Training Manual Application of Genetics and Biotechnology in ...

! 1 

CIBA Special Publication No. 27 

Training Manual 

Application of Genetics and Biotechnology in 

Aquaculture 

Central Institute of Brachshwater Aquaculture 

(Indian Council of Agricultural Research) 

75, Santhome High Road, R.A.Puram, Chennai - 600 028 

March 2006 

WWIY: ciba.tn.nic.in

CONTENTS 

Title Page No I 

I 

1 I 1 

1. Quantitative Genetics in Aquaculture 1 1-6 

I 

I 

G. Gopikrishna, M.S.Shekhar and S. Kannappan i 

2. Genetics in Aquaculture - A Global Scenario 

7-10 

3. Microbial Technique 

G. Gopilaishna 

11-13 

M.S.Shekhar and S.Kannappan 

4. Plasmid DNA Isolation and Gel Electrophoresis 

14-16 

M.S.Shekhar 

5. Gene Cloning 17-18 

M.S.Shekhar , 

I 

6. Mitochondrial DNA Extraction and PCR Amplification of 

Mitochondria1 Genes 

I 

i 

1 

M.S.Shekhar and G.Gopiknshna 

7. Nutrition and Captive Broodstock Development 21-24 

I 

1 

C. Gopal 

8. Probiotics in Aquaculture 25-32 

S. Kannappan and G. Gopilaishna 

9. Bioremediation Of Coastal Aquaculture Water 33-37 

K. K. Krishnani 

10. Drug Sensitivity on Aquatic Pathogens 38-39 

I 

S. Kannappan, K.P. Jithendran and G. Gopi Krishna 

1 11. Immunostimulants for Aquafarming 

I 

S. Kannappan and G. Gopilirishna 

/ 12. Marine Bio-active Compounds and their Bio-therapeutic 

Applications 

S. Kannappan, K. P. Jithendran, G. Gopikrishna and 

M.S.Shekhar 

40-44 

45-55 

I 

I

CHAPTER 1 

Quantitative Genetics in Aquaculture 

G. Gopikrishna, M.S.Shekhar and S. Kannappan 

Genetics is that branch of biology, which deals with heredity and variation. 

Qualitative Genetics deals with traits that are easily classified into distinct phenotypic 

categories and are under the genetic control of only one or a very few genes with 

very little or no environmental modifications to obscure the gene effects. In contrast 

to this, quantitative genetics deals with traits that exhibit continuous variation and the 

phenotypic measurements form a spectrum. Quantitative traits are governed by 

many genes, each contributing a small amount to the phenotype, such that their 

individual effects cannot be detected by Mendelian methods. Genes of this nature are 

called polygenes. The phenotypic variability expressed in most of the quantitative 

traits has a relatively large environment component and a correspondingly small 

genetic component. The task before the quantitative geneticist is to determine the 

magnitude of the genetic and environmental components of the total phenotypic 

variability of each quantitative trait in a population. This can be achieved by 

application of special statistical procedures. 

Dr Ronald A. Fisher (1890-1962) a British Statistician and geneticist was 

instrumental in providing the foundation for modem quantitative genetics by hsing 

the newly emerged Mendelian theory with a biometical approach to the study of 

heredity. 

1.1 Statistics in Quantitative Genetics 

The data on any quantitative trait can be presented graphically as a frequency 

distribution. The horizontal axis or abscissa, measures values of he trait that are 

encountered in a sample from the population. The abscissa is usually subdivided into 

regular intervals. The vertical axis or ordinate measures the frequency of the 

observations in each interval.

1.2 Normal Distribution 

The study of a quantitative trait in a large population reveals that very few 

individuals possess the extreme phenotype and that progressively more individuals 

are found nearer the average value for that population. This type of a symmetrical 

distribution is characteristically bell-shaped and is called the normal distribution or 

Gaussian distribution. 

- 

The average phenotypic value for a normally distributed trait is expressed as X 

or arithmetic mean. 

It is not possible to measure all the individuals in a population, hence, 

measurements are taken on a sample from that population in order to estimate the 

population value (parameter). If the sample is truly representative of the larger 

population of which it is a part, then would be an accurate estimate of the mean of 

the entire population (p). Usually, English alphabets are used to represent statistics 

i.e. measurements derived from the sample, whereas Greek letters are used to 

represent population parameters i.e. attributes of the population from which the 

sample was drawn. Larger the sample size, the more accurately the statistic estimates 

the parameter. 

1.4 Measurement of variability 

Let us suppose that we are comparing two populations for the same quantitative 

trait. Initially, we would compute the respective means. Although the means do 

represent the two populations, they do not tell us anything about the variability of the 

populations. A useful measure of the amount of variability in a population is the 

standard deviation denoted by (a).A sample drawn from this population will have a 

standard deviation (S). It is computed by subtracting the sample mean from each 

individual measurement, squaring the deviations and summing them up for all the 

individuals in the sample. The resulting sum is divided by (n-I) where 'n' is the 

sample size.

If the sample adequately reflects the variability present in the larger 

population, then the statistic 'S' estimates the parameter o. 

One of the important properties of the normal distribution is that 

approximately 68% of the measurements will lie within plus or minus one standard 

deviation from the mean ( pAa). Again approximately 95% of the measurements will 

lie between 2 standard deviations of the mean (prt2o). More than 99% of the 

measurements will be found within plus or minus 3 standard deviations of the mean 

(!JA3o). 

Coefficient of variation 

It is observed that traits with relatively large average metric values generally 

are expected to have corresponding large standard deviations than traits with 

relatively small average metric values. Since different traits may be measured in 

different units, the coefficients of variation are useful for comparing their relative 

variabilities. If we divide the standard deviation by the mean, we get the coefficient 

of variation which is independent of the unit of measurement. 

CV = o / p for a population 

CV = S / 

for a sample 

Variance 

This is the square of the standard deviation. However, unlike the standard 

deviation, the variance cannot be plotted on the normal curve and can only be 

represented mathematically. Variance is used as an expression of variability because 

of the additive nature of its components. Using a technique called the 'analysis of 

variance', the total phenotypic variance (Vp) expressed by a given trait in a 

population can be statistically partitioned into components of genetic variance (Vg), 

non-genetic or environmental variance (Ve) and variance due to genotypeenvironment 

interaction. (Vge)

This technique is useful as it reveals what proportion of the total variance is 

due to genetic factors. As an example, in study on the number of abdominal bristles 

in female Drosophila by Reeve and Robertson (1954), thd total variance for bristle 

number was 5.44 out of which 2.1 1 was genotypic and 3.33 was environmental. 

1.5 Correlations 

The relationship between two sets of variables constitutes correlation. The 

correlation coefiicients range from -1 to +1 with -1 indicating a perfect negative 

correlation and +l indicating a perfect positive correlation. If correlation is zero, it 

indicates that the two sets of variables are independent. Correlations are computed 

using the formula: 

correlations. 

r = Cov (XY) 1 ox. oY 

This general formula is also used for computing genetic and phenotypic 

1.6 Regression 

The change in a dependent variable for a unit change in the independent variable 

is known as regression. Regression equations are also known as prediction equations 

as they can predict the dependent variable for a given value of the independent 

variable. 

b = Cov (XY) / o2 X 

Regression estimates are useful for calculating estimates of heritability. 

1.7 Heritability 

One of the most important factors in the formulation of effective breeding plans 

is a knowledge of the relative contribution which genes make to the trait under 

consideration. Heritability (hZ) is the proportion of the total phenotypic variance due 

to gene effects. The heritability of a given trait may take any value from 0 to 1. 

Broad-sense heritability: 

The proportion of the total variance that is genotypic is called the broad sense 

heritability. However, although this ratio provides information about the importance

of genetic differences among the individuals in a population, it has got very little 

predictive power. It cannot be used to predict the values of a quantitative trait in the 

offspring of specific matings, nor can it be used to study the nature of the genes 

affecting the trait. For theSe purposes, we need a more refined analysis. 

In order to predict an offspring's phenotype, it is necessary to subdivide the 

genotypic variance in the model : Vp = Vg + Ve. The variance due to genotype (Vg) 

encompasses all the genetic factors affecting a trait including interaction among 

genes. Such interactions have little or no predictive value because Mendelian 

segregation breaks up gene combinations. Consequently, the methods for predicting 

phenotypes depend primarily on the effects of individual alleles. 

Let us represent the cumulative sum of the effects by A (for allelic effects) 

and the sum of the interactions by the letter I. 

ThusG=A+IandP=A+I+E 

In this model, the phenotype of an individual is determined by three separate 

components. In a population, this model implies that the phenotypic variance will 

also be determined by three components, provided that the terms A, I and E are 

unconelated. If this is assumed, then: 

oZp = o2 A + o2 I + o2 E 

where o2 A is called the additive genetic variance because it accounts for the 

variability caused by the additive effects of alleles. o2 I is the interaction genetic 

variance, a term that measures the impact of genetic combinations. Sometimes this 

term is subdivided into components for dominance and epistasis. Dominance 

involves combination of alleles at the same locus and epistasis involves combinations 

at different loci. Since neither o2 I nor part of o2 I has much predictive power, 

quantitative geneticists tend to focus their attention on additive genetic variance. This 

leads us to the narrow sense heritability which is defined as the ratio o2 A/ oZP i.e. the 

proportion of the total phenotypic variance that is due to the additive effect of alleles. 

Quantitative geneticists estimate heritability from correlation between relatives. 

This method involves computing the correlation coefficient and then dividing it by 

the fraction of genes that the relatives share by virtue of their common ancestory. 

Half-sibs reared in different environments are useful as they share one-fourth of their 

genes but none of their environmental effects.

1.8 Response to artificial selection: 

The narrow sense heritability has a very important use : to predict the changes in 

the mean of a population under artificial selection. Selection changes the mean of a 

population (of the trait under selection). The superiority of the selected individuals 

over the population is known as selection differential. The difference between the 

mean of the next generation and the whole of the population before selection, is 

termed as the genetic response or genetic gain. It is defined as the product of the 

heritability and the selection differential. 

AG=h2. SD 

In case the heritability is known, the genetic gain in the next generation could 

be predicted. The ratio of genetic gain to the selection differential yields an estimate 

of heritability called the realized heritability. 

References 

Falconer, D. S. and Mackay, T.R.C. 1996 

Quantitative Genetics, 41h Edition, Longman, Essex, UK 

Gardner, E.J., Simmons, M.J. and Snustad, D.P. 1991 

Principles of Genetics, 8'h Edition, John Wiley & Sons, New York. 

Stansfield, W.D. 1969 

Schaum's Outline of Theory and Problems of Genetics, McGraw-Hill Book. Co.

CXAPTER 2 

GENETICS IN AQUACULTURE - A GLOBAL SCENARIO 

G. Gopikrishna 

Aquaculture is gaining importance as an aid to enhance the food requirement 

of people all over the world. It also offers cheap protein to millions of people. 

compared to plants and terrestrial animals where improved varieties are available, 

aquaculture mostly relies on unimproved stock. There are a few examples of 

pioneering selection programs for species such as Atlantic Salmon, Nile Tilapia and 

in India, we have the improved Rohu Carp. It would be desirable to have welldesigned 

breeding programmes in aquaculture with focus on selective breeding, in 

order to reap the benefits ~f conventional selection. 

Selection is the choice of individuals to be chosen as parents for the next 

generation. It does not create new genes but pools the genes responsible for desired 

characters. While running a selection program for a particular trait, the gene 

frequency of that particular trait in a population is increased. This results in a rapid 

response to selection which is translated as improvement. 

