Download 16_Chapter 4

CHAPTER - IV
PHYSIOLOGY OF
EXCRETION OF
Polypheretima elongata
79
INTRODUCTION
The diet of any animal contains multitude of organic compounds;
carbohydrates, fat and protein are among them. From these the animal builds its
own structure and obtains energy for many necessary physiological activities.
The protein is secreted from the amides formed by hydrolysis and degradation of
the proteins in the food. These are absorbed, transported to the sites utilizing
them and the characteristic proteins of the body are then formed. However as
was shown by the classic studies of Schoenheimer and Rittenberg (1963), the
body proteins are in a continual state of flux, changing constantly. Campbell
(1970) observed that nitrogenous compounds are widely distributed in the tissue
and body fluids in the form of protein nitrogen and non-protein nitrogen like urea,
free ammonia , uric acid and creatine.
The material no longer required are removed or broken down to non-toxic
units and excreted. In some cases they may be excreted after little alterations
e.g. creatinine from creatine in vertebrates, but in the majority of cases the
nitrogen fragment is converted to a non-toxic units and removed in the urine
(Andrea and Papini, 2005). If the animal has access to voluminous quantities of
water, ammonia is the most common and most easily voided excretory product
as in amphibians, fish and aquatic invertebrates. For land dwelling species water
conservation is of great importance and the production of small quantities of
urine containing high dose of nitrogenous materials is the rule. The less toxic
products like urea and uric acid make their appearance in these forms (KurtHeininger, 2000).
The nitrogenous excretion of earthworm may be considered as occurring
in two fractions, each accounting for approximately equal portion of nitrogen
removed each day. The first portion consists of a protein amounting to about
0.03mg/100ml urine/day (Bahl, 1947a) and is most probably derived from the
mucus secreted by the body. This mucus act as a lubricant as the worm
proceeds along its burrow. Mucus also helps to bind soil particles together and
preventing the burrow wall from collapsing. Mucus probably acts as a buffer
system outside the body since it is secreted in large amounts when the animal is
80
immersed in a noxious stimulant such as acid. This mucoid protein can account
for about half the total nitrogen lost each day (Needham, 1957; Haggag and ElDuweini, 1959), though this may be a reflection of somewhat unnatural
conditions encountered by the worms. The second fraction, representing the end
products of metabolism, is a fluid comprising a mixture of ammonia and urea. In
annelids the excretory processes are particularly associated with chloragogenous
tissue and nephridia (Mill, 1978).
Function of the nephridia in excretion:
The nephridia are arranged segmentally within the Oligochaete body and
good description of the various modifications and structures are available
(Stephenson, 1930; Goodrich, 1945, Bahl, 1947a). Basically the nephridium is a
tube, often open at both ends, internally and externally, and lined with cilia in
tracts along its length. There is a good blood supply to this organ. The tube is
specialized in to distinct regions and the function of these regions is partially
unknown. Many tracts that line the tubular nephridium are continually beating
such that fluid passes along the lumen from the internal end to the external end
(Mill, 1978).
It is known that the composition of the urine changes as the fluid passes
along the nephridium. Ramsay (1949) showed that the osmotic pressure
changes are mainly a function of wide tube. No work has been published to
show whether salt resorption or water secretion occurs at this stage, although the
phenomenon of granular respiration has been described by Cordier (1934). On
the basis of comparative studies on blood, coelomic fluid and urine of Pheretima
posthuma, Bahi (1947b) concluded that nephridia carryout three functions in the
excretion
of
earthworm
namely
filtration,
reabsorption
and
chemical
transformation.
Physiology of excretion:
The nephridia are abundantly supplied with blood vessels. Their gland
cells extract the excess of water and nitrogenous waste from blood. The septal
nephridia also eliminate the excretory material from the coelomic fluid through
81
nephrostomes. The excretory matter is discharged either directly to the exterior
or into the alimentary canal (Bedre, 1986).
