Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2017, 64: 016 doi: 10.14411/fp.2017.016 http://folia.paru.cas.cz

Research Article Inactivation of proteolytic enzymes by rugosum () from the gut of burbot Lota lota

Galina I. Izvekova, TatyanaV. Frolova and Evgeny I. Izvekov

I.D. Papanin Institute for Biology of Inland Waters, Borok, Russian Academy of Sciences, Russia

Abstract: Parasitic organisms inhabiting the alimentary canal should permanently resist the destructive action of host digestive en- Eubothrium rugosum (Batsch, 1786) (Bothriocephalidea) parasitising the intestine of wild burbot, Lota lota!"#ƽ- cial trypsin and homogenate of intestinal mucosa both being the sources of proteinases. The incubation of live E. rugosum in trypsin ƽ %ƽ & to the incubation medium, the worm extract suppressed over 80% of trypsin activity and nearly half of the proteolytic activity in the mucosa homogenate. Notably, the inhibitory activity of the tapeworms hardly depended on their size characteristics. Finally, the re- search has demonstrated secretion of proteinase inhibitor in E. rugosum, which appears to be essential for its survival in enzymatically hostile environment. Keywords:

Protease inhibitors are important natural regulators of ganism are characterised (Zang and Maizels 2001, Knox ƽ - KVVHKVV>XKV=[# * The inhibitors of serine proteases are among the key inhibitors described in ample publications are currently secretory products of parasites. Being of great importance known (for a review, see Rawlings et al. 2004). In multiple for the parasite’s survival due to their ability to inactivate forms, they occur in various plant and tissues, as the host enzymes, they are normally present in the mi- well as in some microorganisms. Most of the found and \ ƽ characterised protease inhibitors belong to the group of cells (Zang and Maizels 2001). These inhibitors regulate serine proteases (Molehin et al. 2012). protease activity and control a variety of processes related A crucial feature of helminths' adaptation to their envi- to such activity. Particularly, they play an essential role in ronment is the dual relationship with the host proteases. On protection of a parasite from the host’s digestive enzymes the one hand, the parasites use these enzymes to hydrolyse * !] the protein components of food (Izvekova et al. 1997). On Sakanari 1994). Today, the molecular structure of some the other hand, the tapeworms should constantly resist the protease inhibitors is described in nematodes (Grasberger destructive action of proteolytic enzymes (Pappas 1987, et al. 1994, Zang and Maizels 2001, Jin et al. 2011), trem- <=>?H#- ! KVV> X KV=[# tecting them from host’s proteases, including the secretion cestodes (Baig et al. 2005, Li et al. 2006). ƽ Unlike the numerous works devoted to protease in- environment (Soprunov 1987, Pappas and Uglem 1990, hibitors in nematodes and covering quite a wide range of J =>>K# & species, similar works on cestodes are scanty and the list as well as proteases, may participate in suppression of the of studied species is very short (for a review, see Izvek- host’s immune response (Dzik 2006). At present, various ova and Frolova 2017). Up to now only serine protease * inhibitors have been found in cestodes, although it has not always been possible to reveal these inhibitors (Klimenko the vital functions and survival of parasites in the host or- &,=>H={=>H|#-

Address for correspondence: G.I. Izvekova, I.D. Papanin Institute for Biology of Inland Waters RAS, Borok, Nekouzskii Raion, Yaroslavskaya Oblast, 152742, Russia. Phone: +7(48547)24042; Fax: +7(48547)24042; E-mail: [email protected]

