ПАРАЗИТОЛОГИЯ, 2021, том 55, № 2, с. 91-100.

УДК 576.311.344:595.121.3:597.593

LYSOSOMAL ENZYMES IN ADAPTIVE RESPONSES

OF CESTODES OF THE GENUS TRIAENOPHORUS

L. V. Anikieva^а, D. I. Lebedeva^а

^аInstitute of Biology, Karelian Research Centre, Russian Academy of Sciences,

11 Pushkinskaya St., Petrozavodsk, 185910 Russia

*e-mail: vysotskayaru@gmail.com

Received 28.01.2021

Received in revised form 04.03.2021

Accepted 06.03.2021

The activity of seven acid hydrolases (acid phosphatase, DNase, RNase, β-glucosidase,

β-galactosidase, cathepsin В, cathepsin D) and tissue protein content were comparatively studied in

two cestode species of the genus Triaenophorus (T. crassus and T. nodulosus) sampled from pike

(Esox lucius) from Lake Kamennoye (northern Karelia). Differences between the lysosomal enzyme

profiles of these species were identified. Triaenophorus crassus demonstrated higher activities of

acid phosphatase and β-galactosidase. The activities of β-glucosidase, cathepsin В and DNase were

reliably lower than in T. nodulosus. The lower ecological plasticity of T. crassus and the differences

detected in the biochemical reactions in the two helminth species are indicative of a more strenuous

relationships of T. crassus with its definitive host, pike, in comparing with T. nodulosus.

Keywords: Triaenophorus, Esox lucius, host specificity, lysosomal enzymes

DOI: 10.31857/S0031184721020010

The cestode genus Triaenophorus Rudolphi, 1793 is chiefly represented in freshwater

bodies in Northern Europe, Siberia and North America by two species: Triaenophorus

nodulosus Pallas, 1781 and Triaenophorus crassus Forel, 1868 (Kuperman, 1973). Both

cestodes change three hosts over their complex life cycle. The first intermediate hosts for

larval stages of both parasites are Copepoda. The range of second intermediate hosts of

T. nodulosus is extensive, encompassing 57 fish species of 17 families. Plerocercoids of

this helminth are most often found in the liver of perch and ruffe. The diversity of second

intermediate hosts for T. crassusis much narrower, represented by 16 species of Salmonidae

and the related Osmeridae and Thymallidae (Kuperman, 1973). The definitive host for the

cestodes is the Northern pike Esox lucius L., in whose intestines the parasites mature and

complete their development (Kuperman, 1973).

Tapeworms possess some unique features they have acquired while adapting to the para-

sitic lifestyle (Dubinina, 1974). Morphofunctionally and biochemically, they are very well

adapted to their host (Shishova-Kasatochkina, Leutskaya, 1979; Sidorov et al., 1989). The

digestive system being reduced in cestodes, their tegument performs the essential secretory,

excretory, digestion and absorption functions. The helminth tegument is involved in many

physiological and biochemical processes that balance the host-parasite relationship (Davy-

dov, Mikryakov, 1988; Kuperman, 1988; Kuz’mina, 2005). The host organism responds to

helminth invasion by launching versatile protective mechanisms to minimize the damage

inflicted by the parasite (Izvekova, 2001; Sajid, McKerrow, 2002; Vysotskaya et al., 2003;

Dzik, 2006; Dezfuli et al., 2014; Nikishin, 2016).

Adaptations in intestinal cestodes are rendered more complex by the dual environment

of endoparasites, which have to adapt to the host (1^st order environment) as well as respond

to changes in the host’s external environment (2^nd order environment). Changes in the envi-

ronment disrupt the host’s food chains, wherefore facultative hosts get involved, resulting

in the formation of ”nonspecific parasitism” (Kuklin, Kuklina, 2005; Ieshko et al., 2012).

