ISSN 1062-3590, Biology Bulletin, 2007, Vol. 34, No. 5, pp. 468–473. © Pleiades Publishing, Inc., 2007. Original Russian Text © M.M. Kuklina, V.V. Kuklin, 2007, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2007, No. 5, pp. 563–569.
ZOOLOGY
Study of Protein Metabolism of Herring Gulls (Larus argentatus Pontop.) Infected by Trematode Himasthla larina (Trematoda: Echinostomatidae) M. M. Kuklina and V. V. Kuklin Murmansk Marine Biological Institute, Kola Research Center, Russian Academy of Sciences, Vladimirskaya ul. 17, Murmansk, 189010 Russia e-mail:
[email protected] Received July 17, 2006
Abstract—The values and dynamics of some indices of protein metabolism were studied in herring gulls Larus argentatus infected with trematode Himasthla larina in natural populations and in experiment. These indices were compared in infected and uninfected birds. Trematode infection considerably affected host protein metabolism irrespective of the age; however, the changes were more pronounced in nestlings. Increased concentration of γ-globulins, modified albumin, and circulating immune complexes was observed in plasma of infected herring gulls. The experiments demonstrated the most significant changes in protein metabolism of herring gulls 8–11 days after infection with trematode H. larina. DOI: 10.1134/S1062359007050081
Many problems of helminth impact on the body of animal hosts in natural populations remain underexplored. This is also true for marine birds. Some negative impact of parasites on the hosts is assumed a priori; however, little valid data are available on its physiological mechanism. This problem of scientific and practical importance attracted the attention of researchers long ago. There are numerous publications on the impact of helminths on the host organism (Anikieva et al., 1988; Pronina and Pronin, 1988; Sidorov et al., 1989; Izvekova, 1991, 2001; Moskwa et al., 1998; Lunza-Lyskov et al., 2000; Silkina and Mikryakov, 2005a, 2005b; etc.). However, most of these studies were carried out on fishes and domestic or laboratory animals. Little valid data are available on the impact of parasitic worms on the body of marine birds under natural conditions. The goal of this work was to study the impact of infection with trematode Himasthla larina on some indices of protein metabolism in herring gull Larus argentatus Pontop. under natural and experimental conditions. MATERIALS AND METHODS The material was collected during expeditions along the coast of the Kola Peninsula (the Saida Bay of the Kola Bay and the Dal’nezelenetskaya Bay in the East Murman) in June–July 1999–2005. In total, 27 herring gulls were studied including 14 mature and 13 juvenile birds.
Trematodes Himasthla larina (Trematoda: Echinostomatidae) complete their development largely in herring gulls (Galaktionov et al., 1997; Ishkulov and Kuklin, 1998). The extensiveness of herring gull infection with this trematode in the Barents Sea region is 21.2%; the abundance index, 194.4; and intensity of infection, from 3 to 2576 (Kuklin and Kuklina, 2005). The life cycle of H. larina involves three hosts. Littoral gastropods Littorina saxatilis and L. obtusata are the first intermediate hosts, while bivalve Mytilus edulis is the second intermediate host. Experimental studies were carried out at the research center of the Murmansk Marine Biological Institute (Kola Research Center, Russian Academy of Sciences) in Gadzhievo Town (Saida Bay) in July– August 2003. Nestlings of herring gull (n = 10) were collected on the neighboring islands. The experiment lasted 28 days. In the first 2 weeks, the birds were handreared and dewormed (piperazine adipate and alga Fucus vesiculosus) (Dubinina, 1950; Krotov, 1970; Hoppe, 1979). Experimental nestlings (n = 5) were infected on experimental day 14 by feeding mussels with 12, 15, 21, 24, or 30 infective metacercariae of H. larina. Mussels were collected in the littoral zone of the Saida Bay. Preliminary, 100 mussels from this region were studied and the extensiveness and intensity of their infection with H. larina metacercaria were determined (93.0% and 1–42, respectively). Birds were fed the same diet twice a day. Blood plasma of birds was used for biochemical tests. Blood was taken from experimental nestlings 1 day prior to infection and after 4, 8, 11, and 14 days,
