CC BY
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подавляет линейный рост и вегетативное размножение листецов (рис. 1, последующие сутки опыта). Итоговый эффект длительной экспозиции будет соответствовать 3-му выводу, для получения которого потребовалось изучение временной динамики оцениваемых показателей. Мы полагаем, что именно оценка временной динамики различных показателей растительных объектов в ЭМП даёт объективную оценку их действия.
Анализ литературных и собственных данных по действию ЭМП, характерных для ЛЭП, выявил многообразие ответов растений. На наш взгляд, этих данных всё же не достаточно. Описанные результаты, прежде всего, подводят к вопросу: существует ли адаптация растений к длительному обитанию в зонах отчуждения ЛЭП и каковы её возможные механизмы. На решение этого вопроса могут быть направлены последующие исследования.
Работа выполнена в рамках государственного задания (тема № АААА-А18-118012690222-4).
СПИСОК ЛИТЕРАТУРЫ
Плеханов Г.Ф. Основные закономерности низкочастотной электромагнитобиологии. Томск: Изд-во Томского университета, 1990. 188 с.
Aksoy H., Unal F., Ozcan S. Genotoxic effects of electromagnetic fields from high voltage power lines on some plants // Int. J. Environ. Res. 2010. Vol. 4. № 4. P. 595-606.
Aksyonov S.I., Bulychev A.A., Grunina T.Yu., Goryachev S.N., Turovetsky V.B. Effects of ELF-EMF treatment on wheat seeds at different stages of germination and possible mechanisms of their origin // Electromagn. Biol. Med. 2001. Vol. 20. № 2. P. 231-253.
Brayman A.A., Miller M.W., Cox C. Effects of 60-Hz electric fields on cellular elongation and radial expansion growth in cucurbit roots // Bioelecromagnetics. 1987. Vol. 8. № 1. P. 57-72.
Brulfert A., Miller M.W., Robertson D., Dooley D.A., Economou P. A cytohistological analysis of roots whose growth is affected by a 60-Hz electric field // Bioelectromagnetics. 1985. Vol. 6. № 3. P. 283-291.
Dattilo A.M., Bracchini L., Loiselle S.A., Ovidi E„ Tiezzi A., Rossi C. Morphological anomalies in pollen tubes of Actinidia deliciosa (kiwi) exposed to 50 Hz magnetic field // Bioelectromagnetics. 2005. Vol. 26. № 2. P. 153-156.
Davies M.S. Effects of 60 Hz electromagnetic fields on early growth in three plant species and a replication of previous results // Bioelectromagnetics. 1996. Vol. 17. № 2. P.154-161.
Freeman D.C., Graham J.H., Tracy M., Emlen J.M., Alados C.L. Developmental instability as a means of assessing stress in plants: a case study using electromagnetic fields and soybeans // Int. J. Plant Sci. 1999. Vol. 160(suppl.). P. S157-S166.
Handy S.M., McBreen K., Cruzan M.B. Patterns of fitness and fluctuating asymmetry across a broad hybrid zone // Int. J. Plant Sci. 2004. Vol. 165. № 6. P. 973-981.
Inoue M., Miller M.W., Cox C. Growth rate and mitotic index analysis of Vicia faba L. roots exposed to 60-Hz electric fields // Bioelectromagnetics. 1985. Vol. 6. № 3. P. 293-303.
Kellogg C. Effects of electromagnetic fields on the growth and development of bean leaves. MS thesis. Detroit: Wayne State University, 1994.
Kimsey I.J., Szymanski E.S., Zahurancik W.J., Shakya A., Xue Y., Chu C.C., Sathyamoorthy B., Suo Z., Al-Hashimi H.M. Dynamic basis for dG-dT misincorporation via tautomerization and ionization // Nature. 2018. Vol. 554. P. 195-201.
Rajendra P., Sujatha Nayak H., Sashidhar R.B., Subramanyam C., Devendranath D., Gunasekaran B., Aradhya R.S., Bhaskaran A. Effects of power frequency electromagnetic fields on growth of germinating Vicia faba L., the broad bean // Electromagn. Biol. Med. 2005. Vol. 24. № 1. P. 39-54.
Rapley B.I., Rowland R.E., Page W.H., Podd J.V. Influence of extremely low frequency magnetic fields on chromosomes and the mitotic cycle in Vicia faba L., the broad bean // Bioelectromagnetics. 1998. Vol. 19. P. 152-161.
Robertson D., Miller M.W., Cox C., Davis H.T. Inhibition and recovery of growth processes in roots of Pisum sativum L. exposed to 60-Hz electric fields // Bioelectromagnetics. 1981. Vol. 2. № 4. P. 329-340.
Shashurin M.M., Prokopiev I.A., Shein A.A., Filippova G.V., Zhuravskaya A.N. Physiological responses of Plantago media to electromagnetic field of power-line frequency (50 Hz) // Russ. J. Plant Physiol. 2014. Vol. 61. № 4. P. 484-488.
Soja G., Kunsch B., Gerzabek M., Reichenauer T., Soja A.M., Rippar G., Bolhar-Nordenkampf H.R. Growth and yield of winter wheat (Triticum aestivum L.) and corn (Zea mays L.) near a high voltage transmission line // Bioelectromagnetics. 2003. Vol. 24. № 2. P. 91-102.
