Biomarkering metabolic activities of the tapeworm Khawia armeniaca (Cholodkovsky, 1915) in association to its fish host Barbus grypus (Hekle, 1843)

Article history: Received January 07, 2020 Accepted April 25, 2020 Available online November 1, 2020 The present work was aimed to study the relation between the tapeworm Khawia armeniaca infection and some metabolic extents in both the parasitic tapeworm and the parasitized fish Barbus grypus, using LDH and transaminase activities as a vital connotation. 57 adult Barbus grypus fish (Hekle fish) were hunted from Tigris river Rashidiya area -North of Mosul Iraq. The adult tapeworm K. armeniaca were collected from small intestine of the fish. Extract of Liver and intestinal tissues of the infected and uninfected fish in addition to tapeworm tissues were prepared. Some macromolecules concentrations and LD, AST and ALT activities were assayed using colorimetric methods. The results revealed that concentration of proteins, carbohydrates and lipids were lesser significantly at P≤0.05 in the intestinal tissues of infected fish 176.92μg/gm wet weight, 147.21μg/gm wet weight and 112.14 mg/dl respectively than that of uninfected fish 264.70μg/gm wet weight, 223.71μg/gm wet weight and 176.37 mg/dl respectively. Concentration of lipids in the tapeworm tissues was relatively high 130.67mg/dl. As for Liver LDH activity, it was significantly higher in the infected fish 279.90 IU/L than that of the uninfected fish 253.56 IU/L. whereas, liver ALT activity was diminished significantly at P≤0.05 in the liver of uninfected fish. On the other hands, there were no significant different in liver AST activity between the infected and the uninfected fish. There were significant differences at P≤0.05 between activities of the three enzymes in both infected and uninfected fish and tapeworm tissues. On the other hands, AST activity 35.46 IU/L was relatively higher than ALT activity 27.22 IU/L in tapeworm tissues. It is concluded that activities of liver LDH and ALT were significantly affected by intestinal tapeworm infection in Hekle fish and may considered as bioindicators for tapeworm infection in fish.


Introduction
Stress factors like some infections, toxicants, hypoxia and heavy metals pollution could be affecting enzymatic activity in animals. Thus, assessing the alteration in activity of particular enzymes is really helpful to pursue body disorders. When an organ is diseased due to the effect of intrinsic factors, enzyme activity appears to be increased or it may be inhibited due to the active site being either denatured or distorted (1)(2)(3)(4). Proteins, lipids and carbohydrates are abundant and important biomolecules in living organisms including parasites. They have important role in life maintenance in the cell (4). Some researchers were concern with studying metabolism of protein, carbohydrates, lipid and related enzymes in fish cestodes like Aisien and Ogiji those studied lipid metabolism in the tapeworm Oochoristica agamae (5), Malagón et al. those studied carbohydrate metabolism in the fish tapeworm Hysterothylacium aduncum (6), Al-Naftachi that studied protein, carbohydrate and lipids metabolism in snake, bird and fresh water fish tapeworms (7), Waghmare and Chavan those studied carbohydrate metabolites in cestode parasites of Gallus gallus domesticus (8), Dawood et al. those studied energy metabolism in the house frog cestode Ophiotaenia bofonis and the domestic pigeon tapeworm Cotugnea columbae (9), Al-Niaeemi and Dawood those studied lipids and fatty acids metabolism of Bothriocephalus acheilognathi, tapeworm of the common carp (10) and Al-Niaeemi et al. those studied proteins and carbohydrates metabolism in the tapeworm Postgangesia armata that infect Siluris glanis fish (11).
Enzymes like LDH, AST and ALT are present in most tissues and catalyze some steps in the metabolism of carbohydrates and protein; thus, the increase or decrease in their level may be sufficient to provide information of diagnostic value. Actually, alteration in such enzymes activity is necessary for maintaining equilibrium convoy stress effects like endoparasites, which may disrupt physiological and biochemical processes (12).
The level of LDH activity and the functional properties of this enzyme marking the capacity for anaerobic energy production and, thereby, the level of resistance to oxygen deficiency during hypoxia, vigorous exercise or thermal stress, LDH also serves to remove lactate during aerobic recovery, especially in tissues such as liver and heart (13). Furthermore, LDH is one of the guide that could be used to quantify early heart damage (14).
AST is found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells (15). AST catalyzes the reversible transfer of α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism.
AST plays a key role in carbohydrate and protein metabolism especially in the liver, where the higher density of the enzyme exists. This enzyme is released into the blood as the result of liver damages, so its measurement is an index in the evaluation of hepatocellular injury (12). However, they may also be elevated in other conditions such as thyroid disorders, celiac disease, and muscle disorders (16).
Alanine aminotransferase (ALT) or called glutamatepyruvate transaminase (GPT) is one of the aminotransferases (transaminases) enzymes, which catalyzes the transfer of the amino group (NH2) from alanine to α-ketoglutarate. It plays a key role in carbohydrate and protein metabolism especially in the liver, where the higher density of the enzyme exists (12).
ALT is released into the blood as the result of liver damages, so its measurement is an index in the evaluation of hepatocellular injury (17). Fluctuation of ALT levels is normal over the course of the day, and they can also increase in response to strenuous physical exercise (16). Changes in activity of LDH, AST and ALT, added to alterations in the metabolism of protein and carbohydrate were frequently used for evaluating destructive effect of some stress factors on fish tissues (18)(19)(20). Nambati et al. (21) was reported a decrease in total protein and albumin and increase in the hepatic enzymes ALT, AST, and alkaline phosphatase in fish infected with some species of protozoa.
Thus, the present study was aimed to estimate the activity of LDH and some transaminase enzymes in tissues of the intestinal tapeworm Khawia armeniaca and its host, Barbus grypus fish, then combined between enzymes activity and biomolecules level in both the host fish and the parasitic tapeworm, trying to explain relationship between the parasite and its host from the biochemical ambience.

