Plasmid vectors: a brief study

Plasmid vectors: a brief study _______________________________
Presented by:
MANOJ KUMAR
SUKUMAR DANDPAT
VI YEAR__________________________________________________________________________________________________________________________

Genetic engineering stands for the transfer of DNA between hosts (or the species) by in vitro enzymatic manipulation. This means that the DNA to be transferred will be duplicated in the new host. Since most DNA fragments are incapable of self replication in E.coli or any other host cell, an additional segment of the DNA, capable of autonomous replication must be linked to the fragment to be cloned. This autonomously replicating segment is the molecular cloning vectors.
Thus vectors are self replication DNA molecules into which foreign DNA molecules are inserted (Gardner et al. 2001). Vectors have been developed and used in delivery of foreign genes into cells and chromosomes of prokaryotes and eukaryotes. Such a vector plays a central role in recombinant DNA technology.
Most cloning vectors are originally derived from naturally occurring extrachromosomal elements such as bacteriophage and pladmids.
Stanley cohen and his coworkers (1975) first reported the use of bacterial plasmids as molecular cloning vectors. Since that initial report, thousands of cloning vectors have been constructed, and their versatility in terms of cloning sites, host-range and function appears to be limited only by the deftness and imagination of inventor.
Currently specialized cloning vectors are available that enable the investigator to:
1. Identify and isolate regulatory DNA sequences such promoters and terminators;
2. Identify open translation reading frames;
3. Overproduce useful RNAs and proteins; and
4. Determine the nucleotides sequences of genes and segments of DNA.
Primarily a cloning vector is necessary as a carrier of the cloned gene. Without a vector, a DNA molecule introduced into a cell would be diluted out of the cell division and eventually get lost.
Vectors are of following types:
1. PLASMIDS,
2. BACTERIOPHAGE VECTORS,
3. COSMIDS,
4. VECTORS FOT PLANT CELLS,
5. VIRUS VECTORS FOR ANIMAL CELLS,
6. SHUTTLE VECTORS,
7. MINICHROMOSOMES (YAC VECTORS AND BAC VECTORS)
8. EXPRESSION VECTORS AND
9. GENE CARTRIDGES.
THE PRESENT LECTURE WILL BE TOTALLY CENTERED AROUND THE PLASMIDS.
PLASMIDS_____________________________________________________
Plasmids are extra-chromosomal double stranded circular DNA molecules that replicate autonomously. i.e. a plasmids is a replicon (i.e. unit of genetic material capable of independent replication) that is stably inherited (maintained without specific selection) in an extrachromosomal state. Most of the plasmids are not required for the survival of the host in which they reside. But in many cases they are essential under certain conditions, such as in the presence of an antibiotic such as chloroamphenicol, ampicillin. In rhizobium leguminosarum, the agent for nitrogen fixation and module formation are located in the plasmid.
Aspects related to the plasmids:
1. Replication of plasmids: the small sized plasmids use the DNA replicative enzymes of the host cells, while the large-sized plasmids carry genes that code for the special enzymes necessary for their replication, under certain conditions the plasmids can integrate with the bacteria chromosomes. They are called episomes or integrative plasmids.
2. Size of plasmids: the size of plasmids varies from less than 1.0 kb to more than 2000 kb. Small sized plasmids are more advantageous for the gene cloning studies.
3. Plasmid copy number: the copy number is defined as-the number of molecules of a plasmid bound in a single bacterial cell. It ranges from 1 to more than 50 per cell. This is specific for a given plasmid residing in bacteria cell.
4. Amplification of the plasmids: the process of increasing the copy number is called plasmid amplification. When the bacterial culture is at its exponential phase, the antibiotic chloramphenicol is added to the medium. This arrests chromosomal DNA replication and the cell division. The culture is then incubated to another 12 hours for the plasmid molecules to replicate (since plasmid is resistant to the chloramphenicol). This step increases the number of plasmids per cell, sometimes the plasmid copy number may reach to several thousand.