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.14411/fp.2017.016 Izvekova et al.: Protease inactivation by Eubothrium rugosum ity of cestodes to suppress the host proteases are particu- porcine pancreas (MP Biomedicals, Santa Ana, California, USA; activity 250 USP) was used for the experiments. Upon 1 h, 2 h (Reichenbach-Klinke and Reichenbach-Klinke 1970, Mat- and 24 h of worms’ incubation in trypsin solution at 10 °C, its skási 1984, Izvekova et al. 2017). activity was determined and compared with that of the control ƽEubothrium Nybelin, trypsin sample kept under the same conditions. 1922 on their host organisms is actively studied because Sample preparation for measuring the inhibitory activity such as Atlantic salmon, Salmo salar Linnaeus (Saksvik In the second series of the experiment, all the tapeworms et al. 2001), rainbow trout, Oncorhynchus mykiss (Wal- baum), and sockeye salmon, O. nerka (Walbaum) (Bosi above were conditionally divided into two size classes: ‘short’ et al. 2005). Infections with species of Eubothrium rarely ! # QR ! # QR QR tapeworms were 5–7 cm and 10–20 cm long, respectively. The needed for the parasite to complete its life cycle (Mitchell number of parasites in each group varied from four to ten in the =>>|#%Eubothrium ‘short’ worms and from one to three in the ‘long’ ones. Then the ƽ worms were transferred to 5 ml of Ringer’s solution and incubat- eventually increases. Notably, the number of tapeworms K[=V W throughout the incubation. After the incubation, the worms were retardation of the salmon may occur even at low-grade in- homogenised and the homogenates were diluted with Ringer’s !]=>>|<KVV=# solution in the mass-volume ratio of 1 : 9. Eubothrium tapeworms parasitise not only salmonids, In addition, the homogenates were prepared from the intesti- Lota }- lota (Linnaeus), the only freshwater member of the family ural source of proteases. For this purpose, after dissecting the in- "- testine and extracting the worms and the chyme, the mucosa was *} removed with a plastic spatula, homogenised and diluted with years, many burbot populations are threatened, endangered Ringer’s solution in the mass-volume ratio of 1 : 49. All homoge- or have disappeared as a result of anthropogenic impact nates were prepared using glass homogenisers manufactured by and global warming (Stapanian and Myrick 2015). An ad- Sartorius AG (Göttingen, Germany). ƽ The worm and mucosa homogenates were centrifuged at be infection with the large cestode Eubothrium rugosum 6,500× g for 5 min at 4 °C. The incubation media resulting from (Batsch, 1786). This species exclusively parasitises the 24 h keeping the worms in Ringer’s solution and contained their burbots and in some years the prevalence of infection excretory/secretory products, extracts obtained by the centrif- =VV~ !& KV=|# - ugation of their homogenates, as well as homogenised mucosa standing the adaptation of E. rugosum to the enzymatically samples were frozen for later use. The protein content in the incu- hostile intestinal environment is of particular interest. The bation medium and worm extracts was determined using Lowry’s aim of the present study was to assess the ability of this method (Lowry et al. 1951). widespeard cestode to inactivate host proteolytic enzymes. Measuring the inhibitor activity MATERIALS AND METHODS When measuring the inhibitor activity of worms, the protein- ase sources were the homogenate of burbot intestinal mucosa and Study object commercial trypsin. Trypsin was used in concentrations 0.0025, The study was conducted on Eubothrium rugosum from the VVVVVVHVV=\ƽ!H#- intestine of wild burbot, Lota lota. The tapeworms were obtained mine the inhibitor activity, 50–100 μl of worm extract or incuba- from 20 burbots 440–480 mm long caught in the Rybinsk reservoir tion medium were added to the experimental medium containing !€? KKƒ|Vƒƒ„†|? K?ƒV[ƒƒ‡# 0.5 ml of mucosa homogenate or trypsin solution of a certain con- period. The number of worms in a single burbot varied from one centration, and incubated for 15 min. Simultaneously, the same V|==??- ƽ After the incubation, the proteolytic activity in the samples was of worms and sample preparation were performed on the ice bath. measured. Besides, the inhibitor activity of E. rugosum samples was com- thoroughly rinsed in three changes of Ringer’s solution (10 ml, ***\ !J]<€# H#* ƽJ]<€ as a 100 mM solution in dimethyl sulfoxide. For this comparison, Inactivation of trypsin by the live worms 50 μl of the PMSF was added to 0.5 ml of trypsin solution. & - worms from 15 burbots were divided into sixteen groups of Measuring the proteolytic activity ! V?| =|> #% The total activity of alkaline proteases in the homogenate randomly taken groups of worms were transferred to 5 ml of of the burbot intestinal mucosa and the activity of commercial 0.005 mg/ml trypsin solution and the remaining eight groups – to !]JŒ#V|~ 5 ml of 0.01 mg/ml trypsin solution. Commercial trypsin from !\# ƽ H !%

