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22Russian Journal of Nematology, 2012, 20 (2), 113-118
Trehalose metabolism enzymes in Contracaecum
rudolphii (Nematoda)
Krystyna Zoltowska1, Elzbieta Lopienska-Biernat1, Agnieszka Borowa1, Jerzy
Rokicki and Malgorzata Dmitryjuk
1 Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Oczapowskiego 1A, 10-957 Olsztyn, Poland; e-mail: ela.lopienska@uwm.edu.pl 2Department of Invertebrate, Faculty of Biology Geography and Oceanology, University of Gdansk,
Pilsudskiego 46, 81-378 Gdynia, Poland
Accepted for publication 15 July 2012
Summary. Activity of trehalose catabolism enzymes, trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), trehalase and trehalose phosphorylase, as well as the trehalose concentration, were assayed in males, females, and third - (J3) and fourth -stage (J4) juveniles of Contracaecum rudolphii. The trehalose contents in the J3, J4, females, and males were 14.57, 11.55, 10.64 and 6.55 mg g-1 , respectively. All the enzymes assayed were present in the nematodes. Activity of the enzymes was found to be age-dependent; in addition, activity of the trehalose catabolism enzymes were sex-dependent. The highest activity of the enzymes that synthesize and cleave trehalose was typical of the J3. The nematode ability to synthesize trehalose decreases with age: in the adult nematodes it was one-third of that in the juvenile. In C. rudolphii, trehalose was found to be decomposed mostly via phosphorolysis, as the activity of trehalose phosphorylase was particularly high (ca 3 u mg-1 in the juveniles, 2.4 u mg-1 in males, and 1.63 u mg-1 in females). The contribution of trehalase to trehalose catabolism was greater in females than in males, the respective activities amounting to 1.55 ± 0.81 and 0.92 ± 0.24 u mg -1. Key words: Anisakidae, trehalase, trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase, trehalose phosphorylase.
Contracaecum rudolphii is a parasitic nematode of the family Anisakidae (Moravec, 1994). Adult forms occur in the stomach and intestines of piscivorous birds, mostly cormorants and pelicans. A massive invasion may increase mortality of the birds affected, particularly the young ones (Torres et al, 1991, 2000, 2005; Kuiken et al, 1999; Zuchowska, 200; Abollo et al., 2001; Dezfuli et al., 2002). The parasite's dispersal stage (the early second-stage juvenile (J2)) is free-living; the invasive juveniles (the late J2) develops in planktonic and benthic crustaceans, while the third-stage juveniles (J3) parasitise freshwater and marine fishes and are a cause of losses in fisheries (Yang et al., 2000).
The literature concerning the species is limited mainly to the description of its occurrence, its prevalence, development, morphology and taxonomy (Moravec et al., 1988; Okulewicz, 1989; Bartlet, 1996; Szostakowska et al., 2002; Kanarek & Rokicki, 2005; Li et al., 2005; Dziekonska-Rynko & Rokicki, 2007; Szostakowska & Fagerholm, 2007; Zhu et al., 2007). Information on the metabolism of
C. rudolphii is very limited. The available literature contains only data on amino acid profile (Kracmar et al., 2000) and on a preliminary characteristics of hydrolytic enzymes of the nematode, assayed with the API-ZYM semi-quantitative enzymatic test (Dziekonska-Rynko & Rokicki, 2005).