Specimen collection
57 adult Barbus grypus fish were hunted from Tigris river in Rashidiya area in the north of Mosul in Iraq. The collection was made between November 2018 and May 2019. The hunted fish then dissected in the research lab, Biology Department, College of Science, University of Mosul. The adult tapeworm K. armeniaca were collected from the upper part of small intestine of the fish.
The collected helminthes were washed several times with PBS (pH 7.4), put in Petridis, examined under Hamilton dissecting microscope. Wet weight for each helminth was fixed. Then frozen at -18ºC. Some helminthes were prepared for classification (fixed, dehydrated, clarified and mounted by DPX). Classification was performed in Department of Biology, College of Science, University of Mosul, depending on (24). Furthermore, small intestine and liver of the infected and some uninfected Barbus grypus fish were cut, cleaned, washed with PBS pH 7.8, fresh Weight was fixed for each sample then then frozen at -18ºC for the subsequent biochemical assessments.

Preparation of worms and fish liver and intestine extracts
The worms and parts of infected and uninfected fish intestines and liver were suspended in a 0.05M Tris-HCl buffer 7.8 at a concentration of 10% wet weight/volume; then the suspension was homogenized in a tissue grinder.
Cell membranes disrupted using ultrasonic disintegration (MSE), a 12000 vibration/second for 30 second in ice-bath. Four cycles of sonication were applied with the suspension. Ultracentrifugation at 15000g/30minutes was done using MSE super speed cooled ultracentrifuge. The supernatant fraction was chosen for excessive biochemical studies (7).

Estimation of total carbohydrates
Gottschalk method (25) was used to estimate carbohydrates concentration in the worms and fish intestine extracts. Absorbance was measured at 488nm. Carbohydrate concentration were estimated depending on carbohydrate standard curve.

Estimation of total proteins
Schscterle and Pollack colorimetric method (26) was adopted to estimate concentration of protein in the worms and fish intestine and liver extracts. Absorbance was measured at 750nm. Protein slandered curve was used to estimated concentration of protein.

Estimation of total lipids
Chabrol and Chardonnet colorimetric method was used to evaluate whole lipids content (26). 20 µL of worms and fish intestine extracts was heated with concentrated sulphoric acid. Phosphovaniline indicator was added to the mixture to produce purple-red color complex. Absorbance were determined at 540nm. Whole lipids in each sample were estimated depending on the rule: Whole lipids concentration mg/100cm= sample absorbance − blank absorbance standered absorbance − blank absorbance × 500 Assay system for Lactate dehydrogenase (LDH) activity LDH activity were evaluated in K. armeniaca tissues and liver extract of the infected and uninfected B. grypus fish, depending on a method adopted by Wolf (27). LDH reduce pyruvate to lactate in the presence of NADH (cofactor). A specific analysis set, provided by Biomerierux/France was used. Estimation of LDH activity depends on the reaction between the residual pyruvate and 2,4-Dinitrophenyl hydrazine, which produce a reddish brown complex of Pyruvate hydrazone at basic media. Absorption measured at 365 nm.