Yano A., Yoshiaki O., Tomoyuki H., Kazuhiro F. Effects of a 60 Hz magnetic field on photosynthetic CO2 uptake and early growth of radish seedlings // Bioelectromagnetics. 2004. Vol. 25. № 8. P. 572-581.
REFERENCES
Aksoy H., Unal F., Ozcan S. Genotoxic effects of electromagnetic fields from high voltage power lines on some plants // Int. J. Environ. Res. 2010. Vol. 4. № 4. P. 595-606.
Aksyonov S.I., Bulychev A.A., Grunina T.Yu., Goryachev S.N., Turovetsky V.B. Effects of ELF-EMF treatment on wheat seeds at different stages of germination and possible mechanisms of their origin // Electromagn. Biol. Med. 2001. Vol. 20. № 2. P. 231-253.
Brayman A.A., Miller M.W., Cox C. Effects of 60-Hz electric fields on cellular elongation and radial expansion growth in cucurbit roots // Bioelecromagnetics. 1987. Vol. 8. № 1. P. 57-72.
Brulfert A., Miller M.W., Robertson D., Dooley D.A., Economou P. A cytohistological analysis of roots whose growth is affected by a 60-Hz electric field // Bioelectromagnetics. 1985. Vol. 6. № 3. P. 283-291.
Dattilo A.M., Bracchini L., Loiselle S.A., Ovidi E.. Tiezzi A., Rossi C. Morphological anomalies in pollen tubes of Actinidia deliciosa (kiwi) exposed to 50 Hz magnetic field // Bioelectromagnetics. 2005. Vol. 26. № 2. P. 153-156.
Davies M.S. Effects of 60 Hz electromagnetic fields on early growth in three plant species and a replication of previous results // Bioelectromagnetics. 1996. Vol. 17. № 2. P.154-161.
Freeman D.C., Graham J.H., Tracy M., Emlen J.M., Alados C.L. Developmental instability as a means of assessing stress in plants: a case study using electromagnetic fields and soybeans // Int. J. Plant Sci. 1999. Vol. 160(suppl.). P. S157-S166.
Handy S.M., McBreen K., Cruzan M.B. Patterns of fitness and fluctuating asymmetry across a broad hybrid zone // Int. J. Plant Sci. 2004. Vol. 165. № 6. P. 973-981.
Inoue M., Miller M.W., Cox C. Growth rate and mitotic index analysis of Vicia faba L. roots exposed to 60-Hz electric fields // Bioelectromagnetics. 1985. Vol. 6. № 3. P. 293-303.
Kellogg C. Effects of electromagnetic fields on the growth and development of bean leaves. MS thesis. Detroit: Wayne State University, 1994.
Kimsey I.J., Szymanski E.S., Zahurancik W.J., Shakya A., Xue Y., Chu C.C., Sathyamoorthy B., Suo Z., Al-Hashimi H.M. Dynamic basis for dG-dT misincorporation via tautomerization and ionization // Nature. 2018. Vol. 554. P. 195-201.
Plekhanov G.F. Osnovnyye zakonomernosti nizkochastotnoy elektromagnitobiologii [The main regularities of low-frequency electromagnetobiology]. Tomsk: Izd-vo Tomskogo universiteta, 1990. 188 s. [In Russian]
Rajendra P., Sujatha Nayak H., Sashidhar R.B., Subramanyam C., Devendranath D., Gunasekaran B., Aradhya R.S., Bhaskaran A. Effects of power frequency electromagnetic fields on growth of germinating Vicia faba L., the broad bean // Electromagn. Biol. Med. 2005. Vol. 24. № 1. P. 39-54.
Rapley B.I., Rowland R.E., Page W.H., Podd J.V. Influence of extremely low frequency magnetic fields on chromosomes and the mitotic cycle in Vicia faba L., the broad bean // Bioelectromagnetics. 1998. Vol. 19. P. 152-161.
Robertson D., Miller M.W., Cox C., Davis H.T. Inhibition and recovery of growth processes in roots of Pisum sativum L. exposed to 60-Hz electric fields // Bioelectromagnetics. 1981. Vol. 2. № 4. P. 329-340.
Shashurin M.M., Prokopiev I.A., Shein A.A., Filippova G.V., Zhuravskaya A.N. Physiological responses of Plantago media to electromagnetic field of power-line frequency (50 Hz) // Russ. J. Plant Physiol. 2014. Vol. 61. № 4. P. 484-488.
Soja G., Kunsch B., Gerzabek M., Reichenauer T., Soja A.M., Rippar G., Bolhar-Nordenkampf H.R. Growth and yield of winter wheat (Triticum aestivum L.) and corn (Zea mays L.) near a high voltage transmission line // Bioelectromagnetics. 2003. Vol. 24. № 2. P. 91-102.
Yano A., Yoshiaki O., Tomoyuki H., Kazuhiro F. Effects of a 60 Hz magnetic field on photosynthetic CO2 uptake and early growth of radish seedlings // Bioelectromagnetics. 2004. Vol. 25. № 8. P. 572-581.