5. Types of plasmids____________________________________________
Plasmids are classified into following four types:
a) Conjugative or self transmissible plasmids: these plasmids carry a set of transfer (tra) genes that enable the plasmid to transfer from one bacterium to another. Thus tra genes promotes bacterial conjugation. Generally they are of high molecular weight andare present as 1-3 copies per cell
b) Non-conjugative plasmids: these plasmids lack the tra genes. They replicate autonomously but cannot transfer to another bacterium. They generally have low molecular wright and are present in multiple copies. i.e. 20-25 copies per cell.
c) Relaxed plasmids: these plasmids are maintained as multiple copies per cell.
d) Stringent plasmids: these plasmids have a limited number of copies per cell, typically 1-2 copies per cell.
6. Isolation of plasmid DNA: the isolation of the plasmid DNA include the following steps:
a) Growth of bacteria and plasmid amplification.
The bacterial strain containing the required plasmid is grown on LB (=lauria dertani) culture medium (it contains trypton(10 g/l), yeast extract (5g/l) and sodium chloride (10 g/l). the ph of the LB medium is adjusted 7.5. late long, the cultures ae then transferred to LB medium containing proper antibiotic with small amount of chloramphenicol for 12-16 hours of incubation.
b) Breaking of bacterial cells to release their contents.
The bacterial cell are subjected to gentle cell lysis, using lysozyme and then detergent..
c) Treatment of bacterial cells extracts to remove all components except the DNA.
This is followed by the clearing of lysate by centrifugation. Centrifugation tends to sediment high molecular weight DNA (predominantly chromosomal DNA) and cell debris, leaving the small plsmid molecules and RNA in subpernatant.
d) Separation of plasmids from the chromosomal DNA.
Undamaged plasmid is comparatively compact,since plasmid is supercoiled as a consequence of having very few turns of double helix per unit length. Further purification of plasmid is done by caesium chloride density gradient ultracentrifugation of nucleic acid preparation in the presence of the ethidium bromide. Ethidium bromide tends to cause unwinding of DNA as it binds to it and simultaneously decreasing its buoyant density. Since the supercoiled plasmid DNA can unwind to only a very limited extent , it will not bind the dye and have a higher density than the other types of the DNA (which binds to dye). Because of this density difference, plasmid DNA can be separated from other types of DNA (i.e., stained DNA ) by means of isopycnic ultracentrifuge.
7. Criteria for plasmid cloning: for the selection of the suitable cloning vector the following criteria are used
a. A plasmid vector should be small and should contribute as alittle as possible to the overall size of the recombinant molecule. Low molecular weight (small sized) plasmids are easy to handle, and do not get damaged during the process of their isolation.
b. The plasmid should have the ability to confer readily selectable markers (i.e. phenotypic traits on the host cells that can be used to distinguish the transformed cell from the non-transformed ones. (in the process of transformation the naked DNA molecules are taken up by the recipient cell)
c. Plasmid should be easily propagated in the desired host so that the number of recombinant DNA molecules can be amplified.
d. The plasmid should have a large number of copies.
e. A plasmid vector should have an additional genetic marker that can be inactivated by insertion of the foreign DNA. So that the inactivated gene may help in distinguishing the cell harbouring recombinant molecules on the basis of readily altered phenotype.
Design of the future molecular vectors_________________________________
a. The vector should contain dominant genetic marker that can be expressed in a wide range of hosts (e.g. CmR is a dominant genetic marker which does not restrict the vector to particular genetic background.
b. Portioning swquences such as par or cer should be also incorporated into the vector to ensure efficient segregation of plasmids to the daughter cells.
c. Method of controlling the copy number will greatly increase the versatility of the vector.
d. Since many recombinant DNA techniques requires single strands of DNA. A vector with single stranded synthesis capability is desirable.
Refrence: genetics by p.s. verma and v. k. agarwal

the integuments of insect: generalised study (PART 2 FINAL)

Chitin:

It is made up of long chains of acetylated glucosamine residues (N-acetylglucosamine). The adjacent chitin chains are linked to each other by β-glycosidic linkages, particularly the hydrogen bonds to form the microfibrils. It is insoluble in water, alcohol,ethers and other organic solvents. It is soluble in concentrated mineral acids

BIOSYNTHESIS OF CHITIN:

The chitin is synthesized by the insect epidermis. The chitin monomers are secreted into the apolysiat spaces, where they are polymerized to form chain by the enzyme-chitin synthetase. The chitin synthetase is controlled 20-hydroxyecdysone, it is pH and temperature dependent chemical process.

the integuments of insect: generalised study (part 1)

Integuments (part 1)

The integuments of insects like that of other animals are an outer dermo-skeletal covering of the body and are derived from the embryonic ectoderm. It is functionally a composite structure that serves as a skin, skeleton, food reservoir, and switches on post-embryonic development by undergoing moulting repeatedly.

