Download Changes in the Nephridial Structure and Excretory

Chapter - VI
Changes in the Nephridial
Structure and Excretory
Products
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Introduction:
The nitrogen is an essential constituent of all proteins. The
overall metabolism of protein can be assessed in terms of the nitrogen
contained in it. Being a highly concentrated constituent of proteins,
nitrogen forms specialized excretory product like ammonia and urea.
Nitrogen in the food include traces of inorganic nitrogen invested in
the form of nitrates and nitrites and non protein nitrogen derived from
nucleic acid and amino acids. Nitrogenous compounds are widely
distributed in the tissue and body fluid in the form of protein nitrogen
and non-protein nitrogen like urea, free amino acids, uric acids
(Cambell, 1970). Generally aquatic animals excrete either ammonia or
urea and are transported across cell membrane by different
mechanisms having many physiological roles (Wright, 1995).
Oglesby (1969) has reviewed to correlate the morphology of
annelid nephridia with their presumed function in regulating the
composition of the body fluid and to look for analogies with the
vertebrate nephron in structures and function. Apart from the detail
review on nephridial physiology of annelids by Oglesby (1969) there
have been two more important and comprehensive reviews on the
structure and functions of the metanephridia of earthworm Lumbricus
(Reigal 1972) and leech Hirudo (Zerbst-Baroffka Haupt 1975). The
morphological and cytological features associated with different
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physiological functions of metanephridia in polychates like Neries
diversicolour (Delbos 1969) and Sabella pencillus (Koechlin 1969,
1970), in oligochactes Lumbricus terrestris (Boroffka and Haupt 1970,
Haupt 1974) and Dev (1964 a,b,c 1965a) in Hirudinaria granulosa.
Main diet of leech consists of blood having enriched protein
which during digestion is converted to amino acids and absorbed into
body. Some of these amino acids are utilized to resynthesize proteins
for body building processes and most of them are determined to be
used as a source of energy (Campbell 1970). Deamination result in
production of ammonia a substances which is poisonous and readily
diffusible so it has to be excreted quickly or change into some less
toxic substance such as urea and uric acid.
The chemical composition of the urine of Hirudo and
Hirudinaria (Dev, 1963 a, 1964 a,b,c, 1965 ) has been determined in
detail. Histological and histochemical study of excretory apparatus of
Glossoscolex paulistus (Oligochaeta) was studied by Buck (1985).
Dubale and Shah (1984) studied the histopathology of kidney of the
earthworm Pheretima elongata exposed to endosulfan and observed
that the proximal tubules were the first to be affected and that the
functional disorder originated from lesions of proximal tubules.
Chaudhari (2013) studied histological change in the liver and kidney of
Clarius batracus exposed to monochrotophos and fenvalerate.
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The present work has been planned to study the effects of
Deltamethrin and Fenvalerate on the histomorphology of nephridia and
nature of nephridial excreation of the leech Poecilobdella viridis.
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Material and Methods
The leech Poecilobdella viridis were collected from Salim Ali
sarovar near Delhigate Aurangabad. They were acclimatized to
laboratory condition at 27 + 0.50c for 10 to 12 days in plastic tub
containing sufficient quaintly of mixture of tap water and soil. Before
experimentation active and healthy looking leeches of approximately
same size (8 to 11 cm) and weight (8to 10 gm) were taken for
experimentation.
The lethal concentration (Lc50) of Deltamethrin and Fenvelerate
had been determined by static bioassay, for a period of 24 hours as
mentioned in chapter I. To ascertain the effects of pesticides on the
histomorphology of nephridia the leeches were divided into 3 groups of
10 leeches each. 2 groups were exposed separately to lethal
concentration of Deltamethrin (0.035 ppm) and Fenvalerate (1.8 ppm)
for 24 hours. The corresponding control leeches were maintained in
non contaminated freshwater for the experimental period. After
completion of the experiment the leeches were dissected and the
nephridia of both experimental and control group were fixed in
aqueous Bouins fluid separately for 24 hours. After fixation the tissues
were passed through 30% to 100% alcohol for dehydration. They were
embedded in paraffin wax (M.P. 58-600c) and serial sections were cut
at 7 µm. The sections were stained in Delafields haematoxylin and
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counter stained with eosin-Ү (Bancroft and Stevens 1982). Damage to
nephridia of treated leech were recorded by comparing the data
obtained from control.