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Table 1. Biochemical indices of protein metabolism in plasma of herring gulls infected with trematode Himasthla larina Adult birds
Index
uninfected n = 7 44.6 ± 3.3 24.3 ± 1.5 5.5 ± 0.3 7.3 ± 0.8 7.5 ± 0.8 22.8 ± 1.1 39.9 ± 4.1 0.58 ± 0.05 1.0 ± 0.08 1.7 ± 0.1 2.8 ± 0.3 0.78 ± 0.1 57.5 ± 3.7
Total protein, g/l Albumin, g/l α-Globulins, g/l β-Globulins, g/l γ-Globulins, g/l Modified albumin, % of total albumin CIC, OD ALT activity, mmol/(h l) AST activity, mmol/(h l) De Ritis coefficient Urea, mM Uric acid, mM Creatinine, µM
Juvenile birds
infected n = 7 40.2 ± 3.3 18.4 ± 1.2* 5.4 ± 0.5 6.7 ± 0.7 9.7 ± 0.9* 35.6 ± 2.9* 66.6 ± 9.7* 0.7 ± 0.13 1.4 ± 0.26* 2.0 ± 0.19 3.2 ± 0.3 1.0 ± 0.1 41.1 ± 5.2
uninfected n = 7 32.3 ± 2.4 20.1 ± 1.4 3.5 ± 0.3 4.5 ± 0.8 4.2 ± 0.5 22.6 ± 0.7 64.8 ± 2.0 0.59 ± 0.04 0.92 ± 0.07 1.6 ± 0.1 3.7 ± 0.3 0.8 ± 0.06 50.3 ± 4.5
infected n = 6 35.5 ± 2.2 17.3 ± 1.2* 4.5 ± 0.3* 4.8 ± 0.5 8.9 ± 0.4* 38.2 ± 2.1* 114 ± 11* 0.67 ± 0.1 1.0 ± 0.3 1.5 ± 0.18 3.5 ± 0.3 1.2 ± 0.1* 60.7 ± 4.2
Note: * Differences from control (uninfected birds) significant at p < 0.05 (for Tables 1–3).
while single blood samples were taken in the study of the natural bird populations. Blood was collected into sodium heparin tubes and plasma was separated by centrifugation at 3000 rpm for 20 min. The plasma samples were frozen until laboratory analysis. Biochemical tests were carried out using the standard techniques. Protein concentration was determined by biuret assay, while the content of protein fractions was determined by electrophoresis in paper (Kamyshnikov, 2000). The level of modified albumin was determined by the method of Troitskii (1986). Circulating immune complexes (CICs) were assayed by polyethylene glycol precipitation (Laboratornye metody…, 1987). The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined by the method of Reitman and Frankel (Kamyshnikov, 2000). In addition, de Ritis coefficient (the AST/ALT ratio) was calculated. Uric acid was assayed by the method of Muller and Seifert; urea, by the diacetyl monoxime reaction; and creatinine, by the Jaffe reaction (Kamishnikov, 2000). At the time of blood withdrawal, herring gulls were submitted to parasitological analysis including the taxonomic identification of the revealed helminthes and evaluation of the intensity of birth infection. The birds infected with H. larina alone were used in the studies. Later, the results of biochemical tests were compared with the parasitological dissection data. The significance of differences between compared biochemical indices was evaluated using Student’s t-test (Matyushichev, 1990). BIOLOGY BULLETIN
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RESULTS The results of parasitological analysis allowed as to divide herring gulls from natural populations into four groups: I, adult uninfected birds; II, adult birds infected with H. larina (infection intensity: 3–97); III, juvenile uninfected birds; and IV, juvenile birds infected with H. larina (infection intensity: 14–99). A uniform trend of changes in the blood protein pattern was observed in the plasma of infected juvenile and adult gulls: albumin concentration decreased (by 24.3 and 13.9%, respectively) while that of γ-globulins increased (by 29.3 and 112%, respectively) (p < 0.05) (Table 1). The modified albumin level increased 1.6and 1.7-fold in the plasma of adult and juvenile infected gulls, respectively (p < 0.05). The juvenile gulls infected with echinostomatids demonstrated a 28.6% increase in the concentration of α-globulins (p < 0.05). An increased CIC concentration was demonstrated in the plasma of juvenile (1.8-fold) and adult (1.7-fold) gulls relative to uninfected birds (p < 0.05) (Table 1). Adult infected gulls demonstrated a 1.4-fold increase in plasma AST activity (p < 0.05) (Table 1). ALT activity and de Ritis coefficient did not significantly differ from those in intact adult animals. ALT and AST activities as well as de Ritis coefficient were similar in trematode-infected and uninfected juvenile herring gulls. According to our data, the levels of the final products of protein metabolism, urea and creatinine, were similar in the plasma of infected and uninfected juvenile and adult gulls (Table 1). At the same time, echinostomatid infection increased the uric acid level in both adult (1.3-fold, difference insignificant) and juvenile birds (1.5-fold, p < 0.05).
