Control 3.2±0.43 1.3±0.18 - - + CF in the place of 0.5% 5.1±0.21 1.2±0.32 1.3±0.27 - + vitamins 1.0% 4.4±0.18 1.8±0.29 1.7±0.19 - + (T1) 2.0% - - - - -

Control 3.4±0.51 1.5±0.18 - - + CF in the place of 0.5% 1.2±0.27 1.3±0.26 - - + Hormones 1.0% 5.4±0.17 2.4±0.32 2.3±0.27 - + (T2) Aristolochia bracteata bracteata Aristolochia 2.0% - - - - -

CF in the Control 3.4±0.18 - 1.7±0.17 - + place of Hormones 0.5% - 0.5±0.17 - + + and 1.0% 6.4±0.31 3.2±0.28 1.4 ± 0.26 - + vitamins (T3) 2.0% 5.4±0.9 2.9±0.41 1.2±0.19 - +

Table 3.6: Aristolochia bracteata under different concentrations of coelomic fluid of Eudrilus eugeniae

Zone of Inhibition (mm) Conc. µlConc. /disc E. coli E. coli S. aureusS. E. foecalis E. foecalis Bacillus sp. sp. Bacillus S. pyogenes pyogenes S. P. aeruginosa K. pneumoniae pneumoniae K. Salmonella typhi typhi Salmonella Serratia marscecens Salmonella paratyphiSalmonella Aeromonas liquifaciens liquifaciens Aeromonas

20 ------

40 ------

60 - - - - 6±1 - - 8±1 - - 7±1

80 7±1 8±2 - - 10±1 - - 8±1 4±1 - 10±1

100 8± 10±3 4±2 - 11±1 6±2 - 10±2 6±1 - 11±2

Table 2.1: Measurements of zone of inhibition of the coelomic fluid of Eudrilus eugeniae against various bacterial pathogens.

Zone of Inhibition (mm) Conc. µlConc. /disc Fusarium sp., Aspergillus sp. Aspergillus Alternaria solani Sclerotium rolfsii rolfsii Sclerotium Colletotrichum sp Colletotrichum sp Rhizactonia solani Trichoderma viride

20 ------

40 ------

60 ------

80 ------

100 ------7±1

Table 2.2: Measurements of zone of inhibition of the coelomic fluid of Eudrilus eugeniae against various fungal pathogens.

Figure 1.6: 1H-NMR spectrum of coelomic fluid. A: Aromatic region. B: Aliphatic region. AKG: K-ketoglutarate.

Though earthworms are popular among the farmers to scientists for their plant growth promoting activities to anticancer activities, there are not enough studies available that has recorded the molecular basis of these activities. Hence, it was proposed to characterize the coelomic fluid and its various biological activities such as anti microbial and anti-cancer besides its obvious plant growth promoting property. There had been many attempts in producing evidence of earthworms as „Farmers friend‟. However, there is lacuna in mentioning the key molecules or metabolites that are responsible for promoting the growth and yield of the plants in in vitro or controlled environment. In this regard, coelomic fluid was collected by different methods and cold-shock method was standardized as an appropriate way of collection. The coelomic fluid was subjected to 1H-NMR studies to enumerate the biochemicals present in it.

The 1H-NMR result revealed the presence of metabolites which could probably be assigned for promoting plant growth. The coelomic fluid was also demonstrated to have proteolytic and antimicrobial activities against selective bacterial strains.

The coelomic fluid was collected and tested for in vitro micropropagation in five different plant species. This in-vitro plant tissue culture experiment, clearly demonstrated the activity of coelomic fluid promoting faster rate of development of shoots (multiple shoot) and number of leaves than the conventional method as compared with MS media and hormone mixture. Having demonstrated of the proteolytic and antimicrobial activity, the coelomic fluid was subjected to tests that revealed the presence of anti-cancer property or apoptotic inducing metabolite in SiHa – a cervical cancer cell line in-vitro. The coelomic fluid was demonstrated to have anticancer properties against SiHa cell lines through fluorescent staining and ladder assay techniques.

133

As an omnipresent in our soil that is known for its agricultural activities, earthworms were subjected to the presence of textile industrial waste water (effluent). For this, definite number of worms were loaded in a mass of soil providing other necessary conditions and were treated with raw, chemically treated and the biologically treated effluents. Tap water was used as the control. Viability of the worms, cocoon production and hatchlings were recorded for a period of eight weeks.

Raw effluent did not cause any causalities or death of the worms. In turn, biological treatment and the chemical treatment resulted in death of the worms and therefore, the study confirms that the earthworms could serve as a potent tool in the bioremediation of the textile effluent polluted soil.

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