Composition, structure and cytochemical characteristics of peripheral blood cells in Paralichthys olivaceus

doi:10.21203/rs.3.rs-1958174/v1

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Composition, structure and cytochemical characteristics of peripheral blood cells in Paralichthys olivaceus

https://doi.org/10.21203/rs.3.rs-1958174/v1

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The microstructures, classification, counting and size of peripheral blood cells in Paralichthys olivaceus were observed and measured by Wright-Giemsa staining, and the cytochemical characteristics of peripheral blood cells were investigated by different cytochemical staining methods. Besides, the transmission electron microscope was used to observe the ultrastructures of different cells. The results showed that erythrocytes, thrombocytes and leukocytes constituted the peripheral blood cells in Paralichthys olivaceus, and leukocytes were made of monocytes, neutrophils and thrombocytes. More heterochromatins were found in erythrocytes, thrombocytes and neutrophils, however, monocytes had more organelles with fewer heterochromatins. As for neutrophils, endoplasmic reticulums and phagocytic vesicles were abundant. The number of erythrocytes was the highest, and the size of lymphocytes and thrombocytes was the smallest; while neutrophils were the fewest in number but the largest in size. Lymphocytes were the most abundant in leukocytes, followed by monocytes and neutrophils. The cytochemical staining results showed that all leukocytes were positive for SBB. Most of the lymphocytes were positive for PAS, and monocytes were positive for PAS, ACP and POX. As for neutrophils, ACP and POX were positive. Both monocytes and neutrophils showed positive for SBB, ACP and POX, indicating that the two kinds of cells play a vital role in phagocytosis and bactericidal action. Only lymphocytes were positive for ALP, indicating that they were important in inflammation and immune response. Paralichthys olivaceus shared the characteristics similarities in peripheral blood cells with other fishes. However, obvious differences also existed, which reflected the species speciality. These results could enrich the haematological database of bony fish and provide scientific references for researching the cell function and evaluating the health status in Paralichthys olivaceus based on haematological parameters.

Paralichthys olivaceus

Peripheral blood cells

Microstructure

Ultrastructure

Cytochemical characteristics

Flounder Paralichthys olivaceus belongs to the order Pleuronectiformes, genus Bothidae. It is an important mariculture species in China and also the most productive mariculture species in Chile, Korea, Japan and Germany (FAO 2022). Due to the intensive farming of flounder rising, diseases occur frequently, causing economic losses seriously. The peripheral blood cells play an important role in the immune response of fish, which is an important part of the immune system to resist the pathogen infection (Ferdous and Scott 2015).

The peripheral blood cells are sensitive to environmental stimuli and can be used to assess the physiological or pathological status of fish, which is benefit to disease diagnosis (Furtado et al. 2019). Therefore, the study on the morphology, quantity and cytochemical characteristics of peripheral blood cells in fish is of significance for the detection of water environment, diseases diagnosis and production practice. The experimental procedure and judging standard of blood cell detection have been established in human and veterinary medicine, however, there is no uniform index in aquatic animal medicine. Fish is a lower vertebrate with too many species, and the composition and structure of peripheral blood cells vary greatly among species (Han et al. 2021). It is necessary to study the basic details of peripheral blood cells in a certain fish, especially in commercial fish.

Studies have been carried out on the classification, and morphology of peripheral blood cells in many fish species, such as Synechogobius hasta and Sebastes schlegelii (Han et al. 2021), Epinephelus akaara (Mofizur et al. 2019), different freshwater fishes (Ctenopharyngodon idella, Megalobrama amblycephala and Pelteobagrus fulvidraco) (Chen et al. 2019) and Salvelinus fontinalis (Suljević and Mitrašinović-Brulić 2020). The cytochemical characteristics of the peripheral blood cells have also been extensively investigated in Schizothorax prenanti (Fang et al. 2014), Centropomus parallelus (da Silva et al. 2011), Gymnocypris eckloni (Zheng et al. 2017), two Cyprinidae (Carassius auratus and Ctenopharyngodon idellus) (Zhang et al. 2019) and two marine fishes (Synechogobius hasta and Sebastes schlegelii) (Han et al. 2021). These studies have important significance for enriching the blood databases of aquatic animal. In addition, the study of cytochemical properties of peripheral blood cells can explore the composition of various cellular enzymes in different cells, thus revealing and providing functional information on immune defence in peripheral blood cells of different fishes. Nevertheless, the investigations on the cytochemical characteristics of peripheral blood cells in fish were not enough and comprehensive, which hinders the establishment of fish haematological detection systems.