Estimation of Ammonia:
Ammonia content in the nephridia was measured by the formal
titration method as described by Oser (1976). 10% homogenate of
nephridia was prepared in cold distilled water and centrifuged at 1500
rpm for 5 minutes. 1g of finely pulverized potassium oxalate was
added
to
5ml of supernatant.
After adding few drops of
phenolphthalein the mixture titrated against 0.1N NaOH, till pink
colour appears. To this solution 2 ml of neutral formalin solution was
added and mixed well to titrate against 0.5 N NaOH till permanent
pink colour appears. Ammonia content was expressed as mg of
Ammonia per 100 mg of wet tissue (mg %).
Estimation of urea:
The urea content in the nephridia was measured by using urease
A Nesslerisation method as described by Varley (1976). 10%
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 min at 40o-500c. 10% sodium tungstate
and 0.3 ml 2/3 N sulphuric acid were added to incubated mixture and
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mixed well. 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 or the gum
ghati solution was added in case of turbidity. 1 ml of Nessler’s 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 Caraway (1963) as
described by Varley (1976). 0.6 ml of supernatant was added to 5.4 ml
of dilute tengstic acid and centrifuged again at 1500 rpm for 10 min.
To this solution 0.6 ml of sodium carbonate and 0.6 ml of dilute
phosphotungstic acid was added. After mixing well the tubes were
incubated at 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 wet tissue (mg %).
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Observation and Results
General structure of excretory system (Fig 6.1) and details
individual nephridium (Fig 6.2):
In general a typical nephridium has more or less the shape of
horseshoe, forming the main body of nephridium Fig. 6.2. The
horseshoe proper lies in a latero ventral position between two adjacent
caeca of the crop, and is called the main lobe. The main lobe consists
of two unequal limbs an anterior gives rise to vesicle duct which opens
exterior through a rounded aperture called the nephridiopore while the
posterior limb of the main lobe forms a small stout lobe which lies, in
an anterior posterior position lies all along the main lobe and runs
forwards to the apical lobe. The initial lobe is closely associated with
and encircle the apical lobe and its backward extremity joins the main
lobe. The anterior of initial lobe runs towards the testes is sac and ends
by the side of perinephridiostomial ampulla. Within this ampulla lies
the ciliated organ (Bhatia, 1977) which corresponds to the funnel or
nephridiostome of a typical annelid nephridium (Fig 6.2).
The nephridium consists of (a) The Perinephridiostamal ampulla
(b) initial lobe (c) apical lobe (d) main lobe (e) inner lobe and (f)
terminal vesicle.
(a) The perinephridiostama ampulla: It is a compound structure
consisting of a central reservoir, the wall of which has numerous
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perforation like flame cell of helminth. Each has the appearance of
round goblets like cell and freely hang into the middle of the reservoir.
The part of cup like structure attached to the ampullar wall has round
shaped and hollow space in it. There is no ciliated structure observed in
the nephridium of Poecilobdella viridis as it is reported in Hirudinaria
granulosa (Bhatia 1977) (Fig. 6.2).
(b) The initial lobe: It is formed of single row of elongated hollow
cells place end to end by a continuous intracellular canal which gives
off several divert culie in each cell. The canal ends blindly of its inner
or testis sac end, but the other end joins the canal and canaliciuli of the
cells of the main lobe (Fig .6.2).
(c) The apical lobe: The apical lobe cells of are much larger in size
than those of the initial lobe and are with large cellular canals. Several
rows are hollow in nature with a number of intercellular canals
(Fig 6.2).