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Table 2. Biochemical indices of protein metabolism in plasma of herring gull nestlings after experimental infection with trematode H. larina Duration of experiment, days Index
Variant
Total protein, g/l
Experiment Control Albumin, g/l Experiment Control α-Globulins, g/l Experiment Control β-Globulins, g/l Experiment Control γ-Globulins, g/l Experiment Control Modified albumin, % of total Experiment albumin Control CIC, OD Experiment Control
1
4
8
11
14
29.3 ± 0.6 29.4 ± 0.4 15.4 ± 0.2 16.2 ± 0.15 3.7 ± 0.2 3.6 ± 0.1 7.3 ± 0.5 6.7 ± 0.3 2.9 ± 0.2 2.9 ± 0.1 27.1 ± 2.4 25.9 ± 1.8 71.6 ± 5.6 63.3 ± 5.1
31.9 ± 0.4 30.0 ± 0.3 14.8 ± 0.7 16.3 ± 0.2 5.3 ± 0.3* 4.1 ± 0.2 9.3 ± 0.2* 6.4 ± 0.4 3.4 ± 0.4 3.2 ± 0.2 49.4 ± 1.9* 26.5 ± 2.1 97 ± 10* 60.6 ± 5.8
31.9 ± 0.6 30.9 ± 0.3 15.4 ± 0.5 17.2 ± 0.2 4.1 ± 0.5 3.6 ± 0.1 9.3 ± 0.7* 7.1 ± 0.4 3.1 ± 0.4 3.0 ± 0.2 49.5 ± 5.2* 22.3 ± 2.0 113.0 ± 9.8* 65.2 ± 4.7
33.9 ± 0.6 30.5 ± 0.3 13.1 ± 0.9* 16.8 ± 0.2 5.9 ± 0.5* 3.8 ± 0.2 11.1 ± 0.6* 7.0 ± 0.2 3.7 ± 0.3* 2.9 ± 0.2 45.7 ± 7.5* 23.0 ± 2.2 134 ± 11* 62.8 ± 5.7
34.9 ± 0.5 31.4 ± 0.3 16.0 ± 1.0 17.5 ± 0.2 4.4 ± 0.4 4.0 ± 0.1 11.3 ± 0.7* 7.1 ± 0.4 3.2 ± 0.2 2.8 ± 0.2 40.0 ± 2.4* 22.1 ± 2.0 144 ± 12* 70.0 ± 6.7
Table 3. ALT and AST activities and levels of non-protein nitrogen components of plasma in herring gull nestlings after experimental infection with trematode H. larina Index ALT activity, mmol/(h l) AST activity, mmol/(h l) De Ritis coefficient Urea, mM Uric acid, mM Creatinine, µM
Variant Experiment Control Experiment Control Experiment Control Experiment Control Experiment Control Experiment Control
Duration of experiment, days 1
4
8
11
14
0.96 ± 0.1 0.53 ± 0.04 1.28 ± 0.1 0.85 ± 0.04 1.3 ± 0.1 1.6 ± 0.1 3.6 ± 0.3 3.1 ± 0.3 0.56 ± 0.04 0.55 ± 0.1 41.0 ± 4.2 36.8 ± 2.7
1.02 ± 0.1 0.58 ± 0.04 1.4 ± 0.1 0.87 ± 0.05 1.4 ± 0.1 1.5 ± 0.1 3.6 ± 0.4 3.2 ± 0.3 0.64 ± 0.06 0.61 ± 0.06 40.1 ± 3.9 38.4 ± 1.9
1.3 ± 0.1* 0.57 ± 0.04 1.3 ± 0.1 0.91 ± 0.06 1.0 ± 0.1 1.6 ± 0.1 2.8 ± 0.2* 3.0 ± 0.3 0.69 ± 0.1 0.73 ± 0.03 46.1 ± 2.1 45.5 ± 5.1
1.6 ± 0.1* 0.6 ± 0.04 1.3 ± 0.1 0.84 ± 0.05 0.8 ± 0.1* 1.4 ± 0.1 2.8 ± 0.2 * 3.4 ± 0.3 1.4 ± 0.1* 0.78 ± 0.1 35.6 ± 3.8 43.3 ± 3.4
1.2 ± 0.1 0.59 ± 0.04 1.0 ± 0.1 0.83 ± 0.05 0.8 ± 0.1* 1.5 ± 0.1 3.0 ± 0.3 2.9 ± 0.2 1.0 ± 0.08 0.89 ± 0.07 27.4 ± 2.4 42.3 ± 2.9
After experimental infection, mature H. larina were detected in all experimental nestlings; the intensity of infection was 26–97. The changes in protein metabolism of infected nestlings were revealed 4 days after experimental infection. Increased concentrations of α-globulins (by 43.2%), β-globulins (by 27.4%), modified albumin (1.8-fold), and CICs (1.3-fold) were observed (p < 0.05) (Table 2). After 8 days, the concentration of α-globulins slightly decreased, the levels of modified albumin and
β-globulins remained unaltered, while CIC concentration conversely increased 1.6-fold (p < 0.05) (Table 2). At the same time, ALT activity increased 1.3-fold, while the urea concentration conversely decreased by 22.2% relative to baseline (p < 0.05) (Table 3). After 11 days, considerable changes in the protein pattern were observed in the plasma of infected nestlings: the albumin level decreased (by 15.0%) while the concentrations of α-globulins, β-globulins, and γ-globulins increased (by 59.4, 52.0, and 27.6%, respectively) BIOLOGY BULLETIN
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(p < 0.05) (Table 2). Further 1.7-fold increase in ALT activity decreased the de Ritis coefficient 1.75-fold (p < 0.05) (Table 3). The CIC level on day 11 of the experiment increased 1.9-fold relative to baseline (p < 0.05) (Table 2). At the same time, the concentration of uric acid in the plasma of experimental nestlings increased 1.5-fold (p < 0.05) (Table 3). After 14 days, most plasma indices of experimental birds restored to baseline values. This applies to the levels of albumin, α-globulins, γ-globulins, and uric acid (Tables 2 and 3). At the same time, the concentrations of CIC