The Effect of the Incubation Temperature on Morphological and Physical Parameters of Hypophthalmichthys molitrix Erythrocytes and Polymorphonuclear Leukocytes

 

Alena Y. Frolova¹, Svetlana D. Chernyavskikh¹, Huu Quyet Do¹, Van Thanh Vo^1,2

¹Belgorod State University, Pobeda Street, 85, 308015, Belgorod City, Russia

²Ho Chi Minh City University of Education, 280 An Duong Vuongst., Ward 4, Dist. 5, Ho Chi Minh city, Vietnam

 

ABSTRACT:

The influence of temperature on the morphometric and physical features of Hypophthalmichthys molitrix erythrocytes and polymorph nuclear leukocytes was estimated by atomic force microscopy. It was observed that the area, volume and perimeter of Hypophthalmichthys molitrix hemocytes are not changed at low (down to 5°C) and elevated (up to 40°C) incubation temperatures, in comparison with incubation at temperature of 20°C, whereas the elasticity of blood cells – is reduced.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

INTRODUCTION:

Temperature is one of the most important abiotic factors of environment, adaptation to which is carried out through the implementation of a various physiological and biochemical mechanisms in living organisms[1]. The overall picture of changes, occurring in mammals and humans under conditions of acute overheating, was studied [2]. There have been many works that describe the influence of various factors on the morphological characteristics of fish hemocytes [3, 4].However, the issueabout the temperature effect on morphological and physical characteristics of fish erythrocytes and polymorphonuclear leukocytes is still poorly understood.

The purpose of our research is to estimate the temperature influence on morphological and physical parameters of erythrocytes and polymorphonuclear white blood cells in silver carp Hypophthalmichthys molitrix under in vitro conditions.

 

MATERIALS AND METHODS:

Our study utilized the peripheral blood of silver carp Hypophthalmichthys molitrix. Erythrocytes (E) and polymorphonuclear leukocytes (PMNL) served as the objects of the study. Fishes had been in aqueous medium at room temperature (20°C) before having taken out to collect their blood. Blood samples of Hypophthalmichthys molitrix were taken from the caudal vein. Heparin at concentration 10 U/mL was used for prevention of blood clotting. The obtained blood was centrifuged for 10 min at a relative centrifugal force of 400g. The suspensions of erythrocytes and leukocytes were diluted with isotonic saline (0.6% NaCl) [3], then they were incubated within 2 hours using a parallel three variants of temperature conditions: room (20^oC), low (5^oC) and elevated (40^oC).Upon completion of the incubation period, smears were done. From each batch of sample preparation, 20–25 erythrocytes and leukocytes were investigated, using atomic force microscopy (AFM). Hemocytes were scanned on atomic force microscope INTEGRA Vita (configuration based on the inverted optical microscope Olympus IX-71). Morphometric parameters of cells were determined in semi-contact scanning mode with a sweep frequency 0.6–0.8 Hz, using a cantilever of NSG03 series with stiffness 1.1 N/m and the radius of curvature of 10 nm [5]. The area (S, µm²), perimeter (P, µm), volume (V, µm³), large (D, µm) and small (d, µm) diameters of cells were measured from obtained scans. Using the software “Nova” (NT MDT, Zelenograd, 2009), these data were used to build the profile curves of the scanned cells. The cell adhesion (nN) was evaluated using the obtained curves. Elasticity (Young's Modulus, kPa) of erythrocytes and the PMNL was measured using the program “Image Analysis 3.5.0.2070”.

 

The experimental data were presented by arithmetic mean (M) and standard deviation (± m). The significance of differences of the obtained results was determined by the Wilcoxon-Mann-Whitney test (*, p≤0.05).All statistical data were processed using computer program IBM SPSS Statistics 20.

 

RESULTS:

Hypophthalmichthys molitrix erythrocytes after incubation under different temperature conditions are shown in fig. 1a–c.