Folia Parasitologica 2017, 64: 016 Page 2 of 8 doi: 10.14411/fp.2017.016 Izvekova et al.: Protease inactivation by Eubothrium rugosum al. 2002). While preparing the control samples, the equivalent three time intervals tested (p < 0.05). After 24 h incubation, ƽ the level of percent inhibition reached approximately 60% enzymatically active components. The substrate and enzymati- for both trypsin concentrations (Fig. 2). cally active sample were incubated for 60 min at 20–22 °C. The =V|] Weight of worms and protein content in their incubation solution. The sediment of non-hydrolysed protein was removed media and extracts by centrifuging at 6,500× g for 5 min. The colour development In the second series of the experiment, the tapeworms proportional to enzymatic activities was measured in the super- were divided into ‘short’ and ‘long’ groups considering the natant at 440 nm using the Lambda 25 (PerkinElmer, Waltham, heterogeneity of a natural infrapopulation of E. rugosum USA) spectrophotometer. ƽ ƽQRQR!= !%# p > 0.05). At the same time, the average weight of a single versus a blank sample per gram of wet worm, intestinal tissue or QR - !%\\# ‡ - pared to the ‘long’ ones (p–VVV|# chemical assay was conducted in triplicate. the incubation medium was approximately 22 times lower than in the worm extract for both size groups of parasites. Statistical analyses ƽ /<‡&ƽ found between the short’ and ‘long’ groups in their incuba- tested using a one-way ANOVA with a post hoc Dunnet’s test tion medium and extract either (Table 1). when comparing multiple means against a single control mean or with the Tukey’s test for multiple pairwise comparisons. The lev- Inhibitory activity of E. rugosum incubation medium ƽp = 0.05. Statistical analyses When testing the action of 50 μl and 100 μl of incu- were carried out using the software package STATISTICA 6.0 - (StatSoft, Inc., Tulsa, Oklahoma, USA). ƽ QRQR!€|%#% RESULTS trypsin activity (p < 0.1) was observed only using 100 μl of incubation medium from the ‘long’ tapeworms (at both Trypsin inhibition by the live worms trypsin concentrations tested). During all the experiments with trypsin, Eubothrium rugosum remained alive and were not hydrolysed. The incu- Inhibitory action of Eubothrium rugosum extract on bation of the worms in trypsin solution caused a decrease in commercial trypsin sample !€=#ƽ- ƽ tration (Fig. 2). When incubating the worms in 0.005 mg/ VVV\VV=\- ml trypsin solution, their inhibitory action manifested itself tivity decrease in each size group of cestodes when adding only 24 h later. Meanwhile, at a higher concentration of 50 μl or 100 μl of the extract (p—VV#!€|Œ# trypsin (0.01 mg/ml), the enzyme inhibition became appar- extract action was not strongly associated with either en- ent as early as 1 h later, with a minor further increase in the zyme concentration, extract volumes tested or tapeworm ƽK[!€=#% ‘short’ worms’ extract, the percent inhibition of 0.01 mg/ml

Fig. 1 ‡ƽ Eubothrium rugosum (Batsch, 1786) on trypsin activity at concentration of 0.005 mg/ml (A) and 0.01 mg/ml (B).‘Control’ is the activity of trypsin solution at a given time interval after its preparation. ‘E. rugosum’ is the activity of =V W†–?{ /<‡!˜ƽp < 0.05).

Folia Parasitologica 2017, 64: 016 J|? doi: 10.14411/fp.2017.016 Izvekova et al.: Protease inactivation by Eubothrium rugosum

Fig. 2. Percent inhibition of trypsin activity by live Eubothrium rugosum (Batsch, 1786) depending on the incubation period. Inhibitory ƽƽ!VVV\VV=\†–?#{ /<‡!˜ƽp < 0.05).