EFFECTS OF POWER LINE ELECTROMAGNETIC FIELDS ON PLANTS
Yu. G. Izyumov, M. G. Talikina, V. V. Krylov
Papanin Institute for Biology of Inland Waters Russian Academy of Sciences, 152742 Borok, Russia
e-mail: [email protected]. ru
Data on the reaction of plants growing in the zones covered by electromagnetic fields of power transmission lines are given. Researchers noted the suppression of plant's production characteristics in these zones. The effects of magnetic fields that simulated the fields of power transmission lines on plants in laboratories were close to the effects observed in nature. Data on the reaction of aquatic plants to the fields of power transmission lines in natural and experimental conditions were obtained. Duckweed Lemna minor was used as a model species. Based on the obtained results, it was concluded that instead of single estimates it is necessary to study the dynamics of the production and cytological parameters of plants in the zones covered by electromagnetic fields of power transmission.
Keywords: electromagnetic field, power transmission lines, plants, duckweed (Lemna minor)
Труды ИБВВ РАН, вып. 84(87), 2018
Transactions of IBIW, issue 84(87), 2018
УДК 59 7.554.3:577.152.34:59 7-154.31
COMPARATIVE LYSOZYME ANALYSIS IN CARP FISHES (CYPRINIDAE, CYPRINIFORMES)
T. A. Subbotkina1, M. F. Subbotkin1, Vo Thi Ha2
1Papanin Institute for Biology of Inland Waters Russian Academy of Sciences, 152742 Borok, Russia 2Russian-Vietnam Tropical Research and Technological Center, Coastal branch, Nha Trang, Vietnam
e-mail: [email protected]. ru
Eleven species of the family Cyprinidae inhabiting waters in temperate latitudes and tropics were studied on the basis of the lysozyme analysis. Cyprinidae are characterized by low and very low content of lysozyme in the liver, kidneys, and spleen compared to fish of other groups. Lysozyme is not detected in the serum of some carps. Fish cultivated in the tropics do not differ in the enzyme content from fish in temperate latitudes. The related species with the lowest lysozyme content and the species lacking lysozyme in serum were found in different climatic zones. The pattern of the enzyme distribution in the studied organs and tissues of some cyprinid is as follows: kidneys > spleen > liver > serum. Such distribution is also observed in fish from other phylogenetic groups, but it is not the rule for all species. The study demonstrates that cyprinid species in various climatic zones are more similar in the lysozyme level compared to intraspecific differences of common carp Cyprinus carpio and Indian major carp Labeo rohita in experiences of other authors.
Keywords: lysozyme, organs, serum, temperature, climatic zones
DOI: 10.24411/0320-3557-2018-10018
INTRODICTION
Cypriniformes represent the group of freshwater fishes. Some species are capable of surviving from near freezing to high tropical temperatures and exist in a wide thermal range [Nikolsky, 1974; Bowden, 2008]. Many cyprinids from the family Cyprinidae are important in the human diet and their importance has increased as evidenced by their widespread use in aquaculture. In aquaculture rapid fish growth is promoted, but there also exists adverse factors that can quickly lead to large losses in production. Therefore, the development of different approaches for improving the resistance of fish is of interest to many experts. Parameters of immunity are part of the sensitive physiological-biochemical system and are considered as bio-indicators to determine both the condition of fish and habitat [Skouras et al., 2003; Thilagam et al., 2009]. Innate immunity or non-specific resistance is an important part of the mechanisms that support homeostasis and maintain individual organism integrity [Lukyanenko, 1989; Saurabh, Sahoo, 2008].
Lysozyme (EC 3.2.1.17) is an enzyme of the glycosidase group identified in plants and animals including fish, and is an important component of the innate immune defense system. Lysozyme activity or content varies widely in many species and depends on the physiological condition of the fish, the influence of environmental factors and other reasons [Lukyanenko, 1989; Saurabh, Sahoo, 2008; Subbotkin, Subbotkina, 2016; 2018]. It is one of the most studied components of innate immunity of fish [Tort et al., 2003]. However, many questions remain unstudied such as comparative
differences in enzyme activity among various species, differential responses of various species to the same adverse factors or pathogens, among others. There is no consensus among researchers on the nature of these phenomena. Some authors suppose that the similarity in enzyme activity may be caused by the fish's ecology and genetic relationships among species [Lie et al., 1989; Lukyanenko, 1989]. Previously, we examined the levels of lysozyme in fishes from various taxonomic groups [Subbotkina, Subbotkin, 2002; 2003; 2004; 2013]. It was found that the content of enzyme in related species does not depend on the type of feeding (predatory and nonpredatory), as well as of freshwater or saltwater fish. This results support the hypothesis that level and distribution of lysozyme may be associated with fish phylogeny [Subbotkina, Subbotkin, 2003]. Although lysozyme is widely distributed in the body of fish, only serum or blood plasma is usually analyzed. The limited information on the enzyme in the immune organs restrains an understanding of the immune response mechanisms to immunomodulatory factors.