At present, few studies have been reported on the composition, structure and cytochemical properties of peripheral blood cells in flounder (Paralichthys olivaceus). Consequently, the morphology, morphometry and cytochemistry of peripheral blood cells in flounder were studied in this study to enrich the haematology data of flounders, and it also provided basic data for immunological function research in blood cells and health evaluation of flounder.

Animals and staining kits

Healthy adult specimens of Paralichthys olivaceus (35-40 cm, 0.9-1 kg) were obtained from a flounder aquaculture factory, Lian Yungang, Jiangsu Province, China. Then, the fish were transferred to one aquarium and acclimatized for 7 days with flowing seawater at 10-15℃, 30‰ salinity, and the dissolved oxygen concentration at 7 mg.L^-1. After sequential feed restriction for two days, a total of ten individuals were used for the experiment. Neither visible lesions nor gross abnormalities were observed in any of the fish during the experiment.

All the staining kits, including WG (Wright-Giemsa), PAS (periodic acid Schiff), ACP (acid phosphatase), ALP (alkaline phosphatase), NAE (α-naphthol acetate esterase), SBB (sudan black B) and POX (peroxidase), were obtained from a commercial company (Solarbio, Beijing, China).

Blood smear preparation

Fish were anaesthetized with tricaine methane sulfonate (MS-222) for blood collection by caudal venepuncture with a syringe. The blood samples were immediately used to prepare blood smears and air-dried. For each staining method, ten blood smears per fish were prepared and performed with the subsequent staining method.

Staining methods

According to the staining method and judging method established in the laboratory (Han et al. 2021), the prepared blood smears were stained respectively. WG staining results were used for classification count and microscopic observation. PAS positive staining results showed red granules with blue nuclei. SBB positive staining results were black with purplish red nuclei. ACP positive reaction showed red granules with blue nuclei and AKP positive reaction was blue with red nuclei. POX positive reaction showed brown-black with blue nuclei. NAE positive reaction was black with green nuclei.

Microscopic observation

All the staining smears were photographed by Nikon 90i electric microscope to identify the blood cells by the cell size, nucleus size, nucleus morphology, karyoplasmic ratio and cytosolic colouration. The cell smears stained with WG were randomly divided into 3 parts, and 24 view fields were taken to count the cells at 400 magnification each part. 50 cells of different cell types were randomly selected to take pictures for measuring the size of cells and nucleus at 1000 magnification.

Ultramicroscopic observation

The isolation of peripheral blood cells in flounder was conducted according to the established method (Gao et al. 2015). The red blood cells and white blood cells were separated by the Percoll gradient solution. After then, the isolated leukocytes and erythrocytes were respectively fixed with phosphate buffer solution (0.1 M, pH 7.4) containing 1% osmium tetroxide for cell pellets according to the method of Cynoglossus semilaevis (Sha et al. 2017). The electron transmission microscope was used to observe the ultrastructure of erythrocytes and leukocytes.

Data analysis

The results were expressed in the form of mean ± standard deviation.

Morphology of different blood cells

The peripheral blood cells of Paralichthys Olivaceus consisted of erythrocytes, leukocytes and thrombocytes. Leukocytes were classified into lymphocytes, monocytes and neutrophils by WG staining (Fig. 1).

The mature erythrocytes were oval with evenly red cytoplasm, and the oval or round nucleus was located in the centre of the cell with dense and purplish red chromatin (Fig. 1a). The immature erythrocytes were occasionally visible with loose and light purple chromatin (Fig. 1b). The dividing erythrocytes could be observed during the staining (Fig. 1a and 1c).