The integumentary nephridia are termed as exonephric since they
discharge the excretory material directly to the exterior through nephridiopores.
The septal and pharyngeal nephridia, on the other hand, are the enteronephric
nephridial system, since they discharge the excretory products into the lumen of
the gut. The enteronephridia, opening into anterior part of the alimentary canal,
such as pharyngeal nephridia, are sometime called peptonephridia as they are
supposed to function like digestive glands (Morgan, 1981).
The nephridial system also serves for osmoregulation. Water from the
excretory fluid is reabsorbed through gut wall to be utilized again by the worm.
The enteronephric system thus prevents loss of water, which is very useful to
earthworm living in dry regions (Bahl, 1947b). Reabsorption of salt also occurs
during passage of excretory fluid through the nephridia. The excretory fluid of
earthworm generally does not contain uric acid. It contains about 40% urea, 20%
ammonia and 40% amino acid and other nitrogenous compounds. The stage of
the life cycle and the environment also determines the nature of excretory pattern
(Prosser and Brown, 1961). Prosser (1985) determined stage of the life cycle and
the nature of excretory pattern. Prosser (1961) reported that some extrinsic
factors like thermal variations, photoperiodically, desiccation and starvation alter
the nature of excretory products in animals.
In natural conditions earthworms are exposed to a variety of pathogenic
substances, which may cause damage, and infection of the body cavity. When
infection takes place, it is inactivated through non-specific defensive reactions,
among which phagocytes, encapsulation and nodulation are of crucial
importance (Stein and Cooper, 1981). The mechanism of phagocytes in annelids,
being a multi-phase reaction of the immune system, is triggered after
immunological recognition, affected by specialized cells, which are able to
distinguish between the components of organism and alien substances (Affar et
al., 1998). Subsequent stages of phagocytosis are chemotaxy and adhesion, the
intensity dependent on the kind and quantity of phagocyte material, and also on
82
the presence or absence of "plasma" factors. In annelids the factors aiding the
process may include agglutinins and components of activated phenoloxidase
system (Cooper, 1996). Following adhesion a phagolysosome is formed in which
a final enzymatic destruction of biotic factors takes place (Varute and More,
1972; Varute, 1972; 1973).
Some extrinsic factors like thermal variance photoperiodicity, salinity and
desiccation are also known to influence the nature of excretory products in
animals (Posser, 1984). Leverack (1963) has reported altered nitrogenous
excretory pattern from ammonotelic to ureotelic in the earthworm, Lumbricus
terrestris under the condition of starvation and aestivation. Ireland (1978)
observed heavy metal binding properties in chloragocytes of earthworm.
Affar et al. (1998) studied the isolation, purification and partial
characterization of chloragocytes of earthworm. Panda and Sahu (1999, 2000)
studied excretory activity of the worm Drawida willsi in zinc applied soil. Morgan
et al. (2002) observed earthworm excretory product under the stress of heavy
metal. Patil (2002) observed earthworm excretory products under the stress of
organic fertilizers. Morgan et al. (2005) studied metal in the chloragosomes of
Lumbricus terrestris.
A few reports on the toxicity of sodium chloride to excretory product of
temperate worm Eisenia foetida (Tillinghust, 1967, Carley, 1978, Heininger,
1999, 2000; Tribl et al., 2005) are available.
The present study is aimed to understand the effect of salinity on the
excretion of nitrogenous waste product (urea and uric acid) in a tropical
earthworm Polypheretima elongata found in Baramati area.
Excretory system of earthworm Polypheretima elongata:
Excretory system of earthworm consists of segmentaly arranged small,
coiled microscopic structure called nephridia. Such small sized nephridia are
termed micro or mero nephridia their structure and arrangement differ in different
groups of earthworms (Fig. 4.1).
83
In Polypheretima elongata, nephridia occur in the entire segment except
the first three and are distinguished in to three types such as septal nephridia,
pharyngeal nephridia and intugmentary nephridia according to their position in
their body (Fig. 4.1).