A. Structure and chemistry:

It is basically composed of three layers; the inner basement membrane, middle epidermis, and the outer cuticle. The epidermis is commonly called as the hypodermis as it lies below the cuticle. It is thin layer of integument that is cellular, while the inner basement membrane as well as the outer cuticle, both is a cellular structure. The epidermis secretes the cuticle and the cuticle is modified; later on into various skeletal sclerities, appendages, and sensory organs and internal linings of the fore a hind gut, the tracheal system, some organs of reproduction and the exocrine glands:

1. The basement membrane: it is formed from the degenerated epidermal cells, and appears as a non living, amorphous, granular, inner lining of the integument. It separates the epidermis from the hemocoel. It is about 0.5 micron thick. The histochemical studies reveal that it is composed primarily of the neutral mucopolysaccharides. On inner surface of the basement membrane, are attached the muscles, hemocytes and oenocytes. Sometimes, the stellate tracheal cells, collagen fibrils and connective tissue fibrils are imbedded in the basement membrane.

2. The epidermis: it is a unicellular layer formed from the polygonal cells. The polygonal cells are polyploidy, possessing a large number of nucleoli. The cytoplasm is characterized by containing various types of pigment granules. The adjacent epidermal cells are held with one another by means of cytoplasmic process, the desmosomes. Each epidermal cell produces a large number of cytoplasmic processes apically, the pore canals traversing the cuticle and opening above a cuticulin layer. The epidermal cells differentiate in some regions of the integument and constitute various types of mechano and chemoreceptor organs, the dermal glands and particularly in dipteran larvae, the peristigmatic glands around the spiracles. Each dermal gland is formed by a group of three cells; the medial cell constituting the body of gland is formed by a group of three cells, the medial cell constituting the body of the gland and is termed as tormogen cells. During moulting, some epidermal cells modify in to the so called moulting glands which secretes the moulting fluid that digests the old endocuticle to ensure further growth of the new cuticle.

Sometimes, the myofibrils penetrate the epidermis from the site of muscle attachment.

3. The cuticle: the cuticle is secretory product of epidermis. It forms an outermost thick layer of the integument and determines surface pattern and physio-chemical properties of integuments.

It is differentiated into three major regions; outer epicuticle, middle exocuticle and inner endocuticle. The epicuticle is non-chitinous while the exo and the endocuticle are chitinous regions. The exo- and the endocuticles are differentiated from the initially secreted procuticle due to sclerotization or tanning that takes place on the upper region. The exocuticle, therefore represents the sclerotized or tanned and the endocuticle nonsclerotized or untanned, undifferentiated regions of the cuticle.

Besides these three static regions, two more non-static regions develop at the time of moulting. I.e. between the exo- and endocuticles as a transitional semi hard and little darkened region commonly known as the mesocuticle. The other region arises in between the endocuticle and the epidermis in the form of a membrane and is called as the ecdysial membrane or the subcuticle. These structures can be seen only during the larval and pupal moults and are completely absent from the cuticle of the adult insects. The functional significance of these regions is still obscure.

a. The epicuticle: the epicuticle is a very thin outermost layer and varies in thickness from 0.03 to 0.04 micron. It is composed of three or four superimposed layers; outer cement layer, second wax layer, third polyphenol layer and inner one cuticulin. The polyphenol layer is reported only in some insects.

1. The cement layer: it is secreted by the dermal glands or so called verson’s gland in Lepidoptera. It is composed of the lipoprotein complex resembling the natural product, shellac secreted by the lac insect, laccifer lacca. It also contains the carbohydrates like al laccose. It functions as a varnish and provides protective external surface to the integuments. It absorbs the mobile lipids that are used for sealing over the surface abrations in order to prevent water loss from the body. It serves as reservoirs for the lipids too.

2. Wax layer: prominent layer about 0.35 micron thick layer containing partially oriented wax molecules.

Composition:

Hydrocarbons: 48 t0 58 %

Fatty acids: 25 to 18 %

Esters: 9 to 11 %

Cholesterol: 2 to 3 %

Polymers: 12 to 15 %

Note: unlike the plant wax the insect wax lacks the alcohol.