The cytoplasm of the thrombocytes was hardly stained, exhibiting less greyish blue cytoplasm, and the elliptic (Fig. 1c) or short rod-shaped (Fig. 1d) nucleus was observed during the staining.

The cytoplasm of the lymphocytes was stained blue, exhibiting basophilic, and the nucleus was divided into two shapes, oval (Fig. 1e and 1i) or two-leaves shape (Fig. 1d, 1g and 1h) with dense and purplish red chromatin.

The monocytes were round with dark blue cytoplasm, and the nucleus was laterally biased with purple chromatin. The karyoplasmic ratio was more than 0.5 (Fig. 1f).

The neutrophils were round with cytoplasm containing bluish-purple granules, and the nucleus was divided into two shapes, two-leaves shape (Fig. 1i) or oval (Fig. 1j) with purple chromatin. The karyoplasmic ratio was less than 0.5.

The ultrastructure of different blood cells

Under the transmission electron microscope, the surface of erythrocytes was smooth, and the nuclear membrane and nuclear pores were clearly visible (Fig. 2a). The heterochromatin in the nucleus was abundant with sheet-like distribution along the inner side of the nuclear membrane and with a band-like distribution in the centre of the nucleus. The cytoplasm was uniform and compact with high electron density. No organelles were found in the cytoplasm of erythrocytes.

The thrombocytes had a clear nuclear membrane and nuclear pore with abundant heterochromatin in the nucleus. The cytoplasm was even and dense with high electron density, and no organelles were observed (Fig. 2b).

There were protrusions on the surface of the lymphocyte with a clear nuclear membrane (Fig. 2c). Little heterochromatin was present in the nucleus, and it was distributed patchily in the central nucleus and in bands along the inner side of the nuclear membrane. Low electron density and loose cytoplasm were present. The endoplasmic reticulum and vesicles were seen in the lymphocyte.

The surface of the monocyte was rough, and the heterochromatin in the nucleus was few, distributed along the nuclear membrane and scattered in the central nucleus (Fig. 2d). The cytoplasm was loose with low electron density. Mitochondria were abundant and cristae were clear in the Golgi apparatus, and a lot of vesicles were found around the trans-cystic cavity.

The neutrophils were characterized by finger-like protrusions with abundant heterochromatin in the nucleus (Fig. 2e). Golgi apparatus, ribosomes, and granules were observed in the cytoplasm with abundant rough endoplasmic reticulum and numerous vesicles.

Cell measurement and the relative account

The cell size and relative counts of the blood cells in Paralichthys olivaceus were listed in Tables 1 and 2, respectively. The size of peripheral blood cells and nuclei of flounder varied among different cells, with the largest neutrophil (10.01 ± 1.64) μm × (9.26 ± 2.02) μm, followed by monocytes, lymphocytes and thrombocytes, and the smallest erythrocytes were (8.97 ± 1.07) μm × (5.33 ± 0.76) μm. The per cent of erythrocytes in total blood cells was (97.54 ± 0.38)%, and thrombocytes accounted for (1.12 ± 0.19)%. The percentage of lymphocytes in the total number of leukocytes was (49.13 ± 2.67)%, followed by monocytes (36.65 ± 2.56)% and neutrophils (14.22 ± 0.12)%.

Table 1 The cell size and nucleus size of peripheral blood cells in Paralichthys olivaceus

Cell types	Cell size(μm)		Nucleus size(μm)
Cell types	Length	Width	Length	Width
Erythrocytes	8.97 ± 1.07	5.33 ± 0.76	3.94 ± 0.42	2.76 ± 0.47
Lymphocytes	7.76 ± 1.26	6.75 ± 0.97	5.86 ± 1.14	4.16 ± 0.52
Monocytes	9.47 ± 1.66	8.78 ± 1.24	6.46 ± 0.81	4.21 ± 1.07
Neutrophils	10.01 ± 1.64	9.26 ± 2.02	5.66 ± 1.10	4.52 ± 0.66
Thrombocytes	7.60 ± 1.81	4.29 ± 0.56	5.58 ± 0.74	3.92 ± 0.39