Over time, adaptation to new hosts results in speciation and emergence of new parasitic

systems (Kuperman, 1973). The mechanisms of the organism’s adaptation to the environ-

ment at the cellular level are built upon biochemical changes, including the reactions for

supplying the organism with matter and energy, metabolic regulation, and protection against

adverse impacts. An important role in the adaptive and protective responses of aquatic or-

ganisms belongs to lysosomal enzymes - special intracellular organelles containing several

dozens of acid hydrolases (Vysotskaya, Nemova, 2008). Information about the activity of

these enzymes in closely related species of the genus Triaenophorus and their participation

in the process of adapting to the host is in deficit.

The aim was to study in a comparative manner the activity of lysosomal enzymes in tissues

of adult cestodes Triaenophorus nodulosus and Triaenophorus crassus from pike intestines.

MATERIAL AND METHODS

Material for the study was sampled from northern Karelia, from Lake Kamennoye in the Kos-

tomukshsky Strict Nature Reserve (Kem River catchment, White Sea) in June, 2011. The 18 pike

specimens with body length (AC) ranged from 33 to 86 cm (62 ± 3) were investigated. The fish aged

2 - 14 years (7.6 ± 0.7) with body mass 328-5000 g (2184 ± 285).

The captured pikes were examined by partial helminthological dissection, with the prevalence (E)

and intensity (M) of infection with the cestodes T. nodulosus and T. crassus determined as suggested

by Bush et al. (1997). The fish were examined in June; all the retrieved cestodes were mature; data

on Triaenophorus infection rates are given in Table 1.

Pike liver was used for comparisons of biochemical indices in the parasitic systems, since this

lysosome-rich organ is actively involved in the host’s adaptive responses. Whole cestodes were taken

for the analyses. Tissue aliquots were rendered to 10% homogenates in 0.25 М sucrose solution with

EDTA and 0.1% Triton Х-100 non-ionic detergent, which destroys intracellular organelle membranes

releasing the enzymes contained therein. The samples were centrifuged at 10 000 g in centrifuges with

cooling. The supernatant fluid was analysed for the activity of seven lysosomal enzymes (acid phospha-

tase, DNase, RNase, β-glucosidase, β-galactosidase, cathepsin В, cathepsin D) and for protein content.

Analytical studies were done using equipment of the Core Facility of the Karelian Research Centre

of the Russian Academy of Sciences (Tissue Lyser LT homogenizer, Qiagen, Germany; Allegra 64R

centrifuge, Beckman Coulter, USA; spectrophotometer SF-2000, OKB-Spektr, Russia).

The substrate in determinations of the activity of acid phosphatase (EC 3.1.3.2) was sodium

β-glycerophosphate (Barrett, Heath, 1980). The enzyme activity was expressed in micrograms of

hydrolytically generated inorganic phosphorus, whose quantity was calculated based on its reac-

tion with chromogenic reagent (Kahovkova, Odavic, 1969). The activity of acid nucleases - DNase

(EC3.1.22.1) and RNase (EC3.1.4.23) - was determined as suggested by Pokrovskii and Archakov

(1968) and Levitskii et al. (1973), respectively. The substrates were deoxyribonucleic acid (рН 5)

and ribonucleic acid (рН 5.2) solutions in acetate buffer. Hydrolytic reaction products were quanti-

fied by spectrophotometry at 260 nm. The activity of the enzymes was expressed in relative units

D₂₆₀. Determination of the activity of acid β-glucosidase (EC 3.2.1.21) was based on photometric

determination of the para-nitrophenol amount released by the reaction (Pokrovskii et al., 1971). The

substrate was a p-nitrophenyl-β-D-glucopyranoside solution in a citrate buffer (рН 5). The activity of

β-galactosidase (EC 3.2.1.23) was measured as suggested by Barrett and Heath (1980). The substrate

was sodium p-nitrophenyl-β-D-galactopyranoside (рН 4). The activity of both glycosidases was ex-

pressed in micromoles of p-nitrophenol per unit time per mg protein. The activity of acid proteases

was determined by modified spectrophotometric techniques: for cathepsin В (EC 3.4.22.1) - based on

break-up of 0.065 М Na-benzoyl-L-arginine ethyl ester solution in acetate buffer (рН 5), for cathepsin