1. Septal nephridia
They are the largest and attached to both the faces of each intersegmental
septum behind the 15th segment.
a) Structure:
A typical septal nephridium consists of three main parts nephrostome, body
and terminal duct (Fig. 4.2). The nephrostome or ciliated funnel is a rounded
structure opening in the coelome by an elliptical pore surrounded by ciliated cells.
It is followed by a short narrow ciliated tube the neck, which is bent on itself. The
neck leads in to the main body of the nephridium consisting of two parts a short
straight lobe and a long spirally twisted loop with an narrow apical part. The loop
is again distinguishable in to a proximal and distal limb which is spirally twisted
upon themselves. The distal limb is joined with a straight lobe, while the proximal
limb is connected with the next neck following the ciliated funnel and is also
continued of a glandular mass transversed by a coiled tube, having four ciliated
tracts in its course, one in the neck, two in the body and one in terminal duct.
There are four parallel tubules in a straight lobe, three in the basal and two in
apical part of each limb of the twisted loop, and a single tubule in each of the
neck and terminal duct.
b) Arrangement
Each septum behind the 15th segments bears four rows of septal
nephridia, two on its anterior and two on the posterior face. Each row may
content 20-25 nephridia so that there are 80 – 100 nephridia on each septum or
in each coelomic compartment. In each segment the nephridia remains
suspended freely in the coelom while their terminal duct open into a pair of septal
excretory canals which run inward along the posterior face of the septum one on
each side parallel to the commissural vessels of their own side. The canal
discharged their contents dorsally in a pair of supra intestinal excretory ducts
84
situated side by side mid dorsally just above the intestine but beneath the dorsal
blood vessel and extending from 15th to the last body segments through narrow
ductless each having a opening (Fig. 4.2).
2) Pharyngeal nephridia –
These occurs in paired tufts or bunches, one on either side of alimentary
canal in the 4th , 5th and 6th segments. They resemble the septal nephridia in size
but are without nephridiostome. The terminal duct of the pharyngeal nephridia of
each tuft in a segment open into a common thick walled duct. Thus there are
three pairs if common pharyngeal nephridial ducts which run forward parallel to
the nerve cord. The ducts of the 4th and 5th segments open in the buccal cavity.
The pharyngeal nephridia are closely associated the e blood glands (Fig. 4.1).
3) Integumentary nephridia
These are found irregular scattered on either side of the body wall in all
segments except the first two. They are the smallest about half of the size of the
septal nephridia and most numerous, about 200 -250 in each segment. The
clitella region they are 10 times the usual number in each segment forming so to
speak ‘forests of nephridia’. The intugumentary nephridia are V shaped each with
a short straight lobe and twisted loop. Two ciliated canals run through the body.
Like pharyngeal nephridia they are closed internally having no ciliated funnels.
Their terminal ducts open to the exterior independently by separate minute
nephridiopores on the outer surface of the body wall (Fig. 4.1).
MATERIAL AND METHODS
The earthworm Polypheretima elongata having approximately equal size
and weight were collected from an upland non-irrigated field, around Baramati
area. The soil characteristics are the same as described in earlier chapter II.
Earthworms were kept half immersed in glass petriplates containing 30ml of tap
water at 25± 0.5oC for 24 hours to evacuate their guts as proposed by Dash and
Patra (1977). The study was carried out in plastic culture pots under laboratory
conditions following the protocol of Panda and Sahu (2002).
85
The experiments were carried out by using 24 h sublethal dose (1.05g/kg
soil) of sodium chloride to study their effect on excretory products. The
earthworm Polypheretima elongata were exposed for 5 days. Seven sets of
plastic culture pots (30cm, length X 20 cm, breadth X 20 cm, height each with
four replicates and 1 kg of soil) were set up. The sublethal dose of sodium
chloride was sprayed on soil surface. The treated soil was thoroughly mixed to
distribute the sodium chloride evenly and enough water was added to bring the
moisture content up to the field level. The same procedure using distilled water
was applied to prepare a set of control pots. Ten gut evacuated worms were
added in each pots. After 5 days exposure period, Polypheretima elongata were
dissected out separately and nephridia was collected in watch glasses with the
help of fine scalpel, forceps and scissors. Urea and uric acid were estimated by
using the methods described by Varley (1976).
