CONTENTS

1. Introduction 2-10

2. Review of Literature 11-13

3. Materials and Methods 14-21

4. Results & Discussion 22-25

5. Conclusion 26-27

6. References 28-30

7. Photographs 31-36

1

Introduction

Pollution has now become at par with the conventional crimes. As water

is scarce and its demand is likely to increase further, it needs more attention.

Everybody knows that pollution refers to the contamination of the environment

with harmful and undesirable wastes. One of the major agricultural chemical

groups is pesticides which play important role in increasing agricultural

productivity through controlling pests. But on the other hand, they cause much

damage to the non-target organisms both in terrestrial and aquatic environment.

Pesticides are the chemicals, which have posed potential health hazard not only

to livestock and wild life but also to fish, birds, mammals and even human

beings. Aquatic organisms, including fish, accumulate pollutants directly from

contaminated water and indirectly via food chain. The aquatic environment is

continuously being contaminated with toxic chemicals from industrial,

agricultural and domestic activities. In India pesticides are one of the major

classes of toxic substances for management of pests in agricultural sectors and

control of insect vectors of human disease. The runoff from treated areas enters

the river and aquaculture ponds that are supplied by rivers. Water pollution due

to pesticide is posing intricate problems that need our immediate attention. New

chemical formulations are widely used to control pests of agricultural crops.

Overspray and runoff of pesticides from agricultural fields may easily find their

way into the natural water sources and adversely affect the quality of water and

creates hazards for aquatic life resulting in serious damage to non-target

species, including fishes (Magar and Bias, 2013).

2

Pesticides have become an indispensable part of modern agricultural

practices and act as one of the vital factors in increasing food production

(Ganeshwade, 2012). No doubt, the use of pesticides has helped in an

increase in the agricultural production, but their indiscriminate use has also

led to destructions of many plants and animals. Every year, millions of tons of

agrochemicals are applied to soils in and around aquatic ecosystems that

eventually pollute rivers, streams, lakes, wetlands and other water bodies.

Despite the hazards that pesticides cause to the environment and human health,

farmers apply ever increasing amounts of these toxic chemicals to their fields.

The extensive use of pesticides to control pests and to increase agricultural

output has resulted in their adverse effect on non-target species. The poisoning

of agricultural fields by pesticides is a serious pollution problem and its

environmental long term effect may result in the incidence of poisoning of fish

and other aquatic forms. These chemicals create serious ecological problems

particularly water pollution or aquatic pollution. Among these chemicals,

pesticides act as an integral part of present agricultural technology and are

injurious to non-target organisms like fishes. Pesticides, the biologically active

chemicals are used to a great extent for pest control but their spectrum of

activity often extends far beyond the pest. Direct spray of these pesticides into

the paddy fields lead to the contamination of aquatic ecosystem and causes

much stress to the aquatic fauna, especially the edible fishes. This has

necessitated ascertaining the dose response relationship of the pesticide and

fishes for better management of the ecosystem.

Vijayawada is a region in the Krishna District in the State of Andhra

Pradesh, India, and is well known for its picturesque vast paddy fields and its

geographical peculiarities. It is also one of the historically important places in the

3

ancient history of South India .The major occupation in Krishna District is

farming. A variety of fertilizers, herbicides and pesticides are used by farmers

for better yield. Ultimately all these chemicals are directly discharged into the

water bodies in and around the paddy fields. The fishes and other non-target

aquatic organisms are continuously exposed to these unhealthy or toxic

substances dissolved in the medium where they are inhabiting throughout their

life in water. As the farming in the area is increased, farmers felt themselves

constrained by the two cycles a year for rice cultivation. The reason i s the

limited availability of potable water in Kr i shna . During the monsoon seasons,

the water from the mountains flow through the rivers to the sea bringing

potable water to Krishna. But during summer, due to the low level of the

region, seawater enters Krishna and making it unpotable.

The rapid industrialization and green revolution introduced a large

variety of chemicals into the environment. The alteration of the habitat may have

deleterious effects on native flora and fauna. As we modify environment for our

own needs, the destruction of the habitat of various species occurs that directly

leads to the disappearance of many of them. The current global loss of

biodiversity is a process generated by such anthropogenic interventions.

Seasonal utilization of paddy fields for fish culture is quite common in Andhra

Pradesh and West Bengal. In recent years, with the advent of high yielding

varieties of paddy, the use of pesticides has become very popular. Dimecron,

Monocrotophos, Henosan, Thymet, Fernoxan, Nuvacron and Fluben diamide are

the major components of the pesticides being used in Krishna. Hence water

pollution can lead to different changes, ranging from biochemical alterations in

single cell to changes in whole populations. Fishes are one of the most precious

natural resources on earth, and it creates a wide range of benefits to humans,

including fisheries, wildlife, agriculture, urban, industrial, and social

4

development Fishes are much vulnerable to their toxic substances and

bioaccumulation cause serious risk to life. Such toxic substances enter to human

through food chain, as fishes constitute an important part of animal protein in

rural and urban areas. Alteration in the chemical composition of a natural

aquatic environment, due to contact with hazardous substances like heavy

metals, pesticides, and effluents from industries usually affect the behavior,

biochemistry, and physiology of the fauna including fish. However, the

unregulated release of agricultural chemicals especially pesticides into water

bodies have caused environmental problems to all classes of organisms in the

aquatic habitat.