The aim of this work is to make a comparative analysis of lysozyme in tissues: 1) related fish species of the family Cyprinidae from different climatic zones, which differ significantly in the temperature of the environment; 2) cyprinid fish of the order Cypriniformes with that of some species from other orders. To broaden the knowledge about the role of the immune organs, the lysozyme content was determined not only in the serum but in the kidneys, spleen, and liver as well.
MATERIALS European specimens were a wild fishes of the Volga-Caspian basin. Tropical fishes were an aquaculture of Central Vietnam (Table 1). Fishes were obtained from commercial or special scientific fisheries. The caught fish were immediately killed by a sharp blow to the head and bled. Then the fish were transported to the laboratory for sampling organs, where they were
Table 1. Species and collection locations
AND METHODS
measured and weighed. Fish were caught in water bodies of various climatic zones which differ significantly in temperature. The annual temperature varied from 0.4 °C to 23 °C in the Rybinsk reservoir, from 5 °C to 26 °C in the Volga River delta [Litvinov, Roshchupko, 1993] and from 17 °C to 37 °C and higher in the tropics [Kumari et al., 2006; Das et al., 2012].
Species Number, ind. Fork length, cm Mass, g Location of sampling
Ide - Leuciscus idus (L.) 5 24.5-40 180-890 Rybinsk Reservoir, near
Roach - Rutilus rutilus (L.) 12 29-33 270-420 Borok - Sutka River,
Bream - Abramis brama (L.) 15 27-52 490-1410 Verkhne Nikul'skoe
White bream - Blicca bjoerkna (L.) 5 23-24.5 no data
Tench - Tinea tinea (L.) 5 27-30 no data
Rudd - Scardinius erythrophthalmus (L.) 10 19-26 220-400 Volga River Delta, Zhitnoe
Japanese (white) crucian carp - Carassius cuvieri (Temminck & Schlegel) Asp - Aspius aspius (L.) 10 13 25-33 33.5-40 320-800 470-740 North part of Caspian Sea, near Little Pearl Island
Grass carp - Ctenopharyngodon idella (Val.) 5 31-34 280-390 Ponds, Central Vietnam, Nha Trang
Common carp - Cyprinus carpio (L.) 5 33-41 630-1080
Silver carp - Hypophthalmichthys molitrix (Val.) 10 35-54 420-1770
The fishes were killed by a sharp blow to the head and blood was taken from the caudal vein immediately after the capture. The fish were placed in a thermos with ice and transported to the laboratory where they were weighed and the length to the end of the scale cover was measured. Then, their immune organs such as the liver, kidney and spleen were removed for lysozyme concentration determination. Serum was obtained from the blood taken. The lysozyme content was determined by the lysoplate method as described previously [Subbotkina, Subbotkin, 2003]. The method is based on the ability of lysozyme to lyse Micrococcus lysodeikticus cells dispersed in an
RESULTS AND The level of lysozyme varied widely in the organs and tissues of the fish studied (Fig. 1). The highest lysozyme content were found in the kidneys in the asp Leuciscus aspius, and the rudd Scardinius erythrophthalmus, from the Northern Caspian and the Volga River delta, as well as in the tench Tinea tinea, the roach Rutilus rutilus and the ide Leuciscus idus from the Rybinsk reservoir. Lysozyme content in the spleen is lower than the kidneys. The highest levels of enzyme in the spleen were also found in the asp and rudd from the Northern Caspian and the Volga River delta, as well as in the tench and the roach from the
agar gel. By this method, not only clear liquids, such as serum, but also turbid and intensively colored supernatants of tissue homogenates are analyzed similarly successfully. The diameter of cleared zone is proportional to the log of lysozyme concentration [Osserman, Lawlor, 1966]. The lysozyme concentration in the samples was determined by the calibration curve based on the standard preparation from chicken egg protein and was expressed in ^g/g for organ tissue and in ^g/ml for serum.
All results were presented as means ± standard error. Statistical significances were evaluated by the Student's test for p < 0.05.
DISSCUSSION
Rybinsk reservoir. The highest content of lysozyme in the liver was found in the tench and the asp. The serum is characterized by a minimal content of lysozyme in roach, tench, rudd and Japanese crucian carp Carassius cuvieri, or its absence in other species. The fish with the lowest lysozyme were found in those areas that most differed in temperature, such as the Rybinsk Reservoir (bream Abramis brama, white bream Blicca bjoerkna) where the temperature varies from 0.4-1.9 °C in winter to 23 °C in summer [Litvinov, Roshchupko, 1993],
Fig. 1. The lysozyme content in carp species from different climatic zones in ^g/g for liver, kidney, spleen, and ^g/ml for serum. Data are expressed as means values + standard errors. Different lowercase letters on bars indicate significant difference (p <0.05) for the same tissue in various species.
and the ponds in tropics (common carp Cyprinus carpio, silver carp Hypophthalmichthys molitrix, grass carp Ctenopharyngodon idella) with the temperature ranging from 17 °C in cold to 37 °C in warm seasons [Kumari et al., 2006; Das et al., 2012]. Individuals, without enzyme activity in some organs, were observed among them. The species with no serum lysozyme, namely Leuciscus idus, Leuciscus aspius, Abramis brama, Blicca bjoerkna, Cyprinus carpio, Hypophthalmichthys molitrix, Ctenopharyngodon idella were found in all climatic zones.