Assay system for Aspartate aminotransaminase (AST)
activity of AST activity was estimated in K. armeniaca tissues and liver extract of the infected and uninfected B. grypus depending on Reitman and Frankel method (28). AST convert Aspartate to oxaloacetate. A specific analysis set, provided by Biomerierux/France was used. 2,4dinitrophenyl hydrazine (indicator) form reddish brown complex with oxaloacetate during the enzymatic reaction. Absorption of the complex can be measured by spectrophotometer at the wavelength 505nm. Concentration of AST was estimated depending on the slandered curve.

Assay system for Alanine aminotransaminase (ALT)
ALT induce conversion of alanine to pyruvate. Activity of ALT were estimated in K. armeniaca and liver extract of the infected and uninfected B. grypus fish, depending on Reitman and Frankel method (28). A specific analysis set, provided by Biomerierux/France was used. 2,4dinitrophenyl hydrazine (indicator) form reddish brown complex with oxaloacetate during the enzymatic reaction. Absorption of the complex can be measured by spectrophotometer at the wavelength 505nm. Concentration of ALT was estimated depending on the slandered curve.

Statistical analysis
ANOVA-Duncan's test was applied to find the difference in mean values between the tissues of the tapeworm, infected and non-infected fish at P≤ 0.05 significant level (29). The data were processed using Statistical package for society software (SPSS) / version 14 for Windows to analyze the data by computer.

Results
In the present study, we have attempted to assess the exchange influence between the intestinal tapeworm K. armeniaca and its host B. grypus fish considering the metabolic activity in the host and it`s parasite, employing LDH and aminotransferase enzymes as biomarkers. Table 1 showed that proteins have the highest concentration than carbohydrates and lipids in both infected and uninfected fish, were as lipids has the highest concentration than proteins and carbohydrates in K. armeniac tissues. There was significant different at P ≥ 0.05 between average concentration of proteins, carbohydrates and lipids in the intestinal tissues of uninfected fish 264.70 µg/g, 223.71µg/g and 176.37 mg/100m, respectively when compared with that of the infected fish 176.92 µg/g, 147.21 µg/g and 112.14 mg/100m, respectively. Means that presence of the intestinal tapeworm leads to lowering biomolecules concentration in the infected Barbus grypus. The significant different was observed also between the average concentration of protein, carbohydrates and lipids in the tapeworm tissues 114.38 µg/g, 72.56µg/g and 130.67 mg/100m, respectively, when compared with that of the intestinal tissues of both the infected and uninfected Barbus grypus.

Investigating lactate dehydrogenase and transaminase enzymes
In the present work, table 2 illustrated that concentration of protein and activity of ALT in the liver of uninfected B. grypus fish was significantly higher than that of infected B. grypus. On the other hands, LDH activity was higher significantly at P≤0.05 in the liver of infected fish than that of uninfected fish. No significant different were observed in the activity of AST in the liver of infected and uninfected fish. As for Concentration of protein and activity of the three enzymes LDH, AST and ALT in K. armeniaca tissues were lesser significantly at P≤0.05 than that found in both the infected and the uninfected B. grypus fish.