Estimation of urea:
The urea content in the nephridia was measured by using Urease
Nesslerisation method as described by. 5% homogenate of nephridia was
prepared in cold distilled water and centrifuged at 1500 rpm for 10 minutes. To
0.2 ml of supernatant 3.2 ml of distilled water and 20 mg of soyabean meal was
added. The mixture was incubated for 15 minutes at 40-500C. 10% sodium
tungstate and 0.3 ml 2/3 N sulphuric acid were added to incubated mixture and
mixed well to remove protein. After 10 minutes the mixture was centrifuged at
1500 rpm for 10 minutes again. The clear supernatant was used for the urea
estimation. To 2 ml of supernatant 5 ml of ammonia free water of Nesslers
reagent was added. The optical density of the colour was read immediately at
480 nm in spectrophotometer against a reagent blank. The urea content was
expressed as mg of urea per 100 mg of wet tissue (mg%).
Estimation of uric acid:
10% homogenate of nephridia was prepared in distilled water and
centrifuged at 1500 rpm for 10 minutes. The uric acid content in the nephridia
was measured by using the method of Varley (1976). 0.6 ml of supernatant was
added to 5.4 ml of diluted tungstic acid and centrifuged again at 1500 rpm for 10
86
minute. To this solution 0.6 ml of sodium carbonate and 0.6 ml of diluted
phosphotungstic acid was added. After mixing well the tubes were incubated at
a room temperature for 30 minutes. The optical density of the colour was read at
700 nm in spectrophotometer against reagent blank. The uric acid content was
expressed as mg of uric acid per 100 mg of wet tissue (mg%)
Statistical analysis:
The results were expressed as mean of three replicates and data were
analysed statistically by using student ‘t ‘ test (Mungikar, 2003).
RESULTS
Analysis of the nature of nitrogenous excretory products in the nephridia of
the earthworm Polypheretima elongata has clearly evidenced that the major
excretory products are urea and uric acid. The overall effect of tested sodium
chloride at sublethal dose (1.05g/kg soil) was found to increase the urea and uric
acid levels in nephridia after exposure for 5 days (Table No. 4.1, Fig. 4.3).
In the nephridia of control worm urea was found to be 18.75 mg%
whereas sodium chloride (1.05g/kg soil) treated earthworm urea level was found
to be 24.75 mg%. The urea level increased significantly (P<0.05) by 32% after
exposed to sublethal dose of sodium chloride (Table No. 4.1, Fig. 4.3).
In the nephridia of control worm the uric acid was found to be 13.12 mg%.
whereas in sodium chloride (1.05g/kg soil) treated earthworm uric acid level was
found to be 15.75%. The uric acid level increased significantly (P<0.05) by
20.05% in the nephridia after exposed to sublethal dose of sodium chloride
(Table No. 4.1, Fig. 4.3).
DISCUSSION
Salinity is one of the important environmental factors, which has profound
effect on various physiological activities of animal including invertebrates
(Prosser, 1984). Salinity induced changes in central nervous system, respiratory
metabolism, osmoregulation etc. are well reported for other oligochaetes like
Perionyx
excavatus
(Hanumante,
1975),
Octochaetoides
sudershensis
87
(Kodarkar, 1979) and Metaphire houlleti (Bedre, 1986). Salinity is known to
produce
physiologically
the
dehydration
effect
in
many
invertebrates
(Nagabhushanam and Kulkarni, 1983).