The effect of temperature on the fish immune response is an important factor in the studies of non-specific defense. The enhancing of lysozyme serum activity in fish with increasing water temperature has been shown through many experiments [Dautremepuits et al., 2004; Dominguez et al., 2005; Bowden, 2008; Saurabh, Sahoo, 2008]. However, the energy and physiological possibilities of the organism are limited therefore the increase of enzyme activity and the maintenance of it level higher than the physiological norm cannot be continuous. Perhaps the results showing a decrease of lysozyme activity in fish at the water temperature 32.533 °C are due by this [Dautremepuits et al., 2004; Kumari et al., 2006], although such temperature is not extreme for tropical water bodies [Swain et al., 2007; Das et al., 2012]. Increase of lysozyme activity under the impact of rising temperature in an experiment does not mean high levels of enzyme in fish, when a species adapts to constant habitat at high environmental temperatures. Thus, we have not found high values of lysozyme in fishes that have been cultivated in tropical Central Vietnam. It should be noted that these data were found in the fishes in warm period. These species showed lower levels of the enzyme in their organs than other species living in the colder European waters although among them the Abramis brama and Blicca bjoerkna also contain lowest lysozyme. The lysozyme content in Cyprinus carpio, Ctenopharyngodon idella and Hypophthalmichthys molitrix, in the tropics did not differ (p > 0.05) from that of fish grown in the Volga River delta, 2.2-2.9 ^g/g in kidneys, 1.142.0 ^g/g in spleen, 0.46-0.74 ^g/g in liver [Lukyanenko, 1989]. Comparison of the same species from different regions of the Volga River basin indicated that the content of lysozyme is 1.8-2.5-fold in bream, 4-6.2-fold in roach, 6.28.4-fold in ide higher in the Rybinsk reservoir than in the delta [Lukyanenko, 1989]. Taking into account phylogenetic relationships, we have found a similarity in lysozyme levels among related species of cyprinids from different climatic zones.
Physiological norm of variability range for immune parameters may be considered as a result of seasonal changes. The study of seasonal variability in lysozyme activity showed that in the tropical Indian major carp Labeo rohita in winter (7.26±0.87 ml-1) it is lower than in warm periods of the year (12.93±1.66 ^g/ ml-1) [Swain et al., 2007]. Thus, the range of seasonal changes in the enzyme activity in Labeo rohita on average did not exceeded two-fold of the value. However, for another tropical species of carps, Puntius sarana, seasonal variation of serum lysozyme activity (3.46-3.94 ml-1) was not appeared [Das et al., 2012].
The study of Abramis brama in different seasons showed no relationship between water temperature and the content of lysozyme in the liver, kidneys and spleen. The highest lysozyme levels in the organs were observed during the cold months. In contrast, the amount of the enzyme was lower at high water temperatures in summer. The range of variation from the lowest to the highest mean value of enzyme is as follow: kidneys 3.4-12.5 ^g/g; in spleen 1.7-7.4 ^g/g; liver 0.5-3.9 ^g/g. Lysozyme was not detected in serum of bream in all studied periods of the annual cycle regardless of its amount in immune organs [Subbotkin, Subbotkina, 2016]. These results are not unexpected. Previously, we reported the highest content of kidney lysozyme in winter and the lowest in summer in the Russian sturgeon Acipenser gueldenstaedtii and beluga Huso huso from the Volga River. In contrast, the serum had the highest enzyme content in late summer and the lowest level in winter [Subbotkina, Subbotkin, 2012]. Extreme differences have been recorded in sturgeon up to 7-fold in serum, 5-fold in liver and spleen, and 3fold in kidneys, which were observed for one year [Subbotkina, Subbotkin, 2002; 2012]. Muona and Soivio (1992) reported the intra-annual variation of the enzyme activity in the plasma up to 9-fold in Atlantic salmon Salmo salar.
The common pike, Esox lucius, showed a 2fold reduction of lysozyme content in serum from spring to autumn. The highest level of enzyme in liver and spleen was recorded in summer, but it remained unchanged in kidneys through spring, summer and autumn [Izvekova et al., 2010]. Common pike is infected with Triaenophorus nodulosus, which lives in the intestine during the cold season, but emerges from it in the summer. The lysozyme level in the intestine mucosa of fish free parasites increases by a factor of 75 [Izvekova et al., 2010].