Table 2 The percent of the peripheral blood cells in Paralichthys olivaceus

Cell types	The relative percent(%)
Erythrocytes	97.54 ± 0.38
Leukocytes	1.34 ± 0.40
-Monocytes	36.65 ± 2.56
-Lymphocytes	49.13 ± 2.67
-Neutrophils	14.22 ± 0.12
Thrombocytes	1.12 ± 0.19

Note: -indicated the cell percentage in the leukocytes

Cytochemical properties of the blood cells

To further understand the cell populations, six different cytochemical stains were used to investigate the cytochemical characterizations, including a combination of enzymatic stains (ACP, ALP, POX and NAE) and nonenzymatic stains (SBB and PAS). The cytochemical findings of the present study were shown in Figure 3 and Table 3. All of the six cytochemical stainings of erythrocytes and thrombocytes were negative, and the results of cytochemical staining of different leukocytes differed.

The results of PAS staining revealed that monocytes had red glycogen granules in the cytoplasm (Fig. 3, a4), which was PAS-positive. Most lymphocytes were PAS-positive with irregular red glycogen granules distributed at the edges of cells, due to their large karyoplasmic ratio (Fig. 3, a3 L-o), while a few lymphocytes were PAS-negative with no red glycogen particles (Fig. 3, a3 L-n). All of the leukocytes were SBB-positive with obvious brown-black granules in the cytoplasm, showing sites of neutral lipid (Fig. 3, b3-b5). Monocytes and neutrophils showed ACP-positive with red granules in the cytoplasm (Fig. 3, c4-c5). Lymphocytes were ACP-negative with blue cytoplasm (Fig. 3, c3). After ALP staining, only the lymphocytes were positive with blue cytoplasm (Fig. 3, d3), while all other cells were negative with pale yellow. For the POX-staining results, monocytes and neutrophils were POX-positive with brownish black granules distributed in the cytoplasm (Fig. 3, e4-e5), while all other cells were negative (Fig. 3, e1-e3). All of the blood cells were negative for NAE with yellowish cytoplasm (Fig. 3, f1-f5).

Table 3 The cytochemical characteristics of peripheral blood cells in Paralichthys olivaceus

	PAS	SBB	ACP	ALP	POX	α-NAE
Erythrocytes	－	－	－	－	－	－
Lymphocytes	＋/－	＋	－	＋	－	－
Monocytes	＋	＋	＋	－	＋	－
Neutrophils	－	＋	＋	－	＋	－
Thrombocytes	－	－	－	－	－	－

Note: “＋” indicates positive staining, “－” indicates negative staining, “＋/－” indicates both positive and negative reactions

The peripheral blood cells of Paralichthys olivaceus consisted of erythrocytes, thrombocytes, lymphocytes, monocytes and neutrophils, and these results were consistent with previous studies (Chen et al. 2019; Sayed et al. 2020; Han et al. 2021).

The majority of the peripheral blood cells in Paralichthys olivaceus were mature erythrocytes. Occasionally, the immature erythrocytes and the dividing erythrocytes could be seen, these results were similar to those of the studies on Glyptosternum maculatum (Zhang et al. 2011), Acipenser sinensis (Gao et al. 2007), three freshwater fishes (Chen et al. 2019), two carnivorous fishes (Parupeneus forsskali and Thalassoma klunzingeri) (Sayed et al. 2020), indicating that the process of erythrocytes proliferation and maturation existed in the peripheral blood of different fishes. The reason might be explained by the fact that fish did not have bone marrow or other hematopoietic organs, therefore some erythrocytes would enter the peripheral blood to differentiate and maturate. It has also been established that erythrocyte contents determine the capacity of an organism to carry dissolved oxygen (Holland 1970; Val 2000; Witeska 2013; Graham et al. 1985). Under transmission electron microscopy, erythrocytes of Paralichthys olivaceus had no organelles, which were similar to those of Gymnocyprinis eckloni (Zheng et al. 2017), Schizothorax Prenanti (Fang et al. 2014), Ctenopharyngodon idellus, Megalobrama amblycephala and Pelteobagrus fulvidraco (Chen et al. 2019). Besides, the size of erythrocytes in Paralichthys olivaceus was smaller than that in the other two marine fish studies in our previous studies (Han et al. 2021). The size of erythrocytes is inversely proportional to the metabolic activity of an organism (Smith 1925). Therefore, erythrocytes in Paralichthys olivaceus with smaller sizes and no organelles were adapted to the higher oxygen consumption, which was the same as the previous studies (Maciak & Kostelecka-Myrcha 2011; Longeville & Stingaciu 2017; Parrino et al. 2018).