Excretory system of annelids is composed of metanephridia. Unlike
protonephridia found in helminths, metanephridia tubules open at both the ends
into the coelom internally and to the exterior. In annelids, these excretory
processes are particularly associated with the nephridia and chloragogenous
tissue (Mann, 1962). Apart from the detailed review on nephridial physiology of
annelids by Oglesby (1969), there have been one more important and
comprehensive reviews on the structure and function of the metanephridia of the
earthworm Lumbricus rubellus by Riegal (1972). The nitrogenous wastes thus
formed are predominantly from the catabolism of three classes of metabolites:
Pyrimidines, purines and amino acids. Thus the commonly produced nitrogenous
waste products are due to the catabolism of above metabolites.
As far as excretory products are concerned the urea and uric acid were
found predominant in nephridial extract of earthworm than the amount of
ammonia. This accounts for the terrestrial habitat of Oligochaetes and it is not
unnatural. Based on the demonstration of the urea cycle enzyme in the
earthworm and appearance of labeled amidine of arginine in urea, a functional
urea cycle is generally assumed to be present in annelids (Needham, 1970).
When the volume of the water in medium is reduced the urea content of
the tissues also rise in Allolobophora caliginosa (Haggag and El-Duweini, 1959).
The importance of the shift from the ammonotelic to the uretelic mechanism
probably lies in osmoregulatory process. The availability of water in the
environment may obviously vary considerably from time to time and such
variations utilized by Oligochaetes. The changes to terrestrial habitat made by
many Oligochaetes means that water is always at a premium (Ireland, 1978).
Animals living in a great excess of water the highly soluble nitrogenous products
such as ammonia and trimethyamine oxide are very often the sole excretory
product, but with the change over the land and a decreasing volume of available
88
water, first urea and than uric acid becomes the dominant substance, each less
toxic than the proceeding one.
In the present study on Polypheretima elongata salinity found to induced
remarkable changes in the levels of nephridial urea and uric acid. In the control
worm the level of urea and uric acid is very low. Treatment of worm with sodium
chloride produced a significant increase in the urea and uric acid levels after 48h.
This might account for the physiological stress of dehydration due to stress.
Cohen and Lewis (1949) and Needham (1957) found that during drought
and starvation earthworm excrete predominantly urea, and it was established by
Campbell and Bishop (1963) and Bishop and Campbell (1965) that all the
ornithine cyclic enzymes are present in earthworms. Prento (1989) reported
distribution of arginase and other ornithine cyclic enzymes in the gut of the
earthworm Lumbricus terrestris.
Patil (2002) reported a significant increase in urea and uric acid in
earthworm Perionyx excavatus after exposing to soil loaded with fertilizers.
Morgan and Turner (2005) found decreased uric acid in Allolobophora caliginosa
probability due to least disturbance in the formation and extraction of nitrogen
products in response to stress.
Increase in urea and uric acid contents of earthworm Polypheretima
elongata exposed to sodium chloride might be to overcome the toxic stress.
Similar results were reported by some workers (Stein and Copper, 1981; Bilej,
1990; Coutak and Kalac, 1990; Ritting et al., 1996; Kalac, 2002; Kown, 2004;
Frisbie, 2004; Lampi et al., 2005; Jensen and Pedersen 2006) in different
earthworm species for different dose of sodium chloride.
It is quite possible to conclude that the untreated control earthworms are
predominately ureotelic while urea and uric acid found in sodium chloride treated
earthworm Polypheretima elongata may presumably be due to an incomplete
breakdown of uric acid derived from purine metabolism (Homa et al., 2002).
89
Table No. 4.1: Urea and uric acid level in nephridia of Polypheretima elongata
exposed to 24 h sublethal dose of sodium chloride for 5 days.
Tissue
Control
Urea
18.75 ± 0.16
Experimental
(1.05gNaCl/kg soil)
24.75 ±. 0.22
(+32%)
13.12 ± 0.12
Uric acid
15.75 ±. 0.17
(+20.05%)
Fig. 4.3: Urea and uric acid level in nephridia of Polypheretima elongata exposed
to 24 h sublethal dose of sodium chloride for 5 days.