(d) The main lobe: Cells of the lobe are polyhedral and largest in the
nephridun each cell has a very narrow interacellular lumen which
repeatedly branched, thus forming capillary like canaliculi which
ultimately opens at places into the central canal (Fig. 6.2).
(e) The inner lobe: The cell are long and tubular, the lumen in each
cell is very large and makes a big excavation in the cell, leaving only a
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very thin peripheral area of cytoplasm containing a small nucleus. The
intracellular central canal is surrounded by a number of cells (Fig. 6.2).
(f) The terminal vesicle: It is a large, more or less oval sac lying
behind the rest of the nephridium and lies against the ventro lateral
surface of the body wall. It has a thin wall and is filled with a large
quantity of excretory fluid. The vesicle duct, which originates from the
lower end of the anterior limb of the main lobe, runs freely for a short
distance and opens into the wall of the vesicle. In the vesicle wall there
is a layer appears like the pile of a carpet. A canal leads from the
vesicle which perforates the body wall and opens to the exterior
through the nephridopore (Fig. 6.2).
Changes due to pesticidal effects:
Whole nephridial structure was badly damaged. The structure of
nephridial tubules was distorted and large space among tubules were
apparent (Fig.6.4) as compared to control (Fig. 6.3).
Effect of pesticides on excretory products:
Lethal concentration of Deltamethrin (0.035 ppm) produced
6.5%, 38.8%, 48.2% increase in the ammonia level (Table 6.1); 24.8%.
27.6% and 54.8% increase in the urea contents (Table 6.2) and 46%,
3.53% and 6.43% increase in the uric acid (Table 6.3) in nephridia of
leeches exposed for 6, 12, 24 hours respectively.
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Lethal concentration of Fenvalerate (1.8 ppm) produced an
increase of 8.6%. 28.8% and 45.4% in the ammonia content (Table 6.1)
4.8%, 21.9%,and 32.0% in the urea content (Table 6.2) and 1.8%,
3.2% and 32.9% in the uric acid content (table 6.3) were observed in
the nepridia of leech exposed for 6, 12, 24 hours respectively.
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Discussion
Animals belonging to different phyla have employed various
types of excretory devices for regulating their body fluid composition.
Excretory system of annelids, molluscs and arthoropods is known to be
composed of metanephridia. Unlike protonephridia as found in
helminths, metanephridial tubules open by both the ends into the
coelom internally and to the exterior. A detail structure of nephridium
of medicinal leech Hirudo medicinalis was studied earlier by Bourne
(1880) and Goodrich (1945). The structure of nepridial funnels of the
sanguivorous, Indian leech Hirudinaria granulosa and the nephridial
system of a carnivorous leech Haemopis indicus were reported by
Bhatia (1938).
Dev (1963) described the structure of nephridial system of
Hirudinaria granulosa with remarks on the nephridial microflora in the
vesicles which influences the free amino acids. Later studies of
Boroffka et.al., (1970) and Haupt (1974) revealed clearly the
ultrastructure of metanephridia of leech Hirudo medicinalis and
confirmed that such nephridia have morphological features appropriate
for filtration and resorption and secretory in function. Anatomically the
excretory system of Poecilobdella viridis is very much similar to those
of Hirudinaria granulosa and Hirudo medicinalis (Haupt, 1974,
Zerbst Boroffka and Haupt, 1975). But in Hirudinaria granulosa there
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are 17 pair of nephridia, whereas Poecilobdella viridis possesses 16
pairs of nephridia. In Hirudinaria granulose they are arranged
metamerically in segments 6 to 22, where as in Poecilobdella viridis
they are in segments 7 to 22. The histomorphological features did not
have any remarkable variations except the ciliated organ of
Hirduinaria granulosa, which is not observed in perinephridial
ampulla of Poecilobdella viridis. Instead of the ciliated organ there was
a round goblet like cell structure with hollow space in the center
attached with the ampullar wall. These globet like cell are 3 to 4 in
number in the central reservoir of perinephridiostomial ampulla.