In terrestrial animals, the coefficient of variation for growth rate is 7-10% 

while it is 20-35% in fish and shellfish. The fecundity in aquatic animals is very high 

which allows a higher selection intensity in aquaculture. (Gjedrem, 1997) This 

explains the reason for the response to selection being high for growth rate in fishes 

and shellfishes. Hence, it would be worthwhile to go in for well-planned selection 

experiments or selective breeding in fishes/shellfishes. Another very important factor 

governing the selective breeding experiments is that the life cycle of the species of 

fishlshellfish which is to be improved genetically, should be closed. This means that 

the parents should breed, the progeny should be grown upto maturity and the progeny 

should be bred to obtain the next generation. Genetic studies are meaningless if the 

life cycle is not closed. 

There are several selection programmes in aquatic species worldwide. Rye 

(2005) indicated that 36 family based programmes are either in operation or under 

implementation woridwide. Half of these programmes target the salmonid species

and it is assumed that close to 100% of the current world aquaculture production of 

Atlantic Salmon is now based on stocks undergoing systematic genetic improvement. 

The corresponding number for other aquaculture species is quite low, but seems to be 

picking up. There are 11 programmes in Atlantic Salmon in countries such as 

Nonvay, Chile, Canada, Faeroe Islands, Iceland, Scotland and Ireland. Selection 

programmes in Rainbow Trout are in vogue in Norway, Denmark, Finland, Chile and 

USA. There is a programme each for Seabass and Sea bream in Greece. A 

programme for Red Tilapia is on at Ecuador. Selection programme in Nile Tilapia is 

in vogue in Philippines, Vietnam and Malaysia. There are five programmes in 

Marine Shrimp in India, USA, Columbia, China and Thailand. (Rye, 2005) 

In India, the pioneering work carried out at the Central Institute of Freshwater 

Aquaculture, Bhubaneswar, is the first selective breeding programme. The project 

started in 1992 under the Norwegian collaboration. The species taken was Rohu 

(Labeo rohita), as it enjoys consumer preference. The base population was derived 

from different rivers such as Ganga, Yamuna, Sutlej, Gomati and Brahmaputra. The 

first phase was from 1992-1997 and based on the encouraging results obtained during 

the first period, the second phase was launched from 1997-2003. The tagging was 

carried out using PIT (Passive Integrated Transponder) tags. The response to 

selection for growth has been substantial ( 43.5%). ( Iana et a1 2003) 

Growth appears to be the most important trait for selection in aquaculture 

species. Increased growth rate leads to shorter production cycles to market size 

thereby reduced economical and biological risks of aquaculture operations. Growth 

rate is also assumed to be genetically related to feed conversion efficiency. If we 

select for growth rate, we get improvement in feed efficiency also, due to the positive 

relationship. This is known as correlated response. A veq important factor here is 

that if we select for one trait which has got a negative influence on another desirable 

trait, then it would become very difficult to obtain genetic improvement in both the 

traits simultaneously. The more we select for one trait, deterioration starts for the 

other trait. 

In Norway, the pioneering work canied out in Atlantic Salmon since the early 

seventies, is worth mentioning. Salmon had been selected for growth rate and age of 

sexual maturation. The country is stiIl having the programme, of course with 

modifications like incorporation of carcass quality traits and disease resistance as

additional selection criterion. The genetically improved Salmon had improved feed 

efficiency, early sexual maturity and the increased growth rate is mainly due to a 

much higher feed intake per kg body weight. (Gjedrem et a1 2002). 

The genetically improved strain GIFT (Genetically Improved Farmed Tilapia) 

is an important example as to how genetic improvement can contribute to meet the 

protein requirement of the people in five Asian countries such as Philippines, 

Bangladesh, Vietnam, Thailand and Fiji Islands. The genetic gain for growth rate in 

the GIFT project per generation across five generations of selection has been in the 

range of 12-17%. Produ~tion trials and socio-economic surveys in these countries 

revealed that the cost of production per unit of fish produced is 20-30% lower for the 

GIFT strain than for the other Nile strains.(Gjedrem et a2 2002). 

The AKVAFORSK is collaborating with the Columbian Government in a 

genetic improvement project in the Pacific White Shrimp- Litopenaeus vannamei. A 

breeding programme has been established for growth and White Spot Disease 

Resistance. 

Gitterle, (2005) reported that selecting for growth in Pacific White Shrimp will cause 

a positive correlated response on overall survival. He also reported low genotrpe x 

farm interaction for both traits. Resistance to WSSV and harvest weight is under 

additive genetic control and thus would respond to selection. 

In India, an Indo-Norwegian Collaborative project has been started in June 

2004 on 'Genetic Improvement of Penaeus monodon (Tiger Shrimp) Through 

Selection for Growth and White Spot Disease Resistance'. The collaborating 

Institutions are Central Institute of Brackishwater Aquaculture, Chemai, Central 

Institute of Fisheries Education, Mumbai and Institute of Aquaculture Research 

(AKVAFORSK) Norway. This project envisages the rearing of 50 full-sib families of 

Tiger Shrimp, obtaining data on growth rate as well as WSSV disease resistance. 

Tagging is accomplished by Visible Implant Elastomer tags. The challenge tests are 

conducted with individuals represented from each family. The survival data is 

recorded and analysed along with the growth trait. The experiment is progressing at 

CIBA, Chennai. 

There has been considerable interest in 'Transgenic' fishes and shellfish. In 

transgenesis, the fisNshellfish is modified genetically by introducing a gene taken 

from some other source. This results in a geneticalb modzj?ed organism or GMO. As

is terrestrial animals, there are a lot of social and environmental concerns attached to 

transgenic fisWshellfish. Probably, the only area in aquaculture where transgenesis 

would be helpful would be in Ornamental fishes 

It appears that selective breeding is a fairly good technique to genetically 

improve the required traits using conventional selection. First and foremost thing is 

that fishes or shellfish have to be identified that are liked by the customers and have 

their life cycle closed. Thereafter, these aquaculture species could be genetically 

improved through very strong breeding programmes. As selection is applied, the 

improvement would become visible in terms of higher production and benefits 

accruing from it. 

References 

Gjedrem, T. (1 997) 

Selective Breeding to improve aquaculture production. World Aquaculture: 33-45 

Gjedrem, T., Gjerde, B. and Rye, M. (2002) 

Possibilities and Benefits of Selective breeding in Fish and Shellfish. 

In The Fifth Indian Fisheries Forum Proceedings, Eds.S. Ayyappan, S., Jena, J. K. 

and Joseph, M.M. pp 449-455 

Jana, R.K., Mahapatra, K.D., Saha, J.N. and Gjerde, B. (2003) 

Selective breeding of Rohu (Labeo rohita, Ham) An Overview. 

In Final Workshop on Genetic Improvement of Rohu ( Labeo rohita, Ham) For 

Growth Through Selective Breeding. Pp 46-58 

Rye, M. (2005) 

Genetic Improvement Programs in Aquaculture Species-A Global Perspective, 

In Genetics and Health Management in Aquaculture, 

CIFE, Mumbai, India. February 9-10,2005. 

Gitterle, T. (2005) 

Genetic Analyses for resistance to White Spot Syndrome Virus (WSSV), harvest body 

weight andpond survival in the Pacific White Shrimp Litopenaeus vannamei. 

Norwegian University of Life Sciences, Doctor Scientiarum Thesis 2005:9

CHAPTER 3 

MICROBIAL TECHNIQUES 

M.S.Shekhar and S.Kannappan 

1. STERILIZATION: 

Sterilization is the complete elimination or destruction of all forms of 

microbial life and is accomplished by either physical or chemical processes. Steam 

under pressure, dry heat, and liquid chemical are the principal sterilizing agents used 

in the laboratory. 

1.a. Dry Heat: The only items for which dry heat sterilization is appropriate are 

those that cannot be sterilized by steam because they cannot be penetrated, or will be 

damaged by moisture. Dry-heat sterilization actually "bums up" microbial cells." 

Glass wares are generally sterilized by dry heat in Hot air oven. A hot air oven is 

electrically operated and should be equipped with a fan to ensure uniform 

temperature inside. The required temperature for sterilization is generally 160°C for 

one hour. 

1. b. Moist heat : In moist heat (steam) sterilization bacteria die from the 

coagu1,ation or denaturation of the protein constituents. Steam sterilization works 

better than some forms of sterilization because steam destroys most resistant bacterial 

spores in a brief exposure. Cotton, Filters, Instruments, Culture media are generally 

sterilized by Autoclaving Autoclaves can sterilize anything that can withstand a 

temperature of 121°C for 20 minutes. 

2. BACTERIAL CULTURE MEDIA: 

2.a. Luria-Bertani broth: 

LB media formulations have been a laboratory standard for the cultivation of 

Escherichia coli .These media have been widely used in molecular/microbiological 

applications for the preparation of plasmid DNA and recombinant proteins. It

continues to be one of the most common media used for maintaining and cultivating 

recombinant strains of Eschenchia coli. 

There are several common formulations of LB. Although they are different, 

they generally share a somewhat similar composition of ingredients used to promote 

growth, including the following: 

Peptides and casein peptones , Vitamins (including B vitamins) ,Trace 

elements (e.g. nitrogen, carbon, sulfur), Minerals 

The following are some common steps taken to prepare LB broth (assuming a 1 L 

volume): 

Weigh out the appropriate amount of solid to make a 1 L solution 

For example: Weigh 25 g of LB broth (Himedia) 

I. Suspend the solid in approximately 700-800 ml of distilled or deionized water 

2. Heat to boiling (e.g in a microwave) to dissolve the solid completely. 

3. When dissolved, bring the solution to 1 L with distilled water. 

4. Autoclave at 121 "C for 15 minutes. 

5. Cool to 45-50 "C in a waterbath. 

2.b. LB agar plate: 

An agar plate is a sterile petri dish that contains agar plus nutrients, and is 

used to culture microorganisms. Generally, selecting substances are also added to the 

plate, such as antibiotics. Most types of agar are purchased pre-prepared in powder 

form, although it is possible to buy a base agar mix and add nutrients separately. 

They are dissolved in distilled water as per their instructions. It is usually necessary 

to gently boil the mixture to facilitate dissolving: this can be done in a microwave 

oven, or over a gentle flame. Once dissolved, the agar needs to be sterilised, usually 

by pouring it into a conical flask, then sealing the top with a cotton wool wad, and

finally covering the cottop wool with a loose layer of aluminium foil. This is then 

autoclaved for 15 minutes. The sterile agar is then allowed to cool to 50 OC: this is 

just above the setting temperature of agar. Chemical agents (e.g. antibiotics) are 

added at this step to prevent their degradation at the higher temperatures. 

3. STREAKING 

The most common method of inoculating an agar plate is streaking (Fig. 1) 

Figure 1. Streak plates 

With this method, a small amount of sample is placed on the side of the agar plate (as 

a drop from an inoculating loop ). 

1. A sterile loop (flamed until red hot, then cooled by touching the agar away 

from the inoculated sample) is then used to spread the bacteria out in one 

direction from the initial site of inoculation. This is done by moving the loop 

from side to side, passing through the initial site. 

2. This is repeated 2-3 times, moving around the agar plate. 

3. What should happen is that single bacterial cells get isolated by the streaking, 

and when the plate is incubated, discrete colonies are formed that would have 

started from just one bacterium each.