D (EC 3.4.23.5) - based on the hydrolysis of 1% bovine haemoglobin in acetate buffer at рН 3.6

(Alekseenko, 1968). Protease activity was expressed in relative units of change in optical density (_∆D)

per mg protein: cathepsin В - at 525 nm, cathepsin D - at 280 nm. Protein content in the samples

was determined according to the techniques suggested by Bradford (1976).

Data on cestode infection rates in pike were processed and analysed using Past software (Ham-

mer et al., 2001), differences between biochemical parameters were tested for reliability using the

Mann-Whitney U-test (Gubler, Genkin, 1969). Differences were considered significant with p ≤ 0.05.

RESULTS

All the examined pikes from Lake Kamennoye were quite intensively infected with the

cestodes Triaenophorus crassus and T. nodulosus (Table 1). The intensity of the T. nodulosus

(1-73) infection was much lower than the T. crassus (25-175) infection rates.

Table 1. Summary statistics of infection with cestodes of the genus Triaenophorus

in pike from Lake Kamennoye

Index

T. nodulosus

T. crassus

Number of examined pikes

Infection prevalence, %

100

Min intensity

Max intensity

175

Mean intensity

21.57

4.89

13.23

Variance

334.88

2451.53

18.30

49.51

Median

15.5

Variance/mean s2/M

15.52

38.31

The results of the biochemical study of the cestodes are shown in Figures 1-4. It follows

from the reported data that the activity of lysosomal enzymes in parasite tissues was com-

mensurate with the respective indices in the host’s liver. This is indicated by the activity

of the lysosomal marker enzyme - acid phosphatase (Fig. 1). One should remark that the

activity of this enzyme in T. crassus was notably higher than in the other cestode.

The difference between T. nodulosus and T. crassus in tissue acid RNase activity was not

significant (Fig. 2), whereas the other nuclease - DNase, was significantly lower in T. crassus.

The most significant differences between the cestodes concerned the activity of glyco-

sidases (Fig. 3). Firstly, the absolute values of β-galactosidase activity in both worms were

significantly higher than those of β-glucosidase. Secondly, galactosidase activity in T. crassus

was higher and glucosidase activity was lower than in T. nodulosus.

The same was true for the activity of both proteases (Fig. 4). Their levels in T. crassus

were lower than in T. nodulosus. Note also that cathepsin B activity in cestodes was 2-3

times higher than in the host’s liver (1.02±0.04 relative units - ∆D₅₂₅/mg protein per hour).

On the contrary, the cathepsin D activity of cestodes was 4 times lower than in the pike’s

liver (0.91±0.05 relative units - ∆D₂₈₀ /mg protein per hour). The variation of soluble protein

content in the samples was not so significant: this index in T. nodulosus tissues was 74.5 ±

3.7, in T. crassus - 77.5 ± 3.2, and in pike liver - 62.7 ± 4.4 mg/g dry mass.

Figure 1. Acid phosphatase activity in tissues of adult cestodes of the genus Triaenophorus

and the liver of their host - pike. n = 5.

*Differences between variants are significant at p≤0.05.

Figure 4. Protease activity in tissues of adult cestodes of the genus Triaenophorus. n = 5.

*Differences between variants are significant at p≤0.05.

DISCUSSION

It seems a highly aggregated distribution, indicated by the s²/M ratio, was demonstrated

by T. crassus (Table 1), suggesting pike was more susceptible to infestation by this species

as compared to T. nodulosus.