Cirrhinus mrigal or white carp fish is a species of ray-finned fish in

the carp family. Native to streams and rivers in India, the only surviving wild

population is in the Cauvery River,leading to its IUCN rating as vulnerable. It is

widely aquafarmed and introduced populations exist outside its native range. This

species belongs to the family Cyprinidae. This fish occurs in the middle and

lower reaches of the river system extending to the coastal area. It is an omnivore

and it grows to only four inches. The fishes found in estuaries and fresh water of

India is costly and highly nutritive. But now the fish stocks are under gradual

erosion due to over exploitation and alteration of the habitat. Human beings have

been abusing the water bodies around the world by disposing into them all kinds

of waste and agriculture run off. Due to such activities of human beings, the

ponds, lakes, estuaries, streams and rivers are becoming polluted.

According to the data compiled by Krishna water balance study project, 485

tonnes of pesticides are applied in Krishna every year of which 370 tonnes are

used for summer crops alone (KWBSR 1990). Retardation in the natural

propagation of fishes is evident in Krishna from the very low fish yield (Cengiz,

5

E.I. and Unlu, E. 2006).A variety of fertilizers, herbicides and pesticides are used

by farmers for better yield. Ultimately all these chemicals are directly discharged

into the water bodies in and around the paddy fields. The fishes and other non-

target aquatic organisms are continuously exposed to these unhealthy or toxic

substances dissolved in the medium where they are inhabiting throughout their

life.

Water is one of the prime elements responsible for life on earth as two thirds

of earth’s surface is covered by water. Ninety seven percent of the world’s water is

found in Oceans. Only 2.5% of the world’s water is non-saline fresh water.

However, 75% of all the fresh water is bound up in glaciers and ice caps. Of the

remaining 25% fresh water is found in lakes, rivers and 24% is present as ground

water. Water is the essential resource for living system, industrial process,

agricultural production and domestic use. The use of water increases with growing

population, putting increasing strain on these water resources. An adequate supply

of safe drinking water is one of the major pre requisites for a healthy life. Pollution

occurs when a product added to our natural environment adversely affects nature’s

ability to dispose it off. A pollutant is something which adversely interferes with

health, comfort, property or environment of the people. Generally, most pollutants

are introduced in the environment as sewage, agricultural waste, domestic waste,

industrial waste, accidental discharge and as compounds used to protect plants and

animals. As a result of the increasing demand for water and shortage of supply, it is

necessary to increase the rate of water development in the world and to ensure that

the water is used more efficiently.

Drinking water should be suitable for human consumption and for all usual

domestic purposes. The importance of water in daily living makes it imperative

6

that through examinations be conducted on it before consumption. The

determination of drinking water quality guideline value is essential in order to

avoid health risks to the consumers. In developing countries only a small

proportion of the waste water produced by severed communities is treated.

Developing country governments and their regulatory agencies, as well as local

authorities (which maybe city or town councils or specific waste water treatment

authorities or more generally waste and sewage authorities) need to understand that

domestic and other waste waters require treatment before discharge or preferably

recycle and reuse in agriculture or aquaculture. The qualities of water need to be

evaluated thoroughly to generate base line information for welfare of society. It is

necessary to isolate and identify the microorganisms present in the different water

samples. In order to alleviate microbial water pollution first a systematic study on

the types and concentration of microbes present in different sources at different

seasons is to be made. With this objective in view, the present work is planned to

assess the quality of water in and around Vijayawada city from seven different

sites for microbiological parameters and the results are compared with the

standards given by WHO, determined the extent of microbial pollution and

recommendations suggested to improve the quality of Krishna river water.

Species and population genetic assessment requires a reliable source of

biological material. Preserved type specimens labeled with accurate identity and

locality in museums and national repositories serve as authentic materials for

taxonomic studies. These samples have been used for taxonomic studies using

conventional methods of morphometric measurements and meristic counts.

However, recent advancements in molecular biology with approaches like DNA

sequencing have opened avenues ranging from evolutionary biology to forensic

science. Taxonomists and systematists can use genome analysis to work out the

7

relationship within species and branching patterns, hence a molecular approach

targeting the genes encoded by genomic and mitochondrial DNA has been opted

for in systematic and phylogenetic research. Unfortunately, however, the preserved

vertebrates in natural history museums do not have allied tissue samples for DNA

study since they were collected prior to the molecular revolution in systematic

biology, and also collectors during those periods did not opt to preserve tissue

samples parallel to preserved voucher specimens. Molecular systematists were thus

left with the option of trying to recover usable DNA from the preserved specimens,

particularly for evolutionary studies. The ability to extract, amplify, and sequence

DNA from various preserved specimens has opened the possibility of using

museum specimens to address questions pertaining to molecular evolution and

genetic understanding of various species.