Table 2. The lysozyme content and activity in fish of the family Cyprinidae
| Species Localization Activity or content
control experiment
Cyprinus carpió var. Jian serum trace 1.69 iig/mT1
C. carpió serum 0.55-0.73 (ig/ml 0.32-0.41 (ig/ml
0.14-1.13 (ig/ml
C. carpió serum 151.28 (ig/ml 172.4 (ig/ml
C. carpió serum 0.66; 0.72 mg/ml 0.34; 0.74 mg/ml
C. carpió serum 58.14 mg/ml 70.6-80.14 mg/ml
C. carpió serum 0.003-0.012 U 0.012-0.03 U
C. carpió serum 0.060-0.061 U 0.040-0.072 U
kidney 0.31-0.32 U 0.24-0.37 U
spleen 0.17 U 0.11-0.29 U
C. carpió serum 0.6-1 U 0.3-1.7 U
kidney 1.4-1.5 U 1.3-2 U
spleen 0.4-0.6 U 0.15-1.23 U
liver 0.9-1.3 U 0.6-2 U
C. carpió serum 55 U 140 U
C. carpió var. Jian serum no activity 2.2-7.9 U/ml
C. carpió serum 922-975 U/ml 1708-4148 U/ml
C. carpió serum 918 IU/mL 1546-6228IU/mL
C. carpió serum 860-1296 IU/mg 1884-3257 IU/mg
prot prot
C. carpió plasma no activity 0.27-6.11 U/g prot
kidney 2.41-2.49 U/g prot 1.44-3.53 U/g prot
liver no activity no activity
Carassius auratus serum 0.010-0.017 U 0.011-0.072 U
C. auratus serum 58 U/ml 48 and 80 U/ml
C. auratus gibelio serum 102 U/ml 133-309 U/ml
- Acting factor References |
vitamin A trichlorphon, Pseudomonas alcaligenes Monias, Aeromonaspunctata Snieszko Qompsell cadmium apidaecin CpG oligode-oxynucleotides l-methyl-3-octylimidazolium bromide Yang et al., 2008 Siwicki et al., 1990 Wu et al., 2007 Ghiasi etal., 2010 Zhou et al., 2008 Tassakka, Sakai, 2002 Li etal., 2012b
chlorpyrifos Li etal., 2013
nucleotide from yeast RNA, bovine albumin pyridoxine Ocimum basilicum, Cinnamomum zeylanicum, Juglans regia, Mentha piperita ß-(l,3) glucan, Sacharomyces uvarum levamisole Sakai et al., 2001 Feng etal., 2010 Abasali, Mohamad, 2010 Gopalakannan, Arul, 2010 Maqsood et al., 2009
copper, chitosan Dautremepuits et al., 2004
Alcaligenes faecalis cyanobacteria plant extracts Wang etal., 2011a Qiao etal., 2013 Luetal., 2013
C. auratusgibelio^ x Cyprinus
carpioS
Ctenopharyngodon idella C. idella
C. idella C. idella C. idella
C. idella
C. idella
Rutilus rutilus Rutilus frisii kutum
Tinea tinea
Megalobrama amblycephala M. amblycephala
serum
serum serum kidney spleen serum serum serum
serum kidney spleen liver
head kidney spleen liver
serum female: mucus serum male: mucus serum plasma
serum
serum
31 U/100ml
0.82 (ig/ml 10.54-14.56 (ig/ml 4-6 (ig/ml 6-7.1 (ig/ml 49.4 U/ml 109 U/ml 0.12 UL
0.07 U/mg prot 2.94 U/mg prot 0.41 U/mg prot 0.49 U/mg prot
14.08 U/mg prot 13.85 U/mg prot 11.75 U/mg prot
40.0 (ig/ml
0.77-13.92 (ig/ml 0.5-2.35 (ig/ml
0.85-17.89 (ig/ml 0.82-1.45 (ig/ml 0.146-0.186 absorbance 3.9 (ig/ml
10-13 (ig/ml
27-32 U/100 ml
0.52; 1.14 (ig/ml 1.07-14.6 (ig/ml 3.8-6.2 (ig/ml 5.5-9.3 (ig/ml 47.0-60.2 U/ml 101-260 U/ml 0.11-0.53 UL
0.14-0.87 U/mg prot
I.71-4.41 U/mg prot
0.24-4.55 U/mg
prot 0.30-2.00 U/mg prot
25.76 - 38.40 U/mg prot
24.02 - 28.58 U/mg prot
21.41-30.13 U/mg prot
50.25-64.0 (ig/ml
no data no data
no data no data 0.144-0.178 absorbance 2.8-5.6 (ig/ml
II.6-17 (ig/ml
cottonseed meal, gossypol
bacillus diazinon
magnesium Cortisol, cocoa butter carrier lipopolysaccharide, protein of Aeromonas hydrophila (Chester) chitosan
thiamin
fructooligosaccharide season, temperature, reproductive migration, salinity, gonadal growth, sex
testosterone, (3-glucan
anthraquinone extract from Rheum officinale, Aeromonas hydrophila emodin, vitamin C, temperature
Caietal., 2011
Weifen et al., 2012 Soltani, Pourgholam, 2007
Wang etal., 2011b Wang et al., 2005 Sun etal., 2011a
Han etal., 2010
Wen etal., 2015
Soleimani et al., 2012 Ghafoorietal., 2014
Vainikka et al., 2005 Liu etal., 2012 Ming etal., 2012
M. amblycephala
M. amblycephala Mylopharyngodon piceus (Richardson) Labeo rohita
L. rohita L. rohita L. rohita L. rohita
L. rohita
Labeo bata Labeo calbasu Catla catla (Hamilton) C. catla
C. catla
Cirrhinus mrigala (Hamilton) C. mrigala
serum
plasma serum
serum
serum serum serum serum
serum
serum serum serum serum
serum
serum serum
1.9-2.1 U/ml
no data 0.29 U/ml
0.08-16.62 (ig/mL 0.76-8.89 (ig/mL
231 (ig/ml 123.3-125.8 U/l 130-208 U/ml 483.06-612.02 U/mg serum protein no data
7.97-24.09 (ig/ml 3.40-19.62 (ig/ml 2.50-8.05 (ig/ml 549-857 U/ml