CHAPTER 4 

PLASMID DNA ISOLATION AND GEL ELECTROPHORESIS 

Plasmids are (typically) circular double-stranded DNA molecules separate from 

the chromosomal DNA (Fig. 2) and capable of autonomous repIication. Their size 

varies from 1 to over 400 kilobase pairs (kbp). There are anywhere from one copy, 

for large plasmids, to hundreds of copies of the same plasmid present in a single 

bacterial cell. 

Figure 2: Schematic drawing of a bacterium with plasmids enclosed. (1) 

Chro~~iosomal DNA. (2) Plasmids 

Plasmids used in genetic engineering are called vectors. They are used to 

transfer genes from one organism to another and typically contain a genetic marker 

conferring a phenotype that can be selected for or against. Most also contain a 

multiple cloning site (MCS), which is a short region containing several commonly 

used restriction sites allowing the easy insertion of DNA fragments at this location. 

Plasmids serve as important tools in molecular biology, where they are commonly 

used to multiply (make many copies of) or express particular genes. There are many 

plasmids that are commercially available for such uses. Initially, the gene to be 

replicated is inserted in a plasmid. The plasmids are next inserted into bacteria by a 

process called transformation, which are then grown on specific antibiotic(s).

1 .a . Plasmid isolation protocol: 

Solution 1 : 

50mM glucose 

25mM Tris.Cl (pH 8.0) 

1 OmM EDTA 

20pgr'ml RNAse (final concentration) 

Solution 2: 

0.2N NaOH 

1% SDS 

Solution 3: 3M potassium acetate (pH 4.8) 

Procedure 

1. Fill a microcentrifige tube with saturated bacterial culture grown in LB broth 

+ antibiotic. Spin tube in microcentrifuge for 1 minute. Dump supernatant 

and drain tube briefly on paper towel. 

2. Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as 

possible using disposable tip. 

3. Add 0.2 ml Solution 2, cap tubes and invert five times gently. Let tubes sit in 

ice for 2-3 minutes. 

4. Add 0.2 ml ice-cold Solution 3, cap tubes and invert five times gently. 

Incubate tubes on ice for 5 minutes. 

5. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh micro centrifuge 

tube using clean disposable transfer pipette. Try to avoid taking any white 

precipitate during the transfer. 

6. Fill remainder of centrihge tube with 2 volumes of ethanol. Let tube sit at 

room temperature for 2 minutes. 

7. Centrifuge tubes for 5 minutes. Pour off supernatant without dumping out the 

pellet. Drain tube on paper towel.

8. Add 1 ml of ice-cold 70% ethanol. Cap tube and mix by inverting several 

times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump 

out pellet) and drain tube on paper towel. 

9. Allow tube to dry for -5 minutes. Add 50 ul TE or distilled water to tube. 

DNA is ready for use and can be stored indefinitely in the freezer. 

1.b. Gel electrophoresis : 

There are several methods for preparing agarose gels. A common example is 

shown here. Other methods might differ in the buffering system used, the sampIe 

size to be loaded, and the total volume of the gel. 

1. Make a 1 % agarose solution in 1 .Ox TBE (90mM Tris-borate, 2 mM EDTA). 

2. Boil solution. 

3. Let the solution cool down to about 60 OC at rqom temperature. Stir the 

solution while cooling. 

4. Add 1 p1 ethidium bromide per 10 ml gel solution (0.5 pglml). (Wear gloves, 

ethidium bromide is a potent mutacen ) 

5. Stir the solution to disperse the ethidium bromide and then fill it into the gel 

rack. 

6. Insert the comb at one side of the gel. 

7. When the gel has cooled down and become solid, remove the comb. The 

holes that remain in the gel are the slots. 

8. Put the gel, together with the rack, into a chamber with 1.0~ TBE. Make sure 

the gel is completely covered with TBE, and that the slots are at the electrode 

that will have the negative current. 

9. Mix the dye to DNA and load in the slots

CHAPTER 5 

GENE CLONING 

M.S.Shekhar 

Cloning 

Cloning a gene means to extract a gene from one organism (for example by 

PCR) and insert it into a second organism (usually via a vector), where it can be used 

and studied. 

Method of Gene Cloning: 

1. The gene or DNA that is desired is isolated using restriction enzymes. 

2. Both the desired gene and a plasmid are treated with the same restriction enzyme 

to produce identical sticky ends. 

3. The DNAs from both sources are mixed together and treated with the enzyme 

DNA ligase to splice them together. 

4. Recombinant DNA, with the plasmid containing the added DNA or gene is 

formed. 

5. The recombinant plasmids are added to a culture of bacterial cells. Under the 

right conditions, some of the bacteria will take in the plasmid from the solution 

during a process known as transformation. 

6. As the bacterial cells reproduces, the recombinant plasmid is copied. Soon, there 

will be millions of bacteria containing the recombinant plasmid with its 

introduced gene. 

7. The introduced gene can begin producing its protein via transcription and 

translation if cloned in expression vector.

Procedure 

Restriction digestion 

1. Digest the vector and insert DNA with EcoRI and Sal I 

DNA (2 pg) - X p1 

Restriction enzyme buffer - 2.0 p1 

Restriction enzyme (EcoRII Sal I) - 1 unit each 

Distilled water - To 20 ul 

Incubate at 37 * C for one hour. 

2. Gel elute and purify both vector and insert DNA using gel elution kit folIowing the 

standard procedure 

3. Check the concentration of eluted DNA in agarose gel. 

Ligation reaction 

I. Set up the ligation reaction as follows: 

Vector (restriction enzyme digested plasmid) (50 ng) - X p-1 

Insert (restriction enzyme digested ) (1 50 ng) - Y p1 

Ligase buffer - 2.0 p1 

T4 DNA ligase - 1.0 p1 

Water to - 20 pl 

2. Incubate the ligation mixture at 14'~ 

for overnight. 

3. Transform the ligated mixture into DH5u competent cells using standard protocol. 

4. Check for the transfoxmants canying the insert by PCR.

CHAPTER 6 

MITOCHONDFUAL DSA EXTRACTION AND PCR A~TPLIFICATION OF 

MITOCHONDRIAL GENES 

M.S.Shekhar and G.Gopikrishna 

1.a. Procedure for isolation of mitochondrial DNA. 

Mitochondria1 DNA can be extracted from the muscle tissue of shrimps by the 

following procedure: 

1. Wash the muscle tissues (100 mg) in TE buffer (IOmM Tris-HC1, 1mM EDTA, 

pH 8.0). 

2. Homogenize in 1.5 ml Eppendorf tube with 500 p1 of homogenization buffer 

(30mM Tris-HC1, 30mM EDTA, 15 mM NaCI, pH 7.8) containing 100 yglml 

proteinase K. 

3. Mix the homogenized solution with two volumes of SDS 1%, NaOH 0.2 N. 

4. Store samples in ice for 5 min and gently mix with 1.5 volumes of potassium 

acetate (3M potassium, 5M acetate). 

5. After incubating in ice for 5 min, centrifuge for 10 min at 12000 X g. 

6. Extract the supernatant with phenol by adding equal volume of Tris saturated 

phenol. Collect the supernatant after centrifigation for 5 min at 12000 X g. 

7. Precipitate the mitochondrial DNA after adding two volumes of ethanol. 

8. Store at -20°C for 2 hrs. 

9. Pellet the DNA by centrifugation for 5 rnin at 12000 X g. 

10. Add 1 ml of ice-cold 70% ethanol. Cap tube and mix by inverting several times. 

Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) 

and drain tube on paper towel. 

11. Allow tube to dry for -5 minutes. Add 50 ul TE or distilled water to tube. DNA is 

ready for use and can be stored indefinitely in the freezer. 

1.b. Polymerase chain reaction of mitochondria1 genes: 

The PCR reaction mixture contains all 4 cWTPs (200 pM), 30 pmoles 

concentration of each primer, 1 unit of Taq polymerase and 1X polymerase buffer 

containing 1.5 mM MgC12. The thermal program followed was: 93°C for 1 rnin

followed by 30 cycles of 93°C for 1 min, 50°C for 30s, 72°C for 1 min and 72°C for 

7 min as final extension cycle. 

Procedure : 

1. Add 4.0 ul ofdNTP 

2. Add 2.0 ul of Taq DNA polymerse 

3. Add 2.0 ul each of forward and reverse Primers 

4. Add 20.0 ul of 10X Taq buffer 

5. Add 170.0 ul of sterile distilled PCR water 

6. Add the DNA template ( the quantity depends on the concentration of DNA 

being used) 

7. Spin the PCR reaction mixture after distributing into PCR tubes. 

8. Put in thermocycler for amplification. 

9. After the amplification is over, check the PCR product by agarose gel 

electrophoresis.

Nutrition and Captive Broodstock Development 

CHAPTER 7 

Global aquaculture productions of shrimp had been facing immense 

fluctuations through out the World due to number of factors. The decrease in 

production tonnage for all species has been attributed to environmental degradation, 

farm mismanagement and losses due to diseases which are the critical constraints for 

continued development of sustainable shrimp aquaculture (FAO, 1997). In addition 

to these inadequate domestication (poor closed - recirculation system , performance 

of broodstock with the resultant in adequate supply of quality post larvae), limited 

choice of different and economical live feed and inadequate water quality 

management have been identified as further significant constraints to the 

development of shrimp culture. Thus, there is considerable scope of application of a 

wide range of the newly developing biotechnologies in an effort to ameliorate these 

problems. 

Presently wild broodstock availability, quality and cost had been the main 

constraint for the disease free quality seed production. Factors such as transboundry 

transfers of live shrimp - broodstock and post larvae are main causes of rapid 

transmission of epizootic viral diseases in shrimp. Under these circumstances 

conventional methods of controlling aquatic animal pathogens such as chemotherapy 

appears to be less effective in managing newly emerging pathogens. Application of 

molecular biotechnology in screening and detection of pathogens, elucidation of 

pathogenicity, developmental of effective control and preventive measures in 

treatment of disease have'been some of the latest intervention. 

Complimentary approaches: 

Sustainability of shrimp culture depends by using proper broodstock 

management programmes, selection of specific pathogen free (SPF) from the wild, 

raising them under strict sanitary conditions and propagating them through selective 

breeding programmmes in raising specific pathogen resistant (SPR) could sustain

shrimp culture. Thus captive broodstock development programme have been initiated 

in number of countries. However, the success rate in still in infancy. 

Captive broodstock development and management: 

Restricting pathogenicity spread in shrimp culture can be through 

development of captive broodstock. Maintenance of strict vigil against entry of 

pathogen by maintaining high profile sanitary conditions in and around the 

broodstock system could improve the seed quality production. 

Factors that determine the captive broodstock management are: 

Selection of specific pathogen free wild stock with proper screening and 

detection methods. 

Quarantining of selected animal by chemotherapy followed by latency period. 

Transfer and maintenance in biosecured conditions with good quality 

seawater. 

Maintenance of sanitary conditions of materials and human interventions. 

Pre-conditioning of live feed materials by sterilization and proper handling 

and storage before and after feeding respectively. 

* Monitoring and maintenance with minimum fluctuations of physical factors - 

salinity, pH, temperature, ammonia and nitrite levels. 

Time to time screening and segregation of unviable, weak and poorly 

growing animals from the good quality broodstock. 

Selection and segregation of brooders for spawning 

Maintenance of hygienic conditions and feeding high quality lipid rich feeds. 

Collection of eggs followed by chemotherapy and thorough washing before 

transfening. 