 

 

Fig.1. Red blood cells of silver carp Hypophthalmichthys molitrix after incubation at temperature of: a – 5oC; b – 20°C; c – 40oC	Fig. 2. Polymorphonuclear leukocytes of silver carp Hypophthalmichthys molitrix after incubation at temperature of:a – 5oC; b – 20°C; c – 40oC

 

 

As the following figures, regardless of the incubation temperature, the surface of the red blood cells in silver carp are rough, in the nuclear zone – is convex. Hypophthalmichthys molitrix erythrocytes have an oval or elliptical shape, their dimensions are not statistical significant difference after incubating the various temperature conditions. The ratio of the average values of red blood cells’ large and small diameters (D × d) at temperatures of 5^oC, 20^oC and 40^oC were 11.2±0.8 × 7.8±0.6; 10.8±0.8 × 7.5±0.6; and 10.9±0.4 × 8.0±0.4, respectively.

 

Scans of Hypophthalmichthys molitrix polymorphonuclear leukocytes (regardless of the incubation temperature) showed that the roughness of PMNLs’ plasma membrane is more expressed than that in the red blood cells. The shape of white blood cells is more rounded than that in the red blood cells in all incubation temperature conditions. The values of D × d of PMNL at temperature of 5^oC, 20^oC and 40^oC were 6.9±0.5 × 5.8±0.4; 7.4±1.4 × 6.1±1.1 and 7.8±0.9 × 5.1±0.5, respectively (Fig. 2a–c).

Table 1 shows the parameters area, volume and perimeter of Hypophthalmichthys molitrix erythrocytes and PMNL, obtained after incubating at the above-mentioned temperatures.

 

 

Table 1: The morphometric characteristics of Hypophthalmichthys molitrix blood cells by different incubation temperatures

Note:E – erythrocytes; PMNL – polymorphonuclear leukocytes;S – area, V – volume, P – perimeter of blood cells;* – Significant differences in comparison with the temperature of 20^oC (p ≤ 0.05).

 

As it is showed from the table, the significant differences in experimented parameters between the silver carp erythrocytes groups were not found at incubation temperature, decreased to 5^oC, or increased to 40^oC, in comparison with temperature at 20^oC.A similar pattern is observed in Hypophthalmichthys molitrix PMNL, except for the volume, decreased by 38.7% when the incubation temperature increases to 40^oC, compared with those at 20^oC temperature.

 

Table 2 shows the parameters, characterizing shifts of the physical properties of silver carp erythrocytes and polymorphonuclear leukocytes by different incubation temperatures.

 

It was observed that a change in incubation temperature has no effect on the adhesion of Hypophthalmichthys molitrix red blood cells. Adhesion of leukocytes is changed only at low incubation temperature. Therefore, decreasing incubation temperature to 5°C, in comparison with at temperature of 20°C, contributes to increase of the adhesion of PMNL by 31.4%.

 

 

Table 2: Change of the adhesion and elasticity in Hypophthalmichthys molitrix hemocytes by effect of the temperature factor

Note:E –erythrocytes; PMNL – polymorphonuclear leukocytes;* – Significant differences in comparison with the 20^oCtemperature(p≤0.05).

 

The change in incubation temperature affects the blood cells’ elasticity of silver carp. Reducing the incubation temperature to 5°C and rising incubation temperature to 40°C, contribute to decreasing the elasticity of Hypophthalmichthys molitrix erythrocytes by 10.0% and 16.5%, of white blood cells – by 18.6% and 30.1%, respectively, compared with those at 20^oC temperature.

