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Chapter - VI Changes in the Nephridial Structure and Excretory Products 111 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 112 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. 113 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. 114 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 115 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 116 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 %). 117 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 118 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 119 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. 120 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. 121 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 122 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 123 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 124 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 126 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. 127 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. 128 Fig.6.1 Excretery system of Poecilobdella viridis 129 Fig.6.2 Showing the different parts of a typical nephridium of Poecilobdella viridis 130 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. 131 132 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 133 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 134