It is a mono layer in solid or liquid phase and hydrophilic groups of molecules are adsorbed on cuticulin layer. It serves as a water proof layer of integument.

3. the polyphenol layer:

It is most commonly described in the blood sucking bugs. Often appears in liquid state containing various types of polyhydric phenols-homocatechol, protocatechol, dopacatechol etc...

The polyphenols are transported above the cuticulin layer by the pore canals, Secreted by the epidermal cells. At the time of sclerotization the phenols are converted into the quinone in the presence of the phenol oxidase. Quinone gets tanned first. The protein of cuticulin layer and protein of outer procuticle together forms the exocuticle.

4. The cuticulin layer: refractile amber coloured layer formed of a lipoprotein-cuticulin. It is a highly resistant to mineral acids of and most of the organic solvents. It also serves as a growth barrier and determines the surface properties of the integuments.

b. THE CHITINOUS CUTICLE:

It is composed of the exo- and the endocuticle. It is a stratified structure differentiated as an exocuticle and endocuticle. The exocuticle is darkly pigmented, hard, and sclerotized. It provides strong mechanical support to the body size due to its toughness and inelastic properties.

I. The lamellar organisation: the chitin microfibrills lie parallel to each other to form a group of layers about 20 to 25 Å thick. The orientation of the micro fibrils differs from one lamella to the other; hence they can be easily distinguished and counted in the sections. Observations of the locusts have shown that lamellar cuticle is formed during the night and the non lamellar cuticle is formed during the day time. Thus a fully formed cuticle will have alternate bands of the lamellar and non lamellar cuticle. The lamellar regions are elastic in nature hence facilitate the elasticity to the endocuticle for flexibility and stretching properties

II. The pore canals: they run throughout the cuticle. They are about 0.15 to 1.0 micron in diameter. They encase the cytoplasmic fin processes of epidermal cells. In newly synthesised cuticle the pore canals are arranged in a spiral course and contain he cytoplasmic filaments. But in the mature cuticle they become straight and contain cubicula substances. After penetrating the cuticulin layer the pore canals divide. Sometimes the site of division is separated from the pore canal by a plate called the pore plate. The pore plate separates the wax fluid-filled region from the chitin filled region. There are about 15000 canals per square mm in endocuticle of sacrophaga. And about 200000 per cuticle per square mm in periplaneta. Functions: the pore canals transport the secretions of the epidermal cells to the upper surface of the procuticle to facilitate its growth and sclerotization.

(To be continued.................)

Aim of the experiment: to determine the quality of the milk sample by the MBRT (methylene blue reductase test)

Requirements: test tubes, cotton plugs, 10 ml pipette, 1ml pipette, distilled water.
Testing sample: provided sample of the milk.
Procedure:
1. Methylene blue solution prepared by dissolving 1 mg of methylene blue in 25 ml of distilled water.
2. Three test tubes were marked as A, B, C and 10 ml of raw milk sample was taken in test tube A, B, C respectively.
3. 10 ml of methykene blue solution was added to each of samples and was mixed well.
4. The tubes were incubated in hot water bath at 37^.C for 3-6 hours.
5. The test tubes were observed for every 30 minutes for the colour change of the sample from blue to the white colour.
6. The time required for decolouration was recorded

Sample	Time of decolouration
A	180 minutes
B	25 minutes
C	360 minutes

Conclusion:
The milk sample can be classified as per their decolouration time.

Sl. No.	MBRT time	Classification of quality	Approx no. Of bacteria per ml of sample
1	1-30 minutes	Very poor quality	>2 x 1027
2	31-120 minutes	Poor quality	>4 x 106
3	121-60 minutes	Fair quality	>5 x 102
4	>360 minutes	Good quality	<5 x 102

Theory: milk is balanced diet and it contains carbohydrate, fat, minerals, vitamins and proteins. Due to its high nutrient value the milk is often susceptible for the microbial growth due to contamination. Contaminated milk sample when consumed cam spread disease like diphtheria, typhoid etc......
It is therefore essential to determine the microbial quality of milk before processing for consumption.
Rational behind technique:
The bacteria present in the milk utilise the O₂ and produce a reduced environment. Methylene blue is a colour sensitive chemical which is blue in oxidised start and colourless in reduced state. The speed of colour disappearance of MBRT is proportional to the number of bacterial present and taken as an indication of bacterial load in the milk.