25
20
15
(+20.05%)
(+32%)
Urea
Uric acid
%
10
5
0
Control
Experimental
90
REFERENCES
Affar, E., Dufourin, B., Pairier, G.G. and Naderud, A. (1998): Isolation ,purification
and partial characterization of chlorgocytes from the earthworm species
Lumbricus terrestris Mol. Cell Biochem. (1998). 185(1-2) 123-33.
Allan N. Schore (2001): The effect of early relational trauma on right brain
develop affect regulation and infant mental health. Infant mental health Jr. Vlo.22
issue 1-2 Pp.201-269.
Andrea, M.M. and Papini, S. (2005): Influence of soil properties on
bioaccumulation of 14-C-simszine in earthworm Eisenia foetida J.Envn. Sci.
Health. B. 40(1): 55-8.
Bahl , K.N. (1947a): studies on the structure, development and physiology of the
nephridia of the oligocheta. VIII. Biochem. Estimations of nutritive and excretory
earthworm, and the bearing on the role Quart. J.Micr. Sci. 87: 357-371.
Bahl. K. N. (1947b): Excreation in the oloigochaeta. Biol. Revs. 22: 109-147.
Bedre, R.V. (1986): Studies on some physiological aspect of Indian Earthworm.
Ph.D. thesis , Marathwada Univ. Augangabad, India.
Bide R. W., Armour S.G., Yee E. (2000): Allometric respiration body mass data
for animals to be used for estimates of unhalation toxicity to young adult humans
Jr. of Applied toxicity Vol.20, Issue Pp.273-290.
Bleij M., Scheerlick J.P., Vanderiessche T., De Baetselier, P. Vetvica V.( 1990):
the flow cytometric analysis of in Vitro phagocytic activity of earthworm
coelomocytes ( Eisenia foetida, Annelida). Cell Bio. International reports 14 (9):
832-837.
Campbell, J.W. (1970): In: comparitive Biochem. Of nitrogen metabolism Vol.1.
The invertebrates Ed. Campbell, J.W., Acad. Press, New Yrok. 207-297.
Campbell, J.W. and Bishop, S.H. (1963): Urea biosynthesis to invertebrates .
urea formation in the earthworm. Biochem. Biophys. Acta. 77,149-152.
Carway, W. T. (1963): Uric acid estimation by method of Caraway. Edited by
Varely H. Practical Clinical Biochem. Pp.205-207.
Cohen, S. and Lewis, H.B. (1949): Nitrogenous metabolosm of the earthworm
(Lumbricus terristris) Fed. Proc. 8:191.
Comp. Biochem. Physiol. 45A:607-635.
91
Copper, E. L. and Roch, P. (1996): Earthworm Immunity: A model of Immune
competence. Pedobiologia Vol., 47, Pp5-6.
Cordier, R. (1934): Etudes histophysiology sur in nephridle du cambric Arach.
Biol. 45,431-471.
Cotuk A., KalacY (1990): response of the earthworm dendroboena veneta to
bacterial infection. In:Microbiology in poechilotherms. Lesal R (Ed.) Elsevier
press,Amsterdam 245-248.
Dash, M. C. and Patra, U. C. (1977): Density, biomass and energy budget of a
tropical earthworm population from a grassland sitein Orissa, India. Rev. Ecol.
Sol.14 461- 471.
Dowden, B.F. and Benett, H.J.(1965): J.Wat. Pollut. Control Fed. 37:1308-1316.
Edward K. Tillinghast (1967): Excretory pathways of ammonia and urea in the
earthworm Lumbricus terristris L.Jr. of Expt. Zool. Vol. 166, Issue 2, 296-300.
Fingerman, M. (1984): Key note address:Pollution our enemy, All India Symp. On
physiological responses of animals to pollutants, Dec.1982, Marathwada
Univ.,Aurangabad India. Proc. Symp. Physiol.Resp. Anim. Pollut.I-VI.