The preserved specimens in liquid or fluid medium are of the following

categories, viz. ethanol preserved, formalin fixed ethanol preserved, and formalin

(buffered) preserved. Usually most of the national museums hold collections that

are either ethanol preserved or formalin fixed. Formalin-fixed tissues are one of the

popular sources of diagnostic materials as formalin preserved specimens are

commonly available in institutions and in some regional museums. They are often

used as the source of nucleic acids for retrospective molecular analyses based on

DNA amplification by polymerase chain reaction (PCR). The extraction of high-

quality nucleic acid may be problematic in formalin-fixed tissues because of cross-

linking between proteins and DNA as formalin induces DNA fragmentation and

nucleotide alteration. Numerous biological, physical and chemical factors affect

the DNA quality of specimens from natural history collections. There are many

technical challenges to solving the formalin problem starting from the wide

variation in preserving methods adopted for specimen’s storage. Some organisms

8

are fixed in formalin only for short time and then transferred to alcohol for long-

term storage; others are fixed and stored in formalin permanently. The rapid

reaction of formalin with double helical DNA generally is reversible but over the

long term especially with denaturation of the DNA, a variety of reactions can

occur, many of which have not been characterized. Studies have shown that

extraction of DNA from preserved specimens of various forms exists, viz. formalin

fixed, paraffin embedded, air dried and ethanol preserved muscle tissues; however,

reports on extraction of DNA from fish specimens are limited, e.g. DNA extracted

from ethanol specimens and formalin fixed specimens. However, it is known that

nucleic acids from formalin fixed tissues are much worse templates than those

recovered from fresh tissues. Hence, it is necessarily important to quantify and this

study aims to compare the quantity and quality of DNA extracted from formalin

preserved samples. In the present study, genomic DNA was extracted from tissue

samples collected from 2 freshwater polluted water fish species Cirrhinus mrigal

kept preserved in buffered formalin and a comparative analysis has been made on

the quantity and quality of DNA obtained from samples collected during 2

different periods.

DNA markers are being increasingly used for gathering information on the

diversity, conservation biology and population analyses of different organisms. Such

information is useful for planning conservation strategies for great number of fish

species, around 800, which have been designated as threatened in recent years, due

to various anthropogenic stresses.

Species and population genetic assessment requires easy, fast, less expensive

and reliable DNA extraction methodologies. Among different procedures of

9

tissue sampling to obtain DNA, non-invasive sampling seems to be very

attractive and need of the day, since it allows genetic analysis of several

individuals without much handling or sacrificing them. The DNA isolation from

non-invasively collected tissues is particularly useful, when large populations or

threatened species have to be studied. Muscles are the most common tissues used

as sources of DNA, but for collection of tissue the animal needs to be sacrificed.

Another tissue, which is frequently used in vertebrates for DNA extraction is

peripheral blood, but its use in fish present greater difficulties. Although, DNA

can successfully be obtained from muscles samples of fish without the sacrifice

of the animals, it is usually difficult to perform a muscle sampling on many

fishes. Therefore, large-size individuals, specialized staff and high sampling

speed would be necessary for samples’ survival. However, this strategy usually

results in a low quantity and poor quality DNA and also does not provide

individual identification, which limits its potential application

DNA isolation from fish tissue provides a suitable non-invasive procedure

which can overcome the difficulties encountered during tissue sampling from

other tissues and allows the maintenance of the individuals without much

disturbance. In the present paper, an improved and a very rapid DNA extraction

method from the fish muscle tissue has been described that used a modified lysis

buffer. This DNA extraction method provides high-quality and high-quantity

DNA that can serve as a template in p o l y m e r a s e chain reaction (PCR) and

restriction digestion experiments.

10

REVIEW OF LITERATURE

Fishes are very sensitive to a wide variety of toxicants in water, various

species of fish shows uptake and accumulation of many contaminants or

toxicants such as pesticides (Herger et al., 1995). Accumulation of pesticides in

tissues produces many physiological and biochemical changes in the fishes and

freshwater fauna by influencing the activities of several enzymes and

metabolites (Nagarathnamma & Ramamurthi, 1982). Studies on the effects of

organophosphate pesticides on different species of fishes have already been done

by many scientists (Paul and Pant,1987; Maheswari et al.1988; Gopalakrishnan

1990 and Ganguly et al.,1997). Henderson and Pickering (1958) recorded the

LC50 values of malathion for fathead minnow as 25ppm in 24th and 22ppm in

96h. Tarzwell (1958) gave the 96 hour LC50 value for fathead minnows as

12.5ppm. Bhatia (1971) using Puntius ticto determined the 48 hr LC50 value of

malathion as 0.0llppm. Ritakumari and Nair (1978) studied the toxicity of two

organophosphate pesticides on Lepidocephalus thermalis. The magnitude of

pesticide pollution was studied in the Indian fishes by various workers

(Bhattacharya et al., 1997; Munshi et al., 1999, Naveed et al., 2010. Studies on

the effect of different organophosphate pesticides on different species of Channa

(Dubale and Shaw, 1979, Choudhari et al, 1984, Rao et al, 1985, Sastry and

Sharma, 1980) are available. The alteration in biochemical contents in different

tissues of fish due to toxic effects of different heavy metals and pesticides have

been reported by number of workers (James and Sampath (1995), Das et al.