 

DISCUSSION:

Comparative estimation of the study results allowed to detect features of the temperature influence on the physical and morphometric properties hemocytes in silver carp Hypophthalmichthys molitrix under in vitro conditions. As it is showed from the experimental data, at different incubation temperatures, the surface plasmolemma of the red blood cells and polymorphonuclear leukocytes in silver carp is weakly expressed rough, which is typical for other lower vertebrates [3], in particular for fish [6]. We believe that the rough surface of hemocytes associated with disorganization of cytoskeleton elements and the formation of actin-binding domains in the submembrane space, determining generation invaginations or protrusions on plasmalemma [7–10]. As our research, the morphometric parameters of both red and white blood cells Hypophthalmichthys molitrix are not practically changed at increasing and decreasing the incubation temperature. Our results are consistent with Vovk P.S. studies [11] that the temperature tolerance ofHypophthalmichthys molitrix lies within the boundaries of the temperature range from 0^oC to 40^oC. According Opuszynski K. et al. [12], the upper temperature limit of the vital functions for silver carp is 43.5^oC or 46.5^oC, depending on the cultivation method.

Given that the major factors, causing changes in the physical and physiological properties of the blood cells, are the properties of plasmolemma [13, 14], we assume that the decline in its elasticity after incubation of the silver carp erythrocytes occurs due to changes in microviscosity of the lipid bilayers, phase distribution lipid, microenvironment protein, protein-lipid interactions, and other features of the structural membrane organization [14], manifesting small folding on the surface.

 

CONCLUSIONS:

1. The rough surface of the plasma membrane of Hypophthalmichthys molitrix erythrocytes and polymorphonuclear leukocytes, associated with the disorganization of cytoskeleton elements and the formation of actin-binding domains, was observed regardless of the incubation temperature.

 

2. Morphometric parameters of red blood cells and polymorphonuclear leukocytes in silver carp are not changed after the incubation at low (5^oC) and elevated (40^oC) temperatures, in comparison with incubation at temperature of 20^oC, except for the volume of white blood cells at a temperature of 40^oC.

 

3. The reduction of elasticity of Hypophthalmichthys molitrix erythrocytes and polymorphonuclear leukocytes was observed at decreasing or increasing incubation temperature by the background of stable adhesion.

 

PROSPECTS FOR FURTHER RESEARCH:

In the future, we plan to study the influence of temperature on the morphological and functional parameters of Cyprinuscarpio, Ctenopharyngodonidella and Carassiusgibelio hemocytes to find out the adaptation mechanisms of fish blood cells to extreme environmental factors.

 

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2.        Fedorova, M.Z. 2002. Functional properties and reactivity of leukocytes in altered states of the body, caused by factors of a different nature. Author. Dr. Biol. Sciences: 03.00.13; 14.00.16, Moscow.(In Russian).

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8.        Insall, R.H., Machesky, L.M. 2009. Actin dynamics at the leading edge: From simple machinery to complex networks. Developmental cell, 17(3): 310–322.

9.        Itoh, T., Takenawa, T. 2009. Mechanisms of membrane deformation by lipid-binding domains. Progress in Lipid Research, 48(5): 298–305.

10.     McMahon, H.T., Gallop, J.L. 2005. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature, 438(7068): 590–596.

11.     Vovk, P.S. 1979. Temperature and food adaptation of the Far East herbivorous fishes. Pacific Science Congress Proceedings, 41–42.

12.     Opuszyňski, K., Lirski, A., Myszkowski, L., Wolnicki, J. 1989. Upper lethal and rearing temperatures for juvenile common carp, Cyprinuscarpio L., and silver carp, Hypophthalmichthys molitrix (Valenciennes). Aquaculture Research, 3(20): 287–294.

13.     Harakoz, D.P. 2001. The possible physiological role of the phase transition “liquid-solid” in biological membranes. Advances of Biological Chemistry, 41: 333–364.(In Russian).

14.     Chernyavskikh, S.D., Nedopekin, S.V. 2013. Seasonal fluctuations of the relative microviscosity, polarity and sorption capability of Cyprinuscarpio and Rana ridibunda’s erythrocyte membranes. Belgorod State University Scientific Bulletin. Series Natural sciences, 3(146): 99–103. (In Russian).

 

 

 

Accepted on 14.02.2017           © RJPT All right reserved