Table 1. Weight of Eubothrium rugosum (Batsch, 1786) and protein content in their incubation media and extracts.

Size class of E. rugosum Mean weight (g) Protein content group of worms single worm incubation medium (mg/ml) extract (mg/g) ‘Short’ worms 0.89 ± 0.24 V=[/VV| 0.76 ± 0.10 17.28 ± 0.54 ‘Long’ worms =K?/V|= =V|/VK> 0.69 ± 0.17 15.04 ± 1.18

Fig. 3.™ƽ!A) or extract (B) of Eubothrium rugosum (Batsch, 1786). AMVVV\VV=\- spectively. The next bars show the activity of 0.005 mg/ml or 0.01 mg/ml trypsin solution when adding 50 μl or 100 μl of incubation medium obtained from the ‘short’ worms (n = 8) and ‘long’ worms (n = 5); B – the same test conditions and designations as for Part %Œ%Œ/<‡!˜ ƽp < 0.05).

VVV\ reached approximately 80% displaying no clear changes solution, p < 0.05 (Fig. 4). with further increase in trypsin content (Fig. 6). In control samples, the activity of trypsin gradually rose with its concentration (F = 29.754, p < 0.001) (Fig. 5). When adding 50 μl of the worm extract, this activity activity of intestinal mucosa In this series of experiment (Fig. 7), it was determined each trypsin concentration tested. Notably, this reduced that 50 μl of the extract obtained both from the ‘short’ and level of trypsin activity was rather independent on its in- ‘long’ worms considerably inhibit the proteolytic activity itial concentration (Fig. 5). The extracts of worms had of intestinal mucosa. This activity was also suppressed ƽ by PMSF, an inhibitor of serine proteases. The reported size (p > 0.05). When estimating the inhibitory action of ƽ !€ – =[? cestode extract on trypsin solution depending on its con- p–VVVVVK#ƽ centration it was shown that at a lower trypsin content of control. It was also found that PMSF lowers the proteolyt- 0.0025 mg/ml the percent inhibition was about 60%. At ic activity of homogenised mucosa stronger than the ex- a higher concentration of 0.005 mg/ml, the inhibition level tract from the ‘short’ or ‘long’ worms does (in both cases,

Folia Parasitologica 2017, 64: 016 Page 4 of 8 doi: 10.14411/fp.2017.016 Izvekova et al.: Protease inactivation by Eubothrium rugosum

Fig. 4.JƽEubothrium rugosum (Batsch, 1786). Inhibitory ƽƽ!VVV\VV=\#V3=VV3 QR!–?#QR!–#{/<‡!˜ƽ†˜ƽ across the two trypsin concentrations tested, p < 0.05).

Fig. 5.‡ƽEubothrium rugosum (Batsch, 1786) on trypsin activity depending on the enzyme concentration. ‘Con- trol’ is the activity of commercial trypsin at a given concentration. The ‘short’ worm curve and ‘long’ worm curve describe the trypsin activity when adding 50 μl of the extract obtained from the smaller (n = 8) and larger (n = 5) tapeworms, respectively. Data represent /<‡!˜ƽp < 0.05).

Fig. 6. Percent inhibition of trypsin activity by extract from Eubothrium rugosum !Œ=H?œ#ƽ The ‘short’ worm bars and ‘long’ worm bars characterise the trypsin activity when adding 50 μl of the extract obtained from the smaller !–?#!–#{/<‡!˜ƽp < 0.05). p < 0.05). PMSF inhibited 64 ± 4% of mucosal proteolytic 51 ± 4% of the activity (respectively for the ‘short’ and activity, while the extract could inhibit only 45 ± 6% or ‘long’ worms).