Metabolism of carbohydrates, the major energy source of intestinal parasites, has not been studied in the species. Trehalose is a disaccharide consisting of two glucose units connected via an a-1,1-glycoside bond. The sugar is synthesized as trehalose-6-phosphate (T-6-P) from glucose-6-phosphate (G-6-P) and uridinediphosphoglucose (UDpGlc) in a reaction catalysed by trehalose-6-phosphate synthase (TPS). In the subsequent reaction, trehalose-6-phosphate phosphatase (TPP) releases free trehalose from T-6-P. Trehalose metabolism in nematodes was reviewed by Behm (1997). The review is notable by its lack of data on enzymes which synthesize trehalose and decompose it via phosphorolysis. Our earlier publications (Zoltowska et al, 2001, 2002; Dmitryjuk & Zoltowska, 2004; Lopienska-Biernat et al., 2007a) pointed to a
possibility of phosphorolytic trehalose degradation in the nematodes Cystidicola farionis, Hysterothylacium aduncum, Anisakis simplex and Ascaris suum. The initial information on enzymes involed in trehalose synthesis in the parasitic nematodes A. suum and larval A. simplex was supplied by Dmitryjuk et al. (2006a, 2007) and Lopienska-Biernat et al. (2007b).
In nematodes, trehalose, in addition to its role as an energy supplier, is the main sugar circulating in the haemolymph; it also protects the nematode from environmental stress, primarily that caused by abiotic conditions (Behm, 1997). Owing to the importance of trehalose for parasitic nematodes, it was considered necessary to identify trehalose metabolism enzymes present in C. rudolphii extract and to determine their activity. In our opinion, this research is one of the first detailed studies in enzymology of the species. The importance of the study is augmented by the fact that, as reported by Rokicki (2005), the parasite completes its life cycle in Poland now.
MATERIALS AND METHODS
Contracaecum rudolphii individuals were isolated from stomachs of the black cormorants (n = 39) shot in mid-August 2007 on Lake Selment Wielki (the Masurian Lake District, NE Poland). The nematodes, cleaned of the host tissue fragments, were sorted, based on morphological characters (Moravec, 1994; Barson & Marshall, 2004) ), to the J3 and fourth-stage juveniles (J4), males and females, weighed and kept at -800C until assayed.
Preparation of enzymatic extracts. The
nematodes were homogenised with 0.9% NaCl (1 ml to 100 mg tissue). The homogenate was centrifuged for 15 min at 1500 g, at 40C. The supernatant was used for determining the trehalose and protein contents and for assaying the activities
of T-6-P synthase, T-6-P phosphatase, trehalase and trehalose phosphorylase.
Sugar assays. Sugar chromatography was carried out in a SCL-10A Chromatograph equipped with a RID-10A refractometric detector (Shimadzu), on a 250 x 4.6 mm High-Performance Carbohydrate cartridge column, at 35°C. The mobile phase was a 75:25 mixture of acetonitril-deionised water (1.0 ml x min-1 flow rate). The data were processed with the Chromax 2005 software (POL-LAB, Warszawa, Poland).
Enzyme activity assays. Activity of phosphorylase trehalose was assayed as described by Zoltowska et al. (2002); activity of trehalose-6-phosphate synthase was determined following Gaevier et al. (1988); activity of trehalose-6-phosphate phosphatase was assayed as in Kassen et al. (1992). Enzyme activities were related to mg protein, as determined following Bradford (1976).
The data are means of nine replicates. Significance of differences between the means was tested with the analysis of variance (ANOVA) and Tukey's test at P = 0.05..
RESULTS
All the cormorants were found to be infested by C. rudolphii. The mean infestation intensity was 69.43 nematode per bird.
The nematode protein content (related to the wet weight) was significantly higher in the adults than in the younger developmental stages (Table 1). The protein content in J3 was significantly lower (P<0.05) than the contents in all the remaining developmental stages. However, the trehalose contents followed a different pattern (Table 1). The highest trehalose level was typical of the J3, while the trehalose contents in the J4 and in females were about 20% lower. The trehalose content in males was 30% lower than that in females.