Cestodes of the genus Triaenophorus are systematically close species. The ranges of

distribution of both T. nodulosus and its host of the genus Esox are almost the same. The

distribution area of T. crassus is somewhat narrower. It occupies the northern part of pike’s

range and covers circumpolar Holarctic regions. The definitive and the first intermediate

hosts are the same for both helminth species. The most significant difference between them

is the localisation of plerocercoids in the second intermediate host: muscles of salmoniform

fishes for T. crassus, and usually liver of Percidae for T. nodulosus. Adaption to the environ-

ment inside their respective second intermediate hosts has been the key factor for the diver-

gence of these cestode species (Kuperman, 1973). The differences detected in the adaptive

responses of the helminths in our study also suggest that T. crassus is a stricter definitive

host specialist than T. nodulosus. The leading role in the species’ adaptations belongs to

biochemical changes, including changes in the lysosomal enzyme complex activity. These

enzymes are involved in membrane and cellular digestion processes (Vysotskaya, Nemova,

2008). Cestode tegument is rich in structures participating in lysosome formation (Kuperman,

1988). A higher activity of acid phosphatase in T. crassus tissues compared to T. nodulosus

indicates that the former generates more lysosomes and that the costs of its adaptation to

the host are higher. Acid phosphatase is known as a broad-spectrum phosphoric monoester

hydrolase, which has an important role in the metabolism of carbohydrates, lipids, nucleic

acids and phosphorus compounds and, hence, in supplying the organism with energy.

Another enzyme whose activity was significantly higher in T. crassus than in the other

cestode was β-galactosidase. The level of this glycosidase can be elevated when the para-

site’s adaptive reactions involve galactose-containing lipids and proteoglycans, which act

as metabolic regulators (Vdovichenko, Vysotskaya, 2013). Also, considering that carbohy-

drate metabolism is the principal source of energy for helminths, when the stores of energy

substrates have mostly been exhausted, alternative mechanisms can be launched to support

tissue bioenergy, and then the role of lysosomal glycosidases, including β-galactosidase,

will grow (Vysotskaya, Nemova, 2008).

We have previously demonstrated that the activity of the lysosomal protease cathepsin B

in tissues of T. nodulosus cestodes is several times higher than in pike organs (Vysotskaya

et al., 2015). The lysosomal proteolytic system is the main player in protein metabolism.

On top of cleaving proteins to peptides and amino acids, lysosomal proteases perform

a number of specific functions for renewal of proteins, activation of precursors of biologi-

cally active proteins and peptides, including hormones (Turk et al., 2001; Buhling et al.,

2004; Nemova, Bondareva, 2005). Cathepsin В participates in the degradation of many

intra- and extracellular proteins (Brix et al., 2008; Arampatzidou et al., 2011; Yadati et al.,

2020). Cathepsin B was shown to take part in apoptosis and immunoregulation processes

(Turk, Turk, 2009). The high activity of cathepsin B we observed in the tissues of both

cestodes suggests that this enzyme is actively involved in the parasites’ protective response

to impact from the host. Probably that activity of cathepsin B in T. crassus was significantly

lower than in T. nodulosus due to the effect of the peptide antibiotics the host produces to

protect itself against various infection agents, including invasion by helminths (Dezfuli et al.,

2014). Besides, proteases, as well as other acid hydrolases, can be inhibited by own proteins

produced by the parasite to protect against the host’s proteolytic enzymes and excreted at

host-helminth contact sites (Holt et al., 2006; Chen et al., 2017; Izvekova, Frolova, 2019;

Vidak et al., 2019). The assumption that the cestodes in question can respond differently to

the same impact from the host is supported by the recently obtained data on qualitative and

quantitative differences in the protein composition of these species (Kochneva et al., 2018).