Pre treatment of spent shrimp before returning to the common pool for repeat 

spawning. 

Rejection of animals after two successive spawnings.

Role of nutrition in captive broodstock maintenance: 

Nutrition of captive broodstock has a major role in maintenance and quality 

seed production. During development of gonads, quality protein, high profile lipids 

especially PUFA, trace minerals and supplemental vitamins influence the fecundity 

and quality of eggs. Invariably quality live feeds such as squid, clam meat, crab meat 

and polycheates meet the requirements of the developing gonads. However, quality, 

availability and cost of live feeds are the limiting factors in captive broodstock 

maintenance of shrimps. Alternatively artificial pellet feeds having, high quality 

protein, lipids, enriched with trace mineral and vitamins could meet the feed 

requirements. 

The quality of artificial supplemental feed depend on: 

Type of selection of proteins (plant / animal origin) 

- Aminoacids profile of proteins. 

- Plant rich in certain amino acids - soyabean. 

- Animal source proteins are preferred for their richness in certain 

aminoacids and fatty acids. 

Lipidprofile 

- Animal origin (fish oil are rich in omega 3 fatty acids. 

- Plant origin (sunflower oil, corn oil are rich in omega 6 fatty acids). 

- Ratio of w3 : w6. 

- Rancid free oils. 

Balanced mineral requirement 

- Fortification with trace minerals (Cu, Zn, Se, Mn) 

- Ratio of Ca : P 

Vitamin fortification 

- Selected high doses of B-complex, A,C,D,E vitamins 

Use of additives 

- Improve the gustatory stimulus (Glutamic acid, Betain) 

- Certain unknown factors (Squid) enhance quality seed production 

Biotechnological intervention through Nutrition for captive brood stock: 

Shrimp seed quality depends on the type of nutrition that the mother shrimp 

feeds. Live feeds are the most nutritionally balanced feed and if given periodically in 

a day (three times) to satiation, the broodstock maturation development is faster and

eggs produced are good. However, recent studies in aquatic fish nutritional 

requirement, it was found that enriching the live feeds especially during larval 

rearing has given promising results of survival and growth rates. Usually Artemia 

nauplii, rotifers and algal cultures are enriched with highly unsaturated fatty acids 

(PUFA). 

In captive broodstock management bioencapsulated dietary nutrients 

improves the quality of egg production, hatchability and survival of post larvae. 

Using bioencapsulation technique where in the nutrients (vitamins, 

chemotherapeuants and vaccines) are encased in gelatin layer and orally 

administered. Such bioencapsulated feeds are effective, biosecured and can restrain 

pathogenicity entry compared to live feeds. 

Thus, through nutritional manipulation, disease fiee and quality broodstock 

can be maintained under captive conditions.

CHAPTER 8 

PROBIOTICS IN AQUACULTURE 

S. KANNAPPAN AND G. GOPIKRISHNA 

Probiotics can be defined as live microbial food that improves the health of 

man and terrestrial animals. In aquaculture context, it is sometimes termed as a 

biocontrol agent or a bio-remediator, which means that the microbe, when added to 

the water body is taken up by the fish, passes through the intestinal tract, and may 

survive there. The gastrointestinal biota of fish is just a reflection of the water biota, 

as a large volume of water passes through them or is filtered by them. Most of the 

probiotic preparations include microbes belonging to family Vibrionaceae, the 

Pseudornonads, Lactic acid bacteria, Bacillus spp and yeasts. Some of the main 

characteristics expected of a successful probiotic would be its antagonistic activity 

against spoilage and pathogenic organisms and the ability to colonise in the gut. 

Probiotics have got the ability to afford resistance to disease causing agents. 

There is also the competitive exclusion of the pathogens for nutrients or adhesion 

sites and immunostimulatory activity. Though the probiotic concept has been 

extended from humans, terrestrial animals to aquatic animals, there are certain basic 

differences in the embryonic development that should be learnt first in order to decide 

the best age for probiotic treatment. Man and terrestrial livestock undergo embryonic 

development within the amnion whereas the larval forms of fish and shellfish are 

exposed to the external environment at an early ontogenic stage. Hence, these larvae 

get exposed at an early stage to gastrointestinal tract disorders due to microbes as they 

feed even before the tract is fully developed (Timmermans, 1987) and the immune 

system complete (Vadstein, 1997). Probably the best time for probiotic treatment in 

aquatic system is the larval stage to get the desired effect. 

In the digestive tract of fish, the bacterial flora are predominantly Gram- 

negative anaerobes (Clements, 1997). In crustaceans, marine fish and bivalves, the 

two common genera recorded are Yibrio and Pseudomonas (Moriarty, 1990; Prieur et

al., 1990; Sakata, 1990) whereas in fresh water fish, Plesiomorias and 

Enterobacteriaceae members are dominant (Sakata, 1990). Therefore a probiotic 

considered highly efficient in terrestrial system need not necessarily be the choice for 

the aquatic animals. Micorbial flora of the gastrointestinal tract in aquatic animals is 

transient and changes in the flora can be attributed to temperature and continuous 

water flow. The gastic barrier being absent in the larvae, these effects are likely to be 

more pronounced in them. The intestinal microflora in aquatic animals changes 

rapidly with the intrusion of microbes coming from water and food. In larval and 

juvenile fish, the influence of food has been clearly demonstrated (Ringo et al., 1994: 

Tanasomwang and Muroga, 1989). The transience of the flora allows the extension of 

the probiotic concept to living microbial preparations used in treatment of aquaculture 

ponds. Moriarty (1998) extended the definition of probiotics to call it "Microbial 

water additives". Gatesoupe (1999) refers to probiotics as "Microbial cells that are 

administered in such a way as to enter the gastrointestinal tract and be kept alive with 

the aim of improving health". Two studies on improving water quality which 

consequently resulted in higher production of Penaeus monodon consisted of floating 

biofilters pre-inoculated with nitrifying bacteria (Porubcan, 1991a) and seeding 

Bacillus spp near pond aerators (Porubcan, 1991b). The former decreased the amount 

of ammonia and nitrite in the rearing water and the latter reduced the chemical 

oxygen demand. This study underlines the concept that bacteria which improve water 

quality are beneficial to animal health. 

Most commercial probiotics contain nitrifying bacteria and/or bacillus spp. 

Both these groups are very different from one another. Nitrifying bacteria have strict 

ecological niches and are not detected in the gastrointestinal tract of animals. On the 

other hand, there are many reports of Bacillus being isolated fi-om intestinal tract of 

fish, crustaceans and bivalves. Some studies have shown the increased production 

and survival of aquatic animals where a commercial preparation of Bacillus was 

used. Some others have demonstrated the decrease 'of pathogenic Yibrio spp in 

sediment and water. Some of the possible effects could be due to enzymatic 

excretions of the probiotic that has resulted in the emergence of drug-resistant strains. 

This fallout of chemical treatment in aquaculture systems can be overcome by using 

probiotics such as Bacillus spp. Data of Moriarty (1998) showed inhibitory activity 

of Bacillus spp against luminous Vibrio spp in pond sediment and better prawn

survival. It could have been due to the probiotic effect or an effect on the water 

quality by degradation of organic matter. As regards its effect as a probiotic on the 

possible intestinal transience it brings about, more studies are required to be camed 

out. 

Results of the experiments of Moriarty (1998) suggest the Bacillus spp is 

used as a biocontrol agent. The effects could possibly have been due to production of 

antibiotics in relation to sporulation or during proteolysis of the vegetative cells. 

The inclusion of nitrifying bacteria under the term probiotic is rather incorrect. 

This concept is considered as bioremediation which is defined as "Treatment of any 

waste by use of microbes that break down the undesirable substance". Other 

commercial preparations of lactic acid bacteria and Streptococcus faeciunr have also 

been tried in aquaculture but there is not much evidence of its ability to colonise the 

gut. There has been limited success in extending the use of probiotics of terrestrial 

animals to aquaculture. The survival of microbes in the gastrointestinal tract of 

aquatic animals is uncertain, therefore there has been a shift in the research by 

scientists who are now looking for autochthonous strains residing in the 

gastrointestinal tract adhering to the mucus, as possible candidates. 

Antagonism, commonly observed among marine bacteria may be mediated 

by antibiotics, organic acids, hydrogen peroxide and siderophores. However, the in 

vitro activity seen, should be extended to in vivo studies in order to consider it as a 

probiotic. Intestinal colonization is an important potential to consider in a probiotic. 

In a study, it was observed that autochthonous intestinal bacteria adhere specifically 

to intestinal mucus and their adherence potential to mucus was found to be stronger 

than to control surface bacteria. Yeasts are reported to have a greater ability to adhere 

to intestine and colonise by specific adhesions and their application as probionts is 

promising. 

In conclusion one can.see the advantages of probiotics over other chemicals used 

in aquaculture. It is to be expected that probiotic-resistant pathogens will emerge. To 

avoid this situation, one must take care to ensure that probiotics with diversified 

antagonistic properties are used. It is also important to consider the stimulation of the 

immune system by the probiotic. Molecular approaches to analyze bacterial 

communities in the gut may help to look at the influence of specific probiotics on the 

gastrointestinal microbiota.

PROBIOTICS 

MODE OF ACTION 

Adhesion to digestive tract wall to prevent colonization of pathogens. 

Adhesion is acknowledged as the first step of microbes in the process of colonization 

and the intestinal mucous plays a vital role in this process. 

Neutralisation of toxins. 

Bactericidal activity and increased immune response. 

Possible to maintain the LAB population artificialIy at high levels by regular intake 

through feed especially in fishes like Cod, Salmon, Trout 

Similar studies in Crustaceans are lacking. 

Probiotic bacteria may competitively exclude the pathogenic bacteria or produce 

substances that inhibit the growth of the pathogenic bacteria. 

Provide essential nutrients to enhance the nutrition of the cultured animals. 

Provide digestive enzymes to enhance the digestion of the cultured animals. 

Probiotic bacteria directly take in or decompose the organic matter or toxic 

material in the water thereby improving the quality of the water. 

Production of Extra cellular materials 

Production of lactic acid by LAB reduces the pH of the stomach contents in 

endothermic animals. LAB also produce hydrogen peroxide which has bactericidal 

actions in vitro and produces metabolite, thought to neutralize the effect of 

enterotoxin released by other bacteria. LAB is the best studied probiont, however, 

their effect on Crustaceans has not been studied much. LAB is not the dominant gut 

microflora in marine fish as compared to endothermic animals. LAB can be 

introduced into larvae and juvenile fish. Lb acidophilus + Saccharomyces + 

Streptococcus faecium, put together, enhanced the growth and survival of juveniles 

of F. indicus. A challenge with ?Z alginolyticus resulted in low mortality in F. 

indicus juveniles. Upto lo6 cfilgrn shrimp probiotic were detected in the gut of the 

post-larvae.

LAB in endothermic animals 

Raised activities of macrophages and lymphocytes were observed in mice, 

following oral inoculation with LAB. Peptididoglycan derived from Bz~dobacteriunr 

tizernzopizilum would enhance the disease resistance of M. japonicus. Thalassobacter 

utilis have been isolated from the rearing water of larval P. monodon, for use as a 

biocontrol agent. This strain increased the survival rate of the larvae of P. monodolz 

and the swimming Crab (Portuaus trituberculatus). Pseudomonas and Micrococcus 

species of bacteria increased the survival rate of P. nzonodon 

Antagonism of aquatic microbes isolated from crustaceans and its effect 

Biocontrol agent 

Altero~nonas 

-do- 

Source 

Palaenlon 

macrodactylus 

Shrimp 

Tested against 

Lagenidiurn 

(fungus ) 

Vibrio 

Effect 

Protection of crustacean embryos 

from fungal infection 

, Protection from Vibrios 

gainst experimental infections 

Advantages of using bac~llus sp as probiotic agents 

Bacillus can easily move around (motile) because they have whip- like flagella. 