Table 1. Contents of protein and trehalose (mg g-1 fresh tissue) and activities of trehalose metabolising enzymes (u mg-1 ) in _Contracaecum rudolphii_
Enzyme Males (a) Females (b) Juveniles
J4 (c) J3 (d)
Protein 39.4 2 ± 2.71 41.75 ± 4.60 34.52 ± 4.13 ab* 27.31 ± 3.36 abc
Trehalose 6.55 ± 2.39 10.64 ± 4.14 11.55 ± 1.79 a 14.57 ± 3.04 a
T-6-P Synthase 0.37 ± 0.20 0.37 ± 0.22 0.63 ± 0,10 aM 1.21 ± 0.12 abc
T-6-P Phosphatase 0.29 ±0.16 0.25 ± 0.04 0.39 ± 0.17 0.85 ± 0.38 abc
Trehalase 0.92 ± 0.24 bd 1.55 ± 0.81 c 0.99 ± 0.25 bd 1.68 ± 0.27
Trehalose phosphorylase 2.41 ± 0.24b 1.63 ± 0.35 2.97 ±0.76b 3.05 ± 0.87 b
*Letters denote significant (P<0.05) difference between the mean marked and the means in appropriate columns.
The highest trehalase activity was recorded in females. The remaining developmental forms showed similar levels of the enzyme's activity, lower by ca 50% than the level found in females. Trehalose phosphorylase showed significantly higher activity in the C. rudolphii juveniles than in females; the enzyme's activity was also higher in males than in females (Table 1). Activity of the trehalose synthesis enzymes, T-6-P synthase and T-6-P phosphatase was observed to decrease as C. rudolphii progressed in its development. The activity recorded in the J3 was significantly higher than the activities shown by the remaining developmental forms. The adult nematodes showed no sex-dependent differences in the enzyme's activity.
DISCUSSION
Contracaecum rudolphii belongs to widely distributed nematodes parasitising fish-eating birds. Recently, reports have appeared that suggested that C. rudolphii was capable of completing its life cycle in Polish waters (Rokicki, 2005; Szostakowska & Fagerholm 2007). Moreover, Szostakowska & Fagerholm (2007) demonstrated the presence of the J3 larvae of genotypes A and B in a fish dwelling in the brackish Gulf of Gdansk, while only genotype B J3 larvae were found in the freshwater Carassius carassius in Lake Selment Wielki, the place of origin of the birds used in this study. It may be thus presumed that the nematodes examined in this study, and at least their J3 larvae, represent the central-European genotype (Zhu et al., 2007). Contracaecum rudolphii is the dominant stomach nematode in the cormorants. The 100% prevalence found in this study confirms the data reported earlier by Kanarek et al. (2002) and Kanarek & Rokicki (2005) on invasion parameters in the cormorants living in the northern part of Poland. The level of infestation in nestlings and adult cormorants Phalacrocorax brasilianus studied by Torres et al. (1991, 2005) in Chile was similar, however, the mean intensity of infestation in the birds we studied was 50% greater than that reported by these authors.
The trehalose content in the adult C. rudolphii was similar to that reported from A. suum and A. galli, parasitic intestinal nematodes of mammals and birds (Von Brand, 1979); it was 2-3 times that in the fish parasites C. farionis and H. aduncum (Zoltowska et al., 2001, 2002). As in other nematodes, the trehalose content in C. rudolphii was much higher in females than in males (Von Brand, 1979), which is a consequence of the reproduction function of the females requiring them to accumulate large amounts of the disaccharide in the eggs (Dmitryjuk et al, 2006b).
In C. rudolphii, as in other intestinal nematodes (A. suum, H. aduncum, A. simplex), trehalose can be decomposed via hydrolysis and phosphorolysis (Zoltowska et al., 2001; 2002, Dmitryjuk & Zoltowska, 2004; Lopienska-Biernat et al, 2007a). The intensity of both metabolic pathways changes with C. rudolphii development. Trehalose phosphorylase is markedly more active in the juveniles than in the adults (Table 1). A similar pattern was reported from H. aduncum C. farionis, and A. simplex (Zoltowska et al., 2001, 2002; Lopienska-Biernat et al., 2007a). On the other hand, unlike the nematode species mentioned, C. rudolphii did not show any pronounced increase in trehalase activity in the adults, compared to the juveniles. Perhaps this is related to different developmental strategies: H. aduncum and C. farionis use fishes as their definitive hosts, while in the life cycle of C. rudolphii fishes are the second intermediate hosts in which the J3 juvenile develop. Biochemistry of C. rudolphii is thus interesting not only because of the hazard posed by the parasite, but also because of its complex life cycle.