Discussion
In the present work, the results were coming agree with the findings of (11,30,31) in terms of lower protein concentration in infected fish, since they record a higher concentration of protein in the uninfected fish, sheep and pigeon, compared to those infected with Postgangesia armata, Moniezia expansa and Cotugnia cuneata intestinal tapeworms respectively, and also agree with (31,32) in terms of lower protein concentration in tapeworm tissue compared with their host`s tissues. The same as for Dorucu (33) who concluded that infection with Diphyllobothrium spp. were significantly decrease concentration of proteins and lipids in muscles, liver and gonads of the infected Powan fish.
As for the low carbohydrate concentration in K. armeniaca when compared with the infected and noninfected B. grypus fish, our results were consistent with those of (30,32). Irshadullah and Mustafa (34) has been verified that glycogen concentration in Clarias batrachus fish that infected with Caryophyllids parasite were lower 3-4 times than the concentration of glycogen in the uninfected fish. (30) Have been attributed the rise in glycogen concentration in Moniezia expansa that parasitize Caprahircus, to the size of the worm and its location inside the host. Nanware and Bhure (35). Also showed a difference in the glycogen amount in segment of tapeworms that are isolated from Capra hircus.
On the other hands, the present result was not agreeing with that of (9) in term of the relatively low carbohydrate concentration in K. armeniaca tissues, Dawood et al. (9) concluded that concentration of carbohydrates was higher than concentration of proteins and lipids in both Ophiotaenia bofonis, the intestine tapeworm of the house frog Bufo viridis viridis, and Cotugnea columbae, the intestinal tapeworm of domestic pigeon Columba livia domestica. Our result was not agreeing also with that of Dorucu (33) who concluded that concentration of proteins and lipids were higher in the muscle and liver tissues of Powan fish those infected with the tapeworm Diphyllobothrium spp, then that in uninfected Powan fish.
Al-kallak (36) has been referred to the high concentration of lipids and proteins compared with carbohydrates in both tapeworms, Kawia sp (parasitize Barbus fish) and Proteocephalus sp. (parasitize Silurus fish, she added that concentration of proteins in Kawia sp was higher than that in Proteocephalus sp. She has been associated between the variation in macromolecules content in the invader helminthes and the nourishment diversity of their hosts. Al-Egaidy (37) reviled that concentration of protein was higher than concentration of carbohydrates and lipids in adult Faschiola gigantica tissues. She correlated between the low concentration of lipids and the disability of these helminthes to split lipids they have been gotten from host as they grow up; besides, they are dependents highly on carbohydrate catabolism to get energy demands. Al-Niaeem and Dawood (10) stated that concentration of total lipids in the intestinal tapeworm Bothriocephalus acheilognathi was higher than that found in the intestinal tissues of the infected host, Cyprinus carpio fish. And that lipid concentration in the uninfected fish was higher than that in the infected fish. They referred to the role of the invader tapeworm in the consumption of fatty materials found in host intestine, and to the passive effect of the tapeworm on lipids metabolism in the infected fish.
Dorucu (33) explained that the main source of energy in fish are proteins and fats, unlike mammalians, in which the main source of energy is carbohydrates and fats. This may be due to type of fish nutrition, especially the adult. Fish metabolism is adaptive to the type of food added to their ability to remove nitrogenous catabolizes rapidly and continuously, as well as, the activity of lysosomes enzymes is faster in fish compared to mammalians (31). This may explain the higher concentration of proteins and lipids in B. grypus intestine. Regarding tapeworm protein content, Hassan and Hashim (32) revealed that tapeworms secrete protease from their tegument, which decompose surrounding host proteins, producing amino acids that are absorbed subsequently by worm tegument, then used by the tapeworm for subsequent protein synthesis.
As for lipid concentration in K. armeniaca, our result consists with that of Biswal et al. (31) those reported that concentration of total lipids in intestine of the infected fish was lesser than that of the invading intestinal tapeworm. So as Hassan and Hashim (32) those concluded that concentration of total lipids in the tapeworms Davainea shindei, Lytocestus sp. and Postgangesia inarmata were higher than that in their hosts Gallus gallus domesticus, Clarias batrachus and Silurus triostegus (fish) respectively. Furthermore, tapeworms have no De novo pathway for fatty acids metabolism, and thus absorb fatty acids from host intestine in a relatively high amounts (10). This may explain the low concentration of lipids in the intestine of the invaded fish.
Our result was coming agree with that of Hel et al. (38) in term of the relatively high LDH activity in liver tissues of the infected fish. She recorded an increase in blood glucose and LDH activity in tissues of Tilapia fish those infected with metacercariae of the Pyigidiopsis summa and Geneta cestodes. Hel et al. (38) correlated between the increment in LDH activity in the infected fish with the elevation in anaerobic catabolism of blood glucose as a result of liver and muscle tissues damage.