They form endospores, which are useful under stressful conditions. These endospores 

allow the bacillus to reproduce when conditions are favourable. Bacillus produce 

antibiotics examples being bacitracin, polyrnixin, tyrocidin, gramicidin and circulin. 

They produce enzymes that can breakdown polysaccharides, nucleic acids and lipids. 

They are easily transformable (free DNA is easily incorporated to change its genetic 

make up). Certain bacillus are thermophilic, growing at high temperature (50 -70 " 

C). Bacillus can very easily be isolated from soil or air and they grow very well on 

synthetic media.

Safety of novel probiotics 

It cannot be assumed that these novel probioiic organisms share the 

historical safety of traditional strains. Before their incorporation into products, 

new strains should carefully be assessed and tested for the safety and efficacy of 

their proposed use. The following suggestions and recommendation have been 

proposed as suitable models and methods to test the safety of probiotic bacteria. 

1. Determine the intrinsic properties of bacteria and strains selected for probiotic 

use for example: adhesion factors, antibiotic resistance, plasmid transfer, 

enzyme profiles. 

2. Assess the effects of the metabolic products of the bacteria. 

3. Assess the acute and sub-acute toxicity of ingestion of extremely large 

amounts of the bacteria. 

4. Estimate the in vitro infective properties of probiotic bacteria using cell lines 

and human intestinal mucus degradation. 

5. Assess infectivity in animal models like immuno-compromised animals or 

lethally irradiated animals. 

6. Determine the efficacy of ingested probiotic bacteria as measured by dose 

response (minimum and maximum dose required, consequent health effects); 

assess the effects of massive probiotic doses on the composition of human 

intestinal microflora. 

7. Carefully assess side effects during human volunteer studies and clinical 

epidemiological surveilIance of people ingesting large amounts of newly 

introduced probiotic bacteria for infections. 

8. The most rigorous safety testing along the above lines to be undertaken for 

genetically modified strains (GMO) and strains derived from animals. 

Classification of probiotic organisms and their safety status. 

Organisms / Infection potential 

Lactobacillus I mainly non-pathogens, some 

I 1 oppomnistic infections (usually in 1 

I 1 immunocompromised patients) 

I

I 

1 Lacfococcus / mainly non-pathogens 

I 

/ ieucor~ostoc 

I 

1 Streptococcus 

1 

1 Enterococcus 

i 

Bzj?dobacteriunz 

Saccharomyces 

I 

1 mainly non-pathogens, some isolated 1 

case of infections 

oral streptococci mainly non-pathogen 

(including streptococcus thermophilus); 

some may cause opportunistic 

infections 

Some strains are opportunistic 

pathogens with haemolytic activity and 

antibiotic resistance. 

mainly non-pathogens, some isolated 

cases of human infection. 

mainly non-pathogens, some isolated 

cases of human infection. 

References 

Clements, K.D. 1987. Fermentation and gastrointestinal microorganisms in fishes. 

In: Mackie, R.I., With, B.A., Isaacson, R. E. (eds.). Gastrointestinal Microbiology. 

Vol. 1,Gastrointestinal Ecosystems and Fermentation. Chapman & Hall Microbiology 

Series, International Thomson Publishing, New York, pp. 156-98. 

Gatesoupe, F.J.1999. The use of probiotics in aquaculture. Aquaculture. 180, 147- 

165. 

Moriarty, D.J.W. 1990. Interactions of microorganisms and aquatic animals, 

particularly The nutritional role of the gut flora. In: Lesel, R. (ed). Microbiology in 

Poecilotherms, Elsevier, Amsterdam, pp.217-222. 

Moriarty, D.J.W. 1998. Control of luminous Vibrio species in penaeid aquaculture 

ponds. Aquaculture, 164,35 1-358. 

Porubcan, R.S. 1999a. Reduction of ammonia nitrogen and nitrite in tanks of 

Penaeus monodon using floating biofilters containing processed diatomaceous earth 

media pre-inoculated with nitrifying bacteria. Program and Abstracts of the 22nd 

Annual Conference and Exposition, 16-20 June 1991, San Juan, Puerto Rico. World 

Aquaculture Society. 

Porubcan, R.S. 1991b. Reduction in chemical oxygen demand and improvement in 

penaeus monodon yield in ponds inoculated with aerobic Bacillus bacteria. Program 

and Abstracts of the 22nd Annual Conference and Exposition 16-50 June 1991. San 

Juan, Puerto Rico. World~Aquaculture Society.

Pieur, D., Mevel., G., Nicolas, J.L., Plusquellec, A., Vigneulle, M. 1990. Interactions 

between bivalve mollusks and bacteria in the marine environment. Oceanogr. Mar. 

Biol. Annu. Rev. 28,277-352. 

Ringo, E. and Strom, E. 1994. Microflora of Arctic cham, Salvelin us alpinus L. : 

Gastrointestinal microflora of free-living fish and effect of diet and salinity on 

intestinal microflora. Aquacult. Fish. Manage., 25, 623-629. 

Sakata, T.1990. Microflora in the digestive tract of fish and shell-fish. In: Lesel, R. 

(ed.),Microbiology in Poecilothems. Elsevier, Amsterdam, pp, 171-176. 

Tanasomwang, V. and Muroga, K. 1989. Intestinal microflora of rockfish Sebastes 

schlegeli. Tiger puffer Takj%gu rubripes and red grouper Epinephelus akaara at 

their larval and juvenile stages. Nippon Suisan Gakkaishi, 55, 1371-1377. 

Timmermans, L.P.M. 1987. Early development and differentiation in fish. Sarsia, 72, 

331-339. 

Vadstein, 0.1997. The use of immunostimulation in marine larviculture: possibilities 

and challenges. Aquaculture, 155, 40 1-4 17.

BIOREMEDIATION OF COASTAL AQUACULTURE WATER 

K. K. Krishnani 

Aquatic contamination - Health Hazards to aquatic animals 

Aquaculture, particularly culture of tiger shrimp Penaeus nzonodon , has 

extensively been practiced all along coastal regions of India. However, during the 

past 20 years, there has been a revolutionary change in the aquaculture industry. The 

increasing impairment of coastal water quality has affected the aquaculture 

profitability in certain areas. The marine environment has become a dumping ground 

for discharge of wastes of heavy industries carrying metals, chemicals, pesticides and 

other hazardous pollutants, which is likely to result in deterioration of coastal water 

(Sakthivel and Ramamurthy, 2003). Pesticides that are primarily of agricultural 

origin may pose a serious environmental hazard because of their persistence, toxicity 

and lipophilic nature (Krishnani, 1998). Heavy metals which are discharged from 

electroplating industry, metal finishing, leather tanning and chrome preparation, to 

coastal waters has got an adverse impact on the aquatic species as they are among the 

conservative pollutants which are not subjected to bacterial attack or decomposition 

and are permanent additions to the marine environment (Krishnani et al. 2003). The 

pollution of soil and water with xenobiotics is widespread in the environment and is 

creating major health problems. 

Apart from xenobiotics, the culture of marine and brackishwater organisms in 

aquaculture systems may result in metabolic loads in the ponds and may reach the 

environment through drained waste. Accumulation of organic matter, solids, 

nitrogenous compounds and other nutrients may cause stress and DO depletion, 

which are unfavourable tb the animals but favourable to the disease causing agents, 

particularly when the intake and drainage systems of the aquaculture farms are both 

based on the same water source. The control of water quality is therefore the key 

factor for success in shrimp farming. In general, effective management of water

quality in shrimp ponds is therefore a critical pre-requisite not only for maximizing 

productivity, but also for mitigating the adverse impacts of discharging shrimp pond 

water. 

Remediation of coastal aquaculture water 

There is still a lack of effective means for the treatment / removal of 

contaminants from aquatic environment. The most common procedures for removal 

of toxic metabolites of aquaculture water are aeration and water exchange. The 

physical methods are not economically feasible because of odour problems and vast 

area requirements. Water exchange can be effective, if enough, good quality water is 

available to rapidly exchange the pond water. Aeration cannot reduce toxicants 

effectively in brackishwater ponds. Most of the previous work also highlights the use 

of commercially available activated carbons / bioaugmentation materials and ion 

exchange using zeolite, which are relatively expensive and less feasible to use in 

developing countries. Furthermor,e zeolite can not reduce ammonia from 

brackishwater and the large amount of zeolite needed to significantly reduce 

ammonia concentration would be impractical. Activated carbon loaded with toxicants 

is generally incinerated or disposed off on land, thereby causing environmental 

pollution through different routes. 

Bioremediation 

Successful aqua-fanning requires the safe removal of the contaminants from 

aquatic environment. Therefore, development of new technologies 1 methods for their 

safe removal from the environment is essential. The fate of environmental pollutants 

depends on the metabolic activities of indigenous microorganisms (Cook and Hutter 

1981; Erickson and Lee, 1989). The utilization of microorganisms to clean up 

xenobiotics from a polluted environment represents a potential solution to such 

environmental problems. Hence, bioremediation is one of the most rapidly growing 

areas of environmental biotechnology, which involves the use of indigenous microorganisms 

or its enriched culture to break down hazardous substrates to obtain 

chemical energy and nutrient sources for microbial growth. Use of bioremediation 

either by biostimulation or bioaugmentation for environmental clean up is popular 

due to low costs and its public acceptability (Silva et al. 2004). Bioremediation

greatly improves water quality and reduces the pollution level of wastewater before 

its release into the environment. 

Bioremediation techniques were first pioneered by the petroleum industry (Soli 

and Bens, 1972) and have become a popular alternative to chemical or physical 

remediation because of their relatively 'low cost and minimal impact on the 

environment. The interactions between bacteria and pollutant occur through complex 

biochemical and chemical reactions. During the process, micro-organisms, usually 

bacteria, feed on the contaminants and they provide enzymes to enhance 

mineralization of organic matter and to convert toxic complex molecules into water 

and carbon dioxide or other harmless smaller molecules. Bioremediation has many 

other advantages such as improving immunity of cultured animals to pathogenic 

micro-organisms. In addition, beneficial bacteria competitively exclude pathogenic 

bacteria or produce substances that inhibit the growth of the pathogenic bacteria and 

prevent the level of infection and frequent outbreaks of diseases. They provide 

essential nutrients to enhance the nutrition of the cultured animals and also digestive 

enzymes to enhance the digestion of the cultured animals. Hence, bioremediation is a 

constructive approach for decontamination of various toxicants and this can provide a 

low cost alternative to other remediation techniques. Moreover, it is becoming 

evident that indigenous microbial populations rather than introduced populations may 

be ecologically superior for use in natural systems. In situations where indigenous 

degraders cannot rapidly degrade recalcitrant chemicals, bioaugmentation may be the 

only means of successful bioremediation. 