Folia Parasitologica 2017, 64: 016 Page 5 of 8 doi: 10.14411/fp.2017.016 Izvekova et al.: Protease inactivation by Eubothrium rugosum

Fig. 7.‡ƽEubothrium rugosum!Œ=H?œ#***\!J]<€# of the intestinal mucosa of burbot, Lota lota (Linnaeus). ‘Control’ bar is proteolytic activity of homogenate from the burbot intestinal QJ]<€RV)J]<€ V)QR!–?#QR!–# {/<‡!˜ƽ†˜ƽ and PMSF, p < 0.05).

DISCUSSION when adding 100 μl of incubation medium from the ‘long’ The reported suppression of trypsin activity by the tapeworms to trypsin solutions at both tested concentra- live worms is probably associated with the adsorption tions. Similarly, the experiments with H. diminuta failed to of a certain amount of enzyme to the tegument surface reveal excretion of active inhibitor into the ambient medi- with its subsequent inactivation. Trypsin concentration of um (Pappas and Read 1972b). 0.005 mg/ml seems to be too low to cause the inhibitory - ƽ=]ƾ tion medium was reported for the bothriocephalidean ces- of the enzyme becomes adsorbed upon 24 h, which lowers tode nodulosus (Pallas, 1781) parasitising its activity in the external medium. This could be also con- the intestine of pike, Esox lucius Linnaeus (see Izvekova et K[ al. 2017), which might be attributed to the release of a pro- incubation of the worms in trypsin solution. tease inhibitor into the medium due to the regular glyco- It is also noteworthy that trypsin activity grows with calyx replenishment (Oaks and Lumsden 1971). Possibly, its concentration, while the percent inhibition levels are this process in E. rugosum is slower than in T. nodulosus, roughly equal after 24 h incubation of the live worms at ƽ ƽ ™} the former species. absolute amount of inactivated enzyme (proportional to The inhibitor activity found in the tapeworm extracts the inhibitor amount) increases with trypsin concentration. suggests that inactivation of the enzyme occurs after its ad- This is consistent with the data obtained for the cyclophyl- sorption to the tegument. It should be stressed that trypsin lidean cestode Hymenolepis diminuta (Rudolphi, 1819) at both tested concentrations is inhibited incompletely, but from the rat intestine. It was shown that during the in vitro }ƾ- incubation of H. diminuta*!*- asite from proteolysis. According to the data on plerocer- trypsin, these enzymes were inactivated on the tegument coids of the diphyllobothriidean cestode Ligula intestinalis surface (Pappas and Read 1972a,b). Notably, the inactiva- (Linnaeus, 1758), the inhibitory activity of the worm ho- tion proceeded at the same rate even when the worms were mogenate was also higher than that of the cultivation me- successively transferred to fresh enzyme solution (Pappas dium (Matskási and Juhász 1977), which also agrees with and Read 1972b). Since the tapeworms in our experiments our results. stayed alive in trypsin solution, despite the inhibition level Similarly, the attempts to determine the inhibitory ca- being less than 100%, it can be assumed that even the par- pacity of the tegument brush border in H. diminuta (Pap- tial inactivation of the major proteolytic enzyme of the in- pas 1987, Pappas and Uglem 1990) found that the isolated testine, trypsin, ensures the resistance of helminths to their membrane of the tegument in this species is susceptible environment. to the action of proteolytic enzymes. Like the insoluble %ƽ\- fraction of the isolated tegument brush border, this mem- ry products from the worms incubation medium on trypsin brane does not inhibit bovine trypsin (Pappas 1987). At the activity in our studies correlates with the above hypothesis same time, the soluble fraction of the isolated H. diminuta about the partial adsorption of the enzyme to the tegument tegument brush border contains some substances suppress- surface and its inactivation, at least in part. It is also possi- ing the proteolytic and amidase activity of bovine trypsin ble that the amounts of excreted inhibitor were too small and some proteolytic enzymes of the host’s small intestine to detect them by the method used. The latter hypothesis is (Pappas and Uglem 1990). supported by a minor decrease in trypsin activity observed