762.98 U/min/mg
protein 4.65-10.25 (ig/ml 706-731U/m
2.2-2.6 U/ml
247-372 U/ml 0.24-0.28 U/ml
no data
280^100 (ig/ml 132.5-236.8 U/l 130-430 U/ml 677.60-808.74 U/mg serum protein
675.41-1122.34 U/min/mg protein of serum no data no data no data 851-1549 U/ml
470-1711.48 U/min/mg protein no data 572-830 U/m
proteins of A. hydrophila carbohydrate, lipid fish oil, rapeseed oil
normal range
Achyranthes aspera (3-glucan Euglena viridis corn carbohydrate, temperature
starch
normal range normal range physiological normal range
Cynodon dactylon
yeast RNA, co-3 fatty acid, b-carotene
physiological normal range azadirachtin, camphor, curcumin,Aphanomyces invadans
Wang etal., 2013
Li et al., 2012a Sun etal., 2011b
Mohanty et al., 2007
Rao et al., 2006 Misra et al., 2006 Das et al., 2009 Alexander et al., 2011
Kumar et al., 2007
Saurabh, Sahoo, 2008 Saurabh, Sahoo, 2008 Sahoo et al., 2005 Kaleeswaran et al., 2011 Jha et al., 2007
Sahoo et al., 2005 Harikrishnan et al., 2009
Thus, seasonal variation of serum lysozyme does not reflect the full dynamics of the enzyme in fish and it has deeper physiological basis than simply the direct effect of water temperature.
We found very low content or absence of the serum lysozyme in carps. A very low activity or level of enzyme in other studies has also been recorded in Cyprinus carpio [Siwicki et al., 1990; Yang et al., 2008; Ardo et al., 2010; Liu et al.,
2011], Ctenopharyngodon idella [Weifen et al.,
2012], Rutilus frisii kutum [Ghafoori et al., 2014], Megalobrama amblycephala [Liu et al., 2012; Wang et al., 2013]. Also, the tropical carp, Labeo rohita, and olive barb, Puntius sarana, exhibit low lysozyme activity [Dash et al., 2011; Das et al., 2012]. However, the activity of serum lysozyme in other studies was higher. Enzyme activity in Cyprinus carpio increased from trace or very low in both the control and the experimental treatment [Siwicki et al., 1990; Yang et al., 2008; Ardo et al., 2010; Liu et al., 2011] to 151.28 ^g/ml in the control and 172.4 ^g/ml in the experience [Wu et al. 2007]. The range in other units is as follows: from undetectable value-7.9 U/ml [Feng et al., 2010] to 4148-6228 U/ml [Abasali, Mohamad, 2010; Gopalakannan, Arul, 2010], or from 0.0030.03 U to 140 U [Sakai et al., 2001; Tassakka, Sakai, 2002]. The data (Table 2) also indicate a very wide range of variation in the serum enzyme in other species. For example, the normal range of activity in the Labeo rohita is 0.08-16.62 ^g/ml [Mohanty et al., 2007], but has been observed as high as 231-400 ^g/ml [Rao et al., 2006]. The range in other units is as follows: from 123.3236.8 U/l (0.123-0.237 U/ml) to 432.6 U/ml [Misra et al., 2006; Das et al., 2009]. The lysozyme activity in the Megalobrama amblycephala varies from 1.9-2.6 U/ml to 372 U/ml [Li et al., 2012a; Wang et al., 2013].
The data obtained in the experimental conditions in the control fish of various species carps (Table 2) are significantly greater than 5-9-fold of value for the physiological range of seasonal changes in other fish. As a result, the lysozyme content in the serum of various cyprinids species from different climatic zones in our studies are more similar than the values for one species in experiments by many researchers.
Very few studies have assayed lysozyme simultaneously in kidneys, spleen and liver. Most researchers measure serum or plasma lysozyme. There are reports of enzyme measured in organs for seven species of cyprinids [Lukyanenko, 1989], Cyprinus carpio, Ctenopharyngodon idellus, Carassius auratus gibelio [Dautremepuits et al., 2004; Soltani, Pourgholam, 2007; Han et al., 2010; Li et al., 2012b; 2013; Kurovskaya,
Stril'ko, 2016]. In addition, it is difficult to compare results due to differences in assay procedures. Variants of turbidimetric methods and diffusion into agar gel (lysoplate) are the most widespread approaches for the lysozyme assay each with its own units. Moreover, data in the same units may be reported as either lysozyme content or activity. Nevertheless, these studies also show low levels of lysozyme activity or its absence in organs and serum of cyprinids.