11

(1999), Khare and Singh (2002), Sobha et al.(2007) and Hadi et al. (2009).

Organophosphorous pesticides are most preferred because of their low

persistence in the environment as economically useful pesticide by agriculturists

to eradicate insect pests (Gopalakrishnan, K.S. 1990., Wester and Canton,1991;

Hinton et al.,1993; Schwaiger et al.,1997). Dimethoate is one of the organo-

phosphorous insecticide widely used against vegetables and fruit sucking aphids,

mites, saw flies and boring insects on cereals, cotton, chili, tobacco and oil seeds.

During rainy season along with running water dimethoate insecticide enters the

freshwater resources and results in aquatic pollution. Pesticides are also well

known for causing more toxic effects in teleost fishes (Muthukumaravel et al.,

2013) . The toxic effects may include both lethal and sublethal, which may

change the growth rate, development, reproduction, histopathology,

biochemistry, physiology and behaviour(Rand & Petrocelli, 1985). Molecular

biology studies have been widely used as molecular markers in the evaluation

of the fish exposed to the pesticides, both in the laboratory as well as in the field

(Pragna H Parikh et al., 2010; Wester and Canton, 1991; Hinton et al., 1993;

Schwaiger et al., 1997).One of the great advantages of using DNA markers are

being increasingly used for gathering information on the diversity, conservation

biology and population analyses of different organisms. Such information is useful

for planning conservation strategies for great number of fish species. molecular

markers in environmental monitoring is that this category of biomarkers allow

examining specific target organs like gills, kidney, and liver, that are responsible

for vital functions, such as respiration, excretion, and accumulation and

biotransformation of xenobiotics in the fish (Gernhofer et al, 2001); and serve as

warning signs of damage to animal health( Hinton and Lauren,1993).

12

Pesticides wherever applied, they found their way into water bodies

ultimately affecting aquatic fauna in general and fish in particular

(Muthukumaravel et al., 2013). Most of the insecticides were hydrophobic that

they could be easily absorbed by soil particles and could migrate to natural

water systems such as rivers, lakes and ponds through the run-off causing aquatic

pollution. They could enter the food chain when they become accumulated in

aquatic organisms (Madhab Prasad et al., 2002; Sulekha et al., 1999). Malathion,

as well as other pesticides, used in agriculture find their way with runoff water

interfering with all the metabolic processes and get accumulated in vital organs

thereby affecting the functional activity of both exocrine and endocrine systems

of non-target aquatic organisms including fish (Sulekha et al, 2009).

Histopathological alterations could be used as indicators for the effects of

various anthropogenic pollutants on organisms and are a reflection of the

overall health of the entire population in the ecosystem (Mohamad,

2009). The gastrointestinal system of fish is very much vulnerable to ingested

toxic substances (Mandel and Kulshrestha., 1980; Olurin et al, 2002; Pandey et

al, 2005).

Hence the present study is aimed to look into the histoarchitectural

alterations in Fluben diamide induced toxicity in some of the vital organs like

gill, liver, intestine and muscle of the teleost fish, Cirrhinus mrigal, so as to

assess the damage and get an insight into their functional consequences.

13

Materials and Methods

Vijayawada, popularly known as fertilizer city and city of victory is situated in

between 150-43’ and 17-10’ North Latitude and 800 and 81-33’ East Longitude in

Andhra Pradesh with a population of over 86,210,007. The city has an area of

about 5.8 sq km and has a flat altitude with an average ground level of 125m above

sea level. The communities around the river use the water extensively for drinking,

irrigation, industrial and other domestic purposes without prior treatment. The

microbiological parameters, DNA extraction, DNA isolation and DNA

amplifications were determined according to procedures outlined in the Standard

Methods for the Examinations of fresh Water, Wastewaters and muscle tissues of

Cirrhinus mrigal were collected from all the canals, drains and river on a months of

November and December for the year 2015. The samples collected and stored in

clean polythene bottles fitted with screw caps and brought to the laboratory in the

sampling for detailed microbiological analysis.

Study Area:

The present study was conducted in order to analyze the lethal toxic

effects of pollution on Cirrhinus mrigal and to understand the influence of time

over the toxic effect at different periods of exposure. The study area includes

inland waters of lower Krishna in and around 20 Km from Assumption College-

the center of the proposed project. The fishes were collected periodically from

different regions of river Krishna nearby Vijayawada regions which are

14

interconnected by the paddy fields and inland water channels of Krishna.

Collection and Maintenance of Fish:

Healthy fishes were collected from the Krishna area with the help of big

nylon net and hand net. Juveniles of Cirrhinus mrigal(3.5 - 4.5 cm in total length

and 2.5 - 4gm in weight) were brought to the laboratory and acclimatized to the

laboratory conditions for a period of 10 days prior to the experiment. During this

period, they were fed, once a day, on pelleted fish feed and were kept aerated

(Prashanth, 2011). Each trough contained 15 liters of water with uniform number

of fishes. After 10 days, fishes with normal behavioral activity and good health

conditions were selected for further experiment purpose.