Thrombocytes were often thought to be indistinguishable from lymphocytes because of their similar morphology (Ellis 1977). In this study, the morphology of thrombocytes in Paralichthys olivaceus was significantly different from that in lymphocytes. Thrombocytes in fish had nuclei and played an important role in coagulation, antigen presentation and phagocytosis (Morrow and Pulsford 1980; Ferdous and Scott 2015), which was similar to the mammalian platelets. The number of thrombocytes in fish fluctuated greatly between individuals and was significantly influenced by smear preparation methods (Zinkl et al. 1991). In this study, a large number of thrombocytes could be congregated together occasionally, in addition, the number of thrombocytes was more than that in erythrocytes in one vision field. We thought that this was caused by the pressure of blood smear preparation when making the blood smear, which promoted the clotting reaction of thrombocytes. Therefore, according to the previous study (Hrubec et al. 2000), such regions were not selected for cell counting to reduce the effect of preparing pressure on cell counts.

The lymphocytes in the peripheral blood of Paralichthys olivaceus were the most abundant leukocyte (49.13%), the same as the previous results (Pronina et al. 2020). In the work of our previous study (Han et al. 2021), the per cent proportion of lymphocytes in leukocytes of Synechogobius hasta and Sebastes schlegelii reached 66.14% and 72.17%, respectively. It was proposed that variations in components of leukocytes in vertebrate species might be associated with species, age, season, and habitat conditions (Duguy 2010). Some studies have classified fish lymphocytes into big lymphocytes and small lymphocytes, for example, our previous studies have identified the two types of lymphocytes in two marine fishes based only on cell size rather than on functional characteristics (Han et al. 2021). In this study, no lymphocytes of different sizes were observed in Paralichthys olivaceus, however, two different nucleus shapes were found in the lymphocytes, which might be related to the different functional states or the different developmental stages of lymphocytes. In addition, few organelles and a large number of vacuoles were seen in lymphocytes, suggesting that lymphocytes had phagocytosis, which was similar to observations made by Brycon orbignyanus (Tavares-Dias et al. 2006) and Ictalurus punctatus (Tavares-Dias et al. 2007).

Monocytes played an important role in the immune system of fish (Okabe and Medzhitov 2016; Clauss et al. 2008) and they had a chemotactic and phagocytic ability, making them highly sensitive to environmental changes (Zheng et al. 2017). There were vacuoles in monocytes of Paralichthys olivaceus, and some monocytes extended into the surrounding cell space, indicating that monocytes were in the process of phagocytosis. Studies on the peripheral blood of Acipenser sinensis (Gao et al. 2007) and Oncorhynchus Tshawytscha (Lulijwa et al. 2019) have shown that monocytes had the ability to extend pseudopodia and engulf materials. In addition, monocytes had dense nuclear electrons, abundant mitochondria, Golgi apparatus and vesicles, suggesting that monocytes were in the process of protein synthesis.

Fish neutrophils could resist the invasion of extracellular pathogens through degranulation and formation of extracellular antimicrobial peptides, and these cell particles and antimicrobial peptides were able to target microbes more specifically (Rieger and Barreda 2011). The neutrophils of Paralichthys olivaceus were rich in phagocytic vacuoles and organelles, indicating that it had an active phagocytic function.