Many researchers studying innate immunity or nonspecific resistance in fish uses the term "health" or "healthy" fish [Sahoo, Mukherjee, 2002; Das et al., 2009; Dash et al., 2011; Lin et al., 2011; Sun et al., 2011b; Baruah et al., 2012; Devi et al., 2012; Kiron, 2012; Li et al., 2012a]. The condition of these fish is, probably, determined visually by external features. The data in Table 2 show that the values of lysozyme in "healthy" fish of the control vary widely. The notion of "health" or "healthy" fish based on the lysozyme content/activity in the species becomes elusive on the one hand or very narrow for a unique experiment on the other hand, under variability of the parameter such as in Cyprinus carpio or Labeo rohita.
Carp fishes are important for aquaculture. The lysozyme assay has been considered as an indicator of the efficiency for manipulations that should improve viability and sustainability of fish to adverse factors by enhancing nonspecific immunity. However, the difference in the initial condition of "healthy" fishes within the species can make it difficult to evaluate the results of immunostimulatory manipulations. This is due to the fact that the increased lysozyme activity under the influence of immunomodulatory factors in some experiments [Sahoo, Mukherjee, 2002; Yang et al., 2008; Kuang et al., 2012; Sieroslawska et al., 2012] is lower than the control in other studies [Rao et al., 2006; Wu et al., 2007; Jiang et al., 2010]. Moreover, high values of lysozyme in the control fish may require elevated energy expenses for the organism [Kortet, Vainikka, 2008], and therefore the immunomodulatory effect may not be adequate. It has been shown that both water salinity and growth hormone enhance the lysozyme activity in rainbow trout, Oncorhynchus mykiss, acting as individual factors. However, increase of the enzyme activity caused by treatment with growth hormone in fresh water did not continued after moving fish into salt water [Yada et al., 2001].
The comparison among species from different taxonomic groups showed that the cyprinids (order Cypriniformes) are characterized by low or very low content of lysozyme.
Fig. 2. The lysozyme content in different fishes. Data are expressed in ^g/g for liver, kidney, spleen, and ^g/ml for serum as means values ± standard errors.
Research of species from some orders has found the highest content of the enzyme in Acipenseriformes (Fig. 2). The highest concentrations of enzyme, an average exceeding 1000 ^g/g and individual values of more than 1500 ^g/g, were reported in the kidneys of the Acipenser gueldenstaedtii, and the sterlet Acipenser ruthenus [Subbotkina, Subbotkin, 2002; 2003]. The lysozyme content in codfish is even lower than in cyprinids. The minimal level of the enzyme or its absence has been observed in cods inhabiting both salt and fresh water [Fletcher, White, 1973; Lie et al., 1989; Subbotkina, Subbotkin, 2003; 2013]. The Alaska pollock, Theragra chalcogramma (Pallas) (Fig. 2), is the species with the highest content of lysozyme in codfish [Subbotkina, Subbotkin, 2013].
It has previously been shown that lysozyme activity or content distribution in plaice, Pleuronectes platessa, rainbow trout, Salmo gairdnery, and common pike, Esox lucius, is the highest in the kidneys followed by spleen, serum, and liver [Fletcher, White, 1973; Lie et al., 1989; Izvekova et al., 2010]. The sturgeons, inconnu, Stenodus leucichthys, and pike, Esox lucius, have the same pattern of distribution as the cyprinids in the present study [Subbotkina, Subbotkin, 2003]. A similar distribution of enzyme among these organs, with none in the serum, has been shown in 17 species from the Volga River delta [Lukyanenko, 1989]. The higher or the same lysozyme activity in the liver compared to the spleen in carps has been noted by other authors
[Han et al., 2010; Li et al., 2013]. High levels of lysozyme in the kidneys and spleen is not the rule for all fish. In perch fish a high content of the enzyme have been identified in the serum [Subbotkina, Subbotkin, 2003; 2012]. The pattern of lysozyme distribution in these fish is as follows: kidneys > serum > liver > spleen (Fig. 2). Two flatfish species from the Sea of Okhotsk, including starry flounder, Platichthys stellatus, (Fig. 2) have the highest lysozyme levels in the serum [Subbotkina, Subbotkin, 2013]. The pattern of lysozyme distribution most likely reflects the role of the immune organs in functions of nonspecific defense in phylogenetically various fish groups.
The results of the present study of some cyprinids show that low and very low levels of lysozyme are found in the kidneys, spleen and liver and enzyme may be absent in the serum. The pattern of lysozyme distribution in many carp species is as follows: kidneys > spleen > liver > serum, but this will differ depending on the species of other related fish groups. Cyprinus carpio, Ctenopharyngodon idella and Hypophthalmichthys molitrix, and their related species are similar in the level of lysozyme in water bodies which are significantly differ by the temperature regime. The seasonal dynamics of serum lysozyme does not fully explain the changes of this enzyme in fishes. A review of the published data demonstrates a wide range of variability in the content or activity of serum lysozyme.
This research was partly funded by the Russian-Vietnam Tropical Research Centre (Project "Ecolan
E.3.2.6"). Another part of results was obtained in the framework of the state assignment (theme No. AAAA-
A18-118012690222-4). We are deeply grateful Diana Papoulias for improvement in our manuscript and
Nadezhda Ruban for help with English.
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