Several mechanisms which would account for the acclimatization of 

microbial communities in polluted environments have been proposed: a) Induction of 

specific enzymes not present in populations before exposure to the toxicant. b) 

Genetic selection for new metabolic abilities c) Increase in the number of microbes 

that are able to transform. the toxicant to a less toxic form d) Chemical structure of 

the pollutant. The adaption of microbial communities to pollutants results in the 

enhancement of biodegradation and the development of resistance to these noxious 

substances (Barkay and Pritchard, 1988).

Plant assisted bioremediation 

Apart from microbial bioremediation, green remediation or phytoremediation is 

one of the alternatives technologies for removing toxicants from the environment. 

Plants remediate pollutants by direct uptake of contaminants and subsequent 

accumulation of non-phytotoxic metabolites into plant tissue thereby releasing 

exudates and enzymes that stimulate microbial activity and biochemical 

transformation, which subsequently increase the biodegradation potential. 

This process is often referred to as plant-assisted bioremediation. Organisms such 

as algae and zoogleal and filamentous bacteria, growing on aquatic macrophytes and 

other submerged surfaces are termed as periphyton, which has more than one role in 

aquaculture. It improves production and water quality as well. Biofilms, which are 

assemblages of microorganisms and their associated extracellular products are 

typically attached to abiotic or biotic surfaces. Bacterial "consortia1' in the biofilm 

play a key role in the production of enzymes and degradation of organic matter and 

environmental toxicants. 

Summary 

It is clear that water resources will be under increasing pressure for human use 

and future demands for aquaculture-based food supply will increase water demands. 

During culture period, even under emergency situations, many farmers are unable to 

exchange water due to non-availability of good quality water from the source. Often 

shrimp ponds are completely drained to facilitate harvest.' This wastewater may be 

particularly high in organic matter, nutrients, suspended solids and microorganisms 

due to mechanical disturbance of the pond bottom and the large volume of discharge. 

These nutrients and organic matter are biodegradable. Under these circumstances, 

treatment of aquaculture water in the culture pond itself for its reuse is a sensible 

method to support the growth of aquaculture industry without excessive water 

demands that are environmentally unsustainable. Hence, the development of simple 

and cost-effective bioremediation technology for the auwentation of aquaculture 

water may offer advantages of water and area savings, reduced risk of contamination 

and better environmental control. This assists the fanners to improve cuIture 

water/waste water quality and make their farming practices more sustainable.

References 

Barkay, T. and Pritchard, P.H. (1988) 

Adaption of aquatic microbial communities to pollutant stress. Microbial. Sci. 5: 

165-169. 

Krishnani, K.K.(1998) 

Use of Pesticides-Health hazards to Aquatic Animals. Fishing Chimes. 18(7): 26-28. 

Krishnani, K.K, Azad, I.S., Kailasam, M., Thirunavakkarrassu. A.R., Gupta. B.P., 

Joseph, K.O., Muralidhar, M., and Mathew.,A. (2003). 

Acute toxicity of some heavy metals to Lutes calcarifer fry with a note on 

histopathological menifestation. Journal of Erzvironmental Science & Health, A 

38(4): 645-655. 

Sakthivel, M. and Ramamurthy, S. (2003) 

What ails shrimp farming and its future development. Fishing Chin~es. 22(10, 11): 

33-36. 

Silva, E., Fialho, A.M., Sa-correia, I., Richard, G, B., and Shaw, L.J. (2004). 

Combined bioaugmentation and biostimulation to clean up soil contaminated with 

high concentrations of atrazine. Environ. Sci. Technol. 38: 632-637. 

Soli, G. and Bens, E.M. (1972). 

Bacteria which attack petroleum hydrocarbons in a saline medium. Biotechnol. 

Bioeng. 14(3):319-30.

CHAPTER 10 

DRUG SENSITIVITY ON AQUATIC PATHOGENS 

S. Kannappan, K.P. Jithendran and G. Gbpi Krishna 

The ability of various antibiotics to inhibit the growth of sea fish-borne 

pathogens has to be screened in order to use them in suitable situations. At the same 

time, the sensitivity of the same antibiotics or similar effective antibiotics has to be 

determined to find out the Minimum Inhibitory concentrations (MIC). Further, from 

the range of inhibitions observed from the MIC level upwards, a quantitative assay of 

the antibiotic is obtained. These two parameters i.e., MIC and quantitative assay are 

of immense importance in chemotherapy in aquaculture, the lower the MIC, the 

higher will be the potency. 

In order to study the sensitivity, the particular bacterium is inoc~lated onto 

the respective agar plate. Different level of antibiotics is spotted at various points on 

the agar surface with equal distance. The antibiotics diffuse through the agar 

occupying a circuIar zone around the original spot. The bacterium grows on the agar 

surface in all places except in the circular zone, where as antibiotic is present. The 

size of the zone is related to the concentration of the antibiotic used and thus, a 

quantitative assessment can be made 

Materials 

Overnight culture of bacterial strains 

Antibiotic solutions or discs, Nutrient agar 

Ruler or Vernier calipers and sterile Petri plates 

Method 

Melt the respective (nutrient) agar and cool to about 45" C. 

Inoculate about 2.0 ml of the bacterial isolate per 50 ml of agar and mix. 

Pour into petriplates, fairly thick and allow it to solidify for 1-2 h. 

Dilute the antibiotic solution to get serial dilution of the respective antibiotics or 

place the antibiotics discs of known concentration.

Scoop out the agar at various places marked by means of the cork borer. 

If discs are used, they can be placed on the agar surface. 

Pipette out 0.1 ml of the diluted antibiotics solution to the different wells. Remember 

to label them. 

Incubate at 37" C about 12 h. 

Measure the diameter of the inhibition zone and record them in relation to the 

antibiotics concentration. 

Prepare a graph relating these two parameters.

IMMUNOSTIMULANTS FOR AQUAFARMING 

S. Kannappan and G. Gopikrishna 

CHAPTER 11 

Aquaculture is one of the fastest developing growth sectors in the world and Asia 

presently contributes substantially to the global production. However, disease 

outbreaks are a significant constraint to aquaculture production and trade and are 

affecting both economic development and socio-economic revenue in many countries 

in the Asia-Pacific region. 

Problems with present use of antibiotics, .drugs, and chemical treatments to 

prevent diseases in fish, set the stage for a new concept is disease preventionimmunostimulants. 

The adverse effects of antibiotics viz., development of resistant 

strains, residual problems in fish and human carry over, poor knowledge of farmers 

and their cost have been realized in many countries by putting strong strictures on 

their use. The use of drugs/chemicals has also achieved partial success. Vaccination 

in fish, particularly in India will take a long time to come up. Above all, vaccination 

only protects from a single or few diseases for which the antigen it contains. It only 

provides specific form of immunity to the fish. On the other hand, fish rely more on 

the non-specific defence mechanisms compared to mammals. Although, Indian 

farmers have started intensive or super-intensive system of culture practices, many 

are still following semi-intensive culture methods. We cannot recommend 

vaccination to them. Above all, commercial vaccines for fish are rare in India. The 

use of imported vaccines has its own drawbacks. It may not work for the strains 

prevalent in Indian subcontinent. Thus, the only alternative left with farmers is to use 

immunostimulants for sustainable farming. 

The intensive fish culture system is a highly stressful environment for fish. This 

stress in fish suppresses the immune responses, and fish kept under these conditions 

become highly susceptible to diseases. Thus the increase of non-specific immunity in 

stressed fish is important for resistance against many diseases, for which the 

immunostimulants play a major role.

By definition, an immunostimulant is a chemical, drug, stressor, or action that 

elevates the non-specific defence mechanisms or the specific immune response. 

Immunostimulants can be given to activate non-specific defence mechanisms or they 

may be administered with a vaccine for enhancing specific immune response. 

Research on immunostimulants is being intensified particularly in the areas of cancer 

and acquired immunodeficiency syndrome (AIDS). Veterinary practitioners are 

interested in the use of immunostimulants for activation and early protection against 

diseases in domestic animals. Although lot of reports on development of 

imrnunostimulants in fish in abroad are emerging daily, in India, we have a long way 

to go. 

Before a year or so, there was not even a single recommended 

immunostimulant for Indian major carps, which constitute one of the major 

components of Indian fish farming. Although few substances have been 

recommended for use in aquaculture abroad, it cannot be recommended to Indian 

carps because of many reasons. If the dose is not appropriate, it may even lead to 

immunosuppression. Each species has got its own nutritional requirements. Similar 

substances may not be effective for all species. Other problems are time, duration of 

feeding, cost, over-dose and under- dose problems. 

The study and use of immunostimulants in Indian carps first started at CIFA 

with the aim of standardizing low-cost, locally available, cost-effective, user-friendly 

substances to be used in freshwater aquaculture species particularly for Indian major 

carps. Now-a-days, only a few laboratories have taken up work on these lines. 

Immunostimulants will be most effective for short-lived fish, living in 

cool/cold waters as the development of a specific immune response is temperaturedependent. 

Among the non-specific defence mechanisms, the barriers in fish are 

skin and scales, lytic enzymes of mucus and sera and cellular changes are observed in 

monocytes, macrophages, neutrophils and cytotoxic cells. The specific immune 

response is induced by immunogenic stimulation with production of antibodies,

esulting from the increase in numbers of antibody-producing cells. The non-specific 

factors become activated by injury or stress. 

The mechanism of action of various immunostimulants is diverse in nature or may 

be poorly understood. Broadly, they may act through following mechanisms: 

a) Stimulators of T-lymphocytes 

Levamisole, Freund's complete adjuvant, glucans, muramyl dipeptide 

b) Stimulators of B-Cells- Lipopolysaccharides 

c) Inflammatory agents inducing chemotaxis-Silica & Carbon particles 

d) Cell: membrane modifiers - Detergents, sodium dodecyl sulfate, Quarternary 

ammonium 

compounds, saponins 

e) Nutritional factors- vitamins C and E 

f) Cytokines- Leukotriene, Interferon 

g) Heavy metals- Cadmium 

h) Animal and fish extracts and mitogens 

In general, immunostimulants enhance the activity of macrophages, phagocytes, 

lymphocytes and non-specific cytotoxic cells, resulting in resistance and protection to 

various diseases. 

Table 1 .Immunostimulants evaluated in fish and shrimp farming 

A . Synthetic chemicals- Levamisole, FK 565 (lactoyl tetrapeptide from 

Streptomyces olivaceogriseus) 

B. Biological substances: 

Bacterial derivatives- 

MDP (muramyl dipeptide from mycobacterium species), 

LPS (lipopolysaccharide), 

Vibrio vaccine, Achromobacter stenohalis and Clostridium butyricum cells 

Peptidoglucan (from Brevibacterium lactofementum & Yibrio sp) 

b. Yeast derivatives : J3-1-,3 glucan, P-1,6 glucan 

Nutritional factors : Vitamins C, E and A 

Hormone : Growth hormone, Prolactin

Cytokines 

: Interferon, interleukin 

Polysaccharides : Chitosan, chitin, lentinan, schizophyllan, oligosaccharides 

Animal and plant extract - Ete (tunicate), Hde (Abalone), Saponin, glycyrrhizin 

Others 

- Lactoferrin, Soyabean protein, Quil A, Saponin, Spirulina 

A considerable number of natural or synthetic substances have been tried in 

channel catfish Salmon apd Rainbow Trout. The R-1,3 glucan, levamisole, chitosan, 

ascorbic acid and a-tocopherol have already been evaluated in various laboratories 

for carps. At CIFE, Mumbai, ascorbic acid, a-tocopherol and quarternary ammonium 

compounds have also been tried in carps. It has been observed that glucan, a longchain 

polysaccharides extracted from yeast cell wall is the best immunostimulant 

followed by levamisole, ascorbic acid and a-tocopherol in carps. These could work 

efficiently, even in immunosuppressive conditions, to raise the non-specific 

immunity level as well as specific resistance against two common bacterial 

pathogens viz., Aeromonas hydrophila and Edwardsiella tarda. These two pathogens 

cause heavy economic loss in fish farming in India. 