                       
                               Methylene blue-----reduction---------->  methylene blue
                                          ^{(Oxidised state)                                                      (Reduced state)}

aquatic mammals

Aquatic mammals
· Mammals are primarily terrestrial.
· However some mammals are well adapted for aquqtic mode of lide.
· They are not gill breathers but take air through lungs.
· Probably they reverted to water because of extreme competition on land for food and shelter.
· Depending upon the degree of aquatic adaptations the aquatic mammals are of two types: amphibious mammals, completely aquatic mammals.
1. Amphibious mammals:
o They live on land but go into water for food and shelter.
o They show only partial aquatic adaptations such as
a. Small external years.
b. Webbed feet.
c. Flattened tails.
d. Subcutaneous fat.
Ex. Walrus, hippopotamus, beavers.
2. Completely aquatic mammals:
o The members of two orders, cetacea (whales, dolphins, porpoises) and sirenia (manatees and dugong) are essentially aquatic forms.
· Various types of aquatic adaptations in aquatic mammals: the specifications of truly aquatic mammals (cetacea and sirenia) fall into three major categories.
1. Modifications of original structures.
2. Loss of structures.
3. Development of new structures.
a. Modifications of original structures:
1. Body shape: the body shape is one of the most important adaptations to aquatic life. The fish like streamlined body offers very little resistance and allows the animal to swim rapidly through water.
2. Large size body weight: large size reduce skin friction and heat loss. The large size creates no problem for support as water offers buoyancy.
3. Flippers : fore limbs are transformed into skin covered unjointed paddles or flippers having no separate indications for fingers. Flippers can only be moved as a whole at shoulder joint only. The broad and flattened flippers serves as balancers and provide stability during swimming.
4. Hyperdactyly and hyperphalangy: extra digits and extra phalanges, upto 14 or more in some cases, serves to enlarge the surface area of flippers for great utility during swimming in water.
5. High and valvular nostrils: nostrils are placed far back on the top of head so that animal can breathe under water without raising the head much out of water. nostrils are provided with valves which can be closed while diving in warter.
6. Mammary ducts: during lactation, ducts of mammary glands dialate to form large reservoirs of milk. The milk is pumped into the mouth of young by the action of a special compressor muscles. The arrangement facilitates suckling of the young under water.
7. Oblique diaphragm: oblique diaphragm makes thoracin cavity large. This large spaces provides more apace for lungs expansions.
8. Large lungs: large lungs and unlobulated lungs provide a big volume of air to be filled in. the dorsal lung also serves as hydrostatic organ in maintaining a horizontal posture during swimming.
9. Intra narial epiglottis: elongatedtubular and intranarial epiglottis when embraced by th eosft palate provides a continuous and separate air passage, thus allowing breathing and feeding simultaneously.
10. Endoskeleton: cranium becomes small but wider to accommodate the short and wide brain. Cervical vertebrae are fused into solid bony mass because of reduced neck. Sacrum is reduced. Ribs become arched dorsally to increase thoracic cavity. Bones are light spongy and in cetacea they are filled with oil.
11. Teeth: in toothed whales, teeth are monophyodont, homodont and numerous. As many as 250 teeth may be present. The teeth are used for seizing prey, prevent its escape and swallowing it without mastication. Usually the mortality of jaw is reduced as they have no function in mastication.
b. Loss of structures: due to loss of hairs, skin surface is smooth and glistening. Only a few sensory bristles are on snout and lips in some cases. Pinnae are absent. Due to thickening and immobility skin losses its muscles and nerves. Hind limbs are only represented by button-like knobs in the foetus but disappear in adults. Pelvis is also rudimentary. Scrotal sacs are also sbsent and remains inside the abdomen.
c. Development of new structures:
1. Tail flukes: tail flukes develop lateral expansion of skin, called tail fibres. These are not supported by fin rays. They help to propel through water by up and down movements. They enable rapid return to the surface after prolonged submersion.
2. Dorsal fin: most cetacea develop an unpaired adipose dorsal fin without internal skeletal support. It serves as a rudder or keel during swimming.
3. Blubber: it is thick subcutaneous layer of fat acting as heat insulators

TO DETERMINE THE SPECIES AREA CURVE FOR SAMPLING OF POPULATION BY QUADRATE

Aim of the experiment: to determine species area curve for sampling of population by quadrate method.