The lymphocytes of Paralichthys olivaceus showed both positive and negative PAS staining results, and PAS staining revealed the intracellular glycogen. Glycogen was an important source of cellular energy stores and played an important role in innate defence, especially in phagocytosis (Ueda et al. 2001). It was suggested that the intracellular glycogen in Paralichthys olivaceus lymphocytes changed greatly in different functional states, therefore some Paralichthys olivaceus lymphocytes might be PAS-negative. The monocytes of flounder were PAS-positive and the neutrophils were PAS-negative. However, granulocytes generally were phagocytic and PAS-positive (de Oliveira et al. 2021), which was inconsistent with the negative PAS in neutrophils of Paralichthys olivaceus, however, it showed the species specificity of Paralichthys olivaceus.

The SBB staining might display intracellular neutral lipid particles, which was an important structural material and energy source for cells (Chawla et al. 2017). The SBB positive results in lymphocytes of Paralichthys olivaceus suggested that lymphocytes could be supplied by neutral lipid. In addition, both monocytes and neutrophils were SBB positive. Combining the results of PAS staining in monocytes and neutrophils, we suggested that the phagocytic function of Paralichthys olivaceus neutrophils might be mediated by lipid rather than glycogen and that Paralichthys olivaceus monocytes could utilize both glycogen and lipid.

ACP was a lysosomal marker enzyme, with phagocytosis, sterilization and other functions. ALP was an important conserved defence enzyme that fights pro-inflammatory responses (Lallès 2020). POX was a heme-containing enzyme that eliminates the toxic effects of hydrogen peroxide and various small molecules such as methanol, ethanol, nitrite, or formic acid. Both monocytes and neutrophils in flounder were ACP-positive and ALP-negative, indicating that both the two cells lacked ALP enzymes and were rich in ACP enzymes, so they had strong phagocytic and bactericidal abilities. Both monocytes and neutrophils contained large amounts of peroxidase, which fitted their phagocytic and bactericidal functions and was consistent with studies in Ophiocephalus argus Cantor (Wang et al. 2021).

The peripheral blood cells of Paralichthys olivaceus were made up of erythrocytes, leukocytes and thrombocytes, and leukocytes were made of lymphocytes, monocytes and neutrophils, with lymphocytes making up the largest proportion. The number of erythrocytes was the highest, and the size of lymphocytes and thrombocytes was the smallest; while neutrophils were the fewest in number but the largest in size. No organelles in mature erythrocytes and thrombocytes were observed, and there were abundant organelles in leukocytes. Both monocytes and neutrophils were positive for SBB, ACP and POX, indicating that both types of cells played a strong phagocytic and bactericidal role in flounder. Only lymphocytes were ALP positive in the peripheral blood, suggesting that lymphocytes played a role in anti-inflammatory and immune responses. This study can enrich the data of haematology in Paralichthys olivaceus and expand the database of haematology of bony fish, so as to provide basic data for cell biological function and health assessment.

Author contribution

Funding acquisition and project administration: Yingli Gao; Writing-original draft preparation data: Lu Qiang; Methodology and writing-reviewing and editing: Yingli Gao, Xingqiang Wang and Lei Qin; Resources: Binlun Yan and Yingli Gao. All authors read and approved the final manuscript.

Funding

This work was supported by the Policy Guidance Program of Jiangsu Province (SZ-LYG202025; SZ-LYG202133), the Open Research Fund of Jiangsu Key Laboratory of Marine Biotechnology (HS2020003), the Scientific Research Foundation Program of Jiangsu Ocean University (KQ16032) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Data availability

All data generated during this study are included in this published article.

Code availability

Not applicable.

Ethics approval

All animal handling procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals Central of Jiangsu Ocean University, and the protocol was approved by the Institutional Animal Care and Use Committee of Jiangsu Ocean University.

Consent to participate

The authors agree to collaborate and publish this article.

Consent for publication

We will transfer the copyright of the article to editorial office for publishing.

Conflict of interest

The authors declare no competing interests.

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Composition, structure and cytochemical characteristics of peripheral blood cells in Paralichthys olivaceus

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