Tissue samples from the fresh and polluted water of 2 fish species Cirrhinus

mrigal utilized in this study were from the formalin preserved collections of

P.B.Siddhartha College, Molecular biology lab of Zoology department,

Vijayawada. Specimens were preserved in 10% buffered formalin. Two sets of 4

samples each per species were chosen from the collections made during 2015 and

2016 the recent sample that of 2016 was preserved for a short duration, i.e. 1

month prior to the experiment. Cirrhinus mrigal occurs throughout India and is

commonly found in the streams and rivers of southern and southeast Asia.

Cirrhinus mrigal is an endemic species restricted to drainages of the krishna River

in the southern India.

For the present study the muscle tissues have been used from four specimens

of different fresh polluted water fish species namely Cirrhinus mrigala (Fig1&2).

The muscle tissues were collected non-invasively by gentle scrapping on the

middle portion of the body with a forceps, and the detached muscle tissues were

15

collected in 2 ml tube. The scales were either used fresh or preserved in sufficient

amount of 90% ethanol.

Sample Preservation: To minimize the potential for volatilization or biodegradation between

sampling and analysis, the samples are kept in refrigerator and stored at 40C.

Microbial examination of water sample: For the isolation of microorganisms such as fungi, bacteria and

actinomycetes heterotrophic plate technique has been employed. Inoculation were

made and incubated at 370C for 24-48 hours. The colonies developed on agar plate

were counted. For the identification of different organisms present in seven water

samples collected. Different media such as M. Endo agar, Pseudomonas HI. Veg

agar and Thiosulphate Citrate Bile salt sucrose agar (TCBS) were used for sub

culturing.

Detection of fresh water species Cirrhinus mrigal were done by the

enrichment of water samples on Selenite For both, followed by isolation of the

typical organism on selective medium. Detection of Vibrio cholerae was done by

enriching the samples in 1% alkaline peptone water for 6 to 8 hours followed by

isolation on Thiosulphate citrate bile salt sucrose (TCBS) agar medium. Enteric

bacteria isolated on respective selective or differential media were identified on the

basis of their colonial, morphological and biochemical properties followed by

Bergey’s Manual of Determination Bacteriology, 1994.

DNA extraction and analysis:

16

DNA was extracted from different parts of two fresh water two polluted

water fish species, which were collected from Krishna river of Vijayawada

surroundings and local fish markets of Vijayawada. The selected portion for the

DNA extraction was vertebral and muscle tissue of fish (Fig.3&4). The eluted

DNA of Cirrhinus mrigal, of fresh and polluted water showed some variation. The

genomic DNA of eight Cirrhinus species are shown in Fig .1&2. Good quality of

DNA was obtained from all fresh and polluted water fish species. A polymorphic

band of Cirrhinus mrigal was observed in gel picture. Quantity of DNA ranged

from 56.3 to 267.6 μg/μl for all samples. 260/280 ratios which are the indicators of

DNA quality, were ranged from 1.65 to 2.0, indicating that good quality of pure

DNA was obtained, as analyzed in Nanodrop.

The genomic DNA was extracted from tissue samples using the method

devised by Nishiguchi et al. (14). Muscle samples (5 mm3) were initially incubated

in a TE buffer overnight to get rid of fixative. Each tissue sample was digested in

500 μL of STE buff er containing 0.2% SDS and 250 μL of 10 M ammonium

acetate at 55 °C for 10 h. A small amount of tissue (the size of a match-stick head)

was ground in a sterile Teflon Eppendorf grinder (Kontes). After grinding, it was

incubated for 1 h. Samples were then centrifuged at 14,000 rpm for 5 min to pellet

the cell debris and precipitate proteins. The supernatant was transferred to a new

tube and 2 volumes of ice-cold 100% ethanol added to it and then mixed gently by

inverting tubes. The tubes were placed at –20 °C until DNA precipitates. Again

tubes were centrifuged at 4 °C at 14,000 rpm for 15 min, the resulting supernatant

was separated and the same volume of cold 70% ethanol was added. The tubes

were once again allowed to spin at 4 °C at 14,000 rpm for 10 min. Then ethanol

was poured off and the tubes were dried completely. The pellets were resuspended

in 50 μL of TE buffer overnight at 4 °C or for 30 min at 40 °C. The precipitate was

17

centrifuged for 20 min at 12,000 rpm in a micro centrifuge. Ethanol solution was

discarded by decantation and pellet was washed with 1 mL of 70% ethanol. It was

then rotated for 5 min at 12,000 rpm in a micro centrifuge. After discarding the

ethanol solution it was let to dry in a vacuum centrifuge (or at 55 °C). The pellet

was again resuspended in 50-100 μL of TE buffer (pH 7.6) and the sample was

incubated at 45-60 °C to facilitate dissolution of the pellet.