Requirements: quadrate of definite size, graph sheet, pencil, scale.

Theory:The minimum size of the quadrate is equally determined by the species area curve method. The size of the quadrate is very important as too small or too large quadrate may not be representative of the community.

Procedure: the procedure is to lay a quadrate of small area in the sampling plot and the occurrence of the number of the species is observed. The plotting area is independent varieties (X-axis) and the occurrence of number of species, then plotting the area as the dependent variable (Y-axis)

The minimum quadrate size can be determined then the curve takes a horizontal shape indicating that the species number does not increase. The point where the curve flattens is joined with the X-axis. To find out the minimum area corresponding to the occurrence of maximum number of species. This method is very convenient for vegetation and analysis or plant analysis.

a.       A quadrate of definite size is thrown randomly in the area provided for sampling of the species composition at different sites, all the different types of the species and their quantity. It determines the different species concept.

b.      Calculate the total number of individuals of a species and total number of quadrate of occurrence of a species.

c.       Calculate the total species types on each quadrate i.e. quadrate no and the new types of the species to the previous no. of quadrate.

d.      Now plot the species area curve on a graph paper.

Result: the minimum of the two quadrate are required for complete sampling of the given areas curve.

Aim: estimation of protein.

Method: blank standard test.

Principle:

Protein + Cu^{2+        alkaline medium}         Cu-Protein complex

Requirements: three test tubes, micropipette-20 micro liter and 100 micro liter, calorimeter, beaker, micro pipette tips,

Reagents:

Burette reagent, protein, standard, serum, distilled water.

The reagents contain:

1.        Copper sulphate solution (12 mmol)

2.        Potassium iodide ( 20 mmol)

3.        NaOH (600 mmol)

4.        Sodium potassium tartarate (32 mmol)

Procedure:

a.        Calorimeter was switched ON and left ON for 1 hour before the experiment.

b.        Three test tubes were taken and marked as B (blank), T(test tube), S (standard).

c.        Calibration of calorimeter: calorimeter was set to zero i.e. 100% transmittance by the blank solution. Blank solution is prepared by taking 1000 micro liter burette reagent and 20 micro liter of distilled water in test tube.

d.        Now 1000 micro liter of burette reagent was taken in test tube S and T.

e.        Then 20 micro liter of protein standard was added to s marked test tube and 20 micro liter of serum was added in T marked test tube by the help of micropipette.

f.         After mixing the contents of test tube properly, the test tubes were incubated at room temperature for five minutes.

g.        Now the absorbance of T and S test tube solution was measured.

	B	S	T
BIURETE READING	1000 µl	1000 µl	1000 µl
PROTEIN STANDARD	------	20 µl	------
SERUM	------	------	20 µl
DISTILLED WATER	20 µl	------	------

Observation:

Serial number	Absorbance of S	Absorbance of T
1	0.26	0.22
2	0.29	0.27
3	0.12	0.09
4	0.21	0.19
5	0.23	0.21

Calculations:

                                                =

                                                =

                                                =4.4 mg/dl

food chain

Food chain  

Concept:

The transfer of food from plant sources through a series of organisms form a chain called food chain.

Ex: phytoplankton àzooplanktonà small fishà large fishà man

The above food chain is observed in the Indian rivers.

Ex:  grasshopperà melanopusà bufoà a rattle snake

This food chain occurs in Indian pasture

The above two examples clearly show that the base of the food chain is formed by plant( autotrophs) which are grazed by herbivore; which are predated by carnivores; which may be further predated by another higher carnivores.

Food chain relationships are very complex, as one organism may act as a food source of many other organisms and so on. For ex: grass may be eaten by cattle, grasshopper, rabbits, etc…….each of these may be eaten various other carnivores such as snake, toads, birds, or hyenas.

Thus instead of a simple food chain a complex web like structure called food web exists in the environment. Thus plant occupies the first trophic level called producers. The plant grazers occupy the second position called the primary consumers. The flesh eaters are at the third position called secondary consumers. The next trophic level is called tertiary consumer………the different trophic level are arranged in form of a pyramid.