and β-globulins considerably increased (twofold and by 54.5%, respectively) (p < 0.05). Note that no external signs of helminth infection such as torpor, intestinal disorders, and high or low appetite were observed in experimental nestlings throughout the experiment. DISCUSSION A typical pattern of changes in the protein composition of plasma is observed in herring gulls infected with trematode H. larina including hypoalbuminemia, hyper-γ-globulinemia, and, in juvenile gulls, hyper-αglobulinemia. Similar data were obtained in studies of diphyllobothriasis in Arctic fox and gray hamster, hymenolepiasis and schistosomiasis in mouse, and echinococcosis in sheep (Krasov, 1969; Zul’karnaev, 1975; Shimoda et al., 1984; Anikieva et al., 1988; etc.). It was proposed that low albumin levels in plasma can be due to suppressed function of the liver (where this protein is largely synthesized) by toxic metabolites of parasites (Anikieva et al., 1988; Mazur and Pronin, 2006). Changes in the protein pattern of blood can be due to the effect of “nutrients withdrawal” (Ginetsinskaya and Dobrovol’skii, 1978). This can also be due to the advantages for parasitic helminths resulting from accelerated nutrient transport as repeatedly demonstrated for fish–tapeworm pairs (Izvekova, 1991; Kurovskaya, 1991; Kuz’mina et al., 2000). It is worth to note high γ-globulin levels in the plasma of herring gulls infected with H. larina. According to published data (Krasov, 1969; Leikina, 1976; Anikieva et al., 1988; Leutskaya, 1990; Mazur and Pronin, 2006), γ-globulins play a great role in the body’s immune response to helminthiasis. One can propose that toxins and metabolites released by trematode H. larina induce antibody synthesis and general immune response in the host body. It is clearly worth to note a considerably increased level of modified albumin in the plasma of infected herring gulls irrespective of the host age. This can be attributed to changed functional properties of this transport protein induced by excessive metabolites appeared through digestive defects and deficient intestinal absorption in herring gulls after infection with H. larina. BIOLOGY BULLETIN
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As demonstrated previously, the pathological process in helminthiases is accompanied by the formation of CICs possibly containing antibodies (Konstantinova et al., 1998). The main CIC function is to remove foreign antigens from the host’s body. At the same time, CICs often play a significant role in the pathogenesis of helminthiases (in particular, opisthorchiasis and toxocariasis) and the period of their circulation in the host’s body correlates with the period and severity of the pathology (Beklemishev, 1986; Konstantinova et al., 1998). Apparently, CICs recruit eosinophils to the focus, which influence the functions of T and B lymphocytes and macrophages and form the pathological symptom complex (Konstantinova et al., 1998). Gull yearlings proved most susceptible to helminth infection. Infection of juvenile birds at this age with H. larina had a pronounced clinical form: considerably increased levels of α-globulins, γ-globulins, and CICs and decreased albumin level. Juvenile birds have underdeveloped immune system and are much more susceptible to the effect of parasitic antigens and toxins. High levels of the α-globulin fraction can indicate allergic reaction to parasites and stress in juvenile organisms after trematode infection. Although trematode H. larina is parasitic in the digestive tract of birds, numerous studies demonstrated considerable functional impairments in the liver and kidney during helminthiasis (Pathak and Gaür, 1985; Anikieva et al., 1988; Ogorodnikov et al., 1997; Rudneva et al., 2004; etc.). Increased enzyme activities (in particular, ALT and AST) and uric acid level in the plasma of infected gulls can point to such changes. This study demonstrates that the disproteinemia, hyperenzymemia, and hyperurecimia observed during echinostomiasis reflect complex processes in the