A portion of the eluted material approximately 10- to 20-fold was diluted in

DNA Elution Buffer or 10 mm Tris, pH 8.0. The quantification of DNA was done

by UV spectrophotometric analysis (SpectronicR Genesys™ 2). The quantity of

DNA was measured by obtaining the absorbance reading at 260 nm and the purity

of DNA was checked by calculating the ratio of absorbance readings at 260 nm

and 280 nm. Values of 1.7-1.9 generally indicate 85% purity. The concentration of

DNA can be determined as follows: Concentration = 50 μg/mL × Absorbance 260

× {Dilution Factor} and Purity of DNA = Absorbance at 260 nm/Absorbance at

280 nm. After the isolation, DNA samples were taken out and 7 μL of

Bromophenol blue (sample loading dye) was added and then 15 μL of mixed DNA

product was loaded in 1% agarose gel (50 mL) containing ethidium bromide at the

concentration of 20 μL per 50 mL of gel. Th e electrophoresis was carried out for 1

to 2 h at 50 V. After electrophoresis the gel was placed in the UV transilluminator

and bands were visualized and photographed using a digital camera.

DNA was extracted, both from fresh and polluted fish muscle tissues as well

as from more than a month ethanol preserved tissues by using the following

method. The protocol was followed according to Wasko et al. with modifications.

Approximately, 50 mg of muscle tissues were taken from each species and dried

18

on a filter paper. The scales were then cut into small pieces and placed in a 2 ml-

Eppendorf tube containing 940 l lysis buffer (200 mM Tris-HCl, pH 8.0; 100

mM EDTA, pH 8.0; 250 mM NaCl), 30 l Proteinase K (10 mg/ml) and 30 l

20% SDS. The contents in the tubes were incubated at 48C for 45-50 min in a

water bath. The appropriateness of the incubation temperature was studied in a

separate experiment, by incubating scales sample from C. mrigal at different

temperatures viz. 42, 44, 46, 50, 52and 54C. After incubation

(Fig.6), an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was

added to the tube containing lysed scale cells. The contents were then mixed

properly by gently inverting the tube for 10 min to precipitate the proteins and

other part of the nucleic acids. The tube was then rotated for 10 min at 9,200 g.

The top aqueous layer was transferred to a new 1.5 ml-Eppendorf tube (Fig. 5),

leaving interphase and lower phase. The DNA was then precipitated by adding

equal volume of isopropanol and 0.2 volumes of 10 M ammonium acetate and

inverting the tubes gently several times. The precipitated DNA was then pelleted

by centrifugation at 13,200 g for 10 min. The supernatant was removed by

pouring out gently, taking care to avoid loss of DNA pellet. The pellet was then

washed briefly in 500 l chilled 70% ethanol, air-dried and resuspended in 200 l

sterile water/TE buffer (Qualigen).

After ensuring complete solubility of DNA, the purity factor (A260/A280

nm) was measured spectrophotometrically and its integrity was checked by

loading 10 l DNA preparation (2 l extracted DNA, 2 l dye and 6 l sterile

water) on 0.7% agarose gel and stained with ethidium bromide. The quantity

and quality of the DNA were compared by loading 0.2 l Lambda Hind III

DNA standard marker (provided by M/s Bangalore Genei, India, stock conc. 500

19

ng/l) and DNA isolated from blood of Cirrhinus mrigal using Sigma kit (cat #

NA 2000) in the same gel. The DNA quantifications were done using Syngene

Gene Genius gel documentation system. The extracted DNA samples were then

stored at −20C till their further use.

Restriction digestion:For checking the quality, the DNA samples were digested with HaeIII (10 U/ µl)

restriction enzyme (provided by M/s Bangalore Genei, India). The reaction

volume was set up for 10 µl, which contained sterile water, 2 µl 10X RE buffer

(provided with the enzyme), 200 ng template DNA and added 2 U (0.2 µl)

HaeIII restriction enzyme. The reaction mixture was incubated at 37C for 60

min for restriction digestion followed by 15 min incubation at 70C to stop the

reaction. The restriction digested products were tested on 1.2% agarose gel (Fig.

4).

PCR amplification:Polymerase chain reactions (PCRs) for amplification (Fig. 6) of genomic DNA

extracted from fish muscle tissues of different species were carried out with three

random decamer primers (OPAS 12, OPAS 13 and OPAS 14) in a 25 µl

reaction volume. The sequences of these OPAS primers were as follows:

OPAS 12: 5’- TGACCAGGCA-3’ (Mol. Wt. 3037)

OPAS 13: 5’-CACGGACCGA-3’ (Mol.Wt. 3022)

OPAS 14: 5’- TCGCAGCGTT-3’ (Mol.Wt. 3019)

The PCR reaction contained 20 ng genomic DNA, 2.5 µl of 10X PCR

buffer (Fermentas), DDW, 2.0 mM MgCl2 (Fermentas), 0.5 µl of 10 mM

dNTPs mix (Fermentas), 5-6 pmol of each OPAS 12-14 decamer random primers

20

(Operon, QIAGEN) and 0.625 U Taq DNA polymerase (Fermentas). The

amplification was carried out in the Eppendorf Master Gradient Thermal Cycler.

The PCR conditions were initial denaturation at 94C for 4 min followed by 32

cycles of denaturation at 94C for 1 min, annealing at 36C for 1 min,

extension at 72C for 1 min followed by final extension at 72C for 10 min.