In cases where disease outbreaks are cyclical and can be predicted, evaluating the 

non-specific defence mechanisms may reduce losses and the immunostimulants may 

be used in anticipation of events to prevent losses from disease. Indian farmers in 

North can use these the pubstances to prevent losses, where the water temperature 

falls below 15OC in winter and go beyond 35OC in summer. Some of seasonal out 

breaks such as epizootic ulcerative syndrome may be taken care of by using 

immunostimulants. This is an active area of research and holds greats potential. As 

none of these substances have side effects and give a wide range of protection 

(aginst bacterial, protozoal, parasitic, viral and fungal), their use in sustainable 

aquaculture is promising.

Table 2. List of pathogens successfully controlled by immunostimulants 

exposure in 

fishhhrimp 

a. Bacteria: Aeromonas hydrophilla, A. salmonicida, Edwardsiella trada, E. ictaluri, 

Yibrio abguillarum, 7. vulnzficus, K salmorzicida, Yersinia ruckeri, Streptococcus 

SP., 

b. Virus : Infectious haematopoietic necrosis, Viral heamorohagic speticaemia, 

yellow head bacculo viruslwhite spot virus 

c Ichthyopthirius nzultifiliis

CHAPTER 12 

Marine Bio-active Compounds and their 

Bio-therapeutic Applications 

S. Kannappan, K. P. Jithendran and G. Gopikrishna and M.S.Shekhar 

Marine biotechnology can be defined as the use of mechanistic, scientific and 

engineering principles to the processing of materials by marine bioIogica1 agents in 

order to provide chemical compounds for various applications. Under marine 

biotechnology, Archea is a newly defined cIass of organisms that are extremophiles, 

living beyond normal parameters. Examples are thermophiles, halophiles, 

thermoacidic and psychrophiles etc. The ocean has immense, unplumbed resources 

such as, bacterial polysaccharides and peptides, bio-therapeutic agents, neuro-toxic 

compounds, anti-cancer and marine drugs etc. Marine biotechnology has minimum 

pharmaceutical success as compared to terrestrial biotechnology because of 

difficulties of retrieving a sustained and reliable harvest of marine organisms, 

inadequate quantities of qaterial to allow for complete study, difficulties in culturing 

in the experimental laboratory. Many bio-active agents have been identified but none 

in the form of approved pharmaceuticals and clinical trials are available in India. The 

advancement is limited due to the inability to chemically synthesize these compounds 

and raise the organisms in culture and harvest the organism from its natural 

environment. 

Bioactive resources from sea 

Novel Pharmaceutical Compounds from Marine Bacteria 

Recent Developments on Antimicrobial Metabolites from Marine Sponges 

~ioadtive Compounds from Corals 

Pore-Forming Proteins from Sea Anemones 

Bioactive Compounds from Bryozoans 

Novel Alkaloids from Marine Bryozoans

Proteinases from Marine Organisms 

The main fields of interest are enzymes and bio-polymers. Enzymes from 

hyperthermophiles have been a major target of biotechnology programmes in the last 

decade (e.g. EU FP4 and FP5 "Extremophiles as cell factories"). Thermostable 

proteases, lipases, esterases, starch and xylan-degrading enzymes have been actively 

sought and in many cases found in bacterial and archaeal .hyperthennophilic marine 

microorganisms . The best known commercial success of thermostable enzymes is 

the Taq DNA polymerase, obtained from Tfiermus aquaticus (Yellowstone hot 

spring). Marine Thermococcales have been an important source of high fidelity 

thermostable DNA polymerases (Pfu, Vent, Pab, etc.) accounting for 30 % of the 

total sales. Microbial exopolysaccharides (EPS), especially those produced by some 

mesophilic Vibrios and Alteromonas strains isolated from deep-sea hydrothermal 

vents, display interesting therapeutic uses like tissue regeneration and cardiovascular 

diseases (antithrombotic / proangiogenic effects). Characterisation and preliminary 

results on anticoagulant activities of the EPS showed that native EPS were deprived 

of effect 

Over billions of years, marine microbes have moulded the global climate and 

structured the atmosphere. Knowledge of the biochemical processes that adapted, 

diversified and evolved in very different and extreme enyironments is the basis for 

discoveries in biotechnology" (ESF Marine Board, Position Paper 5, 2002). This 

statement reflects the current opinion of most of the scientific community in Europe. 

One salt-loving group of archaea includes Halobacterium, a well-studied archaean. 

The light-sensitive pigment bacteriorhodopsin gives Halobacterium its color and 

provides it with chemical energy. This protein is chemically very similar to the lightdetecting 

pigment rhodopsin, found in the vertebrate retina. 

The newly established genus Pseudoalteromonas contains numerous marine 

species which synthesize biologically active molecules. The production of a range of 

compounds which are active against a variety of target organisms appears to be a 

unique characteristic for this genus and may greatly benefit Pseudoalteromonas cells 

in their competition for nutrients and colonization of surfaces. Species of

PseudoaItero~?torzas are generally found in association with marine eukaryotes and 

display anti-bacterial, bacteriolytic, agarolytic and algicidal activities. 

Pseudoalteronzor2as have been found in association with living surfaces and are 

suggested to produce bioactive compounds against settlement of alga1 spores, 

invertebrate larvae, bacteria and fungi 

Moreover, several Pseudoalteromo~zas isolates specifically prevent the 

settlement of common fouling organisms. While a wide range of inhibitory 

extracellular agents are produced, compounds promoting the survival of other marine 

organisms living in the vicinity of Pseudoalteromonas species have also been found. 

Pseudoalteromonas isolates specifically prevent the settlement of common fouling 

organisms. To determine. the extent by which these antifouling activities and the 

production of bioactive compounds are distributed amongst the members of the 

genus Pseudoalteromonas, 10 different Pseudoalteromonas species mostly derived 

from different host organisms were tested in a broad range of biofouling bioassays. 

These assays included the settlement of larvae of two ubiquitous invertebrates 

Hydroides elegans and Balanus amphitrite as well as the settlement of spores of the 

common fouling algae Ulva lactuca and Polysfphorzia sp. 

Bioluminescence 

The process of bioluminescence is relatively common in both prokaryotic 

cells and complex eukayotic organisms. A biological role for light emission in 

animals (e.g. firefly) may be ascribed to sending signals from one individual to 

another. However, the role of luminescence in bacteria remains unclear. There are 

several known bacterial species that are able to emit light. In fact, light-emitting 

bacteria are the most abundant and widespread of the luminescent organisms found 

in marine, freshwater and terrestrial habitats. The process of luminescence is found in 

symbiotic, saprophytic, parasitic, as well as in free-living bacteria. The ecological 

benefit for a fish or squid living in symbiotic association with luminescent bacteria

has been established. The host organism can use light emitted by bacteria for 

attraction of prey, escape from predators or intraspecies communication. However, it 

is not understood what specific benefit fkee-living or symbiotic bacteria derive from 

producing light. On the other hand, it seems obvious that luminescence must have a 

positive selective value since as much as several per cent of the bacterial cell energy 

is consumed by this process. Some speculations on the potential biochemical role of 

bacterial luminescence were reported (e.g. that the light-emitting system could 

function as an alternative pathway for electron flow) but they have never been 

verified experimentally. 

It has been proven that on the free-living biolumi~lescent marine bacterium 

Vibrio haweyi, many mutants have been isolated. Among these, several mutants 

were very sensitive to UV irradiation. Surprisingly, most of these mutants had also 

lost the ability 10 emit light. There are possibilities that the production of internal 

light ensures effective DNA repair, most probably by photoreactivation, may be at 

Ieast one of the biological functions of bacterial luminescence. 

Marine molecules 

The marine substances are used not only as therapeutic substances but also as 

pharmacological agents and as sources of natural and modified products. Marine 

bacteria are very difficult to work with. Cephalosporins C has been extracted as the 

first marine bio - molecule. It was purified from marine fungi found in the marine 

envt of Sardinia. This molecule is being functioning as antibiotic agent. Antiviral 

molecule, called AZT isolated from thymidine extract of herring milt. The other 

active molecule is the vidarabine phosphate isolated from a sponge in 1987. 

Cyfarabine is an antitumor agent isolated from a sponge during 1976. Fish oil is rich 

in n-3 PUFA eg. DHA, EPA. It has been proven that DHA content of the animal 

brain increases or decreases according to the intake of n-3 PUFA. Dietary PUFA is 

taken up by important organelles in the brain involved in the transmission of 

information to nerve cells and for the energy metabolism and synthesis of membrane 

component essential for the growth of dendrites. Shark and dog fishes do not have a 

swim bladder, instead they do have fatty liver (> 25 % body weight) consisting of 

low density lipids such as diacyl glycerol ethers and squalene which may be

extracted from the liver along with the oil. Squaiene has the ability to regulate 

animal's buoyancy by altering the ratio of glycerol ether to triglyceride in the liver. 

Moreover due to squalene's low density, it enables to compellsate for the weight of 

shark in seawater. A health food called squalene powder is being prepared in Japan 

by supplementing proteins, carbohydrates, flavorings etc. Research has revealed that 

sharks do in need produce a germzapping chemical, which is even terribly potent 

than frog's magainin. This drug is called squalamine, which is an amino steroid, 

inhibiting the angiogenesis of tumors. This seems to explain why sharks rarely get 

cancer and contract infections. Recently, squalamine has been shown to be an 

antibiotic and anti-fungal agent of exceptional activity. Shark bile is used for 

biochemical research to some extent due to its high content of sterols and bile acids. 

A chemical derivative called Chondroitone, extracted from the shark's 

cartilaginous bones, used.as an ingredient in the eye drop preparation. Shark skin 

is rich in a calcareous deposit called shagreen. A metabolic by-product produced 

by the intestine of the blue whale is called ambergris which has wide industrial 

applications. Shark fins mostly consist of cartilaginous tissue used in making 

soup and other Chinese delicacies. Sea cucumber water is reputed to have powers 

to cure asthma, sinus problems and cuts in bums. The water is also believed to be 

good to abate internal injuries and is especially recommended for people who have 

undergone surgical operations and also consumed by women after child birth to 

tone up muscles. Ho1athurin:"A is a toxin in sea cucumber present in specialized 

tubules of the animal, having haemolytic, cytotoxic, neuromuscular and antitumour 

properties in mice. When applied in minute quantities,it is able to initiate a 

powerful contraction of rat diaphragm and frog's muscle. In cephalopods, squid 

ink is usually discarded but the peptidoglycon fraction of the ink exhibited strong 

anti tumor activity in mice. Similarly cuttle fish ink would increase immunologic 

functions of mice. Antibacterial properties from squid ink have been identified. 

Scientists of Kerala Agriculture University, isolated bioactive substances that were 

antibacterial, antiviral and anticancer agents from the ink gland of cuttlefish 

(Sepiapharaonis ehrenberg). An antitumour peptidoglycan was isolated 

from cuttlefish ink and the crude preparation showed antitumour activity against

mice following intraperitonial administration. Horse shoe crab blood contains a 

chemical called amoebocyte lysate, which is used to test sterility, pyrogenicity and 

endotoxins. It can also be used to prepare test kits to diagnose certain diseases like 

meningitis and typhoid. Lysate can be also be used to identify contaminated drugs. 