Functions as that ciliated organs of Hirudinaria granulosa (Bhatia
1977) which revive coelomic corpuscles loaded with excretory
products. But there is no report either contradicting or supporting the
above mentioned function of goblet like cells or perinephridiostomial
ampulla of Poecilobdella viridis. Remaining all parts of the nephridia
almost resemble to the morphological and cytological features of
nephridia of Hirudinaria granulosa. The cells of initial lobe are closely
arranged in a single layer forming a central long canal which leads to
the vary proximal portion of apical lobe which is composed of number
of big cells radically arranged around a central canal. The main lobe
forms major mass of nephridium as observed in Hirudinaria granulose
(Bhatia 1977) with a number of canaliculi and the last portion of main
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lobe gives rise to the inner lobe and opens through nephridium of
Poecilobdella viridis. The nephridial cells secrete primarily urine from
the haemocoelomic fluid. Thus the urine coming from the canaliculae
of the initial lobe and the main lobe, enters the canaliculae of inner
lobe. From there it runs through the canaliculae of the apical lobe into
central canal and finally into the vesicle. While the urine passing
through the central canal its filtration and resorption can be expected
(Bhatia 1977) from the present microscopic studies it was evident that
Deltamethrin and Fenvelerate induced many histomorphological
changes in the nephridia of fresh water leech Poecilobdella viridis. The
concentration of Deltamethrin and Fenvalerate badly damaged the
whole structure of nephridia. The nephridial tubules are destroyed and
the large spaces formed among the tubules. Bhatnagar et al., (1963)
while studying the nephrotoxicity in Clarais batrachus due to
malathion have observed the shrinkage of glomeular tuft along with
prominent desquamation and degeneration of epithelial cells.
The histological disturbance in the nephridial tissue may be due
to the known action of pollutants, on the cellular osmosis, which in
turn leading cells to become inactive and subsequently to death
(Kulkarni et.al., 1979). In the present study histomorphological
changes like distorted nephridial tubules, large space among tubules in
the nephridial tissue of Poecilobdella viridis must be due to known
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action of pesticides on the cellular osmosis and the cell of the tubules
becomes inactive and finally death of cells occur.
As stated earlier the major nitrogenous excretory products like
ammonia, urea and uric acid produced in animals as the result of
protein catabolism are eliminated in different forms and proportions
(Needham 1970) and have many physiological roles including
excretion, acid base regulation, osmoregulation and buoyancy (Wright
1995). Since the leech Poecilobdella viridis is a blood sucking
ectoparasite, its diet (blood) is predominantly proteinaceous. Thus
ammonia and urea are commonly produced nitrogenous wastes perhaps
by the catabolism of its proteinaceous meal. Earlier, ammonia has been
repeatedly confirmed to be the main nitrogenous waste in the excreta
of a temperate leech Hirudo medicinalis (Przylecki 1926, Braconnier
Fayenmedndy 1933). The amount of ammonia excreted by leeches
seems to depend upon the amount of metabolic water available in
surrounding environment. Since the leeches are purinostatic (Needham
1970) the excreted ammonia must come almost entirely from amino
acid.
Arginine synthetase could not be detected in either tissue. It has
been suggested that the hirudineans appear to posses only partial urea
cycle in which they can convert arginine to ornithin and citruline, but
not citruline to arginine. Since the leech Poecilobdella viridis is blood125
sucking animal, therefore, it can be expected that ammonia is the
predominant excretory product of protein catabolism. As it is an
aquatic animal the variation in nephridial excretory products are not
purely depending on the availability or scarcity of water.
The pesticides alter the rate of biochemical reactions involved in
nitrogen metabolism and augment energy demand by entering keto
acids into glycogenesis and tricarboxylic acid cycle (Srinivas, 1994). In
the present study the nephridial ammonia excretion was found to
increase after exposure to lethal concentration of deltamethrin and
fenvalerate (Table 6.1). The increased level of ammonia might be due
to the acceleration of protein catabolism in leeches exposed to the
pesticides.