Thus, according to the biochemical aspects of host-parasite relationships, as well as

data on the distribution, life cycles, and host affiliations, T. crassus appears to be a stricter

specialist than T. nodulosus. It is adapted to living in cold oligotrophic waters. Its first in-

termediate hosts are northern copepodite species. The parasite’s distribution in water bodies

is mainly associated with vendace - a very common species in northern lakes (Potapova,

1978; Valtonen et al., 1989). The other species - T. nodulosus has a broader distribution

and a much wider range of second intermediate hosts, including salmoniform fishes. The

key role in maintaining T. nodulosus abundance belongs to perch and ruffe - the main

items in pike’s diet (Kuperman, 1973). Differences in ecological valence and specialisation

between Triaenophorus species are the reasons for the different resilience of their parasitic

systems through the natural succession in water bodies and under human impact. To wit,

Lake Kostomukshskoye, contaminated by wastes from iron-ore mine and mill, exhibits

a poorer species composition of the biota, including a drastic reduction of the fish fauna.

Vanishing of vendace, the main intermediate host for T. crassus, from the lake entails the

extinction of the parasite, in spite of the presence of other aquatic organisms involved in

its life cycle. In this situation, the decline of aquatic animal diversity similarly leads to

changes in the structure of the T. nodulosus parasitic system in line with changes in the

host’s trophic links, since the main intermediate host (perch) is also absent from the lake.

A typical intermediate host is recruited into the parasite’s life cycle (Ieshko et al., 2012).

Research into the biochemical aspects of relationships in the T. nodulosus-pike parasitic

system in a water body altered by human activities has demonstrated that the main contribu-

tor to the adaptive response to the adverse environmental impact is the host. The parasite,

on the other hand, also contributes to overall homeostasis in the system by adjusting its

metabolism to the host’s condition (Vysotskaya et al., 2015). It is there for ease to say that

the host-parasite relationship in the T. nodulosus-pike system is highly balanced at the fine

biochemistry level. The fact that of the two cestode species of the genus Triaenophorus

only T. nodulosus occurred in the technogenic suppressed lake suggests that this parasitic

system has a higher adaptive potential than that of T. crassus.

Our studies revealed differences between the biochemical and population parameters

of the two cestode species, which evidence a more strenuous process of adaptation to the

host in T. crassus. This, together with a narrower range of second intermediate hosts and

a lower ecological plasticity, corroborates the assumption about a later speciation of

T. crassus compared to T. nodulosus. Of the two ancient and steady parasitic systems, the

original one is the T. nodulosus - pike Esox lucius L. system.

ACKNOWLEDGEMENTS

The studies were financed from the federal budget under state assignments to KarRC

RAS ( No. 0218-2019-0075, 0218-2019-0076 and АААА-А17-117031710039-3).

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ЛИЗОСОМАЛЬНЫЕ ФЕРМЕНТЫ

В АДАПТИВНЫХ РЕАКЦИЯХ ЦЕСТОД РОДА TRIAENOPHORUS

Р. У. Высоцкая, Е. П. Иешко, М. Ю. Крупнова,

Л. В. Аникиева, Д. И. Лебедева

Ключевые слова: Triaenophorus, Esox lucius, гостальная специфичность, лизосо-

мальные ферменты

РЕЗЮМЕ

Проведено сравнительное изучение активности семи кислых гидролаз (кислой фосфатазы,

ДНКазы, РНКазы, β-глюкозидазы, β-галактозидазы, катепсина В, катепсина D) и содержания

белка в тканях двух видов цестод рода Triaenophorus (T. crassus и T. nodulosus) из щуки озе-

ра Каменного (Северная Карелия). Установлены различия в ферментных профилях лизосом

у изученных паразитов. Для Triaenophorus crassus характерны более высокие значения активности

кислой фосфатазы и β-галактозидазы, активность же β-глюкозидазы, катепсина В и ДНКазы была

более низкой, чем у T. nodulosus. Выявленные различия в показателях заражения и параметрах

распределения численности цестод в популяции хозяина (Esox lucius Linnaeus, 1758) свидетель-

ствуют о более напряженном приспособительном процессе к хозяину у T. crassus, что наряду

с более узким кругом вторых промежуточных хозяев и меньшей экологической пластичностью

подтверждает более позднее происхождение этого вида по сравнению с T. nodulosus.

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