The 8 µl amplified product was analyzed on 2.0 % agarose gel (Fig. 6).

The 464 bp fragment of cyt b of fish was amplified with the primers, L14735

(5´-AAA AAC CACCGT TGT TAT TCA ACT A-3´) and H15149 (5´-GCI CCT

CAR AAT GAY ATT TGT CCT CA-3´) by PCR(Fig. 6). The amplified fragments

from eight species of fish from Vijayawada are shown in Fig.2. In all samples

tested, the universal gene fragment was successfully amplified. The amplified band

was resolved using Agilent 2100 Bioanalyzer with good resolution and the 464 bp

band was clearly visible in all eight samples. Dooley et al. 2005, successfully

adopted and validated an earlier PCR-RFLP method using a Cyt b PCR target

sequence and analysis of restriction fragment patterns on the Agilent 2100

Bioanalyzer. RFLP and specific PCR amplification analysis could serve as simple

but powerful tool for screening. The molecular techniques applied in this study

have previously been utilized for salmon (Russell et al.,. 2000) prech (Asensio et

al.,2000) and sardine (Sebastio et al., 2001) species identification in food industry.

Compared with their results, our RFLP patterns would recognize eight species,

with only three restriction enzymes. Application of species specific amplification

to identify fish species are few, however, this technique is frequently applied for

microbiology studies (Zhan et al., 2001). Comparing three recently examined

mitochondrial genes, Lin et al., (2000) illustrated that the cytochrome B gene is

more divergent and was more appropriate for constructing fresh water fish

identification system as indicated in this study.

21

Results & Discussion

In the present study, a comparative analysis of microbiological

characteristics of Krishna river water along with less polluted and more polluted

water systems of four sites selected were made during November-December 2015

and based on microbiological parameters the water quality has been assessed.

Characterisation of bacterial, fungal and actinomycetes isolates: The results of heterotrophic plate count of primary culture are depicted. The

various water and waste water samples showed different morphological

characteristics when cultured on nutrient agar plates and reacted differently to the

diagnostic biochemical tests. However, the isolates from the Krishna river water

sample were gram positive and gram negative, catalase positive and oxidation

fermentation positive, oxidase negative and have the ability to ferment sugar. The

isolates showed different behavioral pattern to iodole test, motility test, voges-

poskauer test, hydrogen sulphide production test and ability to utilize citrate.

Bacteriological analysis: It was clear that the bacterial colonies vary according to the seasons as well

as to the locations. The results of the numerical estimates of bacteria from the

primary culture revealed that the waste water samples contain heavy microbial

load. The highest number of bacterial colonies recorded with the value of

229×104cfu/ml in site-V, this could be attributed to rapid proliferation of

microorganisms which aid in the degradation of organic matter present in the

industrial effluents. The lowest number of bacterial colonies recorded with the

value of 5×104cfu/ml in site-I. Most probable number (MPN) method is used to

22

indicate the fecal contamination in terms of number of coliform bacteria in all the

seven sites and results obtained (2,400cfu/ml) showed that the site-I to III water

sources were highly contaminated. Krishna river water is the main source of

drinking water supply for Vijayawada city, the morphological and biochemical

characterization of the isolates identified the following organisms: Staphyloccus

aureus(Fig.11), Escherichia coli (Fig.11), Vibrio cholera (Fig.11), and

Pseudomonas aeruginosa (Fig.11). The presence of these organisms in water can

change the quality of water. Their presence could be attributed to the ubiquitous

nature of microorganisms and the contaminated state of the river by industrial

effluent which increases the organic content of the river there by providing

excellent nutritional source for the propagation of microorganisms. The presence

of faecal coliform bacterial is an indicator that a potential health hazard exists for

individuals exposed to the source of water.

Fungal analysis: The study revealed that all the seven water samples analysed contained fungi

in relative proportions. Heavy fungal load was obtained in site-V with the value of

12×105cfu/ml. The fungi isolates include Pencillium, Fusarium, Rhizopus, Mucor

and Aspergillus. Health problems associated with these organisms can cause taste

and odor problems thereby affecting the aesthetic properties of water.

Actinomycetes analysis: The population density of actinomycetes varied with different water samples along

with the culture media used for isolation. The highest actinomycetes population

density is recorded in Krishna river water 600×103cfu/ml, this may be because of

presence of runoff water. Actinomycetes in waters collected from water present in

agricultural fields 400×103cfu/ml and canal near agricultural fields 130×103cfu/ml

23

can be attributed to the availability of high amount of organic matter. Less amount

of actinomycetes population in four waters from VTPS and sewage water this is

perhaps due to the incompatibility between surrounding factors.

Genomic DNA of high quality and quantity is required to analyze genetic

diversity by using molecular markers. It becomes one of the major concerns for

DNA based techniques, especially when a large number of samples need to be

processed. A number of simplified protocols for DNA extraction have been

reported, such as salting out procedure, microwave based extraction,

silica-guanidinium.