Ammonia is toxic and can not be retained in the tissue for longer
period, hence it has to be converted to less toxic substance like urea
(Wright, 1995). Present study revealed that nephridal urea content was
increased with increase in exposure period (Table 6.2) Sivaiah and
Ramanarao, (1978) while studying the effect of malathion on excretory
pattern of snail Pila globosa, have found an increase in urea contents in
the tissue.
Under the drastic condition leeches show excretion of uric acid
in traces (Bhaskarrao, 1983). In the present study increase in nephridial
uric acid content are shown in Table 6.3 which revealed that it
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increased with an increase in the period of exposure. In freshwater
leeches Poecilobdella viridis the nephridial uric acid content after
exposure to pesticide was observed first time. This fact shows that the
increased urea and presence of uric acid moieties can be expected as
the reflection of the scarcity of water endogenously, because in
pesticidal media leeches secrete a thick muscus film around their body
disturbing the cellular osmosis the other possibility of the presence of
nephridal uric acid level may be due to an incomplete purine
metabolism as suggested by Campbell (1970).
It is concluded that the untreated control leeches are
predominantly ammonotelic and ureotelic while increased ammonia
urea and uric acid found in pesticide treated leeches Poecilobdella
viridis may be due to an incomplete break down of uric acid derived
from purine metabolism. The histomorophological disturbances
observed in nephridial tissue of Poecilobdella viridis may be due to
known action of pesticides on cellular osmosis, which in turn leading
cell initially to inactive condition and subsequently to death.
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Fig 6.2
Showing the different parts of a typical
nephridium of Poecilobdella viridis.
A. L
-
Apical lobe
AMP
-
Ampulla
N T. LIM
-
Anterior Limb
IN. L.
-
Initial lobe
M. L.
-
Main lobe
N. P.
-
Nephridiopore
POST.L.
-
Posterior limb
TS
-
Testicular sac
VD
-
Vas deferens
VES
-
Vericle
VESD
-
Vesicular duct.
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Fig.6.1 Excretery system of Poecilobdella viridis
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Fig.6.2 Showing the different parts of a typical nephridium of
Poecilobdella viridis
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Fig 6.3: T.S. of the nephridium of control Poecilobdella viridis
maintained in freshwater showing the normal appearance of
tubules [T] x 200.
Fig. 6.4: T.S. of the nephridium of experimental Poecilobdella
viridis treated with pesticides note the damaged structure
with large spaces among the tubules x 200.
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Table No. 6.1
Percent change in nephridial ammonia of Poecilobdella viridis treated
with LC50 values of Deltamethrin and Fenvelerate for different time
period.
Treatment
Change after…… h
6
Control
Deltamethrin
12
24
6.72*
6.51*
5.9*
+ 0.91
+ 0.8
+ 0.8
+ 6.5%
+ 38.8%
48.2%
+ 8.6%
28.8%
45.4%
(0.035 ppm)
Fenvelerate
(1,8 ppm)
+ Standard deviation from mean
* Original values
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Table No. 6.2
Percent change in nephridial urea content of Poecilobdella viridis
exposed for different time period to lethal concentration of
Deltamethrin and Fenvelerate.
Treatment
Change after…… h
6
3.10*
12
3.15*
24
3.10*
+ 0.08
+ 0.07
+ 0.09
+ 24.8%
+ 27.6%
54.8%
+ 4.8%
21.9%
32.9%
Control
Deltamethrin
(0.035 ppm)
Fenvelerate
(1.8 ppm)
+ Standard deviation from mean
* Original values
Table No. 6.3
Percent change in nephridial uric acid content of Poecilobdella viridis
exposed for different time period to lethal concentration of
Deltamethrin and Fenvelerate.
Treatment
Change after…… h
6
2.62
12
2.72
24
2.63
+ 0.06
+ 0.08
+ 0.03
+ 1.46%
+ 3.53%
6.43%
0.4
0.47
+ 0.40
Fenvelerate
1.8%
3.20%
32.9%
(1.8 ppm)
0.08
+ 0.16
+ 0.30
Control
Deltamethrin
(0.035 ppm)
+ Standard deviation from mean
* Original values
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