Scientists of the National Institute of Oceanography, Goa had isolated and 

characterized fluorescent dyes and fluorescent molecules from tissues of sea 

cucumber Holothuria scabra and tested the bio-activity of the compounds for 

therapeutic and industrial applications. 

The thin layer of the epidermal glands of the fish skin has numerous tubular 

or flask shaped openings capable of secreting slime or mucus which consists of 

gfycoprotein with varied gelatinous forms or mucopolysaccharide that covers most 

fishes. The fish odour present in the mucus, play an important role in chemical 

communication among the fishes. Mucus lessens the drag on a fish when it swims 

through water. In some species mucus elongates and precipitaes mud and other 

suspended solids in water. The mucus has various other functions such as decreasing 

the turbulence, drag during swimming, to protect body from microbial or parasite 

invasion, to reduce the water exchange through skin. Skin mucus coat is a physical 

and chemical barrier because it contains enzymes (1y.sozyrnes) and antibodies 

(immuoglobilins), which can kill invading bacteria or other organisms mediated both 

by immune system compounds (IgM, lysozyme etc) and by antibacterial peptides. 

Mucus acts as bandage by covering over a wound caused by trauma of infection. 

Hydrophobic components of epidermal mucus of fresh and seawater fish may exhibit 

strong antibacterial activity against bacteria. Normally any stress in fish cause 

chemical changes in mucus, which decrease its effectiveness against invading 

organisms (Ebran et a1 2000). Fish gill mucous is used as a non-destructive 

biomarker for the detection of heavy metals. 

Bacterial biomolecllle 

Pseudomoans fluorescens bacteria produce antagonistic compounds, may be 

of phenolic base exhibits antifungal, antibacterial, antihelmenthic, herbicidal and 

even phytotoxic activities (Deepti and John 2003). The abalone Haliofis midae 

ingests a diversity of algae, the digestive tract of H. midae is colonized by highly

active polysaccharolytic bacteria which assist in the digestion of seaweed ingested by 

the abalone. Exopolysaccaharide (EPS) produced by wide variety of bacteria 

including fish- borne Alcaligens faecalis are water soluble gum which can be used as 

alternative for synthetic gums due to its wide diversity. EPS have found wide range 

of applications in the food, pharmaceutical and cosmetics, oil and other industries. 

Shell fish have copper containing proteins caIled haemocyanins, has catalase 

like activity (Ghiretti 1956). Antimicrobial peptides are abundant through out the 

animal and plant Kingdom and play an important role in the innate immunity of 

living organisms, especially in organisms that lack adaptive immunity. It is now well 

estabilised that the antimicrobial peptide response is a common feature of innate 

immunity in animals. Penaeidins, a unique antimicrobial peptide family, were first 

purified from haemocytes of the shrimp Litopenaeus vannamei and their genes were 

cloned from the haemocyte cDNA of the shrimps. Penaeidins were active against 

Gram positive bacteria and also filamentous hngi (Destoumieux et a1 1997). 

Lectins have been identified in various marine invertebrates, including 

tunicates, sponges, crustaceans, and clams. The properties of lectins and their roles in 

bivalve host defence are well documented in mussels and oysters due to the 

popularity and economic value of these species. Lectin exhibits strong antibacterial 

activity against Yibrio. A biofilm consists of microbial cells and an extra biopolymer, 

these cells produce exopolysaccharide which provides structure and protection to the 

community. 

In general biofilms show an increased resistance to antimicrobial 

agents. They cause recu~ent microbial infections in humans and animals owing to 

their antibiotic resistance. Fungal biofilm also shows antifingal resistance. Due to 

this heterogenous nature of the biofilms, it is likely that there are multiple resistance 

mechanisms at work within a single community. Biofilm formation collapse 

industrial productivity in terms of forming corrosion, contamination and fouling etc. 

The microbes attached to particles of contaminated soils and aquatic sediments abets 

degrade soil bound contaminants occurring from chemical releases into the 

environment. (Gamini 2003).

Marine organisms are exposed to a wide range of toxins and contaminants 

including pesticides, antifoulants, heavy metals, and hydrocarbons. There are very 

few reliable techniques that are able to link physiological stress responses of aquatic 

organisms to conditions of water quality. Developing tools for rapid diagnosis of 

contaminants in the marine environment is essential. In Antarctica, it has been 

proved by the scientists that sea sponges act as stress indicator organisms. Sponges 

are filter feeders and are ideal for assessing environmental contaminants that enter 

the primary food chain. 

Cyanobacteria in low saline waters 

Cyanobacteria are basically microscopic organism but appear as mats. 

Benthic cyarzobacteria are abundant in mangrove environment due to rich organic 

muddy substratum and shallow water conditions with 15-30 ppt salt. Fifty eight 

species of Cyanobacteria among backwaters and mangrove habitats of East coast of 

India have been reported (Thajuddin et a1 1992). ~~anobacteria are one of the 

potential organisms useful in many ways such as food, feed, fertilizer, fuel, medicine, 

industry and in combating pollution. A cyanobacterium has novel bioactive 

compounds including toxin and pharmaceutical applications. Cyanobacterial species 

has anti-HIV activity, antiviral, antifungal and immunomodulatory activity. 

Medically important gamma linolenic acid is relatively rich in Spirulina and 

Arthrospira sp, which is converted into arachidionic acid in the human body and 

archidionic acid into prostaglandins E2. 

This compound has lowers the blood 

pressure and the contracting function of smooth muscle and thus plays an important 

role in lipid metabolism. 

Global 

A Peruvian company developed a protein based compound called protein01 

which is high in omega fatty acids used to combat malnutrition in developing 

countries. Omega fatty acids have an important role in the development and 

physiology of the human being, due to the fact that they form a part of the structure 

of the neurons, brain, retina and peripheral nerves. Protein01 is elaborated exclusively 

from the muscle of a variety of marine fishes. This product was consumed directly or 

as an ingredient in food preparation. Proteinols have a high concentration of proteins

and exhibit an excellent content of essential aminoacids. The other principal 

advantages of these fatty acids are that they help in fighting cancer, development of 

eye sight and functions of the cellular tissues, regulating blood pressure, the viscosity 

of the blood and preventing cardiovascular diseases, thrombosis, inflammations and 

arthritis etc ( Luis Felipe 2005) 

Novel Healthcare Technologies from marine bacteria 

Australian researchers have discovered novel marine-derived antioxidants 

that may have commercial application in cosmetics and food processing. Several 

indispensable compounds are being evaluated in medicine for use in the prevention 

of neurological disorders, such as Alzheimer's and Parkinson's disease. Tropical 

marine 'bacteria would hold the key for improving human health in anti-ageing 

research. There are UV-tolerant bacteria living on the surface of shallow-water corals 

that are protected by the ability to increase antioxidant enzyme when exposed to 

harmful UV rays. Finding a therapeutic means to regulate this key metabolic enzyme 

could slow the degenerative process of aging, allowing human being to survive 

healthier as they grow old. Microbial enzymes are superior in solubilising fish 

proteins, bacterial alkaliqe protease is 140 % efficient as compared with papain. 

Hale (1969) showed that hydrolysis of raw fish with B. subtilus protease at pH 8.5 

gave high yield of soluble product having an excellent balance of aminoacids. 

Bacterial protease had twice the activity at 50'~ as compared to ficin at 40' C or 

bromelain at 45'C. Alkaline protease from B. subtilus was found to be more efficient 

than two neutral protease of the same bacterium, giving more than 80 % nitrogen 

solubilisation. 

Bivalve mollusk 

The blood cells of bivalve mollusc, Scrobicularia plana, the haemocytes of 

the blood shows phagocytosis and cell free haemolymph shows antibacterial activity. 

The peptides of cell free haemolymph of the mussels M. edulis and M 

galloprovincialis showed antibacterial activity. The cell free haemolymph indicates 

that the antibacterial peptides are present in the serum, which is in agreement with 

other work on antibacterial peptides in bivalve mollusks.

Actinomycetes are important sources of bioactive compounds and more than 

two-thirds of naturally occurring antibiotics come from this single group of bacteria. 

The isolation, distribution and biodiversity of marine actinomycetes is under 

investigation to facilitate screening of these organisms for novel antibacterial, 

anticancer and antiviral compounds. A major research fo.cus is metal resistance in 

actinomycetes. Marine actinomycetes have been isolated that are highly resistant to 

toxic metals, including mercury, cadmium, cobalt and zinc. Mechanisms of 

resistance are under investigation at the molecular level, with the long-term aim of 

using these strains for bioremediation of metal-contaminated soils, waters, and 

industrial waste-streams. Molecular studies are most advanced in the case of mercury 

resistance. 

Marine biotechnology offer chances for revolutionzing human weIfare activities 

Developing bio-therapeutic compounds from marine bacteria like archea and 

using for aquaculture purpose would pave way for eliminating the use of synthetic 

chemicals which in turn gives undesirable effects to the consumers. Good source for 

developing aquaculture drugs from marine plants and seaweeds exists. This 

development would enhance research and development aktivities in the developing 

countries. Establishing bio-tech parks in India for marine resources would be good 

for national development. This provides self employment for collection of various 

marine resources like fungi, corals and other invertebrates. Marine biotechnology 

paves way for new biotechnological industries and entrepreneurs in marine 

proteomics1polysaccharides and biotechnological contract services. 

References 

Deepti., D., Johri, B.N., (2003) Antifungals from fluorescent Pseudomonads : 

Biosynthesis and regulation . Current science, 85 (12) 1693 - 1703. 

Destoumieux, D, Bulet, P, Loew, D., Dorsseluer A.V., J., Bachere, E (1997) 

Penaeidins a new family of antimicrobial peptides in the shrimp Penaeus vannamei 

(Decapods). J. Biol Chem 272 - 28.

Ebran., N JuIien, S, Orange, N Auperin, B Molle G (2000) Isolation and 

characterization of novel glycoproteins from fish epidermis mucus. Bioche. Bioplzy. 

Acta 1467 : 2: 271 - 280. 

Fragkiadakis, G.A ( 2000) Isolation of lectins from haemolymph of decapod 

crustaceans by adsorption-on forrnalinied erythrocytes. J. Biochem. Bioplzys. Methods 

44, 109- 1 14. 

Gamini, S (2003) Development of eco-friendly, beneficial microbial bio-films. 

Current Science, 85 (25) 1395-1396. 

Ghiretti, F (1956) The decomposition of hydrogen peroxide by haemocyanin and by 

its dissociation products. Archives of Bioclzenzisty and Bioplzysics 63, 165 - 176. 

Gustsfson, K.R., Cardellina, J.H., Fuller, R.W., Weislon, O.S., Kiser, R.F., and 

Snader, K.M (2003) Antiviral sulfolipids from Cyanobacteria. J. Nat Cancer Iirst. 

81, 1254. 

Hale (1969) Relative activities of commercially available enzymes in the hydrolysis 

of fish proteins. Food Techno1 23 (1) 107-1 10. 

Luis, F 0 (2005) Protein01 - a new protein product rich in omega acids. Info fish 

Interrzational. 3:49 -5 1. 

Thajuddin, N., Subramanian, G., (1992) Survey of cyanobacterial flora of the 

southern east coast of India. Current Science. Bot. Mar., 35. 305 - 3 11.

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