Charles Elton the distinguished British ecologist realized in 1927 that there must be a limit to the number of links in any food chain. He surveyed many ecosystems and concluded that number of trophic levels very rarely exceeds five because during energy transfer loss of energy takes place in form of heat.

Charles Elton suggested that a small ecosystem cannot have many tertiary consumers because only a small amount of energy reaches the top trophic level.

Types of food chain:

In nature three types of food chain operates in general.

1.   Grazing food chain: the food chain which is herbivore based and the herbivores are considered important consumers is called grazing food chain. Ex: food chain found in the grasslands and the aquatic ecosystems. In grass land ecosystem or the aquatic ecosystem usually up to 50% of primary production is grazed upon by the herbivores and the remaining 50% are consumed by the decomposers as dead organic matter.

2.   Detritus food chain: in a forest ecosystem the insects are usually the dominant primary consumers but they consume less than 10% of the net primary production and the rest 90% of the total food is later on is fed by the small detritus feeding animals such as oligochaetes, and other micro organisms such as protozoa etc…….the animals consume the food by digesting it partially or fully making organic materials available for bacterial and fungal attack. The microorganisms also act as food for other animals. This type of food chain is called detritus food chain.

3.   Parasitic food chain: There must be a limit to he direst grazing of the producers. Because there out grazing may deplete or reduce their productivity therefore besides the food chain a parasitic food chain may also operate in many ecosystem, although the energy passing may be negligible. A parasitic food chain involves a HOST-PARASITE-HYPERPARASITE KINKS.

Vocalization in amphibian.

download in .pdf format

Whatever their musical qualities, vocalization are conspicuous feature of the behavior of most frogs and the toads. Although the function of frogs calls were not well understood until relatively recently most early naturalists realized that calls are given almost exclusively by males are associated with reproduction, and probably serve to attract mates.

Mechanism of sound production:

The basic mechanism of sound production in most anurans is relatively simple system. Air is forced from the lungs by the contraction of muscles in the trunk region and moves through the larynx into the buccal cavity. As the air passes through the larynx, vibrations of the vocal cords and the associated cartilage produce sound. An action of the larynx muscles shapes the sound in variety of forms.

The sound producing system involves three major functional units:

1.       The trunk muscles that powers the system.

2.       The larynx apparatus that produces the sound.

3.       The buccal cavity and the vocal sac that transmit the sound.

Bogert (1960) classified anuran calls into six categories based on the context in which they occur:

1.      Mating calls.

2.      Territorial calls.

3.      Male release calls.

4.      Female release calls.

5.      Distress calls.

6.      Warning calls.

1.       Advertisement calls: bogert used the term mating calls to describe the principal signals given by the males during the breeding seasons. These calls are now called as advertisement calls (wells 1977) because they often serve for more than one function or convey more than one message

2.       Male courtship calls: male frogs often alter vocal behavior when females are nearby , producing calls that render the male more conspicuous in a chorus

3.       Female courtship calls: some female frogs give call in response to the call of male and these are often called as reciprocation calls (littlejohn 1977) they tend to be given at very low intensity and therefore are hard to hear.

4.       Aggressive calls: many male frogs defend their calling sites and have distinctive aggressive vocalization.

5.       Release calls: male anurans usually give release calls when clasped by other males, either when the male being clasped is alone or in complexus with a female. The females that have completed oviposition also give similar calls. Usually these calls consist of a series of rapidly repeated broad spectrum notes.

6.       Distress calls, alarm calls, and defending calls: bogert used the tem distress calls to describe vocalization given by the frogs being attacked by predators. Usually these are loud screams, often with the mouth open.

Energy cost of vocalization by frogs:

Ted taigen and kent wells experimented on hyla versicolor. The rates at which individual frogs consumed oxygen is directly proportional to their rates of vocalization

At low calling rates, about 150 calls per hour, oxygen consumed was barely above resting stage. However at the highest calling rates, about 1500 calls per hour the frogs were consuming oxygen at a rate even higher than they consumed during their highest locomotors activity.

Costs and benefits of vocalization:

The vocalization of male frog is costly in two senses. The actual energy that goes into calls production can be very high and the variation in calling pattern that accompany several interactions among male frogs in a breeding chorus can increase the cost per calls.

                Another cost for the vocalization for a male frog is the risk of predation.

                A critical function of the vocal calls is the permit the female frog to locate the male frog.