Goodrich, E.S. (1945): The study of nephridia and genital ducts since 1895.
Quart. Jr. Micr. Sci., 86: 113-292.
Haggag, G. and EL-Duweini, A.K.(1959): Main nitrogenous constituents of the
excreta and tissues of earthworms. Proc.Egypt. Acad. Sci. 13:1-5.
Hannumante, M. M (1975): Some aspects of physiology of Indian earthworms.,
Ph.D. thesis , Marathwada Univ. Augangabad, India.
Homa, J., Niklinska, M., Polytecyz, B. (2002): Effect of heavy metal on the
earthworm Allolophora chlorotica Abst. Of 7th International Symp. On earthworm
ecology, Pp164-165
Ireland, M.P. and Richards, K.S. (1978): The occurrence and localization of
heavy metals and glycogen in the earthworm Lumbricus rubellus and
Dendrobaena rubida from heavy metals site Histochemistry, 51,153-166.
Jensen, J. and Pedersen,M.B.(2006): Ecological risk assessment ofcontaminated
soil Rev. Environ. Contam. Toxicol. 186:73-105.
Kodarkar, M.S. (1979):Neuroendocrinology of the earthworm, Octochaetoides
sudershensis. Ph.D. thesis , Marathwada Univ. Augangabad, India.
92
Kulkarni,V.D. (1989): Physiological studies on the earthworm Lampito mauritti
(Kingberg, 1987) in relation to the impact of some envn. Factors. Ph.D Thesis,
Marathwada Unvn. Aurangabad., M.S. India.
Kurt Heininger (2000): A unifying hypothesis of Alzheimer’s disease III Risk
factors. Human pharmoclinical clinical and expt Vol. 15, Issue I Pp. 1-70
Kwon T. H., Nielson J. Knepper M. A., Frokiaer J. Nidsens (2005): Angiotensin II
ATI receptor blackade decreases vasopression induced water reabsorption and
AQP2 Levels in Nacl restricted rats A,M.J. Physiol.
Laverack, M.S. (1963): The physiology of earthworms, Pergamon Press, Oxford.
Mann, K.H. (1962): Leeches (Hirudinea): Their structure, Physiology, Ecology
and Embryology, Pergamon Press, London 1-201.
Mann, K.H. (1962): Leeches (Hirudinea): Their structure, Physiology, Ecology
and Embryology, Pergamon Press, London 1-201.
Meinties R.A., Engelbrscht H. (2004): changes in the renal handling of urea in
ship on a low protein diet exposed to saline drinking water Jr. Vet. Res. 71 (3)
165-70.
Milli P. J. (1978): Physiology of Annelids, Academic Press, New York.
Morgan A. J. (1981): A morphological and electron microprobe study of the
inorganic composition of mineralized secretory product of the calciferous gland
and chloragogeneous. Cell Tissues Res. 220(4): 829-844.
Morgan, A. J. and Turner, M.P. (2005): Quantities ultra structure of metal
sequesting cells reflects intersite and interspecies differences in earthworm metal
burdens Envn. Cont. Toxi. 49 (1) 45-52.
Morgan, A. J., Turner, M. P. and Morgan, J. E. (2002): Morphological plasticity in
metal sequestering earthworm chlorgocytes morph metric electron microscopy
provides biomarkers of exposure in field populations. Env. Toxi. Chem.21
(3):610-8
Nagabhushanam, R.and Kulkarni, G. K. (1983): Functional correlates of
neurosecretion in the Hirudinean Annelids. J. Sci. Indus. Res. 42: 287-297.
Needham, A.E. (1957): Components of nitrogenous excreta in the earthworms
Lumbricus terristris and Eisenia foetida. J.exp. Biol. 34(4):425-426.
Needham, A.E. (1970): Nitrogen metabolism in annelids. In: Comparative
Biochem. Nitrogen metabolism Vol. 1. The invertebrates. Campbell, J.W., Ed.