As for the relatively high LDH activity in K. armeneaca tissues, our result consisted with that of (8) who correlate the high activity of LDH in the intestinal tapeworm C. dignopora of Gallus gallus domesticus, with the role of this enzyme in energy supply and in the preservation of the cytoplasmic redox state in tapeworm tissues. Al-Egaidy (37) combined the relatively high activity of LDH (28.66 µ mol/min/mg protein) in Fasciola gigantica tissues with the adaptation of such parasitic trematodes to anaerobic respiration. Dawood et al. (10) referred to the variation and relatively high activity of LDH in the tissues of Ophiotaenia bofonis, the intestine tapeworm of the house frog Bufo viridis viridis, and Cotugnea columbae, the intestinal tapeworm of domestic pigeon Columba livia domestica, they concluded that the variation in level of enzyme activity in the two tapeworms due to the variation in the metabolic rates of their hosts especially during proliferation and activity seasons.
Gluconeogenesis is almost occurring in hepatocytes and in kidney cortex at less extent. it is elevated under certain conditions like starvation, fasting, exercise, stress and in case of some diseases like diabetes and some infectious disease (39). The case that lead to elevation in activity of some enzymes especially the cytosolic enzyme LDH (16). Akinrotim et al. (40) revealed that the elevation of LDH activity under stress conditions (like endoparasites infestation) provides the oxaloacetate that is required for the gluconeogensis pathway to meet the additional supply of glucose for the production of energy under reduced phase of oxidative metabolism. Bao et al. (41) clarified that Sub lethal concentration of copper chloride resulted in significant elevation of LDH activity in muscle and hepatopancreas tissues of the crab Sesarma quadratum. They concluded that the increase in LDH activity may reflect an increased dependence on anaerobic carbohydrate metabolism by the muscle, gills and hepatopancreatic tissues upon exposer to such a toxicant (copper chloride).
In the present work, liver ALT of the infected fish was significantly lesser than that of the uninfected fish, this result was agreeing with Amni et al. (42) who concluded that specific activity of ALT in Fasciola hepatica tissues was higher than that in liver tissues of the infected sheep. But not agree with Ekanem and Yusuf (43) who reported that AST activity was significantly lower in liver of rat infected with Trypanosoma brucei than that of uninfected rats, while there was no significant difference in ALT activity in liver of both infected and uninfected rats.
Our result was not consisted with Al-Naftachi (7) who concluded that ALT activity was approximate in the four cestodes: Bothriocephalus spp. in Heckel fish; Ophiotaenia europaea in snake; Raillietina echinobothrida in gull and Moniezia expansa in sheep.
We hypothesize that the infected fish B. grypus has been stressed and exposed to protein, carbohydrate and lipids deficit in intestinal tissues upon the tapeworm infestation. Such stress factors added to hunger and other factors like tapeworm toxic exudates could change membranes permeability toward membrane bound enzymes like SDH and mitochondrial ALT (41,44). Furthermore, famishing conditions may lead to hypoglycemia and increased NADH/NAD+ ratio and thus increase dependent on gluconeogenesis to maintain blood glucose levels in the infected fish. Alanine and lactate are the major gluconeogenic precursors that enter gluconeogenesis as pyruvate. The high NADH/NAD+ ratio shifts the lactate dehydrogenase equilibrium to lactate, increasing LDH activity. So that, less pyruvate can be formed and therefore, gluconeogenesis is impaired, lead to lessening ALT activity and lowering glucose concentration (45). This explain the low concentration of glucose in intestinal tissues of the infected B. grypus fish that was synchronized with the high LDH and low ALT activity in liver tissues.
On the other hands, Al-Kaabi et al. (46) reviled that B. grypus is omnivores fish, but the adult B. grypus. is incline to predation rather than herbal nourishment. This may explain the relatively higher concentration of both protein and lipids in the tissues of the invading tapeworm K. armeniaca when compared with carbohydrates. At the same time, parasites those living in anaerobic or semi-anaerobic habitat's like intestinal tapeworms, gain their energy almost exclusively through the fermentation of carbohydrate because it is a much better substratum for gaining anaerobic energy than either protein or fat (8). The cytochrome chain in such helminthes is often demand a modified reduction in TCA cycle, the state that demand active LDH enzyme to provide energy and eventually resulted in a low ATP production/mole glucose catalyzed (47). The state that often compensated by high rates of glucose utilization in the parasitic helminthes. And since that tapeworm have no digestive system and get their nutritional demands from host body (48), thus may lead to decrease concentration of these macromolecules in the intestine of the invaded B. grypus.
On the other hands, LDH acts as a pivotal enzyme between the glycolytic pathway and the tricarboxylic acid cycle. Thus, in hypoxic environment (like intestinal lumen), some mitochondrial bound enzymes such as SDH, mitochondrial ALT will have inhibited with simultaneous elevation of LDH. Such condition suggests a bias towards the anaerobic glycolytic pathway (41). The state that may explain a relatively higher LDH activity and lesser carbohydrate concentration and ALT activity in tissues of K. armeniaca tapeworm.

Conclusion
Increase LDH activity and decrease ALT activity in tissues of K. armeniaca tapeworm are related to the anaerobic respiration of the tapeworm. Decrease concentration of total proteins, lipids and carbohydrates in the intestinal tissues of the infected fish is associated with the presence of the intestinal tapeworm K. armeniaca, and so as the diminution in ALT activity and increase LDH activity in the liver cells of the infected fish. This report suggests that LDH and ALT activity in B. grypus. liver is a sensitive index to measure the influence of intrinsic factors such as endoparasites invasion.