Extraction of DNA from formalin preserved tissue samples of the 2 fish species

was successfully accomplished in Figures 1 and 2. The quality of DNA as evident

from the absorbance values at 280/260 nm showed the absence of excessive

proteins (DNA/Protein = 1.6 to 1.8) indicating good quality. Total DNA extracted

from short-term preserved samples of Cirrhinus mrigal species were 71.56- 83.4

(mean = 78.66) and 70.36-78.52 (mean = 74.32) and higher than the amount

extracted from long term preserved samples 43.56-52.38 (mean = 48.44) and

49.23-53.72 (mean = 51.71) respectively (Figure 1). Though there has been a

notable variation in the amount of DNA extracted among the 2 samples of different

duration, the quality of DNA rather showed little variation. Among the recently

preserved samples the quantity of DNA obtained from Cirrhinus mrigal polluted

water species was higher compared to samples of Cirrhinus mrigal fresh water

species though same amount of tissue samples were utilized (Figure 6). Although

there was no signifi cant variation in the quality of DNA among the 2 species

samples, the agarose gel image (Figure 7) of the electrophoresed DNA shows the

set of samples (lanes 1 and 2) kept under preservation for a period of 6 years

24

exhibit DNA of good quality. Studies have shown that the extraction of DNA from

formalin fixed and ethanol preserved samples was possible and the notable feature

was that the quality of DNA varied depending upon the type of tissue utilized for

extraction. Shiozawa et al. (11) reported a similar amount of DNA yielded from

muscle and liver and a higher yield from gut tissue. During this study, DNA was

extracted from muscle tissues from specimens that were fixed and preserved in

formalin. Tissue samples that were kept under long term preservation yielded low

quantity of DNA while a higher quantity of DNA was obtained from short term

preserved samples. The quality (absorbance ratio) of DNA yielded was in the range

1.6-1.8, considered as good, as absorbance ratio values above 1.8 represents good

quality DNA (15,16). Hence, this method could be fruitful for the extraction of

DNA from preserved materials including those collected from type localities

identified as valid voucher specimens and other specimens that remain as un

catalogued collections in national museums and other national repositories. The

ability to extract, amplify, and sequence DNA from formalin preserved museum

specimens increases the information value of museum holdings (11). Further

research should involve standardizing this method to obtain more DNA.

25

Conclusion

The results of the microbiological analysis of water samples collected in and

around Vijayawada investigated have shown that effluents from industries and

agricultural waste is a major source of environmental pollution through the

discharge of the effluent into the water body. The water quality is directly related

to health and is important for determination of water utility. Assessment of water

quality is a critical factor for assessment of pollution levels. The results from the

present study clearly pointed out that waters from sites-II, IV, V, VI and VII are

highly polluted as they contain high levels of dissolved solids, microbiological

values are not within the permissible limits given by WHO. The waters from

agricultural field’s site-III and IV are contaminated with pesticide residues and

agricultural waste, whereas at sites V, VI and VII from industrial effluents, diesel

from rail engines and domestic wastes respectively. Elevated levels of these

pollution indicators, when compared to the control would invariably affect the

taste, smell, appearance and aesthetic properties of the water or could pose a

potential health hazard of varying degrees to various life forms, which depend on

the water for domestic and recreational purposes. Hence these waters need

conventional treatment including disinfection.

Prediction of restriction fragment length polymorphism indictaed that a

combination of three enzymes results for cytochrome b revealed good resolution

power. Cirrhinus mrigal species showed same morphological features and posed

problems to identify the individual species. Therefore, it is essential to identify the

fresh water and polluted water fish species. The rapid molecular technique

developed here can be helpful in fresh water and polluted water fish species

26

identification studies. Fourt species of fresh and polluted water fish were identified

by this rapid PCR-RFLP technique as visualized in Bioanalyzer 2100.

The index of DNA damage assessed by the molecular assays demonstrated

no significant differences in the different sampling sites and in different species.

But efforts should be made to utilize assays for detecting genotoxicity caused by

aquatic pollutants in fishes at DNA level.This will help in formulating long term

strategies for fish conservation programme besides estimating safe level of

pollutants in water. Appropriate screening tests should also be validated for

investigating consequences of genotoxins not only on population but also on gene

pool.

27

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Fig. 1. Fresh water fish sps. Cirrhinus mrigal

Fig. 2. Polluted water fish sps. Cirrhinus mrigal

31

Fig. 3. Removal of muscle tissue from fresh water fish sps. Cirrhinus mrigal

Fig. 4. Removal of muscle tissue from polluted water fish sps. Cirrhinus mrigal

32

Fig.5. was transferred to a new 1.5 ml-Eppendorf tube

Fig. 6. Polymerase chain reactions (PCRs) for amplification

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Fig.7. Agarose gel image.

Figure 8: Agarose (2%) gel electrophoretic profile of PCR products

34

Figure 9: Agarose (0.7%) gel electrophoretic profile of DNA samples obtained from fresh

water fish muscle tissue of Cirrhinus mrigal

Figure 10: Agarose (0.7%) gel electrophoretic profile of DNA samples obtained from

polluted water fish muscle tissue of Cirrhinus mrigal

35

Fig.11 Diversity of bacteria in the water samples collected from Krishna River

E.coli Vibrio cholera

Staphylococcus aureus Pseudomonas aeruginosa

36

37