Acad. Press, New York, 207-297.
93
Oglesby, L.C. (1967): Inorganic components and metabolism; ionic and osmotic
regulation; Annelida Sipuncula and Ehiura. In :Chemical Zool. (M. Florkin and
B,T. Sheer, eds) Acad. Press New York, 4:211-310.
Panda S and Sahu S. K. (1999): Effects of malathion on the growth and
reproduction of Drawida Willsi. Kriskly, N.I. (1992). Proc. Son. Exp. Biol. Med.
200: 248-254.
Panda Smeeta and Sahu Sangtak (2002): Acute toxicity assessment of three
Pesticides to the earthworm Drawida willsi. J.Ecotoxicol.Environ.Monit.12 (3)
215-223.
Patil,N.B.(2002): Impact of agrochemicals on some physiological activities of
earthworm Perionyx excavatus.Ph.D. Thesis Dr. Babasaheb Ambedkar
Marathwada Univ. Aurangabad, MS. India.
Prento,P.(1979): Distribution of arginase and other ornithine cycle enzyme in the
gut of earthworm Lumbricus terrestris L. and some physiological and
comparative implication Comp. Biochem. Physiology. 93(3):509-515.
Prosser, C.L. (1984): Comparative Animal Physiology, W.B. Saunders.
Prosser, C.L. and Brown, Jr. F.A. (1961): Comparative Animal Physiology,
Second Ed Philadelphia, Saunders.
Ramsay, J.A. (1949): The site of formation of hypotonic urine in the nephridium
of Lumbricus J.Exp. Biol., 26:65-75.
Ramsay,J.A. (1949): Osmotic relations of worms J.Exp. Boil.26(1):65-75.
Riegal, J. A. (1972): Comparative physiology of Renal Excreation. Edinburgh,
Oliver and Boyd, 204.
Ritting M. G., Kuhn K. H., Dechand C.A., Gauckler A., Modalell M. (1996):
Phagocytes from both vertebrates and invertebrates species use coiling
phagocytes.dev.comp. Immunol 20 (6):393-406.
Schoenheimer and Rittenberg (1963): cf. The Physiology of earthworms, by
M.S.Laverack, Pergamon Press, Pp.,45.
Steni, E.C. and Copper E. (1981): The role of poisioning in phagocytosis by
coelomocytes of the earthworms, Lumbricus terrestris. Dev. Comp. Immunol.
5:415-425.
94
Stephenson, J. (1930): c. f.: The Oligochaeta, Oxford Univ. Press.978, Biol.Bull.,
144:511-524.
Tribl G.G., Waldhauser F., Drumiul, Sycha T ,Endler G., Zeithhofer J., Auffer E.
(2005): Loss of normal circadian profile of urine excreation in idiopathic restless
legs syndrome sleep mod Issue (5)Pp.391.
Varley, H. (Ed.) (1976): Practical clinical Biochem, Arnol-Heimann, Publ. New
York.
Vartue, A.T. (1973): Lysosomal acids Hydrolases. In the chlorogens cells of
earthworm.
Vartue, A.T. and More, N.K. (1972): Chloragosomes
chloragogenous cells lysosomes Acta Histochem. 44:144-151.
in
earthworms
Vartue, A.T.(1972): Lysosomal enzymes in the chlorogens cells of earthworm.
Comp. Biochem. Physiol. 45A:607-635.
Warren, E.C. (1971): In: Biol. and Water Pollution Control. W. B. Saunders
company, Philadelphia, London, Torendo.
Wayne W. Carley (1978): Water economy of the earthworm Lumbricus terristris
coping with the terrestrial envi Jr. of Expt. Zool. Vol. 205, Issue I, Pp.71-78.
Yefilm Kalac, Ayten Kimiran, Gulruh Uakolu and Ayflon Coutak (2002) The role of
opsonon in phagocytosis by coelomocytes of the earthworm Dendrobaena
Veneta
**********************
95