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The present study on the action of the tapeworm ex- When dividing E. rugosum ƽ - ƽ- es (‘short’ and ‘long’), the working assumption was that centrations has also shown that the inhibitor produced by the inhibitory capacity of helminths may depend on their the worms does not provide 100% inactivation of trypsin. length, and hence the degree of maturity (the latter is ex- When exposed to the tapeworm extract, the activity of # trypsin at each tested concentration decreased. Notably, the level of enzyme activity hardly depended on its con- because the inhibitory action of E. rugosum was inde- tent, while the percent inhibition grew, which suggests an pendent of the worm size. As the average sample weight increase in the absolute amount of inactivated enzyme. QRQRƽ ƽ \ the individual weight of the worms than by their maturi- trypsin solution by the worms’ extract was also found in ty level. A clear dependence of trypsin and chymotrypsin T. nodulosus from the pike intestine (Izvekova et al. 2017). inactivation on the tapeworm weight was also shown for In the current research, the tapeworm extracts had lower H. diminuta by Pappas and Read (1972a,b). These authors ƽ attribute this to the glycocalyx replenishment that occurs in intestinal mucosa than on the commercial trypsin sample. H. diminuta within six hours as demonstrated by Oaks and Similar results were obtained in our previous studies on the Lumsden (1971). inhibitory activity of T. nodulosus from the pike intestine It should be added that in our experiments, not only (Izvekova et al. 2017). This might be due to the fact that the the inhibitory capacity but also the protein content in the proteolytic activity of the intestinal mucosa includes not extract and incubation medium were independent of the only the activity of trypsin, but also chymotrypsin and di- worm size. This is quite natural, as many protease inhib- !‡=>>|„KVV[#& itors are proteins (Rawlings et al. 2004, Knox 2007). In known that serine proteases make the greatest contribution turn, the presence of protein in the incubation medium of ƽ the worms may be explained by the natural renewal of the although this contribution is highly variable (Eshel et al. glycocalyx (Oaks and Lumsden 1971). =>>|„KVV[XKVVH# In summary, as a result of the present study, it was found J]<€ that following the incubation of live cestodes E. rugosum could inactivate only 64% of mucosal proteolytic activity. ƽ On the other hand, Pappas and Uglem (1990) hypothe- - sised that the inhibitor liberated by H. diminuta may have pression depended on trypsin concentration. At the same ƾ- ƽ R teases operating in the small intestine of its rat hosts com- pared to bovine trypsin. Our data indicate the opposite, i.e. studied cestodes is probably associated with the adsorp- the inhibitor produced by E. rugosum appears to be more tion of the enzyme to the tegument surface and its further - inactivation. The extract of E. rugosum inhibits more than ƽ- 80% of commercial trypsin activity and about 50% of the netic position and levels of enzyme activity in the intestines proteolytic activity of the homogenised intestinal mucosa of their hosts, the burbot and rat, respectively. According to Pappas (1980), the inactivated enzyme is incomplete, but even the partial inactivation of intesti- may change the ‘microenvironment’ of the tapeworm in- nal proteolytic enzymes, including trypsin, makes the hel- side the host small intestine, and relatively greater enzyme minths resistant to their environment. Future investigations amounts normally occurring in the small intestine mask the inactivated enzyme. In addition, the portion of trypsin re- characterisation of the proteinase inhibitors liberated by maining active in the intestine despite the inhibitory action E. rugosum to clarify their nature and the mechanism of of the worms and operating along with chymotrypsin and enzyme inactivation. dipeptidases apparently ensure some level of the proteo- ƾ Acknowledgements. This work was supported by the Russian the host and parasite. Foundation for Basic Research (grant number 15-04-02474).

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Folia Parasitologica 2017, 64: 016 Page 7 of 8 doi: 10.14411/fp.2017.016 Izvekova et al.: Protease inactivation by Eubothrium rugosum

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Received 1 March 2017 Accepted 10 April 2017 Published online 9 May 2017

Cite this article as: Izvekova G.I., Frolova T.V., Izvekov E.I. 2017: Inactivation of proteolytic enzymes by Eubothrium rugosum (Batsch, 1786) (Cestoda) from the gut of burbot Lota lota (Linnaeus). Folia Parasitol. 64: 016.

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