Analysis of the results obtained in this study shows that trehalose metabolism in J3 juvenile developing in fishes differs significantly from that of older developmental stages parasitising birds. This conclusion is supported by the highest trehalose contents being found in the J3 juvenile. There is most probably an effect of a higher activity of the enzymes involved in trehalose synthesis, the activity in J3 being twice that in the J4 and three times that in adults. By virtue of their very active trehalose phosphorylase the J3 juvenile cleave trehalose mainly via phosphorolysis. Activity of the juvenile trehalose phosphorylase is about three times that of trehalase, the enzyme involved in trehalose hydrolysis. Noteworthy is the fact that the ratio between activities of both enzymes (trehalase and trehalose phosphorylase) are similar in the two juvenile stages (Table 1), which is perhaps a trait characteristics of the juvenile stage in the parasite's life cycle.
No significant sex-dependent differences in activity of the two enzymes involved in trehalose synthesis in C. rudolphii were observed. On the other hand, trehalose catabolism enzymes do show such differences: the activity of trehalase was significantly higher in females than in males, while trehalose phosphorylase was markedly more active in males than in females. It may be presumed that males catabolism more sugar via phosphorolysis than via hydrolysis. The lower trehalose content in males is perhaps a result of intensive phosphorolysis (Table 1). Sex-dependent differences in C. rudolphii
were previously observed with respect to cadmium and lead accumulation (Barus et al., 2001) and amino acid profiles (Kracmar et al., 2000).
To conclude, it may be stated that trehalose is a sugar important for C. rudolphii, as it is accumulated in particularly high concentration by juvenile stages. This conclusion is supported by the high activity of enzymes involved in both synthesis and decomposition of the sugar in the parasite. Comparison of activities of the trehalose metabolism enzymes shows the metabolism of trehalose to be more intensive in juveniles, particularly in the J3, than in the adult nematodes..
ACKNOWLEDGEMENT
The study was supported by the Polish Ministry of Science and Higher Education grant No. P04C02428.
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Резюме. Исследовали активность ферментов трегалозы: трегалоза-6-фосфат синтазы, (TPS), трегалоза-6-фосфат фосфатазы (TPP), трегалазы и трегалозо-фосфорилазы, а также концентрацию самой трегалозы у самцов, самок и личинок третьей (J3) и четвертой (J4) стадии Contracaecum rudolphii. Содержание трегалозы у лиичнок J3, J4,самок и самцов составляло 14.57, 11.55, 10.64 и 6.55 mg g-1 , соответственно. Были обнаружены все исследуемые ферменты катаболизма трегалозы. Активность ферментов оказалась зависимой от возраста нематод. Кроме того, показана зависимость этой активности от пола нематод. Наиввысшая активность ферментов участвующих в синтезе и расщеплении трегалозы была отмечена у J3. Способность нематод синтезировать трегалозы уменьшалась с возрастом, и составляла у взрослых нематол лишь треть от личиночной. Былопоказано, что у C. rudolphii трегалоза расщепляется в основном за счет дефосфорилирования, поскольку активность именно трегалозо-фосфорилазы оказалась высокой (около 3 u mg-1 у личинок, 2.4 u mg-1 у самцов, and 1.63 u mg-1 у самок). Вклад трегалазы в катаболизм трегалозы был заметнее у самок, чем у самцов, с активностью на уровне 1.55 ± 0.81 и 0.92 ± 0.24 u mg -1 соответственно.