host’s body. This results from the cumulative effect of the proper disease agent (parasite) on bird’s organs and tissues, changes in the function of organs (liver and kidney) exposed to the toxic effect of helminths, and protective responses of the body mediated by immunogenesis going concurrently with the pathologic process. The experimental study demonstrated significant changes in protein metabolism in experimental herring gull nestlings 4 days after infection. In particular, increased levels of α-globulins, β-globulins, modified albumin, and CIC have been demonstrated. Apparently, the development of young tapeworms for four days reached the stage “noticeable” by the host’s body. The fraction of α-globulins includes glycoproteins whose level increase in acute inflammatory and allergic reactions (Entsiklopediya klinicheskikh…, 1997). Considerable changes in protein metabolism of infected nestlings were observed in the period of days 8–11 of the experiment. This can be due to active metabolic activity of mature H. larina forms releasing toxic metabolites and to active antibody synthesis after the exposure to foreign antigens.
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After 14 days, some indices of protein metabolism in the plasma of infected nestlings restored to baseline. Apparently, a dynamic equilibrium in the host–parasite system was established at this stage. The results of the protein metabolism study in herring gulls infected with trematode H. larina indicate complex and ambiguous interactions in the host–parasite system. On the one hand, the dynamics of some indices of protein metabolism clearly confirmed negative impact of helminths on the physiological state of hosts. These indices primarily included the concentrations of γ-globulins, modified albumin, and CICs. On the other hand, the values of many other indices in infected gulls corresponded to the physiological norm. Moreover, no specific pattern has been revealed in the changes in protein metabolism. The experimental study has solved many problems. Stable increase in the levels of modified albumin and CICs was observed in infected nestlings relative to control. Other indices demonstrated a complex pattern of time-related changes; however, by the end of the experiment, nearly all of them restored to the values recorded in intact birds. Parasites can often exist in a healthy host without much harm, which allows helminths to realize their physiological activity and to avoid protective responses of the host’s body. However, parasites cannot completely “mask” their presence in the host, which is reflected in the changes in certain indices protein metabolism. Reciprocal adaptation of the host–parasite interactions and, hence, the helminth pathogenicity for ultimate hosts can often depend on the duration and pathways of their co-evolution. Previous studies demonstrated that trematode H. larina is a usual component of the helminth fauna of herring gulls in the Barents Sea (Kuklin and Kuklina, 2005). This can explain the absence of severe parasite-induced pathogenesis in the birds. Many scientists noted that nestlings are most susceptible to helminths (Kulachkova, 1960, 1979; Grenquist, 1970; Thompson, 1985). The study of the helminth impact on protein metabolism in herring gulls has also demonstrated that yearlings are most susceptible to tapeworm infection. This, apparently, coupled with underdeveloped immune system and insufficient store of endogenous energy sources provided for the extraordinary response of juvenile birds to H. larina infection. CONCLUSIONS To date, the absence of valid data on the immune state of marine birds with echinostomiasis to a certain extent complicates the interpretation of the obtained data. However, the available data suggest a dynamic equilibrium between herring gulls and trematodes H. larina at the level of protein metabolism. This equilibrium is disturbed in the period of 8–11 days after bird infection with trematode metacercariae.
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